spine:

19.48 mm

Mass Screening Techniques for Selecting Crops Resistant to Disease

Mass Screening Techniques
for Selecting Crops
Resistant to Disease
This publication presents an overview of mass screening
techniques for the selection of disease resistant crops. Various
aspects are considered in choosing the most suitable selection
technique for specific plant–pathogen interactions. The careful
selection of the infectious agent is very much dependent on
its origin, method of preparation, content of active substances,
and ease of use. The publication also covers radiation induced
mutations, which in addition to in vitro and in vivo screening
methods, provide a secure and rapid means of developing
resistant genotypes for fruit trees, legumes, vegetables and
tuber crops, with greater emphasis on banana — dessert and
plantain — for its high nutritive value.

INTERNATIONAL ATOMIC ENERGY AGENCY

VIENNA
ISBN 978-92-0-105110-3

Atoms for Peace

P1458 Cover.indd 1 2010-05-18 15:24:49

Atoms for Peace
 Large figure: Sporulation of cucurbit powdery mildew (Golovinomyces cichoracearum)
on cotyledons of susceptible Cucumis sativus cv. Stela F1.

Circles: Detail of leaf discs with different degrees of infection

14 days after inoculation with Golovinomyces cichoracearum.

Photographs courtesy of A. Lebeda, Palacký University in Olomouc, Czech Republic.

P1458 Cover.indd 2 2010-05-18 15:24:49

 Mass Screening Techniques for
Selecting Crops Resistant to Diseases
MASS SCREENING TECHNIQUES
FOR SELECTING CROPS
RESISTANT TO DISEASES
Joint FAO/IAEA Programme of
Nuclear Techniques in Food and Agriculture

INTERNATIONAL ATOMIC ENERGY AGENCY

VIENNA, 2010
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For further information on this publication, please contact:

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International Atomic Energy Agency
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MASS SCREENING TECHNIQUES FOR

SELECTING CROPS RESISTANT TO DISEASES

ISBN 978-92-0-105110-3
© IAEA, 2010

Printed by the IAEA in Austria

May 2010
IAEA-TDL-001
 FOREWORD

Diseases caused by fungal pathogens are the major causes of crop loss. Crop diseases not only reduce
yield but can greatly impair the quality and stability of production year after year, undermining efforts
to promote sustainable agriculture. Environmental and health hazards, resulting from the application of
numerous chemical fungicides, are causing increasing concern. Well established crop breeding
schemes have for a long time allowed the development of tolerant and/or resistant varieties of valuable
food crops. However, these programmes are often long, fastidious and most of the time, fail to identify
the positive mutant. As advances are made in mutation breeding, biotechnology and gene techniques,
more rapid and cost efficient screening methods are needed to enhance the efficiency and success rate
of resistance breeding programmes.

Alternative selection methods rely on scaling up the number of plants screened while reducing the
space and time needed for such. Mass screening methods can be performed with well specified
pathogen inocula, filtrates or purified toxins applied to plant organs or their parts, and/or in vitro
produced plant material. The most important characteristic is that they mimic the infection process
under natural conditions. Whereas purified toxins can be more easily quantified and replicated than
inocula and crude extracts (filtrates), there are pathogens from which toxins have not yet been isolated
(mostly obligate biotrophic parasites). In addition, before utilizing a toxin (mostly produced by
necrotrophic pathogens) as a selection agent for disease resistance, it is important to determine
whether the toxin is an essential component of the pathogenicity and disease development. Some
toxins may not be needed for the full spectrum of pathogenicity and plants may be selected as toxin
resistant but disease susceptible. The use of controlled inoculations and crude extracts on explants
(e.g. leaf disks and detached leaves), or in vitro material, are more laborious than using toxins, but
may be the best choice for plant–pathogen interactions for which reliable toxins are not known.

The source of variation for plant resistance to pathogen can be natural or induced. The use of
mutagenic treatments is an effective way to induce plant variability for resistance to pathogens when
this cannot be obtained by conventional selection procedures and introduced into the cultivated
material through hybridization. The Joint FAO/IAEA Programme on Nuclear Techniques in Food and
Agriculture promotes the use of nuclear techniques in agriculture to sustain food security and rural
development. This book has been sponsored by the Joint FAO/IAEA Programme in recognition of the
importance of minimizing crop losses due to fungal diseases for the effective implementation of
sustainable agricultural practices, and to support mutation breeding programmes.

The impact of induced mutations on breeding of disease resistant cultivars is summarized in Chapter 1.
Different aspects that must be taken into consideration when choosing the most suitable in vitro
resistance screening method for a given plant–pathogen interaction are reviewed in Chapter 2.
Furthermore, in vivo and in vitro mass screening methods developed for fruits, legumes, horticultural
and tuber crops, with emphasis on banana (Musa spp.), are also compiled in this book. Banana and
plantain are an important source of food security and income in many tropical areas of the world.
Their vulnerability to diseases is augmented by the genetic uniformity of the commercial crops and the
absence of efficient resistance sources and genes. Moreover, cultivated triploid bananas are seedless
and breeding through conventional methods is challenging. Methods of resistance screening for two of
the most devastating diseases of banana (Black sigatoka and Fusarium wilt) are shown. Resistance
mass-screening in vivo and in vitro methods for root, vegetable, industrial, legume and fruit crops are
also presented.

3
 The IAEA would like to express its thanks to A. Lebeda (Czech Republic) for the revision of the
chapters. The IAEA officers responsible for this publication are M. Miranda and M.M. Spencer of the
Joint FAO/IAEA Division of Nuclear Techniques in Food and Agriculture.

EDITORIAL NOTE

The papers in this publication (including the figures, tables and references) have undergone only the
minimum copy editing considered necessary for the reader’s assistance.

Although great care has been taken to maintain the accuracy of information contained in this
publication, neither the IAEA nor its Member States assume any responsibility for consequences
which may arise from its use.

The report does not address questions of responsibility, legal or otherwise, for acts or omissions on
the part of any person.

The views expressed in this publication do not necessarily reflect those of the IAEA, the governments
of the nominating Member States or the nominating organizations.

Owing to their number, the references in this publication have been left, as an exception, in the
‘author-date’ style. They have not been converted into the IAEA’s numbered reference style.

The use of particular designations of countries or territories does not imply any judgement by the
publisher, the IAEA, as to the legal status of such countries or territories, of their authorities and
institutions or of the delimitation of their boundaries.

The mention of names of specific companies or products (whether or not indicated as registered) does
not imply any intention to infringe proprietary rights, nor should it be construed as an endorsement
or recommendation on the part of the IAEA.

The authors are responsible for having obtained the necessary permission for the IAEA to reproduce,
translate or use material from sources already protected by copyrights.
 CONTENTS

Chapter 1: Summary: Improvement of crop production and disease resistance through

mutation induction: the IAEA mandate ..............................................................................1
M. Spencer and A. Lebeda
Chapter 2: In vitro screening methods for assessing plant disease resistance ......................................5
A. Lebeda and L. Švábová
Chapter 3: Mass-screening techniques for early selection of banana and plantain crops
resistant to major diseases and pests .................................................................................47
B. Okole
Chapter 4: Mutation induction by gamma-rays and carbon ion beam irradiation in banana
(Musa spp.): a study with an emphasis on the response to Black sigatoka disease….......59
W.O. Reyes-Borja, I. Sotomayor, I. Garzón, D. Vera, M. Cedeño,
K. Degi, S. Nagatomi, A. Tanaka, Y. Hase, Y. Sekozawa, S. Sugaya and H. Gemma
Chapter 5: Rapid and mass screening of banana and plantain for resistance to
Black sigatoka disease using detached leaf and in vitro plantlets .....................................91
P.S. Ojiambo, M. Twizeyimana, A. Tenkouano and R. Bandyopadhyay
Chapter 6: In vitro selection for resistance to Fusarium wilt in Banana ……………….……… .....101
K. Matsumoto, M.L. Barbosa, L.A.C. Souza and J.B. Teixeira
Chapter 7: Selection parameters for resistance to Fusarium oxysporum f. sp.
cubense race 1 and race 4 on diploid banana (Musa acuminata) ....................................115
R. Morpurgo, S. Lopato, R. Afza and F.J. Novák
Chapter 8: Differential chitinase activity in banana cultivars as a response to
Fusarium oxysporum f. sp. cubense infection ................................................................129
R. Morpurgo, M. van Duren, G. Grasso and R. Afza
Chapter 9: Mass screening of mutants resistant to Alternaria blotch from in vitro-cultured
apple shoots irradiated with X-rays .................................................................................135
M. Suzuki and A. Saito
Chapter 10: The pineapple-Fusarium subglutinans interaction: an early selection system
for disease resistance .......................................................................................................159
O. Borrás-Hidalgo and R.S. Bermúdez
Chapter 11: Mass-screening techniques of some tropical crops for resistance to anthracnose
diseases using phytotoxic metabolites ............................................................................173
N.A. Amusa
Chapter 12: Screening of peas for resistance to Fusarium wilt and root rot
(Fusarium oxysporum, Fusarium solani) .......................................................................189
A. Lebeda, L. Švábová and R. Dostálová
Chapter 13: Mass-screening techniques for the early selection of disease resistance
in chickpea (Cicer arietinum) .........................................................................................201
S. Pande, J.N. Rao, M. Sharma, M. Pathak and P. Stevenson
Chapter 14: Improving tolerance to Fusarium oxysporum f. sp. melonis in melon using
tissue culture and mutation techniques ...........................................................................235
Y. Kantoglu, E. Seçer, K. Erzurum, İ. Tutluer, B. Kunter,
H. Peşkircioğlu, Z. Sağel
Chapter 15: Screening for resistance to lettuce downy mildew (Bremia lactucae) ............................245
A. Lebeda and I. Petrželová
Chapter 16: Screening for resistance to tomato powdery mildew (Oidium neolycopersici) ...............257
A. Lebeda and B. Mieslerová
Chapter 17: In vitro and in vivo selection of black pepper (Piper nigrum) mutants
tolerant to Fusariosis .......................................................................................................267
O.F. Lemos, A.Tulmann Neto, J.C. Albino, M.C. Poltronieri and A. Ando
Chapter 18: Screening for resistance to cucurbit downy mildew (Pseudoperonospora cubensis)..…285
A. Lebeda and J. Urban
Chapter 19: Screening for resistance to cucurbit powdery mildews (Golovinomyces
cichoracearum, Podosphaera xanthii)............................................................................295
A. Lebeda and B. Sedláková
Chapter 20: Screening onions and related species for resistance to Anthracnose
(Colletotrichum gloeosporioides) ....................................................................................309
G. Galván
CONTRIBUTORS TO DRAFTING AND REVIEW..........................................................................321
 Chapter 1

SUMMARY

Improvement of crop production and disease resistance through mutation

induction: The IAEA mandate

M. Spencer1, A. Lebeda2
1
Joint FAO/IAEA Division of Nuclear Techniques in Food and Agriculture
International Atomic Energy Agency
Vienna, Austria
2
Palacký University in Olomouc,
Olomouc-Holice, Czech Republic

The post “green revolution” era of the 20th century with the numerous success stories in various parts
of the world has fostered a new “gene revolution”, i.e. a more science based agronomy approach in
order to warrant food security for an increasing world population in the actual context of global
warming, climate variability and change, the sustainable intensification and improvement of crop
production systems regarding the selection of resistance and/or tolerance to stresses should be more
than ever the primary goal in plant breeding [1, 2]. This new vision of agriculture prompts the
scientific community together with the policy makers to reconsider and redefine agriculture practices
for the Third Millennium. Among the multiple threats to agriculture, the increase of temperature
associated with the increased humidity, due to displacement of rain isohyets will have a tremendous
impact on all living creatures including microbial pathogens [3].

The Joint FAO/IAEA Division, by promoting the use of nuclear techniques including mutation
induction as tools for breeding new improved crop varieties, has contributed to enhance the impact of
this science based approach in agriculture in developing countries [4]. The IAEA has provided several
hundreds of agricultural research institutes in Member States enhanced capacity in terms of human
resources as well as in providing high performance germplasm through the development of several
thousands of interesting mutants in crop plants [5, 6]. These mutants with desirable characteristics
have either been directly released for cultivation or used in hybridization programmes with other
mutants and/or cultivars to develop new elite genotypes/cultivars [7]. The success of these mutation
breeding programmes relies on the selection of “positive mutant lines” bearing the trait of interest.

On the other hand, it is clear now that many wild/original plant genetic resources vital for the present
and future agricultural development are threatened by genetic erosion due to the spread of modern
commercial agriculture. This, combined with the climate changes mentioned above, has increased the
vulnerability of existing crops to pests, pathogens and environmental stresses. This situation led the
FAO’s International Technical Conference on Plant Genetic Resources, the World Food Summit Plan
of Action and the International Treaty on Plant Genetic Resources for Food and Agriculture to
strongly recommended that increased attention be given to building capacities to characterize,
evaluate, improve and use plant genetic resources in a sustainable manner. Therefore a project was
designed under the subprogramme on Sustainable Intensification of Crop Production with the overall
objective to: Develop early mass screening techniques to obtain banana plants tolerant to fungal
diseases. In fact, the selection of the proper mutants has been the bottleneck in all mutation induction
breeding programmes, and the difficulty is enhanced when it comes to screening for disease resistance
[8]. It is therefore absolutely necessary to establish reliable, rapid and high throughput screening
techniques for disease resistance in local germplasm as well as in putative mutant lines. Recent

7
1
projects represent a continuation of some previous IAEA activities which were focused on induced
mutations against plant diseases [9].

Thus, papers presented in this book highlight increasing cross-cutting techniques using plant tissue
culture, irradiation-induced mutation, molecular markers technology, isolation and characterization of
mutants for the selection of disease resistant lines. The compilation of this volume has demanded an
active participation of a number of breeders, plant physiology and phytopathology specialists. The task
of correspondence with authors of the chapters in this volume and the cooperation among participants
in the preparation of the manuscripts have led to very extensive and well documented chapters, which
will certainly serve the purpose of facilitating the development of successful disease resistance
mutation breeding programmes.

This book contains a total of 20 chapters. The first two chapters review the impact of induced
mutations and in vitro selection on breeding of disease and pest resistant cultivars. Various aspects
must be considered while choosing the most suitable in vitro selection technique for a given plant-
pathogen interaction. The choice of selection agent is very much dependent on their origin, method of
preparation, content of active substances, and effective use for screening or in vitro selection.
Furthermore, the book covers radiation induced mutations, in vitro and in vivo mass screening
methods developed for fruits, legumes, vegetables, and tuber crops, and with greater emphasis on
banana (Musa spp.) having 6 chapters owing to it as a source of nutrition, food security and great
impact on socio-economic aspects. Methods of screening against the most deadly disease of banana
(Black sigatoka and Fusarium wilt), which are a great threat to sustainable banana production, are well
covered. The book also includes various screening techniques (in vitro selection against Black
sigatoka in banana by using fungal toxin juglone) for the selection against the deadly diseases of crops
such as vegetables (melon and other cucurbits, onion, tomato, lettuce), industrial crops (black pepper),
legumes (chickpea, peas, soybean), fruits (apple, pine apple), and tropical crops (cassava, cowpea,
maize, and yam).

2 8
 REFERENCES TO CHAPTER 1

[1] Collinge DB, Munk L, Cooke BM (2008). Sustainable disease management in European context.
Springer, Dordrecht, Netherlands.
[2] Singh DP, Singh A (2005). Disease and insect resistance in plants. Science Publishers, Enfield
(NH), USA.
[3] Garrett KA, Dendy SP, Frank EE, Rouse MN, Travers SE (2006). Climate change effects on plant
diseases: genomes to ecosystems. Ann. Rev. Phytopathol. 44: 489-509.
[4] http://faostat.fao.org/site/384/default.aspx
[5] Maluszynski M, Nichterlein K, Van Zanten L, Ahloowalia BS (2000). Officially released mutant
varieties – The FAO/IAEA Database. Mutation Breeding Review 12: 1 – 84.
[6] http://www-naweb.iaea.org/nafa/publications/index.html.
[7] http://www-mvd.iaea.org/MVD/default.htm
[8] Strange RL, Scott PR (2005) Plant Disease: A threat to Global Food Security. Ann. Rev.
Phytopathology 43:83-116.
[9] Anonym (1977). Induced mutations against plant diseases. IAEA, Vienna.

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3
 Chapter 2

In vitro screening methods for assessing plant disease resistance

A. Lebeda1* and L. Švábová2
1
Palacký University in Olomouc, Olomouc-Holice, Czech Republic
2
Agritec Plant Research, Šumperk, Czech Republic.
*Correspondence: [email protected]

Abstract

A combination of biotechnological and phytopathological techniques provides an alternative approach

to classical resistance breeding methods. Such techniques have been increasingly used since the 1980s,
in parallel with the progress in plant biotechnology. In the approach of resistance screening and
selection in vitro, both experimental objects, i.e., the plant and the pathogen, must first be transferred
to in vitro conditions, and finally, the plant material must be transferred back to in vivo conditions and
adapted to the outside settings. Specific attention must be paid to the methods of pathogen preparation
for use in screening and selection in vitro. The selection agents are classified according to their origin,
the methods of preparation, nature and content of active substances, and effective utilisation for
screening or selection in vitro. Basic principles and methodological aspects of the in vitro work
(explant cultures, sources of in vitro variability, screening and selection methods, types of selection
agents) as well as examples of practical applications in the breeding of different crops are critically
reviewed in this chapter.

INTRODUCTION

The availability of sufficient genetic variability (i.e., biodiversity) and efficient selection procedures
are two essential prerequisites in plant breeding. Genetic variability can be broadened by utilizing
wild, related species in conventional crosses, whereas novel genetic characters can be obtained
through mutation induction techniques or somaclonal variation. The latter two methods are
particularly interesting if the desirable trait is not present in wild related species or if it cannot easily
be introgressed through conventional breeding. Mutants and somaclonal variants can be selected, for
instance, for disease resistance by using an appropriate selection agent (potentially derived from a
pathogen). DNA alterations (i.e., mutations) that affect the gametes can be transmitted to the progeny;
thus, the selected plant material is a new valuable source of genetic variability for the improvement of
plant resistance to pathogens.

Selection of plants for disease resistance/tolerance is as old as agriculture itself. Plant breeders
working in cooperation with plant pathologists must first decide on the priorities and methodologies in
breeding for resistance to diseases. A basic knowledge about the biology of the causal agent and its
relationship with the host plant is essential. This information is necessary for the development of
suitable methods of screening and selecting for resistance (Russell, 1978). There is a broad range of
different methodological approaches available to detect resistant genotypes and to select plants with
improved resistance (e.g., Király et al., 1974; Dhingra and Sinclair, 1986; Lebeda, 1986; Trigiano et
al., 2004; Singh and Singh, 2005), among which in vitro screening is one of the most high-throughput
and efficient methods (Švábová and Lebeda, 2005).

Tissue culture or in vitro methods are being used effectively in many basic and applied areas of
research in plant biology. The first studies to focus on plant-pathogen interactions in vitro were
performed in the 1940s (Helgeson and Deverall, 1983), and several attempts to apply these methods to

10
5
plant breeding were made (Day, 1980). The possibility of selecting plants for resistance in cultures in
vitro was probably demonstrated for the first time by Carlson (1973), who used methionine
sulfoximine (MSO) as an agent for selecting cells and protoplasts of tobacco. Since that time,
substantial progress has been made in this field, yielding a large number of in vitro-selected
germplasm with the potential for developing novel disease-resistant plants (Daub, 1986). The
development of this methodology is due primarily to the progress of plant tissue-culture methods.
Investigation of host-pathogen interactions in vitro is a very useful and efficient way of gaining a
better understanding of the factors that influence and are responsible for disease development (Ingram
and Helgeson, 1980; Helgeson and Deverall, 1983; Huang, 2001). However, in vitro selections can
also be difficult and time-consuming. In many articles published since 1980, many of the problems
related to the theoretical and practical approaches of in vitro selections and their usefulness for plant
breeding have been addressed (e.g., Shepard, 1981; Wenzel, 1985; Daub, 1986; Buiatti and Ingram,
1991; Graniti, 1991; for a review, see Švábová and Lebeda, 2005). This chapter provides an overview
of the basic principles and methodology of in vitro selections for disease resistance in plants. Data,
strategies and ideas related to the use of in vitro screening that have been published over the last two
decades are summarised and discussed here. This review also includes conclusions and future
prospects in this area.

METHODOLOGY

In vitro cultures of plants

In vitro cultures can be categorised according to: (1) purpose (micropropagation, conservation of
genetic sources, reduction of pathogen transmission, induction of mutations, regeneration after genetic
manipulations, production of various metabolites: e.g., biopharmaceuticals etc.); (2) type of explants
(parts of roots, stems, leaves, apical tissues, floral parts; gametes; meristematic regions – lateral buds,
shoot tips, immature cotyledons, zygotic embryos); and (3) type of culture (protoplasts, cell
suspensions, calli, organogenic, embryogenic) (Bhojwani and Razdan, 1983).

When used in the selection of pathogen resistance, in vitro cultures can be grouped based on: (1)
purpose (screening for stress tolerance, biochemical studies of host-pathogen interactions, selection of
resistant/tolerant lines); and (2) type of selection agent (pathogen, modified pathogen, culture filtrate,
phytotoxin/pathotoxin, elicitor) (Švábová and Lebeda, 2005).

In vitro selection methods utilise a combination of various types of plant organs or in vitro explants
with different kinds of selection agents which may (under optimal conditions) trigger reactions similar
to the responses of the plant to the pathogen. When subjected to the appropriate selection agent, plant
organs or tissues that survive the selection pressure are potential sources of tolerant/resistant
subclones. The difference between the selected tolerant/resistant lines and the original plant material
may originate from somaclonal variation or induced mutagenesis (see below).

Sources of in vitro variability and stability

Tissue culture- or mutation-induced variations are manifested in three ways: (1) variation within cell
cultures, in which individual cells within a culture may vary in morphology and genetic make-up; (2)
primary regenerants showing a non-heritable phenotypic effect, but that can be maintained as
asexually propagated plants if the variation is somatically stable; and (3) heritable variation that is
sexually transmitted to the offspring in a seed-derived population (Kaeppler et al., 1998; cf. Jain,
2001). Depending on the crop, the last two types of variation may be efficiently used for agricultural
purposes.

Somaclonal variability is a very important character in explant cultures where the step of de-
differentiation (callogenesis) is included. Variability of calli subclones and cell lines occurs
spontaneously (Kharabian and Darabi, 2005) or it can be induced and subsequently selected. As a
result of somaclonal variation, plants regenerated from in vitro cultures show miscellaneous variation

6 11
in phenotype, such as in plant height (elongation/dwarfism) and changes in plant architecture
(Tremblay et al., 1999); flower shape, alteration in pigmentation, and number of leaves and branches
(Bhatia and Ashwath, 2004); yield potential and fruit size (Tang and Tai, 2001; Ravindra et al., 2004);
as well as various levels of resistance to abiotic stresses, e.g., drought (Bajji et al., 2004), and biotic
stresses, including diseases and/or pathogens (Isaac, 1991; Evenor et al., 1994; Keller et al., 1994;
Sebastiani et al., 1994; Dan and Stephens, 1995; Jin et al., 1996; Dugdale et al., 2000; Mehta and
Angra, 2000; Mohanraj et al., 2003; Ostry and Ward, 2003). The same range of phenotype variations
can be expected as the result of induced mutations.

The variation in somaclones can be either of genetic or epigenetic origin. The tissue culture system
itself acts as a mutagenic system because cells experience traumatic conditions during explant
isolation and culture, and may undergo a type of re-programming during plant regeneration that is
different to that under natural conditions (Jain, 2001). A situation in which explants were put into
stressful in vitro conditions was nicely illustrated in the paper of Gaspar et al. (2002). Unusual culture
and environmental conditions, mechanical perturbation, wounding, possible air embolism due to
dissection, osmotic shock caused by, for instance, a high sucrose content in the medium, abnormal
mineral nutrition, unusual hormonal treatment, high relative humidity and accumulation of various
gasses (ethylene) in the culture flasks, are all factors that lead to oxidative stress that may result in
spontaneous mutations. Such mutations include hyper- or hypo-methylation, polyploidy/aneuploidy,
chromosome strand breakage, chromosome rearrangements, transposon activity and DNA base
deletions/substitutions. Similar results were also recorded by many other authors; Lopez et al. (2004)
reported chimeric mutants in cocoa (Theobroma cacao) as slippage mutations or allele loss, gene
amplification and gene methylation (Jain, 2001; Martin et al., 2001), random changes in genomic
organisation during differentiation (Sonyia et al., 2001), rearrangements at the DNA level (Sanchez-
Teyer et al., 2003) and chromosomal aberrations (Singh, 2003; Kharabian and Darabi, 2005).

Variability in somaclones can be induced and enhanced by exposure to mutagenic substances, such as
ethyl methanesulfonate (EMS), sodium azide (Hunold et al., 1992), gamma- and X-rays (Saito et al.,
2001; Mangal and Sharma, 2002; Jain, 2005). Genetically stable mutants are considered as a valuable
source of increased genetic variability for various characters, including biotic stress tolerance.

On the other hand, spontaneous somaclonal variation may be disadvantageous in cultures where the
genetic identity must be preserved; that is the case in the micropropagation of commercial material
where high genetic stability is an essential prerequisite. Genetic stability in vitro is closely dependent
on organised ontogenesis in embryonic and meristematic cultures, where the period of de-
differentiation and calli growth is eliminated (Novák, 1990). Genome fidelity in long-term tissue
cultures was confirmed, for example, in silver birch (Betula pendula) (Ryyanen and Aronen, 2005)
and tomato (Lycopersicon esculentum) (Sonyia et al., 2001). Methylation of genes involved in cell
differentiation and progressive elimination of cells that are capable of differentiation is proposed to be
responsible for the progressive loss of organogenic potential (Jain, 2001). In long-term cultures of pea,
such as a 20+ years old multiple-shoot culture of cv. ‘Bohatýr’, the material was thought to be
genetically stable following the use of molecular markers. Repetitive microsatellite sequences did not
show any differences between the cultured and original material (Griga et al., 2004); neither did inter-
retrotransposon amplification polymorphism (Smýkal et al., 2005). Nevertheless, significant
differences were found in the DNA methylation level in this long-term pea culture compared to
controls. The possible consequences of increased methylation in this long-term pea culture include
problematic rooting, and retarded growth and ontogenic development, which were observed after
transfer ex vitro.

In vitro selection

The perfect model of in vitro selection for disease resistance should comprise: (1) an explant culture
able to generate genetic variation (or an in vitro mutation induction system) with high ability to
regenerate resistant/tolerant, genetically stable fertile plants; (2) an easy to obtain selection agent
which induces similar biochemical reactions as the pathogen in vivo. Backward assessment

12
7
(verification) of resistance/tolerance of lines can be proved in vitro via co-cultivation with the
pathogen isolate (reduced growth of hyphae, reduced weight of mycelia as compared to control), and
via greenhouse and field tests; and (3) molecular tools to characterise the selected resistant lines at the
DNA level.

The general scheme for experiments which combines biotechnological and phytopathological
approaches also has to include the following steps: (1) collection of pathogen isolates and their
biological characterisation; (2) establishment of pathogen culture; (3) derivation of an effective
selection agent; (4) establishment of the plant tissue culture; (5) testing of the effect (phytotoxicity) of
the selection agent on the cultures in vitro and a statement of the selection concentrations for a
particular plant-pathogen model; (6) serial selection cycles in vitro; (7) regeneration of explants
surviving selection pressure to plants; (8) testing in vitro, in vivo and heritability analysis; and (9)
assessment in field conditions under natural infection (Figure 2.1).

In vitro screening methods

A major problem associated with the evaluation of somaclonal variation for disease resistance is the
availability of efficient, reliable screening methods (Sebastiani et al., 1994). The effect of the selection
agent (pathogen culture, culture filtrate, phytotoxin, etc.) must be demonstrated in a preliminary
experiment, where a suitable concentration range allows for a comparison of the toxic effects on
susceptible and tolerant/resistant germplasm. The outcome of such preliminary experiments is to
ascertain the precise dosage of selection agent that is optimal for screening resistant material, while
killing or drastically reducing growth of susceptible material. These preliminary experiments must be
performed for each combination of plant tissue culture and selection agent combination.

The effect of the selection agent can be assessed via various parameters, e.g., inhibition of the culture
weight growth, percentage of regenerating explants, percentage of necrotic explants, colour or
morphological changes evaluated with computer image analyses, evaluation of cell viability with 2,3,5
triphenyl tetrazolium chloride (TTC) vital staining (Hollmann et al., 2002) or 3-(4,5-dimethylthiazol-
2-yl)-2,5-diphenyl tetrazolium bromide (MTT) colorimetric assays (Kodama et al., 1991), and staining
of polysaccharides, callose, polygalacturonases and beta-glucanases (Storti et al., 1991; Simoni et al.,
1995; Li et al., 1999). The quantitative values should be further assessed via statistical analyses.

It is necessary to develop suitable techniques for in vitro cultivation of a particular host plant and to
devise the necessary selection schemes. The principles of these techniques have been described in
detail (e.g., Bhojwani and Razdan, 1983; Pierik, 1987). In Table 2.1, the basic methods for aseptic
cultures of plant organ or plant tissues, which may also be used for studies of host-pathogen
interactions and resistance screening are summarised. Different techniques were developed for in vitro
plant resistance screening. In general, intact plants or various parts of plants grown in aseptic culture
can be used for application of one or more selection agents in testing for resistance in vitro. Intact
plants may be grown or co-cultivated in tubes with nutrient solution and agar inoculated with fungus
(Lebeda and Buczkowski, 1986). This method has been efficiently used for resistance screening and
selection of peas (Pisum sativum) against Fusarium spp. (Lebeda and Švábová, 1997), but also for
physiological studies of resistance (Luhová et al., 2002). Cultures of excised organs and/or isolated
cotyledons, leaves, stems, shoots, fruits and roots have frequently been used for resistance screening in
crop-pathogen interactions (Table 2.2, Figure 2.2; Russell, 1978; Barlass et al., 1986; Lebeda, 1986;
Saindrenan et al., 1990; Remotti and Löffler, 1996). The most frequently used in vitro screening
techniques are plant tissue (calli, shoots) cultures (Helgeson and Haberlach, 1980; Storti et al., 1992;
Koike et al., 1993a; Ahmed et al., 1996; Prachi-Sharma and Singh, 2002; Singh et al., 2003); cell
suspension (Ishida and Kumashiro, 1988; Krämer et al., 1988; Koike and Nanbu, 1997; Pedras and
Biesenthal, 2000) and protoplast cultures (Shepard, 1981; Sjödin and Glimelius, 1989; Koike et al.,
1993b; Li et al., 1999); as well as anther and pollen grain culture (Bino et al., 1988) (see Figures 2.3-
2.7). After regeneration, the selected plants typically show a variety of phenotypes (Figure 2.8). These
techniques have been applied to various crops, and the selected regenerants provided a source of
germplasm with improved resistance to important diseases (Table 2.2; Švábová and Lebeda, 2005).

8 13
Selection agents for in vitro screening

There are two essential prerequisites for a pathogen-derived selection agent to be considered useful for
in vitro screening of disease resistance: (1) One or more compounds found in the selection agent
should be present in infected plants; and (2) the ability of the agent to cause at least part of the disease
symptoms when inoculated into healthy plants (modified from Aducci et al., 1997). A comparison
between the plant-pathogen interaction and the reaction of in vitro-cultured material to the selection
agent should, ideally, result in a similar spectrum of pathogenesis-related proteins and/or enzymes
commonly induced in both models (Angelini et al., 1990; Lebeda et al., 2001; Luhová et al., 2002).

Natural pathogen isolates

It is possible to utilise a live pathogen for screening disease resistance in vitro. However, when
explants are exposed to inoculation with a natural pathogen in vitro, the multiple stress factors caused
lead, in most cases, to death of the plant material, which is overgrown and devastated by the pathogen
(Ingram and Helgeson, 1980; McComb et al., 1987; Heath-Pagliuso et al., 1988, 1989; Lebeda and
Švábová, 1997). The conditions in vitro (higher humidity, reduced air velocity, media rich in nutrients)
are very hospitable and favour growth of microorganisms in general. In a number of studies, different
live pathogens were tested as agents for in vitro selection, but were found to be too devastating for the
plant tissues/organs and, therefore, of limited use.

Several successful experiments have been carried out in vitro with live inoculums i.e., by Clavibacter
michiganensis (Bulk et al., 1991), Xanthomonas campestris (Hammerschlag 1990), Plasmodiophora
brassicae (Buczacki, 1980), Mycosphaerella musicola (Trujillo and De Garcia, 1996), Alternaria
alternata (Takahashi et al., 1991), Fusarium solani (Huang and Hartman, 1998), Colletotrichum
trifolii (Cucuzza and Kao, 1986), Peronospora tabacina (Tuzun and Kuć, 1987), Phytophthora
cinnamoni (McComb et al., 1987; Cahill et al., 1992) and powdery mildews (Blumeria graminis,
Erysiphe pisi, Sphaerotheca pannosa, Uncinula aceris) (Webb and Gay, 1980). Therefore, the
pathogen is mostly used more or less modified to various selection agents.

Modified fungal cultures

Inactivation of the pathogen is often applied to circumvent the problems associated with excessive
growth of the pathogen during in vitro selection, which leads to the unselective death of the plant
material. After some time in flasks or Petri dishes, the culture containing the pathogen isolate is
inactivated by autoclaving, and then overlayed with an additional layer of medium; together, these
layers form a so called “double layer” (Ahmed et al., 1991; Švábová and Griga, 1997).

Inactivated cultures represent a complex mixture including small portions of the pathogen isolated.
Crude culture filtrates can be obtained after cultivating agar disks with fungal mycelia on the surface
of the liquid nutrient solution and filtering through a layer of cellulose wadding. Culture filtrates
contain a mixture of fungal metabolites and are very often used for selection, with many examples of
positive results (Cvikrová et al., 1992; Mezzetti et al., 1992; Jayasankar and Litz, 1998; Prachi-
Sharma and Singh, 2002; Thakur et al., 2002; Singh et al., 2003).

Fungal culture filtrates may contain a spectrum of secondary metabolites, such as polysaccharides,
oligosaccharides (Peros and Chagvardieff, 1987), proteins, glycoproteins, unsaturated fatty acids, stem
from the cell walls, cytoplasm of the bacteria or fungi, growth regulators such as auxin, kinetin and
gibberellic acid (Gentile et al., 1992), along with toxins that may play a role as co-determinants of
pathogenicity during disease development (Buiatti and Ingram, 1991; Crino, 1997; Švábová and
Lebeda, 2005). Toxins were found in various filtrates of the two main types of plant-pathogen
interactions, i.e., host-selective and non-host-selective. The application of filtrates to cultures in vitro
can trigger the elicitation of various defence responses, e.g., phytoalexins; activity of certain enzymes
(Saindrenan et al., 1990; Crino, 1997; Lebeda et al., 2001); accumulation of phenolic acids (Cvikrová

14
9
et al., 1992), total phenols, peroxidases and beta 1,3-glucanase (Lebeda et al., 2001; Singh et al.,
2003); and chitinase (Jayasankar and Litz, 1998).

It is well known that inoculation with culture filtrates of some plant pathogenic fungi can produce
disease-like symptoms and may also be used to select for resistance (Wenzel, 1985; Daub, 1986;
Buiatti and Ingram, 1991; Crino, 1997; Švábová and Lebeda, 2005). Culture filtrates are mostly
produced by fungal cultivation in liquid media and subsequent separation of the solid and liquid parts
of the culture. The liquid part of the culture is used as the selection agent. This approach yields a
variety of selection agents whose composition ranges from the absence of any toxin in the filtrate (in
which cases the symptoms are caused by other fungal metabolites) to filtrates that contain
uncharacterised active toxins (Daub, 1986). Crude culture filtrates allow easy bioassay and screening
for toxic effects on plants, cuttings, leaf disks or even cell suspension cultures of the host species
(Isaac, 1991). Crude culture filtrates have been used as selective agents in numerous disease resistance
studies in which they exhibit phytotoxic activity (Chen and Swart, 2002; Švábová and Lebeda, 2005).
Phytotoxicity of cell-free culture filtrates of F. solani f. sp. glycines has been proved, for instance, in
cuttings of soybean (Glycine max) seedlings (Hartman et al., 2004).

The application of metabolites with various levels of toxicity in in vitro selection for resistance has
some advantages when compared with natural conditions (Nedělník and Řepková, 1998): (1)
unfavourable weather and climate conditions are avoided, thus enabling easier and more precise the
assessment of quantitative differences in polygenic traits; (2) a large number of individuals can be
tested in a small space; (3) it is easier to manipulate with large populations of mutants, haploids, and
somaclones with higher variability in the genome; and (4) mass screening of mutants for resistance is
facilitated.

Phytotoxins and pathotoxins

An alternative approach to crude extracts is to purify substances from fungal or bacterial cultures that
have a toxic effect on the plants. The toxic substances, known as phytotoxins, first need to be
identified, then separated from crude extract and purified. Phytotoxins that are known to play a role in
pathogenesis are called pathotoxins (Graniti, 1991; Švábová and Lebeda, 2005). Particularly useful are
the non-host-selective toxins that act primarily by inhibiting active plant defence processes (Buiatti
and Ingram, 1991). A number of different phytotoxic substances have been used as selection agents in
resistance screening, e.g., a toxin from Drechslera teres for selection of barley (Hordeum vulgare)
calli cultures (Hunold et al., 1992), malseccin from Phoma tracheiphila for selection of lemon tree
(Citrus limon) calli and protoplast cultures (Gentile et al., 1992), 2,4,8-trihydroxytetralone from
Mycosphaerella fijiensis in micro-cross section cultures of banana (Musa sp.) (Okole and Schultz,
1997), colletotrichin from Colletotrichum gloeosporioides for the selection of embryogenic mango
cultures (Mangifera indica) (Jayasankar et al., 1999), solanapyrone A,B,C from Ascochyta rabiei for
the selection of shoot cultures of chickpea (Cicer arietinum) (Hamid and Strange, 2000), phomalide
from Phoma lingam in cell suspensions of rape (Brassica rapa) (Pedras and Biesenthal, 2000), AM-
toxin from Alternaria alternata for selection of apple (Malus domestica) shoot cultures (Saito et al.,
2001; Chapter 9), and many others (Švábová and Lebeda, 2005) (Tables 2.3 and 2.4).

Phytotoxins are considered microbial metabolites that may damage or harm plants at very low
concentrations (Graniti, 1991). The idea that metabolites produced by plant pathogens could be
detrimental to plants originated from the experiments of de Bary (1886) and the concept was advanced
by Gäumann (1954) who stated that microorganisms are pathogenic only if they are toxigenic.
Recently, more than 250 phytotoxic metabolites produced by plant pathogenic bacteria and fungi have
been isolated, purified and structurally characterised. Relatively little is known about their
biosynthesis and metabolism, and much less about the genetics underlying their production (Huang,
2001).

Toxin-producers are found among some bacteria (e.g., Pseudomonas, Burkholderia, Clavibacter,
Streptomyces, Xanthomonas) and a number of fungi (e.g., Alternaria, Ascochyta, Bipolaris, Botrytis,

10 15
Ceratocystis, Cercospora, Cochliobolus, Colletotrichum, Drechslera, Fusarium, Phoma,
Pyrenophora, Sclerotium, Septoria, Stemphylium, Verticillium) (Huang, 2001; Švábová and Lebeda,
2005) (Tables 2.3 and 2.4).

There are two main known roles of pathogen-produced toxins: (1) initiation of disease; and (2) disease
development. Toxins are classified based on their mode of action; those that are responsible for the
onset of disease and are essential for causing the disease are known as “pathogenicity factors”, and
those that are required for the development and increase in the extent of the disease are known as
“virulence factors” (Yoder, 1980). The pathological significance of phytotoxins can be assessed by
evaluating the correlation between: (1) the production of toxin and disease occurrence of the pathogen;
and (2) sensitivity to the toxin and susceptibility of the plant to the disease (Yoder, 1981).

The toxins/phytotoxins are either host-selective (HST, or host-specific) or non-host-selective (NHST,

or non-host-specific) according to their specificity in a given plant-pathogen interaction (Mitchell,
1984; Walton and Panaccione, 1993). The HST group comprises a limited number of phytotoxins that
meet the following criteria: (1) the toxin and its producer have similar host specificity; (2) the
virulence of the pathogenic strains is positively correlated to their capacity to produce the toxin; and
(3) the toxin is able to produce, in susceptible plants, symptoms characteristic of the disease caused by
the pathogen (Huang, 2001). HSTs are toxic to plant species or cultivars susceptible to the pathogens
producing these toxins, and there is a correlation between sensitivity to the toxin and susceptibility of
the plant to the pathogen (Knogge, 1996).

NHSTs form a larger group of toxins and include compounds that do not reproduce the patterns of
resistance and/or susceptibility responses of the host to the pathogen infection. They are thought to be
broad-spectrum toxins that affect a large number of crops (Graniti et al., 1991). These toxins act
primarily by inhibiting active defence processes such as detoxification or stimulation of membrane-
localised H+-ATPase activity (Knogge, 1996). However, there are also known toxins with a specific
mode of action, e.g., toxins responsible for plant wilting (Van Alfen, 1989).

Plant pathologists have concerned themselves mainly with the chemistry and biochemistry of
phytotoxins, and their roles in plant pathogenesis (Huang, 2001). However, in the past two decades,
research has been extended to the use of phytotoxins as tools in screening and breeding for disease
resistance (Švábová and Lebeda, 2005). The general strategy is based on considerable evidence that
there is a correlation between tolerance/resistance to phytotoxins and resistance to the pathogen.
Phytotoxins and culture filtrates of pathogens have been used in root growth, leaf chlorosis and lesion
development assays in screening early segregating populations. They have also been used for in vitro
selection of tolerant protoplasts, cells and calli, which are subsequently regenerated into putative
resistant plants (Huang, 2001; Švábová and Lebeda, 2005).

Bacterial phytotoxins

Generally, the bacterial phytotoxins are either glycosides (glycopeptides) or low molecular weight
compounds derived from amino acids and peptides (Strobel, 1976). There are at least six groups of
bacterial phytotoxins: tabtoxin, coronatine, thaxtomins, phaseolotoxin, syringomycin and
rhizobitoxine (Huang, 2001; for examples, see Table 2.3).

Tabtoxin is a dipeptide produced by certain strains and pathovars of Pseudomonas syringae that cause
wildfire or halo blight diseases. Tabtoxin was detected in P. syringae pv. tabaci, causing wildfire of
tobacco (Nicotiana tabacum) plants. At first, crude toxin preparations from P. syringae pv. tabaci
called “wildfire toxin” were produced, and were later characterised as tabtoxins, represented by
tabtoxinine and threonine. According to Strobel (1976), tabtoxins are capable of inducing chlorosis in
plants. However, Kinscherf et al. (1991) found that tabtoxin production alone is not sufficient to cause
disease. The possibility that plasmids may be involved in tabtoxin production has been investigated
(Huang, 2001).

16
11
Coronatine is produced by several P. syringae pathovars and was first detected in liquid cultures of P.
syringae pv. atropurpurea. When applied to leaves of Italian ryegrass (Lolium multiflorum), chlorosis
and browning occurred, similar symptoms as after inoculation with live P. syringae pv. atropurpurea
(Mitchell, 1984).

Thaxtomins are a group of 2,5-dioxopiperazines produced by Streptomyces scabies, S. acidiscabies

and S. ipomoeae, the causal organisms of potato common scab, acid scab and sweet potato pox (Toth
et al., 1998). Thaxtomins induce the formation of scab-like lesions on potato (Solanum tuberosum)
tuber. A positive correlation between pathogenicity and thaxtomin production was recorded on potato
slices (King et al., 1991).

Phaseolotoxin is a non-host-specific and extracellular toxin produced by P. syringae pv. phaseolicola

and the causal agent of halo blight of bean, which induces a chlorotic halo on treated leaves.
Structurally, phaseolotoxin is a tripeptide (Huang, 2001). The primary function of phaseolotoxin is
inhibition of carbamoyltransferase, which is an enzyme involved in chlorophyll synthesis (Patil,
1974). The production of this toxin contributes significantly to the virulence of P. syringae pv.
phaseolicola, whereas avirulent strains are not able to produce toxins in culture (Mitchell, 1984).

Another group of bacterial toxins is formed by syringopeptin, syringomycin and syringotoxin.

Biochemically, these toxins are peptides (Huang, 2001). Syringomycin was characterised in isolates of
P. syringae pv. syringae, a pathogen of stone fruit trees. It has been suggested that syringopeptin
primarily affects cellular membranes (Backman and DeVay, 1971). Syringotoxin, like syringomycin,
is also produced by P. syringae pv. syringae; however, only by strains pathogenic to Citrus spp.,
which do not produce syringomycin. Syringotoxin differs from syringomycin by slower migration on
polyacrylamide gel electrophoresis and by different biocidal activity against bacteria and fungi
(Gonzalez et al., 1981).

Tagetitoxin was identified by P. syringae pv. tagetis, causing a leaf spot of marigolds. Isolated and
purified tagetoxins cause chlorosis of the apical stem part of zinnia plants two to three days after
application (Mitchell and Durbin, 1981).

Rhizobitoxine is produced by certain strains of the Bradyrhizobium (Rhizobium) japonicum bacterium,

which fixes nitrogen in soybean plants, and by Pseudomonas andropogonis, which is the causal agent
of bacterial stripe and leaf spot in corn, sorghum and legumes (Huang, 2001). The toxin was isolated
from chlorotic leaves, nodules and bacterial cultures; and is non-host-specific because it produces
chlorosis in many plant species (Owens et al., 1965). Rhizobitoxines were found to be an
antimetabolic enol-ether amino acid (Owens et al., 1972).

Fungal host-selective toxins (HSTs)

Fungal HSTs comprise a group of structurally complex and chemically diverse metabolites produced
by plant pathogenic strains of certain fungi (Wolpert et al., 2002). HSTs have been mostly described
in necrotrophic pathogens. This group includes fungi such as Alternaria, Bipolaris
(Helminthosporium), Corynespora, Fusarium, Phyllosticta, and others. Most of the known HSTs are
low molecular weight secondary metabolites (e.g., cyclic peptides, terpenoids, oligosaccharides,
polyketides and sesquiterpene glycosides), showing great diversity in their chemical, biological and
physiological effects. The same compound is often produced by several taxonomically different
species and/or single species can produce various toxins (Goodman et al., 1986; Kohmoto and Otani,
1991). HSTs function as essential determinants of pathogenicity or virulence. Investigations into the
molecular and biochemical responses to these disease determinants reveal responses typically
associated with host defence and incompatibility induced by avirulence determinants (Wolpert et al.,
2002).

12 17
However, from the point of view of the host plant, there is no common genetic pattern of response to
HSTs and the fungi producing them. Genetic analyses have shown various genetic patterns of
sensitivity to HST, e.g., nuclear inheritance – monogenic (dominant, semi-dominant, recessive) or
cytoplasmic inheritance (Walton and Panaccione, 1993). The most important and well known HSTs
are produced by Alternaria (Nishimura and Kohmoto, 1983; Huang, 2001), Bipolaris
(Helminthosporium), Drechslera (Huang, 2001) and Phyllosticta maydis (Desjardins and Hohn, 1997;
Huang, 2001). A survey of the most important HSTs used for experimental purposes and resistance
selection is summarised in Table 2.3.

Several host-specific Alternaria toxins are known (Nishimura and Kohmoto, 1983). AAL-toxin, an
amino acid-derived phytotoxin, is produced by A. alternata f. sp. lycopersici, the causal fungus of
tomato stem canker. A detached leaf bioassay showed that AAL-toxin causes necrosis on susceptible
tomato plants. However, electrolyte leakage was not detected prior to the onset of necrosis, indicating
that the plasma membrane is not the primary target site of AAL-toxin (Huang, 2001). Tomato cell
suspension cultures are sensitive to AAL-toxin (Fuson and Pratt, 1988).

Helminthosporoside (HS-toxin) is produced by Drechslera sacchari, the causal organism of eye spot
disease of sugarcane. It is a sesquiterpenoid toxin (Huang, 2001). HS-toxin produces reddish-brown
streaks only on those clones of sugarcane that are susceptible to the fungus. Sensitive clones possess a
membrane protein that recognises and binds the toxin. The disintegration of the outer chloroplast
membrane is the earliest cytological disturbance in susceptible plants, and this is accompanied by a
strong decrease in CO2 fixation (Brown et al., 1982).

Victorin (HV-toxin) is produced by Drechslera victoriae, which causes foot and root rot, and leaf
blight of oat, and only affects susceptible oat cultivars. Chemically, this toxin is a polypeptide (Huang,
2001). The earliest detectable effect of victorin in susceptible plants is an increase in the permeability
of the plasma membrane to electrolytes (Wheeler and Black, 1963). Protoplasts from susceptible
cultivars begin to burst almost immediately after exposure to victorin, whereas protoplasts from
resistant cultivars are not affected (Sammadar and Scheffer, 1968).

HC-toxins are produced by race 1 of Helminthosporium carbonum (Cochliobolus carbonum,

Drechslera zeicola), which is the causal agent of one of the most serious diseases in the recent history
of plant pathology called corn leaf blight (Goodman et al., 1986). HC-toxin is a cyclic tetrapeptide
phytotoxin (Huang, 2001) that causes chlorotic symptoms by inhibiting chlorophyll biosynthesis in
etiolated leaves of susceptible cultivars of maize (Rasmussen and Scheffer, 1988). There are
indications that the HC-toxin inhibits histone deacetylases in maize (Ransom and Walton, 1997).

HMT-toxins (T-toxins) are produced by Helminthosporium maydis (Cochliobolus heterostrophus,

Bipolaris maydis) race T and are highly toxic to cells of corn possessing Texas male sterile (Tms)
cytoplasm. Chemically, HMT-toxins are linear polyketols (Huang, 2001). It has been shown that
HMT-toxins preferentially inhibit root growth of T-cytoplasm but have little effect on resistant corn
with N-cytoplasm (Smedegaard-Petersen and Nelson, 1969).

The PC-toxin is a polypeptide produced by Periconia circinata, which is a pathogen of grain sorghum.
The symptoms produced by this toxin are similar to those seen in plants inoculated with the fungus
(Goodman et al., 1986). The mode of action of this toxin is similar to that of victorin, i.e., it causes an
increased respiration rate, decreased growth, decreased protein synthesis and disturbance of membrane
function (Pringle and Scheffer, 1967).

PM-toxins are produced by P. maydis, which is the causal agent of corn yellow leaf blight. The
pathogen attacks only Tms corn (T-cytoplasm) and produces host-specific, linear polyketol
phytotoxins (Huang, 2001). PM-toxins selectively inhibit seedling root growth, induce leaf chlorosis
and increase leakage of electrolytes in corn leaves with T-cytoplasm (Goodman et al., 1986).

18
13
Fungal non-host-selective toxins (NHSTs)

NHSTs have been identified in various groups of fungi, and are mostly necrotrophic or cause vascular
diseases (e.g., Alternaria tenuis, A. mali, Ceratocystis ulmi, Cochliobolus miyabeanus, Colletotrichum
spp., Fusarium spp., Fusicoccum amygdali, Leptosphaeria maculans, Pyricularia oryzae, etc.).
NHSTs consist of a broad spectrum of toxins affecting various plant species. As for their chemistry,
they comprise mostly macromolecular compounds such as amino acid derivatives, peptides, cyclic
tetrapeptides and diterpenoids (Mitchell, 1984; Ballio, 1991; Huang, 2001). The genetic pattern of
plant responses to NHSTs is not well known. A survey of the most important NHSTs used for
experimental purposes and resistance selection is summarised in Table 2.4.

Many types of NHSTs are produced by Fusarium species. One such example is fusicoccin, which is
produced by F. amygdali, and is involved in wilt disease of almond (Prunus dulcis) and peach (Prunus
persica). Fusicoccin is a diterpenoid glycoside and affects cellular transport processes (Ballio, 1978).

There are also numerous other toxins produced by Fusarium species that cause diseases that are
collectively known as Fusarium wilt; such toxins are fusaric acid (FA), lycomarasmin, moniliformin,
naphthazarin, sambucin and beauvericin (Luz et al., 1990; Řepková and Nedělník, 1998; Desjardins
and Proctor, 2001; Kuzniak, 2001; Zemánková and Lebeda, 2001; Švábová and Lebeda, 2005).
Fusarium species producing FA are represented by F. moniliforme, F. napiliforme, F. thapsinum, F.
nygamai, F. sachari, F. fujikuroi, F. proliferatum, F. subglutinans, F. sambucinum, F. crookwellense,
F. heterosporum, F. oxysporum complex and F. solani complex (Desjardins and Proctor, 2001). The
primary effect of FA is an increase in cell permeability. Lines of banana (Musa sp.), gladiolus
(Gladiolus sp.) and pineapple (Ananas comosus) that were selected with FA also show increased
tolerance to F. oxysporum (summarised by Švábová and Lebeda, 2005).

Generally, species in the F. oxysporum complex are known to produce enniatins, FA, moniliformin,
naphtazarins and sambutoxins, but are not known to produce fusarins, fusarochromanone,
fusaproliferin, fumonisins, trichothecenes or fusaproliferin (Desjardins and Proctor, 2001).
Lycomarasmin is a tripeptide produced by F. oxysporum f. sp. lycopersici and a few other species.
Naphthazarin is produced by F. oxysporum f. sp. pisi and F. solani, and acts by inhibiting the
anaerobic decarboxylation of pyruvate (Isaac, 1991; Baker and Nemec, 1994, 1997). Beauvericin is a
cyclodepsipeptide metabolite, closely related to the enniatins (Moretti et al., 1995) and an important
secondary metabolite of various phytopathogenic Fusarium species (Abdalla et al., 2000). Although
beauvericin did not cause any symptoms in the roots of the tested plants (melon, tomato, wheat and
barley), it showed the highest toxicity towards the protoplasts of all plants tested (Sagakuchi et al.,
2000). Recent data suggest that the production of beauvericin and enniatin is not related to the
pathogenicity of F. oxysporum f. sp. melonis or to the differential specificity of the races (Moretti et
al., 2002). It has been generally concluded that the non-selective toxic activity of beauvericin is due to
its ability to induce pores in biological membranes, resulting in a disturbance of the normal gradients
of physiologically important monovalent cations across membranes (Lemmens et al., 2000).

Species in the F. solani complex produce FA, naphtazarins and trichothecenes. Those species with
Giberella sexual state produce fusarins and moniliformin that are not found in species with Nectria
sexual state (Desjardins and Proctor, 2001).

Trichothecenes are another very important group of Fusarium mycotoxins. The possible role of
trichothecenes in plant pathogenesis was initially assessed with UV-blocked mutant strains of F.
sporotrichoides. The acute phytotoxicity of trichothecenes and their occurrence in infected plant
tissues suggest that this mycotoxin plays a role in the onset of pathogenesis by Fusarium (Desjardins
and Proctor, 2001; Desjardins, 2003). Among the Fusarium toxins identified to date, however, only
trichothecenes and enniatins have been confirmed as virulence factors in plant pathogenesis. The
reduced virulence of F. avenaceum with a disrupted enniatin synthetase gene was proven in
experiments with potato tubers (Herrmann et al., 1996). Localisation studies of trichothecenes
indicated that these toxins could be detected in host tissues at an early stage of infection (Kang and

14 19
Buchenauer, 2002). It was proven that trichothecene production contributes to the virulence of F.
graminearum (Miller et al., 2001; Proctor et al., 2002). Similar results were demonstrated by F.
culmorum where correlations were found between the aggressiveness and deoxynivalenol content
(Hestbjerg et al., 2002; Mesterházy, 2002). Coleoptile tissues of wheat (Triticum aestivum) cultivars
that were resistant to Fusarium head blight were 10 times more resistant to deoxynivalenol and some
other metabolites (including 3 acetyl deoxynivalenol and dihydroxycalonectrin) than disease-
susceptible cultivars (Miller et al., 2001).

There are several NHSTs such as tentoxin, a peptide phytotoxin, that have been found in cultures of
Alternaria tenuis and A. mali (Okuno et al., 1975). These toxins cause chlorosis as the result of
interference with plastid development and a reduction of chlorophyll synthesis. These symptoms were
observed on various plants such as cotton (Gossypium hirsutum), citrus (Citrus limon), cucumber
(Cucumis sativus) and lettuce (Lactuca sativa) (Mitchell, 1984).

The causal agent of Dutch elm disease (Ceratocystis ulmi) produces a high macromolecular weight
NHST called cerato-ulmin (Takai et al., 1983). Ophiobolin (cochliobolin) is a toxin produced by a
number of fungi, in particular by C. miyabeanus (Helminthosporium oryzae) which causes rice leaf
spot disease. Ophiobolin is a sesquiterpene that alters the plasma membrane structure and phenolic
metabolism. The polymerisation of phenolics gives rise to the brown pigments that appear in the
necrotic areas of host tissues (Isaac, 1991).

Fungal cell wall components (elicitors)

Some components isolated from the cell walls of pathogenic fungi are called elicitors and have the
ability to trigger plant defence responses. Fungal elicitors were first used by Buiatti et al. (1985) to
select carnations resistant to F. oxysporum and by Scala et al. (1985) to select resistant tomatoes.
Similar experiments were performed later, also with carnation, by Trillas and Azcón-Bieto (1995).
Other experiments with cell wall components of F. oxysporum were carried out for selection of tomato
(Storti et al., 1992). Alfalfa protoplast and cell cultures were selected with fungal cell-wall
components and heat-released elicitors for resistance to Verticillium albo-atrum (Koike et al., 1993b;
Koike and Nanbu, 1997).

APPLICATIONS

Table 2.2 summarises the practical applications of in vitro selection of disease resistance in order to
obtain breeding lines that are tolerant of or resistant to certain pathogens. The similarities between the
in vitro screening systems and whole plant-pathogen interactions were recently compiled in a
comprehensive review (Švábová and Lebeda, 2005).

The possibility of using pathogen isolates, extracts or toxins as selection agents to screen in vitro
cultures of host plants is now well established. Also, the application of selection agents during plant in
vitro regeneration results in plant lines showing an increased degree of resistance to diseases. In the
past two decades, more than 100 research articles that used in vitro selection methods for the
improvement of resistance to phytopathogens were published. Over 30 plant species and their different
types of in vitro cultures were examined, and various selection agents were obtained from about 40
plant pathogens. Such methodology often utilises some type of explant cultures (excised organ or part
of organ, meristem tip culture, anther or pollen, callus, cell or tissue suspension, protoplast culture) of
the host plants that are treated with various selective agents (natural isolate of pathogen, modified
pathogen culture, culture filtrates, toxins, elicitors), which in turn elicit reactions that parallel those by
the pathogens. Their application successfully resulted in resistant lines in various important crops
including banana (Musa sp.), carnation (Dianthus caryophyllus), grapevine (Vitis vinifera), strawberry
(Fragaria ananassa) and wheat (Triticum aestivum). Nowadays, these techniques are an important
complement to classical breeding methods. Some examples of the application of these techniques in
the improvement of crop resistance are shown in Table 2.2.

20
15
CONCLUSIONS

Molecular studies reveal that stable genetic changes have occurred in plant materials that were
selected in vitro for their increased tolerance/resistance to the selection agents (Kodama et al., 1991;
Knogge, 1996; Loon, 1997; Wojtaszek et al., 1997; Bettini et al., 1998; Jayasankar et al., 2003). The
resistant material often exhibits changes in the expression of pathogenesis-related and/or anti-fungal
proteins. Fundamental knowledge about the role of particular selective components in pathogenesis
and their mode of action are now crucial points for further development in the area of in vitro selection
studies.

This chapter shows that significant progress has been made in phytotoxin research during the last two
decades, particularly in: (1) elucidating the chemical structures of phytotoxins and their relationship to
biological function; (2) the genetics of phytotoxin production; (3) the mode of action of phytotoxins at
the molecular level; and (4) in proving that phytotoxins are efficient tools for screening plants for
disease resistance.

In the case of in vitro selection techniques, which use some of the selective agents mentioned above,
the resulting changes in phenotypic characters (e.g., resistance/tolerance to diseases) can be interpreted
as potential mutations in the plant DNA sequence. Nevertheless, caution is advised when interpreting
the results because similar phenotypes can be caused by epigenetics. Such epigenetic interactions
frequently occur, but are not transmitted to the progeny (Bulk, 1991). Therefore, genetic analyses of
inheritance in selected lines must be performed and the results carefully interpreted.

In vitro selection can considerably shorten the time needed for the selection of desirable traits. In vitro
selection pressure suffers minimal influence from exterior environmental conditions, and can precede
and complement field selections. In vitro-selected putative variants should be tested in the field to
confirm the genetic stability of the selected trait (Jain, 2001), and genetically stable somaclones or
mutants can then be used directly as elite varieties or introduced into plant breeding programmes.

Last but not least, in vitro selection of somaclonal variants and induced mutants is not protected under
intellectual property regulations, nor is it subject to public safety concerns that currently hamper
transgenic (i.e., GMO) approaches for the development of new crop cultivars. Thus, in vitro selection
is a promising, non-transgenic approach, which offers an attractive alternative method for producing
improved cultivars (Jayasankar et al., 2003).

Acknowledgements

This study was supported by the Ministry of Education, Youth and Sports of the Czech Republic,
projects MSM 6198959215 and MSM 2678424601; and by the Ministry of Agriculture of the Czech
Republic, project NAAR QG60099. The authors thank Dr. M. Griga for providing photos used in this
chapter.

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Table 2.1. Methods of aseptic and in vitro culture of plant organs and plant tissues which may be used
for resistance screening*

Culture type Description

Excised organ or part of organ culture The culture of whole or parts of excised roots, leaves,
stems, flowers, fruits etc.

Meristem (tip) culture The regeneration of whole plants from excised stem
apical or axillary meristems, via organogenesis or
embryogenesis

Anther or pollen culture The regeneration of haploid or homozygous diploid

callus or plants from pollen or anther cells

Callus (tissue) culture The culture of disorganised masses of tissue on solid

media. Callus of some species may be induced to
differentiate tissues or organs by changing the hormone
and nutrient balance of the culture medium

Cell or tissue suspension culture The culture of suspensions of single cells and/or cell
aggregates in liquid media. Cells of some species may
be induced to give rise to embryoids and then plants in
culture by changing the hormone and nutrient balance of
the medium

Protoplast culture The enzymatic or mechanical isolation and culture of

cell protoplasts. Protoplast fusion, wall regeneration and
normal or hybrid plant regeneration are all possible with
certain species

*Modified according to Ingram (1976).

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 Table 2.2. Examples of successful application of in vitro resistance screening in selection and plant breeding*

Crop (plant species) Type of in vitro Pathogen (selective agent) Effect on resistance or other results of References
culture the study
Excised organ or part of organ culture
alfalfa (Medicago sativa) separated cotyledons Colletotrichum trifolii (spore useful screening method which correlates Cucuzza and Kao
suspension) with other greenhouse tests (1986)
alfalfa (M. sativa) stem cuttings and leaf Verticillium albo-atrum symptoms on stems and leaves after filtrate Ireland and Leath
infiltration assay (filtrate) treatment were typical for field-infected (1987)
plants; the filtrate has a potential for use in
screening for resistance
cowpea (Vigna unguiculata) leaf disks Phytophthora cryptogea induction of resistant response and Saindrenan et al.
(filtrate) elicitation of phytoalexins (1990)
grape vine (Vitis vinifera) culture of sterile rooted Plasmopara viticola (co- resistance to downy mildew was expressed Barlass et al. (1986)
shoots cultivation with isolate–dual in vitro; technique can be used for
culture) screening and selection for host resistance
onion (Allium cepa) germinating seeds Alternaria porri (filtrate) reduction in seed germination and seedling Gupta et al. (1986)
vigour after filtrate treatment
pea (Pisum sativum and wild roots tube test Fusarium solani, F. broad variation in sensitivity and/or Lebeda and Švábová
species) oxysporum (inoculation) resistance among wild species collection; (1997)
several potential sources of resistance were
selected
pineapple (Ananas comosus) leaf segments and Fusarium subglutinans susceptible cvs. were sensitive to culture Borrás et al. (2001)
wounded plantlets from (filtrate, fusaric acid - FA) filtrate whereas resistant cvs. showed
tissue cultures tolerance; using filtrate allowed the
selection of resistant plants to fungus itself
soybean (Glycine max) intact plants Fusarium solani f. sp. positive correlation between AUDPC of Huang and Hartman
glycines (inoculation, filtrate) inoculated plants and cut seedling test (1998)
using culture filtrate
sugar cane (Saccharum in vitro Ustilago scitaminea correlation between in vitro response and Fereol (1984)
officinarum) micropropagated (inoculation) resistance of plants was proven
culture of intact plants

31
32
tobacco (Nicotiana tabacum) separated leaves Phytophthora cryptogea study of phytotoxicity of individual filtrate Bonnet et al. (1985)
(filtrate) fractions
tobacco (N. tabacum) intact leaves; cell and Alternaria alternata (AT the toxin induced typical symptoms on Ishida and Kumashiro
protoplast culture toxin) leaves of susceptible cultivars; cells of (1988)
resistant cvs. survived toxin treatment
better than those of susceptible cvs; field
tolerance among cvs. is expressed at the
cultured cell level
tomato (Lycopersicon somaclones derived Clavibacter michiganensis limited potential of somaclonal variation as Bulk et al. (1991)
esculentum) from leaves, cotyledons subsp. michiganensis a source of resistance to bacterial cancer
and hypocotyls (suspension ) was shown
Meristem, shoot and embryogenic culture
apple tree (Malus domestica) shoot culture Gymnosporangium juniperi- shoots from resistant variety were not Joung et al. (1987)
virginianae (inoculation) affected by the fungus; differences in
resistance between cotyledonary or embryo
axis shoots were observed
banana (Musa) shoot-tip culture Mycosphaerella fijiensis 2,4,8-THT(trihydroxytetralone) activated Hoss et al. (2000)
(secondary metabolites) phenylalanine-ammonia lyase; necrotic
microlesions and activation of defence
mechanism in resistant cv. led to
incompatible reaction
banana (Musa) multiple-shoot culture fusaric acid tolerant variant selected with FA after Matsumoto et al.
chemical mutagenesis (1995)
chickpea (Cicer arietinum) shoots Ascochyta rabiei, Fusarium sensitivity to the toxins was correlated with Hamid and Strange
oxysporum (filtrates, toxins susceptibility to the disease; selection of (2000)
solanapyrone A, B, C) plants with well expressed
glutathion/glutathion-S-transferase may be
one means of improving resistance
eucalyptus (Eucalyptus micropropagated Phytophthora cinnamomi primary roots of resistant micropropagated Cahill et al. (1992)
marginata) clones (suspension) lines were able to restrict and confine
colonisation by P. cinnamomi
pea (Pisum sativum) tube test, multiple- Fusarium solani, F. comparison of the response using three in Švábová et al. (1998)
shoot culture, dual oxysporum (inoculation, vitro methods
culture culture filtrate, autoclaving
inactivated fungus)
 pea (P. sativum) tube test, multiple- Fusarium spp. filtrates regenerants were tested in field conditions Švábová and
shoot culture, dual and artificially inoculated substrates; Odstrčilová (2001)
culture improved resistance evaluated with
infection degree was observed
strawberry (Fragaria vesca) clones derived from Alternaria alternata resistant lines revealed from surviving Takahashi et al. (1992)
shoot apex calli (inoculation) inoculated calliclones were obtained
wheat (Triticum aestivum) embryogenic culture Septoria nodorum (toxic good correlation between field resistance Keller et al. (1994)
extracts) and embryo resistance in vitro
Callus culture
apple tree (Malus domestica) callus culture Venturia inaequalis (co- study of ultrastructural interactions of Beech and Gessler
cultivation) resistant and susceptible varieties (similar (1986)
relationships)
banana (Musa) callus, intact plants Cercospora musae, both calli and plants showed similar levels Trujillo and Garcia
Mycosphaerella musicola of survival after inoculation (1996)
(inoculation)
barley (Hordeum sativum) callus culture derived fusaric acid plants regenerated from calli selected on Chawla and Wenzel
from immature FA were resistant to the toxin (75-100% (1987)
embryos concentration)
barley (H. vulgare) callus culture derived Drechslera teres (toxin) nine progeny of S2 showed a correlation Hunold et al. (1992)
from immature between toxin tolerance and resistance
embryos against the pathogen
carnation (Dianthus callus culture Fusarium oxysporum resistant calli were used for plant Thakur et al. (2002)
caryophyllus) (filtrate) regeneration – 32% plants had considerable
resistance against pathogen in field
conditions
cauliflower (Brassica callus culture Xanthomonas campestris pv. mutagenised calli were selected on 30% Mangal and Sharma
oleracea var. botrytis) campestris culture filtrate; high level of correlation (2002)
between resistance of calli to the filtrate
and resistance of regenerated plants to
pathogen was observed
celery (Apium graveolens) callus and suspension Septoria apiicola (filtrate) positive response of continual culturing on Evenor et al. (1994)
cultures the percentage of cells surviving in
selections
chickpea (Cicer arietinum) callus culture Fusarium oxysporum inhibition of callus growth; analysis of total Singh et al. (2003)

33
(filtrate) phenols, peroxidase and ß-1,3-glucanase
34
activities
common bean (Phaseolus calli Colletotrichum differential response of bean callus from Fernandez et al. (2000)
vulgaris) lindemuthianum (culture sensitive and/or resistant lines was
filtrate) observed; toxic compounds of the filtrate
were analysed; pathogenicity could be
related with common filtrate substances
corn (Zea mays) calli Helminthosporium carbonum an attempt to regenerate calli resistant to Wolf and Earle (1990)
(toxin HC) HC toxin failed (due to inability to identify
resistant cells among slow growing callus
cells)
eggplant (Solanum callus culture Verticillium dahliae (filtrate) reliable screening system in vitro was Koike et al. (1993a)
melongena) developed; selection for tolerant lines on
cellular level
elm (Ulmus americana) callus culture, stem Ceratocystis ulmi (filtrate) reduction in callus growth of susceptible Pijut et al. (1990)
cuttings variety on media with filtrate; correlation
between callus reaction and cut stem assay
eucalyptus (Eucalyptus callus culture Phytophthora cinnamomi hyphal growth on callus correlated with McComb et al. (1987)
marginata) (agar blocks of fungus susceptibility of the plant
culture)
gladiolus (Gladiolus sp.) shoots and callus Fusarium oxysporum f. sp. shoot assay and ion-release with intact Löffler and Mouris
cultures; intact corms gladioli, fusaric acid cormels gave significantly coinciding (1992), Remotti and
results; part of the Fusarium resistance is Löffler (1996)
based on insensitivity to fusaric acid
oat (Avena sativa) calli Helminthosporium victoriae insensitivity to toxin was heritable; specific Rines and Luke (1985)
(toxin – victorin) resistance can be selected in tissue cultures
of oats
onion (Allium cepa) callus and shoot Pyrenochaeta terrestris calli exposed to the filtrate reflected the Gourd et al. (1988)
cultures (filtrate) degree of whole plant susceptiblity
potato (Solanum tuberosum) calli and bulbs Verticillium albo-atrum hyphal colonisation on tuber disks and Koike et al. (1996)
(inoculation and filtrate) calli; calli development on media
containing culture filtrate are good markers
for in vivo resistance
potato (S. tuberosum) callus culture Erwinia carotovora subsp. calli resistant to E. carotovora were Taylor and Secor
carotovora (inoculation and identified, number of regenerants were too (1990)
bilayer culture) low to determine correlation between in
vitro response and tuber resistance
 tomato (Lycopersicon calli Fusarium oxysporum f. sp. correlation between in vivo resistance and Scala et al. (1985)
esculentum) lycopersici (filtrate and in vitro hypersensitive response and
mycelial cell wall phytoalexin induction, but tolerance to
components-elicitors) toxic filtrate in vitro is not indicator of in
vivo resistance
tomato (L. esculentum) callus and cell Clavibacter michiganensis toxin-tolerant cell lines were developed Krämer et al. (1988)
suspension culture (toxin)
wheat (Triticum aestivum) calli on double layer Fusarium graminearum and 3% of R2 plants were found to be more Ahmed et al. (1991)
F. culmorum (culture resistant than the original cultivars
inactivated by autoclaving)
wheat (T. aestivum) calli Fusarium culmorum and F. 35.7% of lines were more resistant than the Ahmed et al. (1996)
graminearum (filtrate) original cultivars
Cell or tissue suspension culture

alfalfa (Medicago sativa) cell suspension culture Verticillium albo-atrum treatment with conidia and heat-released Koike and Nanbu
elicitors induced a large increase in PAL
(inoculation and heat-released (1997)
elicitors) activity in resistant cell lines
alfalfa (M. sativa) cell culture Fusarium oxysporum cell cultures derived from genotype Cvikrová et al. (1992)
(filtrate) susceptible to F. oxysporum accumulated
double the amount of phenolic acids than
the resistant ones
apple tree (Malus domestica) mesophyll cells Phytophthora cactorum cells exposed to culture filtrate from Mezzetti et al. (1992)
(culture filtrate) virulent strain showed an increase in
fluorescence; possibility of using
fluorescence measurement as a screening
system and filtrate as selective agent
celery (Apium graveolens) somaclones revealed F. oxysporum (inoculation by resistant phenotypes were identified; Heath-Pagliuso et al.
from cell suspension soaking of roots in resistance conditioned by more than one (1988)
cultures suspension) locus
coffee tree (Coffea arabica) cell and protoplast Colletotrichum kahawae selective effect of filtrate on protoplast was Nyange et al. (1997)
culture (filtrate) proven; system can be used for selection
and screening
rape (Brassica juncea, B. cell suspension cultures Phoma lingam (phomalide), differential phytotoxicity of phomalide and Pedras and Biesenthal
napus), mustard (Sinapis Alternaria brassicae direct correlation with plant disease (2000)
alba) (destruxin) resistance; destruxin – less clear

35
relationship to disease resistance
36
soybean (Glycine max) suspension cell culture; Fusarium solani f. sp. positive correlation between foliar Li et al. (1999)
stem cuttings glycines (filtrate) symptom severity and viability staining of
cell cultures
tomato (L. esculentum) cell cultures Alternaria alternata (AL- cells revealed from resistant cultivar were Kodama et al. (1991)
toxins) not affected significantly; host-selective
toxicity was expressed on the cell level as
well as plant tissue
Protoplast culture
alfalfa (M. sativa) protoplast culture Verticillium albo-atrum protoplasts of susceptible variety were Koike et al. (1993b)
(filtrate and cell-wall sensitive to the low molecular weight
components) fractions of the filtrate
grape vine (V. vinifera) protoplasts Botrytis cinerea (filtrate) the plating efficacy and embryogenic Reustle and Matt
competence of regenerated calli decreased (2000)
with increasing filtrate concentration
hop (Humulus lupulus) protoplast and cell Verticillium albo-atrum cytotoxicity of the culture filtrates Connel et al. (1990)
suspension culture (filtrate) correlated both to the virulence of the
isolates and the resistance of the cultivars
rape (Brassica oleracea), protoplasts, calli; intact Phoma lingam (inoculation, cells of the host plant rape were sensitive to Sjödin et al. (1988)
tobacco (N. tabacum), potato plants toxic metabolites, sirodesmin the toxin of Phoma, but the non-host
(S. tuberosum) PL) tobacco and potato cells remained resistant
– toxin has host-selective properties
rape (B. napus, B. juncea, B. protoplasts; cell Phoma lingam (toxin – clear correlation between resistance to P. Sjödin and Glimelius
carinata, B. nigra) aggregates sirodesmin PL) lingam and insensitivity to sirodesmin PL (1989)
is present; toxin can be used to distinguish
resistant and susceptible material both in
vitro and in vivo
tobacco (N. tabacum) protoplast-derived calli Pseudomonas syringae pv. assay of R1 generation derived from toxin- Thanutong et al.
tabaci and Alternaria resistant calli indicated the inheritance of (1983)
alternata (toxins) resistance
tomato (L. esculentum) protoplasts, calli; intact Fusarium oxysporum (fusaric single dominant gene type of resistance to Shahin and Spivey
plants acid, inoculation) Fusarium wilt was obtained after in vitro (1986)
selection on FA

*Modified according to Švábová and Lebeda (2005).

 Table 2.3. Host-selective toxins (HSTs) produced by plant pathogenic fungi and bacteria

Pathogen Host plant Toxin References

Fungal pathogens
Alternaria mali apple, pear AM-toxin Nishimura and Kohmoto (1983), Kohmoto and Otani
(1991), Saito et al. (2001)
A. citri citrus AC-toxin, Gardner et al. (1985), Kohmoto and Otani (1991)
ACRL-toxin Nishimura and Kohmoto (1983)
A. kikuchiana japanese pear AK-toxin Nishimura and Kohmoto (1983), Kohmoto and Otani (1991)
knapweed maculosin Upadhyay and Mukerji (1997)
A. alternata strawberry AF-toxin Nishimura and Kohmoto (1983), Kohmoto and Otani (1991)
A. fragariae tangerine ACT-toxin I, II, Kohmoto and Otani (1991)
ACTG-toxin A,B
A. citri tobacco AT-toxin Nishimura and Kohmoto (1983), Ishida and Kumashiro
(1988), Kodama et al. (1990), Kohmoto and Otani (1991)
A. longipes tomato AL-toxin, Kodama et al. (1991), Kohmoto and Otani (1991), Chen et
AAL-toxin al. (1992), Nishimura and Kohmoto (1983)
A. alternata f. sp. lycopersici
A. brassicae rape destruxin B, Tewari and Bains (1997), Upadhyay and Mukerji (1997),
homodestruxin B, Pedras and Biesenthal (2000)
destruxin B2
A. helianthi sunflower radicin, Nishimura and Kohmoto (1983)
deoxyradicin
A. tenuis pigeonpea tentoxin Nishimura and Kohmoto (1983), Kohmoto and Otani
(1991), Upadhyay and Mukerji (1997)
Corynespora cassiicola tomato CC-toxin Kohmoto and Otani (1991),
Upadhyay and Mukerji (1997)
Drechslera zeicola maize HC-toxin, Buiatti and Ingram (1991), Kohmoto and Otani (1991),
(Cochliobolus carbonum, HC-toxin I, II, III, Wolf and Earle (1990), Walton and Panaccione (1993)
Helminthosporium carbonum) HMT-toxin Yoder (1980)
D. maydis maize HC-toxin Walton and Panaccione (1993)
(C. heterostrophus)
D. sacchari sugarcane helminthosporoside, Yoder (1980), Kohmoto and Otani (1991),
(Bipolaris sacchari, HS-toxin A,B,C Upadhyay and Mukerji (1997)

37
Helmithosporium sacchari)
38
D. victoriae oat HV-toxin (victorin C); Rines and Luke (1985), Kohmoto and Otani (1991),
(Cochliobolus victoriae) victorin Walton and Panaccione (1993)
Periconia circinata sorghum PC-toxin, Kohmoto and Otani (1991), Walton and Panaccione (1993),
peritoxin A,B Upadhyay and Mukerji (1997)
Phyllostica maydis maize PM-toxin A,B,C,D Kohmoto and Otani (1991), Upadhyay and
Mukerji (1997)
Phoma lingam rape phomalide Pedras and Biesenthal (2000)

Phoma tracheiphila lemon malseccin, mellein Gentile et al. (1992), Parisi et al. (1993)

Bacterial pathogens

Pseudomonas syringae pv. tomato syringolide Walton and Panaccione (1993)

lycopersici
P. syringae pv. phaseolicola beans phaseolotoxin Yoder (1980), Moore et al. (1984)
P. syringae pv. tabaci tobacco tabtoxin Yoder (1980), Shaw (1997)
 Table 2.4. Non-host-selective toxins (NHSTs) produced by plant pathogenic fungi

Pathogen Host plant Toxin Reference

Cercospora kikuchii soybean cercosporin Daub and Ehrenshaft (1997)

C. beticola sugar beet CB-toxin, beticolins, cebetins Jalal et al. (1992)
C. arachidicola peanut dothistormin Jalal et al. (1992)
Fusarium spp. barley fusaric acid Chawla and Wenzel (1987)
banana fusaric acid Matsumoto et al. (1995)
alfalfa fusaric acid Řepková and Nedělník (1998)
F. culmorum cereals trichothecenes (DON) Bruins et al. (1993), Hestbjerg et al. (2002),
Kang and Buchenauer (2002), Mesterházy (2002)
F. graminearum wheat DON Mesterházy (2002)
maize DON, Ac-DON Proctor et al. (2002)
F. moniliforme gladiolus, rice, maize fumonisin B Abbas et al. (1995)
F. solani lemon-tree dihydrofusarubin, isomarticin, Nemec et al. (1989)
naphtazarins
pea marticins, fusarubin, Baker and Nemec (1997)
soybean monorden Baker and Nemec (1994)
F. subglutinans pineapple fusaric acid Borrás et al. (2001)
F. oxysporum pea isomarticin, fusaric acid, pyridine Baker and Nemec (1997)
derivative toxin,
jimsonweed moniliformin Abbas et al. (1995)
tomato fusaric acid Kuzniak (2001)
f. sp. gladioli gladiolus fusaric acid Remotti and Löffler (1996)
f. sp. lilii lily fusaric acid Löffler and Mouris (1992)
f. sp. melonis muskmelon beauvericin Moretti et al. (2002)
f. sp. vasinfectum (particular host plants) fusaric acid Luz et al. (1990)
F. verticillioides maize fumonisins Proctor et al. (2002)
Ascochyta pisi pea ascochitine, ascosalitoxin, pinolidoxin Strange (1997)
A. pinodes pea ascochitine, Evidente et al. (1993)
A. fabae faba bean pyrenolide A, Beed et al. (1994)
A. hyalospora lambsquarters hyalopyrone, Venkatasubbaiah and Chilton (1992)
chrysanthone A,B,C
A. chrysanthemi chrysanthemum ascotoxin, Strange (1997)

39
A. imperfecta ascochlorin
40
A. viciae lucerne, clover ascofuranone, Strange (1997)
vetch ascofuranol Strange (1997)
cytochalasins A, B, U, V, T, F, 7-0-
A. heteromorpha oleander acetylachalasin B, Strange (1997)
A. lathyri vetchling cytochalasin A, B Vurro et al. (1992)
A. rabiei chickpea solanapyrone A, C Hamid and Strange (2000)
Alternaria solani potato, tomato solanapyrone Strange (1997)
Leptosphaeria maculans oilseed rape sirodesmin PL, phomalide Buiatti and Ingram (1991),
(Phoma lingam) Pedras et al. (1999)
Mycosphaerella fijiensis banana 2,4,8- trihydroxytetralone Hoss et al. (2000),
Okole and Schulz (1997)
Septoria nodorum wheat mellein Keller et al. (1994)
 PLANT PATHOGEN

Establishment of explant culture: Establishment of the isolate culture:

• organs • purification
• cells • propagation
• protoplasts
• tissues derived in vitro from roots,
stems, leaves, apicals, floral organs Biological characterisation of isolates:
• virulence
• pathogenicity

Treatment with various substances

increasing the somatic variability Isolation of the selection agents:
(mutagens) • inoculation
• double layer
• culture filtrate
Establishment of the regeneration protocol: • crude × purified
• organogenesis • autoclaved × filtered through millipore
• embryogenesis • phytotoxins/pathotoxins
• elicitors

• Establishment of selection concentrations for a

particular explant culture
• Establishment of the evaluation method for the
assessment of the effect of selection agents in
cultures in vitro

Several cycles of in vitro selection

Regeneration of explants that survived

the selection pressure

Biochemical and molecular analyses

of selected plant material

Screening of selected plant material for resistance to

pathogen in greenhouse and/or field conditions

Multiplication of the lines with improved resistance for

further use in breeding programmes or direct farming

Figure 2.1. General scheme of in vitro screening for disease resistance and selection.

41
 Figure 2.2. Culture of pea (Pisum sativum, line HM6) root segments and their response
to Fusarium oxysporum filtrate. Concentration of the filtrate from left to right: 0
(control), 0.1, 1, 2, 5 and 10%. Bar: 10 mm. Photograph: M. Griga.

A B C
Figure 2.3. Culture of pea (Pisum sativum) shoot clusters grown on a “double-layer”
medium. (A) Fusarium oxysporum; (B) Control; (C) F. solani. The first layer was
inoculated with the fungus and grown for four weeks, then inactivated by autoclaving
and overlayed by a second layer of medium suitable for the explant culture. In this
particular case, a CDA medium was used as the first layer for growing the fungus, and an
MS medium with macro- and micro-elements (B5 vitamins, FeEDTA, inozitol, sucrose,
0.1 mM NAA and 20 mM BAP) was used for growing the shoots. Note the presence of
green resistant/tolerant plantlets in (A) and (C). The photograph was taken four weeks
after cultivation. Photograph: M. Griga.

42
 A

Figure 2.4. Differences in rooting of pea (Pisum sativum) shoots on media

supplemented with different Fusarium filtrates (10% [v/v]). From left to right:
control, F. oxysporum, F. poae, F. solani, F. semitectum;.(A) cv. ‘Komet’; (B) cv.
‘Colt’; (C) line DP 1059. Photograph: M. Griga.

43
 A

Figure 2.5. Effect of different concentrations of fusaric acid (FA) on callus culture of
Pisum sativum (line 19/1). Concentration of FA in (A) 0 (control): simultaneous
incidence of green and yellowish calli; (B) 2.5 µM: incidence of explants growing into
normal calli and without any development; and (C) 5 µM: incidence of explants
growing into calli with retarded growth and explants without any development.
Photograph: M. Griga.

0 (control) 0.01 0.1 1 10% (v/v)

Vicia faba

Pisum sativum

Glycine max

Figure 2.6. Effect of Fusarium semitectum filtrate on callus culture of Vicia faba,
Pisum sativum and Glycine max. The concentration of F. semitectum filtrate is shown.
Photograph: M. Griga.

44
 A

Figure 2.7. Multiple-shoot

culture of pea (Pisum sativum,
line HM6) after application of
Fusarium oxysporum filtrate
(10% [v/v]). (A) Variation in
shoot formation; (B) Variation in
rooting intensity of plantlets.
B Photograph: M. Griga.

Figure 2.8. Growing of pea (Pisum sativum) plantlets in non-sterile conditions in the
greenhouse after selection for resistance to Fusarium spp. Photograph: M. Griga.

45
 Chapter 3

Mass-screening techniques for early selection of banana and plantain crops

resistant to major diseases and pests

B. Okole

East Coast Biotechnology Innovation Centre-LIFElab,

Kwazulu Natal, South Africa.
Correspondence: [email protected]

Abstract

Different in vitro selection techniques that are used for selecting banana and plantain plants resistant to
diseases and pests are discussed in this chapter with a focus on Black sigatoka disease. Explants that
are suitable for in vitro selection include shoot meristems, micro-cross sections, callus, cell suspension
and protoplasts. A double selection system using the crude filtrate and the purified host-specific toxin
2,4,8-trihydroxytetralone (2,4,8-THT) is described. The optimum concentration of the host-specific
toxin 2,4,8-THT and the non-host-specific toxin juglone used for selection and the mode of action of
the purified toxins are also shown. A combination of mutation induction and effective selection
methods appears to be the most effective strategy to improve tolerance to diseases in banana and
plantains.

INTRODUCTION

Bananas and plantains (Musa spp.) are among the most important staple food crops for several million
people around the world. They rank fourth among food crops after rice, wheat and maize (CGIAR,
1993). Bananas and plantains play a major role in nutrition and well being, as well as in the cultural
life of millions of people living in the tropics and sub-tropics. In many developing countries, bananas
and plantains are widely used as a food supplement or as a staple food, while dessert bananas are a
common food in Europe, the USA and Canada. Total world production of bananas and plantains is
estimated at 75 million and 33 million metric tons, respectively (FAO, 2005).

The performance of banana and plantain crops, like that of many other crops, is limited by major
diseases and pests. The major diseases and pests that affect banana and plantain production are Black
sigatoka (black leaf streak) caused by Mycosphaerella fijiensis, Panama disease caused by Fusarium
oxysporum f. sp. cubense, moko disease caused by Ralstonia solanacearum, bunchy top disease
caused by the bunchy top virus, nematode diseases caused by Radopholus similis, Pratylenchus spp.,
Meloidogyne spp. etc., and corm borer caused by Cosmopolites sordidus. These diseases and pests
usually lead to significant losses in food production and income. Consequently, the largest single
component in the cost of production of bananas and plantains is disease and pest control. Current
control measures, which comprise approximately 40% of total production costs in commercial
plantations and small holder farms, vary from cultural and biological measures to chemical control
(Ganry, 1993).

Breeding banana and plantain for resistance to diseases and pests is urgently needed by small-scale
and commercial producers. However, these crops have very low clonal multiplication rates and most
of the commercial varieties are polyploid with extremely poor seed production. These barriers impede
sexual hybridisation and consequently slow down genetic improvement of the crop. The ability to
culture bananas and plantains in vitro, is allowing researchers to develop different biotechnological
methods, which they hope to deploy in producing new improved clones that are resistant to diseases

47
and pests. The different non-conventional breeding approaches which may be used to improve these
crops to be disease and pest-resistant include genetic engineering, mutation breeding and in vitro
selection using pathogen culture filtrates or purified toxins from the pathogens.

Genetic engineering would involve the introduction of a well characterised disease- or pest-resistant
gene into banana or plantain by means of Agrobacterium-mediated transformation, microprojectile
particle bombardment or electroporation. The plants would have to be tested, both in the laboratory
and the greenhouse, for gene stability and integration before screening for the major diseases and
pests.

MATERIAL AND METHODS

In the case of mutation breeding, genetic variation is essential for the creation of plants with superior
agronomic traits. However, spontaneous mutations occur at an extremely low frequency. Spontaneous
somatic mutations have played an essential role in the speciation and domestication of plantains and
bananas. Some authors even claim that all the bananas and plantains that we grow and eat were
selected in prehistory from spontaneous mutations (Buddenhagen, 1986). Some of these spontaneous
mutants in Musa (members of the ‘Cavendish’ subgroup and plantains) now play a very important role
in banana and plantain production. Mutagenic agents such as radiation and certain chemicals can be
used to induce mutations at a higher frequency and generate genetic variation from which desired
mutants may be selected (Roux, 2004). Mutation breeding should be encouraged because the
acceptance rate of this technology is higher than that for genetic transformation.

In vitro selection techniques used for banana and plantain are analogous to those used in prokaryotic
systems, where mutations can be efficiently induced and variants selected and isolated at the cellular
level. In vitro selection represents an immediate and inexpensive way of generating and selecting
banana and plantain variants with tolerance to either the pathogen or its toxin from susceptible
varieties, as compared to classical breeding methods (Evans and Sharp, 1986). Pathogens or their
metabolites could be used as selection agents to enhance resistance within tissue cultures and to select
resistant individuals.

The purified forms of these metabolites are known as toxins. Toxins produced by pathogens have been
categorised based on several criteria, such as their chemical nature, mode of action, identity of the
microorganisms producing them, and most definitions take into consideration their involvement as
primary or secondary determinants of pathogenesis (Yoder, 1980). Toxins are primary determinants of
pathogenesis when they act as the key element in infection initiation and symptom development. They
are secondary determinants when they only modify the symptoms’ intensity (El Hadrami et al.,
2005b). In order to assess the involvement of toxins in pathogenesis, commonly used criteria include:
(1) host specificity, (2) presence in infected plants, (3) toxin production at a key step of disease
development, (4) induction of typical disease symptoms and (5) degree of correlation between the
quantity of toxin produced in vitro and the pathogenicity level (Scheffer, 1976; Yoder, 1980;
Markham and Hille, 2001; El Hadrami et al., 2005b; Švábová and Lebeda, 2005). Several
experimental approaches have been developed in order to evaluate the role of toxins in host-pathogen
interactions (Wolpert et al., 2002). The most convenient seems to be the inactivation or specific
elimination of the toxin from the system, followed by the observation of the modifications that occur
during the initiation, establishment or expression of the host-pathogen interaction. For toxins or
culture filtrates to be used as the selection agent in vitro, they will need a competent explant source
that will be used for selection.

In any non-conventional breeding programme of banana and plantain, a suitable, highly regenerative
explant source is needed. Shoot meristem, protoplasts, cell suspensions, callus and micro-cross
sections have been used. Shoot meristems are usually generated from field-grown materials. The
process involves sterilising the shoot tips in 30% commercial bleach solution (Domestos) containing
5% of sodium hypochlorite for 30 minutes. After sterilisation, the shoot tips are rinsed with sterile

48
distilled water or placed directly into Murashige and Skoog (MS) basal salt medium with vitamins
(Murashige and Skoog, 1962). The MS basal salt medium is usually supplemented with ascorbic acid
(80 mg/l), sucrose (30 g/l) and phytohormones: benzylaminopurine (BAP) (2-4 mg/l) and indole acetic
acid (IAA) (0.2 mg/l). The pH is usually brought to 5.8 before 3 g/l of gelrite is added for autoclaving
at 121°C for 25 minutes. After three subcultures, the shoot meristems can be used for transformation,
mutation or in vitro screening.

A second explant source that is used by many laboratories involves the use of micro-cross sections
(Okole and Schulz, 1996). Micro-cross sections are prepared either with a microtome or a scalpel by
cutting a thin section of 300-400 µm from the corm of an in vitro tissue culture plant, which has been
growing on a multiplication medium. The section normally produces several shoot buds with the
addition of the appropriate phytohormones in the medium. These buds will eventually regenerate into
plantlets. With this technique, it is possible to regenerate up to 15 plantlets from one explant without
any morphological changes when compared to the controls. The tissues are also small and flat, and
will have better contact with the pressure source, be it toxins, culture filtrate, or chemical or radiation
mutants. Micro-cross sections can also be used to generate callus and cell suspension cultures with
modifications of the phytohormones added.

The third type of explants used are male flower buds. It has been reported by several researchers
(Escalant et al., 1994; Sági et al., 1995; Becker et al., 2000; Ganapathi et al., 2001) that male flowers
are the most responsive starting material for initiating embryogenic cultures, especially for the
‘Cavendish’ group. The process described by these authors involves culturing the male flower buds on
MS medium supplemented with 18.10 mM 2,4-dichlorophenoxyacetic acid (2,4-D), 5.37 mM
napthalene acetic acid (NAA), 5.71 mM IAA and 4.09 mM d-biotin with 30 g/l sucrose. After 2-3
months, the floral primordial shows enlargement and the development of whitish embryogenic callus.
Embryogenic callus will continue to proliferate when subcultured on MS medium supplemented with
0.22 mM BAP and 1.14 mM IAA. If the cultures stay on this medium, somatic embryos will start
developing during subcultures on the same medium. Green plumule and subsequently complete
plantlets will emerge from the embryos followed by the development of roots within a span of 6 to 8
weeks upon transfer to half strength MS medium supplemented with 0.5 g/l malt extract (ME) and
0.1% activated charcoal (Ganapathi et al., 2001).

Techniques of genetic transformation include: (1) electroporation of protoplasts, which have been
isolated from an embroygenic cell suspension of the cooking banana cultivar ‘Bluggoe’ (Sági et al.,
1995); (2) the procedure of micro-projectile bombardment with DNA-coated particles which has been
applied to embroygenic cell suspensions of ‘Bluggoe’, dessert bananas ‘Williams’ and ‘Grand Naine’
and the plantain ‘Three Hand Planty’ (Sági et al., 1995; Becker and Dale, 2004); and (3) a combined
Agrobacterium and microprojectile bombardment approach recently reported by May et al. (1995) for
the dessert banana ‘Grand Naine’. May et al. (1995) used corm slices as explants and Agrobacterium
infection after wounding of meristematic tissue by using particle bombardment. Transformation by
Agrobacterium was unexpected, because infection with Agrobacterium was formerly considered to be
unsuitable for a monocotyledonous plant like banana.

Application of genetic transformation techniques has already resulted in regenerated transformed

plants from cvs. ‘Bluggoe’ and ‘Grand Naine’ expressing marker genes such as the uidA gene coding
for bacterial β-glucuronidase or an antibiotic resistance gene. However, there are currently no
published reports about the expression of genes in banana or plantain, which might offer protection
against a fungal disease. Sági et al. (1995) recently indicated that they intend to transfer genes into
banana and plantain cultivars coding for cysteine-rich peptides isolated from seeds of different plant
species (Cammue et al., 1993). These peptides have shown high anti-fungal activity to M. fijiensis and
F. oxysporum f. sp. cubense in vitro, while at the same time they are not toxic to human and plant
cells.

49
In vitro selection using culture filtrates and toxins

The two most important diseases studied in this regard are Black sigatoka disease and Panama disease.
For this chapter the focus will be more on Black sigatoka disease.

The strategy is based on the use of more or less purified culture filtrates of M. fijiensis for Black
sigatoka or F. oxysporum f. sp. cubense for Panama disease as a selection agent. Essential conditions
for attributing the function of a disease determinant to a toxin or culture filtrate are: (1) the
demonstration that the toxin or culture filtrate occurs in infected plants, and (2) the ability of the toxin
to cause at least a portion of the syndrome when placed in healthy plants (Aducci et al., 1997).
Although toxins and culture filtrates may play important roles in some plant diseases, their possible
use for selecting resistant genotypes of the host in vitro depends on two observations: (1) all reliably
identified isolates of the fungus produce one or more toxins in culture; and (2) the symptoms of the
disease (chlorosis, necrosis, breakage of the stem, etc.) are also elicited by the toxins alone (Hamid
and Strange, 2000).

The phytotoxicity of M. fijiensis and F. oxysporum f. sp. cubense culture filtrates and toxins has been
demonstrated in bioassays carried out on leaves of banana and plantain (Okole, 1995; Hoss et al.,
2000; Lepoivre 2003; Busogoro et al., 2004; Gimenez and Colmenares, 2004; El Hadrami et al.,
2005a). Most of these compounds are non-host-selective toxins. The symptoms caused by toxins
depends on the chemical nature of the compound and the properties of the plant. The most common
symptoms that can be seen with the naked eye are wilting, necrosis, water-soaking and chlorosis. In
vitro symptoms include slow or rapid cellular collapse, membrane leakages, accumulation of toxic
metabolic intermediates, or ultastructural alterations in organelles (Walton and Panaccione, 1993;
Walton 1996).

Purification of crude extracts of M. fijiensis has allowed identification of a set of phytotoxic

metabolites found in the pathogen culture filtrates. These compounds are 2,4,8-trihydroxytetralone
(2,4,8-THT), 5-hydroxy-1,4-naphtalenedione (commonly called juglone), 2-carboxy-3-
hydroxycinnamic acid, a dimethyl ester of 2-carboxy-3-methoxycinnamic acid, isoochracinic acid and
4-hydroxycytalone (Stierle et al., 1991). Fijiensin, another metabolite of M. fijiensis that is
characterised by a more complex structure than the previous molecules, exhibited no differential
reaction to various cultivars despite the specificity of this molecule on banana (Upadhyay et al., 1990).
Out of the list of compounds produced from the culture filtrate, only 2,4,8-THT has been shown to be
host-specific (Okole and Schulz, 1996; Hoss et al., 2000).

The process normally used for in vitro selection is outlined in Figure 3.1. A double selection system
was applied for selecting banana and plantain resistant plants. This involved selection first in the crude
filtrate of M. fijiensis followed by a second round of selection using the host-specific purified toxin
2,4,8-THT. For that, micro-cross sections (400 µm), from which shoot buds can be regenerated, were
placed in a 9 cm Petri dish. The best concentration when using the crude filtrate was adding 25% of
the crude filtrate into MS multiplication medium described above. The dish containing the micro-cross
sections and the crude filtrate was kept under light conditions of 90 µE/m2/s for four days. After
treatment with the crude fungal filtrate, the sections were transferred to a multiplication medium
without the addition of the crude fungal filtrate. Shoot buds that developed after four weeks on the
brown tissues were regenerated to plantlets.

For double selction, micro-cross sections were again prepared from the plantlets resistant to the crude
filtrate. They were placed for 48 hours under light conditions of 90 µE/m2/s on a banana multiplication
medium containing 20 μg/ml 2,4,8-THT in the medium. A stock solution of the synthetic melanin
inhibitor tricyclazole (5% ethanol [v/v]) (5-methyl-1,2,4-trazolo[3,4-b]-[1,3]benzothiazole, Dow
AgroSciences LLC, Indianapolis, IN, USA) was added to the medium before autoclaving. After
transfer of micro-cross sections onto toxin-free medium, shoot buds produced after four weeks were
regenerated to plantlets and established in soil for in vivo screening using the fungus M. fijiensis.

50
In vivo screening

Banana and plantain plantlets, ~20 cm in height, which were regenerated from micro-cross sections
after the double selection protocol, were screened for fungal resistance with conidial suspension (4 ×
104 conidia/ml) of different isolates collected from four different countries (Cameroon - isolate 200,
Honduras - isolate 400, Papua New Guinea - isolate 309 and Nigeria - isolate 100). Some of these
isolates were kindly supplied by Dr. X. Mourichon CIRAD/IRFA, Montpellier, France and Dr. B.
Fullerton, Mt. Albert Research Centre, New Zealand. Inoculation on the leaves was performed
following the method described in Figure 3.1. Symptoms on the leaves, such as chlorosis and brown to
black lesions resulting from M. fijiensis infection were observed on the plants 40-60 days after
inoculation. Each treatment had a control plant. Resistant plants had no symptoms or chlorosis;
tolerant had 2-3 dark brown spots/flecks, while susceptible plants showed typical Black sigatoka
symptoms.

RESULTS AND DISCUSSION

There were 150 regenerated plants from the micro-cross sections of the three different banana and
plantain cultivars (Table 3.1). The plants that were regenerated from the double selection procedure
were later screened against fungal isolate 100 from Nigeria in the growth room. From Table 3.1, it
could be seen that the different cultivars responded differently to the fungus in the growth room. Of
the three cultivars tested, ‘Petite Naine’ had the highest percentage of resistant plants (19%), while
‘Horn’ plantain had the lowest (10.7%). All the control non-treated plants that were regenerated from
micro-cross sections not treated with the crude filtrate or purified toxin were severely infected and
showed typical Black sigatoka symptoms.

Since the cultivar ‘Petite Naine’ had the highest percentage of resistant plants, it was further multiplied
and tested against four isolates of the fungus from different countries. The results in Table 3.2 show
that the level of resistance of ‘Petite Naine’ plantlets varied with the different isolates used. The
highest percentage of resistant plants came from plants treated with the fungal isolate from Nigeria
(48%), while the lowest percentage of resistant plants (29%) was for plants inoculated with the isolate
from Papua New Guinea.

The resistant plants from both Tables 3.1 and 3.2 were further multiplied (Figure 3.2) for a field
experiment that was carried out in a big commercial plantation in Cameroon (SP&P). Unfortunately,
the results were not conclusive, because of the indiscriminate spraying method used to control disease
in big plantations. Spraying is normally done with small aeroplanes and it was difficult to isolate the
experimental plot from the plantation bananas. There was always a carry over of the chemical spray to
the experimental plot. Although the results looked good, it was not possible to draw conclusions from
the data obtained.

Generation of disease- or pest-resistant plants can be carried out through identification of natural
variants, chemically or by induced mutants or by using a genetic engineering approach. This was the
first report in which Musa micro-cross sections were used for selection to a host-specific fungal toxin
and subsequent regeration of resistant cell lines after challenging with the toxins and the fungus
(Okole and Schulz, 1997). In general, the technique of using micro-cross sections in selection studies
is comparable to other techniques where shoot tips, callus or cell suspension culture have been used in
banana and plantain as an explant source (Matsumoto et al., 1995; Trujillo and De Garcia, 1996; Hoss
et al., 2000; Gimenez and Colmenares, 2004; Reyes-Borja, 2005). Early reports of the ability of M.
fijiensis to produce toxic metabolites in vitro were published by, Molina and Krausz (1989), Natural
(1989, Upadhyay et al. (1989), and Lepoivre and Acuna (1990). In these reports, they did bioassay
studies on leaves of tissue culture banana plants using crude filtrate metabolites and, in some cases, the
purified compounds. In all of their results, they reported biological activity on the leaves with activity
being correlated with the different levels of resistance of the banana or plantain plants.

51
Breeding for resistance is the most promising strategy for fighting Black sigatoka disease, especially
in small farmer plantations. M. fijiensis produces many phytotoxins. The most studied of these
phytotoxins are the pentaketide metabolites, 2-hydroxyjuglone, juglone (a non-host-specific toxin) and
2,4,8-THT (host-specific). Both toxins can be used, jointly with the fungus in field and growth room
controlled conditions, for screening banana and plantain cultivars for Black sigatoka resistance. The
non-host-specific phytotoxin has been shown to act on chloroplasts and disturbs the proton
electrochemical gradient across the plasmalemma membrane. The purified toxin has a direct inhibitory
effect on the electron transfer properties of purified banana chloroplasts. Furthermore, an involvement
of the oxidative burst during the interaction has been suggested (Busogoro et al., 2004; El Hadrami et
al., 2005a). Mora et al. (2002) carried out a study to determine the best concentration to differentiate
two Musa cultivars ‘Fougamou’ (resistant) and ‘Grand Naine’ (susceptible). They reported that the
concentration of 100 mg/l was able to produce necrotic symptoms in both cultivars; nevertheless, with
the 50 mg/l concentration, it was possible to obtain differences between the two cultivars.

The second pentaketide 2,4,8-THT has been summarised by Hoss et al. (2000) as follows: Following
recognition and enhancement of fungal metabolism in a resistant cultivar such as ‘Yangambi Km 5’,
an increase of phenylalanine-ammonia lyase (PAL) activity is considered to be the first detectable
specific reaction of host tissues leading to the biosynthesis of secondary plant compounds. The
elicitation of this process is accompanied by necrotic micro-lesions which are often categorised
phenotypically as a hypersensitive reaction. Other responses, such as phytoalexin accumulation, as
observed in the resistant or tolerant cultivars, are the result of the effects that cause incompatibility
between host and pathogen. Therefore, they concluded that the rapidity and spatial extention of high
2,4,8-THT concentrations at the sites of close contact between host tissue and fungal hyphae are
determinants of the resulting interaction, ranging from susceptibility to resistance inside a host
reaction continuum. The Musa spp.-M. fijiensis pathosystem, therefore, differs from other known
plant-pathogen relationships by the fact that host specificity is determined at the cultivar level due to
an activation of fungal metabolism, which leads on its part to an elicitation of a defence reaction and
results in an incompatible interaction. Stierle et al. (1991) further reported that the most abundant of
the host-specific phytotoxic compounds produced by M. fijiensis is 2,4,8-THT, which induces necrotic
lesions at 5 µg/5 µl in less than 12 hours on sensitive cultivars of bananas.

The application of tricyclazole to healthy leaf tissue did not cause any visible phytotoxic symptoms on
the leaves, but the leaves of the plants treated with tricyclazole and the toxin showed pronounced
necrosis within a few days of incubation. The synthetic compound tricyclazole has been used as an
inhibitor of two reduction systems of melanin biosynthesis, causing accumulation of important
intermediate compounds of pentaketide metabolism. This could hypothetically enhance 2,4,8-THT
biosynthesis (Stierle et al., 1991; Hoss et al., 2000).

CONCLUSIONS

Bananas and plantains (Musa spp.) belongs to the most important staple food crops worldwide.
Production of these crops is limited by many diseases and pests. Resistance breeding is urgently
needed because diseases and pest control measures comprise ca 40% of total production costs. One of
the most efficient banana and plantain breeding approaches is in vitro selection for resistance by using
culture filtrates and toxins produced by most dangerous fungi, i.e. Mycosphaerella fijiensis (causing
Black sigatoka) and Fusarium oxysporum f. sp. cubense (causing Panama disease). Literature data and
our own experiments clearly demonstrated that both fungi produce phytotoxic compounds which are
mostly non-host-selective toxins (e.g. juglone), however, also host-selective toxins (e.g. 2,4, 8-THT).
In this paper we focused on in vitro selection to M. fijiensis. Shoot meristems, micro-cross sections,
callus, cell suspension and protoplasts are suitable for in vitro selection for resistance. A double
selection system showed high efficiency with using: a) the crude filtrate, b) the purified host-specific
toxin 2,4,8-THT, as well as c) the non-host-specific toxin juglone. The most suitable concentrations of
both toxins were suggested for in vitro selection. It is evident that induction of mutations and

52
following selection in vitro by using of toxin technology could be very efficient mass-screening
approach in early stages of banana and plantains breeding for resistance/tolerance to fungal diseases.

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Wolpert JT, Dunkle LD, Ciuffetti LM (2002) Host-selective toxins and avirulence determinants:
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Table 3.1. In vivo response of the toxin-treated banana and plantain plants to Mycosphaerella fijiensis
(isolate 100)

Musa cultivar No. of toxin-resistant Disease reaction1 % Resistance2

plants regenerated
R T S
‘Williams’ 150 22 61 67 14.7
‘Petite Naine’ 150 29 78 43 19.3
‘Horn Plantain’ 150 16 75 59 10.7

1
R = resistant; T = tolerant; S = susceptible; 2 % Resistance represents total number of resistant
plants/total number of toxin-resistant regenerated plants × 100. Source: Okole and Schulz (1997).

Table 3.2. In vivo response of plantlets regenerated from toxin-resitant ‘Petite Naine’ plants to
different Mycosphaerella fijiensis isolates

M. fijiensis No. of fungal toxin- Disease reaction2

isolate1 resistant plants
R T S
100 50 48 2 0
200 50 40 9 1
309 50 29 18 3
400 50 38 10 2

1
Origin of isolates: isolate 100 - Nigeria; isolate 200 - Cameroon; isolate 309 - Papua New
Guinea; isolate 400 - Honduras. 2R = resistant; T = tolerant; S = susceptible. Source: Okole
and Schulz (1997).

57
Figure 3.1. Selection procedure for production of Black sigatoka disease-resistant plants. After
brushing off the waxy layers on the lower surface of the leaves, the conidia and mycelium are sprayed
evenly on both sides of the leaves with a spray gun to allow uniform spread on the leaves. Inoculated
plants are kept in a growth room with the temperature adjusted to 26°C with a 16:8 hour light: dark
photoperiod (light intensity: 45-50 µE/m2/s) and high humidity. Visible disease symptoms of Black
sigatoka usually appear from 40-60 days in control plants (Okole et al., 2000).

58
 Chapter 4

Mutation induction by gamma-rays and carbon ion beam irradiation in

banana (Musa spp.): a study with an emphasis on the response to
Black sigatoka disease

W.O. Reyes-Borja1*, I. Sotomayor2, I. Garzón2, D. Vera2, M. Cedeño2, K. Degi3,

S. Nagatomi3, A. Tanaka4, Y. Hase4, Y. Sekozawa1, S. Sugaya1 and H. Gemma1
1
Graduate School of Life and Environmental Sciences,
University of Tsukuba,
Japan
2
Instituto Nacional Autónomo de Investigaciones Agropecuarias (INIAP),
Ecuador
3
Institute of Radiation Breeding,
National Institute of Agrobiological Sciences,
4
Department of Ion Beam Applied Biology,
Japan Atomic Energy Agency,
Japan.

*Correspondence: [email protected]

Abstract

Gamma-rays and carbon ion beam irradiation methods were applied to study critical doses, genetic
variability and the response to Black sigatoka disease. ‘Cavendish Enano’, ‘Williams’, ‘Orito’ and
‘FHIA-01’ cultivars of banana were studied. Both gamma-rays and carbon ion beam irradiation
methods had different biological effects when banana explants were exposed to them. In both
methods, increased dose caused increased mortality. ‘FHIA-01’ tolerated high doses of gamma-rays
but was susceptible to high doses of carbon ion beam irradiation. The results suggest that the response
in ‘FHIA-01’ can be explored using other dose intervals between 150 and 300 Gy. Weight and height
were also reduced drastically when high doses of gamma-rays and carbon ion beams were applied.
The LD50 of cultivars ‘FHIA-01’ and ‘Orito’ revealed high sensitivity to both gamma-rays and carbon
ion beams. DNA deletion in ‘FHIA-01’ occurred by using gamma-rays at doses of 200 and 300 Gy,
suggesting that ‘FHIA-01’ is definitely a promising cultivar with a high sensitivity response to
gamma-ray exposure, and that there is a high chance of improving its fruit quality by mutation
induction. Sigmoid drooping leaf, a putative mutation of ‘FHIA-01’, was generated. This mutation is
heritable as mother plant and sucker showed the same characteristics. Future research could be
conducted on the relationship of leaf shape to fruit quality and production. Hexaploid cells were
detected by flow cytometry (five plants in ‘Cavendish Enano’ and one in ‘Williams’), signifying that
chromosome duplication can be induced by carbon ion beams. Variation in the leaves such as being
abnormal, double, long, rudimentary, spindled and yellow spotted leaf was visible, suggesting that
long-term chronic irradiation (gamma-rays) directly affects active cell division at the meristem level,
resulting in severe damage or even death of the meristems. During the juglone toxin experiment on
gamma-ray-irradiated plants, 20 plants were selected from the ‘Orito’ lot, eight in ‘Williams’ and five
in ‘Cavendish Enano’. In the carbon ion beam experiments, six plants of ‘Williams’ and two of
‘Cavendish Enano’ were selected as possible candidates with a better response to Black sigatoka
disease. In addition, following irradiation with a carbon ion beam, a fast growing plant was observed
and selected as earliness is an important characteristic for shortening the crop life cycle. Finally, field

59
experiments throughout the whole plant cycle are needed to evaluate mutated traits for fruit quality,
yield and post-harvest characteristics for a final selection.

INTRODUCTION

Due to low reproductive fertility and high polyploid levels in banana, traditional hybridisation
breeding techniques remain difficult (Rowe, 1984). Diseases are among the most important factors
seriously affecting banana production worldwide. Among them, Black sigatoka disease caused by
Mycosphaerella fijiensis Morelet, being the most destructive disease to attack the leaves, is one of the
most serious constraints of banana cultivation (Craenen and Ortiz, 1996). It causes yield losses of 50%
or more. Most research focuses on generating cultivars tolerant/resistant to this disease. Thus,
radiation breeding could prove to be a viable method in banana breeding work. The use of mutagens
has been acknowledged to be a reliable method for breeding plants with improved characteristics in
many crops. In this study, nuclear techniques, especially the use of 60Co and 137Cs as sources of
gamma-rays and carbon ion beams, have been coupled with in vitro techniques, to induce mutation, as
a source of genetic variability for potentially improved and more desirable traits. At the same time,
genetic variation has been noted when tissue culture techniques are used and to a greater extent when
these variations are induced by the use of mutagens (Bermúdez et al., 2002; García et al., 2002).

Using gamma-rays, Roux (2004) reported different banana mutants with improved morphological
characteristics of bunch size and cylindrical shape (mutant line name: ‘Klue Hom Thong KU1’) in
Thailand, and plant height (dwarfness) (mutant line names: ‘SH-3436-L9’ and ‘6.44’) in Cuba. Mutant
lines with increased tolerance to Fusarium oxysporum (‘Mutiara’ and ‘Novaria’) developed in
Malaysia, and to the toxin of M. fijiensis (‘GN35-I to GN35-VIII’) developed by the IAEA were also
reported.

The ion beam technique has recently been used rather than gamma-rays to produce a wide range of
mutants. Fukuda et al. (2004) mentioned that ion beams can frequently produce large DNA alterations
such as inversions, translocations and large deletions rather than point mutations, resulting in the
production of desirable characteristics. Yu (2006) stated that the most important application of
artificially-induced mutations is in mutation breeding, using sexual or asexual offspring to induce ideal
genotypes. Since the biological effects of ion beams, as a new mutagen, were discovered, this
technique has progressively received increasing attention. Ion beams integrate the factors of mass,
energy and charge, inducing damage to the biological materials, thereby displacing, recombining and
compounding the biological molecules and atoms.

The objectives of this research, conducted to find mutations, especially in response to Black sigatoka
disease, are listed below:

(1) To apply both tissue culture and irradiation techniques to induce genetic variability for mutant
selection, and to determine irradiation sensitivity (60Co, 137Cs and carbon ions) in both explant and
plantlet stages of banana.
(2) To conduct studies on irradiated materials with the aim of finding a Black sigatoka disease-
tolerant or resistant banana mutant, by using juglone toxin and conidial cultures of M. fijiensis as
inoculation techniques to evaluate disease resistance at early stages of banana in vitro.

MATERIAL AND METHODS

Plant material

Four cultivars of banana were irradiated using gamma-rays and carbon ion beams. ‘Cavendish Enano’
(M. acuminata AAA) belongs to the ‘Cavendish’ subgroup, has an intermediate plant height, a normal
bunch and an acceptable commercial fruit quality. This triploid cultivar is susceptible to Black

60
sigatoka disease and is one of the most important ‘Cavendish’ subgroup cultivars cultivated
worldwide. It is also well known as ‘Grand Naine’. ‘Williams’ (M. acuminata AAA) is also a triploid
belonging to the ‘Cavendish’ subgroup. It has a normal plant, a good bunch and good fruit quality, but
is highly susceptible to Black sigatoka disease. ‘Orito’ (M. acuminata AA) is a favourite cultivar in
Ecuador. It grows in humid areas and most of the plantations are organic, since the farmers cultivate
with a minimum of cultural practices. This cultivar possesses small finger, sweet and delicious and has
a high carotenoid content compared to ‘Cavendish’. It is susceptible to Black sigatoka disease but less
so than the ‘Cavendish’ type. This cultivar is also called ‘Baby banana’ on the organic banana market.
‘FHIA-01’ (M. acuminata AAAB) is a tetraploid hybrid, and very resistant to Black sigatoka disease.
Bunch weight ranges from 39 to 56 kg, with the number of functional leaves at the harvest stage
ranging from 9 to 11. Plant height is ~4 m, with finger number ranging from 170 to 229. The fruit
possess a very different taste to that of the ‘Cavendish’ type. This cultivar was included in this
experiment as a control to compare cultivars resistant or susceptible to Black sigatoka disease.

Gamma-ray and carbon ion beam characteristics and irradiation doses

Gamma irradiation was carried out in collaboration with the National Institute of Radiation Breeding,
National Institute of Agrobiological Science, located in Hitachiohmiya, Ibaraki Prefecture, Japan.
Regenerated plants and explants were treated using gamma-rays provided in facilities such as the
“Gamma room” (60Co), “Gamma field” (60Co) and “Gamma greenhouse” (137Cs). In the “Gamma
room” facility, the doses were: 0 (control), 50, 100, 150, 200, 300 and 500 Gy. In the “Gamma field”
facility, plantlets were treated with irradiation doses of 0.5, 1 and 2 Gy, and in the “Gamma
greenhouse” facility, plantlets were irradiated with three doses: 0.25, 0.50 and 0.75 Gy (20 hours/day).

Regarding the research on carbon ion beams, irradiation was conducted at the Takasaki Ion
Accelerators for Advanced Radiation Application (TIARA), Japan Atomic Energy Agency (JAEA),
Japan. Carbon ions with a total energy of 320 million electron volts (MeV) were generated by an
Azimuthally Varying Field (AVF) cyclotron. The physical properties of the carbon ions were as
follows: the incident energy at the target surface was 311 MeV (25.9 MeV/u), the range of the ions in
a target was 2.2 mm, and the mean linear energy transfer (LET) in a target was estimated to be 137.6
keV/µm. ‘Cavendish Enano’ and ‘Williams’ samples were irradiated with doses of 0, 0.5, 1, 2, 4, 8,
16, 32, 64 and 128 Gy as a first trial to scan the best dose, and later on a second irradiation was
conducted using doses of 0, 0.5, 1, 2, 4, 8 and 16 Gy on ‘Williams’, ‘Orito’ and ‘FHIA-01’.

Plant and explant conditioning pre- and post-irradiation

The initial material consisted of in vitro micro-propagated plants prior to the establishment of the
experiments. Four week old in vitro shoot tips from the four banana cultivars were used as a source of
explants for both “Gamma room” and carbon ion beam irradiations (Figure 4.1A and D, respectively).
These shoot tips were grown in a multiplication solid medium that consisted of Murashige and Skoog
(MS) medium (Murashige and Skoog, 1962) supplemented with 6-benzylaminopurine (BAP; 2.25
mg/l), indole-3-acetic acid (IAA; 0.05 mg/l), sucrose (20 g/l) and agar (9 g/l) at pH5.6. For the
“Gamma room” experiment, ‘Cavendish Enano’, ‘Williams’, ‘Orito’ and ‘FHIA-01’ varieties were
used. The irradiation doses were applied six days after planting the explants in the solid medium. The
survival rate (%), height (cm) and weight (g) of the shoots were determined one month after
irradiation. For fast propagation, during pre- and post-irradiation, the explants were transferred to
multiplication liquid medium (same as above without agar) by dividing the corm of each.

For the carbon ion beam irradiation, banana explants as thin as 2 mm were requested to allow total
penetration of the ion beam. Prior to irradiation, an experiment was conducted to clarify the
regeneration rate of the thinner banana explants. Cultivars ‘FHIA-01’ and ‘Williams’ were used for
that experiment. Two types of slicing or cutting methods (vertical and horizontal slices) were applied
to the corms (i.e., banana rhizome). The slices were placed in a regeneration liquid medium,
containing MS medium supplemented with BAP (5 mg/l) and sucrose (20 g/l) at pH 5.6. 25 slices

61
were placed in a 300 ml Erlenmeyer flask containing 100 ml of medium. Five flasks (replications),
containing the same number of explants, were prepared. The explants were stirred in a shaker at
100 rpm. The regeneration rate (%) and weight (g) of explants were recorded. From the results, the
slicing method allowed the selection of the highest number of regenerated plants which were applied
in the establishment of the experiment for carbon ion beam irradiation.

For the first irradiation with the carbon ion beam, vertical slices from four week old shoot tips of
‘Cavendish Enano’ and ‘Williams’ were placed in 6 cm diameter plastic dishes containing
multiplication solid medium and were covered with sterilised Kapton films (thickness: 8 μm, Toray-
Dupont, Japan) in order to prevent the loss of energy of the carbon ions. The explants were planted on
a Petri dish two days before irradiation. For this purpose, 20 explants/dish × 2 dishes (40 explants per
dose) were used, giving a total of 400 explants per cultivar (800 explants considering both cultivars).
Two days after irradiation, the explants were transferred to a 100 ml Erlenmeyer flask containing
50 ml of MS liquid medium. 19 days later, growth of the explants was evaluated using the following
parameters: weight (g), height (cm), survival rate (%) and LD50 (lethal dose 50%; i.e., irradiation dose
that causes 50% of mortality in test explants).

In the case of the chronic irradiation, both “Gamma field” and “Gamma greenhouse” facilities were
used (Figure 4.1B, and C). The experiments were carried out using acclimated plantlets ~20 cm in
height growing in 40 cm diameter pots. Plants at the “Gamma field” facility were irradiated for a
period of 34 days. One plant per treatment and per cultivar was used, except for the cultivar ‘Orito’ for
which the dose of 1 Gy was omitted. At the “Gamma greenhouse” facility, the banana plants were
irradiated for a period of nine months. One plant per dose was irradiated in ‘Williams’. Two plants per
treatment at doses of 0.25 and 0.5 Gy, and only one plant at a dose of 0.75 Gy were irradiated for the
cultivars ‘Orito’, ‘Cavendish Enano’ and ‘FHIA-01’. To propagate the irradiated plants, the meristems
were excised in aseptic conditions and cut into several explants. The meristems were placed into a
multiplication solid medium for in vitro culture. Explants from this experiment were sub-cultured
three times to increase plant numbers for post-irradiation studies.

In general, after irradiation either by gamma-ray or by carbon ion beam methods, the explants were
immersed in the multiplication liquid medium and stirred in a shaker at 100 rpm. During all the
maintenance stages in the tissue culture room, the temperature was kept at 27°C with a 16:8 hour light:
dark photoperiod (light intensity: 65 µmol/m2/s). After irradiation, initiation solid medium,
multiplication liquid medium and regeneration solid medium were used to propagate the explants into
a large population to study the mutations. For a better understanding of the sequence of this
experiment, a schematic drawing is shown in Figure 4.2.

Relative DNA content measured using flow cytometry

To analyse the relative DNA content of banana leaf samples, a PAS Flow Cytometer (Partec) equipped
with a mercury arc lamp that was suitable for analysis of samples stained with a CyStain UV kit was
used. The samples were prepared using 400 µl of nuclei extraction buffer and approximately 0.5 cm2
of banana leaf tissue on a plastic 55 mm Petri dish. Then, using a sharp razor blade, the samples were
chopped for 30 to 60 s and filtered through a Partec 50 µm cell-trics disposable filter using a test tube
to collect the filtrate. Samples were incubated in ice for 30 s to 5 min. After incubation with the
extraction buffer, 1.6 ml of staining solution was added to the test tube and the sample was
immediately analysed in the flow cytometer to obtain the fluorescent index (FI).

Non-irradiated ‘Cavendish Enano’, ‘Williams’, ‘FHIA-01’ and ‘Orito’ were used as controls to
compare with the irradiated material. In order to assess FI values, an internal control must be used that
has a ploidy level different than that of the test sample whose relative DNA content is being
calculated. In this case, non-irradiated ‘Orito’ (diploid) was used as an internal control for the
irradiated triploids ‘Williams’ and ‘Cavendish Enano’, and tetraploid ‘FHIA-01’. To compare
irradiated samples of ‘Orito’, non-irradiated ‘Cavendish Enano’ was used as the internal control.

62
The Relative Nuclear DNA Content (RDC) was calculated using the FI peak mean value of the test
sample divided by the FI peak mean of the internal control. The resulting values of RDC were
analysed by frequency distribution. Flow cytometry analysis of gamma-ray-irradiated plants was
carried out using 20 plants for each dose and cultivar, plus 20 non-irradiated and ‘Orito’ as control
plants. Only the plants resulting from the “Gamma room” experiment were analysed by frequency
distributions. Regarding the carbon ion beam-irradiated samples, 115 samples were analysed using
‘FHIA-01’ as the control.

After analysis of the relative DNA content, ‘FHIA-01’ was found to be the most affected cultivar
among the irradiated plants at the higher doses (200-300 Gy). Plants with a reduced DNA content
were selected. The plants were kept in big pots with soil and later on were transferred to the
greenhouse and maintained under controlled temperature conditions.

Juglone toxin screening

Prior to screening the irradiated banana plants obtained from gamma-ray and carbon ion beam
irradiation, trials using juglone toxin were conducted on non-irradiated plants of ‘Cavendish Enano’,
‘Williams’ and ‘Orito’, which are susceptible to Black sigatoka disease. ‘FHIA-01’, which is resistant,
was included in this experiment as a resistance indicator control. Various concentrations of juglone
were applied, in order to select the most suitable concentration for producing differentiable necrotic
area between non-irradiated susceptible cultivars and the resistant control. Juglone (5-hydroxy-1,4-
naphthoquinone) is one of the most active toxins of the seven compounds produced by M. fijiensis
Morelet, that induce the formation of necrotic lesions on plants leaf cells (Stierle et al., 1991; Strobel
et al., 1993). This toxin was used in this experiment to screen young plants as an indicator of
resistance/tolerance to Black sigatoka disease.

Non-irradiated in vitro derived plants, bearing 4-6 leaves and with a height of 15-20 cm, were used for
this experiment. Four plants per cultivar and four leaf disks for each concentration were used to
evaluate extended necrosis induced by juglone on the leaf disks of banana. Low concentrations of
juglone (10, 15, 20, 25, 30, 35, 40, 50, 55 and 60 ppm) were initially tested; however, these
concentrations did not induce any necrosis on the disks 24 and 48 hours after inoculation. A second
trial using concentrations of 100, 150, 200, 250, 300 and 350 ppm was conducted. Leaf disks were
immersed in 2 ml of juglone solution contained in a small Petri dish (diameter: 40 mm) and kept in an
illuminated incubator at 26ºC for 24 and 48 hours. The disks were obtained from the second expanded
leaf with a cork borer of radius 6 mm (113.09 mm2). Leaf disks from ‘FHIA-01’ (Black sigatoka
disease-resistant) were included as a resistance indicator to compare with the irradiated susceptible
cultivars (‘Cavendish Enano’, ‘Williams’ and ‘Orito’).

After inoculation using the toxin, leaf disk photos were taken with an Olympus Camedia Digital
Camera C-5050 Zoom. The Leaf disk necrotic area = LDNA (%) was measured using the GIMP 1.2
software by counting the number of pixels of the full disk and the number of pixels of the green area
by selecting hand-drawn regions and obtaining the value for the necrotic area by subtraction
(Figure 4.3) as is expressed in the following formula:

LDNA (%) = Pixels of the full disk - Pixels of the green area × 100
Pixels of the full disk

Based on the findings of the preliminary experiments, juglone at concentrations of 100 or 150 ppm
was sufficient to obtain clear differences between the resistant and susceptible cultivars. This served as
an indicator to differentiate the juglone-tolerant response among irradiated plants. Thus, using this
criterion, irradiated plants were screened using 150 ppm of juglone. In the present experiment, we
confirmed that juglone at different concentrations is able to induce marginal necrosis in banana leaf
disks, based on the application of the method to black spot disease of Japanese pear (Sanata, 1988).

63
For the gamma-irradiated population, the disks of a 6 mm in diameter were taken from the second
expanded leaf of each cultivar and immersed in a Petri dish (diameter: 30 mm) containing 10 ml of
juglone solution. Samples were kept in an illuminated incubator at 26°C for 24 hours. ‘FHIA-01’ was
also included as the indicator of resistance. Photographs of the leaf disks were processed as mentioned
above. A total of 208, 179 and 307 plants were screened in ‘Cavendish Enano’, ‘Williams’ and
‘Orito’, respectively. Data were analysed by frequency distributions, and values divided into nine
classes. The lowest LDNA (%) class values (necrosis produced by using 150 ppm of juglone on the
leaf disks) were selected, considering that those values were around 9% of LDNA (%) which was the
value obtained for the resistance indicator ‘FHIA-01’.

In the case of the plants irradiated with carbon ion beams, the leaf disks (five disks per plant) were
taken from the second expanded leaf of cultivars ‘Williams’ and ‘Cavendish Enano’ from the field-
planted experiment. Leaf disk samples of ‘FHIA-01’ (indicator of resistance) were obtained from
sucker plants available in the banana collection of the Estación Experimental Tropical Pichilingue,
Ecuador. The value of the resistance indicator ‘FHIA-01’ was calculated at ca. 42.2%. A solution
containing 150 ppm of juglone was prepared using distilled water. 10 ml were dispensed into a Petri
dish (90 mm) and the leaf disks were immersed in the solution. The leaves were rinsed with distilled
water and disks with a radius of 7.5 mm (176.71 mm2) were removed using a cork borer. Petri dishes
with leaf disk samples were kept in the light and at room temperature for 24 hours. A total of 435 leaf
disks were analysed (87 plants × 5 leaf disks/plant). After 24 hours, photographs of the leaf disks were
taken and processed in the same way as described above.

The lowest values of LDNA (%) of the gamma-ray population were selected; however, in the case of
the population irradiated with carbon ion beams, the LDNA (%) data were combined with the values
for the Disease Development Period (DDP; measured in days) and Infection index (II; measured in %)
in order to select candidates using a regression analysis.

Plant regeneration and acclimatisation for Black sigatoka disease assessment

Most of the plants evaluated for Black sigatoka disease were from the carbon ion beam-irradiated
plants due to the high mortality of the plant material. After the irradiated explants were sub-cultured
three times to regenerate shoots, they were planted individually into a test tube containing 10 ml of
solid MS medium, and 0.5 mg of activated charcoal for rooting. A total of 1707 rooted plantlets were
transferred to sterilise plastic bags and transported to Ecuador (Station Experimental Tropical
Pichilingue; Instituto Nacional de Investigaciones Agropecuarias, INIAP) for field experiments.

Immediately after arrival in Ecuador, plantlets were kept for two days in a tissue culture room at 26°C
with a 16:8 hour light: dark photoperiod (82 µmol/m2/s) to recover photosynthesis. They were then
transferred to a soil substrate bed (with a 1:1 mixture of soil and decomposed rice husk). The plantlets
were covered with a plastic sheet to avoid dehydration under greenhouse conditions. Only 87 plants
survived the nursery acclimatisation. The high mortality might have been caused by unfavourable
transportation conditions and pathogen attacks. These materials were used for the experiments for
Black sigatoka inoculation at the nursery and, later on, in field conditions.

On plants of ~30 cm in height, three leaves per plant were inoculated, and the younger expanded leaf
was marked as the first leaf for inoculation by a conidial solution (Figure 4.4). The second and third
successive young emitted leaves were inoculated by fragments of the diseased banana leaves
(Figure 4.5). Conidial cultures of M. fijiensis at a concentration of 1.5 × 106/ml were produced. After
inoculation, the plants were kept at 26°C with a high relative humidity (~85%) in a dark incubation
room for 48 hours. For second and third leaf inoculation, leaf fragments from plants severely infected
by Black sigatoka were placed at the base and inside the canopy of each plantlet as a potential natural
inoculum. A fickle cotton sheet moisturised three times a day was provided to cover all the plantlets to
ensure sporulation and to enhance the inoculation.

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Tolerance to Black sigatoka disease on inoculated banana plants was evaluated by two indices: DDP
(days) and II (%). DDP (days) was expressed as days until the full development of a spot with a dry
grey centre, using the stages of symptoms described by Fouré’s scale from the time of inoculation
(Orjeda, 1998). The disease severity determined by II (%) was calculated using the values obtained
from the Stover’s scale modified by Gauhl (Orjeda, 1998).

Putative mutants and factor of effectiveness-FE (%)

Several putative mutants were produced by gamma-ray and carbon ion beam irradiation. Mutants
showing traits such as juglone tolerance, low relative DNA content, dwarfism, sigmoid drooping leaf,
fast growth of sucker, good response to Black sigatoka disease, fast growth of plantlet, hexaploid
plants and several kinds of leaves were observed.

A factor of effectiveness-FE (%) was used to measure the efficiency of the mutagens based on data
obtained from phenologic and phenotypic variations. FE (%) was calculated with a modified formula
by Walther (1969) cited by Bhagwat and Duncan (1998) and described as follows:

FE (%) = Total number of variations × 100

Total number of plants treated

Statistical analysis

The statistical one-way analysis of variance by Tukey-Kramer (www.jmp.com, JMP, Version 5) was
used to analyse data from the gamma-ray population. For carbon ion beam irradiation, the data were
processed using analysis of variance (General AOV/AOCV, analytical software Statistics for
Windows version 2.0) followed by Tukey analysis (p ≦ 0.05). The radiosensitivity was evaluated as
the survival rate-lethal dose (LD50), and analysed by exponential regression. The LD50, which
determines the dose (Gy) necessary to kill half of the irradiated population, is the value used to assess
acute toxicity.

RESULTS AND DISCUSSION

Explant slicing conditioning prior to ion beam irradiation

The analysis of variance for regeneration rate (%) reported high significance for the two slicing
methods; however, there were not significant differences between cultivars. In contrast, the weights of
the explants were different between cultivars; however, no significant differences were found between
the two slicing methods. These results suggest that the regeneration rates (%) of ‘Williams’ and
‘FHIA-01’were affected by the type of slicing method to the same extent, but that the weight of the
explants was not affected by this factor.

Figure 4.6 shows the relationships of the explants’ weight (A) and regeneration rate (B) with the
slicing methods (vertical and horizontal) in ‘Williams’ and ‘FHIA-01’. Vertically-sliced cuttings
showed the highest regeneration rate (%) in both cultivars as is shown in Figure 4.7. Regeneration
rates of 60-70% were observed in ‘Williams’ and ‘FHIA-01’ using the vertical cutting method, in
contrast to only 37-43% in horizontally-sliced cuttings. The weights of the explants were similar in
both cultivars when the corm shoot tips were cut vertically. Horizontal slicing slightly affected
‘Williams’ although there were no significant differences between the two slicing methods. Explants
from this cultivar weighed, on average, 0.6 g when using the horizontal slicing method, compared to
0.9 g when using the vertical slicing method. Finally, the vertical slicing method was selected as the
best method for regenerating banana explants as thin as 2 mm and this type of slice was obtained from
shoot tips as a material for carbon ion beam irradiation.

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Gamma-ray and carbon ion beam biological effects on banana shoots

The application of radiation in the “Gamma room” facility showed that all the cultivars were affected
by the irradiation doses as reported by Reyes-Borja et al. (2005) (Figure 4.8A-C). The survival rate
decreased with increasing doses, and is cultivar-dependant. Differences in the sensitivity to the doses
of gamma-rays between ‘Cavendish Enano’ and ‘FHIA-01’ were significant at the 5% level.
‘Williams’ and ‘Orito’ showed a higher significance at the 1% level. ‘Williams’ and ‘FHIA-01’
showed a superior height, weight and survival rate when the dose reached 150 Gy, while ‘FHIA-01’
tolerated doses of up to 500 Gy, but with a very low survival rate (Figure 4.8A). ‘Cavendish Enano’
and ‘Orito’ appeared to be more sensitive to gamma-ray exposure, as evidenced by the low values of
plant height and weight recorded in these cultivars. Figure 4.9 shows the biological effect of gamma-
rays significantly reducing the growth of the explants as the doses increased.

Irradiation had a similar effect on plantlet height and weight across all cultivars with increasing
radiation doses. According to the Tukey-Kramer analysis, the four cultivars showed significant
differences (p ≦ 0.01). Doses >150 Gy strongly affected the weight of the shoots. Plantlet height
decreased in all cultivars with increasing dose rate. The difference between ‘Cavendish Enano’ and
‘FHIA-01’ was significant at the 5% level (p ≦ 0.05), and that between ‘Williams’ and ‘Orito’ was
significant at the 1% level (p ≦ 0.01). ‘Cavendish Enano’ was significantly different (p ≦ 0.05)
across dosages. The effect was more pronounced in ‘Cavendish Enano’, even at the lowest dosage,
resulting in low growth when >150 Gy was applied. ‘FHIA-01’ at a dose of 200 Gy still showed
vigorous growth, but this was reduced strongly when dosage was increased to 500 Gy. ‘Williams’ and
‘Orito’ could not tolerate radiation dosages >200 Gy. However, the height in ‘Williams’ remained
similar even when doses were increased to 50, 100 and 150 Gy (Figure 4.8C).

Figure 4.10A shows the optimum range dose of gamma-rays obtained in ‘Cavendish Enano’,
‘Williams’, ‘Orito’ and ‘FHIA-01’ through the LD50. Survival rate (%) values were used to calculate
the LD50 by exponential regression as an analysis to assess acute sensitivity in all four cultivars. The
LD50 was highest in ‘Williams’ (83.94%) and lowest in ‘Orito’ (65.0%); ±5% was aggregated to the
LD50 value of each cultivar to select the optimum irradiation doses of gamma-rays. Consequently, the
optimum doses for ‘Cavendish Enano’ ranged from 74.0 to 81.8 Gy, for ‘Williams’ from 79.7 to
88.1 Gy, for ‘Orito’ from 61.8 to 68.3 Gy, and for ‘FHIA-01’ from 73.2 to 80.9 Gy.

These results are in agreement with those obtained by Novak et al. (1990), who reported that Musa
cultivars exhibit significant differences in radiosensitivity and post-irradiation recovery. These
differences depend on the ploidy level and the hybrid constitution (genomes A and B). In their study,
the tetraploid cultivar ‘SH-3436’ (AAAA) exhibited the lowest level of radiation damage, whereas the
diploid cultivar ‘SH-3142’ (AA) was the most sensitive to gamma irradiation, which is in agreement
with the results obtained in the present study because ‘Orito’ (diploid AA) also showed high
sensitivity. However, triploids seemed to be unstable, since ‘Williams’ was less affected by irradiation
than ‘Cavendish Enano’. A similar trend was observed by Novak et al. (1990), who showed that the
highest dose (35-40 Gy) of gamma-rays was suitable for mutation induction in the triploid cultivar
‘Highgate’, but also in the tetraploid cultivar ‘SH-3436’.

Regarding carbon ion beam irradiation, the biological effects in both ‘Cavendish Enano’ and
‘Williams’ (first irradiation) were as follows. The analysis of variance for the height of explant
showed significant differences between cultivars and among ion beam doses (p ≦ 0.05). As shown in
Figure 4.8E,F, the weight and height of plantlets were affected when the Gy doses were increased
irrespective of cultivar. The highest growth behaviour in terms of weight and height was observed
when doses ≦2 Gy were applied. These parameters decreased when 4 Gy or higher were used.

The survival rate (%) of the explants is presented in Figure 4.8D. In general terms, the higher the
irradiation dose used the higher was the mortality rate, but doses of 8 Gy and lower did not seem to

66
have any effect on survival rates (Reyes-Borja et al., 2007). Figure 4.11 shows banana plantlets of
‘Cavendish Enano’ and ‘Williams’ affected by different doses of ion beam (Gy), 19 days after
irradiation. Hase et al. (2002) reported that high-LET radiation such as heavy-ion beams have greater
biological effects than low-LET radiation such as gamma-rays and X-rays. High-LET causes a
reduction in survival and a linear increase in the frequency of aberrant cells. Thus, it is possible that
the induction of chromosome aberrations depended on the LET.

The LD50 values for both ‘Cavendish Enano’ and ‘Williams’ (carbon ion beam first irradiation) were
obtained by exponential regressions. The LD50 for ‘Williams’ was established at 13.5 Gy and for
‘Cavendish Enano’ at 15.0 Gy indicating that the former would be more sensitive to the ion beams
than the latter. We only discuss the survival rate and LD50 for the batch of explants irradiated second
with carbon ion beams in ‘Williams’, ‘Orito’ and ‘FHIA-01’. The analysis of variance for survival rate
showed significant differences between the ion beam doses, but there was no difference between
cultivars. The survival rate of the explants was similar when 0-8 Gy were applied, but with 16 Gy this
variable was affected. The LD50 values for ‘Williams’, ‘Orito’ and ‘FHIA-01’ were estimated at 9.0,
3.8 and 4.6 Gy, respectively, indicating that ‘Orito’ and ‘FHIA-01’ were more sensitive to the ion
beams than ‘Williams’. Even though ‘Williams’ was irradiated twice, with LD50 values equal to
13.5 Gy and 9 Gy for the first and second irradiations, respectively, it was still more resistant to ion
beams than ‘Orito’ and ‘FHIA-01’.

As a consequence, when considering the data from the first and second irradiations, 5% were added
and subtracted from each LD50 value of each cultivar to designate the optimum irradiation dose range
of carbon ion beams. When this was done, the optimum doses for ‘Williams’ ranged from 12.8 to
14.2 Gy, for ‘Cavendish Enano’ (average of the two irradiations) from 14.3 to 15.8 Gy, for ‘FHIA-01’
from 4.4 to 4.8 Gy, and for ‘Orito’ from 3.6 to 4.0 Gy. Figure 4.10B shows the optimum dose range
for carbon ion beam irradiation in each cultivar.

Plants irradiated over a period of 34 days in the “Gamma field” facility did not show any phenotypic
variation; however, plants kept at the “Gamma greenhouse” facility for a period of nine months
showed unique characteristics as reported by Reyes-Borja et al. (2005) (Figure 4.12). Novak et al.
(1990) reported the presence of considerable phenotypic variation among the plants regenerated from
shoot-tips after mutagenic treatment. At the early stages of plant development, the irradiation affected
the emergence and expansion of the younger leaves, and several plants formed compact leaf rosettes.
Aberrant morphology of the laminae was observed mainly in younger leaves due to damage of the
apical meristem. The results from the present study in the “Gamma greenhouse” facility are in
agreement with the findings reported by Novak et al. (1990). We also found damage to meristematic
cells as shown in Figure 4.13. Double and spindled leaves were the most relevant characteristics
(Figure 4.12B,D), while yellow spot leaf was the most frequently observed characteristic in
‘Cavendish Enano’, ‘FHIA-01’ and ‘Orito’ (Figure 4.12E), except for ‘Williams’.

Measuring relative DNA content variation with flow cytometry

A flow cytometer was used to measure the relative DNA content of plants irradiated with gamma-rays.
The relationships between the irradiation doses and the relative DNA content showed that the values
from the three cultivars (‘Cavendish Enano’, ‘Williams’ and ‘Orito’) were almost the same, with an
imperceptible variation for all the applied doses. However, a perceptible variation was observed in cv.
‘FHIA-01’ when the dose was increased. The variation in ‘FHIA-01’ was better observed when a
frequency distribution of the relative DNA contents was analysed (Figure 4.14). The analysis detected
a reduction in relative DNA content with increasing irradiation dose (Gy). The lowest value was 1.947
(96% of donor), observed at 300 Gy. These results suggest that deletion of DNA occurred when high
doses were applied in ‘FHIA-01’, exhibiting a shift to the left, indicating a loss of chromosomes or
chromosomal parts.

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A report by the FAO/IAEA (2002) states that flow cytometer screening was started to identify
aneuploid mutants in irradiated banana plants. Aneuploid mutants can be detected through
chromosome counts, but this is a rather time-consuming process. Results obtained by flow cytometry
were compared to chromosome counting in meristem shoot-tip cells. It was shown that flow cytometry
is sensitive enough to detect aneuploidy in Musa. Dolezel et al. (2002) reported that flow cytometry
was used to determine ploidy levels of Musa accessions. Among 890 accessions, 2% of mixoploid
plants were detected. In the annual report published by the National Institute of Agro-biological
Sciences (2002), it was reported that the rates of the hairless mutation in sugar cane obviously
increased with increasing irradiation dose. A very high mutation rate was observed at 200 Gy and all
the hairless mutants had 1.4% less DNA than the donor. DNA deletions frequently occurred at doses
>100 Gy.

The relative DNA content was measured in 115 samples from the population subjected to carbon ion
beam irradiation. The flow cytometer reported hexaploid cells among the analysed samples (Figure
4.15). In ‘Cavendish Enano’, five hexaploid plants were found when the applied ion beam dose was
4 Gy. In ‘Williams’ variety just one hexaploid plant was observed. These results suggested that carbon
ion beam irradiation at 4 Gy can produce a duplication of the chromosomes. According to Yu (2006),
if a normal chromosome is increased by a segment with the same sequence, it is said that duplication
occurred. If the duplicated segment follows the original sequence, it is called tandem duplication; if
the segment is linked to the reverse sequence, it is called a reverse duplication. The same author also
reported that a large amount of chromosomal lagging can definitely induce changes in chromosome
number and can, thus, possibly result in aneuploidy. Using N-ion implantation doses of D3 (3 × 1016
ions/cm2) and D4 (4 × 1016 ions/cm2), monomers with a chromosome number of 41 appeared in wheat
‘Premebi’. In ‘Rye AR1’ with dose D4, monomers with chromosome numbers of 19 appeared, 15 of
them being normal chromosomes and 4 being trisomic B chromosomes.

Screening of sensitivity to juglone toxin

In the experiment using juglone at concentrations of 100, 150, 200, 250, 300 and 350 ppm, and
evaluating 24 and 48 hours after inoculation, no significant differences with respect to time or dose
were noted among susceptible and resistant (‘FHIA-01’) cultivars as the toxin continued to induce
necrosis even 48 hours after treatment (Figure 4.16). The results suggest that all the damage caused to
cells by the juglone occurred just after 24 hours in the susceptible cultivars. The figures show that an
increase in concentration of juglone was concomitant with an increase in the extent of necrosis in the
leaf disks. The mechanisms of the stopped necrosis after 24 hours in susceptible cultivars and the
increased necrosis after 48 hours in the resistant cultivar ‘FHIA-01’ are still unknown. Thus, an in
depth study of the cellular interaction with juglone at the morphological, molecular and biochemical
levels should be carried out.

As a consequence, embedding leaf disks for 24 hours was the selected time to inoculate the irradiated
materials. In relation to this, significant differences across various concentrations of juglone using the
Tukey-Kramer (p < 0.05) test (Table 4.1) were detected. Differences were also observed among the
cultivars tested. ‘FHIA-01’ was the least affected across the concentrations tested in contrast to the
susceptible cultivars ‘Cavendish Enano’, ‘Williams’ and ‘Orito’ (Figure 4.16 E, F). The effectiveness
of the juglone concentrations is shown in Figure 4.17.

This experiment was undertaken to determine a concentration of juglone sufficient to induce necrosis
on banana leaf disks and allow discrimination among the irradiated plants. As previously mentioned,
juglone is one of the seven toxins produced by M. fijiensis Morelet as it attacks the leaf cells of the
plant, resulting in necrotic tissues. In this experiment, we confirmed that various concentrations of
juglone can induce necrosis in banana leaf disks. The results showed that a concentration of 150 ppm
of juglone was sufficient to induce differentiable necrotic areas among susceptible and resistant
cultivars. Thus, 150 ppm of juglone solution was used for screening the irradiated population. At a
concentration of 350 ppm, juglone induced necrosis among susceptible and resistant cultivars at a

68
similar intensity. Thus, the use of high concentrations of juglone is not ideal for conducting screening
trials since even the highly resistant cultivar ‘FHIA-01’ was affected. Molina and Krausz (1988)
observed a clear variation between the Black sigatoka disease-susceptible cultivar ‘Grand Naine’ and
the highly Black sigatoka disease-resistant line ‘IV-9’. This variation was observed with the needle-
piercing method using phytotoxic extract from the pathogen, and measuring the diameter of the area of
necrotic tissue. The toxic activity of the extracts can, apparently, be used to screen banana and plantain
breeding materials rapidly even at a very early stage of plant growth.

In the present study, regarding the plants irradiated with gamma-rays, a total of 208, 179 and 307
plants were screened in ‘Cavendish Enano’, ‘Williams’ and ‘Orito’, respectively. Plants showing low
levels of necrosis (~4-9% produced by using 150 ppm of juglone on the leaf disks) were selected. Out
of these, 5, 8 and 20 plants were selected in ‘Cavendish Enano’, ‘Williams’ and ‘Orito’, respectively.
Figure 4.18 shows the frequency distribution as expressed by class limits of LDNA (%) in the three
cultivars. The values in these plants were lower than the resistance indicator value (9.5%) of ‘FHIA-
01’ that was previously calculated.

In studies reported by the FAO/IAEA (2002), it was found that of ~4000 irradiated ‘Grande Naine’
plants screened, 19 putative mutants were selected for their tolerance to 25 ppm of juglone; however,
new screening for resistance to M. fijiensis in these plants still has to be confirmed through inoculation
with the fungus, which is a very slow process. On the other hand, the results obtained by Lepoivre et
al. (2002) confirm the possibility of selecting banana plants resistant to M. fijiensis metabolites;
however, in their research this approach did not result in a higher level of resistance to black leaf
streak disease.

Lepoivre et al. (2002) reported that chloroplasts are a target site of juglone. When juglone was used,
swelling chloroplasts were observed by electron microscopy in ethyl acetate crude extract (EaCE)-
treated leaves. Upon observation, ‘Fougamou’ (a partially resistant cultivar) chloroplasts appeared to
be less affected by juglone than those in ‘Grand Naine’. These results suggest that the chloroplasts are
one of the primary action sites of juglone.

Different screening methods have been mentioned by different authors (Molina and Krausz, 1988;
Harelimana et al., 1997; Lepoivre et al., 2002). The leaf puncture bioassay has been widely used to
assess host tolerance/resistance to Black sigatoka disease or juglone; however, although detached
banana leaves or the injection of crude toxic extract into the leaves is easy, neither method is sensitive
or quantitative. In this study, since the leaf disks of the resistant cultivar (in this case ‘FHIA-01’) were
less affected by 150 ppm juglone compared to the susceptible cultivars, the leaf disk immersion
method was a simple and rapid method to measure tolerance/resistance to juglone. Limited necrosis
was observed in susceptible irradiated cultivars (selected ones); however, new studies to reconfirm the
tolerance/resistance to juglone on the selected material must be undertaken. On the other hand, Van
Harten (1998) has mentioned that selection using phytotoxins and culture filtrate are more effective
than the use of the pathogen itself. Nevertheless, the selected materials in the present study must be
assayed under field conditions, applying natural inoculums of Black sigatoka to confirm that the use of
juglone is an adequate means of evaluating resistance/tolerance or susceptibility at early stages of
plant growth.

The results of the LDNA (%) obtained from carbon ion beam-irradiated plants are shown in
Figure 4.19C, F in both ‘Cavendish Enano’ and ‘Williams’, respectively (Reyes-Borja et al. 2007). In
this case, the results of LDNA (%) were combined with DDP (days) and II (%) for candidate selection.
As shown in Figure 4.19C,F, lower LDNA (%) values varied from 38.0 to 44.9% in irradiated
‘Cavendish Enano’ and from 33.0 to 39.9% in ‘Williams’ (indicated between dotted vertical lines).
Conversely, LDNA (%) values from non-irradiated plants did not show such low values. Therefore,
the low LDNA (%) values in irradiated plants might be attributed to tolerance to Black sigatoka
disease, possibly due to slower penetration of the toxin into the cells as the result of mutations. Leaf-
disk bioassays have been reported to be effective for evaluating resistance in several crops. Ostry et al.

69
(1988) working with Septoria musiva, a disease of Populus spp., described that this method was
sufficiently sensitive to distinguish between clones with high, moderate or low resistance. Etame
(2003) inoculated banana genotypes showing different reactions to Black sigatoka with juglone toxin
and the pathogen for selecting resistant plants. The genotypes resistant to juglone toxin (‘Fougamou’,
‘Pisang madu’, ‘M53’ and ‘Klutuk’) also showed resistance to the pathogen, although some cultivars
resistant to M. fijiensis were susceptible to juglone. In conclusion, this method should only serve as a
preliminary screening technique before field tests.

Combining DDP (days) and II (%) with LDNA (%) to select plants tolerant/resistant to Black
sigatoka disease

As shown in Figure 4.19A, D, the frequency distribution by class limits of the DDP (days) in both
cultivars, the higher class limit values were clearly separated from the lower ones, as indicated
between dotted vertical lines, ranging from 53.0 to 59.9 days and from 50.0 to 54.9 days in ‘Cavendish
Enano’ and ‘Williams’, respectively. The lowest II (%) values in ‘Cavendish Enano’ corresponded to
25.0-34.9 days and, in ‘Williams’, to 27.0-36.9 days, as marked between dotted vertical lines, showing
a slight variation contrasting greatly with the higher values (Figure 4.19B,E).

Using the II (%) to evaluate a hybrid population of plantain, Cohan et al. (2003) demonstrated that
‘CRBP-39’ (AAAB) was extremely resistant to Black sigatoka disease in three developmental phases:
the vegetative phase at 6 months, the flowering phase and the harvest phase. II (%) is, therefore, a very
useful parameter for evaluating the resistance of plants to this disease. In this research, the II (%)
allowed us to observe inter-individual variation in response to Black sigatoka disease, which varies
from very susceptible to tolerant among the plants regenerated from irradiated explants, when
subjected to the inoculum. The effect of the irradiation probably causes DNA alteration as mentioned
by Fukuda et al. (2004), and results in expanding the variation in relation to this pathogen.

The appearance of fewer symptoms following infection is closely related to a response by the plant
defence mechanism. By crossing two susceptible triploid plantain cultivars (‘Bobby Tannap’ and
‘Obino 1 Ewai’) as female parents with the resistant wild, diploid banana ‘Calcutta 4’, Ortiz and
Vuylsteke (1994) obtained segregated progeny with a durable horizontal resistance. In the case of the
progeny with a partially resistant response, slow lesion development and, ultimately, reduced
sporulation were observed. Ortiz and Vuylsteke (1994) also discussed that the possible mechanisms of
Black sigatoka disease resistance were expressed by different pathways such as the synthesis of
phytoalexins, the production of lignin or suberin, polyphenolic content (higher in resistant cultivar),
low stomata density and increased epicuticular wax. However, the mechanism still remains obscure.

Taking the results of the three parameters DDP (days), II (%) and LDNA (%) together, variations in
relation to tolerance to Black sigatoka disease have been expanded in the banana population
established from in vitro plantlets irradiated with carbon ion beams, suggesting that ion beam
irradiation is a useful tool for mutation breeding in banana.

For selecting tolerant/resistant plants, the variables DDP (days), II (%) and LDNA (%) were combined
by linear regression to assess the response to Black sigatoka disease in the irradiated materials. The
linear regression permitted us to categorise the plants that showed a better response to this disease.
LDNA (%) regression versus II (%) showed high significance (p ≦ 0.01) compared to LDNA (%)
versus DDP (days), and DDP (days) versus II (%) that were significant at the 5% level (p ≦ 0.05) in
‘Williams’ (Figure 4.20). The regression among the three combined variables permitted us to select six
plants in ‘Williams’ (with code numbers ‘W 16 II 74’ (1), ‘W 128 I 67’ (2), ‘W 1 II 148’ (3), ‘W 8 II
13’ (4), ‘W 1 II 19’ (5) and ‘W 1 II 31’ (6) showing high relationships) and two plants in ‘Cavendish

70
Enano’ (with code numbers ‘CE 4 II 30’ and ‘CE 64 I 5’ - data not shown) as candidates with
increased tolerance to Black sigatoka disease.

Regarding the complete assessment of the candidate plants, field experiments based on the whole plant
cycle are necessary to evaluate not only the response to Black sigatoka disease but also the fruit
quality, potential production and post-harvest parameters, which are valuable components for final
selections.

Putative mutants and factor of effectiveness-FE (%)

Summarising the effectiveness of both gamma-rays and carbon ion beams, putative mutants such as
juglone tolerance, low relative DNA content, dwarfism, sigmoid drooping leaf, fast growth of sucker,
good response to Black sigatoka disease, fast growth of plantlet, hexaploid plants and several kinds of
leaves were obtained (Table 4.2). An interesting sigmoid drooping leaf mutant was produced in
cultivar ‘FHIA-01’ by applying high doses of gamma-rays. The characteristics were observed in the
first and second generations (mother plant and sucker), suggesting that this characteristic is heritable.
Sigmoid drooping leaf is a peculiar characteristic in certain Musa cultivars such as ‘FHIA-03’, which
is a cooking banana (AABB) with high production and good vigour (Reyes-Borja, 1995). Figure 4.21
shows a comparison of the putative mutant sigmoid drooping leaf with the normal growth of ‘FHIA-
01’. A fast growing plantlet (Figure 4.22) was also observed among the irradiated cultivars. This plant,
selected as fast growing, has a very useful characteristic as it may result in earliness of fruit
harvesting. Regarding the hexaploid cells that we found by measuring the relative DNA content, this
could be a way of causing chromosome duplication, efficiently and fast, especially on improved
diploids possessing resistance that could be used as parental lines for banana breeding. Gamma-ray
irradiation also produced a putative dwarf of ‘FHIA-01’ but, unfortunately, resulted in death during
development. The low relative DNA content that resulted in ‘FHIA-01’ is evidence for the
effectiveness of the irradiation. This directly affects phenologic and phenotypic characteristics as
confirmed by the variations obtained in ‘FHIA-01’.

CONCLUSIONS

The methodologies applied in the course of this research were highly efficient in developing the main
process related to mutation induction in banana. The technique used to measure relative DNA content
was very useful for detecting alterations at the DNA level caused by irradiation, and allowed us to
select plants that during cropping expressed good putative mutations as shown for cultivar ‘FHIA-01’
irradiated with gamma-rays. Still more useful were the results, by which this technique detected
hexaploid cells, suggesting that chromosome duplication had occurred. This kind of material can be
interesting as parental lines for breeding by crossing, especially when developed from diploids
resistant to Black sigatoka disease. The technique using leaf disks exposed to juglone toxin permitted
to discriminate between cultivars that are resistant or susceptible to Black sigatoka disease.
Additionally, it also allowed selecting candidates from plants irradiated at earlier stages. Combining
DDP (days) and II (%) with LDNA (%) produced more accurate results. Nevertheless, an additional
selection method must involve subjecting the selected material to natural inoculum to study the disease
indices, especially at the flowering and the harvest stages for at least 3 or 4 cycles for final selection.

Gamma-ray and carbon ion beam irradiation produced the same trends in survival rate when banana
explants were subjected to irradiation. However, the methods differ in terms of LET, with carbon ion
beam irradiation being effective at low doses. The cultivars most sensitive to the irradiation among
those used in this experiment were ‘Orito’ (gamma-rays and carbon ion beam) and ‘FHIA-01’ (carbon
ion beam). A sigmoid drooping leaf plant of ‘FHIA-01’ and plants weakly affected by Black sigatoka
disease and juglone toxin were obtained. In addition, a single plant of ‘Cavendish Enano’ irradiated
with a carbon ion beam under greenhouse conditions showed fast growth among a group of plants.
Regarding the selected plants only weakly affected by Black sigatoka disease, field experiments
considering the whole plant cycle must be undertaken to confirm not only the Black sigatoka response

71
but also fruit quality and potential production and post-harvest evaluations as necessary components
for final selections. Additionally, field studies of mutant plants such as sigmoid drooping leaf, fast
growth, hexaploids and diminished DNA content plants must also be carried out to confirm whether
the new traits involve single or linked genes.

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Table 4.1. LDNA (%) of banana leaf disks in different concentrations of juglone

Juglone LDNA (%) of banana leaf disks affected by juglone

(ppm)
‘Cavendish ‘Williams’ ‘FHIA-01’ ‘Orito’
Enano’

0 0.0 0.0 0.0 0.0

100 11.0 ± 1.8 cA 11.1 ± 1.9 cA 6.0 ± 2.6 dB 14.5 ± 1.5 dA
150 14.9 ± 1.2 bcA 13.6 ± 1.8 cA 9.5 ± 1.9 cdB 17.1 ± 1.9 cdA
200 19.6 ± 1.7 abA 14.7 ± 0.9 bcB 11.5 ± 3.8 bcdB 19.4 ± 2.0 bcdA
250 22.1 ± 5.8 abA 18.1 ± 1.9 abAB 12.3 ± 1.1 bcB 22.4 ± 1.8 abcA
300 24.7 ± 4.5 aA 20.2 ± 1.5 aAB 15.5 ± 2.0 abB 23.3 ± 3.1 abA
350 24.4 ± 3.9 aA 21.7 ± 1.7 aA 19.3 ± 3.2 aA 25.7 ± 4.7 aA

Values followed by the same letter in a column do not differ significantly according to Tukey-Kramer
(p < 0.05). Values in a row followed by the same capital letter do not differ significantly according to
Tukey-Kramer (p < 0.05). Values ± mean standard deviation.

Table 4.2. Factor of effectiveness-FE (%) and putative mutations produced by gamma-ray (60Co,
137
Cs) and carbon ion beam irradiation in four cultivars of banana

Facility/mutagen Cultivar Total No. of Putative Gy dose FE

no. of plants with mutation inducing (%)
plants variation mutation (n)
Gamma room/60Co ‘C. Enano’ 282 4 Tolerant of 100 (3) 1.63
juglone 150 (1) 0.35
Gamma field/60Co ‘C. Enano’ 26 1 Tolerant of 2 (1) 3.84
juglone
Gamma ‘C. Enano’ 5 5 AL1 0.25 (1) 20.0
greenhouse/137Cs AL, DL 0.25 (1) 20.0
RL, LL, 0.5 (1) 20.0
SL, YSL, LL 0.5 (1) 20.0
YSL, LL 0.75 (1) 20.0
Gamma room/60Co ‘Williams’ 156 7 Tolerant of 50 (2) 1.28
juglone 150 (5) 3.20
Gamma field/60Co ‘Williams’ 14 1 Tolerant of 0.5 (1) 7.14
juglone
Gamma ‘Williams’ 3 3 AL 0.25 (1) 33.3
greenhouse/137Cs AL, RSSL 0.5 (1) 33.3
AL 0.75 (1) 33.3
Gamma room/60Co ‘Orito’ 282 20 Tolerant of 50 (8) 2.83
juglone 100 (8) 2.83
150 (4) 1.41
Gamma ‘Orito’ 5 5 SSL, LL, YSL 0.25 (1) 20.0
greenhouse/137Cs RSSL, AL 0.25 (1) 20.0
LL, YSL 0.5 (1) 20.0
LL, YSL, AL 0.5 (1) 20.0
LL, YSL, AL 0.75 (1) 20.0
Gamma room/60Co ‘FHIA-01’ 120 18 Low DNA 200 (10) 8.33
content 300 (7) 5.83
Gamma room/60Co ‘FHIA-01’ 120 1 Dwarfism 300 (1) 0.83

75
 Facility/mutagen Cultivar Total No. of Putative Gy dose FE
no. of plants with mutation inducing (%)
plants variation mutation (n)
Gamma room/60Co ‘FHIA-01’ 120 1 Sigmoid 200 (1) 0.83
drooping leaf
Gamma room/60Co ‘FHIA-01’ 120 2 Fast growth of 200 (1) 0.83
sucker 300 (1) 0.83
Gamma ‘FHIA-01’ 5 5 YLS, LL 0.25 (1) 20.0
greenhouse/137Cs YLS, LL 0.25 (1) 20.0
YLS 0. 5 (1) 20.0
YLS 0. 5 (1) 20.0
YLS 0. 75 (1) 20.0
Carbon ion beam ‘C. Enano’ 42 2 Good response 4 (1) 2.38
to Black 64 (1) 2.38
sigatoka
disease
Carbon ion beam ‘C. Enano’ 85 1 Fast growth of 4 (1) 1.17
plantlet
Carbon ion beam ‘C. Enano’ 29 5 Hexaploid 4 (5) 17.24
Carbon ion beam ‘Williams’ 40 6 Good response 1 (3) 7.5
to Black 8 (1) 2.5
sigatoka 16 (1) 2.5
disease 128 (1) 2.5
Carbon ion beam ‘Williams’ 84 1 Hexaploid 4 (1) 1.69
1
LL = long leaf; YSL = yellow spotted leaf; SL = spindled leaf; SSL = spindled short leaf; RL =
rudimentary leaf; AL = abnormal leaf; RSSL = right side short leaf; DL = double leaf.

76
 A B

C D

Figure 4.1. Irradiation of banana. (A) explants in 90 mm dishes (indicated by the arrow) in
the “Gamma room” facility; (B) plants in the “Gamma field” facility; (C) plants in the
“Gamma greenhouse” facility; and (D) a Petri dish (indicated by the arrow) containing the
samples in the carbon ion beam irradiator.

77
 B
Laboratory
assessment

E
Carbon
ion-beams

In vitro F
plants
C
Gamma
A Room 2-month
old plants
Nursery
assessment

Gamma
G
Field

D
Field
assessment
Gamma
Greenhouse
H

Figure 4.2. Schematic diagram for the improvement of banana lines resistant to Black
sigatoka disease through mutation induction with gamma-rays and carbon ion beams. (A)
Propagation in liquid medium, solid medium for regeneration; (B) Induction of mutation
through irradiation; (C) Propagation (three times) and regeneration; (D) Meristem
propagation (three times) and regeneration; (E) Assessment of weight, height, survival rate
and LD50; (F) Leaf disk toxin screening using juglone; (G) Assessment of relative DNA
content by flow cytometry; (H) Assessment of Black sigatoka disease resistance: DDP
(days), II (%) and putative mutations.

A B C

Figure 4.3. Digital pictures calculating LDNA (%) by using the GIMP 1.2 software,
measuring the full disk area in pixels (A) minus the cut green area pixels by selecting
hand-drawn regions (B), and obtaining the value for the necrotic area by subtraction (C).

78
 A B

C D

Figure 4.4. Mycosphaerella fijiensis Morelet colonies (A), filtration (B) and inoculation of
the irradiated banana plants (C) kept in an incubation room for 48 hours (D). A concentration
of 1.5 × 106 conidia/ml (inoculation of the first leaf) was used.

A B

C D
3
(June 6, 2006)
2
(May 24, 2006)

1
(May 16, 2006)
E F

Figure 4.5. Inoculation method using diseased leaf fragments. (A) Collected leaves from a
banana collection kept in the Estacion Experimental Tropical Pichilingue (INIAP), Ecuador.
(B, C, D) Diseased leaf fragment moistening before placement among the plants to be
inoculated. (E) Plants covered by a cotton sheet for 48 hours, and moistening three times a
day. (F) A plant showing the inoculated leaves and inoculation dates.

79
 80 Williams
A

Regeneration rate (%)

70 FHIA-01
60
50
40
30
20
10
0
Vertical Horizontal
80
B
Regeneration rate (%)

Slicing methods
70
60
50
40
30
20
10
0
Vertical Horizontal

Figure 4.6. Relationships of the explants’ weight (A) and regeneration rate (B) with the
two slicing methods (vertical and horizontal) in ‘Williams’ and ‘FHIA-01’ 13 days after
culture.

Figure 4.7. Regenerated plantlets of ‘Williams’ from both vertical (A) and horizontal (B)
slicing methods 13 days after culture.

80
 100 100 C. Enano
A C. Enano
Williams D Williams
80 FHIA-01 80

Survival rate (%)

Survival rate (%)

Orito

60 60

40 40

20 20

0 0
0 50 100 150 200 300 500 0 0.5 1 2 4 8 16 32 64 128
1.8 1.8
B E
Weight of plantlets (g)
Weight of plantlets (g)

1.6 1.6
1.4 1.4
1.2 1.2
1 1.0
0.8 0.8
0.6 0.6
0.4 0.4
0.2 0.2
0 0.0
0 50 100 150 200 300 500 0 0.5 1 2 4 8 16 32 64 128
5 5
C
Height of plantlets (cm)

Height of plantlets (cm)

F
4 4

3 3

2 2

1 1

0 0
0 50 100 150 200 300 500 0 0.5 1 2 4 8 16 32 64 128
Gamma ray dose (Gy) Carbon ion beams dose (Gy)

Figure 4.8. Survival rate (A, D), weight (B, E) and height (C, F) of the explants in banana
cultivars one month after gamma-ray irradiation and 19 days after carbon ion beam
irradiation.

81
 Control 50 100 150 200 300 500 (Gy)

D
Figure 4.9. Biological effects produced by different gamma-ray doses (Gy) in four cultivars
of banana. (A) ‘Williams’; (B) ‘FHIA-01’; (C) ‘Cavendish Enano’; (D) ‘Orito’.

90
Optimum range of gamma ray

A
85

80
dose (Gy)

75
70

65
60
C. Enano Williams Orito FHIA-01
Optimum range of carbon ion

16 5
B
beam dose (Gy)

15

14 4

13

12 3
C. Enano Williams Orito FHIA-01

Figure 4.10. Optimum range of gamma-ray (A) and carbon ion beam (B) doses obtained
with ±5% of the LD50 values. Squares represent the LD50 values.

82
 A

B
Figure 4.11. Biological effects produced by different carbon ion beam doses (Gy) in two
cultivars of banana. (A) ‘Cavendish Enano’; (B) ‘Williams’. Carbon ion beam doses for
each cultivar from left to right: 0 (control), 0.5, 1, 2, 4, 8, 16, 32, 64 and 128 (Gy).

A B C

D E F
Figure 4.12. Unique characteristics observed at the “Gamma greenhouse” facility on
irradiated plants after nine months. (A) abnormal leaf (AL); (B) double leaf (DL); (C)
rudimentary leaf (RL); (D) spindled leaf (SL); (E) yellow spotted leaf (YSL); (F) long
leaf (LL).

83
 A B

C D

Figure 4.13. Banana meristems affected by long period-chronic irradiation (137Cs)

applied in the “Gamma greenhouse” facility. ‘Cavendish Enano’ at 0.50 Gy (A, B),
and ‘FHIA-01’ at 0.50 Gy (C, D).

12 0 Gy
No. of plants

9
6
3
0
121.75 1.80 1.85 1.90 1.95 2.00 2.05 2.10
No. of plants

9 50 Gy
6
3
0
121.75 1.80 1.85 1.90 1.95 2.00 2.05 2.10
No. of plants

9 100 Gy
6
3
0
121.75 1.80 1.85 1.90 1.95 2.00 2.05 2.10
No. of plants

9
150 Gy
6
3
0
121.75 1.80 1.85 1.90 1.95 2.00 2.05 2.10
No. of plants

9
200 Gy
6
3
0
121.75 1.80 1.85 1.90 1.95 2.00 2.05 2.10
No. of plants

9 300 Gy
6
3
0
1.75 1.80 1.85 1.90 1.95 2.00 2.05 2.10
Relative DNA content

Figure 4.14. Frequency distributions of relative DNA contents in a population of ‘FHIA-

01’ irradiated with different gamma-ray doses.

84
 Hexaploid
cells

Figure 4.15. Relative DNA content measured by flow cytometry showed hexaploid
cells in ‘Cavendish Enano’ (encircled, peak 3) produced by an ion beam dose of 4 Gy.
‘Cavendish Enano’, code no. CE4I69 (peak 1) and ‘FHIA-01’ used as standard (peak 2).

30 Cavendish Enano 25 Williams

A B
25 20
20
LDNA (%)

LDNA (%)

15
15
10
10 24 h after 24 h after
48 h after 48 h after
5 5

0 0
25 0 100 150 FHIA-01
200 250 300 350 30 0 100 150 Orito200 250 300 350
C Juglone toxin concentration (ppm) D Juglone toxin concentration (ppm)
20 25
20
LDNA (%)

LDNA (%)

15
15
10
24 h after 10 24 h after
5 48 h after 48 h after
5

0 0
30 0 100
24 h150 200 250 300
after inoculation 350 30 0 10048 h150 200 250 300
after inoculation 350
E Juglone toxin concentration (ppm) F Juglone toxin concentration (ppm)
25 25

20 20
LDNA (%)

LDNA (%)

15 15

10 C. Enano 10 C. Enano
Williams Williams
5 FHA-01 5 FHA-01
Orito Orito
0 0
0 100 150 200 250 300 350 0 100 150 200 250 300 350
Juglone toxin concentration (ppm) Juglone toxin concentration (ppm)

Figure 4.16. Relationship between LDNA (%) and juglone toxin concentration (ppm) in four
cultivars of banana, 24 and 48 hours after inoculation.

85
 Juglone toxin concentration (ppm)
0 100 150 200 250 300 350

D
Figure 4.17. Leaf disk screening technique using juglone toxin in several
concentrations (ppm) in four cultivars of banana. The highlighted disks (box) indicate
the selected dose for subsequent screening. (A) ‘Cavendish Enano’; (B) ‘Williams’; (C)
‘Orito’; (D) ‘FHIA-01’.

160

140 C. Enano
Williams
120 Orito
No. of plants

100

80

60
40

20
0
<4 4.0- 9.0- 14.0- 19.0- 24.0- 29.0- 34.0- >39.0
8.99 13.99 18.99 23.99 28.99 33.99 38.99
Class limits of LDNA (% )

Figure 4.18. Mutagenesis efficiency in regenerated plants of banana after gamma-ray

treatment. The frequency distribution was expressed by class limits of LDNA (%) in
‘Cavendish Enano’ (n = 208), ‘Williams’ (n = 179) and ‘Orito’ (n = 309). Plants in the
lower values (encircled area) were selected.

86
 30
A Cavendish Enano 30
D Williams
25 25

20
Plants (%)

20

Plants (%)
15 15

10 10

5 5

0 0
<41.0 41.0- 46.0- 48.0- 50.0- 52.0- 53.0- 53.5- 54.0- 58.0- <40.0 40.0- 42.0- 45.0- 47.0- 49.0- 50.0- 51.0- 52.0- 53.0- 54.0-
45.9 47.9 49.9 51.9 52.9 53.4 53.9 57.9 59.9 41.9 44.9 46.9 48.9 49.9 50.9 51.9 52.9 53.9 54.9
Class limits of DDP (days) Class limits of DDP (days)
25 20
B E
20 16
Plants (%)

Plants (%)

15 12

10 8

5 4

0 0
25.0- 27.0- 31.0- 33.0- 34.0- 35.0- 36.0- 39.0- 42.0- 45.0- 47.0- 27.0- 28.0- 32.0- 34.0- 35.0- 37.0- 39.0- 43.0- 45.0- 48.0- 50.0- >52
26.9 30.9 32.9 33.9 34.9 35.9 38.9 41.9 44.9 46.9 47.9 27.9 31.9 33.9 34.9 36.9 38.9 42.9 44.9 47.9 49.9 51.9

Class limits of II (% ) 20
Class limits of II (%)
25
C F
20 Irradiated population 16 Irradiated population
Non-irradiated Non-irradiated
Plants (%)
Plants (%)

15 12

10 8

5 4

0 0
<38 38.0- 40.0- 42.0- 44.0- 45.0- 50.0- 70.0- 85.0- 90.0- 95.0- <33.0 33.0- 36.0- 38.0- 40.0- 43.0- 45.0- 50.0- 60.0- 75.0- >80.0
39.9 41-9 43.9 44.9 49.9 69.9 84.9 89.9 94.9 99.9 35.9 37.9 38.9 42.9 44.9 49.9 59.9 74.9 79.9

Class limits of LDNA (%) Class limits of LDNA (% )

Figure 4.19. Mutagenesis efficiency following carbon ion beam irradiation. Frequency
distributions are expressed as class limits of DDP (days), II (%) and LDNA (%) in
‘Cavendish Enano’ (A-C) and ‘Williams’ (D-F). The range between dotted vertical lines
indicates the best values of the three parameters.

87
 100 Williams
A
y = 1.3195x + 1.6644
80 R2 = 0.2455

LDNA (%)
60

1
40 5
2 4
3 6
20
25 30 35 40 45 50 55
II (%)

100
B
y = -1.9706x + 147.69
80 R2 = 0.1293
LDNA (%)

60

1
40 5
6 2
3 4
20
38 41 44 47 50 53 56
DDP (days)

60
y = -0.1803x + 54.317
C
R2 = 0.1362
55
1 5
2
4
DDP (days)

50 6
3
45

40

35
25 30 35 40 45 50 55
II (%)

Figure 4.20. Statistical analysis following irradiation of ‘Williams’ banana. (A)

LDNA (%) regression versus II (%); (B) LDNA (%) regression versus DDP (days);
(C) DDP (days) regression versus II (%). Encircled values with bold triangles belong
to the code numbers ‘W 16 II 74’ (1), ‘W 128 I 67’ (2), ‘W 1 II 148’ (3), ‘W 8 II 13’
(4), ‘W 1 II 19’ (5) and ‘W 1 II 31’ (6) that clearly show relationships within the three
combinations of the variables.

88
 A B

C D

Figure 4.21. Putative mutant of ‘FHIA-01’ obtained by gamma-ray irradiation (A) and
its petioles (B). Normal leaf shape of ‘FHIA-01’ and its petioles (C-D) growing under
greenhouse conditions.

A B
Figure 4.22. A ‘Cavendish Enano’ plant following carbon ion beam irradiation (4 Gy)
showing fast growth (A-B in the centre) compared with the other plants in the group.

89
 Chapter 5

Rapid and mass screening of banana and plantain for resistance to

Black sigatoka disease using detached leaf and in vitro plantlets

P.S. Ojiambo1, M. Twizeyimana1, A. Tenkouano2 and R. Bandyopadhyay1*

1
International Institute of Tropical Agriculture,
Ibadan, Nigeria
2
International Institute of Tropical Agriculture,
Humid Forest Ecoregional Center,
Yaounde, Cameroon.

*Correspondence: [email protected]

Abstract

We describe the use of in vitro plantlets in tubes and detached leaves in screening Musa genotypes
against Mycosphaerella fijiensis, the causal agent of Black sigatoka disease. The utility of these two
assays was evaluated using eight genotypes with different levels of disease resistance. Differences in
disease response were observed when in vitro plantlets in tubes and detached leaves of selected Musa
genotypes with different levels of disease resistance were inoculated with M. fijiensis. The response of
genotypes depended on their level of disease resistance, with resistant genotypes having longer
incubation times and smaller infected leaf areas compared to the susceptible cultivar ‘Agbagba’ and to
relatively less resistant genotypes. A similar pattern in cultivar response was observed for symptom
evolution time. Although symptom stages appeared earlier on in in vitro plants compared to detached
leaves, genotypes were consistently ranked based on their level of disease resistance. Disease severity
data from the two assays were strongly correlated, with higher severity being observed on the
susceptible cultivar ‘Agbagba’ and lowest severity on resistant land race ‘Calcutta-4’. The remaining
genotypes had disease severity levels corresponding to their levels of resistance to Black sigatoka
disease. Based on these results, we conclude that these two rapid assays are reliable for screening
banana and plantain for resistance to Black sigatoka disease.

INTRODUCTION

Black sigatoka disease, also known as Black leaf streak, is caused by Mycosphaerella fijiensis
(teleomorph of Pseudocercospora fijiensis) and is regarded as the most economically important leaf
disease of bananas and plantains worldwide (Carlier et al., 2000; Marín et al., 2003). The fungus
reduces the green leaf area and impedes photosynthesis, which is needed for fruit filling. The disease
results in heavy losses (Marín and Romero, 1992; Mobambo et al., 1993; Craenen and Ortiz, 1998)
and, in highly susceptible varieties, the disease can lead to the total collapse of the plant. Fungicides
and planting of resistant genotypes are the main strategies for controlling the disease. In subsistence
production of bananas and plantains, use of fungicides is impractical (Craenen and Ortiz, 1998). For
commercial growers, banana genotypes resistant to Black sigatoka disease may not be a priority;
however, they are more environmentally friendly.

Breeding for resistance to Black sigatoka disease in banana and plantain has taken a high priority
worldwide. However, the evaluation of resistance has been slow due to the necessity of avoiding
escapes by evaluating resistance in whole plants over several years and in a wide range of
environments. Furthermore, effective field screening depends on the presence of proper environmental
conditions, is time-consuming and expensive, and can often be carried out only once a year. Similarly,

91
early screening in a greenhouse (Mobambo et al., 1994) depends on ensuring ambient conditions
conducive for disease development, and a limited number of genotypes can be evaluated at a time.
Screening for resistance requires methods to unambiguously discriminate resistant and susceptible
genotypes at different stages of plant development. Such techniques should be robust, efficient and
amenable to mass screening of genotypes. Thus, there has been interest in developing in vitro assays
as methods for screening banana and plantain for Black sigatoka disease resistance. Direct inoculation
of in vitro plantlets in tubes and inoculation of detached leaves in moist chambers are two assays that
overcome the limitations associated with field screening. Recently, Twizeyimana et al. (2007)
developed and evaluated the utility of in vitro techniques to screen Musa species for resistance to M.
fijiensis. In that study, M. fijiensis inoculum concentrations, incubating conditions and plating medium
for detached leaves were optimised. Our objective here is to provide a step-by-step protocol for rapid
and mass screening of banana and plantain for resistance to Black sigatoka disease using detached
leaves and in vitro plantlets in tubes.

MATERIAL AND METHODS

Screening banana and plantain genotypes using in vitro plantlets in tubes

Preparation of tissue culture plants

Tissue culture plants were prepared using standard shoot-tip culture protocols. In this procedure, we
adopted a protocol described by Vuylsteke (1998). Briefly, to initiate cultures, 1 cm3 pieces containing
the apices were excised from suckers, surface-sterilised in 95% ethanol for 15 s, soaked for 15 minutes
in 0.75% NaOCl solution to which a drop of Tween 20 was added per 50 ml, and then rinsed three
times with sterile distilled water. Shoot tips 1-2 mm in length with 2-4 leaf primordia were isolated
aseptically and placed on modified Murashige and Skoog (MS) medium basal salt mixture (Murashige
and Skoog, 1962), supplemented with 30 g/l sucrose and 0.4 mg/l thiamine. Cultures were maintained
at 28-30°C on a 12:12 hour light:dark cycle. Shoot tips and meristem were induced to proliferate by
adding 0.18 mg/l indole acetic acid (IAA) and 4.5 mg/l benzylaminopurine (BAP) to the basal
medium. Plant regeneration was accomplished on the same basal medium supplemented with 0.19
mg/l naphthalene acetic acid and 0.23 mg/l BAP. It takes 7 to 10 weeks to establish rooted plantlets
from isolated shoot tips and about three weeks for in vitro plantlets to develop leaflets with enough
material for inoculation.

Inoculum preparation

M. fijiensis inoculum was prepared by isolating the pathogen from diseased leaves as described by
Stover (1976). Briefly, leaf fragments bearing Stage 6 necrosis were placed in moist bags for 48 hours
to allow maturation of pseudothecia. Pieces of leaves with mature pseudothecia were stapled on 9 cm
diameter filter paper disks, dipped in sterile water for 5 minutes and placed inside the lids of Petri
dishes. The lids were then placed on Petri dishes containing 3% water agar. Within one hour,
ascospores forcibly discharged naturally on the agar surface. Single germinating ascospores were
transferred onto V8 juice agar after 24 hours. The V8 juice agar was prepared by mixing 100 ml of V8
juice, 0.2 g of CaCO3, 20 g of agar and 900 ml of sterile distilled water to make the resultant solution
up to 1 l. The resultant solution was then autoclaved using standard procedures. Culture plates were
incubated at 19-20°C under continuous UV light for pathogen development. Inoculated cultures
started to produce conidia approximately 26 days later.

Inoculation of plantlets in tubes

Following sporulation of cultures, sterile distilled water was added to V8 agar on Petri dishes, and the
spore suspension was filtered through two layers of cheesecloth. A drop of Tween 80 was added to the
resulting spore suspension and using four hemocytometer counts, sterile distilled water was added to
adjust spore concentrations to 5 × 105 conidia/ml. This spore concentration was previously reported to

92
be optimum for inoculation in growth chamber assays (Twizeyimana et al., 2007). Prior to inoculation,
a drop of 1% Triton X-100 was added to the inoculum suspension to enable mycelia and spores to
adhere to the leaf surface. The three largest leaves of each in vitro plantlet (one spot per leaf) were
aseptically inoculated by pipetting 40 μl droplets of spore suspension. Droplets were placed carefully
to avoid trickling from the leaves to the plant growth medium, on which the pathogen can grow well
and kill the plantlets. Leaves touching the inner wall or the lid of the tubes or the medium were
excluded from inoculation. Inoculated plantlets were incubated at 25°C with a 12:12 hour light:dark
cycle for disease development (Figure 5.1).

Disease assessment

Observations on inoculated leaflets were made from 2 to 32 days after inoculation, and included
incubation time, disease severity (% of leaf area infected), symptom evolution time and stages of
symptoms. Incubation time is defined as the time between inoculation and appearance of the first
symptoms (Stage 1), whereas symptom evolution time is defined as the number of days between Stage
1 and occurrence of mature lesions (Stage 6). Symptom stages were recorded as described by Fouré
(1987): Stage 1 = first visible symptoms are yellowish specks <1 mm in diameter on the abaxial
surface of the leaf; Stage 2 = initial speck elongates and widens to form a reddish brown streak up to 2
mm in length and streaks are visible on both sides of the leaf; Stage 3 = red-brown streaks reach a
length of 20-30 mm and the colour starts to change from red to dark brown; Stage 4 = the streak
broadens and develops into an elliptical spot, dark brown on the abaxial surface and black on the
adaxial surface of the leaf; Stage 5 =: the central area of the dark spot becomes totally black and
necrotic, the lesion is slightly depressed and the black spot is surrounded by a bright yellow halo;
Stage 6 = the centre of the spot dries out, fades and becomes whitish to grey, and the spot is
surrounded by a dark brown to black border and further depressed.

A schematic representation of the above steps involved in the use of the in vitro plantlets in tubes for
screening Musa genotypes is shown in Figure 5.2.

Screening banana and plantain genotypes using the detached leaf assay

Preparation of detached leaves

Newly opened leaves were cut into large pieces that could easily be cleaned in a 1 l beaker. These leaf
pieces were surface-sterilised in 1% NaOCl solution for 90 s, and washed in 5 to 6 changes of sterile
distilled water. Thereafter, the pieces were cut into smaller segments, each measuring 4 cm in length
by 3 cm in width.

Medium preparation, plating and inoculation of leaf pieces

Medium containing 1% agar technical (product number LP0013, Oxoid Ltd., Hampshire, England)
was autoclaved, and 5 mg/l of gibberellic acid was added to the medium immediately before
dispensing in Petri dishes. Gibberellic acid at this concentration is able to maintain a green colour in
banana leaves for 45 to 52 days, which is needed to differentiate disease severity levels among
cultivars (Twizeyimana et al., 2007). Two of these pieces were placed in plastic Petri dishes with the
adaxial side on 1% agar technical medium amended with gibberellic acid. Inoculum was prepared as
described above; prior to inoculation, a drop of 1% Triton X-100 was added to the spore suspension.
Leaf pieces were inoculated (two droplets per leaf piece) by pipetting 40 μl droplets of spore
suspension (5 × 105 conidial/ml) onto the abaxial side of the leaf. Plating and inoculation of detached
leaves were conducted under aseptic conditions. Petri dishes with inoculated leaf pieces on amended
medium were sealed and incubated at 25°C with a 12:12 hour light:dark cycle.

93
Disease assessment

Observations on inoculated detached leaves began two days after inoculation and lasted 32 days
(Figure 5.3). Long days of assessment are required for genotypes that have higher levels of resistance
to Black sigatoka disease. Data on incubation time, disease severity, symptom evolution time and
symptom stages as described above were recorded.

A schematic representation of the above steps involved in using the detached leaf assay for screening
Musa genotypes is depicted in Figure 5.4.

Utility of the rapid screening methods

Musa genotypes with contrasting resistance levels under field conditions were evaluated using two
rapid methods to determine the correspondence of resistance levels in detached leaves and in vitro
plantlets. The genotypes (with field resistance reactions in parenthesis) were: ‘Agbagba’ (highly
susceptible), ‘FHIA-25’ and ‘PITA-21’ (moderately susceptible), ‘PITA-17’ (resistant) and ‘Calcutta-
4’ (highly resistant). Data on incubation time, symptom evolution time, time to Stage 4 and disease
severity collected from the two assays were analysed as described previously (Twizeyimana et al.,
2007) to determine the utility of these assays as rapid screening methods for Black sigatoka disease.

RESULTS AND DISCUSSION

Disease severity was significantly (p < 0.01) affected by genotypes evaluated for resistance using both
assays. For example, 32 days after inoculation, ‘Agbagba’ had disease severity levels of 81 and 31%
when evaluated using in vitro plantlets and detached leaf assays, respectively, while ‘Calcutta-4’ had a
corresponding severity level of 1.2 and 0.8% in the in vitro and detached leaf assays, respectively
(Table 5.1). Moderately susceptible cultivars had intermediate levels of disease. In both assays,
disease severity assessed 32 days after inoculation separated cultivars based on resistance. Similarly,
evaluated genotypes significantly (p < 0.001) affected incubation time, symptom evolution time,
disease severity and symptom stages in both assays.

Resistant cultivars had longer incubation times than susceptible cultivars (Table 5.1) and much longer
symptom evolution times compared to relatively more susceptible genotypes. Cultivars did not
complete their symptom evolution time in the detached leaf assay, but a consistent pattern was
observed in both assays, with susceptible cultivars having shorter symptom evolution times (e.g., 17
days for ‘Agbagba’) or days to Stage 4 (for the detached leaf assay) than the moderately resistant
cultivars (e.g., 32 days for ‘PITA-21’). Disease severity was not significantly different (p = 0.2997 for
the two-tailed t-test) when assessed using the two assays. In addition, Spearman’s rank correlation
showed that disease severity in the two assays was strongly correlated (r = 0.88, p < 0.0001). Although
incubation time was significantly different (p = 0.0096 for the two-tailed t-test) between the two
assays, Spearman’s rank correlation indicated a strong correlation between the two assays (r = 0.70, p
< 0.0001).

Development of symptoms was observed much earlier on in in vitro plantlets than on detached leaves
(Table 5.2), irrespective of the cultivar evaluated. Highly significant differences (p < 0.0001) in
symptom stages were observed 16, 24 and 32 days after inoculation between the two assays. Ranking
of cultivars based on symptom stage was similar for both assays, with susceptible and moderately
resistant cultivars having correspondingly more advanced symptom stages compared to resistant
cultivars, except at eight days after inoculation (Table 5.2). In the detached leaf assay, the genotypes
evaluated did not show any symptoms after eight days of inoculation, except for the highly susceptible
‘Agbagba’ that had Stage 2 symptoms.

94
CONCLUSIONS

The screening process has been one of the difficulties in breeding banana and plantain for black leaf
streak resistance, with field evaluation being the principal procedure (Mobambo et al., 1997;
Nwauzoma et al., 2002; Oluma et al., 2003). However, field screening has limitations, since it depends
on the presence of proper environmental conditions, can often be carried out only once a year and is
time-consuming and expensive. Inoculation of in vitro plantlets in tubes and inoculation of detached
leaves in moist chambers are two assays that overcome the limitations associated with field and other
early screening (Mobambo et al., 1994) of banana and plantain for resistance to Black sigatoka
disease.

Detached leaves in moist chambers have been used primarily for screening for rusts (Asnaghi et al.,
2001; Herath et al., 2001; Browne et al., 2005) and other diseases (Xie and Mew, 1998; Foolad et al.,
2000; Huang et al. 2005). Recently, Twizeyimana et al. (2007) conducted studies to evaluate the use
of detached leaves for screening Musa species for resistance to black leaf streak. A critical aspect of
this assay is the prevention of senescence of leaf pieces for the duration of the period required to
express symptom stages and disease severity levels necessary to differentiate between cultivar
responses (Xie and Mew, 1998). Benzimidazole (Asnaghi et al., 2001) and cytokinin (Browne et al.,
2005) have been used in the incubation medium to prevent chlorophyll degradation in detached leaves
of other plants. Based on studies by Twizeyimana et al. (2007), agar medium amended with 5 mg/l of
gibberellic acid is able to prevent chlorophyll degradation up to 52 days after plating.

Development of Black sigatoka disease on in vitro plantlets and detached leaves was well correlated,
and cultivars were consistently ranked using the two assays. Symptoms appeared much earlier when
cultivars were screened using in vitro plantlets than with detached leaves as indicated by the
significant difference in incubation time between the two assays. The shorter incubation times
observed for in vitro plantlets may be related to the age of in vitro tissues, which are much more tender
with limited secondary thickening as opposed to detached leaves from the field. The appearance of
symptoms on in vitro plantlets differed from that observed on older plants in the field (Capó et al.,
2003). Primarily, spots were slightly circular, possibly because young plants derived from tissue
culture have limited vein development and black leaf streak lesions tend to be spherical (Mourichon et
al., 2000). Although a paired analysis of disease severity indicated no significant difference between
the two assays, this difference in morphology of the tissues may also explain the higher severity levels
observed on in vitro plantlets compared to the detached leaves for a similar set of cultivars. Higher
disease severity on young tissue culture plants has been reported previously (Mobambo et al., 1997).

Disease development on young tissue culture plants has been reported to be highly correlated (r =
0.98) with disease development on adult plants of the same genotype under field conditions
(Mobambo et al., 1997). A similar correlation has been reported by Romero and Sutton (1997) who
observed that banana hybrids, ‘FHIA-1’ and ‘FHIA-2’, and the susceptible ‘False Horn’ cultivar
expressed the same level of disease reaction in both growth chamber and field evaluations. As such,
disease parameters from these two assays reported here are expected to correspond to disease reactions
under field conditions. An added advantage of these two assays is that they allow for simultaneous
testing of multiple fungal isolates on the same plant, which could shorten the time for development of
broad-spectrum resistance to black leaf streak. The assays are reliable and efficient alternatives to
greenhouse- and field-screening methods, and may be employed for mass screening of Musa species
to facilitate breeding for Black sigatoka disease resistance. Resistant genotypes identified by these
rapid screening methods must be evaluated under field conditions as the final confirmatory test.

95
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Capó YA, Mora ML, Rodríguez MAD, Acosta M, Cruz M, Portal N, Kosky RG, García L, Bermúdez
I, Padrón J (2003) Early evaluation of black leaf streak resistance by using mycelial suspensions of
Mycosphaerella fijiensis. In: Jacome L, Lepoivre P, Marín D, Ortiz R, Romero R, Escalant JV (eds.)
Mycosphaerella Leaf Spot Diseases of Bananas: Present Status and Outlook. Proc 2nd Int. Workshop
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France, pp 169-175.

Carlier J, Fouré E, Gauhl F, Jones DR, Lepoivre P, Mourichon X, Pasberg-Gauhl C, Romero RA

(2000) Sigatoka leaf spots: Black leaf streak. In: Jones DR (ed.) Diseases of Banana, Abacá and Enset.
CAB International, Wallingfold, UK, pp 37-77.

Craenen K, Ortiz R (1998) Influence of Black sigatoka disease on the growth and yield of diploid and
tetraploid hybrids. Crop Prot 17: 13-18.

Foolad MR, Ntahimpera N, Christ JB, Yin GY (2000) Comparison of field, greenhouse, and detached-
leaflet evaluation of tomato germplasm for early blight resistance. Plant Dis 84: 967-972.

Fouré E (1987) Varietal reactions of banana and plantains to black leaf streak disease. In: Persley GJ,
de Langhe E (eds.) Banana and Plantain Breeding Strategies. ACIAR Proc No 21 Canberra, Australia,
pp 110-113.

Herath IHMHB, Stoddard FL, Marshall DR (2001) Evaluating faba beans for rust resistance using
detached leaves. Euphytica 117: 47-57.

Huang S, Vleeshouwers VGAA, Jacobsen E (2005) An accurate in vitro assay for high-throughput
disease testing for Phytophthora infestans in potato. Plant Dis 89: 1263-1267.

Marín DH, Romero RA (1992) El combate de la Sigatoka negra. Boletín No. 4, Departamento de
Investigaciones, Corporación Bananera Nacional, San José, Costa Rica, 22 pp.

Marín DH, Romero RA, Guzmán M, Sutton TB (2003) Black sigatoka: An increasing threat to banana
cultivation. Plant Dis 87: 208-222.

Mobambo KN, Gauhl F, Vuylsteke D, Ortiz R, Pasberg-Gauhl C, Swennen R (1993) Yield loss in
plantain from black sigatoka leaf spot and field performance of resistant hybrids. Field Crops Res 35:
35-42.

Mobambo KN, Pasberg-Gauhl C, Gauhl F, Zuofa K (1994) Early screening for black leaf streak/Black
sigatoka under natural inoculation conditions. Infomusa 3: 4-16.

Mobambo, KN, Pasberg-Gauhl C, Gauhl F, Zuofa K (1997) Host response to Black sigatoka in Musa
germplasm of different ages under natural inoculation conditions. Crop Prot 16: 359-363.

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Mourichon X, Lepoivre P, Carlier J (2000) Host-pathogen interactions. In: Jones DR (ed.) Diseases of
Banana, Abacá and Enset. CAB International, Wallingfold, UK, pp 67-72.

Murashige T, Skoog F (1962) Revised media for rapid growth and bioassays with tobacco tissue
cultures. Physiol Plant 15: 473-497.

Nwauzoma AB, Tenkouano A, Crouch JH, Pillay M, Vuylsteke D, Daniel Kalio LA (2002) Yield and
disease resistance of plantain (Musa spp., AAB group) somaclones in Nigeria. Euphytica 123: 323-
331.

Oluma HOA, Onekutu A, Onyezili FN (2003) Reactions of plantain and banana cultivars to Black
sigatoka leaf spot disease in three farming systems in Nigerian guinea savanna. J Plant Dis Prot 111:
158-164.

Romero RA, Sutton TB (1997) Reaction of four Musa genotypes at three temperatures to isolates of
Mycosphaerella fijiensis from different geographical regions. Plant Dis 81: 1139-1142.

Stover RH (1976) Distribution and cultural characteristics of the pathogens causing banana leaf spot.
Trop Agric (Trinidad) 53: 111-114.

Twizeyimana M, Ojiambo PS, Tenkouano A, Ikotun T, Bandyopadhyay R (2007) Rapid screening of

Musa species for resistance to Black leaf streak using in vitro plantlets in tubes and detached leaves.
Plant Dis 91: 308-314.

Vuylsteke DR (1998) Shoot-tip Culture for the Propagation, Conservation, and Distribution of Musa
Germplasm. International Institute of Tropical Agriculture, Ibadan, Nigeria, 82 pp.

Xie GL, Mew TW (1998) A leaf inoculation method for detection of Xanthomonas oryzae pv.
oryzicola from rice seed. Plant Dis 82: 1007-1011.

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Table 5.1. Incubation time, symptom development and affected leaf area due to Black sigatoka
disease on Musa genotypes evaluated using in vitro plantlets in tubes and detached leaves

Cultivara In vitro plantlets Detached leaves

Symptom
Incubation development Affected leaf Incubation Days to Leaf area
time (days) (days) area (%) time (days) Stage 4b infected (%)
‘Agbagba’ (HS) 5.0 17.3 82.3 8.0 21.4 32.7
‘FHIA-25’ (MS) 6.3 31.2 29.8 14.0 29.1 14.5
‘PITA-21’ (MS) 6.8 32.0 30.8 12.3 28.9 11.0
‘PITA-17’ (R) 14.0 N/Ac 5.7 23.1 N/Ac 3.0
‘Calcutta-4’ (HR) 26.7 N/Ac 1.2 29.5 N/Ac 0.8
LSD (α = 0.05) 7.0 4.8 16.2 2.7 5.8 10.2
a
Disease reaction of cultivars under field conditions: HS = highly susceptible; MS = moderately
susceptible; R = resistant; HR = highly resistant. bNone of the cultivars evaluated using the detached
leaf technique completed their symptom development and thus, number of days to Stage 4 were
recorded instead. c‘Calcutta-4’ and ‘PITA-17’ did not complete their symptom development when
evaluated using in vitro plantlets, and similarly symptoms did not reach Stage 4 when evaluated using
the detached leaf technique. Within each column, mean values followed by similar letters are not
significantly different (p ≤ 0.05) based on Fisher’s protected least significant difference (LSD) test.

Table 5.2. Symptom stages of Mycosphaerella fijiensis on Musa cultivars on three assessment periods
evaluated using in vitro plantlets in tubes and detached leaves

Cultivara In vitro plantlets in tubes Detached leaves

8 days 16 days 24 days 32 days 8 days 16 days 24 days 32 days
‘Agbagba’ (HS) 4 6 6 6 2 3 5 5
‘FHIA-25’ (MS) 3 4 5 6 NS 1 3 5
‘PITA-21’ (MS) 2 4 5 6 NS 1 3 4
‘PITA-17’ (R) 1 1 2 3 NS NS 1 3
b
‘Calcutta-4’ (HR) NS NS 1 2 NS NS NS 1
a
Disease reaction of cultivars under field conditions: HS = highly susceptible; MS = moderately
susceptible; R = resistant; HR = highly resistant. bNS denotes that no symptoms were observed on the
respective cultivar. Symptom stages were based on the 1-6 Fouré scale (Fouré, 1987) in which Stage 1
= minute yellowish specks, <1 mm in length only seen on the lower leaf surface and not visible in
translucent light; 2 = specks are 2-4 mm in length, brown and visible in translucent light; 3 = brown
specks elongate, with streaks reaching 2-3 cm; 4 = broader, round or elliptical spots, brown underneath
and black on the leaf surface; 5 = spots are totally black, surrounded by a yellow halo; and 6 = centre
of spots dries out and fades to grey and a black ring surrounds the yellow halo.

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Figure 5.1. Symptoms of Black sigatoka disease on in vitro plantlets after being
inoculated with Mycosphaerella fijiensis. ‘Agbagba’ and ‘Calcutta-4’ are highly
susceptible and highly resistant to Black sigatoka disease, respectively, while ‘FHIA-23’
and ‘PITA-17’ (shown as 44791) have intermediate levels of disease resistance. No
symptoms are visible on ‘Calcutta-4’, while ‘Agbagba’ has high disease severity.

Preparation of tissue culture plants

A

Figure 5.2. Schematic representation

of steps involved in the use of in
vitro plants for screening banana and
B plantain for resistance to Black
sigatoka disease caused by the
Incubation of inoculated plantlets at 25ºC fungus Mycosphaerella fijiensis. (A)
with a 12:12 hour light:dark cycle Prepare tissue culture plantlets using
standard protocols. (B) Prepare M.
C fijiensis conidial suspension and
aseptically inoculate leaflets (one
drop per leaflet) with 40 μl of 5 × 105
conidia/ml. Avoid leaflets touching
the inner wall of the tube, lid or
medium. (C) Observe disease
symptoms starting 2-32 days after
inoculation. Record data on
incubation time, symptom evolution
time, symptom stages and final
disease severity.

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 Figure 5.3. Symptoms of Black sigatoka disease on detached leaves on medium
amended with gibberellic acid 32 days after inoculation with Mycosphaerella fijiensis.
‘Agbagba’ and ‘Calcutta-4’ are highly susceptible and highly resistant to Black sigatoka
disease, respectively, while ‘FHIA-23’ has an intermediate level of disease resistance.
No symptoms are visible on ‘Calcutta-4’ as opposed to ‘Agbagba’ and ‘FHIA-23’.

Preparation of plating medium Preparation of detached leaves

A B
Plating of leaf pieces on amended medium
C

D
Incubation of inoculated leaf pieces at
25°C with a 12:12 hour light:dark cycle

Figure 5.4. Schematic representation of steps involved in the use of detached leaves for
screening banana and plantain for resistance to Black sigatoka disease caused by the
fungus Mycosphaerrella fijiensis. (A) Prepare medium containing 1% agar technical
and add 5 mg/l of gibberellic acid after autoclaving prior to dispensing into Petri dishes.
(B) Collect clean, newly opened leaves, surface sterilise in 1% NaOCl, rinse in sterile
distilled water and cut into pieces measuring 4 cm in length by 3 cm in width. (C)
Aseptically place leaf pieces in Petri dishes with the adaxial side on the amended
medium. (D) Prepare M. fijiensis conidial suspension and aseptically inoculate detached
leaves (two drops per leaf piece) with 40 μl of 5 × 105 conidia/ml. (E) Observe disease
symptoms starting 2-32 days after inoculation. Record data on incubation time,
symptom evolution time, symptom stages and final disease severity.

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 Chapter 6

In vitro selection for resistance to Fusarium wilt in Banana

K. Matsumoto*, M. L. Barbosa, L.A.C. Souza and J.B. Teixeira

Embrapa Genetic Resources and Biotechnology,

Brasilia,
Brazil.

*Correspondence: [email protected]

ABSTRACT

Fusarium wilt or Panama disease, caused by the fungus Fusarium oxysporum f. sp. cubense (FOC), is
one of the most serious diseases of banana in tropical and subtropical countries. Although there are
banana cultivars resistant to the disease, transfer of the resistance trait to susceptible cultivars by
traditional cross-breeding is difficult. In vitro selections of banana mutants tolerant to race 1 of FOC
were carried out with fusaric acid and culture filtrate on multiple bud clumps. Regenerated plants
showed tolerance to the disease in the greenhouse, and some of the plants also showed resistance in
the field. Although the level of tolerance observed was not sufficiently high, in vitro selection by toxic
substances extracted from pathogenic fungi resulted in an improvement of plant tolerance to the
pathogen. Other selection factors for in vitro selection were reviewed, and a protocol for obtaining
disease tolerant plants of banana is suggested.

INTRODUCTION

Fusarium wilt or Panama disease is caused by the fungus Fusarium oxysporum f. sp. cubense (FOC).
The fungus can survive in the soil for a long period without host plants. Cultural practices for control
of this disease, such as fungicide applications or culture rotations, are not economically practicable.
Biological control is still in the initial phases of development but does have limited applications
(Jaizme-Vega et al., 1998; Ting, et al., 2003). The only effective control available is based on the use
of resistant or tolerant cultivars to the disease (Cordeiro, 1997; Cordeiro and Matos, 2000). Genetic
improvement is, thus, indispensable for the sustainability of banana crop production. Genetic
improvement by cross-breeding has been intensively conducted for the last three decades, and has
produced some results in bananas of the ‘Prata’ subgroup (Musa sp. AAB group). However, the
expansion of the disease and the emergence of a new fungal race in the ‘Cavendish’ subgroup (Musa
sp. AAA group) still pose a threat to many banana plantations. Efforts to produce improved
germplasm must be intensified and a larger number of new varieties must be obtained. Besides, the
‘Cavendish’ subgroup shows a high level of male and female sterility; thus, improving it by traditional
cross-breeding is extremely difficult (Dantas et al., 1997). Given these conditions, in vitro selection
techniques coupled to mutation induction or somaclonal variation could complement conventional
breeding methods.

In vitro selection is an in vitro cultivation technique under controlled stress conditions that favours or
disfavours the growth of a cell or tissue type of interest. Compared with selection in an experimental
field, in vitro selection can considerably reduce the space needed for improving crops. However, some
factors influencing in vitro selection may differ from those in field selection, as for instance, resistance
to the fungus FOC. In order to select plants resistant to Fusarium wilt, FOC can directly be applied to
plants in field selections, but cannot be applied to plantlets in vitro. This is because the fungus grows
much faster than plant tissues in in vitro culture conditions. The fungus quickly dominates the culture
media and flasks, and the growth of the plant tissue is impeded by a lack of nutrients or space, rather
than due to susceptibility to the disease. That is the biggest problem of in vitro selection. Furthermore,

101
the fungus does not attack in vitro tissues equally, thus allowing susceptible plantlets to escape from
the selection pressure. To overcome this problem, techniques using several selection factors (agents)
have been created, such as fusaric acid, culture filtrates of the pathogenic fungus and other substances
that cause similar effects as the fungal attack (Švábová and Lebeda, 2005). In this study, techniques
for in vitro selection using specific agents are reviewed.

MATERIAL AND METHODS

In vitro selection with fusaric acid

Fusaric acid is the most abundant toxin produced by several species of the genus Fusarium, and is
responsible for the symptoms of Fusarium wilt disease (Davis, 1969). Once the fungus uses the toxin
to destroy vegetable cells and to invade the interior of the plant, plants composed of toxin-resistant
cells may block the invasion of the fungus and show tolerance to the disease. In vitro selection of
mutants tolerant to fusaric acid seems to be a viable strategy for obtaining plants tolerant to Fusarium
wilt disease.

The general process for in vitro selection is shown in Figure 6.1. We present the methods used and the
results obtained in our laboratory. As the first step of in vitro selection, multiple bud clumps (see
Figure 6.3A) were obtained by cultivating shoot tips of the banana plant cultivars ‘Maçã’ (Musa sp.
AAB group) and ‘Nanicão’ (Musa sp. AAA group, ‘Cavendish’ subgroup) that are susceptible and
resistant, respectively, to Fusarium wilt disease caused by race 1 of the fungus FOC. These bud
clumps were maintained in a proliferation medium consisting of Murashige and Skoog (MS) medium
with salts and vitamins (Murashige and Skoog, 1962), 5 mg/l 6-benzylaminopurine (BAP), 30 g/l
sucrose, solidified by 2 g/l phytagel (Sigma Chemical Co., St. Louis, MO, USA). The cultures were
maintained in a culture room (28 ± 2°C, 14 hour photoperiod, 56 µmol/m2/s light intensity). In the
second step, concentrations of fusaric acid that inhibit growth of multiple buds were determined. The
multiple bud clumps were cultured on the above mentioned proliferation medium supplemented with
fusaric acid (Sigma Chemical Co.) at concentrations ranging from 0.05 to 1.6 mM. The fusaric acid
was added to the autoclaved culture medium after filter-sterilisation (0.22 or 0.45 µm pore size
membrane filter). After one month of culture, the fresh weights of the bud clumps were measured.

The effect of fusaric acid on the growth of multiple bud clumps is shown in Figure 6.2. The growth of
both cultivars (‘Maçã’ and ‘Nanicão’) was drastically inhibited by 0.1 mM fusaric acid. There was no
significant difference in susceptibility to fusaric acid between the disease-susceptible ‘Maçã’ and
disease-tolerant ‘Nanicão’, suggesting that the degree of disease resistance between these two cultivars
cannot be distinguished by fusaric acid.

In the third step of selection, i.e., selection of tolerant Material, we chose 0.1 mM as the appropriate
concentration of fusaric acid for the selection of resistant mutants of ‘Maçã’. Following treatment with
the chemical mutagen ethyl methanesulfonate (EMS) for 2 hours to induce mutations (Matsumoto and
Yamaguchi, 1984), multiple bud clumps were transferred into a selection medium, which consisted of
proliferation medium supplemented with 0.1 mM fusaric acid. They were cultivated in selection
medium for 3 months, with monthly transfers to fresh media. After the selection period, the multiple
bud clumps were transferred to the culture medium for rooting (MS salts and vitamins, 0.25 mg/l α-
naphthalene acetic acid [NAA], 30 g/l sucrose and 2 g/l phytagel) without fusaric acid, and
regenerated plantlets were transferred to polyethylene bags containing 2 l of soil in a greenhouse for
acclimatisation (Matsumoto et al., 1995).

Tests of tolerance to the disease, which were performed in the fourth and final step of the process,
were carried out in a greenhouse by artificially inoculating plantlets (15-20 cm in height) acclimatised
for one month or rhizomes (~50 cm in height) from plantlets acclimatised for four months with the
fungus FOC. The artificial inoculation was performed by submerging the roots of uprooted plantlets or
rhizomes in a spore suspension of the fungus FOC (107 conidia/ml). They were then again transplanted

102
to polyethylene bags with soil previously infested by the fungus FOC (Sun and Su, 1984; Matsumoto
et al., 1995). The results of the tolerance test on selected 15-20 cm plantlets and rhizomes are shown
in Tables 6.1 and 6.2, and in Figure 6.3. At the growth stage of the 15-20 cm plantlets, disease
symptoms appeared in as many selected as in unselected plantlets. Thus, the selection did not seem to
be effective. However, when rhizomes from 50 cm plantlets were inoculated with the fungus, the
selected plantlets showed higher tolerance to the disease than those not selected. These results showed
that selection by fusaric acid was effective: however, the acquired tolerance was not visible at the
early stages of development of the acclimatised plantlets.

In vitro selection with culture filtrate

The culture filtrate consists of a liquid culture medium in which microorganisms have been cultivated
for a certain period of time and eliminated by filtration through a membrane filter. The culture filtrate
is expected to contain several metabolic substances produced by the microorganisms, including
several toxins besides the fusaric acid that was discussed previously. Microorganisms are usually
eliminated by filtration rather than by autoclaving because some metabolic substances are unstable at
high temperature. Compared with the use of fusaric acid, selection using culture filtrate may be more
efficient for obtaining disease-tolerant plants, because the filtrate contains some toxins that have not
yet been identified, but that may be responsible for defining the degree of disease susceptibility or
tolerance in the host plants. In some species, Fusarium-tolerant plants have already been obtained
using culture filtrates as a selection factor (Arcioni et al., 1987; Binarova et al., 1990; Borrás et al.,
1997). Therefore, we speculated that the technique could be useful for the assessment of resistance to
Fusarium wilt in banana plants as well.

Details of the culture filtrate preparation are described in Matsumoto et al. (1999b). Race 1 of the
fungus FOC was isolated from a disease-susceptible banana plant that showed symptoms of Fusarium
wilt disease, propagated once in potato dextrose agar (PDA) medium and conserved in the refrigerator
(4°C). A piece of the fungal colony with the PDA medium was inoculated in 100 ml of Czapek Dox
Broth (CZD) medium in a 300 ml Erlenmeyer flask, with a multiple bud clump (~10 × 10 × 10 mm) of
a disease-susceptible banana variety (co-cultivation technique). The culture was maintained in a
culture room without agitation for 21 days. It was then filtered through a four-layer gauze (or
cheesecloth) and centrifuged at 8000 rpm for 20 minutes. The supernatant was finally filtered through
a 0.45 µm membrane filter to eliminate the fungus.

Figure 6.4 shows the growth inhibition caused by the culture filtrate on in vitro multiple bud clumps of
disease-susceptible ‘Maçã’ and disease-tolerant ‘Nanicão’ cultivars. Inhibition of growth was higher in
‘Maçã’ than in ‘Nanicão’ (Figure 6.4). Since ‘Maçã’ is a susceptible cultivar and ‘Nanicão’ a resistant
one, we could expect to obtain disease-tolerant plants using culture filtrate for selection. Therefore, in
contrast to the experiment using fusaric acid, where no differences in growth inhibition were observed
between the two cultivars (Figure 6.2), the use of FOC filtrates seems to be efficient for the selection
of tolerance to Fusarium wilt.

To induce mutations, the same treatment was carried out as for selection with fusaric acid. After the
mutagenic treatment, the multiple bud clumps were cultivated on proliferation medium (MS, 5 mg/l
BAP, 30 g/l sucrose and 2 g/l phytagel) for 5 days, and then transferred to the medium for the first
round of selection (MS, 5 mg/l BAP, 10% [v/v] culture filtrate, 30 g/l sucrose and 2 g/l Phytagel).
After one month of cultivation, the growing bud clumps were subdivided and transferred to a new
medium for the second round of selection (MS, 5 mg/l BAP, 15% [v/v] culture filtrate, 30 g/l sucrose
and 2 g/l Phytagel). Another two rounds of selection were undertaken using the same medium. After
the successive rounds of selection, the clumps of multiple buds tolerant to the culture filtrate were
propagated, and plantlets were regenerated on rooting medium. The plantlets were acclimatised and
their tolerance to the fungus was evaluated in the greenhouse as described previously.

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Tables 6.3 and 6.4 show the results of the selection using the 10 to 15% (v/v) culture filtrate, after
mutations were induced by the chemical mutagen EMS. When acclimatised plantlets (15-20 cm) were
artificially infested with FOC and disease symptoms were observed in pseudostem, the selected
plantlets did not show any significant increase in disease tolerance. However, symptom observation in
rhizomes revealed that the selected plantlets were significantly more tolerant than those not selected
(Table 6.3). The selection efficiency was also confirmed by evaluation of pseudostem height and root
fresh weight (Table 6.4). Although selection with the culture filtrate was very efficient, the level of
tolerance did not reach that of the ‘Nanicão’ cultivar, which is well-known for its resistance to
Fusarium wilt (Table 6.3).

Evaluation methods for in vitro-selected plants

The evaluation method for disease resistance is as important as the selection method itself for
achieving successful results. As a rule, in vitro selection for Fusarium wilt tolerance does not utilise
live FOC fungus. Consequently, an evaluation of the selected plants by the fungus itself is
indispensable as a final step of the selection process. Such evaluation is carried out on field plants or
acclimatised plantlets in the greenhouse. Although field tests are ideal for this purpose (Hwang, 1990;
Smith et al., 2006), they require a huge experimental area, and plants are frequently damaged by
diseases not-targeted or other climatic factors. To overcome these problems, Sun and Su (1984)
developed a method for acclimatised plantlets as described in section 2 (in vitro selection by fusaric
acid). With some modification and adaptation, it was also applied for ex vitro selection (Bhagwat and
Duncan, 1998; Bermúdez et al., 2002; Mak et al., 2004). Acclimatised small plantlets in the
greenhouse or net-house were infested by the fungus FOC for the evaluation, and symptomless
plantlets were selected to be transplanted in the field. This method can be used to analyse a limited
number of plantlets and is feasible as a pre-field-selection for tolerance to the disease. Even though the
method is very useful, care must be taken in its application, because the disease susceptibility or
tolerance of the selected Material somewhat depends on the plantlet stage and evaluation parameter
(see Tables 6.1-6.4).

Inoculation of fungus on both acclimatised plantlets and field plants may kill plants that, although
susceptible to the disease, might have excellent agronomic characters. More recently, a non-
destructive evaluation method for disease resistance was developed using fungal culture filtrate. It was
based on concentrated culture-filtrate inoculation onto needle-mediated wounds of middle-aged
banana leaves (Companioni et al., 2003, 2005). Evaluating seven banana clones, three of which were
disease-susceptible and four of which were disease-resistant, the susceptible clones always showed
major lesion areas after 48 hours of incubation. This evaluation method is of great interest not only
because it is non-destructive but also due to the very short time needed for evaluation.

Protocol for in vitro selection of Fusarium wilt-tolerant plants

The in vitro selection protocol used in our laboratory is as follows:

Plant material preparation

(1) Small and apparently healthy suckers are collected from a Fusarium wilt-susceptible plant.
(2) Suckers are washed with tap water and cut into 10 × 10 × 10 mm3 blocks with shoot tip.
(3) The tissue blocks are surface-sterilised in a laminar flow cabinet with 70% alcohol for 15 s,
followed by 1% sodium hypochloride with a few drops of Tween 20 for 15 minutes.
(4) External tissues are removed leaving blocks of 5 × 5 × 5 mm3.
(5) They are transferred to banana proliferation medium consisting of MS (Murashige and Skoog,
1962) salts and vitamins, 5 mg/l BAP, 30 g/l sucrose, 2 g/l phytagel and maintained in a
culture room (28 ± 2°C, 14 hour photoperiod, 45-60 µmol/m2/s light intensity).
(6) After 3 to 8 months of culture (subcultures each of 30 to 45 days), multiple bud clumps will be
obtained.

104
Production of culture filtrates

(7) FOC fungus is isolated on PDA medium from a disease-susceptible banana plant that shows
disease symptoms.
(8) The fungus is proliferated once on the same medium in Petri dishes for two weeks (28 ± 2°C,
45-60 µmol/m2/s of continuous illumination) and stored in a refrigerator (4°C) until use.
Pathogenicity of the isolates should be checked using, for instance, the method of Sun and Su
(1984).
(9) A piece of the fungal colony (2-3 mm in diameter) and a tissue (~10 mm in diameter) of the
banana multiple bud clump from Step 6 are inoculated into 100 ml of CZD medium in a 300
ml Erlenmeyer flask.
(10) The culture is incubated at 28 ± 2°C and 45-60 µmol/m2/s illumination with a 14 hour
photoperiod for 21 days without shaking.
(11) The cultured liquid medium is then filtered through a four-layer gauze or cheesecloth and
centrifuged (8000 rpm, 20 minutes) to precipitate mycelium and conidia.
(12) The supernatant is passed through a membrane filter (0.45 or 0.22 µm pore size) to remove the
remaining fungi. This cultured fungus-free medium is used as the culture filtrate for the
following selection. The culture filtrate should be used immediately; it is not recommended to
store it.

Mutagen treatment and selection

(13) At least 500 pieces, each ~3 × 3 × 3 mm3, of the banana multiple bud clumps from Step 6 are
incubated in a 500 ml Erlenmeyer flask with 200 ml of aqueous solution supplemented with
4% (v/v) dimethylsulphoxide (DMSO) and 0.3% (v/v) EMS.
(14) The flask is kept on a gyratory shaker (100 rpm) for two hours at 28 ± 2°C.
(15) After washing three times with sterile distilled water, the multiple bud clumps are transferred
to banana proliferation medium (Step 5) in Petri dishes and cultured for one week.
(16) The multiple bud clumps are then transferred onto the selection medium, which consists of
proliferation medium supplemented with 10-15% (v/v) culture filtrate or 0.1 mM fusaric acid.
(17) After 30 to 45 days of culture, growing multiple bud clumps are separated into smaller pieces
(3 × 3 × 3 mm3) and transferred to the newly prepared selection medium.
(18) The above process is repeated once more.
(19) The selected multiple bud clumps are propagated on proliferation medium for 2-3 months until
multiple shoot formation.
(20) The multiple shoots are separated and transferred to rooting medium composed of MS salts
and vitamins, 0.25 mg/l NAA and 30 g/l sucrose solidified by 2 g/l of phytagel.
(21) After 20 to 30 days of culture, regenerated plantlets are transferred to black polyethylene bags
or containers with a substrate mixture and acclimatised in a greenhouse equipped with a mist
system.
(22) The disease resistance of the acclimatised plantlets is evaluated in the greenhouse and/or in the
field.

RESULTS AND DISCUSSION

Perspectives of the in vitro selection for disease resistance

The results mentioned in this chapter show the applicability of fusaric acid and culture filtrate in the
selection of plants tolerant to the fungus FOC. However, the level of tolerance obtained by both
methods was always lower than the tolerance level of ‘Nanicão’ that was used as a resistant control for
the disease. Some of selected lines of ‘Maçã’ plants showed resistance in the field. At the same time,
however, they became more susceptible to another disease, namely Yellow sigatoka disease
(Matsumoto et al., 1999a). This effect might have been caused by secondary mutations or somaclonal
variations induced by the successive rounds of selection, which were carried out with the aim of

105
avoiding occurrence of escapees and chimeras. Since many of the mycotoxins in the culture filtrates
are mutagens (Jimenez et al., 1997; Sewram et al., 2005; Švábová and Lebeda, 2005), successive
selections could induce secondary mutations. Undesirable mutations can be eliminated by new
selection steps addressing the correction of the undesired character (Tang and Hwang, 1998).
However, to avoid the secondary mutations in the first place, it is advisable to perform a single round
or a few successive rounds of selection. The risk of occurrence of escapees and chimeras can be
reduced by the use of smaller-sized explants, suspension cells or protoplasts. A reliable protocol of
plant regeneration from the cells or protoplasts must become an important tool.

The history of plant breeding for disease resistance has shown that the acquired resistance will always
be broken by the emergence of new microbial races. This means that we have to continuously breed
new plants with different genes or mechanisms of disease resistance. In vitro selections with different
selection factors make this strategy possible. Morpurgo et al. (1994; Chapter 7 of this book) showed
that in vitro explants from disease-resistant and disease-susceptible banana cultivars were susceptible
to both culture filtrate and fusaric acid irrespective of their known field resistance/susceptibility
response. Therefore, they concluded that the use of culture filtrate or fusaric acid was not feasible for
selecting a novel resistant genotype of Musa to FOC. However, contrary to their observations, the
disease tolerances of susceptible banana cultivars were improved after selection with metabolic
substances (Matsumoto et al., 1995, 1999a; Cardenas et al., 2003; Saravanan et al., 2003). We could
now draw the conclusion that the use of culture filtrates or fusaric acid for selecting a novel genotype
of Musa resistant to FOC is quite feasible. The disease tolerance mechanism of in vitro-selected plants
may then be different from that of pre-existing resistant cultivars, since both resistant and susceptible
cultivars show equal susceptibility to the metabolic substances. Considering that the mechanisms of
tolerance to one selection factor are different from another, tolerances to several selection factors can
be accumulated in one genotype by cumulative selection processes (Tang and Hwang, 1998). The in
vitro selection system may produce new plants that confer several resistance mechanisms regulated by
several genes. The acquired tolerance would, then, be a horizontal resistance characteristic and would
be difficult to be broken by the emergence of new races of the fungus. It could, therefore, be
maintained for a longer period.

Besides pathogen-produced metabolic substances, many other substances or chemicals can be used for
in vitro selection of tolerance to Fusarium wilt. New selection factors or gene markers can facilitate in
vitro selections and increase their applicability. Elicitors from fungal cell wall fractions are promising
substances, since plant defence responses were induced by the elicitor treatments (De Ascensao and
Dubery, 2000). Although we still do not have supporting data, plants selected by a proline analogue,
such as hydroxyproline (Matsumoto et al., 1987), may over-produce proline and increase the
absorption capacity of water from the soil. This could reduce damage caused by Fusarium wilt.
Transgenic plants with an introduced glucanase gene have increased tolerance to Fusarium wilt
(Mahmood et al., 2003). Glucanase over-producing plants may also be obtained by selecting explants
that grow on a culture medium containing glucan as a substitute for part of the sucrose.

CONCLUSIONS

Reliable protocols for in vitro selection of banana plants resistant or tolerant to diseases are an
important tool for the improvement of this crop, particularly in combination with in vitro mutagenesis.
Our in vitro selection protocol based on the use of culture filtrates or fusaric acid for selecting novel
genotypes of Musa resistant to FOC was shown to be quite feasible. In addition to the substances used
in this study, many other substances or chemicals, such as fungal elicitors, may be useful for in vitro
selection of tolerance to Fusarium wilt in bananas. Gene markers may also facilitate in vitro selections
and increase their applicability.

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Cardenas JE, Pocasangre L, Riveros AS, Rosales F (2003) Early selection of in vitro plants of ‘Gros
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growth inhibition by L-hydroxyproline in a banana. Environ Control Biol 25: 63-66.

Morpurgo R, Lopato SV, Afza R, Novak FJ (1994) Selection parameters for resistance to Fusarium
oxysporum f. sp. cubense race 1 and race 4 on diploid banana (Musa acuminata Colla). Euphytica 75:
121-129.

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culture. Physiol Plant 15: 473-497.

Saravanan T, Muthusamy M, Marimuthu T (2003) In vitro establishment of tolerant clones of banana

against race 1 Fusarium oxysporum f. sp. cubense. J Appl Hort Lucknow 5: 81-84.

Sewram V, Mshicileli N, Shephard GS, Vismer HF, Rheeder JP, Lee YW, Leslie JF, Marasas WFO
(2005) Production of fumonisin B and C analogues by several Fusarium species. J Agric Food Chem
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Smith MK, Hamill SD, Langdon PW, Giles JE, Doogan VJ, Pegg KG (2006) Towards the
development of a Cavendish banana resistant to race 4 of Fusarium wilt: gamma irradiation of
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107-113.

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Sun EJ, Su HJ (1984) Rapid method for determining differential pathogenicity of Fusarium
oxysporum f. sp. cubense using banana plantlets. Trop Agric (Trinidad) 61: 7-8.

Švábová L, Lebeda A (2005) In vitro selection for improved plant resistance to toxin-producing
pathogens J Phytopathol 153: 52-64.

Tang CY, Hwang SC (1998) Selection and asexual inheritance of a dwarf variant of Cavendish banana
resistant to race 4 of Fusarium oxysporum f. sp. cubense. Aust J Exp Agric 38: 189-194.

Ting ASY, Meon S, Jugah K, Anuar AR (2003) Effect of artificially induced suppressive soil on
Fusarium wilt. Infomusa 12: 33-34.

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Table 6.1. Comparison of susceptibility of selected and unselected ‘Maçã’ banana plantlets to
Fusarium wilt*

Variety Selection Pseudostem Root fresh Fusarium wilt symptoms (%)

height (mm) weight (g)
Pseudostem Rhizome

‘Maçã’ Not selected 1435 11.88 76 88

‘Maçã’ Selected 1860 19.58 90 90
t-test ** 0.0002 0.0003 0.2531 0.6332

*15-20 cm plantlets of ‘Maçã’ bananas were infested artificially with race 1 of FOC in the greenhouse.
17 samples per treatment were analysed. **Values from a two-sided t-test for the two values
immediately above. Source: Matsumoto et al. (1995).

Table 6.2. Comparison of susceptibility of selected and unselected ‘Maçã’ banana rhizomes to
Fusarium wilt*

Variety Selection Pseudostem Root fresh Fusarium wilt symptoms (%)

height weight (g)
(mm) Pseudostem Rhizome

‘Maçã’ Not selected 264a 0.46A 57a 100A

‘Maçã’ Selected 825b 2.48B 14b 57B
‘Nanicao’ Not selected 852b 3.69B 0b 29B

*Rhizomes from 50 cm plantlets were infested artificially with race 1 of FOC in the greenhouse. 14
rhizome samples were analysed. The ‘Nanicão’ variety was used as a control for the disease-tolerant
variety. In each column, values followed by the same letter are not significantly different from each
other at the 5% level using Duncan’s multiple-range test. Source: Matsumoto et al. (1995).

110
Table 6.3. Frequency of plants with symptoms of pseudostem and rhizome vascular discolouration
caused by race 1 of FOC on banana plantlets of the ‘Maçã’ cultivar selected or not selected with
culture filtrate in the greenhouse test

Fusarium wilt symptoms (%)

Variety Selection Number of plants
observed Pseudostem Rhizome

‘Maçã’ Not selected 34 47.1 82.4

‘Maçã’ Selected 31 41.9 67.7
‘Nanicão’1 Not selected 34 2.9 29.4
1
Nanicão was tested as a control for a resistant plant to race 1 of FOC. A comparison between selected
and not selected ‘Maçã’ showed a significance level of 0.4346 for pseudostem and 0.1406 for rhizome
(t-test). A comparison between ‘Nanicão’ and other treatments showed a significance level <0.01.
Source: Matsumoto et al. (1999a).

Table 6.4. Comparison of pseudostem and root growth between culture-filtrate selected and not
selected ‘Maçã’ banana, infested by race 1 of FOC in greenhouse tests

Variety Selection Pseudostem height (mm) Root fresh weight (mg)

‘Maçã’ Not selected 26.42 ± 6.251 460.71 ± 109.96
‘Maçã’ Selected 43.63 ± 10.83 1077.27 ± 313.43
two-sided significance level by t-test 0.1613 0.0536
1
Mean ± standard error. Source: Matsumoto et al. (1999a).

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 Development of
protoplast, cell or tissue Establishment of
culture techniques selection factors

Susceptibility test of
explanted materials Mutation
against selection factors induction

Selection of tolerant
materials against
selection factors

Plant regeneration

Disease tolerance
tests of selected plants

Figure 6.1. In vitro selection process to obtain disease-tolerant plants.

Figure 6.2. Effects of fusaric acid on growth of banana multiple bud clumps.

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 A B

C D

Figure 6.3. (A) Multiple bud clumps; (B) FOC-inoculated rhizomes from unselected
‘Maçã’ banana; (C) FOC-inoculated rhizomes from from ‘Maçã’ banana selected with
fusaric acid; (D) FOC-inoculated rhizome of ‘Nanicão’ banana.

Figure 6.4. Effects of culture filtrate on growth of banana multiple bud clumps.

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 Chapter 7

Selection parameters for resistance to Fusarium oxysporum f. sp.

cubense race 1 and race 4 on diploid banana (Musa acuminata)*

R. Morpurgo, S. Lopato, R. Afza* and F.J. Novák†

Joint FAO/IAEA Programme, Plant Breeding Unit,

Agency’s Laboratories, Seibersdorf,
Austria

*Correspondence: [email protected]

ABSTRACT

Shoot tip cultures from banana clones susceptible and resistant to Fusarium oxysporum f. sp. cubense
(FOC) race 1 and race 4 were grown in vitro in the presence of different concentrations of fusaric acid
and fungal crude filtrates or inoculated with a conidial suspension of FOC to assess correlation
between in vivo and in vitro behaviour. Explants were susceptible to both filtrate and fusaric acid
irrespective of their known field resistance/susceptibility response. No clear linkage between in vivo
and in vitro behaviour was observed, and our results suggest that the use of crude filtrate or non-host-
specific toxin (fusaric acid) in a screening programme for selecting a novel resistant genotype of Musa
to FOC is not feasible. When peroxidase activity was used as a parameter to discriminate between
susceptibility and tolerance, the results were in good agreement with the field response of host plant to
pathogens. Early enzymatic activity increased in the incompatible host-pathogen interaction but not in
the compatible interaction.

INTRODUCTION

According to the FAO Trade Yearbook 1990 (FAO, 1991), Musa (banana, plantain and cooking
banana) is cultivated in 117 countries worldwide, mostly in the tropics and subtropics of South and
Central America, Asia and Africa. Less than 10% of the whole production of 70 million tons of fruit
enters world trade; the majority is locally consumed as staple starchy food. Several factors, edaphic,
physical and biotic limit Musa production. Among the latter, the most important are two fungal
diseases, the Sigatoka complex, caused by three related Mycosphaerella spp.; and Panama disease
caused by FOC. The susceptible cultivar ‘Gros Michel’ was gradually replaced by the resistant
‘Cavendish’ clones but a new race of FOC has recently developed that has broken the resistance of
‘Cavendish’ to race 1. The new race 4 of FOC appeared, presumably as independent events, in four
different regions of the world, namely the Canary Islands, Australia, Taiwan and South Africa (Stover,
1990) and is now spreading rapidly in other banana producing countries. Breeding for banana
resistance is particularly difficult due to the sterile and polyploid nature of the plant and to the
saprophytic – pathogenic nature of the fungus (Novak, 1992). The relationship of the pathogen with
the plant host and the complex interplay occurring between them and environmental parameters results
in an extremely complex situation (Beckman et al., 1962; Marois, 1990). Selecting plant genotypes
resistant to pathogens has become one of the major tools of combating agricultural losses and
increasing productivity. However, traditional plant breeding is not able to keep pace with pathogen
evolution. Field screening is time consuming and requires a large amount of space and intensive
manpower inputs. Furthermore, disease symptoms develop only after a long period of incubation and
the results can be misleading due to non-uniform distribution of pathogens in soil.

Plant tissue culture techniques have been used for investigating mechanisms of host pathogen
relationship (reviewed in Chapter 2) and have been proposed as an aid in banana breeding
programmes (Novak, 1992). The most widely used system for in vitro evaluation of

115
resistance/susceptibility is the use of purified toxins and/or fungal crude filtrate (Daub, 1986).
However, the use of filtrate produced in culture by Fusarium spp. was questioned by Beckmann
(1987). Results obtained by Buiatti et al. (1987) showed that in F. oxysporum f. sp. dianthi cell
culture, a better correlation exists when biochemical parameters, i.e., phenolic metabolites, are
considered. Peroxidase (EC 1.11.1.7) is a multi-purpose enzyme that has been correlated with active
plant defence mechanisms by condensing phenolic compounds to form lignin. In this paper, we
compared the effect of crude fungal filtrates on banana plants and the production of peroxidase after
inoculation with FOC race 1 and race 4 conidial suspensions in order to identify suitable selection
criteria.

MATERIAL AND METHODS

Plant material

Two diploid (AA) clones, namely ‘SH-3362’ and ‘Pisang Mas’ (syn. ‘Sucrier’), showing different
resistance to FOC race 1 and race 4 were used. ‘SH-3362’ is a product of cross-breeding with high
resistance to both races of FOC (Rowe, 1991) while ‘Pisang Mas’ is a sterile diploid cultivar, widely
cultivated for small sweet fruit, moderately resistant to race 1 and fully susceptible to race 4 of FOC.

Fungal culture

FOC race 1 strain 2264 (vegetative compatibility group [VCG] 0125) and race 4 strain YO 604 (VCG
01290) were provided by Dr. K. Pegg, DPI, Queensland. Small plugs of mycelium were transferred to
Petri dishes filled with solid Czapek-Dox medium (Difco, Becton Dickinson, Sparks, MD, USA) and
incubated in the dark at 28°C. After a week, plugs of actively growing mycelium were transferred to a
250 ml Erlenmeyer flask containing 150 ml of liquid Czapek-Dox and further incubated under the
same conditions as above for a maximum of 21 days. The culture broth was then filtered through a
double layer of cheesecloth and sterilised through a 0.22 µm Millipore membrane. The crude filtrate
was then stored at -20°C.

In vitro plant culture

Shoot tips of in vitro grown banana plantlets were excised and transferred to 250 ml Erlenmeyer flasks
containing 25 ml of culture medium composed of MS salts (Murashige and Skoog, 1962),
supplemented with Gamborg’s B5 vitamins (Gamborg et al., 1968), 5 μM 2iP, 40 mg/l cysteine and
40 g/l sucrose. The pH was adjusted to pH5.8 and the medium was autoclaved at 120°C and 1 bar for
20 minutes. After a one week incubation at 25°C on a rotary shaker at 80 rpm under continuous light,
the culture medium was removed and an equal amount of fresh medium was added in order to avoid
toxic activity due to phenolic compounds produced by the explant.

Fusaric acid assay

Shoot tips of in vitro grown banana were excised and transferred to the same culture medium
supplemented with different concentrations (0.02, 0.045, 0.06, 0.09 mM) of fusaric acid (Sigma). The
control and treated plants were screened after three weeks for growth parameters such as shoot and
root fresh weight, shoot height and explant survival.

In vitro assay of crude filtrate

Banana shoot tips were transferred to the same medium described above with or without different
percentages (4.5, 6, 9 and 12% [v/v]) of crude filtrate obtained 21 days after fungal culture. Plants
were screened after three weeks of culture for shoot and root fresh weight, shoot height, explant
survival.

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Ion leakage

Leaf disks were obtained from fully expanded leaves of the same genotypes grown in the greenhouse
using a cork borer. Leaf disks were held in pure crude filtrate of both races for 30 minutes under
vacuum to facilitate filtrate penetration, washed three times with distilled water and placed in test
tubes filled with 20 ml of an isotonic sucrose solution. Conductivity was measured (YSI Model 34,
YSI, Yellow Springs, USA) at two minute intervals to follow ion release from the cell.

Plant inoculation

Banana plantlets were grown and rooted under axenic conditions as described elsewhere (Novak et al.,
1987). Roots were trimmed and plantlets were treated with a microconidial suspension (5 × 105
conidia/ml) as described by Hwang and Ko (1987). Controls were treated with distilled water only.
Treated and non-treated plants were transplanted in sterile vermiculite and placed in a growth chamber
at 29°C. At weekly intervals, three plantlets were removed and assayed for peroxidase activity.

Peroxidase assay

Corm tissue was ground in a mortar adding to the tissue 0.1 M sodium phosphate buffer pH 6.8 (1.2
[w/v]). The resulting homogenate was centrifuged at 14000 rpm in an Eppendorf centrifuge and
assayed for protein content according to Bradford (1976). In addition, the method described by
Garraway et al. (1989) was used to extract ionically bound peroxidase. Activity was measured in 1 ml
final volume reaction mixtures. Five µl of corm extract were incubated in 0.3% guaiacol, 2 mM
hydrogen peroxide in 0.01 M sodium phosphate buffer pH 6. After two minutes, absorbance was read
at a wavelength of 470 nm and protein content assessed.

Isoelectrofocusing (IEF)

Samples containing equal protein content were loaded on a polyacrylamide gel (T = 7.5%, C = 3%)
containing Pharmalyte pH 3.5-9.5 (Amersham Pharmacia Biotech, Piscataway, NJ, USA) as a carrier
ampholyte. The gel was prefocused for 30 minutes. Running conditions were 8 W fixed, 33 mA and a
voltage up to 2500 V. After running, the gels were stained in sodium phosphate buffer pH 6,
containing 0.3 % guaiacol and 2 mM hydrogen peroxide or 0.6 mg 4 chloro-1-naphtol per ml and 2
mM hydrogen peroxide (Ye et al., 1990).

RESULTS AND DISCUSSION

Effect of fusaric acid

No conclusive results were obtained using fusaric acid as a selection agent in tissue culture media. A
concentration of 0.45 mM of fusaric acid stimulated the growth of the resistant clone ‘SH-3362’ while
at twice the concentration (90 μM), plant growth was slightly inhibited. The clone ‘Pisang Mas’,
susceptible to race 4, showed little response to different levels of fusaric acid (Figure 7.1).

In vitro assay of crude filtrate

Production of toxic substances in the filtrate was assayed by reading 2 ml aliquots in a

spectrophotometer set at 272 nm at weekly intervals. Absorbance was then correlated with biological
effects of the same filtrate on banana shoot tip culture. A significant correlation (r = 0.92) was found
between these two methods. Crude filtrate obtained at weekly intervals exhibited maximum toxic
activity three weeks after inoculation. Figures 7.2 shows the inhibition of shoot growth of both
susceptible and tolerant clones cultured in media with different concentrations of crude FOC filtrate.
The addition of the filtrate from both races of FOC in the medium at a concentration of 9%
significantly reduced the growth of treated plants; at a concentration of 12%, explants were not able to

117
develop a new shoot and most died. A direct effect of the filtrate on the rate of mortality on the
cultured plantlets of both genotypes was observed. Both susceptible and tolerant clones responded in
the same way to the filtrate, i.e., inhibition of all the growth parameters (i.e., total fresh weight, plant
height, root fresh weight and shoot fresh weight). No differential response was observed between
banana clones with filtrates of race 1 or race 4. The same tendency was observed by determination of
ion leakage (Figure 7.3). In this case, the addition of filtrate to detached leaf disks caused an increase
in ion leakage as compared with the non-treated leaf tissue. The release of ions increased with time but
it was not correlated with known fungal race pathogenicity. On the contrary, in ‘SH-3362’, the ion
leakage after treatment with filtrate of race 1 was higher than that observed with race 4.

Growth of inoculated plants

In all plants inoculated with FOC conidia from race 1 and 4, no wilting symptoms were observed up to
28 days of culture in vermiculite at 29°C. However, internal symptoms, i.e., browning of xylem
vessels were observed on dissection of the susceptible clone ‘Pisang Mas’ inoculated with race 4. The
same clone showed reduced root growth.

Peroxidase activity

Different tissues, i.e., root, corm and leaf of both banana genotypes were analysed for constitutive
peroxidase activity before fungal inoculation. In non-infected plants of both clones, the highest
peroxidase activity was found in root tissue while the lowest activity was recorded in leaf tissue.
However, the two clones showed marked differences in constitutive activity. This was at least five
times higher in the root and corm tissues of the resistant ‘SH-3362’ clone than in the susceptible
cultivar ‘Pisang Mas’ (Figure 7.4). In plants infected with FOC race 1, both clones showed a prompt
increase in peroxidase activity with seven days of inoculation (see Figure 7.5). The general response
of the two banana clones to race 1 was essentially similar, with fast induction and a high level of
activity over the whole experimental period. The reaction of host plants to race 4 provided a
completely different picture. After seven days, the resistant clone ‘SH-3362’ showed a 10-fold
increase in peroxidase activity, declining slowly over the next 21 days (Figure 7.5A). The susceptible
cultivar ‘Pisang Mas’, on the other hand, responded to the infections with a small increase, although
not statistically significant, in enzymatic activity over the first seven days after infection (Figure
7.5B). Thereafter, the enzymatic activity in this clone either decreased to a lower level comparable
with the non-inoculated plants (14 and 28 days after inoculation) or slightly increased (21 days after
inoculation).

In addition to the qualitative aspects and the kinetics of induction, it was interesting to note that at any
evaluation point there were marked differences between the resistant and the susceptible genotypes. In
fact, the total peroxidase activity in the resistant clone ‘SH-3362’ was always higher at least by a
factor of six than the enzymatic activity induced by the infection with FOC race 4 in the susceptible
cultivar ‘Pisang Mas’.

Isolectrofocusing

4 chloro-1-naphtol gave better band resolution than guaiacol and a higher number of isozymes were
detected. Using this stain, it was possible to count at least 12 bands of isozymes, common to all the
clones. Among them, seven anionic bands were present. We did not detect differences in isozyme
patterns between induced and non-induced plants; intensity of bands increased in both control and
treated plants with time. However, the intensity of some bands increased earlier in inoculated plants
than in controls (Figure 7.6).

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CONCLUSIONS

Phytotoxins in crude fungal culture filtrates have previously been used as selection agents in in vitro
cultures and a correlation between in vivo and in vitro responses has been reported in several plant-
pathogen systems (Daub, 1986). However, the involvement of purified non-host-specific toxins or
crude filtrate in wilt disease has been questioned (Beckmann, 1987; Buiatti and Ingram, 1991). In our
work, crude filtrate reduced all growth parameters tested and finally led to the death of explants
without typical wilting symptoms in both tolerant and resistant Musa genotypes. Production of toxic
secondary metabolites during culture of Fusarium spp. depends not only on the species of the forma
specialis but also on culture and growth conditions such as temperature, inoculum concentration and
age of culture as well as on the physiological stage of the pathogenic organism itself (Durbin, 1983).
Usually, the activity of toxic compounds present in a crude filtrate is measured by bioassay of living
material. A major drawback in this procedure is the long time needed to observe effects. The
spectrophotometric method described in this work provides a faster way of determining both the
biological effects and the filtrate concentration to be added in the medium.

Our results are in agreement with those described by Epp (1987), who was not able to discriminate
between resistant and susceptible banana plants using fusaric acid as a selection agent in culture
media. Resistance to FOC, therefore, does not seem to be correlated with resistance to a non-host-
specific toxin. No conclusive response was obtained using banana shoot tips in culture on a medium
containing crude filtrate; this suggests that toxic compounds excreted by the fungus while in culture
are not responsible for the breakdown in resistance caused by FOC race 4 in previously resistant
banana clones. Since in the literature (e.g., Daub, 1986) no host-specific toxins have been reported for
the genus Fusarium, it appears quite unlikely that toxins could act as determinants of virulence. Toxic
effects suggest that crude filtrate may play some role in pathogenesis but this should act rather in the
post-infection processes. This assumption is in agreement with the in vitro behaviour of alfalfa where
virulence of Fusarium could not be correlated with the toxic activity shown by the filtrate on explants
(Binarova et al., 1990).

Previous work conduced on the Dianthus caryophyllus-Fusarium oxysporum f. sp. dianthi interaction
using two different systems, i.e., culture filtrate and fungal cell wall elicitor, showed that the latter is a
more efficient experimental system for the identification and characterisation of susceptible and
tolerant responses in the host plant (Buiatti et al., 1985, 1987).

Resistance to FOC can be accomplished by the whole plants’ defence mechanism. Indeed, the only
reported mechanism that acts as a defence against FOC in banana is a prompt response to infection
mediated by the build-up of a mechanical barrier that may prevent the spread of the pathogen into the
plants’ vascular system (Beckman et al., 1961; Beckman, 1990).

Peroxidase is a multi-purpose enzyme that catalyses the condensation of phenolic compounds into
lignin. The current model that involves peroxidase in defence mechanisms considers the condensation
of phenolic monomers derived from the phenylpropanoid pathway into insoluble polymers (Robb et
al., 1991). This step is catalysed by anionic peroxidase. It has been demonstrated by different authors
(Seevers et al., 1971; Graham and Graham, 1991; Peng and Kuc, 1992) that peroxidase plays an
important, early and specific role in the hypersensitive containment of the pathogen. The results
presented in this work demonstrate that the resistant plant responded actively to infection of both FOC
races. The fact that in the compatible interaction, peroxidase activity increases rapidly suggests that in
banana this enzyme might be involved in the defence response. These observations are in agreement
with the model proposed by Beckman (1990), which involves the rapid build-up of mechanical
barriers and with the role of phenolic metabolite infusion as proposed by Strobel and Sinclair (1991).
The speed and magnitude for activation of the defence mechanism appear critical for the expression of
resistance (Reuveni et al., 1992). Moreover, the measurement of constitutive peroxidase activity
shows a positive correlation between high enzymatic activity in non-infected plants and the resistance
to FOC. This fact, jointly with the activation of enzymatic activity in tissues that are not the primary

119
site of infection, is in agreement with the systemic defence mechanism proposed by Hammerschmidt
and Kuc (1982) and by Hammerschmidt et al. (1982). To be effective, a defence mechanism based on
peroxidase activity should be constitutively present or rapidly elicited in response to pathogen
infection. In the case of the race 4 susceptible cultivar ‘Pisang Mas’, this threshold is probably not
reached, while in the resistant ‘SH-3362’ this is already present in uninfected plantlets. On the
contrary, ‘Pisang Mas’ is able to reach the threshold level when infected with the incompatible race 1.
However, this work was done using only two clones and the actual role of peroxidase in the active
containment of Fusarium should be confirmed by a more extensive screening using different clones of
Musa. To conclude, whereas this work shows that peroxidase activity can be used as a parameter to
discriminate between susceptible and tolerant clones of banana, it emphasises the need for more
extensive research on the physiological mechanism that underlines the resistance/susceptibility
response of the banana to FOC.

REFERENCES TO CHAPTER 7

Beckman CH (1987) The Nature of Wilt Diseases of Plants. APS Press, St. Paul, Minnesota, USA,
182 pp.

Beckman CH (1990) Host responses to the pathogen. In: Ploetz RC (ed.) Fusarium Wilt of the Banana.
APS Press, St. Paul, Minnesota, USA, pp 107-114.

Beckman CH, Halmos S, Mace ME (1962) The interaction of host, pathogen and soil temperature in
relation to susceptibility to fusarium wilt of bananas. Phytopathology 52: 134-140.

Beckman CH, Mace ME, Halmos S, McGahan MW (1961) Physical barriers associated with
resistance in fusarium wilt of bananas. Phytopathology 51: 505-515.

Binarova P, Nedelnik J, Fellner M, Nedbalkova B (1990) Selection for resistance to filtrate of

Fusarium spp. in embryogenic cell suspension culture of Medicago sativa L. Plant Cell Tiss Organ
Cult 22: 191-196.

Bradford MM (1976) A rapid and sensitive method for the quantitation of microgram quantities of
protein using the principle of protein-dye binding. Anal Biochem 72: 248-259.

Buiatti M, Bettini P, Mourgo R, Bogani P, Nascari G, Pellegrini GM, Gimelli F, Venturo R (1985)
Correlation between in vivo resistance to Fusarium and in vitro response to fungal elicitors and toxic
substance in carnation. Theor Appl Genet 70: 42-47.

Buiatti M, Ingram D (1991) Phytotoxin as tools in breeding and selection of disease resistant plants.
Experientia 47: 811-819.

Buiatti M, Marcheschi G, Venturo R, Bettini P, Bogani P, Morpurgo R, Nacmias B, Pelligrini MG

(1987) In vitro response to Fusarium elicitor and toxic substances in crosses between resistant and
susceptible Carnation cultivars. Plant Breeding 98: 346-348.

Daub ME (1986) Tissue culture and the selection of resistance to pathogens. Annu Rev Phytopathol
24: 159-186

Durbin RD (1983) Future prospects in toxin research. In: Daly JM, Deverall BJ (eds.) Toxins and
Plant Pathogenesis. Academic Press, Sydney, Australia, pp 159-177.

Epp MD (1987) Somaclonal variation in bananas: a case study with Fusarium wilt. In: Persley GJ, De
Langhe EA (eds.) Banana and Plantain Breeding Strategies. ACIR Proceedings No. 21, Canberra,
Australia, pp 140-150.

120
FAO (1991) Trade Yearbook 1990, FAO Statistics Series. Food and Agriculture Organization, Rome,
Italy, 377 pp.

Gamborg OL, Miller RA, Ojima K (1968) Nutrient requirements of suspension cultures of soybean
root cell. Exp Cell Res 50: 473-497.

Garraway MO, Akhtar M, Wokoma ECW (1989) Effect of high temperature on peroxidase activity
and electrolyte leakage in maize in relation to sporulation of Bipolaris maydis race T. Phytopathology
79: 800-805.

Graham MY, Graham TL (1991) Rapid accumulation of anionic peroxidase and phenolic polymers in
Soybean cotyledon tissues following treatment with Phytophthora megasperma f. sp. glycinea wall
glucan. Plant Physiol 97: 1445-1455.

Hammerschmidt R, Kuc J (1982) Lignification as a mechanism for induced systemic resistance in

cucumber. Physiol Plant Pathol 20: 61-71.

Hammerschmidt R, Nucklers E, Kuc J (1982) Association of enhanced peroxidase activity with

induced systemic resistance of cucumber to Colletotrichum lagenarium. Physiol Plant Pathol 20: 73-
82.

Hwang SC, Ko WH (1987) Somaclonal variation and banana screening for resistance to Fusarium
wilt. In: Persley GJ, De Langhe EA (eds.) Banana and Plantain Breeding Strategies. ACIR
Proceedings No. 21, Canberra, Australia, pp 182-185.

Marois JJ (1990) Biological control of diseases caused by Fusarium oxysporum. In: Ploetz RC (ed.)
Fusarium Wilt of Banana. APS Press, St. Paul, Minnesota, USA, pp 77-82.

Murashige T, Skoog R (1962) A revised medium for rapid growth and bioassay with tobacco tissue
culture. Plant Physiol 15: 473-497.

Novak FJ (1992) Musa (bananas and plantains). In: Hammerschlag FA, Litz RE (eds.) Biotechnology
of Perennial Fruit Crops. CAB International, Wallingford, UK, pp 449-487.

Novak FJ, Donini B, Hermelin T, Micke A (1987) Potential for banana and plantain improvement
through in vitro mutation breeding. ACORBAT, Memorias VII Reunion CATIE, Costa Rica, pp 67-
70.

Peng M, Kuc J (1992) Peroxidase generated hydrogen peroxide as a source of antifungal activity in
vitro and on tobacco leaf disks. Phytopathology 82: 696-699.

Reuveni R, Shimoni M, Karchi Z, Kuc J (1992) Peroxidase activity as a biochemical marker for
resistance of Muskmelon (Cucumis melo) to Pseudoperonospora cubensis. Phytopathology 82: 749-
753.

Robb J, Lee SW, Mohan R, Kolattukudy PE (1991) Chemical characterization of stress induced
vascular coating in tomato. Plant Physiol 97: 528-536.

Rowe PR (1991) Breeding bananas and plantains for resistance to Fusarium wilt: the track record. In:
Ploetz RC (ed.) Fusarium Wilt of Banana. APS Press, St. Paul, USA, pp 115-120.

Seevers PM, Daly JM, Catedral FF (1971) The role of peroxidase isozyme in resistance to wheat stem
rust disease. Plant Physiol 48: 353-360.

121
Stover RH (1990) Fusarium wilt of banana: some history and current status of the disease. In: Ploetz
RC (ed.) Fusarium Wilt of Banana. APS Press, St. Paul, Minnesota, USA, pp 1-8.

Strobel NE, Sinclair WA (1991) Role of flavanolic wall infusion in the resistance induced by Laccaria
bicolor to Fusarium oxysporum in primary roots of Douglas-fir. Phytopathology 81: 420-425.

Ye SZ, Pan OS, Kuc J (1990) Activity, isozyme pattern and cellular localization of peroxidase as
related to systemic resistance of tobacco to blue mold (Peronospora tabacina) and to Tobacco Mosaic
Virus. Phytopathology 80: 1295-1299.

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 Chapter 8

Differential chitinase activity in banana cultivars as a response to Fusarium

oxysporum f. sp. cubense infection

R. Morpurgo, M. van Duren, G. Grasso and R. Afza*

Joint FAO/IAEA Programme, Agriculture and Biotechnology Laboratory,

International Atomic Energy Agency,
Seibersdorf, Austria.

*Correspondence: [email protected]

Abstract

Six banana clones with varying levels of resistance were inoculated with conidial suspension of races
1 and 4 of Fusarium oxysporum f. sp. cubense (FOC). Chitinase activity in the corm and root tissues
was monitored before and after infection to relate with the field resistance or susceptibility of banana
cultivars. Resistant clones showed high constitutive chitinase activity in roots and a rapid response to
infection. The results suggest that chitinase could be considered as part of a complex mechanism
leading to disease resistance.

INTRODUCTION

Bananas (Musa spp.) are important crops, providing carbohydrate-rich food to large populations in the
tropics and subtropics. In South and Central America, bananas are also important export crops.
Protection against fungal diseases represents a major challenge to banana production. In many areas of
the world, losses from diseases reduce income and pose a threat to the economic survival of small-
holding farmers. At present, disease control is based mainly on three strategies: application of
pesticides, breeding for disease resistance and improvement of agronomic practices. However,
chemical control of Fusarium wilt is not yet available. Banana breeding is time consuming, expensive,
and constrained by the sterility of most cultivated bananas. Thus, a better understanding of the
resistance mechanisms is required to develop a reliable early screening method.

Like many other species, bananas employ a diverse array of defence mechanisms to resist pathogen
attack. For example, callose deposition and phenolic compounds have been associated with resistance
to Fusarium oxysporum f. sp. cubense (FOC) (Wardlaw, 1930; Beckman, 1990), but little is known
about the physiology of the defence response.

Chitinases hydrolyse chitin, a homopolymer of 6-1-4 linked N-acetyl-D-glucosamine, which is a major

component of the cell wall of most fungi. Chitinases have been isolated and purified from bacteria,
fungi and plants, and are able to inhibit fungal growth by degrading the newly synthesised chitin at the
hyphal tips. Chitinases are synthesised in low amount in many plants species and crops. Most evidence
for the role of chitinases in the plant defence response is based on data from in vitro systems or from
complementation tests involving plant transformation and scoring of resistance. Purified enzymes have
been shown to hydrolyse isolated fungal cell walls and inhibit the growth of pathogen in culture.

The present investigations were undertaken to study the chitinase response of bananas clones with
different ploidy levels and resistance to F. oxysporum. Previous studies showed that in several banana
clones, chitinase was expressed constitutively. In this paper, we report evidence on the induction of
chitinase following infection of banana plants with FOC conidial culture.

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MATERIAL AND METHODS

Plants, pathogen and inoculation

FOC race 1 strain 2264 (VCG 0125) and race 4 strain Yo 604 (VCG 01290) were kindly provided by
Dr. K. Pegg, Queensland Department of Primary Industries (QDPI), Indooroopilly, Australia. The
strains were cultivated on Czapek-Dox medium (Difco, Becton Dickinson, Sparks, MD, USA) for one
week. Actively growing hyphae were removed, transferred to Potato Dextrose Agar (PDA), and
incubated at 28°C under constant light to promote conidia formation. Conidial suspensions in distilled
water were collected by scratching the mycelial surface.

The following banana clones which differ in genome, ploidy, and susceptibility to FOC race 1 and 4
were investigated: ‘SH-3362’ (AA) resistant to race 1 and 4; ‘Grand Naine’ (AAA), ‘Pisang Mas’
(AA), ‘SH-3142’ (AA), ‘Dwarf Parfitt’ (AAA), susceptible to race 4 and resistant to race 1,
‘Highgate’ (AAA) susceptible to both races. Plants were multiplied in vitro and rooted as described
elsewhere (Novák et al., 1989). After one month, rooted plantlets were obtained and agar was washed
off with tap water, the roots were trimmed and the plants were inoculated by dipping the roots in spore
suspensions containing 105 conidia/ml for 10 minutes under air flow to facilitate the mechanical
uptake of conidia by root vessels. The controls were treated in the same manner but inoculated only
with distilled water. Immediately after inoculation, the plants were transferred to pots in a greenhouse
and samples were taken at intervals for chitinase activity.

Chitinase analysis

Plantlets were sampled at random and the corm tissue collected under cold water by trimming the
roots and the pseudostem. The tissue was weighed and ground with a mortar and pestle in liquid
nitrogen. Phosphate buffer pH6.8 was added to the resulting powder, and after homogenisation
incubated for 30 minutes at 4°C. The homogenate was collected with a micropipette, centrifuged for
15 minutes at 14000 rpm at 4°C, and the supernatant was assayed immediately for protein content
according to the method of Bradford (Bradford, 1976). Extracts with equal amount of protein were
incubated in 100 ml of glycol chitin, and variable amounts of distilled water were added to bring the
incubation mixture to 200 ml and incubated overnight at 40°C. The final reaction mixture was brought
to 1 ml and read at 510 nm using a spectrophotometer, and expressed as unit protein content.

Isoelectrofocusing (IEF)

Equal amounts of protein were applied on a 4.6% polyacrylamide gel containing 5% Pharmalyte as a
carrier ampholyte. Running conditions were as follows: 8 W fixed, 12-33 mA, 2500 V. After running,
the gel was washed, and the bands were differentiated using an overlay gel (7.5%) with 1.1 ml of
3 M NaAc buffer pH5 and 1.3 ml glycol chitin in a final volume of 33 ml.

RESULTS AND DISCUSSION

No wilting symptoms were observed in the inoculated material up to 28 days of culture. However,
internal symptoms, i.e., tissue discolouration, were observed on dissection of the susceptible clones
inoculated with both FOC races. The clones were analysed for constitutive chitinase activity before
fungal inoculation. No differences were observed between susceptible and resistant clones in
constitutive chitinase.

After inoculation with a conidial suspension of FOC race 1, all clones except ‘Highgate’ showed
increased chitinase activity but with a different time course (Figure 8.1). The clone ‘Dwarf Parfitt’
showed a rapid and sustained increase in enzymatic activity during the first two weeks, while in
‘Highgate’ chitinase did not increase. The clone ‘Pisang Mas’ exhibited a slow increase in enzymatic

130
activity with time. When the same banana clones were incubated with FOC race 4, clone ‘SH-3142’
showed a higher increase in chitinase activity (Figure 8.2). An increase in chitinase activity was
particularly evident in ‘Pisang Mas’, with a peak activity after five weeks. Clone ‘SH-3142’ showed a
different pattern of enzymatic activity, with a burst induction after 48 hours, followed by another peak
5 weeks after infection (Figures 8.3 and 8.4).

The resistant clone ‘SH3142’, infected with race 1 and 4 showed root chitinase activity comparable to
corm tissue (Figures 8.5 and 8.6). However, in plants inoculated with race 4, there was a three- to
four-fold increase when compared with plants inoculated with race 1. Moreover, root chitinase activity
was already evident 24 hours after infection, while in corm tissue the activity increase was evident
after 48 hours (Figures 8.7 and 8.8). In ‘Pisang Mas’, the enzymatic activity increased both after
infection with race 1 and 4 but in a transient manner, i.e., after 48 hours when infected with race 1 and
after 5 weeks when inoculated with race 4; however, the increase was not statistically significant. As
far as the response to race 1 is concerned, a four-fold increase over the control was observed but only
at a single point. In the highly susceptible clone ‘Highgate’, the chitinase activity did not increase at
all. Clone ‘SH-3142’ showed a more prompt response than the other clones when inoculated with
race 4.

At least 12 isochitinases have been recorded in diploid bananas (Morpurgo et al., 1994). To determine
which isozymes contribute to the rise in total activity, IEF was used to separate the isochitinases
extracted from the banana clones. At least four bands, two cationic and two anionic, concomitantly
increased in the incompatible reaction between race 4 and ‘SH-3362’. However, this increase was not
observed in the incompatible reaction of the same clone with race 1. The activity of most of the other
isozymes remained unchanged with time (data not shown).

The current model on a chitinase-based defence mechanism considers that the enzyme acts as an anti-
fungal compound as well as a producer of chitin fragments, which act as elicitors or messengers to
activate other metabolic compounds for defence against the pathogen. The results presented in this
paper confirm that the resistant clones of banana respond actively to infection (Morpurgo et al., 1994)
with a dramatic increase in the chitinase activity, but this response is more tissue-specific than the
previously studied peroxidase. However, total chitinase activity by itself should not be considered an
absolute parameter. The rate of production appears to be more important, i.e., the activation response
of the host to the infection. The speed and magnitude of the activation of the mechanism appear to be
critical for expression of resistance.

Resistance of plants to invasion by potential pathogens is the result of a multiple defence reaction
comprising both constitutive and inducible mechanisms. The use of biochemical markers to select for
resistance may expedite breeding programmes by reducing the number of field trials. The main criteria
for practical use of such markers are reliability in predicting resistance and the ease of handling the
assay. This would have a comparative advantage over traditional field screening. For any marker, a
number of prerequisites must be fulfilled. Firstly, it should positively correlate with the level of
resistance or susceptibility of the breeding population. Secondly, it should be able to distinguish
between F2 segregants or between individuals among a mutagenised population. Thirdly, it should be
applicable to a large breeding population.

The results of this study showed that the plant reaction to pathogens can be estimated by the inducible
chitinase activity during the time course of active chitinase production after infection. However, to
follow the enzymatic activity over a long time involves intensive work and careful control of
environmental conditions. Thus, predicting plant response on the basis of inducible chitinase becomes
a long procedure.

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CONCLUSIONS

Based on the present study, it was possible to divide the experimental population into two broad
classes of resistant and susceptible sub-populations. On the basis of chitinase activity in root tissues, it
would be possible to identify suitable parents for breeding for resistance to Fusarium. The differential
response based on chitinase activity confirms our previous results (Morpurgo et al., 1994) obtained
with peroxidases, and suggests that such an approach could be useful in screening for variation among
the cultivars, wild species and sub-species to identify sources of resistance.

REFERENCES TO CHAPTER 8

Beckman CH (1990) Host response to the pathogen. In: Ploetz RC (ed.) Fusarium Wilt of the Banana.
APS Press, St. Paul, Minnesota, USA, pp 93-105.

Bradford MM (1976) A rapid and sensitive method for the quantification of microgram quantities of
protein using the principle of protein-dye binding. Anal Biochem 72: 248-259.

Morpurgo R, Lopato SV, Afza R, Novák FJ (1994) Selection parameters for resistance to Fusarium
oxysporum f. sp. cubense race 1 and race 4 of diploid banana (Musa acuminata Colla) Euphytica 75:
121-129.

Novák FJ, Afza R, van Duren M, Perea-Dallos M, Conger BV, Tang X (1989) Somatic embryogenesis
and plant regeneration in suspension cultures of dessert (AA and AAA) and cooking (ABB) bananas
(Musa spp.). Bio-Technology 7: 154-159.

Wardlaw CW (1930) The biology of banana wilt (Panama disease). III. An examination of sucker
infection through root bases. Ann Bot 45: 381-399.

132
 1800 1400
Highgate Highgate
1600
SH3142 1200 SH3142

% increase over control

% increase over control

1400
Pisang Mass 1000 Pisang Mass
1200
Dwarf Parfitt Dwarf Parfitt
1000 800

800 600
600
400
400
200
200

0 0
1 2 21 35 1 2 21 35
Days after inoculation (dai) Days after inoculation (dai)

Figure 8.1. Differential response of banana Figure 8.2. Differential response of banana
clones to inoculation with Fusarium clones to inoculation with Fusarium oxysporum
oxysporum f. sp. cubense race 1 measured as f. sp. cubense race 4 measured as chitinase
chitinase activity (A470) increase over the activity (A470) increase over the control.
control. Relative activity is described in Relative activity is described in terms of the
terms of the change of absorbance per minute change of absorbance per minute per 5 μ1 of
per 5 µl of extract. Each point represents the extract. Each point represents the average of
average of three replicates and two three replicates and two independent
independent experiments. experiments.

600 700
Resistant Resistant
Susceptible 600 Susceptible
500
% increase over control
% increase over control

500
400
400
300
300
200
200

100 100

0 0
1 2 21 35 1 2 21 35
Days after inoculation (dai) Days after inoculation (dai)

Figure 8.3. Chitinase activity in corm tissue Figure 8.4. Chitinase activity in corm tissue of
of susceptible and resistant bananas after susceptible and resistant bananas after infection
infection with FOC race 1. with FOC race 4.

133
 140 250
Highgate Highgate

120 SH3142 SH3142

% increase over control

% increase over control

200
100 Pisang Mass Pisang Mass

Dwarf Parfitt 150 Dwarf Parfitt

80

60
100

40
50
20

0 0
1 2 21 35 1 2 21 35
Days after inoculation (dai) Days after inoculation (dai)

Figure 8.5. Chitinase activity in root tissue of

Figure 8.6. Chitinase activity in root tissue of
‘Highgate’, ‘SH-3142’, ‘Pisang Mas’ and
‘Highgate’, ‘SH-3142’, ‘Pisang Mas’ and
‘Dwarf Parfitt’ bananas after infection with
‘Dwarf Parfitt’ bananas after infection with
FOC race 1.
FOC race 4.

70 250
Resistant Resistant

60 Susceptible Susceptible
200
% increase over control

% increase over control

50

150
40

30
100

20
50
10

0 0
1 2 21 35 1 2 21 35
Days after inoculation (dai) Days after inoculation (dai)

Figure 8.7. Chitinase activity in root tissue of Figure 8.8. Chitinase activity in root tissue of
susceptible and resistant bananas after infection susceptible and resistant bananas after infection
with FOC race 1. with FOC race 4.

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 Chapter 9

Mass-screening of mutants resistant to Alternaria blotch from in vitro-

cultured apple shoots irradiated with X-rays

M. Suzuki* and A. Saito

Aomori Green BioCenter,

Aomori
Japan.

*Correspondence: [email protected]

Abstract

We have established mass screening methods for producing mutants resistant to Alternaria blotch
disease in several cultivars of apple (Malus domestica) by irradiation with X-rays. An in vitro assay
system using chemically-synthesised AM-toxin I of Alternaria alternata (Fr.) Keissler is successful for
screening the resistant mutants. The degree of resistance to the disease was assayed by the number of
necrotic lesions induced by applying various concentrations of AM-toxin I to leaf disks of the first,
third and fifth leaves from the shoot apex of plants. Following the establishment of the optimal
screening conditions, we produced several disease-resistant mutants by irradiating in vitro shoots with
various doses of X-rays. After resistant mutants were selected by the first and second screening tests
with AM-toxin I, they were tested by spraying them with a spore suspension of the pathogen. The
resistance to the disease was maintained for five years during field cultivation of the mutants, implying
that the resistance was not due to environmental factors but due to genetic fixation. No differences
were observed in the number of fruiting trees or colouration of fruits between mutants and the original
plants.

INTRODUCTION

Inducing mutation by exposure to radiation is one of the most significant tools in breeding, especially
for vegetatively propagated crops and perennial orchard trees. When breeding of a tree is performed
via crossing, a long time is needed to eliminate unnecessary traits by back-crossing. Compared with
crossing, only a few traits are usually affected by breeding through mutation induction. Many cultivars
of orchard trees have been produced by natural mutation. Most of these are derived from mutants of
bud mutation (Shamel, 1943; Soost and Cameron, 1975). Since Stadler (1930) utilised X-rays and
radium irradiation for inducing artificial mutations, irradiation of seeds or dormant scions has been
commonly attempted for producing useful mutants. When the genetic background of the traits is clear,
the efficiency of mutation breeding by irradiation is enhanced. Traits controlled by polygenes are not
easily altered, but traits controlled by a single gene are good candidates for genetic improvement. In
orchard trees, disease resistance against scab and rust in apples is known to be controlled by a single
dominant gene; whilst, canker in peach, black spot in pears and Alternaria blotch in apples are each
controlled by a single recessive gene.

As irradiation with gamma- or X-rays usually induces recessive mutations, mutation breeding with
irradiation is effective for diseases controlled by recessive genes. One such example is the breeding of
the Japanese pear (Pyrus pyrifolia Nakai) cv. ‘Nijisseiki’ resistant to black spot disease, which was
first carried out by chronic irradiation with gamma-rays (Sanada et al., 1988). Black spot disease is
caused by the Japanese pear pathotype of A. alternata (Fr.) Keissler (Kozaki, 1973), and homozygous

135
recessive genes result in resistance to the disease. Masuda et al. (1997) also produced mutants of the
Japanese pear cultivar ‘Osanijisseiki’ that were resistant to this disease.

In apples, attempts were also made to select mutants resistant to Alternaria blotch in the susceptible
cultivar ‘Indo’ (Tabira et al., 1993a; Sanada et al., 1994; Masuda and Yoshioka, 1996). Alternaria
blotch is a very serious disease of apple in Japan caused by the apple pathotype of A. alternata (Fr.)
Keissler (previously described as A. mali Roberts). Upon infection by this fungus, necrotic lesions
appear on leaves followed by defoliation as the disease progresses, resulting in a decreased fruit yield.
The disease can usually be controlled by spraying the apple trees with fungicides many times during
the growing season. However, controlling the disease is difficult if the primary leaf infections are
already abundant by the time of the first spraying. Furthermore, spraying many times is laborious,
represents a health hazard to farmers and is detrimental to the environment. Saito and Takeda (1984)
found that susceptibility to the disease is controlled by a single dominant gene; susceptible cultivars
are heterozygous and resistant cultivars are homozygous recessive. This mode of inheritance is similar
to that of black spot disease resistance in Japanese pear.

It is known that host-specific toxins, which are produced by the pathogen at the time of spore
formation, cause damage to the plant cytoplast and chloroplasts (Park et al., 1981). These toxins can
be purified (Ueno et al., 1975) and classified into three groups, namely AM-toxin I, II and III. In order
to evaluate the degree of plant susceptibility to Alternaria blotch, filtrates of fungal cultures or crude
toxins partially purified from filtrates have been previously used. However, these are sometimes
troublesome since the titre of the toxins or filtrates may vary among cultures. In addition, intermediate
types of resistance are not easily distinguished when using a culture filtrate or crude toxin (Tabira et
al., 1993a). Thus, to eliminate the variability of filtrates, we decided to use a chemically-synthesised
toxin (AM-toxin I).

The classification of cultivars according to degree of susceptibility to a pathogenic toxin is directly

correlated to the actual degree of susceptibility to the live pathogen in plants grown outside (Tabira et
al., 1993a; Masuda, 1995). Therefore, we usually use in vitro shoots as testing material, because these
can be prepared throughout the year and are easy to handle as compared with shoots grown outside.
When the screening is first carried out with in vitro-grown shoots, the correlation between susceptible
and intermediate resistant cultivars may not be as clearly observed as on in vivo material. It is,
therefore, important to establish an assay method for determining the degree of susceptibility when
using in vitro-grown shoots for the selection of resistant mutants.

Until recently, acute or chronic gamma-ray irradiation was generally used for inducing mutation as
was done for Japanese pear. However, in our experience, gamma-ray irradiation may not always be the
best choice. For instance, gamma-irradiation requires facilities that may not be available; in Japan, for
example, gamma-ray irradiation facilities are only available at the Institute of Radiation Breeding in
the National Institute for Agrobiological Resources (NIAR) under the Ministry of Agriculture,
Forestry and Fisheries (MAFF). Moreover, in acute gamma-ray irradiation, in addition to the desired
mutations, the frequency of unfavourable mutations may be much higher than that of chronic gamma-
ray irradiation. On the other hand, for the induction of mutations, chronic gamma-ray irradiation
requires a longer period of time than acute gamma-ray irradiation. Moreover, in the case of a gamma-
field such as the one used in Japan, in order to obtain mutants exhibiting useful traits, it is important to
produce mutants on a mass scale, which requires a large area for selection of the mutants with
desirable traits after irradiation. Therefore, we chose a more convenient system, and used X-ray
irradiation as an alternative tool to gamma-ray irradiation. X-ray facilities are small and the irradiation
can be performed in small laboratories and can be carried out on a smaller scale.

As for the irradiation doses, the sensitivity to irradiation varies between different kinds of fruit trees.
Generally, persimmons and chestnuts show high sensitivity, whereas citrus fruits and loquats show
high resistance (Shamel and Pomeroy, 1936). Some traits, such as skin colour and fruit size, tend to be
affected more easily by irradiation, whereas fruit shape and ripening time are more recalcitrant to

136
mutation induction (Shamel and Pomeroy, 1936). In apples, the sensitivity also differs between
cultivars; therefore, we first had to establish the optimal dose and conditions for irradiation for each
cultivar. As a comparison to X-ray irradiation, we also performed gamma-ray irradiation of shoots
from several cultivars. In parallel, we established an optimal screening method for Alternaria blotch
disease (Saito et al., 2001). After optimisation of both irradiation conditions and screening protocol,
we produced apple mutants resistant to Alternaria blotch disease from several susceptible cultivars
through a highly efficient regeneration procedure for apple shoots (Saito and Suzuki, 1999).

MATERIAL AND METHODS

Irradiation of shoots with various doses

In vitro-grown apical shoots were cut into 5 mm pieces and sub-cultured at intervals on shoot
proliferation medium (Murashige and Skoog [MS] medium containing 1.0 mg/l 6-benzylaminopurine
[BAP], 30 g/l sucrose and 0.8% agar, pH5.8) (Murashige and Skoog, 1962). 10 shoots were inoculated
onto a fresh proliferation medium 30 days after subculture. These shoots were irradiated with various
doses of X-ray in a soft X-ray irradiation unit (Soft X-ray unit, OHMICRON OM-100RAL, Ohmic
Ltd., Tokyo, Japan). Shoots of cv. ‘Hokuto’ were irradiated with doses of 5, 6, 8, 10, 12, 15 and 20 KR
at a dose rate of 0.01, 0.1, 0.5 and 1 KR/min, whereas, shoots of cv. ‘Aori 10’ were irradiated with the
same dose at 0.01, 0.1 and 1 KR/min.

Concerning gamma-ray irradiation, irradiation with doses of 60, 80 or 120 Gy at a dose rate of 5
Gy/hour was carried out in the gamma-room of the Institute of Radiation Breeding, NIAR, MAFF.
After irradiation, Petri dishes containing shoots were cultured at 25°C with a 16:8 hour light:dark
photoperiod. The numbers of surviving shoots and morphological properties were investigated 30 days
after irradiation.

Method of assaying susceptibility to pathogenic toxins on leaf disks and apical shoots with AM-
toxin

The degree of susceptibility to AM-toxin was evaluated for several apple cultivars including resistant,
intermediately resistant and susceptible cultivars. 10 leaves of the first, third and fifth leaves from the
top shoot were collected, respectively. Leaves were washed first with detergent and then with tap
water. After drying off the water, disks (diameter: 8 mm) were cut out with a cork-borer from the
central parts of the leaf.

Chemically-synthesised AM-toxin I (Hashimoto et al., 1996) was dissolved in 1 ml dimethylsulfoxide

(DMSO), suspended in sterile water and stocked at 100 μM. This solution was sequentially diluted to
10, 1 and 0.1 μM, and sterilised by filtration through a 0.45 μM membrane filter. The toxin solution
was diluted and 0.5 ml was dispensed into 24 hole-titre plates. Leaf disks were placed into each
solution and immersed for 48 hours in darkness at 28ºC. Thereafter, the degree of necrotic lesions was
evaluated.

The first screening test of susceptibility in irradiated shoots

After irradiation, shoots of the surviving plants were allowed to elongate, and each shoot was divided
into 10 segments and transferred onto a proliferation medium. Multiple shoots were produced one
month after culture. These shoots were each separated into 10 pieces, and sub-cultured onto a fresh
medium. This procedure was repeated 3 times to propagate shoots and eliminate chimeras. Finally, 10
shoots (~2 cm long) were cut off and transferred onto a rooting medium (MS medium containing 1.0
mg/l 3-indolebutyric acid [IBA], 30 g/l sucrose and 0.8% agar, pH5.8).

Rooted plantlets were produced after ~1 month, and were then acclimatised and transferred to pots
containing soil (Prime Mix TKS-2, Sakata Seed Ltd., Yokohama, Japan) after eliminating agar

137
attached to roots with tap water. Pots were covered with polystyrene bags to maintain humidity and
plants were cultivated in the shade for two weeks. After new leaves developed, the polystyrene bags
were removed to acclimatise plants to the open air; the plants were cultivated in a sunny place for ~1
month.

Thereafter, these plants (~15 cm) were screened with toxin to evaluate the degree of susceptibility. The
third leaf of each shoot was cut into 8 mm diameter disks with a cork borer. Two disks from each leaf
were immersed with the toxin solution and incubated at 28ºC in darkness for two days, and the
occurrence of necrotic lesions observed.

The second and third screening tests in selected resistant mutants

Resistant mutants selected by the first screening test were grown in a greenhouse and the third leaves
from the tops of shoots of elongated plants (~30 to 40 cm) were collected. Leaf disks (diameter: 8 mm)
from the central region of these leaves were cut out with a cork borer. To re-evaluate the degree of
resistance, the leaf disks were immersed in the high concentration (100 μM) of toxin solution used for
the titre plates, and incubated for two days at 28ºC in darkness. Highly resistant plants showing no
necrosis were selected.

After selection by the second screening test, the third screening by spores of the pathogen was carried
out. Pathogens (A. alternata strain FIV-C-45) were cultured on solid potato dextrose agar medium
(Difco, MD USA) for 5-7 days. Proliferated pathogens were collected with a brush and suspended in
small amounts of distilled water. The suspension was then filtered through gauze to eliminate cell
debris and agar. The suspension containing spores of the pathogen was adjusted to 3.3 × 104/ml with
distilled water. Spore suspension was then sprayed onto the leaves of selected mutant plants. Leaves
inoculated with pathogen were kept for two days at 25ºC in the inoculation box, in which humidity
was maintained at 90-95%. Thereafter, plants were transferred to a greenhouse, and disease symptoms
and number of necrotic lesions were investigated for the upper 10 leaves from the top shoots seven
days after inoculation. The procedure of producing mutants resistant to Alternaria blotch is illustrated
in Figure 9.1.

Establishment of an assay method for evaluating the degree of susceptibility

The degree of susceptibility to AM-toxin I was different among cultivars (Figure 9.2A). A resistant
cultivar (‘Jonathan’), an intermediate cultivar (‘Fuji’), a susceptible cultivar (‘Oorin’) and a highly
susceptible cultivar (‘Indo’) were used as standard material for evaluating the degree of susceptibility
(Tables 9.1 and 9.2). Samples were classified into four groups by the degree of necrosis as follows:
grade 0 = no necrotic lesions; grade 1 = necrotic lesions on the leaf disk periphery; grade 2 = necrotic
lesions on <50% of the leaf disk; grade 3 = necrotic lesions on 50-75% of the leaf disk; grade 4 =
necrotic lesions on 75-100% of the leaf disk. Thereafter, the average grades were calculated based on
the degree of necrotic lesions on each leaf. The results of the screening with AM-toxin were mostly
consistent with those obtained using the culture filtrate (Saito et al., 1989) and the grade of necrotic
lesions of leaf disks properly corresponded to the degree of susceptibility as previously ascertained
from field tests, when applying toxin concentrations of
0.1-1 μM. The degree of susceptibility for the resistant cultivar (‘Jonathan’) was from grade 0 to 0.5,
for the intermediate cultivar (‘Fuji’) from grade 0.5 to 3.5, and for the susceptible cultivars (‘Oorin’
and ‘Indo’) from grades 2.0 to 4.0. Thus, the degree of susceptibility to Alternaria blotch was
successfully evaluated in vitro by measuring necrotic lesions of leaf disks exposed to chemically-
synthesised toxin.

For the screening test, another important factor was the age of the leaves. Young leaves were more
susceptible to toxin or fungal filtrate than older leaves (Figure 9.2B) (Sawamura and Yanase, 1963;
Saito and Takeda, 1984). The first leaves from the top of the shoot were too susceptible to the toxin,

138
whereas the upper 3rd and 5th leaves from the top of the shoots were appropriate Material for the
evaluation of the degree of susceptibility. Therefore, the upper 3rd leaves were usually used for the
evaluation with toxin at the concentrations of 1 and 10 μM.

Survival of irradiated shoots with various dose rates of irradiation

Cultivars ‘Hokuto’ and ‘Aori 10’ were mainly used as material for production of resistant mutants.
They varied in their percentage survival for various dose rates of irradiation (Figure 9.3). Up to 12 KR,
no plant died at a dose rate of 0.01 KR/min irradiation. However, the survival rate decreased gradually
as the dose rate increased above 0.1 KR/min. In cv. ‘Hokuto’, no plant died at a dose rate of 0.01
KR/min and at doses of up to 12 KR, whereas the survival rate declined to 50% at a radiation dose of
13 KR with a dose rate of 0.1 KR/min (Figure 9.3A). In cv. ‘Aori 10’, no plant died at the dose rate of
0.01 KR/min, whereas only 50% survived a radiation dose of 6 KR with a dose rate of 0.1 KR/min
(Figure 9.3B). This means that the extent of irradiation damage varied between the two cultivars. The
survival rate was always higher in cv. ‘Hokuto’ than in cv. ‘Aori 10’ (Figure 9.3C). Under conditions
of high irradiation doses, many of the surviving plants showed rosette-type shoots or abnormal leaf
morphology (Figure 9.4D). Survival rate increased when chronic radiation was given at low dose rates
compared with those at high dose rates for the same radiation dose. The survival rate also decreased
with an increase in total dose.

Efficient radiation dose and dose rate for production of mutants

Leaf disks of cultivars ‘Hokuto’ and ‘Aori 10’ were screened with the toxin and the corresponding
resistant shoots were selected. Subsequently, shoots were elongated and rooted, resulting in the
formation of plantlets. In cv. ‘Hokuto’, non-irradiated plants showed severe necrotic lesions (>grade 3)
at a toxin concentration of 10 μM (Table 9.3). At radiation doses < 4 KR, almost all of the irradiated
plants showed the same number of necrotic lesions as non-irradiated plants. A radiation dose of 4 KR
was, thus, insufficient for the production of resistant mutants. The number of necrotic lesions
decreased on several plants irradiated with a radiation dose of 6 KR and some of them showed no
necrotic lesions, the same as for the resistant cultivar ‘Jonathan’, which was graded as 0. This implies
that irradiation with a 6 KR dose was efficient for mutant production.

Thus, the first screening of resistant mutants was carried out in plants irradiated with radiation doses
between 6 and 12 KR. During the first screening, plants whose leaf disks remained green in solution
containing 10 μM of the toxin (Figure 9.5A) were selected for the second screening test. Leaf disks
were prepared from the third leaves from the top of the shoots of juvenile plants selected in the first
screening. These leaf disks were screened by immersion in a solution containing 100 μM toxin (Figure
9.5B). Highly resistant mutants, showing grades 0 or 1, were obtained from plants irradiated with 8
and 10 KR. These plants were more resistant than the resistant cultivar, ‘Jonathan’.

Besides the susceptible cultivars ‘Hokuto’ and ‘Aori 10’, other susceptible cultivars, namely ‘Fuji’ and
‘Oorin’, were also used as Material for the production of resistant mutants (Table 9.4). In the first
screening of cv. ‘Fuji’, the frequency of occurrence of resistant mutants did not differ greatly in
irradiated plants at radiation doses between 8 and 10 KR. However, more resistant mutants were
obtained in plants irradiated with a 0.1 KR/min dose rate than with a 0.01 KR/min dose rate. Resistant
mutants were also obtained in cv. ‘Oorin’ irradiated with 8 and 10 KR, whereas only a few resistant
plants were obtained with a radiation dose of 6 KR. In the gamma-ray irradiated plants, resistant plants
were also obtained from cultivars ‘Hokuto’ and ‘Oorin’ in the first screening with irradiation doses of
60, 80 and 120 Gy (Table 9.5). No resistant mutants were obtained for cv. ‘Aori 10’ at any irradiation
dose of either X- or gamma-rays.

139
Evaluation of phenotypes of resistant mutants

In the second screening, resistant mutants showing the highly resistant grades of 0 and 1 appeared in
cv. ‘Fuji’ plants irradiated with 6, 8 and 10 KR at a dose rate of 0.1 KR/min, whereas no resistant
mutants were obtained at 0.01 KR/min. As for cv. ‘Oorin’, the same tendency was observed as for cv.
‘Fuji’, although more resistant mutants were obtained compared to cv. ‘Fuji’. After the second
screening selection, the frequency of disease symptoms was tested in resistant mutants by inoculation
with a spore suspension of the pathogenic fungus (Table 9.6). Many of these mutants were more
resistant than the original plants (Figure 9.6), showing a degree of resistance the same as that of the
resistant cultivar, ‘Jonagold’ or ‘Jonathan’, and the highly resistant cultivar ‘Sansa’, which showed
evaluation grades of 0. Four resistant mutants were obtained in cv. ‘Hokuto’ by X-ray irradiation (dose
rate 0.1 KR/min, radiation dose 10 KR); 13 and 20 resistant mutants were obtained by X- and gamma-
ray irradiation, respectively, in cv. ‘Fuji’; and 12 resistant mutants were obtained by X-ray irradiation
in cv. ‘Oorin’. Additionally, several intermediate resistant mutants were obtained from cv. ‘Aori 10’
(data not shown).

In resistant mutants of cv. ‘Hokuto’, the frequency of the occurrence of Alternaria blotch was
investigated for five years to test the variability of disease resistance. Five scions of each of the four
mutants and original plants were grafted onto rootstocks of the dwarf cultivar ‘EM 26’ and cultivated
in the farm. The disease occurrence was greatly repressed in these mutants compared with the original
plant over five years (Table 9.7). No morphological abnormality has so far been observed in the
mutants (Figure 9.7). Moreover, there was no difference in the number of fruiting trees and colouration
of fruits between these mutants and the original plants. As for diseases including ‘stem end cracking’,
‘fruit splitting’ and ‘core rot’, the occurrence was less in the mutants than in the original plants (Table
9.7). The observation that there was no alteration in fruit properties except for disease resistance
coupled to the stability of the disease resistance opens the possibility that these mutants could be
candidates for new cultivars. This study has demonstrated the usefulness of X-ray irradiation, besides
gamma-ray irradiation, in breeding of orchard trees.

Calculating the severity of symptoms

The severity of symptoms was calculated based on the conversion formula below:

Severity of symptoms = Σ(G × n)/N × 6 × 100

Where: G = Disease grade is calculated as follows:

Number of disease spots per leaf

0 1-5 6-10 11-30 31-50 >51 defoliation
Disease Grade (G) 0 1 2 3 4 5 6

n = Number of leaves corresponding to the disease grade (G)

N = Number of leaves used in tests (10 leaves per plant)

RESULTS AND DISCUSSION

In Japanese pears, mutants resistant to Black spot disease have been obtained for cultivars ‘Nijisseiki’
(Sanada, 1986; Kotobuki et al., 1992) and ‘Osanijisseiki’ (Masuda et al., 1997) by chronic gamma-ray
irradiation, and for cultivars ‘Shinsui’ and ‘Osanijisseiki’ (Murata et al., 1994) by acute gamma-ray
irradiation (dose rate 2.5 Gy/hour, total dose 80 Gy). Of 2168 and 2335 dormant shoots of ‘Shinsui’
and ‘Osanijisseiki’, respectively, one resistant mutant each was obtained by acute irradiation. One
resistant mutant was also produced from 671 in vitro-grown shoots of ‘Osanijisseiki’ by acute gamma-
ray irradiation (dose rate 5 Gy/hour, total dose 80 Gy) (Tabira et al., 1993b). In apple, Tabira et al.
(1993a) produced one resistant mutant from 453 in vitro shoots of cv. ‘Indo’ by acute gamma-ray

140
irradiation (dose rate 5 Gy/hour, total dose 120 Gy) and this resistant mutant showed 10000-fold
higher resistance than did the original plants. Using almost the same procedure of acute gamma-ray
irradiation at a dose rate of 5 Gy/hour (total dose 80 Gy), Masuda and Yoshioka (1996) selected one
resistant mutant from 3602 in vitro shoots by screening with a pathogenic toxin. A mutant of
‘Nijisseiki’ resistant to black spot disease was registered as ‘Gold Nijisseiki’ and commercialised.
Subsequently, a self-compatible and resistant cultivar called ‘Gold Osanijisseiki’ was developed and
registered, for which the cultivated area in Tottori prefecture, the major district of its production, in
2005 was up to 999 ha and the yield was 23300 tones. This implies that half of the total production of
‘Nijisseiki’ was converted to cultivars derived from mutants.

In this study, we demonstrated that X-ray irradiation was effective in apple breeding as well as
gamma-ray irradiation. We obtained mutants resistant to Alternaria blotch of the cultivars ‘Hokuto’,
‘Fuji’ and ‘Oorin’ through screenings with the toxin and spore suspension of pathogenic fungus. Five
mutants of 1153 plants were obtained for cv. ‘Hokuto’ at a dose rate of 0.1 KR/min (Table 9.3),
whereas for cv. ‘Fuji’, 27 resistant mutants were selected from 1340 plants by X-ray irradiation (Table
9.4). In the case of gamma-ray irradiation, 33 mutants were obtained from 1017 plants (Table 9.5),
indicating that the frequency of mutation was almost of the same order between X- and gamma-ray
irradiation. For ‘Oorin’, 39 resistant mutants were obtained from 530 plants by X-ray irradiation.
Among them, highly resistant mutants appeared that had the same or higher resistance compared with
the resistant cultivars ‘Jonathan’ and ‘Jonagold’.

The frequency of occurrence of resistant mutants of cv. ‘Oorin’ was on the order of 10-1 in the first
screening, although the general frequency of mutation is 10-3 to 10-4, demonstrating that the
frequencies of this study are higher. Whereas, the frequency of resistant mutants in Japanese pears
produced by acute gamma-ray irradiation is estimated at 10-3, which is almost the same as that of
chronic gamma-ray irradiation (Sanada et al., 1988), the frequency of mutation generation by acute
gamma-ray irradiation is generally suggested to be higher than that by chronic irradiation (Sparrow et
al., 1961). However, irradiation at high dose rates tended to cause chromosomal rearrangements and
chimeras, resulting in the occurrence of inferior traits. In apples, shoot mutations occurred so
frequently that they could have been induced by an intrinsic mutator. A retrotransposon was reported
to be an intrinsic mutator in plants (Hirochika, 1993; Kobayashi et al., 2004). If irradiation induced the
activation of a retrotransposon in apples, these retrotransposons might move into chromosomal breaks
resulting in a mutation.

In the case of resistant mutants of Japanese pear, all the mutants that have been obtained until now are
intermediate resistant types. A possible reason to explain these partially resistant mutants in Japanese
pear is that the L-2 layer of the apical dome of shoots has been genetically changed by irradiation and
became resistant, whereas the L-1 layer remained unchanged and susceptible. The resulting plants
would, therefore, be chimeric having different genetic composition in the L-1 and L-2 cells. These
chimeras could not be separated and were transmitted to progeny. In orchard trees, the operation called
‘cutting back’ has been carried out to eliminate chimeras and produce stable mutants; however, this is
a long-term process. To avoid the formation of chimeras in combination with X- or gamma-ray
irradiation, plant tissue culture techniques are utilised (Daub, 1986; Novak, 1991). In this study, we
obtained no chimeric mutants by using in vitro shoot culture.

Tabira et al. (1998) compared resistant mutants of Alternaria blotch and the original apple cultivar by
2D protein gel electrophoresis. They found that among 1400 proteins, a specific spot of molecular
mass 60 kDa (PI 5.5) was lost in resistant mutants. In their results, 9 susceptible cultivars, excluding
‘Oorin’, had this spot, whereas, it was not present in any of 12 resistant cultivars. This spot seems to
be correlated with the susceptibility, and resistant mutants produced by irradiation were derived from
the loss of this protein. The data presented in this paper showed that the gene responsible for
susceptibility to Alternaria blotch might be lost by gamma-ray irradiation, which resulted in the
production of resistant mutants. In our study, Random Amplification of Polymorphic DNA (RAPD)
markers linked to susceptibility to Alternaria blotch were absent in resistant mutants of irradiated cv.

141
‘Hokuto’ although these markers were found in the original cv. ‘Hokuto’ (Fukasawa-Akada et al.,
1999). In the case that target traits are controlled by a single recessive gene, X-ray irradiation is
demonstrated to be a useful tool in mutation breeding, owing to its property of gene destruction.
However, if aimed traits are controlled by polygenes or a single dominant gene, another approach will
be necessary.

CONCLUSIONS

A simple mass screening technique was developed for the selection of disease resistance to Alternaria
blotch in apples using a small X-ray irradiation unit and in vitro-cultured shoots. The frequency of
mutation was nearly identical to that caused by gamma-ray irradiation. Mutants exhibited stable
disease resistance to Alternaria blotch for five years and no abnormal phenotype was observed,
demonstrating that this technique is effective for the production of induced mutants if the desired traits
are controlled by recessive genes.

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 Table 9.1. Effect of AM-toxin I on leaf disks of cultivars with various degrees of susceptibility to Alternaria blotch

Degree of Leaf Concentration of toxin

Cultivar susceptibility a position 100 μM 10 μM 1 μM 0.1 μM
1 4.0 ± 0.00*a 4.0 ± 0.00a 4.0 ± 0.00a 4.0 ± 0.00a
Highly
‘Indo’ susceptible 3 4.0 ± 0.00a 4.0 ± 0.00a 4.0 ± 0.00a 3.4 ± 0.48b
5 4.0 ± 0.00a 4.0 ± 0.00a 4.0 ± 0.00a 3.2 ± 0.32b
1 4.0 ± 0.00a 4.0 ± 0.00a 4.0 ± 0.00 a 3.6 ± 0.48a
‘Oorin’ Susceptible 3 4.0 ± 0.00a 4.0 ± 0.00a 3.6 ± 0.48a 2.7 ± 0.42d
5 3.8 ± 0.32a 3.8 ± 0.32a 3.2 ± 0.48b 2.0 ± 0.20e
1 4.0 ± 0.00a 4.0 ± 0.00a 3.5 ± 0.50b 3.3 ± 0.42b
‘Fuji’ Intermediate 3 4.0 ± 0.00a 3.4 ± 0.48b 2.6 ± 0.48d 1.2 ± 0.32g
5 3.2 ± 0.32b 2.1 ± 0.18e 1.1 ± 0.36g 0.5 ± 0.50i
1 4.0 ± 0.00a 3.2 ± 0.64b 0.5 ± 0.50i 0.0 ± 0.00j
‘Jonathan’ Resistant 3 2.8 ± 0.48c 0.4 ± 0.48i 0.0 ± 0.00j 0.0 ± 0.00j
5 2.1 ± 0.36e 0.2 ± 0.32i 0.0 ± 0.00j 0.0 ± 0.00j
a
The grade of susceptibility is calculated based on the occurrence of necrotic lesions on leaf disks as follows: 0 = no necrotic lesion; 1 = a few necrotic lesions
at the cut end of leaf disks; 2 = necrotic lesions on <50% of a leaf disk; 3 = necrotic lesions on 50-75% of a leaf disk; 4 = necrotic lesions on 75-100% of a
leaf disk. Each grade was a mean of 10 leaves. Values are means ± SE (n = 10 leaves). Means followed by the same letter are not significantly different (p <
0.05).

145
 Table 9.2. Effect of AM-toxin I on in vitro shoots of cultivars with various degrees of susceptibility to Alternaria blotch

146
Degree of Concentration of toxin
Cultivar susceptibility a 10 μM 1 μM 0.1 μM 0 μM
‘Starking Highly
Delicious’ susceptible 4.0 ± 0.00*a 3.5 ± 0.50a 3 .0± 0.00a 0.0 ± 0.00c
‘Aori 10’ Susceptible 3.5 ± 0.50a 2.5 ± 0.50a 2.0 ± 1.00b 0.0 ± 0.00c
‘Hokuto’ Susceptible 2.0 ± 0.00b 2.0 ± 0.00b 1.0 ± 0.00b 0.0 ± 0.00c
‘Fuji’ Intermediate 1.0 ± 0.00b 0.0 ± 0.00c 0.0 ± 0.00c 0.0 ± 0.00c
‘Jonagold’ Resistant 0.0 ± 0.00c 0.0 ± 0.00c 0.0 ± 0.00c 0.0 ± 0.00c
a
The grade of susceptibility is calculated based on the occurrence of necrotic lesions on in vitro shoots as follows: 0 = no necrotic lesion; 1 = a few necrotic
lesions near the end of the shoots; 2 = necrotic lesions on <50% of the shoot; 3 = necrotic lesions on 50-75% of the shoot; 4 = necrotic lesions on 75-100% of
the shoot. Each grade was a mean of two shoots. Values are means ± SE (n = 2 shoots). Means followed by the same letter are not significantly different (p <
0.05).
 Table 9.3. Production of resistant mutants in cv. ‘Hokuto’ by X-ray irradiation and selection with Alternaria alternata toxin

No. of shoots according to No. of shoots

Radiation Toxin No. of the degree of No. of shoots 2nd
Dose rate dose concentration screened susceptibilitya 1st screening screening
Cultivar (KR/min) (KR) (μM） shoots 0 1 2 3 4 testb testc
Non-
‘Hokuto’ irradiated - 10 49 0 0 0 6 43 - -
0.01 6 10 263 1 33 86 63 80 1 0
8 10 223 3 25 58 52 85 3 0
12 10 232 5 48 51 63 65 5 0
0.1 1 10 92 0 0 0 3 89 0 -
2 10 88 0 0 8 21 59 0 -
4 10 82 0 1 17 29 35 0 -
6 10 276 4 55 54 79 84 4 0
8 10 312 5 30 87 103 87 5 1
10 10 303 8 38 116 78 63 8 4
Non-
‘Jonathan’ irradiated - 100 10 0 2 2 2 4 - -
(resistant) 10 10 8 2 0 0 0 - -
a
Same as described in Table 9.1; bShoots evaluated as grade 0 after immersion in 10 μM toxin in the first screening test; cShoots evaluated as grade 0 or 1 after
immersion in 100 μM toxin in the second screening test.

147
 Table 9.4. Production of resistant mutants in cultivars ‘Fuji’, ‘Oorin’ and ‘Aori 10’ by X-ray irradiation and selection with Alternaria alternata toxin

148
No. of shoots according to
Radiation No. of the degree of No. of shoots
Dose rate dose screened susceptibilitya 1st No. of shoots
Cultivar (KR/min) (KR) shoots 0 1 2 3 4 screeningb 2nd screeningc
‘Fuji’ Non-irradiated 38 0 0 0 13 25 - -
0.01 6 99 2 11 33 28 25 2 0
8 111 1 32 29 18 31 1 0
10 102 2 33 29 27 11 2 0
0.1 6 280 22 67 73 66 52 22 6
8 527 53 164 132 81 97 53 12
10 533 44 125 146 103 115 44 9
‘Oorin’ Non-irradiated 20 0 0 0 1 19 - -
0.1 6 189 12 41 66 52 18 12 10
8 185 27 70 64 10 14 27 18
10 156 21 61 57 11 6 21 11
‘Aori 10’ Non- irradiated 40 0 0 0 0 40 - -
0.01 6 123 0 0 0 0 123 0 -
12 125 0 0 0 0 125 0 -
0.1 6 121 0 0 0 0 121 0 -
8 110 0 0 0 1 109 0 -
10 98 0 0 0 0 98 0 -
‘Jonathan’ Non-irradiated - 10 8 2 0 0 0 - -
(resistant)
a
Evaluation of susceptibility marked with grades from 0 to 4 was the same as described in Table 9.1; bShoots evaluated grade 0 after immersion in 10 μM
toxin in the first leaf disk screening test; cShoots evaluated grade 0 or 1 after immersion of 100 μM toxin in the second leaf disk screening test.
 Table 9.5. Production of resistant mutants in cultivars ‘Hokuto’, ‘Fuji’ and ‘Aori 10’ by gamma-ray irradiation and selection with Alternaria alternata toxin

No. of shoots according

Toxin No. of to the degree of No. of shoots
Dose rate Radiation concentration screened susceptibilitya No. of shoots 2nd
Cultivar (Gy/hour) dose (Gy) (μM) shoots 0 1 2 3 4 1st screeningb screeningc
‘Hokuto’ Non-irradiated - 10 49 0 0 0 6 43 - -
1 0 0 0 9 40
5 60 10 181 3 14 26 29 109 3 0
1 7 23 31 32 88
80 10 168 5 19 38 35 71 5 0
1 13 27 42 35 51
120 10 203 4 14 35 40 110 4 0
1 10 22 41 58 72
‘Fuji’ Non-irradiated - 10 38 0 0 0 13 25 - -
5 60 10 116 5 15 33 38 25 5 1
80 10 410 35 115 93 81 86 35 12
120 10 491 46 164 165 71 45 46 20
‘Aori 10’ Non-irradiated - 10 40 0 0 0 0 40 - -
5 60 10 96 0 0 0 0 96 - -
80 10 87 0 0 0 1 86 - -
120 10 45 0 0 0 0 45 - -
‘Jonathan’ (resistant) Non-irradiated - 100 10 0 2 2 2 4 - -
- 10 10 8 2 0 0 0 - -
- 1 10 8 2 0 0 0 - -
a
Same as described in Table 9.1; bShoots evaluated as grade 0 after immersion in 10 μM toxin in the first screening test; c Shoots evaluated as grade 0 or 1
after immersion in 100 μM toxin in the second screening test.

149
 Table 9.6. Symptoms after inoculation of control and resistant mutant plants with a spore suspension*

150
Rate of leaf No. of
Susceptibility No. of symptoms Severity of resistant
Cultivar level Radiation Dose rate Dose plants (%)a symptomsb mutants
‘Hokuto’ Susceptible control Non-irradiated - - 5 84.0 ± 4.8 45.5 ± 5.3 -
Selected mutant X-rays - 10 KR 4 20.0 ± 5.0 6.7±4.2 4
‘Oorin’ Susceptible control Non-irradiated - - 2 75.0 ± 5.0 49.2 ± 0.9 -
Selected mutant 6 KR 5 56.0 ± 12.8 15.3 ± 7.7 3
0.1
X-rays KR/min 8 KR 9 37.8 ± 30.9 10.7 ± 10.1 6
10 KR 4 20.0 ± 25.0 3.4 ± 4.2 3
‘Fuji’ Susceptible control Non-irradiated - - 5 54.0 ± 4.8 19.3 ± 3.5 -
Selected mutant 6 KR 2 40.0 ± 0.0 6.7 ± 0.0 2
0.1
X-rays KR/min 8 KR 8 25.0 ± 22.5 4.8 ± 4.4 7
10 KR 4 5.0 ± 5.0 0.9 ± 0.9 4
60 Gy 1 10.0 ± 0.0 1.7 ± 0.0 1
gamma-rays 5 Gy/hour 80 Gy 8 16.3 ± 17.8 4.2 ± 4.8 5
120
Gy 15 20.0 ± 12.9 4.1 ± 3.1 14
‘Jonagold’ Resistant control Non- irradiated - - 4 15.0 ± 10.0 3.3 ± 1.7 -
‘Jonathan’ - - 3 3.3 ± 4.4 0.6 ± 0.8 -
‘Sansa’ - - 3 0.0 ± 0.0 0.0 ± 0.0 -

*The mutant plants shown here had been screened with the toxin method. aRate of symptoms appeared within group after the inoculation of spore suspension;
b
Severity of symptoms appeared within group after the inoculation of spore suspension; cIn cvs. ‘Hokuto’ and ‘Oorin’, we selected resistant mutants showing
<40% infected leaf symptoms and severity of symptoms <15. In cv. ‘Fuji’, we selected resistant mutants showing <40% rate of infected leaf symptoms and
severity of symptoms <10. Values are means ± SE (n = 1-15 plants).
Table 9.7. Fruit characteristics of the mutants and original cv. ‘Hokuto’

Mutant/ No. of damaged fruits

No. of fruit Fruit colouration Core rot
original Stem end Fruit Fruit
samples (1-5)a (0-3)b
line cracking splitting drop
5-1 11 0 1 0 4.0 0.7
5-3 7 0 0 1 4.0 1.0
3-1 6 0 0 1 4.3 0.2
3-2 9 0 0 0 4.2 0.8
3-3 47 0 1 0 4.1 0.9
3-4 10 0 0 3 4.9 0.4
19-1 35 0 0 2 4.0 0.9
18-2 3 0 0 0 4.7 1.3
18-3 7 0 0 0 4.0 0.7
original 1 59 2 3 4 4.1 1.1
original 3 3 0 1 0 3.0 0.7
original 4 4 0 0 0 4.3 2.0
a
Samples were rated from 1-5 according to the degree of fruit colouration from lowest to highest colouration;
b
Samples were rated from 1-3 according to the degree of core rot from lowest to highest core rot.

151
 A
Shoot irradiation

I B
Surviving shoots
Resistant plants

H
Evaluation of resistance C

２
③
D
Shoot
G proliferation

F E
Potted Rooted
plants plantlets

Figure 9.1. Schematic representation of the procedure of producing mutants resistant to

Alternaria blotch. (A) Irradiation to shoots with various doses and dose rates. (B)
Investigation of surviving shoots. (C) Culture of surviving shoots in proliferation medium.
(D) Proliferation of surviving plantlets. Shoot clumps were divided and transferred into
fresh medium to proliferate (repeated at least three times). (E) Elongated shoots were
transferred to rooting medium. (F) Plants were acclimatised. (G) The third leaves (from
the top) were collected and disks were cut out with a cork borer from the central parts of
the leaf. (H) Evaluation of resistance was carried out by immersing the leaf disks in AM-
toxin solutions and evaluating the degree of necrosis on the leaf disks. Plants whose disks
remained green were selected for a second screening with the AM-toxin. (J) Selection of
resistant plants.

152
Figure 9.2. Susceptibility of cultivars to Alternaria blotch AM-toxin I. (A) In vitro
shoots of cultivars ‘Starking Delicious’, ‘Aori 10’, ‘Jonagold’, ‘Hokuto’ and ‘Fuji’
were directly immersed in an AM-toxin solution. Browning of shoots of susceptible
cultivars is the result of necrosis, whereas shoots of resistant cultivars remained green.
The AM-toxin I solution was used at concentrations of 0 (control), 0.1, 1 and 10 µM.
(B) Leaf disks of the first, third and fifth leaves of cultivars ‘Jonathan’ (J), ‘Fuji’ (F),
‘Oorin’ (O) and ‘Indo’ (I) immersed in AM-toxin solution. The susceptibility to AM-
toxin I varied among cultivars and leaf age, i.e., older leaves were slightly more
resistant to the toxin. The AM-toxin I solution was used at concentrations of 0.1, 1, 10
and 100 µM.

153
 Figure 9.3. Survival rate of X- and gamma-ray irradiated shoots of cultivars
‘Hokuto’ and ‘Aori 10’. (A) In vitro-cultured shoots of cv. ‘Hokuto’ irradiated with
increasing doses of X-ray irradiation. (B) In vitro-cultured shoots of cv. ‘Aori 10’
irradiated with increasing doses of X-ray irradiation. (C) In vitro cultured shoots of
cultivars ‘Hokuto’ and ‘Aori 10’ irradiated with increasing doses of gamma-ray
irradiation.

154
Figure 9.4. Appearance of in vitro-cultured shoots after gamma-ray irradiation. (A)
In vitro-cultured shoots of cv. ‘Oorin’ (after being cultured for 30 days on shoot
proliferation medium). (B) Non-irradiated in vitro cultured shoots of cv. ‘Hokuto’.
(C) Appearance of in vitro-cultured shoots one month after irradiation on cv.
‘Hokuto’ (radiation doses of 60, 80 and 120 Gy). Shoots appeared as normal as non-
irradiated ones. (D) Appearance of in vitro-cultured shoots one month after
irradiation on cv. ‘Aori 10’ (radiation doses of 60, 80 and 120 Gy). Callus formation
from shoots appeared in parts of the irradiated shoots. (E) Magnified detail of (C).
Normal phenotypes were seen in 120 Gy-irradiated cv. ‘Hokuto’. (F) Magnified
detail of (D). Rosette-type shoots and abnormal leaf morphology appeared one month
after gamma-ray irradiation on cv. ‘Aori 10’ (radiation doses of 120 Gy). Bar: 2 cm
(A, B, E, F). Bar: 3 cm (C, D).

155
 A

Figure 9.5. Screening tests of mutants with AM-toxin I solution. (A) First screening
test for selecting ‘Fuji’ mutants resistant to Alternaria blotch. Leaf disks were cut from
the third leaves and immersed in 10 μM AM-toxin I solution. Leaf disks of resistant
mutants remained green, whereas necrotic lesions appeared on the susceptible ones.
(B) Second screening test for mutants selected in the first screening. Leaf disks from
the third leaves of each selected mutant were immersed in 10 and 100 µM AM-toxin I
solution. Leaf disks of 12 mutants were treated per plate.

156
 A B

Figure 9.6. Regenerated plant of the original plant of cv. ‘Fuji’ (A), which showed
necrotic lesions 10 days after inoculation with a spore suspension of the apple pathotype of
Alternaria alternata, and of a mutant resistant to Alternaria blotch (B).

A B

C D

Figure 9.7. Evaluation of fruits of mutants resistant to Alternaria blotch. Fruits of

mutant lines 21 (A), 3 (B) and 19 (C) of cv. ‘Hokuto’ compared to fruits of the
original cv. ‘Hokuto’ (D). All fruits were harvested in 2005. No morphological
changes were observed between the mutants and the original cv. ‘Hokuto’.

157
 Chapter 10

The pineapple-Fusarium subglutinans interaction: an early selection system

for disease resistance

O. Borrás-Hidalgo1* and R. Santos Bermúdez2

1
Center for Genetic Engineering and Biotechnology (CIGB),
Havana, Cuba;
2
Bioplants Centre, Ciego de Avila, Cuba.

*Correspondence: [email protected]

Abstract

This chapter summarises a study on the effects of selective agents from Fusarium subglutinans on
seedlings, calli and in vitro plantlets of pineapple (Ananas comosus [L.] Merr) as well as their
applications for breeding programmes in order to increase the resistance to fusariose disease in
pineapple. The phytotoxic effects of culture filtrates from F. subglutinans, the causal agent of
fusariose in pineapple, were examined in several pineapple cultivars differing in resistance level. The
phytotoxic effects were assessed by electrolyte leakage and by placing the culture filtrate on wounds
in seedlings, by inhibition of growth and regeneration of calli, and by inhibition of multiplication of in
vitro plantlets. As a proof of concept, the susceptible cultivars were the most sensitive in each test,
whereas resistant cultivars showed resistance to the culture filtrate and their calli grew well even in the
presence of high concentrations of culture filtrate, which were highly toxic to the susceptible cultivars.
In addition, culture filtrates reduced shoot formation in in vitro plantlets and when plants were
regenerated from calli derived from the susceptible cultivars but not from resistant ones. These results
show the potential of culture filtrates of F. subglutinans for in vitro screening of pineapple for
resistance to the pathogen. Furthermore, the possibility of using culture filtrates as a selection agent
for resistance in F1 hybrids was also evaluated. Using culture filtrate as a selection criterion allowed
resistant plants to be obtained that behaved quite similarly to those when the fungus itself was used as
a selection agent.

INTRODUCTION

Fusariose, caused by Fusarium subglutinans, constitutes the most serious pineapple disease in Brazil
where it was first reported in the State of Sao Paulo (Kimati and Tokeshi, 1964). Besides causing
losses as high as 80% of marketable pineapple fruits (Robbs et al., 1965), the pathogen infects
approximately 40% of the asexually propagated material and kills about 20% of the pineapple plants
prior to harvesting (Matos, 1995; Matos et al., 1997).

The pathogen is able to infect all parts of the pineapple plant and the development of the disease
results in a characteristic gum exudation (Matos, 1995). A pineapple plant propagated from an infected
asexual propagative part can show, besides gum exudation, one or more of the following alterations:
bending of the stem, usually to the side where the lesion is located; changes in plant phyllotaxis,
increasing the number of leaves per spiral; changes in plant architecture, looking like a funnel;
shortening of the stem; death of the apical meristem; chlorosis (Pissarra et al., 1979). Due to the
disease development in the stem and base of the leaves, water flow is reduced, the plant stops growing
and the leaves show a reddish colour (Matos, 1995). In a later stage of infection, the plant wilts and
dies.

159
The control of pineapple fusariose has been mainly based on the use of pathogen-free propagating
material, evading the pathogen and direct protection by chemical control (Matos, 1995). Chemical
control is costly and often ineffective, and in time may lead to the occurrence of pathogen isolates
resistant to fungicides. Moreover, the application of chemicals for crop protection should be reduced
for environmental and health reasons (Pinho et al., 1997).

The potential for disease resistance as a control measure for pineapple fusariose has been suggested in
studies based on observations carried out either under field conditions or with artificial inoculation
techniques (Matos et al., 1991). Considering that growing resistant cultivars constitutes one of the
cheapest and most efficient control measures of plant disease, the identification of sources of
resistance to F. subglutinans is a very important step in obtaining commercial pineapple cultivars
resistant to the pathogen (Cabral and Matos, 1995).

Many plant pathogens produce toxins related to a number of different diseases (Wood et al., 1972;
Durbin, 1981). For example, race T of Bipolaris maydis (Nisikado and Miyake) Shoemaker (syn.
Helminthosporium maydis), the causal agent of southern corn leaf blight, produces T-toxin, which acts
specifically on the mitochondria of susceptible cells (Miller and Koeppe, 1971). Fungi of the genus
Fusarium produce a variety of biologically-active metabolites in culture filtrates reported to be toxic to
cotyledons, germinating seeds and plants of many vegetal species (Jin et al., 1996; Matsumoto et al.,
1999a). Fusaric acid (5-butylpyridine-2-carboxylic acid), produced by several fungi of the genus
Fusarium, is associated with wilt of banana, cotton, pea, tomato, and other plants, and affects
membrane permeability, increasing leakage of potassium and other electrolytes and inhibits respiration
in this interaction (McLean, 1996; Kuzniak et al., 1999). Treatment of tomato cells with fusaric acid
resulted in decreased cell viability, but in increased activities of intracellular superoxide dismutase,
catalase, guajacol peroxidase and ascorbate peroxidase. An increase in extracellular pH and oxygen
production could reflect both the phytotoxic effect as well as fusaric acid action as a plant-pathogen
recognition factor (Kuzniak, 2001).

Experiments with pathogen culture filtrates have shown that the in vitro tissue response correlates with
the disease reaction of the host variety. Thus, in these cases, selection using a phytotoxin might offer
an alternative to field selection as reported for a number of host-pathogen interactions, and may allow
selection of important traits in disease resistance (Daub, 1986). Advantages of using in vitro plantlets
and calli rather than whole plants for assaying phytotoxins have been reviewed elsewhere (Chapter 2;
Botta et al., 1994; Remotti et al., 1997). Screening for disease resistance in the field is time-
consuming, costly and dependent upon natural fluctuations in the inoculum abundance and weather
factors that influence pathogen spread, infection, disease development and disease expression (Ostry,
1997). The creation of genetic variability and a suitable procedure for reliably identifying resistant
genotypes constitute two prerequisites for disease resistance improvement.

Progress in the field of in vitro selections is often hampered by the fact that: (1) the substances
contained in filtrates may not yet have been completely characterised; (2) the expression of resistance
to the toxin in vitro may vary from that shown to the pathogen in planta; and (3) the level of desired
resistance is not obtainable via toxin influence (Chapter 2; Švábová and Lebeda, 2005).

The aim of this chapter is to summarise our main results and knowledge about toxins and other
substances produced during the interaction between pineapple and F. subglutinans, their potential role
in pathogenesis mechanisms, and possible screening and selection for in vitro and in vivo resistance.
The chapter covers basic research studies and methods that elucidate the phytotoxic effects of culture
filtrate and toxins produced by F. subglutinans on in vitro and in vivo plants, but also try to develop
practical applications to obtain resistant breeding lines. Their application successfully resulted in
resistant lines. Nowadays, these techniques are an important complement to classical breeding
methods.

160
MATERIAL AND METHODS

Culture filtrates and fusaric acid production

An isolate of F. subglutinans (isolated by the Bioplants Centre, Ciego de Avila, Cuba) was grown on
potato dextrose agar (PDA) dishes for seven days at 26 ± 2°C with a 12 hour photoperiod at 4000 lux,
provided by warm white fluorescent tubes. Dishes of fungal mycelium were inoculated in Czapek-Dox
broth (1 l) supplemented with 2 g of NaNO3, 1 g of K2HPO4, 0.5 g of MgSO4 × 7H2O, 0.5 g of KCl,
10 mg of FeSO4, 30 g of sucrose, 1 ml of 1% (w/v) ZnSO4 and 1 ml of 0.5% (w/v) CuSO4. The
cultures were grown in 250 ml Erlenmeyer flasks with 100 ml of medium at 26 ± 2°C under the light
conditions mentioned above. After three weeks of stationary incubation, mycelia and conidia were
filtered through Whatman No.1 filter paper (Whatman, Clifton, NJ, USA) and a 0.2 µm Millipore
membrane (Sartorius AG, Goettingen, Germany). The culture filtrate was evaporated under reduced
pressure at 40°C to 10, 20, 30, 40, 50, 60, 70, 80, 90 and 100% (v/v) using a rotary evaporator
(Heidolph, Bioblock Scientific, Strasbourg, France). The isolation, extraction and purification of
fusaric acid were performed following the methodology described by Bacon et al. (1996).
Alternatively, commercial fusaric acid may also be used (e.g., product number F6513, Sigma
Chemical, St. Louis, MO, USA).

Electrolyte leakage bioassay

Ion leakage was used as an indicator of cell membrane damage in response to the presence of
phytotoxic culture filtrate on pineapple seedling (30 days old) of resistant (‘Perolera’) and susceptible
(‘Smooth Cayenne’) cultivars. Leaf disks (diameter: 10 mm) of each cultivar were taken using a cork
borer, and placed in a tube with 1 ml of 80% culture filtrate. Disks were infiltrated under a vacuum for
24 hours at room temperature, rinsed with distilled water several times and placed in 20 ml of distilled
water for 24 hours to allow electrolyte leakage. The conductivity of this latter solution was measured
with a 660 Metrohm conductivity meter (Metrohm AG, Herisau, Switzerland) equipped with a
temperature-compensating electrode (K = 0.77). Leaf disks infiltrated with distilled water and fungal
culture media were used as controls. Each test was repeated five times.

Applying the culture filtrate or fusaric acid on wounded leaf segments

To determine the phytotoxic effects of the culture filtrate or fusaric acid, they were applied at different
concentrations onto wounded leaf segments. Fusaric acid concentrations of each culture filtrate
concentration were used. The cultivars ‘Perolera’ (resistant), ‘Spring’ (resistant), ‘Perola’ (susceptible)
and ‘Smooth Cayenne’ (susceptible) were used. Leaf segments (~20 mm) of 30 day old seedlings were
excised, placed on moist filter paper in Petri dishes, and punctured with a fine needle, and 5 µl of the
culture filtrate or fusaric acid (in different concentrations) were placed onto the wound area. All leaf
segments were incubated on moist filter paper in Petri dishes (continuous fluorescent light,
approximately 22 ± 2°C). Sterile water and fungal culture medium were used as negative and positive
controls, respectively, and the assays were performed using five leaf segments per plant and 25 plants
per cultivar. The reactions of leaves were noted after 35 hours of incubation and necrotic spots were
measured using the following equation:

A = π/4 a × b

Where: A = Necrosis spot area (mm2), a = radium longer (mm), b = radium shorter (mm)
and π = 3.1415.

161
Effect of culture filtrate on calli and in vitro plantlets

Effect of culture filtrate on growth of calli

Calli were established aseptically from two pineapple cultivars according to Daquinta et al. (1996),
and placed on Murashige and Skoog (MS) basal medium (Murashige and Skoog, 1962) supplemented
with 2.5 mg/l of Dicamba (3,6-dichloro-o-anisic acid) and 0.5 mg/l of 6-benzylaminopurine (BAP)
(medium A) per litre and incubated for five to six weeks under dark conditions at 26 ± 2°C. The
induced friable calli were subcultured monthly. Culture filtrates (without concentrate) adjusted to
pH5.8 were added to autoclaved medium A at three concentrations (10, 20 and 30%) and poured into
100 mm diameter dishes.

Ten pieces (~10 mg) of calli from each cultivar were transferred to medium A supplemented with
culture filtrate. The experiment consisted of three dishes (replicates) for each cultivar in a completely
randomised design. Dishes were incubated under the conditions mentioned above and two weeks later,
calli were visually ranked in five categories: 1 = no browning; 2 = callus surface slightly brown; 3 =
whole tissue brown; 4 = whole tissue deeply brown with restricted growth; and 5 = whole tissue
deeply brown with no growth. In addition, callus growth rate was measured as recommended by Arai
and Takeuchi (1993). This experiment was repeated once.

Effect of culture filtrate on plant regeneration and shoot multiplication from calli and in vitro plantlets

Two cultivars resistant to F. subglutinans, ‘Perolera’ and ‘Spring’, and two susceptible ones, ‘Smooth
Cayenne’ and ‘Perola’, representing a range of disease reactions, were used in all the experiments.
Calli and in vitro plantlets were established aseptically according to Daquinta et al. (1997). The in
vitro plantlets were placed on MS basal medium supplemented with 2.1 mg/l BAP and 0.3 mg/l
naphthaleneacetic acid (NAA) (medium for shoot multiplication) and incubated with a photoperiod of
12 hours at 26°C. For plant regeneration, the calli were placed on MS basal medium supplemented
with 0.5 mg/l BAP (plant regeneration medium) and incubated under the same conditions.

Culture filtrates adjusted to pH5.8 were added to autoclaved medium at five concentrations (4, 8, 12,
16 and 20% [v/v]) to assess their effect on shoot multiplication and plant regeneration. The plant
regeneration medium was poured into 100 mm diameter Petri dishes and the shoot multiplication
medium was transferred to 250 ml Erlenmeyer flasks. 100 shoots of in vitro plantlets (~240 mg) from
each cultivar were transferred to the shoot multiplication medium supplemented with the different
concentrations of the culture filtrate. The incubation conditions were as described above. The
multiplication coefficient was determined from the mean number of shoots per explant. A similar
number of calli (~100 mg) from each cultivar was placed on the plant regeneration medium and
regeneration was calculated from the mean number of shoots per callus. The multiplication coefficient
was recorded after two months of culture, and plant regeneration was determined after three months.
All the experiments were repeated twice.

Selection procedures

The culture filtrate was applied onto wounded leaves to select susceptible and resistant F1 hybrids. The
culture filtrate and fusaric acid concentrations used were 80% (v/v) and 0.75 mg/ml, respectively. F1
hybrids of ‘Spring’ × ‘Smooth Cayenne’; ‘Smooth Cayenne’ × ‘Spring’; ‘Smooth Cayenne’ ×
‘Perolera’ and ‘Perolera’ × ‘Smooth Cayenne’ were used. The resistance level of the F1 hybrids and
cultivars under natural conditions was measured in greenhouse field tests. 25 plantlets per cultivar and
F1 hybrid were wounded with a stainless needle. A puncture wound was produced at the basal region.
Inoculation was performed by dipping wounded plantlets in an inoculum of 105 conidia/ml for three
minutes. After inoculation, the plantlets were kept under greenhouse conditions at 25-30°C, for two
months, and then inspected for disease development.

162
Evaluation was based on a 0-6 numerical rating system in which 0 = no disease development in the
stem; 1 = 2% of the stem infected by the pathogen; 2 = 3-5%; 3 = 6-10%; 4 = 11-20%; 5 = 21-50%;
and 6 = 51-100% (Disease Index [DI]). A resistant reaction is defined as no disease development in
inoculated plantlets. F1 hybrids and cultivars in which infection takes place but the colonisation of the
host tissue proceeds slowly, reaching a DI of 3 or less two months after inoculation and showing
significantly less disease severity than the susceptible control are considered as tolerant of the
pathogen (Matos et al., 1991). The experiment was conducted with a completely randomised design.

Data analysis

The experimental data were analysed by analysis of variance conducted according to Duncan’s new
multiple range test using SPSS/PC (Statistics Package for Social Science) (1992). Means were
compared by least significant differences at p < 0.05 unless otherwise stated.

RESULTS AND DISCUSSION

The results of the studies presented here indicate that F. subglutinans isolates that cause fusariose
disease produce one or more phytotoxins in culture that are also phytotoxic to leaves, calli and in vitro
plantlets of pineapple. The assays shown here provide a good measure for determining the phytotoxic
effects of F. subglutinans culture filtrate and the potential of using the filtrate for in vitro screening of
pineapple for resistance to this pathogen.

Electrolyte leakage bioassay

The change in the conductivity (µS/cm) of the solution over a 24 hour incubation period was used as
the measure of electrolyte leakage. Table 10.1 shows how electrolyte leakage depends on the cultivar
used. ‘Smooth Cayenne’ (susceptible to fusariose) showed a highly significant electrolyte leakage in
the presence of the culture filtrate. In contrast, ‘Perolera’ (resistant) showed no significant electrolyte
leakage, even in the presence of culture filtrate. The distilled water and fungus culture medium did not
induce significant electrolyte leakage in the washing solution (Borrás et al., 1998).

One of the most vulnerable parts of the cell is the plasma membrane, the first subcellular component
likely to encounter the toxin (Batchvarova et al., 1992). A rapid and nearly universal response of
susceptible plants or tissue to toxin treatment is an alteration of membrane permeability, which is
usually detected as an efflux of solutes or as a depolarisation of electropotential across the plasma
membrane (Dunkle and Wolpert, 1981). Regardless of the primary site of action of host-specific
toxins, the results of studies in which electrolyte leakage is taken as an indication of toxicity must be
carefully interpreted, and the relationship of electrolyte leakage to other disease symptoms induced by
the toxin must be clearly established (Dunkle and Wolpert, 1981).

Applying the culture filtrate or fusaric acid onto wounded leaves

Figure 10.1A describes the effect of different culture filtrate concentrations on the development of
necrotic spots from pineapple leaves. F. subglutinans filtrate induced necrotic spots on pineapple
leaves from resistant and susceptible cultivars after a 35 hour treatment. The necrotic spot areas in
susceptible pineapple leaves treated with the filtrate were larger than those of the resistant cultivars.
‘Smooth Cayenne’ and ‘Perola’ (both susceptible) developed necrotic lesions with an area of up to 10
mm2 (Borrás et al., 2001).

A significant increase in necrotic spot area was directly correlated to an increase in the concentration
of culture filtrate in susceptible cultivars. The ‘Perolera’ and ‘Spring’ resistant cultivars showed a
lower degree of necrotic spot area when treated with culture filtrate in comparison to susceptible
cultivars. Significant differences between susceptible and resistant cultivars were observed when the
culture filtrate was applied at a 50% concentration and higher (Borrás et al., 2001).

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On the other hand, all cultivars were sensitive to fusaric acid regardless of their resistance level and
the sensitivity increased as the concentration used increased (Figure 10.1B). These results corroborate
our observations that fusaric acid has a non-specific behaviour in the response of susceptible and
resistant pineapple cultivars (Borrás et al., 2001).

This study represents the first attempt to assess the potential of using culture filtrate as a selection
agent for fusariosis resistance. The role of toxins in pathogenesis is usually assessed by evaluating the
correlation between toxin production and pathogenicity of the organism, and between the sensitivity of
different genotypes to the toxins and their susceptibility to the disease (Yoder, 1980). Our results have
shown that the cultivars’ responses to the culture filtrate of F. subglutinans are related to the responses
of the cultivars to the pathogen. This behaviour was also found by Jin et al. (1996) who used a
phytotoxic culture filtrate from F. solani on wounded leaves and showed that the susceptible cultivars
were more sensitive to culture filtrate than the resistant ones. Furthermore, their assay showed that the
response of soybean plants to crude fungal culture filtrate was correlated with the severity of Sudden
Death Syndrome in inoculated soybean (Glycine max L.) plants grown in a greenhouse.

In our case, fusaric acid produced phytotoxic effects on all cultivars. Therefore, the response of
different pineapple genotypes did not correlate with the response to F. subglutinans under natural
conditions. However, since fusaric acid has been classified as a non-selective toxin (Gäumann, 1957;
Matsumoto et al., 1995), it is likely that there must be other components present in the culture filtrate
that are responsible for the selectivity observed. Extracellular molecules such as fungal elicitors of
plant resistance have been reported in a number of studies related to host-pathogen interactions
(Knogge, 1996; Abad et al., 1997; Bailey et al., 1997; Ellis et al., 2000).

Effect of culture filtrate on calli and in vitro plantlets

Effect of culture filtrate on growth of calli

Calli of ‘Smooth Cayenne’ and ‘Perolera’ grew well on the subculture medium and their growth was
not affected by fungal culture media. However, there was a general trend for increased calli browning
in the callus pieces of ‘Smooth Cayenne’ at higher levels of culture filtrate in medium A (Figure 10.2).
The mean calli browning rate increased from 2.2 to 4 and 5 when the amount of culture filtrate in the
medium was 10, 20 and 30%, respectively. In contrast, ‘Perolera’ calli had a lower browning rate,
especially when higher levels of culture filtrate were added to medium A. On the other hand, the
growth of ‘Smooth Cayenne’ calli decreased on subculture medium containing a high concentration of
the culture filtrates. Compared to the other cultivars, ‘Perolera’ calli were not greatly affected by the
culture filtrate (Table 10.2). In our study, culture filtrates of F. subglutinans were toxic to susceptible
pineapple calli, as they caused a decrease in callus growth and caused browning. It was also observed
that resistant calli had lower browning rates, even at higher concentrations of F. solani culture filtrate
(Jin et al., 1996).

As in our previous study (Borrás et al., 1998), callus browning rate and callus fresh weight were found
to be good measures of callus sensitivity to culture filtrate. The correlation between pineapple cultivar
susceptibility and the toxicity of culture filtrates suggests that filtrates could be used for in vitro
screening of disease resistance. Tissue culture systems can provide a means of rapid screening for
disease resistance when a toxin is involved in disease development. However, it would still be
premature to conclude that any of these characteristics could be used for in vitro screening of
germplasms for fusariose disease as has already been attempted in several host-parasite systems (Kaur
et al., 1987).

Effect of culture filtrate on plant regeneration and shoot multiplication from calli and in vitro plantlets

The regeneration of plantlets from calli of the susceptible cultivars ‘Smooth Cayenne’ and ‘Perola’
was inhibited at high concentrations of culture filtrate in the regeneration medium. Even at the lowest

164
culture filtrate concentration, regeneration was considerably reduced (Table 10.3). Calli from the
resistant cultivars ‘Perolera’ and ‘Spring’ were less affected at the same concentrations of culture
filtrate (Hidalgo et al., 1999).

In vitro shoot multiplication from plantlets of the susceptible cultivars ‘Smooth Cayenne’ and ‘Perola’
was highly inhibited in media containing high concentrations of culture filtrate. Resistant in vitro
plantlets (‘Perolera’ and ‘Spring’) were less affected by the same concentrations, demonstrating a
higher tolerance compared with untreated controls (Table 10.4).

The present results support previous observations that F. subglutinans culture filtrates applied at
various concentrations can reduce growth and development of pineapple tissue cultures and that the
effect is correlated with the resistance of the cultivars from which the tissue cultures were derived
(Hidalgo et al., 1999). Mendes et al. (1993) found that the addition of filtrates of F. oxysporum f. sp.
cubense to culture medium had toxic effects on the growth of banana shoot tips. Ahmed et al. (1991)
tested wheat calli for tolerance to toxic metabolites of F. graminearum and F. culmorum, and observed
that the regeneration ability of the tolerant calli was lower than that of the unselected calli. A similar
response was observed when susceptible soybean calli were exposed to high levels of F. solani culture
filtrate, showing reduced growth and regeneration (Jin et al., 1996).

In contrast, in tests on the effect of Alternaria solani culture filtrate on adventitious shoot regeneration
from tuber disks of five potato cultivars, which had been selected on the basis of their field reaction to
A. solani and represented a range of disease reactions, the filtrate actually stimulated regeneration
(Lynch et al., 1991).

The toxicity of the F. subglutinans culture filtrate on in vitro plantlets and calli of the selected
cultivars of pineapple reflected the performance of the isolate in greenhouse tests on whole plants,
suggesting a possible role for fungal extracellular toxic compounds in this disease. The results indicate
that these characteristics can be used for in vitro screening of germplasm for resistant lines, as has
already been attempted in several other host-parasite systems (Ludwig et al., 1992; Song et al., 1994).

Selection procedures

Susceptible F1 hybrid pineapple leaves treated with culture filtrate developed necrotic spots of between
8 and 10 mm2 (Table 10.5). The culture filtrate produced the highest phytotoxic effects on the
susceptible F1 hybrids. Accordingly, the mean DI registered in pineapple plants inoculated with the F.
subglutinans pathogen was significantly lower in F1 hybrids resistant to the culture filtrate than in
susceptible ones. In total, eight F1 hybrids showed resistance to the culture filtrate, one F1 hybrid had
partial resistance and four were sensitive to it. The fusaric acid had phytotoxic effects on all the
cultivars and F1 hybrids, producing necrotic spot areas of 14 mm2 without any significant genotypic
difference (Borrás et al., 2001).

There are no effective chemicals for preventing fusariose disease. The only and best way to eliminate
this disease is to develop cultivars that are resistant to or at least tolerant of F. subglutinans. In cross
breeding programmes, new resistant cultivars can be developed using resistant cultivars or wild
species as breeding material. The reaction of pineapple plants to F. subglutinans infection can vary
within and among cultivars, indicating that differences in resistance to fusariose disease exist in
pineapple genotypes under some conditions (Cabral and Coppens, 1997).

Our results show that it is possible to select resistant plants using the culture filtrate of F. subglutinans
and this method reflects infections under natural conditions. A number of efficient protocols have been
developed to select for resistance in plants to various pathogens by using culture filtrate and purified
toxins in selection experiments (Litz and Lavi, 1997; Alarcon et al., 1998; Matsumoto et al., 1999b).
A prerequisite for success is the sensitivity of the plant to the toxin, reflecting the susceptibility of the
intact plant to the pathogen. Resistance selection can be performed in several ways. One method is

165
one-step selection, in which a lethal concentration of the selective agent is used; another method is
stepwise selection, in which the concentration of the selective agent is increased gradually until it
reaches a lethal concentration (McLean, 1996).

CONCLUSIONS

The basic advantages of using in vitro cultures as compared to natural conditions are: (1) unfavourable
weather and climate conditions are avoided, which enables the assessment of quantitative differences
in polygenic traits more easily and precisely; (2) a large number of individuals can be tested in a small
space; (3) it is easier to manipulate mutants, haploids, somaclones with higher variability in the
genome; and (4) mass screening of mutants for resistance is facilitated (Nedelnık and Repkova, 1998).

Although there are not many reports of cultivars whose resistance is based on in vitro selections
(although many resistant breeding lines were obtained), interest in utilising in vitro methods for
improving resistance to plant pathogens remains (Upadhyay and Mukerji, 1997). Thus, selection of
resistance in vitro must be considered as one of the methods which, in combination with conventional
resistance screening and plant breeding methods including biotechnological procedures, may offer
plant breeders a new approach to accelerate the development of disease-resistant plants (Crino, 1997).

These results suggest that the selection approach could have two major limitations: (1) the lack of
knowledge concerning the genotypic dependence of the characterised toxins or extra-cellular
metabolites that confer host-pathogen selectivity and (2) no assurance that the susceptibility and/or
resistance of cultured tissues to the culture filtrate or toxins reflect those of the whole plant. The
evaluation of genotypes produced within pineapple breeding programmes using culture filtrate could,
therefore, offer a first step towards a simplification of selection procedures, which should be
complemented by a second step of selection using artificial inoculation with the pathogen as
previously reported by Matos et al. (1991).

Acknowledgements

We thank Blackwell Publishing and Springer Science and Business Media for granting permission to
use previously published material.

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Table 10.1. Electrolyte leakage from leaf dishes of cultivars ‘Smooth Cayenne’ and ‘Perolera’

Conductivity (µS/cm)
Treatment
‘Perolera’ ‘Smooth Cayenne’
80% culture filtrate 3.9a 9.8b
Water (control) 2.1a 2.2a
Fungal culture medium (control) 3.4a 3.7a
Least significant difference (0.05) 0.7 5.7
Coefficient of variation (%) 20 23

a: Treatments not significantly different at p < 0.05; b: Treatment significantly different at p < 0.01.

Table 10.2. Pineapple calli browning rate and calli fresh weight of two pineapple cultivars grown on
Fusarium subglutinans culture filtrate-amended medium A for two weeks

Cultivars Concentration of Calli browning rate1 Calli fresh weight (g)2

culture filtrate (%)
‘Perolera’ 0 1a 16.8a
10 1a 15.9a
20 1.1a 15.3a
30 1.2a 14.9a
‘Smooth Cayenne’ 0 1a 14.2a
10 2.2b 10.9b
20 4.5c 6.4c
30 5c 3.9d
Coefficient of variation (%) 23 32
1
Browning rate: 1 = no browning; 2 = callus surface slightly brown; 3 = whole tissue brown; 4 =
deeply brown with restricted growth; 5 = deeply brown with no growth. 2Measured according to Arai
and Takeuchi (1993). a: Treatments not significantly different at p < 0.05; b: Treatment significantly
different at p < 0.01.

Table 10.3. Effect of different concentrations of Fusarium subglutinans culture filtrates on pineapple
plant regeneration from callus*

Concentration of Susceptible cultivars Resistant cultivars

culture filtrate (%) ‘Smooth Cayenne’ ‘Perola’ ‘Perolera’ ‘Spring’
0 9.6a 7.1a 8.6a 6.8a
4 3.3b 2.8b 7.4ab 5.1ab
8 1.5c 0.4c 5.0bc 3.7bc
12 0.0c 0.0c 3.3cd 2.0cd
16 0.0c 0.0c 1.6de 1.1de
20 0.0c 0.0c 0.0e 0.0e
Standard error 0.78 0.95 1.40 1.34

*Calculated as the mean number of shoots per callus. Data within a column followed by the same
letter are not significantly different at p < 0.05 using Duncan’s new multiple range test.

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Table 10.4. Effect of different concentrations of Fusarium subglutinans culture filtrates on pineapple
plant multiplication in vitro*

Concentration of Susceptible cultivars Resistant cultivars

culture filtrate (%) ‘Smooth Cayenne’ ‘Perola’ ‘Perolera’ ‘Spring’
0 7.5a 7.9a 7.2a 6.2a
4 2.9b 4.1b 6.4b 5.8b
8 2.1c 2.8c 5.4c 4.6c
12 1.5d 1.7d 4.7d 2.4d
16 0.8e 0.8e 2.7e 1.5e
20 0.3f 0.2f 1.9f 0.9f
Standard error 0.24 0.20 0.21 0.19

*Calculated as the mean number of shoots per explant. Data within a column followed by the same
letter are not significantly different at p < 0.05 using Duncan’s new multiple range test.

Table 10.5. Behaviour of F1 hybrids and cultivars treated with culture filtrate, fusaric acid and
Fusarium subglutinans pathogen under laboratory and greenhouse conditions

Necrotic spot area (mm2)

F1 hybrid and cultivars Culture filtrate Fusaric acid (0.75 Disease
(80%) mg/ml) Index
‘Spring’ × ‘Smooth Cayenne’ no. 24 0.3c1 14.2 0c
‘Perolera’ × ‘Smooth Cayenne’ no. 52 1.2c 12.8 0c
‘Perolera’ × ‘Smooth Cayenne’ no. 56 0.2c 12.7 0c
‘Perolera’ × ‘Smooth Cayenne’ no. 58 0.7c 13.4 0c
‘Perolera’ × ‘Smooth Cayenne’ no. 60 0.6c 13.8 0c
‘Smooth Cayenne’ × ‘Spring’ no. 02 4.6b 12.1 3.9b
‘Spring’ × ‘Smooth Cayenne’ no. 08 8.7a 14.1 5.4a
‘Spring’ × ‘Smooth Cayenne’ no. 23 9.6a 13.6 6.0a
‘Smooth Cayenne’ × ‘Perolera’ no. 03 0.8c 14.5 0c
‘Smooth Cayenne’ × ‘Perolera’ no. 12 1.5c 13.7 0c
‘Smooth Cayenne’ × ‘Perolera’ no. 22 9.5a 12.6 6.0a
‘Smooth Cayenne’ × ‘Perolera’ no. 32 9.1a 12.3 5.5a
‘Smooth Cayenne’ × ‘Perolera’ no. 15 0.4c 14.5 0c
‘Perolera’ 1.2c 13.1 0c
‘Spring’ 1.8c 13.0 0c
‘Smooth Cayenne’ 8.8a 13.8 6.0a
‘Perola’ 9.4a 12.9 5.6a
Standard deviation 0.98 NS 0.46
1
Data followed by the same letter within a column are not significantly different. The data were
analysed by single analysis of variance (Duncan, p < 0.05). NS: not significant.

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 Figure 10.1. Effect of culture filtrate (A) and fusaric acid (B) produced by Fusarium
subglutinans on pineapple leaves of ‘Smooth Cayenne’ (◊), ‘Perola’ (), ‘Perolera’ (○)
and ‘Spring’ (U) cultivars under laboratory conditions. The data points represent the mean
value of five leaf segments and the vertical bars the standard deviation.

Figure 10.2. Phytotoxic effects of different concentrations of culture filtrate from Fusarium
subglutinans on pineapple calli. (A-D) Resistant cultivar ‘Perolera’; (E-H) Susceptible
cultivar ‘Smooth Cayenne’. (A, E) No culture filtrate (control); (B, F) 10% culture filtrate;
(C, G) 20% culture filtrate; (D, H) 30% culture filtrate.

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 Chapter 11

Mass-screening techniques of some tropical crops for resistance to

anthracnose diseases using phytotoxic metabolites

N.A. Amusa

Institute of Agricultural Research and Training,

Obafemi Awolowo University,
Ibadan, Nigeria

Correspondence: [email protected]

Abstract

Toxic metabolites, also known as chemopathogens, are one of the weapons used by pathogens to
induce a disease condition in susceptible host plants. Many pathogens are known to produce toxins
both in vitro and in vivo, and these toxins have been implicated in the development of disease
symptoms in the host tissues. Among such pathogens are various Colletotrichum spp., the causal
agents of anthracnose. Anthracnose is one of the most devastating diseases of many tropical crops
such as cereals, legumes, tuber/root crops and fruits. Colletotrichum f. sp. manihotis, C.
gloeosporioides, C. lindemuthianum, C. truncatum and C. graminicola are the causal agents of
cassava, yam, cowpea, soybean and maize anthracnose, respectively, and produce toxic metabolites in
culture that fluoresce between 254 and 366 nm. Disease symptoms induced on these crops by the
extracted metabolites of the respective pathogens are similar to those induced by the pathogens during
natural infections. Bioassays of the purified culture filtrate of these pathogens using the respective host
plants produced different sizes of lesions. Results of the in vitro screening of these crops using toxic
metabolites produced by the respective pathogens showed a positive correlation with field screenings
based on natural epidemics. Toxic metabolites appear to be a more effective technique for screening
crops for resistance to anthracnose and other diseases caused by toxigenic pathogens than screening
methods based on natural disease infections.

INTRODUCTION

Diseases constitute a major set back in crop production worldwide and especially in the tropics.
Diseases affect plants from the planting stage through harvesting, as well as during the storage of their
produce. Sinclair (1988) reported that diseases accounted for an estimated loss of 7 million tons of
agricultural produce worldwide. Anthracnose diseases of cassava and yam have resulted in an annual
loss of 30-70% in both crops (Nwankiti, 1982; Ikotun and Hahn, 1991). The effect of plant diseases is
not only on the market value of the produce but also on the availability of planting material. Hahn et
al. (1989) reported that the most significant effect of cassava anthracnose disease is the reduction of
healthy planting material available to the farmers.

Several methods have been employed to manage plant diseases in crops (Hahn et al., 1988, 1989), but
the most effective approach has been to breed and select for disease-resistant cultivars (Nwankiti et
al., 1987). Most of the existing techniques for selecting resistant cultivars include evaluation for
disease incidence and severity in the field and in the greenhouse. However, these screening procedures
are very cumbersome, time consuming and labour intensive. In addition, field screening requires the
availability of large planting areas.

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Symptoms produced by some pathogen species of Colletotrichum, Curvularia, Helminthosporium,
Pseudomonas, Cochliobolus, Alternaria and Mycosphaerella include spots and blights with a limiting
pathogen-free yellow halo in leaves (Byther and Steiner, 1972; Daub, 1982; Nwankiti, 1982;
Adebitan, 1984; Theberge, 1985; Hartman et al., 1986); dark-brown to black lesions in cotyledons
(CIAT, 1980); symptoms like die-bark, canker and stalk rot in stems; and black canker in pods
(Sharma and Sharma, 1969; Chandraskhanran and Ramakrishnan, 1973; Singh and Allen, 1979;
Theberge, 1985; Singh and Rachie 1985). These symptoms suggest a role for toxic metabolites
secreted by the pathogen during the development of the disease.

Toxic metabolites isolated from some species of Colletotrichum cause symptoms similar to those
caused by the live pathogens. Such toxic metabolites include colletotin from C. fuscum (Goodman,
1960; Lewis and Goodman, 1962), colletotrichin and colletopyrone from C. nicotianae (Masatoshi et
al., 1976, 1978). Identified metabolites from other pathogens include piricularin from Piricularia
oryzea, victorin from Cochliobolus vitoriae, phaseolotoxin from Pseudomonas syringae pv.
phaseolicola, cercosporin from Cercospora spp. and toxins from Periconia circinata and
Helmithosporium sacchari (Samddar and Scheffer, 1968; Byther and Steiner, 1971, 1972; Strobel and
Hapner, 1978; Daub, 1982).

The phytotoxic metabolites of most of these pathogens have been reported to play a significant role in
pathogenesis (Chandraskhanran and Ramakrishnan, 1973; Walker and Templeton, 1978; Amusa 1991,
Amusa et al., 1993). Some of these metabolites, also known as pathotoxins, are chemopathogens of
biological origin that can be used to replace the producing pathogens in studies to investigate the
nature and the development of pathogenesis (Wheeler and Luke, 1963). This is because the
pathotoxins play a causal role in the onset of diseases and produce symptoms characteristic of the
disease in susceptible plants. Thus, phytotoxic metabolites have been used to screen crops for disease
resistance (Wheeler and Luke, 1955; Hartman et al., 1986; Amusa, et al., 1994; Amusa, 1998, 2000).

This chapter reports the development of a method using toxic metabolites of Colletotrichum species
that infect some economic crops in Africa. This method allows for rapid screening of disease
resistance in cultivars of the host crops. It is believed that, if properly employed, this method could be
used as a reliable tool for the rapid screening of large numbers of clones produced by plant breeders
every year and also to predict possible reactions of plants to infections by pathogens.

MATERIAL AND METHODS

Plant material

Cowpea, soybean, maize, cassava and yam plants showing anthracnose symptoms were obtained from
various demonstration plots at the International Institute of Tropical Agriculture (IITA), Ibadan,
Nigeria. Infected sorghum plants were obtained from a private garden located at the University of
Ibadan. Stems and leaves of cassava and yam exhibiting symptoms of anthracnose were also collected
from the Tuber Root Improvement (TRIP) experimental research field located at IITA.

The infected plant parts were excised, cut into 2 mm pieces and surface-sterilised with 10% sodium
hypochloride for 30 s. They were then plated on Acidified Potato Dextrose Agar (APDA) and
incubated for 6 days at 26°C.

The pathogens were identified using cultural, morphological, pathogenicity tests as well as comparing
them with confirmed representatives of the different species. Further confirmation of the identity of
the pathogens was undertaken by the International Mycological Institute, New Surrey, UK.

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Toxin production in culture and extraction

Two media were used for toxin production: modified Richard’s medium (Sharma and Sharma, 1969)
and Czapek-Dox medium (Lewis and Goodman, 1962). 100 ml of each of the media were dispensed
into 250 ml Erlenmeyer flasks and autoclaved. A total of 10 flasks (250 ml Erlenmeyer) were used for
each medium. Each flask was inoculated with three mycelial disks (diameter: 5 mm) cut from the
margin of young growing colonies of the pathogen. Half of the flasks were incubated in a shaker at
100 rpm for 30 days at 26°C with a 12:12 hour photoperiod (Masatoshi et al., 1976). The other half of
the inoculated flasks was incubated statically for 30 days at 26°C with a 12:12 hour photoperiod.
Three culture flasks were removed from the incubator every seven days and the contents were filtered
using six layers of cheesecloth to remove the mycelial mat. The culture filtrates were adjusted to pH3
using 1 N HCl and were then extracted three times with a 1:1 (v/v) of ethylacetate for 48 hours. The
pH of the extracted metabolites was readjusted to pH7 using 1 N NaOH after which they were dried
out at 40°C under reduced pressure using a rotary evaporator. The extracts were then weighed, and the
weight of extractable metabolites from each pathogen and culture medium was recorded.

Partial purification of the toxic metabolites

The extracted phytotoxic metabolites of each pathogen were dissolved in a small quantity of
ethylacetate and then spotted on preparative thin layer chromatography (TLC) on Merck silica gel 60
F254 plates (0.5 mm) that were pre-coated using ethylacetate, acetic acid and water (3:3:1) as the
solvent system (Lewis and Goodman, 1962; Frantzeen et al., 1982). The phytotoxic components were
detected on TLC plates under ultraviolet (UV) light at 254 and 366 nm. The zones that fluoresced
under the UV light were marked and scraped from the plates, and eluted with 250 ml of ethylacetate.
After evaporation of the ethylacetate, the resulting metabolites became an oily brownish semi-solid
substance, which was then tested for bioactivity. Non-fluorescing zones were marked, scraped and
subjected to the same test for bioactivity.

Detached leaf bioassay

Cowpea, soybean, sorghum and maize plants were grown in 1.2 dm3 pots for six weeks in the
greenhouse. These plates were brought to the laboratory 12 hours before being used. Mature leaves
were excised with a sterile razor blade at the petiole and then covered with wet sanitary cotton. 10 µl
of the partially purified toxic metabolites were individually spotted on one half of the leaf. Sterile
Richard’s medium was spotted as a control on the other leaf half. A pin was used to prick through the
centre of each spot to allow infiltration of the solutions. Treated leaves were placed in sterile Petri
dishes and incubated in a high humidity chamber at 28°C and observed after 24 hours. Three leaves of
each test plant were used for each bioassay, and replicated three times.
Seed bioassay

3 ml of the partially purified toxic metabolites at different concentrations (100 µg/ml, 10 µg/ml, 1
µg/ml and 0.1 µg/ml) were poured onto sheets of sterile filter paper placed in Petri dishes. The solvent
was allowed to evaporate completely by leaving the plate open for 12 hours, and then 1 ml of sterile
distilled water was added to each dish. 20 seeds each of cowpea, soybean, maize, sorghum and millet
were placed on the sterile filter papers impregnated with toxic metabolites. After incubation at 30°C
for 24, 48, 72 and 96 hours under continuous light, the number of germinated seeds was counted.
Control experiments were also conducted in the same manner but without the Colletotrichum toxic
metabolites.

Seedling bioassay

3 ml of the toxic Colletotrichum metabolites at concentrations of 100, 1 and 0.1 µg/ml were gently
spotted onto filter papers placed in Petri dishes. After complete evaporation of the solvent, 1 ml of
distilled water was poured into each of the Petri dishes. 10 seedlings each of cowpea, soybean and

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maize and 24 of sorghum and millet were transferred onto the filter papers containing the toxic
metabolites. After incubation at 30°C for 3 days under continuous light, the lengths of the hypocotyls
and roots were measured. The seedlings used in these experiments were obtained by germinating seeds
on wet filter paper at 30°C for a period of two (sorghum and millet) or four days (cowpea, maize and
soybean).

Whole plant bioassay

Test plants were grown on vermiculite for 21 days in a greenhouse. After carefully uprooting, test
plants were placed in test tubes containing 10 ml of the toxic metabolites (100 µg/ml) of the respective
Colletotrichum species pathogenic to the crop. This assay was replicated three times. In the control
experiments, uninoculated sterile Richard’s medium was used. The experiment continued for 24 hours
and observations were made and recorded every 12 hours.

In vitro screening of legumes (cowpea and soybean) for anthracnose disease resistance

14 cultivars of cowpea and soybean were screened with the toxic metabolites of C. lindemuthianum
and C. truncatum. Screening for resistance to anthracnose and brown blotch in both cowpea and
soybean was carried out using leaf, stem and pod puncture bioassay techniques. Eight week old
cowpea and soybean leaves, stems and pods from the greenhouse showing no symptoms of infection
were excised from the shoots and brought to the laboratory. They were rinsed in running tap water,
dipped in 10% NaOCl for 30 s and then rinsed in five changes of sterile distilled water. The plant parts
were individually placed in sterile Petri dishes lined with moistened sterile filter paper. Subsequently,
three 10 μl droplets of the concentrated phytotoxic metabolites were placed on these plant parts, which
were then punctured with sterile needles at the centre of each droplet to allow infiltration. Ten leaves,
stems and pods were inoculated per cultivar of each crop type and these were replicated three times.

In vitro screening of maize (Zea mays) for anthracnose diseases using toxic metabolites

To establish an in vitro screening method for anthracnose resistance in cereals, the maize inbred line
1787 obtained from IITA germplasm that is known to be susceptible to C. graminicola, i.e., the
causative agent of anthracnose leaf blight (ALB) and anthracnose stalk rot (ASR), was used. When
plants were 12 weeks old, the leaves were excised with razor blades, cut into 8 cm long pieces, and
covered with wet sanitary cotton. 10 μl of the metabolites of the pathogen were spot inoculated on one
half of each leaf piece. On the other half, spots of uninoculated Richard’s medium were placed as a
control. A sterile pin was used to prick though the centre of each spot. The treated leaves were placed
in sterile, moist Petri dishes and incubated for 24 hours. Three leaves were inoculated per plant, a total
of five plants were selected and these were replicated three times.

Subsequently, seven other maize inbred lines raised in pots in the greenhouse were used for the
bioassay. Six week old maize leaves from the greenhouse showing no symptoms of infection were
excised from the shoot and brought to the laboratory. The leaves were cut into 8 cm long pieces, rinsed
in running tap water, dipped in 10% NaOC1 for 30 s and then rinsed in five changes of sterile distilled
water. The surface-sterilised maize leaves were individually placed in sterile Petri dishes lined with
moistened sterile filter paper. Three 10 μl droplets of the concentrated phytotoxic metabolites were
placed on these plant parts, which were then punctured with sterile needles at the centre of each
droplet. Five leaves were inoculated per maize inbred line and these were replicated four times. This
experiment was repeated three times.

In vitro screening of tuber root crops (cassava and yam cultivars) for disease resistance

Screening for resistance in both cassava and yam clones was performed using leaf and stem puncture
bioassay techniques. 45 clones of cassava and 24 clones of yam were evaluated using toxic
metabolites of the pathogens. Stems and leaves from two month old plants of cassava and yam

176
showing no symptoms of infection were excised from the main shoots and taken to the laboratory.
They were rinsed in running tap water, dipped in 10% NaOCl for 30 s, and then rinsed again in sterile
distilled water. Cassava and yam stems were cut into pieces (10 cm). Excised leaves and stems were
placed in sterile Petri dishes lined with moistened sterile filter papers. The partially purified phytotoxic
metabolites (10 μg/ml) were spotted on the stem cuttings and excised leaves. A total of four droplets
were placed on each leaf or stem. Five leaves and stems per clone were used, and these were replicated
three times. The punctured leaf surfaces were encircled with petroleum jelly to avoid the spread of the
metabolites and incubated for 12 hours. Inoculated stems were incubated for 10 days. Distilled water
and uninoculated Richard’s medium were used as controls.

Rating resistance and susceptibility levels in the in vitro toxic metabolite assays

The reaction to the toxic metabolites was rated according to a modified lesion diameter scale (Goodie,
1958; Sudi and Podhardizky, 1959) to determine the level of resistance of the plant material to
anthracnose. The diameter of the induced necrotic lesions was recorded and transformed to log values.
The degree of resistance or susceptibility was determined using the following scale: <7.0 mm = highly
resistant; 7.1-11.0 mm = resistant; 11.1-15.0 mm = moderately susceptible; and >15.1 mm =
susceptible. The data were then subjected to analysis of variance (ANOVA) and the Duncan’s multiple
range (DMR) test to separate the means at p < 0.05.

Field screening of tuber root crops (cassava and yam cultivars)

45 cassava and 24 yam clones were planted in 1996 and 1997 in the Tuber Root Improvement
Programme (TRIP) experimental research field located at the IITA, Ibadan, Nigeria. The institute lies
in the lowlands rain forest belt with a mean annual rainfall of 1000-1500 mm, and temperatures
ranging around 23-32°C and 19-35°C during the wet (April to October) and dry (November to March)
seasons, respectively. The IITA is located to the north of Ibadan at latitude of 7° 31' N and a longitude
of 3° 45' E, and at an altitude of 210 m above sea level.

Cultivation of cassava germplasm

Each cassava cultivar was planted in a 10 × 10 m sized plot and spaced at 1 × 1 m between and within
rows. A cassava stem cutting ~12 cm in length was planted by partial burial of the stem on the ground,
leaving about two nodes unburied. The design used was the completely randomised block design with
four replicates for each cultivar, with each plot containing 100 plants. The plots were weeded
manually three times during the course of the experiments.

All the cassava plants in each plot, except those at the edge, were assessed at 3, 6 and 9 months after
planting (MAP) for the incidence and severity of cassava anthracnose disease (CAD). The cassava
plants were examined from top to bottom for symptoms of CAD infection. The severity score was
based on a scale of 1-5 (Ikotun and Hahn, 1991): 1 = no visible symptoms; 2 = development of
shallow canker on the lower part of the plant; 3 = development of successive canker higher up the
plant with the older canker becoming larger and deeper; 4 = development of a dark brown lesion on
green shoot, petiole and leaves, young shoot and young leaves; 5 = death of part of or whole plant.

Disease scores for field tests were calculated as the mean value between the severity score as follows:
< 2.0 = resistant; 2.0-3.0 = moderately susceptible; > 3.0 = susceptible. The data collected were
subject to ANOVA, with inferences drawn from the analysed data using DMR tests.

Cultivation of yam germplasm

Each yam cultivar was planted in a 10 × 10 m sized plot and spaced at 1 × 1 m between and within
rows. In each plot, mounts were made and on each mount, a yam minisett was planted by burying it
under the soil within the mount. The design used was the completely randomised block design with

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four replicates for each cultivar, with each plot containing 100 plants. The plots were weeded
manually three times during the course of the experiments.

All the yam plants in each plot, except those at the edge, were assessed at 3, and 6 MAP for the
incidence and severity of yam anthracnose disease (YAD). The yam foliage and the stems were
examined for symptoms of infection. The severity score were based on a scale of 1-5 (IITA, 1993): 1 =
no symptoms (highly resistant); 2 = necrotic spots 1.00-2.00 mm in diameter (resistant); 3 = necrotic
spots 2.01-3.00 mm in diameter (moderately susceptible); 4 = necrotic spots of 3.01-4.00 mm in
diameter (susceptible); 5 = necrotic spots >4.00 mm in diameter (highly susceptible).

The results were pooled and subjected to ANOVA. Comparisons were made using the DMR test.

RESULTS AND DISCUSSION

Conditions for the production of toxic metabolites in vitro

Isolates of C. truncatum, C. lindemuthianum, C. gloeosporioides, C. gloeosporioides f. sp. manihotis

and C. graminicola used in this study were tested for the production of toxic metabolites in both
Richard’s and Czapek-Dox media. Colletotrichum cultured in Richard’s medium under constant
shaking produced the highest concentrations of toxic metabolites, whereas cultures in this medium
under still incubation ranked second in the production of toxic metabolites. Cultures in Czapek-Dox
medium incubated with and without shaking ranked third and fourth, respectively, as shown in Table
11.1. Colletotrichum species have been previously reported to produce phytotoxic metabolites when
cultured in vitro (Goodman, 1960; Sharma and Sharma, 1969; Masatoshi et al., 1976). Richard’s
medium was found to support higher production of toxic metabolites than the Czapek-Dox medium.
This might be connected to the nutrients present in the Richard’s medium (Frantzeen et al., 1982). The
presence of these nutrients, at the required concentration, is necessary for effective toxin production in
culture (Amusa, 1991). Greater concentrations of toxic metabolites were produced when cultures were
shaken. Shaking incubations provide suitable aeration to the culture medium, supporting growth of
fungi and, as such, this condition has been well exploited for toxin production (Masatochi et al., 1976,
1978; Frantzeen et al., 1982; Amusa, 1991).

When subjected to TLC chromatography, the bands containing the phytotoxic metabolites fluoresced
under ultraviolet light at 254 and 366 nm. The toxic metabolites of C. truncatum and C.
lindemuthianum had a band corresponding to a retention factor (RF) of 0.70-0.75; toxic metabolites of
C. gloeoesporioides and C. gloeosporioides f. sp. manihotis had a band at RF 0.65-0.70; the RF band
obtained for C. graminicola was at 0.75-0.80. Metabolites from the non-fluorescent bands induced
neither chlorotic nor necrotic lesions on test plants.

Toxic metabolites were produced in seven day old cultures of all Colletotrichum species whereas, 28
day old cultures were found to be more effective in inducing symptoms on susceptible hosts because
they contained larger amounts of extractable metabolites of the pathogens (Figure 11.1A-C). There
were no significant differences (p < 0.05) between the amounts of the extractable metabolites obtained
from 21 and 28 day old cultures. Phytotoxic metabolite activity detected in seven day old cultures
suggests that the production of toxic metabolites probably commences early during infection and may
play an important role in pathogenicity and expression of symptoms. The production of fluorescent
compounds such as colletotrichin and colletopyrone produced by C. nicotianae (Goodman, 1960;
Lewis and Goodman, 1962; Masatochi et al., 1976, 1978), and collectotin, a glycopeptide from C.
fuscum, has been previously reported (Goodman, 1960).

Toxic activity of Colletotrichum metabolites

In order to assess the activity of the partially purified metabolites produced by the Colletotrichum
species, tests with detached leaves and stems were conducted. The toxic metabolites induced necrotic

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lesions of varying sizes on leaves and stems of susceptible hosts. Chlorotic lesions were induced and
became visible on the leaves of test plants within 3 hours of inoculation. About 10 hours after
inoculation, the chlorotic lesions become necrotic surrounded by a yellow halo (Figure 11.2). The
toxic metabolites of these Colletotrichum species also affected the germination of the test crops as
shown in Table 11.2. No germination was recorded when seeds were treated with 100 µg/ml of the
metabolites, while at 10 µg/ml of the toxic metabolites, germination of 7-30% was recorded. The
percentage of germination increased when lower concentrations of metabolites were used, suggesting
that the toxic metabolites are responsible for the inhibition of seed germination and prevention of
seedling growth. It is speculated that the phytotoxins produced by these Colletotrichum species have a
lethal effect on the seeds and seedling tissues. Colletotrichum species are known to infect seeds and
prevent germination and, in cases where germination occurs, the seedlings soon die.

Results of the seedling bioassay revealed that seedlings treated with a 100 μg/ml solution showed
symptoms of blight disease and ceased to grow. Although the severity of symptoms caused by the
metabolites decreased with increased dilutions, at a concentration of 0.1 µg/ml, there was a slight
increase in the length of both roots and shoots of the cowpea and soybean seedlings as compared to
controls without the phytotoxins (Figure 11.3A, B). These observations suggest increased seedling
vigour at low concentrations of the phytotoxins for reasons not yet understood.

The effect of the Colletotrichum toxic metabolites (100 µg/ml) on the test crops was very rapid and
could be observed already within 12 hours of exposure. The first observable symptom on these young
test crops was the yellowing of the leaves, especially those at the base. This was followed by drooping
and wilting of the leaves; the plants then collapsed and eventually died. At 100 µg/ml, the metabolites
kill young plant shoots, perhaps by affecting the function of the vascular tissues. A high degree of
correlation was seen between the response of cassava clones to natural infection by C. gloeosporioides
f. sp. manihotis and to the toxic metabolites in vitro (Table 11.3).

In vitro screening technique to determine resistance and susceptibility of legumes to anthracnose

The metabolites of C. truncatum and C. lindemuthianum in culture were also phytotoxic to cowpea
and soybean explants in vitro, and the symptoms produced by these metabolites were similar to those
induced by the pathogens themselves. Leaf explants in vitro showed much larger lesions than stem
cuttings and pods (Table 11.4). Due to the large variation in lesion sizes between these explants, the
leaf lesion size was found to be more suitable for calculating the level of resistance/susceptibility of
the cowpea cultivars. Among the 14 cowpea cultivars treated with C. truncatum phytotoxins, three
cultivars (‘IT82D-994’, ‘IT82D-699’ and ‘IT81D-773’) exhibited induced necrotic lesions averaging
between 11.6 and 13.9 mm in diameter in the leaves, and were considered to be moderately
susceptible to the toxic metabolites. The remaining 11 cultivars had leaf lesions >15.1 mm and were
considered to be susceptible. Mean lesion values between leaves, stems and pods of the cultivars
‘IT82D-994’, ‘IT82D-699’ and ‘IT81D-773’ were found to be statistically (p < 0.05) lower than for
the other cultivars (Table 11.4).

All 14 cowpea cultivars displayed large leaf necrotic lesions and were considered susceptible to the
metabolites of C. lindemuthianum. Averages between leaf, stem and pods lesions suggest that the
cultivars ‘IT82D-994’, ‘IT81D-773’, ‘IT82D-699’, ‘TVU-3232’, ‘848-2245-4’, ‘IT82D-60’ and ‘IFE
BROWN’ may be slightly less susceptible than the other cultivars; however, these findings were not
statistically significant (Table 11.4).

The cowpea cultivars ‘IT82D-994’, ‘IT82E-32’ are reportedly susceptible to anthracnose and brown
blotch diseases (Adebitan, 1991). This corresponds well to the results of in vitro tests in this study.
Similarly, field and screen-house studies showed that ‘IFE BROWN’ and ‘IT82D-60’ are susceptible
to C. lindemuthianum and C. truncatum (Emechebe and Soyinka, 1985), which also corresponds to the
reaction of these two cowpea cultivars to the metabolites of the pathogens in vitro.

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Explants of soybean treated with toxic metabolites showed less variance in the lesion sizes in leaves
and pods; therefore, mean lesion values between these two types of explants were suitable to estimate
level of resistance and susceptibility. ‘PI-17144’ and ‘TGM236’ had mean necrotic lesions of 5.3 and
10.3 mm, respectively. ‘PI-17144’ was considered highly resistant, and ‘TGM236’ was resistant to
soybean anthracnose The remaining 12 cultivars were found to be susceptible with mean lesions >15.1
mm (Table 11.5). The soybean cultivars ‘PI-17144’ and ‘TGM236’ have been reported to be resistant
to anthracnose (IITA, 1987), whereas the reaction of the susceptible cultivars to the in vitro test with
toxic metabolites of C. truncatum corresponded to the greenhouse and field-screening experiments
(IITA, 1987). Therefore, the results of the present study show that in vitro screening for disease
resistance using toxic metabolites obtained from C. trunctum and C. lindemuthianum is practicable
and a good indicator of resistance to these pathogens. Also, the use of these metabolites allows for the
rapid screening of a large population of crops and cultivars within a short period of time (24 hours), as
also shown by Hell and Weber (1986).

In vitro screening technique to determine anthracnose resistance in maize and cereal crops

Initial in vitro tests using the maize inbred line 1787, which is known to be susceptible to anthracnose,
and the toxic metabolites of C. graminicola revealed the induction of necrotic lesions on leaves within
24 hours of inoculation. On the other hand, in control experiments, the leaves of line 1787 were
inoculated without the toxic metabolites and did not show any symptoms.

Similar tests were then performed with six other maize inbred lines in comparison to 1787. Inoculation
of lines 5057, 1787, 9030, 1201 and 5012 with the toxic metabolites of C. graminicola induced the
development of necrotic lesions ranging from 18.8 to 22.6 mm in diameter (Table 11.6). These maize
lines were rated as susceptible to the development of necrotic lesions caused by toxic metabolites of C.
graminicola. On the other hand, Ku 1414 and 9450 exhibited necrotic lesions of 11.2 and 12.9 mm,
respectively, and were considered moderately susceptible to the toxic metabolites of C. graminicola
(Table 11.6). In 1988, IITA reported the high susceptibility of maize inbred lines 9030, 1787, 1201
and 5057 among others developed at the IITA to anthracnose stalk rot, while Ku 1414 and 9450 were
found to be resistant to both stalk rot and leaf blight phases of the disease in the field and in
greenhouse experiments (IITA, 1988). Based on the similar responses of these seven maize inbred
lines to in vitro inoculation with toxic metabolites of C. graminicola and to the causal agents (i.e., live
pathogens), we conclude that it is possible to screen for resistance to anthracnose disease in maize
using a fast and reliable in vitro method with toxic metabolites of this pathogen. Likewise, toxic
metabolites of some fungal pathogens have been used as a tool for rapid screening of a large number
of cereals, such as rice, oats, sorghum and maize (Wheeler and Luke, 1955; Schertz and Tai, 1969;
Byther and Steiner, 1971, 1972; Gengenbach et al., 1977) for resistance against some fungal diseases
of these cereal crops.

Tuber and root crops (in vitro screening technique)

The 24 clones of yam (Dioscorea sp.) reacted differentially to the induction of necrotic lesions by the
toxic metabolites of C. gloeosporioides. Based on these reactions, yam clones were categorised as
susceptible, moderately susceptible and resistant (Table 11.7). Out of the 24 clones, only TDc750,
TDr179 and TDr89/01750 were completely resistant. TDe, TDa87/01117 and TDr87/00211 were
moderately susceptible, with necrotic lesions <15.0 mm. The remaining 18 clones exhibited varying
degrees of susceptibility (Table 11.7). Yam clones TDr 89/01750, TDr 179 and TDr 750, with scores
between 1.0 and 1.60, were regarded as resistant based on the field disease scores. The other 18
clones, with disease scores of between 2.00 and 3.62, were susceptible to varying degrees (Table
11.7). Moreover, there was a positive and significant correlation (r = 0.890) between the reactions of
the yam clones to natural infection in the field and to the toxic metabolites of the pathogen.

11 of the 45 clones of cassava (Manihot esculenta) treated with toxic metabolites of C.

gloeosporioides f. sp. manihotis had necrotic lesions <7.0 mm and were considered to be highly

180
resistant (Table 11.8). Eight other clones were considered resistant, with lesion sizes ranging between
7.1 and 11.0 mm. Nine other clones were considered moderately susceptible, while 17 clones
exhibited varying degrees of susceptibility (Table 11.8). Based on the field disease scores of the same
45 cassava clones, 17 clones were found to be resistant, 10 moderately susceptible and the remaining
17 clones were susceptible to varying degrees (Table 11.8). The resistant clones in the field included
those that were rated resistant and highly resistant in the in vitro assay. Likewise, the 17 susceptible
clones in the field were also found to be susceptible in vitro. Therefore, a positive and significant
correlation existed between the results of the field and the in vitro screening tests of the 45 cassava
clones (Table 11.8).

Taken together, the present results reveal a strong positive correlation between field disease score,
based on natural infections, and necrotic lesion sizes induced on the plant tissue using toxic
metabolites in vitro. Moreover, the disease reactions of these cassava and yam clones were similar to
those obtained from several other field screenings using natural infection in previous studies (IITA,
1993). For instance, yam clones TDr89/01750 and TDr179 were reported as resistant to yam scorch
(anthracnose), while TDa289 and TDa291 were regarded as very susceptible (IITA, 1993). These
results correspond well with the results of this study. Similarly, in field and screen-house studies, the
cassava clones Tms30211, Tms63391 and Tms30572 were found to be resistant to cassava
anthracnose (Ikotun and Hahn, 1991), while 4(2)1443 was reported to be susceptible (IITA, 1993).
Furthermore, the in vitro screening experiment was completed within 24 hours of inoculation. Hence,
several thousand cassava and yam germplasms can be screened within a short period of time by this
method.

In cases where toxins act as the sole determinants of the diseases in plants, knowledge of such toxins
may be used to control the diseases. Quchi et al. (1989) described the detoxification of fusaric acid
produced by Fusarium spp., that cause wilt of tomato plants, with the use of Pseudomonas
solanacearum A-16 and Cladosporium werneckii. These two organisms produced detoxifying
compounds and these, in combination with the organisms, have been used to protect young tomato
plants against Fusarium wilt. Since then, this method has been useful in other crops for disease
screening (Wheeler and Luke, 1955; Sudy and Podhardszky, 1959; Rines and Luke, 1985; Hartman et
al., 1986; Amusa, 1991). The results of this experiment suggest that screening for disease resistance
with toxic metabolites extracted from Colletotrichum spp. is feasible. This method also has the
potential to be more efficient and less expensive than field screening.

CONCLUSIONS

Effective field screenings using the causative pathogens of anthracnose disease take, on average, two
to three years of activities, and depend on several environmental factors that may affect the
pathogenicity level of the inoculum. Therefore, field screenings are not only very cumbersome and
time consuming, but also highly variable.

The production of phytotoxic metabolites by the five Colletotrichum species in this study has given an
insight into one of their modes of attack on plant tissues and, hence, could help breeders in breeding
for resistance. Since there are variations in the mode of reaction of the host plant tissues to phytotoxic
metabolites of the pathogens, the phytotoxin-tolerant host crops could serve as a marker in breeding
for resistance. However, since the metabolites of the pathogen affect plant tissues directly, and
resistance and susceptibility to diseases in plants is usually genetically controlled, environmental
factors that normally affect conventional field-screening methods are totally eliminated. Positive and
significant correlation was found to exist between the results of the field screening of the 45 cassava
and 24 yam clones using natural infections and necrotic lesion sizes induced on the plant tissues using
toxic metabolites of the pathogen. The in vitro screening experiment was completed within 36 hours of
inoculation. Hence, several thousand cowpea, soybean, maize, cassava and yam germplasms can be
screened within a short period of time by this method. The success of this experiment may, however,
depend on the virulence of the strains of the pathogen used.

181
The future looks bright as new, more effective and safer management techniques find their way into
plant disease management. Some of these new techniques actually involve the development of new
cultivars with high and dependable levels of resistance to important diseases within a very short time.
Traditional breeding techniques will continue to play a role in varietal development, but the use of
new and rapid techniques of evaluation promise to accelerate the development of resistant cultivars.
The chemical industry should devote more time and resources to producing some of these phytotoxins,
which are environmentally friendly, highly dependable bio-control Material that will greatly improve
our ability to manage some of the more important diseases affecting important crops.

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Andrus and More. Dissertation, University of Ibadan, Nigeria, 89 pp.

Adebitan SA (1991) Factors influencing infection, spread and severity of two Colletotrichum species
on cowpea (Vigna unguiculata (L) Walp) under different cropping systems. PhD thesis, Department of
Agricultural Biology, University of Ibadan, Nigeria, 255 pp.

Amusa NA (1991) Extraction, characterization and bioassay of metabolites of some plant pathogenic
species of Colletotrichum. PhD thesis, University of Ibadan, Nigeria, 210 pp.

Amusa NA (1996) Responses of corn (Zea mays) to in vitro inoculation with toxic metabolites of
Colletotrichum graminicola Int. J. Trop. Plant Dis. 14: 107-111.

Amusa NA (1998) Evaluation of cassava clones for resistance to anthracnose disease using phytotoxic
metabolites of Colletotrichum gloeosporioides f. sp. manihotis and its correlation with field disease
reactions. Trop. Agric. Res. Ext. 1: 116-120.

Amusa NA (2000) Screening of cassava and yam cultivars for resistance to anthracnose using
phytotoxic metabolites of Colletotrichum species Mycopathology 150: 137-142.

Amusa NA, Ikotun T, Asiedu R (1993) Extraction of a phytotoxic substance from Colletotrichum
gloeosporioides infected yam leaves. Int J Trop Plant Dis 11: 207-211.

Amusa NA, Ikotun T, Osikanlu YOK (1994) Screening cowpea and soybean cultivars for resistance to
anthracnose and brown blotch diseases using phytotoxic metabolites. Afr. Crop Sci. J. 2: 221-224.

Byther FC, Steiner GW (1971) Partial characterization and use of a host-specific toxic from
Helminthosporium sacchari on sugar cane. Phytopathology 61: 691-695.

Byther SR, Steiner GW (1972) Use of Helminthosporoside to select sugar cane seedling resistant to
eye spot disease. Phytopathology 61: 691-695.

Chandraskharan MN, Ramakrishnan K (1973) Production of toxic metabolites by Colletotrichum

capsici (Syd.) Butl. and Bisby and its role in leaf spot disease of turmeric. Curr. Sci. 47: 362-363.

CIAT (1980) Annual Report. Centro Internacional de Agricultura Tropical (CIAT), Cali, Columbia, 96
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Daub EM (1982) Cercosporin a photosensitizing toxin from Cercospora species. Phytopathology 72:
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Emechebe AM (1981) Brown blotch of cowpea in Northern Nigeria. Samaru J. Agric. Res. 1: 20-26.

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Rachie KO (eds.) Cowpea Research Production and Utilization. John Wiley and Sons, New York,
USA, pp: 173-192.

Frantzeen KA, Johnson LLB, Stitevile DL (1982) Partial characterization of phytotoxic

polysaccharides produced in vitro by Colletotrichum trifolii. Phytopathology 72: 568-573

Gengenbach BG, Green CE, Donova CM (1977) Inheritance of selected pathotoxin resistance in maize
plants regenerated from cell cultures. Proc. Nat. Acad. Sci. USA 74: 5113-5117.

Gengenbach BG, Rines HW (1986) Use of phytotoxin in selection of disease resistance mutants in
tissue culture. IOWA State J. Res. 60: 449-476.

Goodie MJ (1958) Physiological specialization in Colletotrichum lagenarium. Phytopathology 48: 79-

83.

Goodman KN (1960) Colletotin a toxin produced by Colletotrichum fascum. Phytopathology 50: 325-
327.

Hahn SK, Isoba JCG, Ikotun T (1989) Resistance breeding in root and tuber crops at the International
Institute of Tropical Agriculture (IITA), Ibadan, Nigeria. Crop Protec. 8: 147-168.

Hahn SK, Isoba JCG, Ikotun T, Theberge RL, Swenne R (1988) Major economic disease of Cassava
and plantains in Africa. Trop. Agric. Res. Ser. 22: 106-112.

Hartman CL, Scor GA, Venette, JR, Albaugh DA (1986) Response of bean calli to filtrate from
Pseudomonas syringae pv. phaseolicola and correlation with whole plant disease reaction. Physiol.
Mol. Plant Pathol. 28: 353-358.

Hell WH, Weber DJ (1988) Assay for determining resistance and susceptibility of onion cultivars to
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IITA (1987) Annual Report. International Institute of Tropical Agriculture, Ibadan, Nigeria, 92 pp.

IITA (1988) International Institute of Tropical Agriculture Strategic Plan 1989-2000. International
Institute of Tropical Agriculture, Ibadan, Nigeria, 89 pp.

IITA (1993) Root and Tuber Improvement Program Archival Report (1998-1993) Part 111.
International Institute of Tropical Agriculture, Ibadan, Nigeria, 118 pp.

Ikotun T, Hahn SK (1991) Screening cassava cultivars for resistance to the cassava anthracnose
disease. In: Proceedings of the 9th Symposium of the International Society of Tropical Root Crops,
African Branch pp: 178-183.

Lewis RB, Goodman RM (1962) Morphological effects of Colletotin on tomato and digitalis foliage.
Phytopathology 52: 1273-1276.

Masatoshi C, Kosuge Y, Sunao Y, Suzuki A, Tamora S (1978) Isolation and structure and biological
activities of phytotoxic substance from Colletotrichum nicotianae. Agric. Biochem. 42: 1037-1043.

Masatoshi C, Svong-be Hyeon, Suzuki A, Tamora S (1976) Isolation and structure elucidation of
colletopyrone from Colletotrichum nicotianae. Agric. Biochem. 42: 1453-1455.

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Nwankiti AO (1982) Studies on the etiology and control of anthracnose/brown blotch disease complex
of Dioscorea alata in Nigeria. PhD thesis, University of Nigeria, Nsukka, Nigeria, 140 pp.

Nwankiti AO, Okoli OO, Okpala EU (1987) Screening of water yam (Dioscorea alata) cultivars for
tolerance to anthracnose blotch disease. Fitopathologia Brasilensis 12: 35-39.

Quchi SH, Toyoda R, Utsimi H, Hashimoto T, Hadana (1989) A promising strategy for the control of
fungal disease by the use of toxin-degrading microbes. In: Graniti A, Durbin P, Ballio A (eds.)
Phytotoxin and Plant Pathogenesis. Proceedings of the NATO Advanced Research Workshop on
Phytotoxins and Plant Pathogenesis, Capri, Italy, 30 May-3 June 1988 (NATO ASI Series. Series H:
Cell biology, vol 27). Springer-Verlag, Berlin: 319-335

Rines HW, Luke HH (1985) Selection and regeneration of toxin sensitive plants from tissue culture of
oats. Theor. Appl. Genet. 71: 16-21.

Samaddar KK, Scheffer RP (1968) Effect of the specific toxin in Helminthosporium victoriae on host
cell membrane. Physiology 43: 21-28.

Schertz KF, Tai YP (1969) Inheritance of reaction of Sorghum bicolor (L) Moench to toxin produced
by Periconia circinata Mang. Sacc. Crop Sci. 9: 621-624.

Sharma MC, Sharma BC (1969) Toxic metabolites produced by Colletotrichum gloeosporioides

causing citrus die-back in India. Phytopathology 22: 67-72.

Sinclair JB (1988) Anthracnose of Soybean. In: Wyllie TO, Scoof DH (eds.) Soybean Disease of the
North Central Region, 187 pp.

Singh SR, Allen DJ (1979) Cowpea pests and diseases, IITA, Ibadan, 89 pp.

Singh SR, Rachie KO (1985) Cowpea Research Production and Utilization. John Wiley and Sons,
New York, USA, 460 pp.

Strobel GA, Hapner KD (1975) Transfer of toxin susceptibility to plant protoplasts via the
helminthosporoside binding protein of sugarcane. Biochem. Biophys. Res. Commun. 63: 1151-1156.

Sudi J, Podhardszky J (1959) Rizsjajtank Piriculariareziszten ciajanakgyors et biraiasa U. Yeghazi

Vizsgalattial (Rapid test for determining resistance of Pyricularia disease in greenhouse). Kise-koz
52/A: 3-16.

Theberge LR (1985) Common African Pest and Diseases of Cassava, Yam, Sweet Potato and
Cocoyam. IITA, Ibadan, Nigeria, 85 pp.

Walker HL, Templeton GE (1978) In vitro production of phytotoxic metabolites by Colletotrichum

gloeosporioides f. sp. aeschynomenes. Plant Sci. Lett. 12: 91-96.

Wheeler HH, Luke HH (1955) Mass screening for disease resistance mutants in oats. Science 122:
1220-1229.

Wheeler HH, Luke HH (1963) Microbial toxin in plant diseases. Annu. Rev. Microbiol. 17: 223-242.

184
Table 11.1. Effect of media and incubation on the production of toxic metabolites by some species of
Colletotrichum

Pathogen Extractable metabolite (mg)

Colletotrichum spp. Medium Shaking Still
C. graminicola Richard’s 590 360
Czapek Dox 298 200
C. lindemuthianum Richard’s 540 325
Czapek Dox 285 210
C. truncatum Richard’s 390 275
Czapek Dox 290 210
C. gloeosporioides f. sp. manihotis Richard’s 500 300
Czapek Dox 260 205
C. gloeosporioides Richard’s 410 295
Czapek Dox 235 190

The amount of toxic metabolite recorded above was obtained from a pool of 750 ml of each of the five
pathogens. Source: Amusa (1991).

Table 11.2. Percentage of germination of cowpea, soybean, maize, sorghum and millet seeds with
varying concentration of metabolites from Colletotrichum species

Germination (%)
Pathogen producing
Test plant Concentration of toxic metabolite (µg/ml)
toxic metabolites
100 10 1 0.1 Control
Cowpea (IT82-60) C. lindemuthianum 0 10 60 100 100
Cowpea (IT82-60) C. truncatum 0 7 50 95 100
Cowpea (TVx-2336) C. lindemuthianum 0 8 58 100 100
Cowpea (TVx-2336) C. truncatum 0 8 52 100 100
Soybean (Samsoy) C. truncatum 0 10 49 93 100
Maize C. graminicola 0 20 60 100 100
Sorghum C. graminicola 0 38 68 100 100
Millet C. graminicola 0 21 64 100 100

Source: Amusa (1991).

Table 11.3. Pearson correlation coefficient of the response of cassava clones to natural infection of
Colletotrichum gloeosporioides f. sp. manihotis and to the toxic metabolites

Field disease Necrotic lesion sizes Necrotic lesion

score on leaves (in vitro) sizes on stem
(in vitro)
Field disease score - 0.9038 0.9586
Necrotic lesion sizes on leaves (in vitro) - 0.9053
Necrotic lesion sizes on stem (in vitro) -

Values are significant at p < 0.001 (n = 45). Source: Amusa (2000).

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Table 11.4. Response of 14 cowpea cultivars to in vitro inoculation with phytotoxic metabolites of
Colletotrichum truncatum and C. lindemuthianum

Mean size of necrotic lesion (mm)

Cultivar C. truncatum C. lindemuthianum
Leaves Stems Pods Χ Leaves Stems Pods Χ
IT282E-16 22.2 16.2 14.7 17.7a 20.7 16.3 14.6 17.2a
TVU3236 21.7 16.0 13.4 17.0a 21.6 15.2 14.1 16.9ab
TVU-300 20.4 15.4 14.6 16.2ab 17.5 15.9 14.9 16.1bc
IT82E-32 19.2 15.4 14.4 16.3ab 15.9 15.5 14.6 15.0cd
TVU-1994 18.3 16.2 14.9 16.4ab 18.3 15.2 13.9 15.8cd
TVU-1990 18.2 15.5 14.4 16.2ab 18.5 14.1 12.8 15.3cd
IT81D-1137 16.2 15.7 13.8 15.2bc 16.9 16.9 14.1 15.3cd
IFE BROWN 16.5 16.4 14.7 15.9bc 18.6 15.6 13.9 14.7d
IT82D-60 16.4 15.9 14.7 15.7bc 18.2 15.3 13.9 14.5d
848-2245-4 16.6 14.7 13.7 15.0bc 16.2 14.4 14.1 14.2d
TVU-3232 15.4 14.5 13.4 14.4c 15.2 14.2 13.8 14.0d
IT82D-699 13.9 13.7 11.1 12.9d 17.2 14.9 13.1 14.0d
IT81D-773 13.5 13.0 11.5 12.7d 15.5 14.8 13.7 14.2d
IT82D-994 11.6 12.1 11.2 11.6d 15.7 14.2 13.3 13.7d

Each value is a mean of five replicates (5 measurements/plant) and is a transformation from the loge
value used for the analysis. Mean values between leaves, stems and pods (X) followed by the same
letters are not significant at p < 0.05 by Duncan’s multiple range test. Source: Amusa et al. (1994).

Table 11.5. Response of 14 soybean cultivars to in vitro inoculation with phytotoxic

metabolites of Colletotrichum truncatum

Mean necrotic lesion sizes (mm)

Cultivars Leaves Pod Χ
536D 23.6 19.4 21.5a
932-2E 23.6 18.9 21.3a
TGX923D 22.9 18.8 20.9ab
SAMSOY 25.6 18.3 20.3ab
1614-1E 21.9 19.3 20.6ab
TGM-337 20.7 18.9 19.8bc
TGM-298 20.3 18.5 19.4bc
1-851D 19.2 16.7 18.2cd
TGM-297-2 18.1 17.2 17.7de
TGM-623 18.3 15.3 16.8def
TGX-536-02D 17.8 15.2 16.5ef
TGM-705 15.2 15.0 15.4f
TGM-236 10.6 10.1 10.3g
PI-17144 3.5 7.2 5.3h

Each value is a mean of five replicates (5 measurements/plant) and is a transformation from the loge
value used for the analysis. Each value within the same column is a mean of three replicates
(30 plants/clone). Mean values between leaves and pods (X) followed by the same letters are not
significant at p < 0.05 by Duncan’s multiple range test. Source: Amusa et al. (1994).

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Table 11.6. Response of seven maize inbred lines to in vitro inoculation with phytotoxic metabolites
of Colletotrichum graminicola

Maize inbred lines Mean necrotic size (mm)1

5057 22.6a
1787 22.1ab
9030 21.9b
1201 20.9b
5012 18.8c
9450 12.9d
Ku1414 11.0d
1
Mean values followed by the same letters are not significant at p < 0.05 by Duncan’s multiple range
test. Source: Amusa (1996).

Table 11.7. Response of 24 yam clones to in vitro inoculation with phytotoxic metabolites of
Colletotrichum gloeosporioides in comparison with field screening

In vitro screening Field screening

Clone Necrotic lesion Disease score Necrotic lesion Disease score
size on leaves size on leaves
TDa87/01116 22.62a S 3.61a S
TDc289 22.49a S 3.61a S
TDa86/001115 22.49a S 3.60a S
TDa86/00258 22.45a S 3.57a S
TDa85/01093 22.11b S 3.43a S
TDa85/00272 20.11b S 3.32ab S
TDa85/00601 20.07b S 3.19abc S
TDr85/00272 19.91b S 2.54cd MS
TDr87/00203 18.87c S 2.51cd MS
TDa291 18.81c S 2.39de MS
TDr86/00057 18.56c S 2.47de MS
TDa87/00293 18.447c S 2.16de MS
TDa297 17.23d S 2.26de MS
TDa86/00057 17.01d S 2.08ef MS
TDr87/00340 16.79d S 2.12e MS
TDr293 15.77e S 2.10e MS
TDa294 15.35e S 2.01ef MS
TDe 15.08e S 2.00ef MS
TDa87/01117 12.58f MS 1.60fg R
TDr87/00211 12.58f MS 1.58fg R
TDa5 12.35f MS 1.58fg R
TDc750 9.86gh R 1.54g R
TDr179 8.75h R 1.41g R
TDr89/01750 8.59h R 1.30g R

Each value is a mean of five replicates (% measurement/plant), each is a transformation from the loge
value used for the analysis. Each value within the same column is a mean of three replicates (30 –
plants/clone). Mean value followed by the same letters are not significant at p < 0.05 by Duncan’s
multiple range test. S = susceptible; MS = moderately susceptible; R = resistant. TDa = Tropical
Dioscorea alata; TDr = Tropical Dioscorea rotundata; TDe = Tropical Dioscorea esculenta; TDc =
Tropical Dioscorea cayenesis. Source: Amusa (2000).

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Table 11.8. Response of 45 cassava clones to in vitro inoculation with phytotoxic metabolites of
Colletotrichum gloeosporioides f. sp. manihotis

In vitro screening Field screening

Cassava clone Stem Leaves Average size Disease Average Disease
score size score
87/00004 24.98 21.47 23.22a S 3.85ab S
87/00038 23.33 20.89 22.11ab S 3.72ab S
87/00010 22.94 20.78 21.86ab S 3.51ab S
84/00065 23.90 19.68 21.79ab S 3.51ab S
87/00011 22.01 20.33 21.79ab S 3.45ab S
87/00028 21.27 19.51 20.39bcd S 3.40ab S
84/00460 20.17 20.55 20.36bcd S 3.40ab S
87/00052 20.64 18.47 19.55cde S 3.38ab S
83/01162 20.01 18.15 19.08def S 3.36ab S
84/00535 17.84 19.45 18.38def S 3.38ab S
87/00015 17.28 19.48 18.38ef S 3.42ab S
4(2)1443 23.41 13.09 18.23ef S 3.35ab S
87/00133 20.58 15.67 18.12ef S 3.28ab S
86/00079 16.81 17.84 17.33fg S 3.18b S
84/00083 15.85 16.91 16.38gh S 3.12b S
83/01438 16.17 16.00 16.08gh S 3.04bc S
87/00069 15.26 16.16 15.71ghij S 3.00bc S
86/00368 14.90 15.11 15.01hijk MS 2.71bc MS
85/00537 14.82 14.88 14.85hijklm MS 2.64c MS
80/02496 14.25 14.81 14.53ijklm MS 2.5lc MS
CB/71693 14.34 13.54 13.94jklm MS 2.48c MS
84/00115 13.76 13.73 13.47klm MS 2.32c MS
81/00065 12.81 13.76 13.29imn NIS 2.45c MS
83/01137 12.80 12.40 12.60mno MS 2.34cd MS
86/00133 12.91 11.49 12.20mnop MS 3.21d MS
87/00007 11.47 12.27 11.27nop MS 2.25d MS
82/01258 10.83 11.16 11.00opq R 2.15d MS
84/00182 10.69 10.65 10.67pq R 2.04d MS
84/00064 11.74 9.38 10.56pq R 1.64d R
87/00043 9.88 11.21 10.54q R 1.61de R
87/00050 8.57 11.17 9.87qr R 1.55de R
87/00072 8.41 10.17 9.24qrs R 1.59de R
87/00038 4.12 12.73 8.43rs R 1.60de R
30572 3.21 12.97 8.09t R 1.61de R
81/0006S 5.07 3.58 4.32t HR 1.52de R
63397 5.26 3.19 4.23t HR 1.41e R
87/00017 4.28 4.16 4.22t HR 1.26e R
87/00110 3.31 4.81 4.06t HR 1.15c R
87/00146 3.71 4.30 4.00t HR 1.12e R
86/00143 4.31 3.76 3.95t HR 1.12de R
89/00072 3.74 3.01 3.82t HR 1.13de R
CBRS-10- 3.42 3.99 3.7t HR 1.12de R
80411
30211 3.03 4.03 3.67t HR 1.14de R
87/00072 4.21 2.90 3.56t HR 1.12de R
CB/81/00202 3.15 3.96 3.56t HR 1.12de R
Each value is a mean of five replicates (five measurements/plant) transformed to loge values. Each
value within the same column is a mean of three replicates (30 plants/clone). Mean values followed by
the same letters are not significant (p = 0.05) by Duncan’s multiple range test. S = susceptible; MS =
moderately susceptible; R = resistant; HR = highly resistant. Source: Amusa (2000).

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 Chapter 12

Screening of peas for resistance to Fusarium wilt and root rot

(Fusarium oxysporum, Fusarium solani)

A. Lebeda1*, L. Švábová and R. Dostálová2

1
Palacký University in Olomouc,
Faculty of Science, Department of Botany,
Olomouc-Holice,
2
Agritec Plant Research,
Šumperk,

Czech Republic

*Correspondence: [email protected]

Abstract

In the introduction to this chapter, two fungal pathogens (Fusarium solani and F. oxysporum) are
reviewed from the viewpoint of their incidence, importance, symptoms, mode of infection,
harmfullness for peas, and known resistance resources. Further, this chapter gives detailed information
about the screening of germplasm and plant breeding material, including the methods of pathogen
isolation, cultivation and maintenance, preparation of plant material for screening and inoculum
preparation. Three types of laboratory screening methods (tube tests, seed soaking test, root
submersion test) are described. The results of testing are assessed via root and shoot symptom
expression and further evaluated as a degree of infection. In F. oxysporum f. sp. pisi, six races are
described, including the reaction to them on a set of differential genotypes. Finally, practical
applications of these screening methods are summarised.

INTRODUCTION

Fusarium is an anamorph genus with worldwide distribution and a remarkable degree of diversity. Its
species are common in soil and have been found to be important pathogens on a very wide range of
agricultural crops (Zemánková and Lebeda, 2001). F. solani and F. oxysporum are both cosmopolitan
species (Backhouse et al., 2001). Root rot, caused by F. solani (Mart.) Sacc. is a serious problem for
many plants, and is a troublesome and often serious disease in both dryland and irrigated areas (Kraft,
2000). Fusarium root rot of peas, caused by F. solani f. sp. pisi, was first reported as a serious
pathogen in the USA (Bisby, 1918; Jones, 1923). This disease is distinct from Fusarium wilt, caused
by F. oxysporum f. sp. pisi, but sometimes occurs in conjunction with other diseases of peas
(Zaumeyer and Thomas, 1957). Fusarium wilts are among the most important diseases affecting grain
legumes throughout the world (Allen and Lenné, 1998). Fusarium wilt diseases are caused by formae
speciales of F. oxysporum which are pathogenic to selected genera or species of crop plants.
Futhermore, Fusarium wilt pathogens are specialised in the process of systemic infection and
colonisation of the plant vascular system (Jiménez-Díaz, 2000). F. oxysporum Schlecht. f. sp. pisi
(Van Hall) Snyd. et Hans. causes vascular wilt of pea, because of its mycelium plugs in the tracheas.
F. solani (Mart.) App. et Wr. causes root and stem rot followed by necrosis and death of leaves (Kraft,
1994). Both pathogens very often occur simultaneously. Pathogenic forms of Fusarium spp. penetrate
a host root either through wounds or directly through root apices, and the fungus moves into the

189
vascular tissue. The pathogen is spread throughout the plant by means of mycelia growth or conidia,
primarily microconidia, produced in infected xylem vessel elements. One of the earliest responses to
Fusarium spp. infection is the deposition of additional wall callose material (papillae) within contact
cells. The vascular plugging was found to seal off xylem elements of resistant pea cultivars in the
same way as physical barriers (lignification) can retard or prevent vascular invasion (Kraft, 1994).

Breeding of grain legumes and peas for disease resistance is considered to be a basic prerequisite for
improving and stabilising yield (Ranalli, 2003). The most prominent diseases are root fungal diseases;
however, most of these cannot be controlled by chemicals. Some previous literature data concerning
the variation in resistance/susceptibility of peas to Fusarium spp. have been summarised by several
authors (e.g., Hagedorn, 1984; Jacobsen, 1992). Ali et al. (1994) summarised the available information
on valuable sources of resistance to soil-borne root disease of peas. From this review, it is evident that
sources of resistance in peas are rather limited. Most of these sources have been reported in cultivated
forms and cultivars of Pisum sativum. Evaluations of current pea cultivars show that resistance to
Fusarium root rot and vascular wilt is not very common (Hwang et al., 1995; Jedryczka, 1995).
Detailed data about sources of resistance to Fusarium diseases in wild Pisum species and accessions
are not available. A set of 10 accessions of wild P. sativum subspecies and varieties was studied for
resistance to F. solani and F. oxysporum under controlled inoculation (Lebeda and Švábová, 1997).
The observation of visual symptoms on roots and stems showed substantial differences in the response
among the P. sativum accessions under study. Complete resistance to both pathogens was not
recorded. A very high level of resistance was observed in four accessions (W 1957, W 1824, W 1828,
W 1872) (Lebeda and Švábová, 1997). Screening for resistance to Fusarium wilt race 2 of a P. sativum
core collection (altogether 452 accessions, including two wild progenitors: P. sativum ssp.
abyssinicum and P. sativum ssp. elatius) was undertaken in the USA (McPhee et al., 1999). However,
only 62 (14%) of the accessions were resistant. The resistant accessions included accessions from P.
sativum ssp. elatius originating from 24 countries. Of the screened accessions, 39 were also resistant to
race 1. One wild progenitor of P. sativum ssp. elatius (PI 344012) possessed resistance to races 1 and
2 (McPhee et al., 1999).

Resistance to Fusarium wilt in peas is race-specific. There are some data available about the genetics
of resistance to four US races (1, 2, 5 and 6), while the genetics of resistance to races 3 and 4 is not
understood. The genetics of resistance to races 1, 2, 5 and 6 is conferred by different single dominant
genes (Hagedorn, 1984; Muehlbauer, 1992) and is available in numerous germplasms (McPhee et al.,
1999). Grajal-Martin and Muehlbauer (1992) studied the link between the resistance genes for races 1
(Fw) and 2 (Fnw), and reported a recombination frequency of 46% indicating independent assortment.
Recent advances in pea genetics and genetic mapping have been summarised by Jing et al. (2005) and
Loridon et al. (2005).

MATERIAL AND METHODS

Pathogen isolation, cultivation and maintenance

The isolation of Fusarium spp. from soils and plant debris is carried out on various selective media,
e.g., potato peptone agar (PPA), peptone-pentachloronitrobenzene (PCNB) or selective Fusarium agar
(SFA) media (Brayford, 1993; Summerell et al., 2003), containing antibiotics to prevent bacterial
contamination. The Petri dishes and flasks with medium are sterilised by autoclaving and the hot
content of the flask with agar medium is evenly poured into Petri dishes under sterile conditions (flow-
box). After cooling and solidification, the Petri dishes can be packed, marked and stored in a
refrigerator until further use. The samples of soils or plant debris with symptoms of fusarioses are
placed onto the surface of selective media and further incubated in the dark at room temperature (20-
22°C) for 8-10 days. The colonies of Fusarium spp. are further subcultured (purified) until uniform
growth and expected mycelia phenotypes are attained. Pure cultures are placed on media for the
induction of sporulation, e.g., carnation leaf agar (CLA) or potato dextrose agar (PDA) (Summerell et
al., 2003), Komada’s medium (Dhingra and Sinclair, 1985), sucrose nutrient agar (SNA) or potato

190
sucrose agar (PSA) (Brayford, 1993), and checked under the microscope to determine the species. The
isolates are then multiplied and maintained on various media in Petri dishes (mostly on Czapek-Dox
Agar [CDA]) depending on how they will be used.

Cultivation of F. oxysporum and F. solani for fungal identification must be done on a nutrient-poor
medium such as as CLA (Nelson et al., 1983). Optimal development of sporodochia and sporodochial
macroconidia requires exposure of colonies, at least, to fluorescent light, and preferably also to near
UV or black light. Culturing the fungus on media with high sugar levels (i.e., PDA, PSA) should be
avoided as such media tend to promote mutations leading to degeneration of strains. Degenerated
strains may show slower growth, abundance of aerial mycelium, reduced pigmentation, paucity of
macroconidia (Jiménez-Díaz, 2000) or reduced (lost) pathogenicity. For long-term preservation of
cultures, lyophylisation or storage in liquid nitrogen is recommended.

Preparation of plant material for screening

Seeds are surface-sterilised with 96% ethanol for 30 s and subsequently for 20 minutes in 5-10%
chloramine (Bochemie Ltd., Bohumín, Czech Republic), or by another chemical (hypochlorite) used
for seed surface-sterilisation. The seeds are then rinsed three times in sterile deionised water and
germinated aseptically in the dark at room temperature (20-22°C) in flasks on a layer of cellulose
wadding, soaked with sterile water. Seedlings about eight days old, with well developed roots, are
transplanted into glass tubes (for the tube test) containing CDA with Knop’s solution (1 g of
Ca(NO3)2, 0.25 g of KH2PO4, a trace amount of FeCl3, 1000 ml of H2O) (Lebeda and Buczkowski,
1986).

Inoculum preparation, inoculation and incubation

Tube test

Isolates of Fusarium spp. are cultivated on the surface of CDA in glass tubes (diameter: 16 mm).
Inoculated glass tubes are incubated in the dark at room temperature. A seven day old pathogen
culture is used for resistance screening. Pre-germinated seeds are transferred to tubes with a fungal
culture and ~10 ml of Knop’s solution in sterile conditions (Johnston and Booth, 1983). The plants in
tubes are cultivated for 21 days at a 20/22°C day/night temperature with a 12 hour photoperiod and an
irradiance of 100 μmol/m2/s in a growth chamber. During the incubation, Knop’s solution must be
regularly supplemented into the tubes.

An adapted tube resistance screening test, according to Lebeda and Buczkowski (1986), was used for
resistance screening of peas (Lebeda and Švábová, 1997). Isolates of F. solani and F. oxysporum were
cultivated on the surface of CDA in glass tubes (diameter: 16 mm) in the dark at 23°C. Glass tubes
with seven day old pathogen cultures were used for growing and screening pea seedlings. The control
plants were grown in glass tubes with CDA and Knop’s solution; however, without pathogen. At least
10 pea plants of one genotype were used for screening and disease evaluation (see Figure 12.6).

Whaley (1984) developed a similar test for rapid in vitro screening of peas for resistance to F. solani f.
sp. pisi. Seeds are surface-disinfected and suspended in 0.1% water agar containing 1 × 106 conidia/ml.
Seeds are germinated on moist filter paper until the plumules are 30 mm long, and the seedlings are
transfered to the test tubes. Inoculation is conducted by filling the test tube with a conidial suspension.
Peas are incubated in the inoculum for 14 days in a growth chamber at 24°C. The reaction of seedlings
was evaluated according to the scale described below.

Seed soaking test

Another screening procedure was described by Kraft and Kaiser (1993). It involves soaking high
vigour seed for 4 hours in a conidial suspension adjusted to 1 × 106 conidia/ml. Inoculated seeds are

191
then planted into perlite in plastic boxes and incubated in the growth chamber or greenhouse. The
perlite is maintained in wet conditions throughout the two week incubation. The plants examined
(tested) are scored for disease resistance. In addition, a technique of evaluating resistance of peas in
artificially-infested soil has been developed (Kraft, 1975).

Root submersion test

Several screening methods have been described for evaluating pea seedlings for resistance to F.
oxysporum f. sp. pisi. In general, these methods are based on the following steps: (1) uprooting of
seedlings; (2) root washing and cutting; (3) submersion of roots in a spore suspension and cutting; and
(4) transplantation of inoculated seedlings into a growing medium (Dixon and Doodson, 1970;
Haglund, 1974; Kraft and Haglund, 1978). Haglund (1989) described a new submersion “tray method”
of screening pea seedlings for resistance to F. oxysporum f. sp. pisi. This method involves growing pea
seedlings on Styrofoam trays containing 72 cells filled with vermiculite (or agroperlite). Six seeds
were planted in each cell for 16-20 days at 16-18°C until the peas were at the 4-5 node stage of
development. At this time, the plants were removed from individual cells, around one-third of the root
plugs were cut off, the remaining roots were submerged into the inoculum for ~5 s and the plugs were
transplanted into 10 cm diameter plastic pots filled with vermiculite (or agroperlite). The reaction of
the roots was evaluated ~21 days after transplanting using a 0-3 scale (see below) or a 0-4 scale
modified from Vishnyakova (2000). The modified “tray method” of Haglund (1989) is also used in the
laboratory of the authors of this chapter (Figures 12.1-12.5).

Disease assessment

In the case of the tube test, evaluation of the degree of infection (DI) could be carried out continuously
at two-day intervals (until 21 days after inoculation); for other tests (seed soaking, root submersion),
evaluation is performed at the end (21 days after inoculation). Infected and control plants are mostly
grown in three replications (each replication has 10 plants). The symptoms on roots and shoots (stems
and leaves) are assessed on a 0-3 scale (Lebeda and Buczkowski, 1986; Luhová et al., 2002).

Roots 0-3 scale (Lebeda and Buczkowski, 1986; Luhová et al., 2002): 0 = symptomless, roots free of
any visual symptoms; 1 = limited occurrence of local necrosis, discolouration (browning) on the main
and lateral roots; 2 = mild necrotisation and reduced development of main and lateral roots; 3 = severe
necrotisation and growth depression of main root, no development of lateral roots, complete collapse
of roots (Figures 12.6 and 12.7).

Roots 0-4 scale (Vishnyakova, 2000): 0 = symptomless or very weak symptoms (≤10% of tissue is
affected); 1 = weak symptoms (10-25% of tissue is affected); 2 = medium symptoms (26-50% of
tissue is affected); 3 = strong symptoms (51-75% of tissue is affected); 4 = very strong symptoms
(>75% of tissue is affected).

Shoots (above ground): 0 = symptomless, stems and leaves free of any visual symptoms; 1 = limited
growth depression and wilting, plant is more or less turgid; 2 = mild growth depression, wilting and
chlorosis of leaves; 3 = severe wilting and chlorosis, complete collapse of plant (Lebeda and
Buczkowski, 1986). The final degree of infection (DI = P) was expressed in percentage (0-100%)
according to Towsend and Heuberger (1943) using the following formula:

P = Σ(n × v) × 100/x × N

Where: P = the total degree of infection (DI); n = number of plants in each assessed category
(infection degree); v = infection degree (0-4); x = scale range (in this case = 4) and N = total number
of assessed plants.

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Determination of pathogenic variability

Altogether, six races of F. oxysporum f. sp. pisi have been reported until now (Haglund, 1974; Kraft,
2000). Race 1 was identified in the USA in 1924. Since then, a further five races have been
recognised: race 2 in the Netherlands; races 3 and 3A in continental Europe and England; race 4 in
central Canada (Haglund, 1984); races 5 and 6 in Washington State in the USA in the 1970s (Haglund
and Kraft, 1970, 1979). Races 1 and 2 occur in all growing areas of the world, whereas races 5 and 6
pose a threat only in the western USA and Canada (Haglund and Anderson, 1987). The differentiation
of the most important races is shown in Table 12.1.

The symptoms of races 1, 5 and 6 include discolouration of the roots and basal parts of stems, yellow
to orange colour in vascular tissues, and progressive yellowing of lower leaves. Race 2 has similar
symptoms, but moreover with curling of leaves and stupules, and yellowing of the leaves from the
stem base to the top part of the plant (apex) (Haglund, 1984).

CONCLUSIONS

Resistance screening methods described in this chapter have broad applications not only for resistance
studies of peas (P. sativum) to Fusarium spp., but also for other crops as the methodology may be
easily adapted. Resistance to Fusarium spp. is an important characteristic for the creation of
germplasm core collecions of pea genetic resources, as well as in breeding programmes focused on the
improvement of resistance to root pathogens. Lines with confirmed resistance are used as standards
during: (1) the selection process for determination of the resistance level in hybrid populations and
their selection for production of resistant varieties; (2) testing of commercial cultivars from the List of
Declared Cultivars that are regularly tested for resistance to the complex of root pathogens (including
Fusarium spp.) in laboratory and field tests; and (3) determination of F. oxysporum f. sp. pisi races as
a part of studies focused on virulence structure of pathogen populations using the differential set of
pea lines (Table 12.1; Haglund, 1974; Kraft, 1994). The methods described here allow for the
simultaneous screening of large sets of genotypes and are reproducible.

Acknowledgements

This study was supported by the Ministry of Education, Youth and Sports of the Czech Republic,
projects MSM 6198959215 and MSM 2678424501; and by the Ministry of Agriculture of the Czech
Republic, projects NAAR QG60099 and QF3071.

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Table 12.1. Reaction of Fusarium oxysporum f. sp. pisi races

F. oxysporum f. sp. pisi races

Line
1 2 5 6
M 410 S S S S
Vantage R S S S
Mini S R S S
Mini 93 R R S S
Sundance II R S R S
Grant R S S R
WSU 23 R R R S
WSU 28 R S R R
74 SN 5 R R R R

R = resistant; S = susceptible.

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 Figure 12.1. Reduction of the roots before inoculation with Fusarium oxysporum
races 1 and 2 according to Haglund’s method. Photograph: R. Dostálová.

Figure 12.2. Evaluation scale (0-4) of root symptoms for Fusarium oxysporum f. sp. pisi.
Photograph: R. Dostálová.

Figure 12.3. Detail of cv. ‘Gotik’ with a tolerant response (left), ‘Terno’ with complete
collapse (middle) and ‘Concorde’ with a sensitive response (right). Photograph: R.
Dostálová.

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Figure 12.4. Control and inoculated pea plants – Fusarium oxysporum f. sp. pisi race 2.
Photograph: R. Dostálová.

Figure 12.5. Resistance screening of pea plants in perlite and culture boxes, cultivation
room. Photograph: R. Dostálová.

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 Figure 12.6. Tube test. Response of pea plants to Fusarium oxysporum f. sp. pisi (left:
resistant response; right: susceptible response). Photograph: A. Lebeda.

Figure 12.7. Tube test – response of pea plants to Fusarium solani (from left to right:
control, filtrate (10% [v/v]), inoculation by pathogen culture). Photograph: M. Griga.

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 Chapter 13

Mass-screening techniques for the early selection of disease resistance in

chickpea (Cicer arietinum)

S. Pande1, J.N. Rao1, M. Sharma1, M. Pathak1 and P. Stevenson2*

1
International Crops Research Institute for the Semi-Arid Tropics,
Andhra Pradesh, India;
2
Natural Resources Institute, University of Greenwich,
United Kingdom & Royal Botanic Gardens, Kew, Richmond, Surrey,
United Kingdom

*Correspondence: [email protected]

Abstract

Host plant resistance offers the most sustainable and effective disease management option to combat
diseases in chickpea. In this chapter, we briefly describe the distribution, economic importance and
symptoms of the most important diseases of chickpea including Ascochyta blight, Botrytis grey
mould, Fusarium wilt, dry root rot, collar rot, black root rot and stem rot. Screening methods
developed by different research institutes to select germplasm and breeding lines resistant against
these diseases are reviewed. A variety of field, greenhouse, growth room and laboratory screening
techniques along with disease rating scale to facilitate the identification of resistant breeding material
are discussed in detail. Coloured illustrations for each technique are provided as well as extensive lists
of available resistant sources against all diseases. For the selection of sources of resistance to various
diseases in chickpea, and for breeding high yielding cultivars with improved levels of resistance, this
chapter describes current techniques for the simultaneous screening of a large number of chickpea
plants.

INTRODUCTION

Chickpea (Cicer arietinum L.) is an important food legume crop of the semi-arid tropics, particularly
in the rainfed ecology of the Indian subcontinent, the Mediterranean region, the West Asian and North
African region (WANA), Eastern Africa and Latin America. In the recent past, this crop has
experienced an export-driven expansion into new niches such as Australia and Canada. Globally,
chickpea is cultivated on an area of about 11.12 million ha, adding 8.62 million tons of grains to the
global food basket (FAO, 2005). As many as 45 countries grow chickpea but a dozen countries, viz.,
India, Turkey, Pakistan, Iran, Mexico, Myanmar, Ethiopia, Australia, Spain, Canada, Syria and
Morocco, contribute 96% to global production. Chickpea is an important source of protein for millions
of people; it especially provides essential amino acids for vegetarian populations and for those for
whom meat is a scarce luxury. It is also important in cropping systems because of its ability to fix
atmospheric nitrogen and to build sustainability in soil fertility.

Chickpea is attacked by 172 pathogens including 67 species of fungi, 3 bacterial species, 22 viruses
and mycoplasma, and 80 species of nematodes (Haware, 1998). Some of the major diseases of global
importance are: Ascochyta blight (Ascochyta rabiei [Pass.] Labr.), Fusarium wilt (Fusarium
oxysporum Schlecht: Fries emend Synd. And Hans. f. sp. ciceri [Padwick] Matuo and Sato), Botrytis
grey mould (Botrytis cinerea Pers. Ex Fr.), dry root rot (Rhizoctonia bataticola [Taub.] Butler), black
root rot (Fusarium solani [Mart.] Sacc.), collar rot (Sclerotium rolfsii Sacc.), stem rot (Sclerotinia
sclerotiorum [Lib.] de Bary) and foot rot (Operculella padwickii Kheswalla). The incorporation of

201
resistance against these diseases in host plants is the most economical and effective method of
combating these diseases, and could be utilised in hybridisation programmes for the development of
resistant cultivars for commercial cultivation of chickpea. While effective resistance to some of these
diseases is available in some areas, adequate levels of resistance against all of these diseases is not
available in the cultivated germplasm and breeding material of chickpea. However, high levels of
resistance are available in wild Cicer species, and these can be identified as sources of useful
resistance characters with the use of the reliable screening techniques described in this chapter.

Host plant resistance is the most economical, environmentally acceptable and sustainable means of
controlling most biotic constraints, especially diseases. For exploitation of host plant resistance, the
first step is to develop a reliable screening technique for large-scale screening of germplasm and
breeding material. Techniques to screen chickpea germplasm and breeding material against various
diseases have been developed by different research institutions, and are being used to screen for
resistance in cultivated and wild Cicer species, and their segregating breeding material across the
world.

Since the late 1970s, pathologists at the International Crops Research Institute for the Semi-Arid
Tropics (ICRISAT), Patancheru, India, have focused their attention on developing techniques to
screen germplasm and breeding material, to develop disease-resistant cultivars and to assist breeders.
Most of these early approaches have been briefly reported (Reddy and Nene, 1978; Nene and Haware,
1980; Singh et al., 1981). Procedural details of these techniques were reported comprehensively by
Nene et al. (1981). In general, the field techniques are used for large-scale screenings of germplasm
and breeding material, and greenhouse/laboratory techniques are used to confirm resistance identified
in the field screening as well as to carry out inheritance and race identification studies, and studies on
plant material for which supply is restricted or limited.

I. Ascochyta blight (Ascochyta rabiei)

Distribution and economic importance

Ascochyta blight (AB) of chickpea is the most devastating disease in many chickpea-growing areas of
the world. It has been reported from 35 countries across six continents, i.e., Asia (Bangladesh, China,
India, Iran, Iraq, Israel, Jordan, Lebanon, Pakistan, Syria and Turkey); Africa (Algeria, Egypt,
Ethiopia, Kenya, Libya, Morocco, Sudan, Tanzania and Tunisia); Europe (Bulgaria, Cyprus, France,
Greece, Hungary, Italy, Portugal, Romania, Spain and the Ukraine); North America (Canada and the
USA); South America (Columbia and Mexico); and Australia (Nene et al., 1996). The economic
importance of the disease is evident from the frequent occurrence of epidemics in the past in several
chickpea-growing areas of the world. More than 20 epidemics have been reported and most of these
epidemics have occurred in Pakistan, India and European countries. The disease occurred in epidemic
form in 1981-1983 in the north-western states of India and Pakistan, resulting in total loss of the crop
(Singh et al., 1982, 1984). The occurrence of severe epidemics of AB has also caused substantial
losses in yield in the Mediterranean region (Hawtin and Singh, 1984). More than $1 million US dollars
of financial losses in the Palause region of the USA were reported by Kaiser and Muehlbauer (1988).
Elsewhere, the disease is currently the most important yield-limiting factor, potentially affecting 95%
of the chickpea area in Australia (Knights and Siddique, 2002).

Symptoms of Ascochyta blight

The initial symptoms appear as water soaked lesions on upper leaves. Later, these lesions become dark
brown spots and spread rapidly on aerial parts of the plant: leaves, petioles, flowers, pods, branches
and stem (Figure 13.1). Pycnidia are arranged in concentric rings in the lesions, which is the
characteristic symptom of the disease. The spots on leaves and pods are circular, while on the stem
and branches they are elongated. The apical twigs, branches and stem often show girdling, and the
plant parts above the girdled portion are killed or break off even before drying. On the seed coat, dark

202
lesions are formed with pycnidia, which often lead to seed infection through testa as well as the
cotyledons.

Methodology

Different screening techniques have been developed and modified at various research centres for
artificial resistance screening of chickpea germplasm against A. rabiei in controlled environment and
field conditions.

Pot culture

Chickpea plants are grown in polythene pots (15 cm) in the greenhouse. The pots containing one
month old test plants and susceptible controls are placed in an 8-10 cm deep circular pit. Water is
added to the pots before inoculation. The plants are inoculated by spraying spore suspension (1 × 105
spores/ml) and are covered with a moist muslin cloth chamber. After 48 hours, cloth chambers are
removed and the plants are kept wet during the day time by spraying water daily for up to 13 days
from 10 am to 4 pm. Leaf wetness is maintained for 21 days to ensure maximum disease severity
(Singh et al., 1982).

Controlled environment screening techniques (CEST)

A controlled environment facility (CEF) with adjustable temperature, humidity and photoperiod was
developed at ICRISAT for screening chickpea germplasm and breeding material for AB resistance
(Figure 13.2). Several screening techniques were developed and standardised using the CEF and are
explained below.

Whole plant screening technique (WPST)

Chickpea seedlings are grown in plastic trays (35 × 25 × 8 cm) filled with a mixture of sterilised river
sand and vermiculite (3:1) in a greenhouse for 10 days. The susceptible control ‘Pb 7’ is sown in each
tray along with test entries (Figure 13.3A). Trays are transferred to the CEF, maintained at 20 ± 1°C
and ~1500 lux light intensity for 12 hours a day and allowed to acclimatise for 24 hours. The seedlings
are inoculated by spraying a conidial suspension (5 × 104 conidia/ml) of A. rabiei. The conidia are
produced on chickpea seed and harvested into sterile distilled water. After inoculation, the seedlings
are allowed to partially dry for 30 minutes to avoid dislodging of spores, and 100% relative humidity
(RH) is maintained for four days, and thereafter, 6-8 hours a day until the completion of the
experiment (Pande et al., 2005). Disease severity is scored on a 1-9 rating scale at 14 DAI (Haware et
al., 1995a).

Cut twig screening techniques (CTST)

In comparison to the above method, the CTST is relatively simple and requires less time, resources
and effort although it is ultimately less reliable since it does not evaluate effects on whole plants.
Furthermore, test plants are used when damaged and this may induce chemical changes in the test
material that could affect the results or perhaps exaggerate the effect of defence chemicals such as the
phytoalexins, medicarpin and maackiain, that are known to be produced in chickpea (Stevenson et al.,
1997; Stevenson and Aslam, 2006). In this technique, the tender shoots of chickpea plants (30-60 days
after sowing) are cut with a sharp edge blade in the evening and inoculated by keeping them alive in
water and sand. Details of the technique are given below.

Cut twig screening technique in water (CTST-W)

In the CTST-W, the lower portion of the detached twigs is wrapped with a cotton plug and transferred
to a test tube (15 × 100 mm) containing fresh water. These tubes are placed in a test tube stand,

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transferred to the CEF maintained at 20 ± 1°C and ~1500 lux light intensity for 12 hours a day, and
allowed to acclimatise for 24 hours (Figure 13.3B). The seedlings are inoculated by spraying a
conidial suspension (5 × 104 conidia/ml) of A. rabiei. The conidia are produced on chickpea seed and
harvested into sterile distilled water. After inoculation, the seedlings are allowed to partially dry for 30
minutes to avoid dislodging of spores, and 100% relative RH is maintained for four days, and
thereafter, 6-8 hours a day until the completion of the experiment. The disease symptoms appear on
the susceptible control six days after inoculation. This method of screening is very useful in a wide
hybridisation programme, where every plant may be valuable for other parameters. Resistant plants
identified by this method can be further used in crosses and back crosses in the same crop season
(Sharma et al., 1995). Disease severity is scored on a 1-9 rating scale at 10 and 14 DAI (Pande et al.,
2005).

Cut twig screening technique in sand (CTST-S)

In this technique, instead of placing the detached twigs in tap water in test tubes, they are planted in
sterilised moist sand filled in plastic trays (35 × 25 × 8 cm) (Figure 13.3C). Twigs of susceptible
cultivars along with test entries are also kept in each tray for comparison. Trays are transferred to the
CEF, maintained at 20 ± 1°C, allowed to acclimatise for 24 hours and inoculated following standard
procedures as mentioned earlier. Disease severity is scored on a 1-9 rating scale at 10 and 14 DAI
(Pande et al., 2005).

Detached leaf/leaflet technique

The leaves of chickpea plants are detached from the test plants, sterilised with sodium hypochlorite
solution (5%) or mercuric chloride (0.1%) and aseptically transferred to Petri dishes containing water
agar. These leaves are inoculated by spraying spore suspension (1 × 105 spores/ml) of A. rabiei. The
lids of the Petri dishes are sealed with paraffin wax and incubated at 20 ± 1°C with a 12 hour
photoperiod. The development of lesions is initiated in 2-3 days. Lesions become apparent in 4-6 days
and the observations on disease development are recorded on day 8 (Singh and Sharma, 1998).

Leaflets from the most recent fully expanded leaves are collected from 15 day old chickpea plants.
The detached leaflets are allowed to float; lower surface down on tap water inside 90 mm Petri dishes,
and the upper surfaces of the leaflets are inoculated with 5 µl of A. rabiei. The leaflets are incubated
for 14 days at 20 ± 1°C, with a 12 hour photoperiod. Disease severity scores are based on the number
of leaflets infected and lesion size (Dolar et al., 1994). The disease ratings are scored as described
previously for whole plant screening methods.

Field screening

The field screening technique consists of planting test material in 3 to 5 m rows, spaced 40 cm apart in
replicated trials. Indicator-cum-infector rows of a highly susceptible variety (‘L 550’ or ‘Pb 7’ or ‘ILC
1929’) are planted after 4-8 rows, depending upon the material to be tested. At the flowering stage,
plants are inoculated in the evening by spraying with a spore suspension (1 × 105 spores/ml). Inoculum
is multiplied on potato dextrose broth or chickpea extract dextrose broth medium. High RH (>85%) is
maintained by running a perfo-spray system for 10-15 minutes every hour from 10 am to 4 pm daily.
There should be 100% mortality in susceptible material/control 15 days after inoculation. The
observations are recorded 21 days after inoculation (Singh et al., 1982; Reddy et al., 1984).

Nene et al. (1981) gave a detailed account for developing screening techniques for chickpea
germplasm against AB (Figure 13.4A). The procedures included:

(1) Collecting debris of infected chickpea plants and storing it dry for use in the following season.
For 1 ha, six bags (100 × 75 cm) should be sufficient.

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(2) Planting 2-4 rows of a susceptible cultivar (‘ICC 460’, ‘Syrian Local’ and ‘Pb 7’) all around the
plot.
(3) Planting test lines following normal agronomic operations. Ensure that a susceptible cultivar is
planted after every 2-4 test rows. These rows will serve as indicator-cum-spreader rows.
(4) Identifying the normal time of infection. In many countries, favourable (cool and wet) weather
is common around flowering time. At such time, scatter the infected plant debris (step 1) all
over the plot.
(5) Arranging for a sprinkler irrigation system as a standby. This must be used if dry weather
prevails at the normal time of infection.
(6) If the disease development is not uniform, spraying spore suspension prepared either from
infected plants from the field itself or from a pure culture of the fungus. For a 1 ha plot, prepare
25-40 flasks (250 ml) of inoculum; 150-250 l of diluted inoculum will be enough for 1 ha.
(Prepare chickpea flour-dextrose broth by mixing 40 g of chickpea flour and 20 g of dextrose in
1000 ml of water. Pour 30 ml of broth into each flask. Autoclave at 15 lb for 20 minutes.
Inoculate with a pure culture of A. rabiei and incubate for 10 days with 12 hours of light at 20-
25°C. Dilute as indicated above).
(7) Recording disease rating when the susceptible control lines show the maximum rating (9 on the
1-9 rating scale). Record the disease rating again when the crop is close to maturity.

Note: Closer spacing, with rows across the normal wind direction, enhances uniform disease
development.

Isolation plant propagator

Nene et al. (1981) gave a detailed account of this technique for screening chickpea germplasm against
AB (Figure 13.4B). The procedures included:

(1) Using units of the isolation plant propagator (Burkard Manufacturing Co. Ltd., Rickmansworth,
Herts, England).
(2) Whenever necessary, operating evaporative coolers around the propagators to maintain
temperatures below 30°C.
(3) Arranging for artificial light (cool day light; eight 120 cm tubes, 40 W each) in the lower
sections of the propagators.
(4) Filling pots with autoclaved fine riverbed sand and vermiculite (3:1).
(5) Growing at least 10 seedlings of one accession in one pot. In addition, raising seedlings of a
susceptible control (ICC 460) in one pot in each subsection of the propagator.
(6) Obtaining a pure culture of an aggressive isolate of A. rabiei. Prepare chickpea flour-dextrose
broth (40 g chickpea flour and 20 g dextrose in 1000 ml distilled water) and place 30 ml in each
of the 250 ml flasks. Autoclave at 15 lb for 20 minutes. Inoculate the medium with the fungus.
Incubate at 20-25°C for 10 days with 12 hours of artificial light per day.
(7) Removing fungal growth from the flasks and diluting with sterile distilled water to 2 × 104
spores/ml.
(8) Using a hand sprayer to spray inoculate on the two week old seedlings with the fungus
inoculum. Cover plants with a plastic cover for 10 days.
(9) Recording the incubation period, percentage infection and percentage mortality.
(10) Scoring the disease severity on a 1-9 rating scale twice, once when the susceptible check shows
a rating of 9, and again 10 days later.

Disease rating scale

The rating scale for AB on chickpea seedlings is given in Table 13.1. Based on the disease score,
the test lines are categorised for their reaction to AB infection as follows: 1 = immune (I); 1.1-3 =

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resistant (R); 3.1-5 = moderately resistant (MR); 5.1-7 = susceptible (S); and 7.1-9 = highly
susceptible (HS).

Test lines showing a rating of 1-3 are considered acceptable for the breeding programme; ratings
of 3.1-5 are only acceptable if lines with a rating of 1-3 are not available; ratings of 5.1-9 are not
acceptable.

Sources of resistance

Several sources of resistance to AB have been identified in studies conducted in different chickpea-
growing areas of the world (Table 13.2).

II. Botrytis grey mould (Botrytis cinerea)

Distribution and economic importance

Botrytis grey mould (BGM) of chickpea is an important disease in the South Asian countries,
Bangladesh, Nepal, India and Pakistan. It has also been reported from Argentina, Australia, Canada,
Columbia, Hungary, Mexico, Myanmar, Spain, Turkey, the USA and Vietnam. The grey mould
appeared in epidemic form in 1968 in the chickpea crop of the sub-mountainous region of Uttar
Pradesh, Terai area (Joshi and Singh, 1969), and again in 1981-1983 along with AB in north-western
states of India, viz. Punjab, Haryana, Himachal Pradesh, Jammu and Kashmir, Uttar Pradesh,
Rajasthan, parts of Bihar and West Bengal, where the crop was completely destroyed (Singh et al.,
1982; Garewal and Laha, 1983). This disease caused a 100% loss in yield in the Terai region of Nepal
(Reddy et al., 1988). Since the epidemic in the early 1980s, the disease has occurred regularly in a
moderate to severe form depending upon the environmental conditions and has become a major
production constraint where winters are characterised by cool, wet and foggy days.

Symptoms

All aerial plant parts are attacked by this disease. Initial symptoms are water soaking and softening of
affected plant parts, viz., leaf, flowers and tender shoots. On these plant parts, brown spots are
produced, which are readily covered with dense fungal growth in the form of sporophores and
mycelium (Figure 13.5). Plant parts covered by dense foliage and in wet conditions are heavily
covered with sporophores. On stem, the grey mould symptoms are gradually replaced by dark grey to
black sporodochia. When the relative humidity is low, irregular brown spots on leaves appear without
any fungal growth. Occasionally, small and tiny black sclerotia are produced on dead tissues and on
water soaked lesions on pods. Under congenial environmental conditions, all the flowers are attacked,
resulting in the complete failure of the crop. The infected pods either do not produce any seeds or
produce only small, shrivelled seeds (Haware and McDonald, 1992). The disease can appear at any
time during plant growth, but maximum development of the disease is observed during the
reproductive phase.

Methodology

Techniques to screen chickpea germplasm and breeding material for BGM resistance have been
developed by different research institutes. The screening techniques, viz., growth room and field are
being used for screening germplasm and breeding material for BGM resistance.

Growth room screening technique

This technique was developed at ICRISAT, Patancheru. Seedlings of test genotypes, along with the
susceptible control ‘H 208’ are raised in 10 cm diameter plastic pots (5 seedlings/pot) filled with a

206
sand and vermiculite mixture (4:1) in a greenhouse (Figure 13.6). The pathogen, B. cinerea, is
multiplied on potato dextrose broth and incubated at 25°C with a 12 hour photoperiod in a Percival
incubator. 10 day old seedlings are transplanted to a plant growth room and inoculated with a 10 day
old conidial suspension (5 × 104 conidia/ml) of the pathogen. The growth room is maintained at 24 ±
2°C and 95-100% RH with a 12 hour photoperiod until the end of the experiment. Disease symptoms
start appearing 24 hours after inoculation. BGM severity is scored on a 1-9 rating scale (1 = no
infection and 9 = all plants killed) three days after inoculation and subsequently every alternate day for
nine days (Pande et al., 2002).

Growth chamber screening technique

This screening technique was developed by Punjab Agricultural University (PAU), Ludhiana, Punjab,
India. The test lines are planted in polythene bags (15 × 10 cm) containing sandy-loam soil in a
greenhouse. Test plants along with susceptible controls (‘G 543’ or ‘H 208’) are transferred, 25 days
after sowing, to a growth chamber with controlled environmental conditions (approximately 20°C,
>90% RH, and alternate light and dark periods). After watering the pots, plants are inoculated by
spraying spore suspension (5 × 104 spores/ml) of B. cinerea, and are enclosed for six days within a
moist chamber prepared with polythene sheets supported by iron frames (46 × 46 cm). During the
incubation period, an 8:16 hour light: dark photoperiod is provided. The disease symptoms appear
after 24 hours, and 100% mortality of susceptible lines and the control are recorded six days after
inoculation (Singh et al., 1982).

Controlled environment screening techniques (CTST)

Using the same CEF mentioned for AB, the following screening techniques have been developed for
screening resistance against BGM.

Whole plant screening technique (WPST)

This technique was developed at ICRISAT, Patancheru. Seedlings of the test material are grown in
plastic trays (35 × 25 × 8 cm) filled with a mixture of sterilised river sand and vermiculite (4:1) in a
greenhouse for 10 days (Figure 13.7A). A susceptible control, ‘JG 62’/’H 208’, is sown as an indicator
in each tray along with test entries. Trays are transferred to a CEF, maintained at 15 ± 1°C and ~1500
lux light intensity for 12 hours a day, and allowed to acclimatise for 24 hours. The seedlings are
inoculated by spraying a conidial suspension (3 × 105 conidia/ml) of B. cinerea. The conidia are
produced on autoclaved marigold flowers and harvested into sterile distilled water. After inoculation,
the seedlings are allowed to partially dry for 30 minutes to avoid dislodging of spores and 100% RH is
maintained until the end of the experiment. Disease severity is scored on a 1-9 rating scale at 14 and
20 DAI.

Cut twig screening technique (CTST)

The cut twig screening technique (CTST) proved very effective and efficient for screening breeding
material derived from a wide range of hybridisation programmes, particularly for back crossing (Singh
et al., 1997, 1998), even though this technique may affect plant chemistry and, thus, resistance as
mentioned above. Details of the techniques are given below.

Cut twig screening technique in water (CTST-W)

This technique involves cutting 10-15 cm long tender chickpea shoots in the evening with a sharp-
edged blade. The lower portion of the single twigs are wrapped with a cotton plug and transferred to a
test tube (15 × 100 cm) filled with water. The tubes are placed in a test tube stand and transferred to a
CEF at 15 ± 1°C and a 12 hour photoperiod one day prior to inoculation for acclimatisation (Figure
13.7B). Twigs are then spray inoculated with the inoculum (3 × 105 conidia/ml) of B. cinerea. Twigs

207
of a susceptible variety (‘H 208’, ‘G 543’ or ‘L 550’) are used as susceptible controls. Post-inoculation
incubation conditions are similar to those used for the whole plant screening technique. Disease
symptoms appear 24 hours after inoculation. Complete mortality is observed six days after inoculation
in the susceptible lines (Singh et al., 1998).

Cut twig screening technique in sand (CTST-S)

The procedure for screening BGM using CTST-S (trays) is similar to that described for AB CTST-S
screening (Figure 13.7C). Inoculum concentration, inoculation method and post-inoculation incubation
conditions are those described for the whole plant screening technique for BGM. Data on disease
severity is recorded eight days after inoculation (Pande et al., unpublished).

Field screening technique

This technique is efficient for large-scale screening of germplasm and breeding material in segregating
generations. The test lines are sown in 2-3 m long rows spaced at 30 × 10 cm. Indicator-cum-infector
rows of the susceptible cultivar ‘H 208’ are sown after every two test rows. When the plants are 70-80
days old (at the onset of flowering), the field is irrigated in the morning and plants are inoculated by
spraying a spore suspension (50000 spores m/l) of 10 day old culture of B. cinerea. From the morning
of the following day, sprinkler irrigation or the perfo-spray system is run every day for about 15
minutes every 1 or 2 hours from 9 am to 5 pm depending upon the environmental conditions (Figure
13.8). The inoculation of plants is repeated twice at 10-day intervals after the first inoculation. The
disease severity is scored on a 1-9 rating scale in mid-February, and in the first and last weeks of
March (Pande et al., 2002).

Disease rating scale

A 1-9 rating scale is used at ICRISAT for scoring BGM. The interpretation of the scale is given in
Tables 13.3 and 13.4.

Sources of resistance

Host-plant resistance available to BGM infection in chickpea, determined by screening programmes

conducted at various locations in India and other countries, is given in Table 13.5.

III. Fusarium wilt (Fusarium oxysporum f. sp. ciceris)

Distribution and economic importance

Fusarium wilt (FW) is a serious disease in many chickpea-growing parts of the world. It has been
reported from 32 countries on six continents, i.e., Asia (Bangladesh, China, India, Iran, Iraq,
Myanmar, Nepal, Pakistan, Sri Lanka, Syria and Turkey); Africa (Algeria, Egypt, Ethiopia, Kenya,
Malawi, Morocco, Sudan, Tunisia, Uganda and Zambia); Europe (Hungary, Italy, Spain and the
former USSR); North America (the USA); South America (Argentina, Chile, Columbia, Mexico and
Peru); and Australia.

It is estimated to cause a 10-15% yield loss annually in India, but can result in 100% loss under
specific conditions (Jalali and Chand, 1992). In Spain, a 12-15% loss due to wilt and root rot was
reported (Trapero-Cases and Jimenez-Diaz, 1985). The production of chickpea in California has
declined in recent years and FW is a major constraint in the production of kabuli chickpea in
Mediterranean areas (Haware, 1990).

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Symptoms

The disease can affect the crop at any growth stage. The whole seedlings collapse and lie flat on the
ground. Affected seedlings do not show rotting on the root. The actual affected plants show typical
wilting, i.e., drooping of petiole, rachis and leaflets. The lower leaves are chlorotic, gradually turn
yellow and then light brown or straw coloured (Figure 13.9A). Dried leaflets of the infected plants are
not shed at maturity. When the stem of the infected plant is split open vertically, black discolouration
of xylem vessels can be seen (Figure 13.9B). Sometimes, only a few branches are affected, resulting in
partial wilt. Drooping of petioles and vascular discolouration are characteristic symptoms of the
disease.

Methodology

Chickpea wilt pathogen is mainly soil-borne and for successful management of the disease, it is
important to exploit host plant resistance since control with fungicidal spray into the soil is impractical
and good resistance is available to this pathogen. To identify sources of resistance and development of
wilt-resistant genotypes, wilt sick plots are required. Techniques to develop these are described below
along with techniques for screening against wilt.

Field screening

Test lines are planted in 5 m long rows, 40 cm apart, in a wilt sick plot using a randomised block
design. A susceptible control, ‘JG 62’ (‘ICC 4951’), is planted after every two test rows (Figure
13.10A). Observations on seed germination are recorded 20 days after sowing and on wilt incidence
after a one month interval and until harvesting. Detailed procedures for development of a wilt sick plot
and identification of sources of resistance have been given by Nene et al. (1981, 1989). The
procedures included:

(1) Selecting a plot of adequate size and ensuring that it is isolated from other chickpea fields to
avoid spread of the fungus inoculum from this plot to others. The plot should have been cropped
in the previous year with chickpea, and at least traces of wilt incidence should have been
observed.
(2) Collecting as many wilted plants from other fields as possible, chopping them into small pieces
and incorporating these uniformly into the surface soil of the plot.
(3) Planting a sole crop of a highly susceptible cultivar (‘JG 62/T-3’) in this plot. Ensure a good
plant population and carry out normal agronomic operations.
(4) By the end of the season, at least 20% of the plants should show wilt symptoms. After
harvesting and threshing, scatter the debris uniformly all over the plot and incorporate it by
dicing. In addition, repeat step 2; this will help to increase the level of the inoculum to make the
soil “sick”.
(5) Repeating steps 3 and 4 in the next season. By the end of this season, you should see >90% wilt
incidence. If the incidence is <70%, repeat steps 3 and 4 once more.
(6) Initiating screening in the next season. Plant a susceptible cultivar after every two test rows in
the whole field. These rows will serve as controls, and will help in monitoring and maintaining
the wilt sickness of the plot. The susceptible control rows should show >90% wilt.
(7) From the 4th or 5th year onwards, plant a susceptible control in every fifth row. This will
provide space for more breeding material and, at the same time, maintain the level of sickness.
(8) Planting any other crop in this plot is not recommended.

It must be emphasised here that by following all of these steps, a sick plot in which F. oxysporum f. sp.
ciceris will be the most predominant pathogen can be developed. However, the presence of other soil-
borne pathogens cannot be avoided.

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Pot culture

Wilt resistant lines identified by the field screening technique are further tested in pot culture under
controlled conditions to confirm resistance. The inoculum of the fungus is multiplied on sand-maize
medium (9:1) in 250 ml flasks for 15 days at 25°C. Half of the content of the flask is mixed in a pot
containing 2 kg of sterilised soil. The fungus is allowed to become established in the soil mixture in
clay pots for four days and chickpea seeds are then planted in the infested soil. In a modification of
this method, 20 day old seedlings are transplanted into the inoculated pots after dipping their roots in
the spore suspension of F. oxysporum f. sp. ciceri. Observations on the percentage of plant mortality
are recorded 15 and 45 days after inoculation (Singh et al., 1987).

Nene et al. (1981) gave a detailed account for developing the pot screening technique and screening
chickpea germplasm against wilt (Figure 13.10B):

(1) Obtain a pure culture of F. oxysporum f. sp. ciceris from infected chickpeas in your area by
following standard isolation procedures.
(2) Prepare a sand-maize meal medium by placing 90 g of riverbed sand, 10 g of maize meal and 20
ml of distilled water in each 250 ml Erlenmeyer flask. Autoclave the medium in the flasks at 15
lb for 20 minutes. Inoculate each flask with a bit of fungal growth from tubes and incubate at
25°C for 15 days.
(3) Prepare a fungus-soil mixture by hand-mixing contents of each flask with 2 kg of non-
autoclaved field soil. The soil must come from a chickpea field where wilt normally occurs; for
example, the field test plot (sick plot).
(4) Fill large (diameter: 30 cm) earthen pots with the inoculated soil from step 3. Approximately 10
kg of soil will be required to fill each pot. Water the pots and wait for four days before
proceeding to the next step.
(5) Sow 40-50 seeds of a highly susceptible cultivar in each pot at a 2-3 cm depth. Water
adequately and regularly. Most plants should show wilting after 10 days.
(6) Remove healthy plants after 30 days. Chop and incorporate all the wilted plants into the soil.
(7) Repeat steps 5 and 6 until 90% wilt is observed. These pots are then ready for screening.
(8) Divide a pot into two sections. Plant 10 seeds of a test line in one section and 10 seeds of a
susceptible control in the other.
(9) These pots can be used for several successive screenings.

Note: Screening in pots requires up to only 60 days at any time of the year, compared with 4-6 months
of the season in a sick plot.

Water culture

Nene et al. (1981) gave a detailed account for developing this technique and screening chickpea
germplasm against wilt (Figure 13.11):

(1) Obtain a pure culture of F. oxysporum f. sp. ciceris from infected chickpeas in your area.
(2) Sow 15 surface-sterilised (5 minutes in 2.5% sodium hypochlorite) seeds of the test as well as
susceptible control lines in autoclaved riverbed sand placed in 15 cm pots. Use one pot for each
line. Approximately 1 kg of sand will be required for each pot. Nurse the seedlings until these
are required for transplanting.
(3) Prepare potato dextrose broth (peeled and sliced potato: 200 g; dextrose: 20 g; distilled water:
1000 ml). Place 100 ml of broth in a 250 ml flask and prepare as many flasks as needed. One
flask of inoculum will be sufficient for testing 18 lines (10 seedlings per line). Autoclave at 15
lb for 20 minutes.
(4) Inoculate the medium (step 3) with a bit of the fungal growth from tubes. Incubate on a shaker
(eight hours each day) at room temperature (25-30°C) for 10 days.

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(5) Dilute the entire contents of a flask with sterile distilled water to get a final inoculum dilution of
2.5% (~3.5 l of water is needed to attain the desired dilution of the contents of one flask). This
will ensure approximately 6.5 × 105 spores/ml.
(6) Pour 20 ml of inoculum into each sterilised 150 × 15 mm glass tube (step 5).
(7) Remove the 10 day old (from sowing) seedling from the sand (step 2). Wash the root system in
running water and then rinse in sterile distilled water.
(8) Transplant one seedling into each tube (step 6) and hold it in position with a cotton plug.
(9) Add sterile distilled water to the tubes every two days to make up the loss.
(10) Use 10 seedlings for each line. With each batch of test line, use seedlings of a susceptible line
(‘JG 62’) as a control. In addition, keep a non-inoculated seedling as another control for each
line.
(11) Keep tubes in a specially designed box.
(12) The susceptible control usually wilts in 7-10 days. Record data 15 days after inoculation (step
8). Non-inoculated seedlings should remain green for up to three weeks.

Root-dip technique

(1) Obtain a culture of F. oxysporum f. sp. ciceris from infected chickpea plants and purify by
single spore isolation.
(2) Sow surface-sterilised seeds of the test plants and a susceptible control in sterilised polythene
bags for eight days.
(3) For multiplication of inoculum, a 7 mm disk of actively growing culture of F. oxysporum f. sp.
ciceris is inoculated into 100 ml of potato dextrose broth in 250 ml flasks and incubated for five
days at 25°C in a shaker at 125 rpm.
(4) Dilute the entire contents of a flask with sterile distilled water to get a final inoculum
concentration of 6.5 × 105 conidia/ml for use as inoculum.
(5) Eight day old seedlings grown in sterile sand from step 2 are uprooted, the lowermost portion of
the roots cut and the roots dipped for 30 s in inoculum (step 4) (Figure 13.12).
(6) Inoculated seedlings are transplanted to pre-irrigated vertisol and sand (4:1) in plastic pots
(diameter: 15 cm) and the temperature is maintained at 25 ± 3ºC in a greenhouse. Inoculated
seedlings are observed for mortality from 15 to 30 DAI (Nene et al., 1981).
(7) 15 seedlings of each accession are tested in three replications with five seedlings in each pot.

Disease rating scale

The disease rating scale presented in Table 13.6 is used. Test lines showing a disease incidence of
≤10% are considered acceptable for the breeding programme, while test lines showing 10.1-20.0% are
only considered if lines with ≤10% are not available.

Sources of resistance

Seven races of Fusarium are known to cause wilt of chickpea. Races 1, 2, 3 and 4 were identified from
India (Haware and Nene, 1982c); race 0 and 5 from Spain (Jimenez-Diaz et al., 1989); and race 6 from
California (Phillips, 1998). A large number of good sources of resistance have been identified (Haware
et al., 1992a, 1992b; Pundhir et al., 1998; Dua et al., 2001). The two most important sources of
resistance are the germplasm line, ‘WR 315’ (‘ICC 8933’), and the cultivar, ‘JG 74’. The former is
resistant to all races except race 3, while the latter is resistant to all races except race 2 (Haware,
1998). A twin-podded variety, ‘JG 62’, is highly susceptible to all races, except race 0, and widely
used as a susceptible control for races 1 to 4 in India. The availability of a good source of resistance to
FW and the availability of easy and effective field screening methods have made breeding for
resistance to FW an easy task.

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Using these techniques, over 150 wilt-resistant lines at ICRISAT and 100 lines at PAU, Ludhiana,
India have been identified (Singh et al., 1984, 1986, 1991; Nene, 1988; Haware et al., 1990). Some of
the sources of resistance to FW identified in different countries are listed in Table 13.7.

Resistance to wilt is more common in desi chickpea germplasm (Haware et al., 1990). A large number
of chickpea genotypes having a moderate to high level of resistance against wilt have been identified
through multi-location testing in wilt sick plots under the All India Coordinated Pulses Improvement
Project since 1985 (Table 13.8). Some of these have been utilised to develop wilt-resistant varieties.

IV. Dry root rot (Rhizoctonia bataticola)

Distribution and economic importance

Dry root rot (DRR) has been recorded in Egypt, Ethiopia, India, Iran, Kenya, Lebanon, Mexico,
Myanmar, Pakistan, Spain, Sri Lanka, Sudan, Syria, Tanzania, Turkey, Uganda, the USA and Zambia
(Nene et al., 1996). It causes considerable yield losses that vary from 5% to >50% in badly infested
fields. The disease has been reported from all the chickpea-growing areas of India, but is more serious
in central and southern parts of the country, particularly in rainfed and poor soils.

Symptoms

The petioles and leaflets of the affected plants droop only at the top of the plant. The tap root turns
black, shows sign of rotting, and is devoid of lateral and finer roots (Figure 13.13). A greyish
mycelium can sometimes be seen on the tap root. The dead roots are quite brittle and show shredding
of bark. The tip of the root is easily broken on touching. With the aid of a magnifying glass, minute
sclerotia can be seen on the exposed wood of the root and inner side of the bark or whenever split open
at the collar region vertically.

Methodology

The field and pot screening techniques described for FW are also applicable for screening against this
disease. Nene et al. (1981) gave a detailed account for developing and screening chickpea germplasm
against this disease. The steps mentioned for the development of a wilt sick plot also hold good for
DRR. The only difference is to plant a cultivar that is susceptible to R. bataticola, but resistant to F.
oxysporum f. sp. ciceris, i.e., ‘BG 212’ and ‘ICC 229’. As pointed out in step 9 under wilt screening
techniques, it will not be possible to have a sick plot only for R. bataticola. We can, however,
encourage its multiplication to ensure its predominance over other soil-borne pathogens. To confirm
resistance specifically to R. bataticola, it would be necessary to follow the blotter paper technique (see
below).

Blotter paper technique

(1) Obtain a pure culture of R. bataticola from infected chickpea plants in your area.
(2) Sow 20 surface-sterilised (5 minutes in 2.5% sodium hypochlorite) seeds of the test lines in
autoclaved riverbed sand placed in 15 cm pots. Use one pot for each line. Sow seeds of control
lines as well. Approximately 1 kg of sand will be required for one pot. Nurse the seedlings until
five days after sowing.
(3) Prepare potato dextrose broth (peeled and sliced potato: 200 g; dextrose: 20 g; distilled water:
1000 ml). Place 100 ml of broth into one 250 ml flask and prepare as many flasks as needed.
Autoclave at 15 lb for 20 minutes. One flask of inoculum will be needed to test 10 lines at once.
(4) Inoculate the medium (step 3) with the fungus. Incubate for five days at 25ºC.

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(5) Remove the mycelial mats from the flasks at the end of the incubation period. Add two mycelial
mats to 100 ml of sterile distilled water and macerate these in a blender for 1 minute (operate
the blender intermittently). Place this inoculum in a beaker of a suitable size.
(6) Uproot the five day old seedlings of the test lines (step 2). Wash the root system in running
water and rinse in sterile distilled water.
(7) Hold all seedlings of a test line in your hand and dip the roots into the inoculum (step 5) with an
up and downwards movement for about 30 s. Remove excess inoculum by touching the edge of
the beaker.
(8) Place 20 seedlings of the test line side by side on a blotter paper (size 45 × 25 cm with one fold;
any colour; thin) so that only the cotyledons and roots are covered, and the green tops of the
seedlings remain outside the blotter paper after it is folded. Fold the blotter paper and moisten it
adequately but not excessively. One folded blotter paper will have seedlings of one test line
(Figure 13.14).
(9) Inoculate seedlings of a susceptible control (‘BG 212’) with each batch of test seedlings.
(10) Keep the folded blotters, one on top of the other, in heaps of 10 in a tray. One of these 10
blotters should have the seedlings of the susceptible control (step 9).
(11) Place the trays in an incubator at 35ºC for eight days. Provide 12 hours of artificial light.
Moisten the blotters adequately every day.
(12) At the end of the incubation period (eight days), examine the seedlings for the extent of root
damage, and score for the disease.

The DRR severity is recorded on a 1-9 rating scale as shown in Table 13.9.

Sources of resistance

ICRISAT and NARS in India screened >10000 chickpea accessions and breeding lines in various
segregating generations. These centres have identified several sources of resistance such as ‘ICC
2867’, ‘9023’, ‘9032’, ‘1003’, ‘10803’, ‘11550’ and ‘11551’, having combined resistance against
DRR and FW (Nene et al., 1989).

Several chickpea lines, viz., ‘GG 588’, ‘589’, ‘609’, ‘GL 782’, ‘GF 734’, ‘ICC 4969’, ‘G 543’, ‘F 61’,
‘GL 84254’, ‘85058’, ‘86059’, ‘86071’, ‘PPL 41’, ‘41-1’, ‘57’, ‘PGL 81-1’, ‘GG 763’, ‘773’ and
‘774’, have been found to be resistant to root rot. These lines also had multiple disease resistance to
wilt and foot rot, and some of them were also resistant to AB. The mechanisms responsible for this
activity may well be the same as those for medicarpin and maackiain that have been found to be
responsible for resistance to both AB (Daniel and Barz, 1990) and Fusarium wilt (Stevenson et al.,
1997). In addition, several wilt-resistant lines, viz., ‘GL 84170’, ‘84200’, ‘84254’, ‘85058’, ‘86059’,
‘86071’, ‘86072’, ‘90134’, ‘90145’, ‘PPL 41’, ‘41-1’, ‘57’, ‘146’, ‘155’, ‘GG 773’ and ‘774’, were
also found to be resistant to root rot (Singh et al., 1987, 1991a, 1991b). Although the genotypes
‘Kranti’ (‘ICCC 37’), ‘Bharti’ (‘ICCV 10’), ‘JG 130’ and ‘Sadbhawana’ (‘WCG 1’) are tolerant of
DRR disease in chickpea (Dhar et al., 2004), stable sources of resistance to DRR have not yet been
found and are eagerly being sought.

V. Collar rot (Sclerotium rolfsii)

Distribution and economic importance

Collar rot (CR) of chickpea is widespread in moist tropics and warm countries including Bangladesh,
Colombia, Egypt, Ethiopia, India, Kenya, Mexico, Nepal, Pakistan, the Philippines, Sudan, Syria,
Uganda and Zambia (Nene et al., 1996).

213
Symptoms

The disease usually occurs at the seedling stage, particularly in wet soil conditions. The affected plants
turn yellow and show signs of rotting at the collar region (Figure 13.15A). Whitish mycelial strands
can be seen on dried tap root (Figure 13.15B). On seedlings uprooted from wet soil in the early stages
of infection, rapeseed-like sclerotia (diameter: 1 mm) can be observed. The disease is usually seen in
patches in the field.

Methodology

Greenhouse screening technique

(1) Isolate a culture of S. rolfsii from CR-infected chickpea plants following standard isolation
procedures and purify by single sclerotial culture on potato dextrose agar (PDA).
(2) For multiplication of S. rolfsii, prepare a groundnut (Arachis hypogea) shell medium (soak
partially broken groundnut shells in water for two hours separately and autoclave at 121ºC for
45 minutes). Inoculate each flask with a mycelial bit (1 cm2) from a 10 day old culture of S.
rolfsii grown on PDA and incubate at 25 ± 1ºC with a 12 hour photoperiod for 20 days.
(3) The potting medium is prepared by mixing groundnut shell inoculum from step 2 with
autoclaved soil (medium vertisol, pH7) at a rate of 100 g of inoculum per 4 kg of soil.
(4) The potting medium from step 3 is filled in metal trays (70 × 30 × 16 cm).
(5) 10 seeds of each cultivar are sown in infested soil in a completely randomised block design in
three replications at 2-3 cm depths. Water adequately and regularly.
(6) Observations are recorded on collar rot incidence 30 days after inoculation.
(7) The temperature in the greenhouse should range between 28-30ºC.

Sources of resistance

Resistance in available germplasm seems to be scarce; however, tolerance has been reported (Gurha et
al., 1982). Germplasm accessions ‘ICC 1696’, ‘ICC 4709’ and ‘ICC 14391’ (S. D. Singh, personal
communication); breeding lines ‘RSG 130’, ‘132’ and ‘191’ (Chitale et al., 1990); and cultivar ‘SAKI
9516’ (Dua et al., 2001) have shown low incidence of CR.

VI. Black root rot (Fusarium solani)

Distribution and economic importance

Black root rot (BRR) is a minor disease reported from Argentina, Chile, India, Mexico, Spain, Syria
and the USA. However, losses are significant and there is a need to identify sources of resistance to
this disease.

Symptoms

The disease can occur at any stage but more often at the seedling stage. The affected plants turn
yellow and wilt. Dead plants are seen scattered in the field. The root system is rotten, most of the finer
roots are shed and the remaining roots turn black (Figure 13.16A). Affected plants dry prematurely but
may go on producing new roots if sufficient moisture is available. Excessive moisture and moderately
high temperatures (25-30°C) encourage disease development (Nene and Reddy, 1987).

214
Methodology

Greenhouse screening

(1) F. solani isolated from chickpea roots is multiplied on potato dextrose broth (100 ml in a 250 ml
flask) for seven days at 25°C in a shaker at 125 rpm.
(2) Seedlings are raised in plastic pots (15 cm) in an autoclaved sand-soil (vertisol) mixture (1:1).
(3) The inoculum is diluted by adding 100 ml of sterile water and mixed thoroughly.
(4) For inoculation, about 3 cm of soil around the seedlings is removed and 5 ml of inoculum is
poured near the collar region. The soil surface is re-levelled. The soil is kept moist before and
after inoculation.
(5) 25 days after inoculation, the seedlings are carefully removed from each pot and the soil is
washed from the roots. Data is recorded on a 1-9 rating scale.

Root-dip technique

The root-dip technique described for Fusarium wilt is also applicable for screening against this
disease. The steps mentioned for growing the seedlings and inoculum preparation are the same. The
only difference is that the culture used is F. solani and the disease score is recorded on a 1-9 rating
scale based on root blackening.

The severity of BRR is recorded on a 1-9 rating scale as shown in Table 13.10.

VII. Stem rot (Sclerotinia sclerotiorum)

Distribution and economic importance

Stem rot (SR) of chickpea is a devastating disease in many chickpea-growing parts of the world. It has
been reported from Algeria, Australia, Bangladesh, Chile, Egypt, Hungary, India, Iran and Morocco
(Nene et al., 1996).

It is particularly severe in the north-western states of India. It appeared in epidemic form and caused
heavy damage in 1947-1948, 1948-1949, 1953-1954, 1977-1978, 1980-1981 and 1982-1983, and the
crop was completely damaged in some of the chickpea-growing areas (Singh and Singh, 1979; Singh
et al., 1989). It again appeared in a severe form in 1986-1987 and 1987-1988 (Singh et al., 1989).

Symptoms

The disease rarely attacks younger plants. It normally attacks adult plants near flowering and podding
stages, when they produce a dense canopy and the soil remains wet for a longer period due to rain. The
characteristic symptom of the disease is the production of a web of white mycelial strands at the collar
region and above (Figure 13.16B). Black irregular sclerotial bodies can be seen mingled with mycelial
strands. Sometimes, lesions and fungal growth can be seen on aerial parts of the plant. This is due to
aerial infection by the pathogen. The leaves of affected plants turn yellow, dry up and turn straw
coloured.

Methodology

Several workers have tried different screening techniques using various types of inocula such as SR
sick pots, stem base inoculation with colonised chickpea seeds, stem pieces, carrot slices, PDA disks,
sclerotia and ascospores. The technique consists of inoculating chickpea plants with any of the inocula
mentioned above and maintaining high moisture/RH in a greenhouse or covering with polythene bags
or dasuti cloth chambers for about 144 hours at a temperature of around 20-25ºC. Fresh inoculum

215
(four days old) is more virulent than older samples. The screening technique consists of inoculation of
soil with 14 day old chickpea meal medium (4:1) in the ratio of 1:16 in polythene bags (75 × 10 cm).
The seeds of test lines are planted in these pots in a greenhouse.

Chickpea plants, 25-30 days old, are placed outside the greenhouse in 8 cm deep circular pits. Water is
added in and around the pit, and plants are covered with moist dasuti cloth chambers for 10 days. High
moisture/RH is maintained by daily spraying the chamber with water from 10 am to 4 pm. There is
substantial production of apothecia on the base of the plants that release ascospores inside the chamber
and rapidly cause secondary infection. The disease can effectively be produced with the application of
ascospores in place of sick pots as described above (Kapila and Singh, 1995).

Sources of resistance

In view of the nature of the disease, it is difficult to identify sources of resistance. Under natural
epiphytotic conditions, ‘GL 84102’, ‘GL 88223’, ‘GLK 88114’ and ‘GF 89-75’ were moderately
resistant to SR, in addition to a few breeding lines which escaped attack by the disease (Singh et al.,
1987). Among 11 accessions of eight Cicer species screened, Cicer judaicum (‘ILWC 19-2’), C.
reticulatum (‘JM 200’), C. pinnatifidium (‘199’) and C. yamashitae (‘210’) were found to be
moderately resistant to SR.

CONCLUSIONS

Cultivation of disease-resistant chickpea cultivars is the most effective method of disease control. Host
plant resistance provides economical, environmentally acceptable and sustainable disease
management. For identification and development of sources of host plant resistance in the available
germplasm and breeding material, reliable and reproducible screening techniques are essential.
Techniques to screen chickpea germplasm and breeding material against various chickpea diseases
have been developed by different research institutions and are described here. However, there is a
need to correlate field, greenhouse, growth room and laboratory screening techniques for disease
resistance in chickpea.

Although chickpea research workers have tried to generate adequate information on chickpea diseases
and their management, efficient and complete control measures are not yet available for use by
farmers poor in resources in South Asia and elsewhere. Substantial progress has been made in the
development of screening techniques to identify sources of resistance, but there has been little success,
especially in identifying high levels of resistance in cultivated Cicer species. However, high and stable
levels of resistance have been identified in wild Cicer species. It is expected that chickpea disease-
resistant and high yielding cultivars endowed with other desirable characteristics will be made
available to farmers in the near future. Meanwhile, high priority should be given to participating in
farm validation of the available components (including moderate levels of host plant resistance with
high yield) of disease management and their integration.

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Table 13.1. Disease rating scale for Ascochyta blight

Rating Symptoms
1 No symptoms
2 Minute lesions prominent on the apical stem
3 Lesions up to 5 mm in size and slight drooping of the apical stem
4 Lesions obvious on all plant parts and clear drooping of apical stem
5 Lesions obvious on all plants parts; defoliation initiated; breaking and
drying of branches slight to moderate
6 Lesions as in 5; defoliation; broken, dry branches common; some
plants killed
7 Lesions as in 5; defoliation; broken, dry branches very common; up
to 25% of the plants killed
8 Symptoms as in 7 but up to 50% of the plants killed
9 Symptoms as in 7 but up to 100% of the plants killed

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Table 13.2. Sources of resistance to Ascochyta blight in chickpea germplasm

Genotype Remarks Reference

ICC 3634, ICC 4200, ICC 4248, ICC 5124, ICC Reddy and Singh
6981, ILC 196, ILC 3346, ILC 3956 and ILC 4421 (1984)

ILC 72, ILC 191, ILC 3279 and ILC 3856 Resistant in eight Singh et al. (1984)
chickpea-growing
countries (including
India, Pakistan and the
Mediterranean region)

ICC 76, ICC 187, ICC 607, ICC 1121, ICC 1136, ILC 482 and ICC 1903 Katiyar and Sood
ICC 1416, ICC 1754, ICC 1762, ICC 1903, ICC were always rated 1 on a (1985)
7773, ILC 236, ILC 482, ILC 484, ILC 2548 and 1-9 scale
ILC 2956

ICC 4000 and 4014 Both foliage and pods of Singh and Kapoor
ICC 4000 were resistant (1985)
to Ascochyta blight

ILC 3864, ILC 3870 and ILC 4421 Pal and Singh (1990)

ILC 190, ILC 201, ILC 202, ILC 2506, ILC 3856, Resistant to 3 to 6 races Singh and Reddy
ILC 5928, ICC 3996 and FLIP 83-48 of A. rabiei (1990)

ILC 5586, ILC 5894, ILC 5926, ILC 6482, ILC Resistant both in Singh and Reddy
7795, ICC 4475, ICC 6328 and ICC 12004 greenhouse and field (1992)

ILC 3287 Rate-reducing Reddy and Singh

phenomenon of (1993)
Ascochyta blight
observed

CG 715, ACC 76, H 86-8, H 86-100 and HK 86-120 Singh and Pal (1993)

ILC 3896, ICC 7514, NEC 123, P 1279-2 and P Gaur and Singh
4268-1 (1996)

ICC 8161 Shukla and Pandya

(1988)

ICC 1278, ICC 1284, ICC 1285 and ICC 1304 Wadud and Riaz
(1988)

FLIP 92-262C, FLIP 92-110C and FLIP 92-154C Toker et al. (1999)

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 Table 13.3. Description of different parameters of the Botrytis grey mould scoring scale

Rating Leaf (%) Stem and branches Flower (%) Pod (%)
1 No infection No infection No infection No infection
2 Up to 1 No infection Up to 1 Up to 1
3 2-5 No infection 2-5 2-5
4 6-15 Small lesion on few branches 6-15 6-15
5 16-25 Moderate lesions on stem and 16-25 16-25
branches
6 26-40 and defoliation Expanding lesions on stem and 26-40 26-40
is common branches
7 41-60 and defoliation Large lesions; stem girdling and 41-60 41-60
is very common drying of branches
8 61-80 and nearly Very large lesions; stem 61-80 61-80
complete defoliation girdling; extensive drying of
branches
9 81-100 and complete Complete drying of stem and 81-100 81-100
defoliation branches

Table 13.4. Rating scale of Botrytis grey mould of chickpea for field and controlled environment
screening

Rating Field screening Controlled environment screening

1 No infection on any part of the plant No infection on any part of the plant
2 Minute lesions on lower leaves, flowers and Minute water soaked lesions on emerging
pods covered under dense plant canopy; usually tender leaves; usually not seen
not visible
3 Lesions on <5% of the leaves, flowers and pods Minute water soaked lesions on 1-5% of
covered under dense plant canopy emerging and uppermost tender leaves;
usually seen after careful examination
4 Lesions and some fungal growth (conidiophores Water soaked lesions on 6-10% of uppermost
and conidia) can be seen on up to 15% of the tender leaves and tender shoots
leaves, flowers, pods and branches covered
under dense plant canopy
5 Lesions and slight fungal growth on up to 25% Water soaked lesions; soft rotting of 11-25%
of the leaves, flowers, pods, stems and branches of tender leaves and shoots
covered under dense plant canopy
6 Lesions and fungal growth on up to 40% of the Water soaked lesions; soft rotting of 26-40%
leaves, flowers, pods, stems and branches; of top leaves and shoots
defoliation; 25% of the plants killed
7 Large lesions and good fungal growth on up to Soft rotting; fungal growth on 41-55% of the
60% of the leaves, flowers, pods, stem and leaves and branches
branches; defoliation common; drying of
branches; 50% of the plants killed
8 Large lesions and profuse fungal growth on up Soft rotting; fungal growth on 56-70% of the
to 80% of the leaves, flowers, pods, stems and leaves, branches and stems
branches; severe defoliation; drying of
branches; 75% of the plants killed
9 Large lesions; very profuse fungal growth on up Extensive soft rotting; fungal growth on
to 100% of the flowers, pods, stems and >70% of the leaves, branches and stems
branches; almost complete defoliation; drying of
plants; 100% of the plants killed

224
Table 13.5. Sources of resistance to Botrytis grey mould resistance in chickpea

Resistance Genotype Reference

Wild species ILWC 35/S-1 (C. echinospermum) and ILWC 9/S-1 (C. Singh et al. (1991a)
pinnatifidum)
C. judaicum 182, C. judaicum ILWC 19-2, C. Singh et al. (1998)
pinnatifidum 188, C. pinnatifidum 189, C. pinnatifidum
199, C. pinnatifidum ILWC 9/S-1, C. bijugum ILWC 9/S-
1, C. bijugum ILWC 7/S-1, C. echinospermum ILWC
35/S-1 and C. echinospermum ILWC 39

Land races GPC 14, HIMA and P 6223 Singh and Kant (1999)
ICC 1069, 6250, 7574 and 10302 Rathi et al. (1984a;
1984b)
ICC 466, ICC 478, ICC 662, ICC 755, ICC 756, ICC 799, Tripathi and Rathi (2000)
ICC 800, ICC 1069, ICC 1591, ICC 7574, ICC 10302,
ICCL 87322
GL 84212 and ICC 1905 Singh and Kaur (1989)
GNG-3, C-235 and BG-249 Pandey et al. (1982)
P 919, CPI 56566, JM 995 and E 100 Y Singh and Kapoor (1985)

Table 13.6. Disease rating scale for Fusarium wilt

Disease incidence (%) Disease reaction

0-10 Resistant
10.1-20.0 Moderately resistant
20.1-40.0 Moderately susceptible
40.1-100 Susceptible

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Table 13.7. Sources of resistance to Fusarium wilt in chickpea germplasm

Genotype Remarks Reference

GL 84170, 84200, 84254, 85058, 86059, Moderately resistant in India Singh et al. (1991b)
86071, 86072, 90134, 90145, PPL 41, 41-
1, 57, 146, 155, GG 773 and 774

Avarodhi (ICC 14344), BG 246, ICCC Resistant at several locations in Nene et al. (1989)
32, ICC 42 India in multi-location testing

Kabuli cultivars (Surutato 77, Senora, UC Resistant in Mexico and Haware et al. (1990)
15 and UC 27) California

FLIP 84-43 (ILC 5411, FLIP 85-20, FLIP Highly resistant in Spain Jimenez-Diaz et al.
85-29C, ILC 127, 219, 237, 267 and 513 (1989)

IC 10149, ICC 9023, ICC 11550, ICC Resistant source reported from Gaur and Chaturvedi
902, ICCC 42, ICCV 10, Phule G 95007, India against FW (2004)
KWR 108, GPF 2, IPC 99-13, IPC 99-1,
IPC 2000-14, IPC 2000-41, IPC 99-10,
IPC 2000-18, IPC 2000-52, IPCK 9-3,
CPS 1, WR 315, JG 74, JG 1265, GL
8834, GL 87079, GL 91061, GL 86123, H
86-72, H 86-18, KPG 259-4

Accessions of several wild species Resistant Haware et al.

including C. bijugum, C. echinospermum, (1992a)
C. judaicum and C. pinnatifidum

Table 13.8. Chickpea genotypes with a moderate to high level of resistance against Fusarium
oxysporum f. sp. ciceris

Chickpea type Genotype

Desi Avarodhi (ICC 14344), BG 209, 246, 313, 307, BGM 418, 419, 432, 436, 439, 443,
444, 451, GF 89-36, GL 86114, 86123, GNG 468, 469, H 82-2, 83-18, 83-23, 83-60,
83-84, 84-8, 85-65, 85-124, 86-8, 86-18, 86-20, 86-21, 86-39, 86-42,86-72, 86-73,
86-91, 86-100, 86-142, 86-143, 86-156, 86-158, 86-170, 88-123,89-50, JG 315, 317,
322, KPG 63, 142-1, 143-1, PDG 83-39, 84-10, 84-14, 85-7, 85-18, Phule G 87207

Kabuli ICCC 32, ICCC 42, BG 287

Source: Consolidated Report on Rabi Pulses, Plant Pathology, AICRP, Kanpur (1985-1997).

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Table 13.9. Rating scale for dry root rot disease

Rating Observation Disease reaction

1 No infection on roots Immune
>1 and <3 Very few small lesions on roots Resistant
>3 and <5 Lesions on roots clear but small; new roots Moderately resistant
free from infection
>5 and <7 Lesions on roots; many new roots generally Susceptible
free from lesions
>7 Roots infected and completely discoloured Highly susceptible

Table 13.10. Rating scale for black root rot disease

Disease rating Symptoms

1 Plant healthy; no root infection
3 Plant healthy; slight infection in hypocotyl region along with restricted
lesions on few roots
5 Plant stunted; black root rotting on 50% of roots
7 Plant stunted accompanied by yellowing of leaves; 75% of roots
affected
9 Plants with severe stunting and yellowing of leaves; completely rotted
roots

227
 pycnidia

A C
Figure 13.1. Ascochyta blight lesions on stems (A), leaves (B) and pods (C).

Figure 13.2. Controlled environment facility at ICRISAT, Patancheru, Andhra

Pradesh, India.

A B C

Figure 13.3. Controlled environment screening techniques. (A) Whole plant screening
technique; (B) Cut twig screening technique in water; (C) Cut twig screening
technique in sand.

228
 A B

Figure 13.4. Field screening (A) and plant propagator screening technique (B) for
Ascochyta blight.

A B
Figure 13.5. Botrytis grey mould on flowers, twigs (A) and pods (B).

Figure 13.6. Growth room screening technique (pots) for Botrytis grey mould.

229
 A

C
Figure 13.7. Controlled environment screening techniques at ICRISAT, Patancheru 502
324, Andhra Pradesh, India. (A) Whole plant screening technique; (B) Cut twig
screening technique in water; (C) Cut twig screening technique in sand.

Figure 13.8. Field

screening technique
using the perfo-
irrigation system for
Botrytis grey mould.

230
 A B
Figure 13.9. Fusarium wilt. (A) Cowpea plants killed by fusariose; (B) Internal
browning of the root.

A B
Figure 13.10. Screening for tolerance to Fusarium wilt in the field (A) and pot (B).

Figure 13.11. Water culture technique for Fusarium wilt.

231
 A B

C D E

Figure 13.12. Root-dip technique for Fusarium wilt screening. Eight day old seedlings
are grown in sterile sand (A); uprooted (B); washed and the lowermost portion of the
roots cut (C); roots are dipped for 30 s in the Fusarium inoculum and transplanted into
plastic pots (diameter: 15 cm) containing pre-irrigated vertisol and sand (4:1 [v/v]) and
maintained at 25 ± 3°C in a greenhouse (D). Inoculated seedlings are observed for
mortality from 15 to 30 days after inoculation (DAI) (E).

Figure 13.13. Dry root rot symptoms in detail.

232
 Figure 13.14. Blotter paper technique for dry root rot screening.

A B
Figure 13.15. Collar rot. (A) Field symptoms; (B) White mycelial growth at the
collar region.

A B
Figure 13.16. Symptoms of black root rot (A) and Sclerotinia stem rot (B).

233
 Chapter 14

Improving tolerance to Fusarium oxysporum f. sp. melonis in melon using

tissue culture and mutation techniques

Y. Kantoglu1*, E. Seçer1, K. Erzurum2, İ. Tutluer1, B. Kunter1,

H. Peşkircioğlu1, Z. Sağel1
1
Division of Agriculture,
Saraykoy Nuclear Research and Training Center,
Turkish Atomic Energy Authority,
Ankara,
2
Ankara University,
Agricultural Faculty,
Department of Plant Protection,
Ankara,

Turkey.

*Correspondence: [email protected]

Abstract

Fusarium wilt is a vascular disease of the Cucurbitaceae family, especially in muskmelon (Cucumis
melo L.), caused by the soil fungus Fusarium oxysporum f. sp. melonis (FOM). This pathogen persists
in the soil for extended periods of time, and the only effective control is the use of resistant cultivars.
During the last three decades, tissue culture techniques have been utilised in crop improvement to
generate changes in the genetic material of plants via in vitro somaclonal variations (by organogenesis
or somatic embryogenesis) and induced mutagenesis. More recently, researchers have been using in
vitro techniques to investigate the effects of fungal culture filtrates or toxins on susceptible and
resistant genotypes of different plant species or cultivars to assess disease resistance. This method is
effectively used for cucumber and melon. There are various in vitro culture techniques that can be
used for cucumber (Malepszy, 1988). In this chapter, we show a method for mass-selection of melon
mutants resistant to Fusarium wilt. In vitro selection of resistant cells, from both irradiated and non-
irradiated explants, is performed using culture filtrates of different FOM races. This research could
lead to the development of new melon cultivars resistant to Fusarium wilt.

INTRODUCTION

Fusarium wilt is a vascular disease of the Cucurbitaceae family caused by the soil fungus FOM, which
is detrimental to muskmelons (C. melo L.). Fusarium wilt of melon is prevalent in temperate and
tropical regions and is a problem worldwide. FOM can survive in the soil for extended periods of time
as chlamydospores, and is capable of colonising crop residues and the roots of most crops grown in
rotation with melon. The only effective control is the use of resistant cultivars. Four races of FOM
have been identified, namely 0, 1, 2 and 1.2 (Risser et al., 1976; Mas et al., 1981). Race 1.2 has been
further subdivided into races 1.2y and 1.2w, which cause yellowing and wilt symptoms, respectively.
Two resistance genes (Fom-1 and Fom-2) have been identified in melons (Mas et al., 1981; Martyn
and Gordon, 1996; Joobeur et al., 2004). Fom-1 confers resistance to FOM races 0 and 2, and Fom-2
confers resistance to races 0 and 1. These two genes are extensively used in breeding programmes,
which can be assisted by marker assisted selection using markers linked to these resistance genes

235
(Wang et al., 2000; Zheng and Wolff, 2000; Burger et al., 2003). No genes have been identified that
confer resistance to race 1.2 (Zink and Thomas, 1990; Wechter et al., 1995). However, polygenic
recessive genes have been found to confer resistance to race 1.2 in Piboule genotypes (Messiaen et al.,
1962; Risser et al., 1976). Table 14.1 shows some sources of resistance to FOM among melon
genotypes.

Turkey is the second largest world producer of cantaloupes and other melons, behind China only. In
2004, Turkey produced 1700000 tons and accounted for 6.1% of the worldwide production of melons
(FAO, 2005), but its production is declining year after year because of Fusarium wilt. Therefore,
combating Fusarium wilt in the cultivation of melons is of high economic importance for Turkey. In
some parts of Turkey, the prevalent races of this pathogen have been determined. Fantino and Zengin
(1974) isolated race 1.2 from wilted plants showing intensive root rot in Eastern Thrace. In the Aegean
region, Yildiz (1977) recovered three races of the pathogen, race 1 being the most common (57%),
followed by race 1.2 (35%) and race 0 (6%). Yücel et al. (1994) obtained races 0, and 1.2 in the East
Mediterranean region. Erzurum et al. (1999) isolated races 0, 1.2 and 2 in Central Anatolia. Based on
these results, Fusarium wilt is a widespread disease all over Turkey. FOM has caused severe losses for
farmers as our native cultivars are not resistant to this disease. It is believed that our native cultivars
will disappear if resistance to FOM is not introduced into the cultivated material. For this reason,
many scientists in Turkey are focusing on research to develop new resistant cultivars via conventional
and biotechnological breeding methods.

In vitro techniques became widely spread during the 20th century, and their potential to make
important contributions to plant breeding was quickly understood. In vitro techniques for crop
improvement first consisted of micropropagation and plant regeneration, and then in vitro methods
were also found to be useful for eliminating disease and selecting for resistant cells or explants.

Over the last three decades, in vitro tissue culture techniques have been used to generate genetic
changes via somaclonal variations (by organogenesis or somatic embryogenesis) that can be used for
breeding purposes. In vitro selection using specific chemical compounds and pathogens is another
useful aspect of tissue culture (Figure 14.1). Selection with phytotoxins and culture filtrates appears to
be more effective than the use of the pathogen itself (Van Harten, 1998). Researchers now use fungal
culture filtrates or toxins to investigate the response of susceptible and resistant genotypes of different
plant species or cultivars to disease factors. The use of in vitro methods for the evaluation of resistance
is dependent upon a positive correlation between in vitro culture filtrate resistance and whole plant
disease resistance. Gray et al. (1986), Chawla and Wenzel (1987), Connell et al. (1990), Malepszy and
El-Kazzaz (1990), and El-Kazzaz and Malepszy (1992) developed protocols for in vitro determination
of resistance. In comparison with field screening and other biotechnological methods, selection
techniques are more cost and labour effective, and do not require large experimental fields.

More recently, in vitro techniques have been combined with mutation induction for generating genetic
variation, including novel disease-resistant mutants. Mutation induction can be caused by chemical or
physical mutagens that alter the structure of the DNA (Smith, 1985). Treatment of in vitro tissues with
physical or chemical mutagens may increase the frequency of genetic variation considerably. The
physical mutagens most commonly used are X-rays, gamma rays and UV light, whereas ethyl
methanesulfonate (EMS) is the chemical mutagen most used in crop improvement. Irradiation
treatments may be a suitable choice of mutagen for a number of reasons including the fact that
application is fast and that, in contrast to chemical mutagens, there is no risk that residues remain in
the medium. The in vitro mutation frequencies are much higher than for somaclonal variation.
Somaclonal variation is also important for in vitro plant breeding and desirable cells or plantlets,
which are disease resistant, can be obtained by this method. Finally, in vitro techniques are also useful
in classical mutation breeding programmes by vegetative propagation before or after mutagenic
treatment, by in vitro selection or by clonal propagation of selected mutants (Figure 14.1).

236
According to Malepszy and El-Kazzaz (1990), in vitro selection using FOM filtrates can be effectively
used for the selection of cucumber and melon (Megnegneau and Branchard, 1991). This method can
also be applied for the selection of various mutants, which are disease resistant. In this chapter, we
describe a method to screen for resistance in melon, from both irradiated and non-irradiated explants
by using FOM culture filtrates of various races in in vitro conditions. If successfully applied, this
methodology can lead to the identification of new melon cultivars resistant to FOM within a relatively
short period of time. The same methodology can also be adapted for screening other crop species for
resistance to Fusarium spp.

METHODOLOGY

The present work was performed with in vitro plantlets obtained from seeds of melon cv. ‘Yuva’
which is an important commercial cultivar in Turkey. Two types of explants (cotyledon and
hypocotyls with leaf and cotyledon explants) were used for callus and suspension culture initiation
(Taner et al., 2004).

Preparation of fungal culture filtrate and selection medium

In this research, we followed the procedure developed by Megnegneau and Branchard (1991). Petri
dishes containing potato dextrose agar (PDA) medium were inoculated with FOM isolates (A-1)4 and
(A-6)4 obtained from the Plant Protection Department at Ankara University in Turkey (Erzurum et al.,
1999). Petri dishes were incubated at 26°C in the dark. 15 days later, 2 × 106 conidia were transferred
to 200 ml of liquid Richard’s medium. Cultures were kept at 26°C in the dark. After 20 days, fungal
cultures were filtered twice through filter paper to remove mycelia. The pH of the filtrate was adjusted
to pH5.7 with 1 N HCl or 0.1 N NaOH. Subsequently, the cultures were sterilised through a 0.22 μm
filter unit attached to a syringe. During filtrate preparation, it is very important to avoid thermal
degradation of toxic compounds in the fungal culture filtrate. Filtrates should, therefore, be aliquoted
and frozen for long-term storage. The working filtrate aliquot can be kept in the refrigerator. Filtrates
were added to autoclaved modified Murashige and Skoog (MS) basal medium. According to Taner
(2002) and Taner et al. (2004), non-irradiated and irradiated explants were transferred to Petri dishes
containing half strength MS medium with basal salts (Murashige and Skoog, 1962) supplemented with
0.5 mg/l 2,4-dichlorophenoxyacetic acid (2,4-D), 0.5 mg/l kinetin, 250 mg/l casein enzymatic
hydrolysate (Sigma Chemical Co., St. Louis, MO, USA) and 15% sucrose (for cotyledon explants) and
MS basal medium, which was supplemented with 0.5 mg/l indole-3-acetic acid (IAA), 0.5 mg/l 6-
benzylaminopurine (BAP) and 15% sucrose (for hypocotyls with leaf and cotyledon explants). For
both media, the pH was adjusted to pH5.6 and the fungal culture filtrate was added at different
percentages (4, 6, 8, 10, 12, 14, 16, 18 and 20% [v/v]). Control plates contained only the modified MS
basal medium without fungal culture filtrate. The steps for fungal culture filtrate preparation are
shown in Figure 14.2 and described in detail elsewhere in this book.

Preparation of in vitro plant material

Figure 14.3 shows the steps for the initiation of the callus cultures. Seeds of melon genotypes were
surface-sterilised, rinsed and germinated in vitro as previously described by Çürük (1999) and Taner
(2002). Briefly, seeds were surface-sterilised for 20 minutes in 20% sodium hypochloride solution
containing 1% (v/v) Tween 20, and washed three times in sterile distilled water. After removal of the
seed coats, seeds were placed in “baby food culture jars” with Magenta B-caps (product numbers
V8630 and B8648, Sigma) that contained 50 ml of solid MS medium with 0.7% Difco agar and 15%
sucrose. The cultures were maintained at 25°C, under fluorescent illuminescence with a light intensity
of 10000 lux and a 16 hour photoperiod. For resistance screening, two kinds of non-irradiated and
irradiated explants (i.e., cotyledons and hypocotyls containing leaves and cotyledons) were used
according to Taner et al. (2004). Cotyledon explants were transferred to Petri dishes containing half
strength MS medium with basal salts (Murashige and Skoog, 1962) supplemented with 0.5 mg/l (2,4-
D), 0.5 mg/l kinetin and 15% sucrose. Hypocotyls with leaf and cotyledon explants were transferred to

237
MS basal medium, which was supplemented with 0.5 mg/l IAA, 0.5 mg/l BAP and 15% sucrose. For
both media, the pH was adjusted to pH5.6 with 1 N HCl or 0.1 N NaOH. It is very important that the
experiments contain >50 explants for each combination.

In vitro mutagenic treatment

Irradiation doses and their effects on plants are known to be genotype-dependent. Even with the same
genetic material, the optimal dose for mutation induction depends on the type of plant material to be
irradiated. In the case of in vitro irradiation, the optimal dose is typically lower than that used for seed
irradiation. Therefore, every experiment should start with radiosensitivity tests to determine the
optimal irradiation dose for the plant material being used. The LD50 (i.e., the dose that will kill 50% of
the test organisms within a designated period) is often used as the optimal dose for mutation induction.
The lower the irradiation LD50, the more sensitive the plant material is.

After seed germination, seven day old in vitro plantlets which contained true leaves and cotyledons
were irradiated with a 60Co gamma source. Five different doses of gamma rays were used (10, 20, 30,
40 and 50 Gy) for the radiosensitivity assays. In our experiments, a gamma irradiation dose of 25 Gy
was found to be the most effective dose for in vitro plantlets of the ‘Yuva’ cultivar. Moreover, after
irradiation, it is important to transfer the explants onto fresh regeneration medium containing the
fungal culture filtrate as described in the “Preparation of in vitro plant material” section of this chapter
to avoid any toxicity of the medium components due to the irradiation.

In vitro screening and evaluation

The fungal culture filtrate was added to the media at different concentrations as indicated above.
Cotyledon cultures were incubated at 26°C in the dark for three weeks. Hypocotyl explants with leaves
and cotyledons were incubated with a light period of 16 hours at 15000 lux and at 26°C for three
weeks according to Taner et al. (2004). At the end of this three week period, explants were assessed
according to their regeneration capacity (Figure 14.4). Formation of white/yellow callus from the
explants indicated survival capacity of the explants in the presence of the filtrate, suggesting that the
plant material was resistant to the disease. On the other hand, a brownish colour indicated that the calli
were dead, pointing to susceptibility.

The resistant calli were selected and subcultured for an additional two weeks on the same medium
containing the filtrate for further confirmation of resistance. The resistant calli were then transferred to
hormone-free MS medium supplemented with the fungal filtrate for induction of somatic
embryogenesis (Taner and Yanmaz, 2003). For plantlet regeneration, MS medium supplemented with
1.0 mg/l IAA was used according to Taner (2002). After this period, the rate of mortality of the
explants was estimated. The regeneration of shoots and roots, growth performance and number of
shoots were used as indicators for measuring the degree of resistance. Plantlets were then transferred
to greenhouses and advanced to the M2 generation for further selection and evaluation.

CONCLUSIONS

Seven doubled-haploid (DH) melon lines, originating from parthenogenesis using irradiated pollen
were produced. Two promising DH lines were selected for resistance/tolerance to Fusarium wilt
following inoculations with race 1.2w. These two DH lines represent a source of resistance/tolerance
that is commercially exploitable either as rootstocks or as lines for conventional breeding (Ficcadenti
et al., 2002).

Table 14.2 shows the results of previous studies using in vitro methods for the selection of melon and
cucumber resistant to Fusarium wilt. This technique is very effective for the rapid screening of melon
and cucumber. We are currently applying in vitro mutagenesis combined to in vitro selection for the

238
improvement of the commercial melon cultivar ‘Yuva’ to expand the genetic variation of muskmelon
in Turkey and to select resistant types of this cultivar.

REFERENCES TO CHAPTER 14

Burger Y, Katzir N, Tzuri G, Portnoy V, Saar U, Shriber S, Perl-Treves R, Cohen R (2003) Variation
in the response of melon genotypes to Fusarium oxysporum f. sp. melonis race 1 determined by
inoculation tests and molecular markers. Plant Pathol 52: 204-21.

Chawla HS, Wenzel G (1987) In vitro selection of fusaric acid resistant barley plants. Plant Breeding
99: 159-163.

Connell SA, Legg T, Heale JB (1990) Sensitivity of cells and protoplasts of Hop cultivars to cytotoxic
components of culture filtrates of Verticillium albo-atrum isolates from Hop. Plant Pathol 39: 92-101.

Çürük S (1999) Investigations on in vitro plant regeneration and genetic transformation on melon
cultivars. Çukurova University Graduated School Of Science, Horticultural Science, Adana, Turkey, p
217.

El-Kazzaz AA, Malepszy S (1992) Selection of resistant Cucumis sativus regenerated plants to
Fusarium oxysporum via tissue culture. The First Egyptian-Italian Symposium on Biotechnology,
Assiut, Egypt, 21-23 November, pp: 121-129.

Erzurum K, Taner KY, Seçer E, Yanmaz R, Maden S (1999) Occurrence of races of Fusarium
oxysporum f. sp. melonis causing wilt on melon in Central Anatolia. J Turk Phytopathol 28: 87-97.

Fantino MG, Zengin H (1974) Ricerche sull’agente dell’avvizzimento del cocomero e del melone.
Informatora Fitopatologica XXIV, 7: 7-10.

FAO (2005) Provisional 2004 Production and Production Indices Data. Food and Agriculture
Organization of the United Nations (FAO), Rome, Italy. http://faostat.fao.org/faostat/collections?
subset=agriculture

Ficcadenti N, Sestili S, Annibali S, Campanelli G, Belisario A, Corazza L (2002) Sources of tolerance

to Fusarium oxysporum f. sp. melonis race 1,2 in DH lines of muskmelon (Cucumis melo). Acta Hort
(ISHS) 588: 133-136.

Gray LE, Guan YQ, Wildholm JM, Gray LE (1986) Reaction of soybean callus to culture filtrates of
Phialophora gregata. Plant Sci 47: 45-55.

Joobeur T, King JJ, Nolin SJ, Thomas CE, Dean RA (2004) The fusarium wilt resistance locus Fom-2
of melon contains a single resistance gene with complex features. Plant J 39: 283-297.

Malepszy S (1988) Cucumber (Cucumis sativus L.). In: Bajaj YSP (eds.) Biotechnology in Agriculture
and Forestry, Vol. 6. Crops II. Springer-Verlag, Berlin, Heidelberg, Germany, pp: 277-293.

Malepszy S, El-Kazzaz AA (1990) In vitro culture of Cucumis sativus XI. Selection of resistance to
Fusarium oxysporum. Acta Hort (ISHS) 280: 455-458.

Martyn RD, Gordon TR (1996) Fusarium wilt of melon. In: Zitter TA, Hopkins DL, Thomas CE (eds.)
Compendium of Cucurbit Diseases. APS Press, St. Paul, Minnesota, USA, pp: 11-13.

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Mas P, Molot PM, Risser G (1981) Fusarium wilt of muskmelon. In: Nelson PE, Toussen TA, Cook
RJ (eds.) Fusarium: Disease, Biology and Taxonomy. Pennsylvania State University Press, University
Park, Pennsylvania, USA, pp: 169-177.

Megnegneau B, Branchard M (1991) Effects of fungal culture filtrates on tissue from susceptible and
resistant genotypes of muskmelon to Fusarium oxysporum f. sp. melonis. Plant Sci 79: 105-110.

Messiaen CM, Risser G, Pecaut P (1962) Etude des plantes resistantes au Fusarium oxysporum f. sp.
melonis dans la variété de melon Cantaloup Charentais. Ann Amélior Plant 12: 157-164.

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cultures. Physiol Plant 15: 473-497.

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melonis races and resistance genes in Cucumis melo. Phytopathology 66: 1105-1106.

Smith M (1985) In vitro mutagenesis. Annu Review Genet 19: 423-62.

Taner KY (2002) Plant formation by somatic embryogenesis in melon (Cucumis melo L.). PhD thesis,
Ankara University, Ankara, Turkey, 145 pp.

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Horticulture Congress, 8-12 September 2003, Antalya, Turkey, pp: 361-364.

Taner KY, Yanmaz R, Yazar E, Alper A (2004) The effects of sucrose concentration and pH level on
different explant types for in vitro organogenesis in melon (Cucumis melo L.). Alatarim J 3: 11.

Van Harten AM (1998) Mutation breeding theory and practical applications. Cambridge University
Press, Cambridge, UK, 353 pp

Wang YH, Thomas CE, Dean R (2000) Genetic mapping of fusarium wilt resistance gene (Fom-2) in
melon (Cucumis melo L.). Mol Breeding 6: 379-389.

Wechter WP, Whitehead MP, Thomas CE, Dean RA (1995) Identification of a randomly amplified
polymorphic DNA marker linked to the Fom-2 Fusarium wilt resistance gene in muskmelon MR-1.
Phytopathology 85: 1245-1249.

Yildiz M (1977) Researches on Fusarium oxysporum f. sp. melonis races in the Mediterranean region
of Turkey and response of some national melon genotypes to the disease. Ege University of
Agriculture, Department of Plant Protection, Ege, Turkey, 112 pp.

Yücel S, Pala H, Sarı N, Abak K (1994) Determination of Fusarium oxysporum f. sp. melonis races in
the East Mediterranean region of Turkey and response of some melon genotypes to the disease. 9th
Congress of The Mediterranean Phytopathological Union, Kuşadası, Turkey, pp: 87-89.

Zheng XY, Wolff DW (2000) Randomly amplified polymorphic DNA markers linked to fusarium wilt
resistance in diverse melons. HortSci 35: 716-721.

Zink FW, Thomas CE (1990) Genetics of resistance to Fusarium oxysporum f. sp. melonis races 0, 1,
and 2 in muskmelon line MR-1. Plant Dis 80: 1230-1232.

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Table 14.1. Muskmelon genotypes resistant to Fusarium oxysporum f. sp. melonis*

Genotype Resistance to FOM races Type and origin

‘Védrantais’ 0 and 2 Cultivar (INRA, France)
‘Dulce’ 0 and 2 Cultivar (USA)
FM 025 0 and 2 Breeding line (ARO, Israel)
FM 018 0 and 2 Breeding line (ARO, Israel)
FM 004 0 and 2 Breeding line (ARO, Israel)
FM 023 0 and 2 Breeding line (ARO, Israel)
FM 014 0 and 2 Breeding line (ARO, Israel)
FM 024 0 and 2 Breeding line (ARO, Israel)
‘Doublon’ 0 and 2 Cultivar (INRA, France)
‘Hemed’ 0 and 2 Commercial cultivar (Hazera, Israel)
‘Freeman cucumber’ 0 and 1 Breeding line (ARO, Israel)
PI 161375 0 and 1 Breeding line (ARO, Israel)
F65 0 and 1 Breeding line (ARO, Israel)
I4-6-2-B 0 and 1 Breeding line (ARO, Israel)
‘Maqdimon F1 Hybrid’ 0 and 1 Commercial cultivar (Hazera, Israel)
‘Omega (5080) F1 Hybrid’ 0, 1 and 2 Commercial cultivar (Nunheru Zaden,
The Netherlands)
‘Caruso (5093) F1 Hybrid’ 0, 1 and 2 Commercial cultivar (Nunheru Zaden,
The Netherlands)
‘Piboule’ 1.2 Cultivar (France)
‘Dinero’ 1.2 Commercial cultivar (Syngenta Seeds,
Milano, Italy)
ASR04993033 1.2 Commercial cultivar (Asgrow Seeds,
Latina, Italy)

*Modified from Burger et al. (2003).

Table 14.2. In vitro screening for resistance to Fusarium wilt in Cucumis spp

Species and cultivar Pathogen Result Reference

Melon (Cucumis melo cv. ‘Yuva’) Fusarium oxysporum Ongoing study
f. sp. melonis
Cucumber (C. sativus cv. F. oxysporum f. sp. 7% of regenerated Malepszy and El-
‘Borszczagowski’ and Gy-3) cucumerinum plants were resistant Kazzaz (1990)
Melon (C. melo) F. oxysporum f. sp. Correlation between Megnegneau and
melonis filtrate density and Branchard (1991)
explant growth
established
Cucumber (C. sativus cv. F. oxysporum f. sp. Selected plants El-Kazzaz and
‘Borszczagowski’ and Gy-3) cucumerinum showed various Malepszy (1992)
modes of resistance
expression when
exposed to 100%
culture filtrate

241
 Germplasm

Somaclonal Mutation In vitro mutation by chemical

variation or physical mutagen

Genetic Variation

Selection In vitro selection with

culture filtrates
Genetic mapping
(TILLING, AFLP, etc.)
Field evaluation

New variety

Figure 14.1. Plant breeding scheme by induced mutations and somaclonal variations
(modified from Van Harten, 1998).

A B

C D

Figure 14.2. The steps of filtrate preparation. (A) Fungal cultures were filtered through
filter paper to remove mycelium; (B) Fungal cultures were sterilised with a 0.22 µm
Sartorius filter unit with a vacuum for complete removal of fungal cells; (C) After
sterilisation; (D) Fungal culture filtrate, which was added to autoclaved basal medium.

242
 A B

C D E

Figure 14.3. The steps for culture initiation. (A) In vitro seed culture. Seeds were cultured
after removal of the seed cover; (B) In vitro plantlet production; (C) Hypocotyls with leaf
and cotyledon explants; (D) Cotyledon explants; (E) Incubation period.

A B

Figure 14.4. Selection for resistant melon calli with fungal culture filtrate. (A) Live
callus (resistant); (B) Dead callus (susceptible).

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 Chapter 15

Screening for resistance to lettuce downy mildew (Bremia lactucae)

A. Lebeda* and I. Petrželová

Palacký University in Olomouc,

Faculty of Science,
Department of Botany,
Olomouc-Holice,
Czech Republic

*Correspondence: [email protected]

Abstract

Screening methods of Lactuca spp. for resistance to Bremia lactucae (lettuce downy mildew) are
described in this chapter. Procedures of isolation, cultivation and maintenance of B. lactucae isolates
are summarised. Resistance screening can be carried out either on lettuce seedlings, detached
cotyledon leaves, leaf disks or parts of true leaves from adult plants. Methodology of inoculum
preparation, inoculation and incubation conditions is described. For the assessment infection degree, a
qualitative or quantitative approach is used. A basic differential set of Lactuca spp: used for testing B.
lactucae isolates and virulence phenotypes of the most important B. lactucae races was established,
including the denomination of races by the sextet code. The methods described have a broad
application both in practice and research. They are useful for resistance screening of new breeding
material, for searching for new sources of resistance in Lactuca spp. germplasm, for investigating
plant resistance mechanisms and also for the study of virulence variation in B. lactucae isolates and
populations.

INTRODUCTION

Lettuce is one of the most important vegetable crops and has a long history of germplasm research and
resistance breeding (Lebeda et al., 2007). Lettuce downy mildew, caused by the peronosporaceous
fungus B. lactucae Regel (Figure 15.1A, B), is a serious disease of cultivated lettuce (Lactuca
sativa L.) distributed worldwide wherever the lettuce crop is grown (Marlatt, 1974). However, it can
also attack more than 200 other species from about 40 genera of the family Compositae (Crute and
Dixon, 1981). The most common wild host species of this pathogen is Lactuca serriola (prickly
lettuce) and it can also be frequently found on some Sonchus species (Petrželová and Lebeda, 2004).
However, it has been proven that B. lactucae is highly specific and mostly limited to the same genus
of plants (Crute and Dixon, 1981; Lebeda and Syrovátko, 1988; Lebeda et al., 2002). Thus, except for
some Lactuca species, weedy growing Compositae cannot serve as a source of inoculum for cultivated
lettuce (Lebeda and Syrovátko, 1988).

Several different mechanisms of resistance to B. lactucae have been identified in cultivated lettuce
(Lebeda et al., 2001). Most of the resistance is considered to be race-specific (Lebeda et al., 2002).
Race specificity is also common in some wild Lactuca species and some closely related genera
(Lebeda and Boukema, 1991; Lebeda, 1998; Lebeda and Petrželová, 2001, 2004b). The interaction
between L. sativa, L. serriola and B. lactucae conforms to the gene-for-gene relationship (Illot et al.,
1989; Crute, 1992a), in which resistance is determined by dominant Dm resistance genes (R-factors) in
the hosts, and matched by dominant factors for avirulence (Avr) in the pathogens (Hammond-Kosack

245
and Jones, 1997). More than 40 resistance Dm genes or R-factors have been identified in L. sativa and
L. serriola so far (Reinink, 1999; Sicard et al., 1999; Lebeda et al., 2001, 2002; Michelmore et al.,
2002), and the same number of complementary avirulence (Avr) genes or factors are thought to exist
in B. lactucae. The existence of many additional resistance genes in populations of L. serriola is also
expected (Lebeda, 1986a; Farrara and Michelmore, 1987; Bonnier et al., 1994; Lebeda and Petrželová,
2004b, 2004c). Cross inoculation experiments with B. lactucae originating from L. sativa and wild L.
serriola may contribute to unveiling novel sources of resistance in lettuce. However, so far there is
only limited information available on the distribution of race-specific resistance in natural populations
of L. serriola (Lebeda and Petrželová, 2004b, 2004c). More detailed research in this field is needed.

Only a limited number of race-specific resistance genes have been utilised in commercial lettuce
breeding programmes. In Europe, the most frequently used resistance genes in lettuce are Dm2, Dm3,
Dm4, Dm5/8, Dm6, Dm7, Dm11, Dm16, R18 and their combinations (Crute, 1992b; Lebeda and
Zinkernagel, 1999, 2003a).

Race-specific resistance has a big disadvantage as it does not provide durable protection against
lettuce downy mildew, and the introduction of new resistant cultivars is mostly followed by the
appearance of new virulent races of the pathogen (Lebeda and Zinkernagel, 1999, 2003b). During the
last few decades, lettuce resistance breeding has been focused on searching for and utilisation of novel
sources of resistance to B. lactucae from wild Lactuca spp. and closely related species, especially L.
serriola, L. saligna and L. virosa (Norwood et al., 1981; Bonnier et al., 1992; Lebeda, 1998;
Maisonneuve et al., 1999; Jeuken and Lindhout, 2002; Lebeda et al., 2007). The transfer of genes for
resistance from wild species to lettuce may, however, make the latter species vulnerable to attack by
races more usually associated with original, wild host species (Lebeda, 1984).

Reliable methods for screening for resistance/susceptibility in lettuce species are, therefore, extremely
important for the success of breeding programmes (Lebeda et al., 2007). Ideally, these methods should
allow a large number of plants to be screened simultaneously. This chapter describes the use of
seedlings and leaf disks inoculated with live isolates of downy mildew.

MATERIAL AND METHODS

Pathogen isolation, cultivation and maintenance

One or two leaves with symptoms of lettuce downy mildew (i.e., chlorotic lesions surrounded by veins
and covered with pathogen conidiophores) are usually taken from infected lettuce plants. The leaf
samples should be placed in small, closed plastic boxes lined with moistened cellulose cotton-wool,
and they must be transported to the laboratory as soon as possible. If the samples are sporulating, the
spores may be used directly for pathogen isolation. However, leaves with chlorotic spots must most
often be incubated for 1-3 days at 10-15°C and with a 12 hour photoperiod until profuse sporulation
occurs (Lebeda, 1986a, 1986b; Lebeda and Petrželová, 2004a).

Isolates of B. lactucae can be obtained either from localised leaf spots or monosporic isolates, i.e.,
inoculum is prepared by isolation and cultivation of one conidium. Nevertheless, isolates that originate
from one lesion are not necessarily genetically homogeneous. For detailed genetic studies (e.g.,
genetics of virulence, hybridisation between virulence phenotypes etc.), it is better to use monosporic
isolates (Lebeda, 1986b).

For isolation of spores from localised spots, the spots are cut out of infected leaves with sterile
scissors. There are two ways of isolating the pathogen from these leaf pieces. The first one is based on
washing off spores from the leaf surface with tweezers into a small amount (~5 ml) of distilled water
in a beaker. Prepared conidial suspension is sprayed onto seedlings of universally susceptible
cultivars/accessions, e.g., L. sativa cvs. ‘Cobham Green’ (Lebeda and Blok, 1991) or ‘Hilde’ (Lebeda,
1997), or L. serriola line ‘LSE/57/15’ (Lebeda and Petrželová, 2004a) by a glass chromatography

246
sprayer. The second and more efficient method is based on direct contact of cut leaf pieces covered
with conidiophores with a pre-moistened surface of the seedlings. This way, one can avoid dilution of
inoculum and the probability that conidia will reach the leaf surface of seedlings is much higher.

Isolates are maintained and multiplied on seedlings of susceptible plants. Re-inoculation is needed
every 10-12 days. Between individual experiments, isolates can be stored frozen at –20°C on infected
seedlings. However, the vitality of conidia decreases rapidly at this temperature after 2-3 months of
storage. For longer storage periods, isolates can be safely maintained up to 6-7 months in a –80°C
freezer (Lebeda, 2002). Inoculum from frozen conidia is then prepared by re-suspending them in
distilled water.

Preparation of plant material for screening

The test must be carried out in containers or dishes where high air humidity and optimal conditions for
incubation can be maintained throughout the experiment. The best way to carry out extensive
resistance tests with lettuce is to use plastic photographic boxes (320 × 265 × 60 mm) lined with three
layers of moistened cellulose cotton-wool and one layer of filter paper, and closed with a glass cover
(Figure 15.2). These boxes are suitable for tests performed with seedlings or leaf parts (Figures 15.2
and 15.4). In the case of lettuce seeds, the seeds to be tested are sown into lines with a spacing of ~2.5-
3 cm, and the boxes should be kept in a cultivation room under a night/day temperature regime of
10/15°C and a 12 hour photoperiod until the seedlings have fully expanded cotyledonary leaves (~5-7
days after sowing). Before applying the inoculum, it is necessary to remove the seed coats from the
seedlings (Lebeda, 1986b).

In addition to intact seedlings, the tests can be performed on detached cotyledon leaves that are placed
with their axial side downwards on moistened filter paper. It is also possible to use parts of true leaves
from adult plants (2-3 months after transplantation). Leaf disks (diameter: 10 mm, Figure 15.4) or
segments of approximately the same size are cut out of leaves by the use of a cork borer and treated
the same way as detached cotyledon leaves (Lebeda, 1986a, 1986b). Seedlings and parts of leaves can
also be grown in Petri or Drigalski dishes (90, 150 or 200 mm), or in small plastic boxes (Lebeda and
Pink, 1998) lined with moistened cellulose cotton wool and filter paper under the conditions described
above. They are good for testing smaller numbers of samples and should be prepared a short time
before inoculation.

Inoculum preparation, inoculation and incubation

The inoculum is prepared by washing 1-2 day old spores (~9-12 days after inoculation) from infected
seedlings of susceptible plants with distilled water. The optimal concentration of a conidial suspension
is approximately 105 spores/ml (Lebeda, 1979, 1986b). Once the spores are placed in the solution, the
inoculum must be applied to seedlings or leaf disks as soon as possible.

The inoculation may be performed either by spraying or soaking. Conidial suspension is usually
sprayed onto tested plants with a glass chromatography sprayer (Lebeda, 1984). For one plastic
photographic box, approximately 5-6 ml of inoculum is needed as the surface of the leaves must be
completely wet. This method is not well applicable, for instance, to some wild Lactuca species due to
the wax layer on the leaf surface causing the sprayed inoculum to agglomerate into small droplets.
Crute and Dickinson (1976) described a method of soaking seedlings or leaf disks in prepared
inoculum; however, the spraying method is still more efficient (Lebeda, 1986b).

B. lactucae requires specific conditions for its growth. The optimal temperature for conidial
germination and penetration ranges from 10-15°C. For successful infection, high relative humidity
(90-100%) and a dark period during penetration are also needed (Lebeda, 1986b). Boxes or dishes
with inoculated seedlings or leaf disks (or parts of leaves) are incubated in a cultivation room at a
night/day temperature of 10/15°C. Temperatures >20°C inhibit the development of the pathogen. For

247
the first 12-24 hours after inoculation, the boxes must be covered with black foil. After that, they are
kept under a 12 hour photoperiod. Under optimal conditions, sporulation usually occurs 7-9 days after
inoculation. The final assessment of infection is usually carried out on day 13 or 14 after inoculation.
In the case of incompletely compatible responses, the assessment of infection may be performed even
later, at approximately 17-21 days after inoculation (Lebeda, 1986b).

Disease assessment

There are two methods (qualitative and quantitative) for assessing resistance/susceptibility of lettuce to
lettuce downy mildew (Lebeda, 1986b). The qualitative assessment is good for the screening of
breeding material, whereas the quantitative method is usually used for research purposes.

Qualitative assessment

The following scale is mostly used for the assessment of infected seedlings, but it can also be used for
leaf disks (Lebeda, 1986a, 1986b; Lebeda and Pink, 1998):
+ susceptible response: macroscopically visible sporulation on 80-100% of seedlings;
- resistant response: no visible sporulation on seedlings;
(-) incompletely resistant response: limited sporulation often followed by macroscopically visible
necrotic response or chlorosis (Lebeda et al., 2001);
(+) heterogeneous response: mixture of completely susceptible and some resistant plants in tested
sample; it may be caused by a segregation or impurity of used seed, or by a heterogeneity of an
isolate (Crute and Dickinson, 1976).

Quantitative assessment

The main criterion for the quantitative or semi-quantitative assessment is the intensity of pathogen
sporulation (Figures 15.3 and 15.4). The degree of infection is usually assessed at two day intervals 6-
14 days after inoculation.

For the assessment of sporulation intensity on cotyledon or true leaves, a scale ranging from 0-3 is
used (Figure 15.3) as described by Dickinson and Crute (1974):
0 = no visible conidiophores on leaves;
1 = limited sporulation, sporadic conidiophores present;
2 = < 50% of cotyledon area covered with conidiophores;
3 = > 50% of cotyledon area covered with conidiophores.

For the assessment of sporulation intensity on leaf disks (Figures 15.2 and 15.4), a scale ranging from
0-4 is used (Lebeda, 1983, 1986a):

0 = no visible conidiophores;
1 = ≤ 25% of leaf disk surface is covered with conidiophores;
2 = > 25 - ≤ 50% of leaf disk surface is covered with conidiophores;
3 = > 50 - ≤ 75% of leaf disk surface is covered with conidiophores;
4 = > 75 % of leaf disk surface is covered with conidiophores.

For both scales, the final value of sporulation intensity (degree of infection, DI) is expressed as the
percentage of the maximum score according to Towsend and Heuberger (1943):

P = Σ(n × v) × 100/x × N

Where: P = the total degree of infection (DI); n = number of plants in each assessed category
(infection degree); v = infection degree (for leaf disks 0-4); x = scale range (for leaf disks = 4) and N =
total number of assessed plants or leaf disks.

248
Each test usually comprises 20-25 seedlings per tested sample in one replication. In the case of a
variable reaction, the test must be repeated. When testing adult plants, five leaf disks or segments
originating from one plant are included in one test, and a total of five plants are tested (or more
depending on the uniformity of the material used).

Determination of pathogenic variability

Virulence of isolates is examined by screening the differential set of Lactuca accessions with well-
characterised race-specific Dm genes and/or R-factors. The basic differential set of Lactuca spp.
genotypes used for B. lactucae is listed in Table 15.1. Isolates’ virulence and the sextet codes of the
most important races in the recent past are summarised in Table 15.2.

CONCLUSIONS

The methods described in this chapter have broad applications, not only for research purposes but also
in lettuce resistance breeding programmes (Lebeda et al., 2007). They are used for the routine
resistance screening of new breeding Material and for searching for new sources of resistance among
Lactuca spp. germplasm but also for screening of virulence of pathogen populations occurring on
cultivated lettuce (Lebeda and Zinkernagel, 2003a, 2003b). The method using lettuce seedlings
especially has a big advantage as it allows testing of huge numbers of genotypes simultaneously.

Acknowledgements

This study was supported by the Ministry of Education, Youth and Sports of the Czech Republic,
project MSM 6198959215, and by the project QH 71229 (NAZV).

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Lebeda A, Pink DAC, Mieslerová B (2001) Host-parasite specificity and defense variability in the
Lactuca spp.-Bremia lactucae pathosystem. J Plant Pathol 83 (2, Special issue): 25-35.

Lebeda A, Ryder EJ, Grube R, Doležalová I, Křístková E (2007) Lettuce (Asteraceae; Lactuca spp.),
Chapter 9. In: Singh RJ (ed.) Genetic Resources, Chromosome Engineering, and Crop Improvement;
Volume 3 -Vegetable Crops. CRC Press, Francis & Taylor, Boca Raton, Florida, USA, pp: 377-472.

Lebeda A, Syrovátko P (1988) Specificity of Bremia lactucae isolates from Lactuca sativa and some
Asteraceae plants. Acta Phytopath Entom Hung 23: 39-48.

Lebeda A, Zinkernagel V (1999) Durability of race-specific resistance in lettuce against lettuce downy
mildew (Bremia lactucae). In: Lebeda A, Křístková E (eds.) Eucarpia Leafy Vegetables ’99. Proc.
Eucarpia Meeting on Leafy Vegetables “Genetics and Breeding”. Palacký University, Olomouc,
Czech Republic, pp: 183-189.

Lebeda A, Zinkernagel V (2003a) Characterization of new highly virulent German isolates of Bremia
lactucae and efficiency of resistance in wild Lactuca spp. germplasm. J Phytopathol 151: 274-282.

Lebeda A, Zinkernagel V (2003b) Evolution and distribution of virulence in the German population of
Bremia lactucae. Plant Pathol 52: 41-51.

Maisonneuve B, Bellec Y, Souche S, Lot H (1999) New resistance against downy mildew and lettuce
mosaic potyvirus in wild Lactuca spp. In: Lebeda A, Křístková E (eds.) Eucarpia Leafy Vegetables
’99. Proc. Eucarpia Meeting on Leafy Vegetables “Genetics and Breeding”. Palacký University,
Olomouc, Czech Republic, pp: 191-197.

Marlatt RB (1974) Biology, morphology, taxonomy and disease relations of the fungus Bremia.
Florida Agricultural Experiment Station Technical Bulletin 764: 1-25.

Michelmore RW, Ochoa OE, Truco MJ, Ryder EJ (2002) Lettuce Breeding. Annual Report (April
2001-March 2002). Salinas, CA, USA: California Lettuce Research Board, pp: 52-66.

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Norwood JM, Crute IR, Lebeda A (1981) The location and characteristics of novel sources of
resistance to Bremia lactucae Regel (Downy mildew) in wild Lactuca L. species. Euphytica 30: 659-
668.

Petrželová I, Lebeda A (2004) Occurrence of Bremia lactucae in natural populations of Lactuca

serriola. J Phytopathol 152: 391-398.

Reinink K (1999) Lettuce resistance breeding. In: Lebeda A, Křístková E (eds.) Eucarpia Leafy
Vegetables ’99. Proc. Eucarpia Meeting on Leafy Vegetables “Genetics and Breeding”. Palacký
University, Olomouc, Czech Republic, pp: 139-147.

Sicard D, Woo SS, Arroyo-Garcia R, Ochoa O, Nguyen D, Korol A, Nevo E, Michelmore RW (1999)
Molecular diversity at the major clusters of disease resistance genes in cultivated lettuce and wild
Lactuca spp. Theor Appl Genet 99: 405-418.

Towsend GR, Heuberger W (1943) Methods for estimating losses caused by diseases in fungicide
experiments. Plant Dis Rep 27: 340-343.

Van Ettekoven K, Van der Arend AJM (1999) Identification and denomination of “new” races of
Bremia lactucae. In: Lebeda A, Křístková E (eds.) Eucarpia Leafy Vegetables ’99. Palacký University,
Olomouc, Czech Republic, pp: 171-175.

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Table 15.1. Basic differential set of Lactuca spp. used for testing B. lactucae isolates*

Sextet number Differential genotype Dm gene/R-factor

(see Table 15.2.) (see Table 15.2.)
Cobham Green Dm 0/R?
1 Lednický Dm1
2 UCDM2 Dm2
3 Dandie Dm3
4 R4T57D Dm4
5 Valmaine Dm5/8
6 Sabine Dm6
7 L. serriola (LSE/57/15) Dm7+R?
8 UCDM10 Dm10
9 Capitan Dm11
9 Hilde × L. serriola (H×B) Dm11
10 British Hilde R12
11 Pennlake Dm13
12 UCDM14 Dm14
13 L. serriola (PIVT 1309) Dm15
14 L. serriola (LSE/18) Dm16
15 L. serriola (LS-102) R17
16 Colorado R18
17 Ninja R36
18 Discovery R37
19 Argeles R38

R?: Resistance factor (R-factor) not known or not well characterised.

*Based on Van Ettekoven and Van der Arend, (1999).

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 Table 15.2. Virulence characteristics of most important Bremia lactucae races and their sextet codes

254
Dm gene/R-factor 0 1 2 3 4 5/8 6 7 10 11 12 13 14 15 16 17 18 36 37 38
Sextet number 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19
Sextet value 1 2 4 8 16 32 1 2 4 8 16 32 1 2 4 8 16 32 1

B. lactucae race Reaction1 Sextet code2

Bl 1 + + + - + - - - + - + + + - (-) - - - - (-) 11/58/00/00
Bl 2 + + + + + + + - + (-) + + + - - - (-) - - + 63/58/00/01
Bl 4 + + + - + + m + + - + + + - (-) - (-) - - (-) 27/59/00/00
Bl 5 + + - + - - - + + - + + - + (-) - - - m - 05/27/01/00
Bl 6 + + + - + + m - + + + + + - (-) - - - - - 27/62/00/00
Bl 7 + + + + + - (+) + + (-) + + + (-) - - - - - - 47/59/00/00
Bl 10 + + + + + + + + + (-) + + + m - - - - - (-) 63/59/00/00
Bl 12 + + - - + + + + + + + + + + + - - - - - 57/63/03/00
Bl 13 + + - + - + m + + + + + + - (-) - - - - - 21/63/00/00
Bl 14 + + + + + + + - + + + + + - - - - - - - 21/63/00/00
Bl 15 + + + + + + - + + + + + (-) - (-) - - - - (-) 31/31/00/00
Bl 16 + + + + + + + + + + + + - - + (-) - - - (-) 63/31/02/00
Bl 17 - - + + - + - + + - + + + + - - + (-) (+) - 22/59/41/00
Bl 18 + + + - + + + + + + + + - - + - + - - - 59/31/10/00
Bl 20 + + + + + + + + + + + + - - + - + - - - 63/31/10/00
Bl 21 + + + + + + + + + + + + - + + - - + + - 63/31/51/00
Bl 22 + + + - + + + + + + + + + + (-) - + - - - 59/63/09/00
Bl 23 + + + + + + + + + + + + (-) (-) + (-) - (-) - + 63/31/02/01
Bl 24 + + + - + + + + + + + + - - + - + - - + 59/31/10/01
Bl 25 + + + - + + + + + + + + - - + - + - + - 59/31/42/00

1
Reaction rating: - = resistant; (-) = incomplete resistance; + = susceptible; (+) = almost susceptible; m = moderate necrotic, leaking.
2
Denomination for each race. Modified from Van Ettekoven and Van der Arend (1999), and IBEB (2005).
 A

Figure 15.1. (A) Microphotograph of Bremia lactucae conidiophore and conidia. (B)
Abaxial leaf surface of Lactuca sativa with sporulating Bremia lactucae.

A B

Figure 15.2. Boxes with inoculated seedlings (A) and leaf disks (B) of Lactuca spp.

255
 A

Figure 15.3. Detail of seedlings with different degrees of infection (DI) 12 days after
inoculation by Bremia lactucae. (A) DI = 0; (B) DI = 1; (C) DI = 2; and (D) DI = 3.

A B
Figure 15.4. Comparison of resistant and susceptible responses in seedlings (A) and
leaf disks (B) of Lactuca sativa 14 days after inoculation with Bremia lactucae.

256
 Chapter 16

Screening for resistance to tomato powdery mildew (Oidium neolycopersici)

A. Lebeda* and B. Mieslerová

Palacký University in Olomouc,

Faculty of Science, Department of Botany,
Czech Republic.

*Correspondence: [email protected]

Abstract

This paper provides basic knowledge about a devastating disease of greenhouse tomato plants, namely
tomato powdery mildew (Oidium neolycopersici), including information about its host range, sources
of resistance to this pathogen within wild Lycopersicon spp., its pathogenic variability (a preliminary
differential set is presented), the genetic background of resistance and mechanisms of resistance. The
basic methods of pathogen isolation, cultivation and maintenance, and methods of preparation of
experimental plants are briefly described. Two methods of resistance screening are described, leaf disk
method (as modified from Mieslerová et al., 2000) and the whole plant method (as modified from Bai
et al., 2003), including a description of the scales used for assessment of the degree of infection (DI).

INTRODUCTION

Tomato powdery mildew, caused by Oidium neolycopersici (Kiss et al., 2001), is a relatively new
disease, which occurs predominantly in greenhouse raised tomato crops. Since the mid 1980s, when
epidemics first started in Western Europe, the pathogen has rapidly spread throughout Europe and also
to the New World (Mieslerová and Lebeda, 1999). Only the anamorph of this fungus has been found
until now (Figures 16.1. and 16.2).

The symptoms of tomato powdery mildew infection are white circular pustules, which appear
primarily on the upper sides of leaves, and can also affect stems and petioles. The colonies of O.
neolycopersici are initially small, 3-10 mm in diameter, then enlarge and can cover the whole leaf
surface. In heavily infected plants, powdery mildew also develops on the lower side of leaves, but
never on the fruit. O. neolycopersici can be morphologically easily distinguished from Leveillula
taurica (Lév.) Arnaud/Oidiopsis sicula Scalia, syn. O. taurica (Lév.) Salmon, which causes heavy
infections of tomatoes and red pepper in tropical and semi-arid areas (Palti, 1988).

Until now, experimental studies have been concentrated mainly on the morphological and molecular
characterisation of this pathogen (Whipps et al., 1998; Lebeda and Mieslerová, 1999; Jones et al.,
2000; Kiss et al., 2001; Mieslerová et al., 2002), its host range (Whipps et al., 1998; Lebeda and
Mieslerová, 1999; Lemaire et al., 1999; Huang et al., 2000a) and on searching for resistance sources
within the genus Lycopersicon (Lindhout et al., 1994a; Kumar et al., 1995; Ignatova et al., 1997;
Milotay and Dormanns-Simon, 1997; Ciccarese et al., 1998; Mieslerová et al., 2000).

Based on a comprehensive study including light microscopy, scanning electron microscopy (SEM)
analysis and internal transcribed spacer (ITS) sequence analysis, Jones et al. (2000) placed the fungus
to Erysiphe sect. Erysiphe very close (nearly identical) to E. aquilegiae var. ranunculi and clearly
distinct from E. orontii and E. cichoracearum. Kiss et al. (2001), in their detailed studies, identified
and described the powdery mildew on tomatoes from Australia (O. lycopersicum) as a species distinct
from the tomato powdery mildew (O. neolycopersici) widespread in Europe, Africa, North and South
America, and Asia.

1
257
A detailed study of the host range of O. neolycopersici revealed that this fungus mainly infects some
species of the families Solanaceae and Cucurbitaceae although the experiments gave different results,
mainly concerning representatives of the Cucurbitaceae family (Fletcher et al., 1988; Lebeda and
Mieslerová, 1999; Lemaire et al., 1999; Huang et al., 2000a). No susceptible taxa have been found in
such families as Brassicaceae, Compositae, Leguminosae and Poaceae, while some susceptible species
have been reported from the families Apocynaceae, Campanulaceae, Crassulaceae, Cistaceae,
Linaceae, Malvaceae, Papaveraceae, Pedaliaceae, Scrophulariaceae, Valerianaceae and Violaceae
(Whipps et al., 1998).

The most valuable donors of resistance were detected in accessions of Lycopersicon hirsutum, L.
parviflorum, L. peruvianum and L. pennellii (Lindhout et al., 1994a; Ignatova et al., 1997; Milotay and
Dormanns-Simon, 1997; Ciccarese et al., 1998; Mieslerová et al., 2000), while only a few resistant
genotypes were found in L. esculentum and L. pimpinellifolium (Kumar et al., 1995; Ciccarese et al.,
1998; Mieslerová et al., 2000), which are considered to be the closest relatives of tomato.
Macroscopically, resistance to O. neolycopersici in wild tomato species is characterised by strongly
restricted mycelial growth and lack of sporulation (Lindhout et al., 1994a). Microscopically, the
hypersensitive reaction (HR) is the major mechanism of resistance to O. neolycopersici in
Lycopersicon species (Huang et al., 1998; Mieslerová et al., 2004). A biochemical study of the
generation of reactive oxygen species and peroxidase activity during O. neolycoersici infection of
Lycopersicon species has been performed (Mlíčková et al., 2004; Tománková et al., 2006).

However, limited information is available on the pathogenic variability of O. neolycopersici. Host

range experiments have revealed considerable differences, mainly in the ability of different O.
neolycopersici isolates to infect representatives of the Solanaceae family, which suggests the existence
of different pathotypes (formae speciales) (Huang et al., 1998; Mieslerová and Lebeda, 1999).
Because nearly all recent commercial tomato cultivars are considered to be highly susceptible (Kozik,
1993; Lindhout et al., 1994a; Mieslerová et al., 2000), these are not useful for the differentiation of O.
neolycopersici isolates. Based on pathogenicity studies of isolates of O. neolycopersici originating
from different European countries, Lebeda and Mieslerová (2002) proposed a preliminary differential
set of genotypes (Table 16.1).

Intensive study of the genetic basis of resistance, mainly in wild Lycopersicon species (L. hirsutum, L.
peruvianum), has revealed the existence of six genes responsible for resistance to O. neolycopersici
(Ol-1-6) (Lindhout et al., 1994b; Ciccarese et al., 1998; Huang et al., 2000b; Bai et al., 2004, 2005).
According to these results, the near-isogenic lines were produced from crosses which contained the
different dominant Ol genes in an L. esculentum genetic background. The disease tests with local O.
neolycopersici isolates demonstrated that the resistance conferred by different Ol genes was isolate-
dependent and may be race-specific (Table 16.2).

MATERIAL AND METHODS

Pathogen isolation, cultivation and maintenance

Leaves of tomato highly infected by O. neolycopersici are used as the source of inoculum. An
important feature for the successful isolation of the pathogen is the quality of the inoculum. The leaves
with pathogen should not be too old (i.e., they should not exhibit seriously necrotised leaf tissue), and
should not be collected in wet weather, because this causes the conidia not to be released from the
conidiophores. The isolates of O. neolycopersici are maintained on plants of highly susceptible
cultivars of tomato (e.g., cv. ‘Amateur’) that are about two months old. The upper side of two or three
leaves of a susceptible tomato plant are inoculated by surface contact (dusting/tapping) using infected
plants (Lebeda and Mieslerová, 1999).

These plants are kept under plastic covers in a growth chamber at a temperature of 20-25°C with a
12 hour photoperiod. In two- or three-week intervals, new tomato plants are re-inoculated with the
isolate.

258 2
Preparation of plant material for screening

Seeds of tested plants are treated with 6% chloramine for 20 minutes and then sown into plastic boxes,
8 × 11 × 2.5 cm, containing perlite. Seedlings with fully developed cotyledons are transferred to 7 cm
plastic pots containing garden soil/peat (2:1 [v/v]). Plants are grown in a growth chamber at 20°C with
a 12 hour photoperiod, and are used for experiments at the three to five true leaf stage (about eight
weeks old) (Lebeda and Mieslerová, 1999).

Inoculum preparation, inoculation, incubation and disease assessment

Leaf disk method (modified from Mieslerová et al., 2000)

The leaf disks (diameter: 14 mm) are cut using a cork borer from true leaves and placed with the
adaxial side up on water agar (15 g/l, Agar ST Imuna, Imuna Pharm Ltd., Šarišské Michalany,
Slovakia) in Petri dishes. The upper side of each leaf disk is inoculated by surface contact
(dusting/tapping) using leaves of tomato cv. ‘Amateur’ that have 80-100% of the surface covered with
fresh sporulating mycelium of O. neolycopersici. The average number of powdery mildew conidia
delivered to leaf disks is 65 ± 15/mm2. After inoculation, the Petri dishes are placed in a growth
chamber and incubated at 18/20°C (night/day) with a 12 hour photoperiod.

The method of spraying leaf disks with a suspension of powdery mildew conidia, which is frequently
used in experiments with downy mildews (of the order Peronosporales), was also tested. However,
poor results were achieved. Although the very high humidity inside the Petri dishes usually supports
the germination of conidia, the water drops on the leaf surface inhibited the development of tomato
powdery mildew mycelium, resulting in weak sporulation even in the positive control accession.

The degree of susceptibility of the tested accessions to the O. neolycopersici isolates was evaluated
macroscopically 5-14 days after inoculation at two-day intervals. For assessing the DI, a 0-4 scale was
used (Figure 16.3): 0 = without symptoms of pathogen development; 1 = mild development of
mycelium without sporulation; 2 = well-developed mycelium with mild sporulation; 3 = intensive
sporulation and well-developed mycelium covering <50% of the leaf disk area; 4 = intensive
sporulation and well-developed mycelium covering 50-100% of the leaf disk area. For each accession,
the mean and standard deviation (SD) of the P (percentage of the total degree of infection) was
calculated according to the formula of Towsend and Heuberger (1943) as follows:

P = Σ(n × v) × 100/x × N

Where: P = the total degree of infection (DI); n = number of plants in each assessed category
(infection degree); v = infection degree (0-4); x = scale range (in this case = 4) and N = total number
of assessed plants.

Data recorded on the 14th days after inoculation were used to calculate the DI. Plants were considered
to be resistant when the % max DI was between 0 and 30, as susceptible when the % max DI was
between 60 and 100, and as incompletely resistant when the % max DI was between 30 and 60. The
presence or absence of an HR was macroscopically assessed.

Whole plants method (modified according to Bai et al., 2003)

The 6-8 week old plants are sprayed with a suspension of 2 × 104 conidia/ml. The inoculum is
prepared by washing conidial spores from freshly sporulating leaves of heavily infected tomato plants
in tap water and is used immediately. Inoculated plants are placed under plastic covers at 18/20°C
(night/day) with a 12 hour photoperiod. At least five plants per accession are used.

The inoculated plants are evaluated according to the following DI scale (Figure 16.4): 0 = no visible
sporulation; 1 = very few fungal spots (weak sporulation); 2 = a moderate number of fungal spots

3
259
(heavy sporulation); 3 = a very high number of fungal spots (heavy sporulation). The plants are first
evaluated five days after inoculation and for the last time on the 18th day following inoculation. The
last evaluation (if plants are not totally destroyed) is used for determining the susceptibility/resistance
of the tested accession. Plants are considered to be resistant when the DI ≤ 1, as susceptible when the
DI > 2 and as incompletely resistant when 1 < DI ≤ 2. In addition, the presence of necrotisation in
infected leaf tissue is macroscopically assessed.

CONCLUSIONS

Tomato powdery mildew (O. neolycopersici) has started to cause serious infections in tomato crops
since the late 1980s. Vast amounts of information have been obtained about this pathogen, including
its interaction with the host Lycopersicon spp. Studies on the host range and sources of resistance have
produced a considerable amount of data that can be applied to the breeding of resistant tomato
cultivars and disease management. However, there is still only limited data available about the
pathogenic variability of this fungus. It is assumed that the interactions of Lycopersicon spp. and O.
neolycopersici are based on race-specificity. However, only detailed research on these interactions will
elucidate the exact mechanisms of resistance and their genetic background. The methods described in
this paper have a broad potential for application both in screening Lycopersicon spp. germplasm and in
subsequent tomato breeding programmes. These methods can also be applied to the research of
pathogenic variability of O. neolycopersici. The methodology described is useful for basic research of
host-pathogen interactions, pre-breeding and selection mass screening, as well as for characterisation
of resistance of commercial tomato cultivars.

Acknowledgements

This study was supported by the Ministry of Education, Youth and Sports of the Czech Republic,
project MSM 6198959215.

REFERENCES TO CHAPTER 16

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262 6
Table 16.1. List of Lycopersicon spp. accessions recommended as a basis for a preliminary
differential set of genotypes and differentiation of pathogen races

Oidium neolycopersici race/response

Lycopersicon spp. Accession OL 1 OL 2 OL 3
L. esculentum cv. ‘Amateur’ S1 S S
L. hirsutum LA 94 R S M
L. hirsutum LA 1738 R R S
L. hirsutum LA 1731 R R M
L. hirsutum f. sp. glabratum LA 2128 R R R
1
Reaction (DI = degree of infection): R = resistant (DI = 0-30%); M = moderately susceptible (DI =
30-60%); S = susceptible (DI = 60-100%). Source: Lebeda and Mieslerová (2002).

Table 16.2. List of Lycopersicon spp. accessions and their reactions with local isolates of Oidium
neolycopersici

Oidium neolycopersici isolate/response

Lycopersicon spp. Accession On-Ne On-Hu On-Cz On-It On-Fr On-Fl
L. esculentum ‘Moneymaker’ S1 S S S S S
Crosses based on L. NIL Ol-1 R MR R R R R
esculentum NIL F3-Ol-2 R R R R (MR) R R
NIL Ol-3 R R R R R R
NIL Ol-4 R R S R R R
NIL Ol-5 R MR R R R R
NIL Ol-6 R R S R R R

1
Reaction: R = resistant (DI ≤ 1); MR = moderately resistant (1 < DI ≤ 2); S = susceptible (DI > 2)
(see Figure 16.4). Source: Bai et al. (2005).

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 Figure 16.2. Electron micrograph showing a
germinating conidium and appressorium formation
of Oidium neolycopersici.

Figure 16.1. Electron micrograph

showing conidiophores of Oidium
neolycopersici.

A B C

D E F

Figure 16.3. Assessment of the degree of infection (DI) of tomato powdery mildew on
leaf disks. (A) DI = 0: without symptoms of pathogen development; (B) DI = 1: mild
development of mycelium without sporulation; (C) DI = 2: well-developed mycelium
with mild sporulation; (D) DI = 3: intensive sporulation and well-developed mycelium
covering <50% of the leaf disk area; (E) DI = 4: intensive sporulation and well-
developed mycelium covering 50-100% of the leaf disk area; (F) HR: Hypersensitive
response (dark spots at the site of fungal penetration).

264
 A B

C D E

Figure 16.4. Assessment of the degree of infection (DI) on leaves of the whole plants. (A)
DI = 0: no visible infection; (B) DI = 1: very few fungal spots (weak sporulation); (C) DI
= 2: a moderate number of fungal spots (sporulation); (D) DI = 3: a very high number of
fungal spots (heavy sporulation). (E) Pustules of sporulating Oidium neolycopersici
accompanied by necrotic surrounding cells.

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 Chapter 17

In vitro and in vivo selection of black pepper (Piper nigrum) mutants

tolerant to Fusariosis

O. Filgueira de Lemos1, A. Tulmann Neto2*, J. de Cássia Albino2,

M. Costa Poltronieri1 and A. Ando2

1
Empresa Brasileira de Pesquisa Agropecuária (EMBRAPA),
Amazônia Oriental,
Pará
2
Centro de Energia Nuclear na Agricultura (CENA), Universidade de São Paulo,
São Paulo

Brazil.

*Correspondence: [email protected]

Abstract
The cultivation of black pepper plants in Brazil is severely limited by Fusariosis, a disease caused by
Fusarium solani f. sp. piperis that leads to plant death. Considering the fact that the black pepper
plants in Brazil are derived from a few plants introduced from Singapore, and that introduction of
other germplasm has been extremely difficult, the genetic variability found within the populations is
very low. Conventional breeding methods have, therefore, been highly ineffective for obtaining
resistance or tolerance to Fusariosis disease. In order to generate genetic variability and selection of
plants resistant to Fusariosis, the objective of the present study was to establish useful methodology
using gamma irradiation in association with in vitro and/or in vivo techniques. For this purpose, it is
suggested that: (1) a dose of 20 Gy of gamma-rays be applied for in vitro mutation induction of buds
from young plants; (2) generation advancement be done at least three times by subculturing
(micropropagation) in order to eliminate chimerism; (3) in vitro selection be carried out with a filtrate
of F. solani f. sp. piperis; (4) in vivo selection be perfomed through the application of a suspension of
fungal spores onto the plants; and (5) agronomic evaluation of selected plants be carried out in an area
with a high incidence of F. solani f. sp. piperis.

INTRODUCTION

Black pepper (Piper nigrum) originates from India and belongs to the Piperaceae family, whose
members have different numbers of chromosomes, 36, 48, 52, 60, 104 and 128 (Sharma and
Bhattacharyya, 1959; Dasgupta and Datta, 1976). The plant is considered autogamic, the inflorescence
is a pendulous spike 5-20 cm long, and there are female, male and hermaphrodite plants. Natural
pollination is performed by geitonogamy through the dispersion of pollen by water, dew or rain
droplets; fruit formation occurs six months after pollination (Nambiar et al., 1978; Poltronieri et al.,
1999).

The crop thrives in hot and moist weather, requiring an average rainfall of > 2500 mm/year, moisture
of > 80%, average temperatures of ~ 23-28°C, and soils of good drainage (Albuquerque et al., 1989).
The main producing countries are India, Indonesia, Brazil and Malaysia. Seeds as well as vegetative
cuttings are used for propagation. Propagation with seeds is used mostly in breeding programs, while
propagation with cuttings is more commonly used for commercial cultivations (Nambiar et al., 1978;
Albuquerque et al., 1989).

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Given the narrow genetic base of the Brazilian cultivars, black pepper cultivation has been severely
attacked and limited by a disease known as Fusariosis, which is caused by the fungus Nectria
haematococca f. sp. piperis (F. solani f. sp. piperis). The symptoms of Fusariosis include root decay
and drying of the branches. The disease spreads quickly all over the planting regions, destroying large
areas of cultivation in a short period of time (Albuquerque and Duarte, 1977; Duarte and Albuquerque,
1999).

Tissue culture techniques can be used to create genetic variation through a process called somaclonal
variation, which causes changes similar to those induced by physical and chemical mutagenic agents,
and which can be incorporated into genetic breeding programmes. In vitro selection allows selection
of mutants tolerant to abiotic factors and resistant to diseases in a short period of time, and can be used
as an auxiliary to field selection. The use of molecular markers is ideal for distinguishing genetic
somaclonal from epigenetic changes (Jain, 2001).

In vitro culture techniques were first applied for the formation of calluses and differentiation of
multiple sprouts and roots of black pepper plantlets. More recently, regeneration of black pepper
plants via somatic embryogenesis has been achieved (Mathews and Rao, 1984; Khoon and Talib,
1985, Philip et al., 1992; Joseph et al., 1996).

Conventional and non-conventional breeding programmes produce genetic variability in the

population followed by selection of the desired genotypes (Wenzel, 1985). The use of mutation
induction techniques is an alternative because it allows the generation of genetic variability and has
sped up success in breeding cultivated species (Maluszynski et al., 1995; Donini and Sonnino, 1998).
Regarding black pepper cultivation in Brazil, conventional methods for obtaining genotypes with
resistance or tolerance to the Fusariosis disease have brought little progress, for no tolerance to the
disease has been observed within the species (Poltronieri et al., 1999). Elsewhere, the best results
through conventional breeding were obtained with the hybrid Panniyur-I (Nambiar et al., 1978).

For plants with an unknown genetic source of a desirable feature or even for apomictic or sterile plants
in which species hybridisation is not an option, the induction of mutations is an alternative method of
plant breeding (Tulmann Neto et al., 1998). The selection of variants is an important step in plant
breeding by mutation and, for the most part, is harder than the induction itself (IAEA Technical
Reports, 1977). According to Ahloowalia and Maluszynski (2001), more than 1800 cultivars were
obtained directly or indirectly through mutation induction in plants with seed propagation, such as
wheat, rice, barley, peanuts and beans. For vegetatively propagated plants, 465 mutants were obtained,
most of them for ornamental plants (chrysanthemum, rose, begonia, azalea, dahlia, etc.) and a few for
fruit-bearing plants (apple, Japanese pear, grapefruit, banana, pineapple, date).

In contrast to conventional in vivo cultivation, in vitro cultivation results in a quick dissolution of

chimeras and the recovery of genetically stable mutants (Tulmann Neto et al., 1998). For in vitro
selection, phytotoxic effects of fungal filtrate have been proven in the selection process for resistance
to the disease caused by Fusarium in pineapple (Hidalgo et al., 1998, 1999; Borrás et al., 2001),
soybeans (Jin et al., 1996) and banana plants (Matsumoto et al., 1999a,b). Variations in the level of
tolerance to the toxins produced by Fusarium in vitro are mostly due to the plant variety or genotype,
which is similar to the responses observed following pathogen attacks under field conditions. Such
variations reflect the different resistance mechanisms of the host that operate at various levels, from
the prevention of fungal invasion and expansion to tolerance or degradation of the fungal toxin
(McLean, 1996).

MATERIAL AND METHODS

In the present study, we conducted: (1) tests for radiosensitivity of in vitro buds to gamma radiation;
(2) cultivation of F. solani f. sp. piperis fungus; (3) tests using the culture filtrate as a selective agent;
(4) in vitro and in vivo selection of variants; and (5) agronomic evaluation of V5 plants in areas of
Fusariosis occurrence.

268 11
Radiosensitivity of in vitro buds to gamma radiation

Doses ranging from 0 to 80 Gy were tested, and an entirely randomised experimental design was used
with treatment doses of 0, 20, 30, 40 and 50 Gy, with seven replications, each of which consisted of 20
buds per Petri plastic sterile plate (diameter: 8.5 cm), totalling 700 buds. Each plate contained agar
water (0.7% w/v) and was individually irradiated at a dose rate of 1297 kGy/hour with a gamma-cell at
CENA/USP, Piracicaba, São Paulo. After irradiation, buds were cultivated in Murashige and Skoog
(MS) semi-solid basal medium (Murashige and Skoog, 1962) supplemented with 0.5 mg/l 6-
benzylaminopurine (BAP) and 0.2 mg/l indole-3-acetic acid (IAA). After 45 days of cultivation at 25
± 3ºC with 16 hours of daylight, with a light intensity of ~3000 lux, the survival rate, number of buds
per explant and explant weight (g) were evaluated. The mean values were subjected to linear
regression analysis using the software Microsoft Excel to determine the dose of gamma radiation
necessary to reduce the survival rate, number of buds and explant weight to 30-50% (Figure 17.1).

In vitro phytotoxicity of the culture filtrate of F. solani f. sp. piperis

The influence of the Czapek-Dox medium components in the multiplication culture – basic MS culture
medium supplemented with 0.5 mg/l BAP and 0.2 mg/l IAA – was evaluated by examining the
performance of in vitro buds in a range of Czapek-Dox concentrations (0, 20, 30, 40 and 50% [v/v]) in
the bud multiplication medium. The test was performed in an entirely randomised experimental design
with three replications, each replication represented by a flask with five buds. The data on explant
survival and buds per explant after 45 days of cultivation were subjected to variance analysis and
Tukey tests for a comparison of the averages.

On the other hand, isolates of F. solani f. sp. piperis of infected plants were cultivated on potato
sucrose agar (PSA) culture medium. Two weeks later, 1 cm disks containing fungal mycelium were
cultivated for 35 days on Czapek-Dox medium, and an evaluation of the number of micro- and
macroconidia per ml was carried out on the 8th, 11th, 14th, 17th, 23rd, 29th and 35th days after
inoculation. At the end of the period, hyphae were dried in the greenhouse at 40°C for 48 hours and
weighed. The data was used to generate a growth curve using the software Microsoft Excel.

In order to test the filtrate phytotoxicity, an experiment was conducted with a filtrate from a 28 day old
fungal culture at different concentrations (0, 20, 30, 40 and 50% [v/v]) in basal MS medium
supplemented with 0.5 mg/l BAP and 0.2 mg/l IAA – bud multiplication medium – and two forms of
filtrate sterilisation. The first form of sterilisation was filter sterilisation (FS) using a sterile filter with
a pore size of 0.22 μm coupled to a vacuum pump, and adding the filtrate to the autoclaved bud
multiplication medium in an aseptic flux chamber. In the second sterilisation method, the fungal
culture filtrate was autoclaved for 20 minutes, and added to the bud multiplication medium at the
desired concentration, and then autoclaved for a second time (AII). The evaluation was done by
calculating the percentage of bud lethality through variance analysis and a Tukey test for comparison
of the averages, and linear regression analysis.

In vitro selection of variants that survived the treatment with fungal culture filtrate

The buds that were subjected to gamma irradiation and surviving buds underwent several
multiplication cycles in order to eliminate chimerism and to obtain homogeneous mutants. They were
subjected to in vitro selection on a culture medium supplemented with a selection agent: fungal culture
filtrate at concentrations of 50 and 55% (v/v) of the total filtrate obtained after 28 days of fungal
cultivation in Czapek-Dox medium. A total of 4117 buds were subjected to in vitro selection, of which
2332, 532, 160 and 1093 buds originated from doses of 10, 20, 30 and 40 Gy, respectively. Survival
was evaluated and converted into percentages of selection, and the selection index for each of the
selection agent conditions applied was determined using chi-square tests.

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Preparation of spores and plant inoculation

The fungus F. solani f. sp. piperis was cultivated on PSA medium for two weeks, and spores were
collected by adding sterile water and filtering through four layers of gauze. The spores where then
counted in a Neubauer chamber, and adjusted to the desired concentration with sterile water.
Susceptible black pepper plants cultivated in black polyethylene bags in the greenhouse were
inoculated with different fungal concentrations (0, 2 × 102, 2 × 104 and 2 × 106 spores/ml) either
applied to the soil or to aerial parts of the plant. 5 ml of the inoculum for each concentration, and five
plants per treatment were used.

Seedlings regenerated from the buds selected in vitro were planted in soil, acclimatised for six months,
and used for inoculations with the fungal spores. The inoculation was performed by spraying the aerial
plant parts and watering the soil. Evaluations of seedling mortality were performed visually starting
two months after inoculation. Re-isolation of the pathogen was done for confirmation based on Koch’s
postulate that “the microorganism must be re-isolated from the diseased organism and correspond to
the original microorganism in pure culture”.

Field selection and evaluation of plants in areas of Fusariosis incidence

Evaluated and selected black pepper lineages were obtained from an experiment started in 1978 (Ando
et al., 1984). 428 cuttings, originating from the cultivar ‘Singapore’, were subjected to gamma
irradiation with doses of 20 and 25 Gy, and planted in a recently deforested area in the municipality of
Tomé-Açu, State of Pará, Amazonia, Brazil, where irradiated and surviving plants were grown to
produce young plants V1. V1 plants were pruned and a total of 500 cuttings with 2-3 internodes were
prepared from which V2 plants were grown individually in 5 l plastic buckets containing a soil
substrate compound. In the third month after planting, the soil of the V2 plants was inoculated with a
spore suspension (5 × 104 spores per ml; 10 ml per 5 l bucket) of F. solani f. sp. piperis grown in PSA
for 15-20 days. Surviving V2 plants (43 individuals) were transplanted to an area of high levels of
Fusariosis occurrence in order to undergo natural selection. After three years of cultivation, three
plants had survived from which V3 plants were produced and preliminary characterisation of the
materials was undertaken. V4 plants were produced from the V3 plants with the best performances
regarding the occurrence of the disease, growth vigour, shape and productivity of fruits in the fourth
year of cultivation. The V4 plants with the best field performances based on evaluations by technicians
after five years of cultivation were used to produce lineages of V5 plants.

Agronomic evaluation of the V5 plants

The experiment was started in March 1994 in an area of natural occurrence of Fusariosis. Lineages
originating from both 16 V5 cuttings and one non-irradiated cutting (cultivar ‘Singapore’) were used.
The evaluation was conducted in the field, each consisting of a repetition of eight plants, varying from
one to seven repetitions per lineage.

All V5 lineages were evaluated for the occurrence of Fusariosis and survival, length and weight of the
spike, number of seeds per spike, wet and dry weight of 100 fruits, and individual plant production. In
addition, the blooming period and the number of orthotropic and plagiotropic branches were also
evaluated.

Averages and variation of the variables were obtained. A comparison made using the Tukey test was
conducted based on three years of data collected in the field, as well as an analysis of genetic
divergence through main components and canonical variables.

270 13
RESULTS AND DISCUSSION

Radiosensitivity of in vitro buds to gamma radiation

Buds subjected to different doses of gamma radiation (0 to 80 Gy) showed different responses. It was
observed that the higher the dose, the greater the physiological effects, both in terms of survival rates
of explants and in terms of differentiation of buds and explant weights. For doses > 40 Gy (60 and 80
Gy) the survival rates were 0%; and for the 10 Gy dose, the irradiation effects were not visible as the
in vitro responses were similar to those of non-irradiated buds.

Determination coefficients (R2) were > 0.91, representing a > 95% correlation, and the resulting
equations allowed us to calculate the gamma radiation doses necessary to achieve a 30-50% reduction
in the parameters evaluated (survival rate, number of buds and explant weight) in comparison to non-
irradiated material. The optimal doses varied from 14.17 to 18.47 Gy for a 30% reduction and 23.62 to
30.78 Gy for a 50% reduction (Table 17.1). The data revealed a direct relationship and functional
response between the applied dose and the results obtained, which are shown in Figures 17.2. It should
be noted that the most accurate correlation was observed for explant weight, followed by survival rate
and number of different buds per explant.

Based on the physiological effects of gamma irradiation (Figure 17.3) and on suggestion by Predieri
(2001), a dose of 20 Gy was chosen, as this dose would cause a determined physiological effect of >
30% and < 50% reduction of the evaluated parameters. The same dose was established by Ando et al.
(1984), from which three surviving plants were selected after artificial and natural selection against
Fusariosis. Likewise, higher doses caused more DNA alterations, which are mostly prejudicial and
may compromise other desirable features of the original cultivar (Przybyla, 1994).

After selecting the dose, 1204 buds were subjected to 20 Gy of gamma radiation, and 741 buds
survived, which represents 72.36% of the total number irradiated, close to the 72.29% determined
before by the equation. As to the number of differentiated buds per explant, an average of 1.64 against
the expected mean of 1.56 was obtained. Therefore, it can be said that the results obtained with a dose
of 20 Gy were very similar to those expected (Ahloowalia and Maluszynski, 2001).

In vitro toxicity of the culture filtrate of F. solani f. sp. piperis

Buds cultivated in a multiplication culture medium at different Czapek-Dox concentrations (0 to 50%)

presented a similar response, for both bud proliferation and survival, after 45 days of cultivation. The
average number of buds per explant varied from 2.67 to 3.20 and the survival rate from 69.79 to
100%, respectively at concentrations of 50% and 0% Czapek-Dox medium (Table 17.2). It should be
emphasised that buds considered as non-survivors presented green tissue even though there was no
differentiation of new buds. It is believed that the constituent compounds in the Czapek-Dox medium
had no noticeable negative effect on the in vitro responses of cultures. This was a key condition for the
use of tests with the fungal culture filtrate in the multiplication medium of buds to determine its
concentration as a selection agent, in order to avoid the selection of other culture medium constituents
different from the fungal culture filtrate, as suggested by Daub (1986).

In fungal cultures grown to produce the filtrate to be used as a selection agent, the average conidium
production from three flasks followed the fungus growth curve equivalent to the estimated number of
conidia per ml. On the 8th day of cultivation, conidium production was about 8 × 104, and 3 × 105 on
the 29th day, and decreased thereafter. Likewise, the production of hyphae varied from 1070 to 1153
mg dry weight after 35 days of cultivation, reaching an average of 1098 mg for every 200 ml of
culture medium.

The resulting growth curve indicated that the most intense growth phase happened between 0 and 20
days after the beginning of cultivation, and that a stationary phase started from the 23rd day, with a
decline occurring after the 29th day (Figure 17.4). This allowed us to infer that the tendency of more

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271
intensive production of secondary metabolites and consequent phytotoxins might take place between
the 23rd and 29th day of cultivation. F. solani f. sp. piperis culture filtrate was also produced by
Duarte (1993) between 20 and 25 days of cultivation.

To assess the possibility of using the culture filtrate of F. solani f. sp. piperis as a selection agent, tests
were performed to determine the toxicity of the culture filtrates to in vitro buds. For this purpose, two
types of sterilisation of the culture filtrates were tested: FS and AII (Table 17.3). After FS, no bud
differentiation was observed at concentrations > 30% FS, while 40% FS was estimated to be lethal to
buds of plants susceptible to Fusariosis (Figures 17.5A and 17.6A, B). When the culture filtrate was
sterilised by AII, no evidence of bud differentiation was found for 50% AII. 80, 60 and 20% of bud
differentiation was observed in 20, 30 and 40% AII, respectively. Regression analysis allowed us to
estimate that 52.1% AII concentration, as a selection agent, would cause 100% mortality of
susceptible buds (Figures 17.5B and 17.6C, D). Matsumoto et al. (1995) and Hidalgo et al. (1999)
established that the optimal concentrations of culture filtrates to be used as selection agents for in vitro
selection of banana and pineapple were 15% (F. oxysporum f. sp. cubense) and 20% (F. subglutinans)
(see Chapters 6 and 10).

In vitro selection of variants which survived the fungal culture filtrate

The concentration of the culture filtrate of F. solani f. sp. piperis played an important role in the
survival rate of buds, considering that lower survival rates of buds were observed when a 55% filtrate
concentration was used as a culture medium for all buds, independent of the gamma radiation dose
applied to them previously. In buds that originated from irradiation with 30 Gy, no survival was
observed with a 55% concentration. Only three buds were selected with a 50% concentration among
160 buds initially transferred to the selection medium. On the other hand, a higher percentage of
selected buds on a 50% filtrate concentration was obtained from buds subjected to gamma irradiation
with a 10 Gy dose (11.35%), followed by 20 Gy (9.13%) and 40 Gy (4.22%) as shown in Table 17.4.

It is important to point out that a 50% concentration of the selection agent was not very effective in the
selection process, considering that > 40% of buds survived, except for those originating from a 30 Gy
dose (4.76%). Finally, fungal culture filtrate at a 55% concentration proved significantly more
effective as a selection agent and could be used for screening materials for potential resistance to
Fusariosis (Figure 17.7).

Determination of spore concentration and method of inoculation

The form of fungal infection that proved to be the most efficient in the process of selection was that of
soil inoculation where a concentration of 2 × 106 spores/ml caused 100% of plant death after six
months (Figure 17.8). Inoculation by spraying caused plant death, but survivors could still be found. In
the case of soil inoculation, symptoms of the disease initially appeared within one to two months of
planting, with leaves showing chlorosis, a yellowish colour and consequent necrosis. The leaves and
stem dried, and plant death occurred about six months after planting, under the weather conditions at
Piracicaba, State of São Paulo, in greenhouse cultivation. A similar artificial selection procedure was
used by Ando et al. (1984) to select plants resistant to Fusariosis using soil inoculation with a
suspension of 4 × 105 spores/ml of the fungus.

Agronomic evaluation of plants originating from gamma-irradiated cuttings

The selection of three surviving V2 plants from 428 irradiated V1 plants in 1978 allowed six
generations to be propagated in the past 20 years. During the whole process, plants had been cultivated
in areas of Fusariosis incidence. Through continuous selection of plants of good architecture, normal
morphology as well as vigorous and favourable production features, propagation was attained through
cuttings. From the fourth generation on (V4 plants), cuttings were planted in lineages. Such lineages
planted in areas of Fusariosis incidence were evaluated for their agronomic traits, principally based on
production characteristics, and lethality/survival after contact with the disease (Figure 17.9).

272 15
Agronomic evaluation of the V5 plant lineage

Based on evaluations conducted up to the year 2000, from the lineages consisting of three or more
repetitions, C25, C45, C132 and C170 stood out by presenting lower death rates caused by Fusariosis.
Nevertheless, other climatic or physiological factors such as drought and an excess of water in the soil
affected the survival of lineages originating from the V1 plants more significantly than the control
(Table 17.5). It should be emphasised that death by Fusariosis incidence was less pronounced (19.3%)
in lineages originating from irradiated cuttings than from the control (47.4%). In principle, the effect
of gamma irradiation and selection for resistance or tolerance to Fusariosis favoured higher survival
rates of plants in areas of fungal occurrence.
Plant flowering varied greatly, both in the V5 plants of all lineages and in the control plants, beginning
in mid-November when the rain season had just started, until March. Fructification also varied,
beginning six months after the first flowering, verified from late May through September, with its
peak in August, when most spikes presented fruits with a colour ranging from yellowish to reddish, a
common situation for black pepper plants in the region according to Poltronieri et al. (1999).

Production characteristics evaluated in the year 2000 presented variation with regard to spike
characteristics. Spike length varied from 6.60 to 9.28 cm (C163 and C26, respectively), with an
average of 7.91 cm; spike weight between 3053 and 5870 g (C163 and C70, respectively) with an
average of 4589 g; and a number of fruits from 27.3 to 49.6 per spike (C163 and C70, respectively)
with an average of 38.1.

As for the weight per 100 fruits, variation between 8.66 g (N108) and 14.84 g (C137) was found, with
an average of 11.61 g. Regarding average production per plant, lineage C45 presented the smallest
production (1540 g) while the highest production (8900 g) was observed in lineage N135 (Table 17.6).
The range of variation in spike length, spike weight and weight per 100 fruits was small, whereas the
variation range for the number of fruits per spike was more prominent. The general production average
per plant in the mutant lineages was 3912 g, while in the control plants (CT), originating from the
traditional cultivar ‘Singapore’, the average per plant reached 2700 g.

Better performance was found for the V5 plants than for the control, which provides evidence that the
V5 plants cultivated in an area of Fusariosis incidence show a more favourable response when
subjected to unfavourable cultivation conditions.

Mutagenesis through gamma irradiation aiming at genetic breeding of black pepper plant against
Fusariosis disease seems to be a more viable alternative when associated with in vitro technology such
as in vitro selection and generation advancement through micropropagation and with
genetic/molecular evaluation for detection of new allelic forms as well as adequate methods of
artificial and natural selection against the fungus. All these integrated factors enable the manipulation
of large populations under mutagenic treatment and reduce the time for stabilisation of chimeras,
allowing in vitro screening of potential materials for resistance or tolerance to the disease, whereas
genetic variation of selected plants can be detected through genetic and molecular analysis, confirming
the expression of the character by appropriate methods of selection during greenhouse cultivation as
well as in the field. Therefore, once a mutant possessing disease resistance or tolerance and potential
productivity is obtained, it can be cloned in vitro in a shorter period of time and in a larger quantity. A
new cultivar can be launched and/or incorporated into genetic breeding programmes of black pepper
plants. Technologies thus generated and integrated will be powerful tools, especially regarding time
and cost reduction, in the development of new cultivars.

CONCLUSIONS

The use of mutation induction through gamma in vitro technology is a viable alternative for genetic
breeding of black pepper plant that is aimed at obtaining genotypes resistant and/or tolerant to
Fusariosis, since no source of genetic resistance has yet been found within the species. Procedures of

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micropropagation, mutagenesis and in vitro selection of variants tolerant to metabolites of the fungus
should be coupled with adequate strategies of artificial selection with fungal spore suspension and
natural selection in areas of incidence of the disease as well as with evaluations of the agronomic traits
of the selected material with regard to the production features.

This study has led to the following conclusions: (1) A dose of 20 Gy of gamma radiation is appropriate
for mutagenesis of in vitro cultivated buds; (2) In vitro selection of variants against Fusariosis is
possible with a 55% concentration of F. solani f. sp. piperis culture filtrate cultivated for 28 days in
Czapek-Dox medium; (3) A concentration of 2 × 106 spores/ml of fungus in suspension applied to the
soil is adequate for artificial selection against Fusariosis under greenhouse conditions; and (4) V5
plants selected from gamma-irradiated cuttings show higher longevity and productivity in areas of
Fusariosis incidence.

REFERENCES TO CHAPTER 17

Ahloowalia BS, Maluszynski M (2001) Induced mutations: a new paradigm in plant breeding.
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Albuquerque FC, Duarte MLR (1977) Pimenta-do-reino e suas doenças na Região Amazônica.
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Albuquerque FC, Veloso CAC, Duarte MLR, Kato OR (1989) Pimenta-do-reino: recomendações
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Ando A, Albuquerque FC, Poltronieri MC, Tulmann Neto A (1996) Obtenção de mutantes resistentes
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276 19
Table 17.1. Effect of radiation dose on survival, number of buds and weight per explant, linear
equation, determination coefficient and adequate dose for a reduction of 30 and 50% of the evaluated
parameters

Dosage Average
(Gy) Survival Buds/Explant Weight/Explant
0 100.0 2.96 1.18
20 87.86 1.47 0.93
30 51.57 0.51 0.59
40 32.83 0.34 0.31
50 19.60 0.22 0.20

Equation y = –1.7395x + 107.08 y = –0.0572x + 2.7024 y = –0.01198x + 1.1847

R2 0.9322 0.9152 0.9501
30%1 74.956 = 18.47 Gy 1.8917 = 14.17 Gy 0.8293 = 17.95 Gy
50%2 53.54 = 30.78 Gy 1.3512 = 23.62 Gy 0.5924 = 29.91 Gy
1
30% reduction for each evaluated parameter according to the linear equation. 250% reduction for each
evaluated parameter according to the linear equation.

Table 17.2. Effect of different concentrations of Czapek-Dox medium in the bud multiplication
medium on the survival and proliferation of buds per explant

Mean square F test Czapek-

No. of
Coefficient Degree of Dox Survival
buds per
of variation freedom Survival Buds/ Survival Buds/ medium (%)1
explant2
Explant Explant (%)
0 100.0a3 3.20a
NS NS
Treatments 4 0.0065 0.1441 2.06 1.32 20 93.19a 3.00ab
30 89.83a 3.15ab
Residue 14 0.0032 0.1094 40 79.48a 2.78ab
50 69.79a 2.67b
1
General survival average = 87.62%; standard deviation = 4.197; CV = 4.79%. 2General survival
average = 2.99; standard deviation = 0.331; CV = 11.06%. 3Values within a column followed by the
same letters are not significantly different at < 0.05. NS = Not significant.

20
277
Table 17.3. Effect of the filtrate added to bud multiplication medium on bud differentiation per
explant after 45 days of cultivation

Coefficient of Degree of Mean F test Filtrate Non-differentiation (%)1

variation freedom square (%) FS AII
Sterilisation (A) 1 0.142 19.961** 0 22.47b2 A 9.58c A
20 89.83a A 19.02c B
Filtrate (%) (B) 4 0.118 16.639** 30 100.00a A 40.00bc B
40 100.00a A 78.30ab A
Factor A × B 4 0.024 3.395* 50 100.00a A 100.00a A

Treatments 9 0.079 Equation y = 1.526x + 39.728 y = 1.892x – 2.973

R2 0.7474 0.8829
Residue 13 0.007 100% 40.0% FS 52.1% AII
mortality3
1
General non-differentiation average = 62.74%; standard deviation = 4.98; CV = 7.94%. 2Values
followed by the same letter do not differ between each other. Lower case letters: comparison within
the column; Capital case: comparison. 3Filtrate concentration that should cause 100% mortality
according to the linear equation. *, ** Significant at p < 0.05 and p < 0.01, respectively.

Table 17.4. In vitro selection of buds surviving in culture medium containing two concentrations of
fungal filtrate as a selection agent after six weeks of cultivation

Dosage Filtrate 55% Filtrate 50% General

(Gy) Live Dead Total % Live Dead Total % Live Dead %
10 123 961 1084 11.35 550 698 1248 44.07 673 2332 28.86
20 24 239 263 9.13 119 150 269 44.24 143 532 26.88
30 0 97 97 0.00 3 60 63 4.76 3 160 1.88
40 43 977 1020 4.22 54 19 73 73.97 97 1093 11.27

Total 190 2274 2464 7.71 726 927 1653 43.92 916 4117 22.49

278 21
Table 17.5. Fusariosis incidence and plant survival during 1999 and 2000

Lineage1 Total No. Replication Surviving plants (%) Plants killed by

of plants Fusariosis (%)
1999 2000 1999 2000
C25 22 3 7 (31.8) 7 (31.8) 1 (6.7) 1 (6.7)
C26 40 5 18 (45.0) 12 (30.0) 2 (9.1) 8 (28.6)
C45 24 3 7 (29.2) 6 (25.0) 1 (5.9) 1 (11.1)
C70 56 7 29 (51.8) 19 (33.9) 0 (0.0) 9 (24.3)
C123 40 5 19 (47.5) 15 (37.5) 1 (4.8) 4 (16.0)
C132 24 3 9 (37.5) 8 (33.3) 0 (0.0) 0 (0.0)
C137 24 3 13 (54.2) 10 (41.7) 1 (9.1) 3 (21.4)
C163 56 7 23 (41.1) 12 (21.4) 3 (9.1) 11 (25.0)
C166 56 7 19 (33.9) 11 (19.6) 4 (10.8) 11 (24.4)
C170 48 6 18 (37.5) 15 (31.3) 0 (0.0) 3 (9.1)
N27 8 1 4 (50.0) 4 (50.0) 1 (25.0) 1 (25.0)
N108 16 2 7 (43.8) 5 (31.3) 1 (11.1) 3 (27.3)
N121 8 1 5 (62.5) 3 (37.5) 0 (0.0) 2 (66.7)
N135 8 1 4 (50.0) 3 (37.5) 0 (0.0) 1 (25.0)
N148 8 1 3 (37.5) 3 (37.5) 0 (0.0) 0 (0.0)

Total 438 185 (42.2) 133 (30.4) 15 (5.9) 59 (19.3)

Control 24 3 13 (54.2) 5 (20.8) 2 (18.2) 9 (47.4)

1
Lineages selected from irradiated cuttings and plants from non-irradiated cuttings (control) in Tomé-
Açú, Brazil.

Table 17.6. Spike features, weight per 100 fruits and average production per green pepper plant of V5
lineages after six years of cultivation

Lineage Spike1 Weight/100 Production (g)

Length (cm) Weight (g) Fruits fruits (g) average/plant
C26 9.28 4.197 43.0 10.68 4661.11
C132 7.85 4.842 32.6 12.36 3650.00
C166 8.03 3.874 32.5 12.45 4521.43
N135 8.42 5.054 43.6 10.79 8900.00
C70 8.79 5.870 49.6 12.07 4258.89
N148 7.33 4.686 42.8 8.80 2925.00
C25 7.94 4.581 35.5 10.52 3000.00
C45 6.90 4.624 33.7 14.40 1540.00
N1A 7.47 4.897 39.3 10.55 5400.00
C163 6.60 3.053 27.3 12.52 3072.22
C123 7.70 4.759 41.8 10.47 4629.17
N121 9.07 5.614 45.4 12.44 3950.00
C170 8.72 4.758 31.8 13.90 2722.22
C137 7.36 4.087 34.8 14.84 2566.67
N27 7.68 4.321 36.4 10.33 3203.33
N108 7.49 4.206 39.3 8.66 3600.00

Average 7.91 4.589 38.1 11.61 3912.50

1
Average of 10 spikes.

22
279
 Gamma rays (0-80 Gy)

In vitro cultures Irradiation Survival rate

Figure 17.1. Radiosensitivity test of in vitro buds irradiated with various doses of gamma-
rays ranging from 0 to 80 Gy and cultivation in a bud multiplication medium.

1.4
1.2
Explant weight (g)

1
0.8 y = -0.0198x + 1.1847
2
0.6 R = 0.9501
0.4
0.2
0 A
0 10 20 30 40 50 60
Dose (Gy)

3.5
Mean of buds/explants

3
2.5
2
y = -0.0572x + 2.7024
1.5
2
1
R = 0.9152
0.5
0
B
-0.5 0 10 20 30 40 50 60
Dose (Gy)

3.5
3
Mean of buds/explant

2.5
2
y = -0.0572x + 2.7024
1.5 2
R = 0.9152
1
0.5
0
C
-0.5 0 10 20 30 40 50 60
Dose (Gy)

Figure 17.2. Effect of gamma radiation on explant weight (A), survival (B) and number of
buds per explant (C).

27

280
 0 Gy 20 Gy 30 Gy 40 Gy 50 Gy

Figure 17.3. Effect of gamma radiation, doses ranging from 0 to 50 Gy, on in vitro buds.

350 000
23 29
300 000

250 000 35
Conidium/m l

17
200 000
14
150 000 11
100 000 2
8 y = -643.88x + 34949x - 168193
50 000 2
R = 0.9812
0
0 5 10 15 20 25 30 35 40
Days

Figure 17.4. Growth curve of Fusarium solani f. sp. piperis cultivated on Czapek-Dox
medium for 35 days.

28

281
 140
120

Mortality (%)
100
80
60
y = 1.5256x + 39.728
40 2
20
R = 0.7474
0
0 10 20 30 40 50 60
FS culture filtrate (%)
A

120
y = 1.8919x - 2.973
100 2
R = 0.8829
Mortality (%)

80
60
40
20
0
-20 0 10 20 30 40 50 60

AII culture filtrate (%) B

Figure 17.5. Effect of two different forms of culture filtrate sterilisation on bud
differentiation. (A) filter sterilisation (FS); (B) Double autoclaving (AII).

40% 20% 0% 30% 50%

A B

C 40% 20% 0% 30% 50% D

Figure 17.6. Effect of different filtrate concentrations (0, 20, 30, 40 and 50%) as a selective
agent in bud multiplication medium. (A-B) Filter sterilisation (FS) after 45 days of
cultivation; (C-D) Double autoclaving (AII) after 45 days of cultivation.

29

282
Figure 17.7. In vitro selection of variants originating from gamma irradiation of buds
which survived the culture filtrate of Fusarium solani f. sp. piperis cultured for six weeks.

Figure 17.8. Plants subjected to inoculation with fungal spores (0 to 2 × 105, × 104 and
× 106 spores/ml). (A) One month after inoculation; (B) Six months after inoculation:
Left (E) via spraying and right (D) via soil inoculation.

30

283
 A B C

D E F

Figure 17.9. Mortality and survival of 5V plants in an area of high incidence of

Fusariosis. (A-C) death by Fusarium solani f. sp. piperis attack; (D-E) surviving V5
plants; (F) control plant originated from non-irradiated cuttings.

31

284
 Chapter 18

Screening for resistance to cucurbit downy mildew (Pseudoperonospora

cubensis)

A. Lebeda* and J. Urban

Palacký University in Olomouc, Faculty of Science, Department of Botany,

Czech Republic

*Correspondence: [email protected]

Abstract

Basic methods of screening for resistance to Pseudoperonospora cubensis, causing cucurbit downy
mildew, are described in this chapter. Procedures of pathogen isolation, cultivation and maintenance in
in vitro conditions are summarised. The preparation of plant material suitable for such tests is also
given. The methodology of inoculum preparation, inoculation and incubation is described. Both
qualitative and semi-quantitative methods for disease assessment exemplified by using leaf disks are
described. A well characterised set of differential germplasm for determination of P. cubensis
pathotypes (Lebeda and Widrlechner, 2003), including 12 cucurbitaceous genotypes from six genera,
is presented. The denomination of pathotypes using unique (tetrad) numerical codes and following the
differential set is proposed. Methods described in this chapter have broad applications in research and
practical breeding.

INTRODUCTION

The Cucurbitaceae is a remarkable plant family, deserving of our attention because of its economic,
aesthetic, cultural, medicinal and botanical significance. In the Old and New Worlds, cucurbits have
been associated with human nutrition and culture for more than 12000 years. Thus, the Cucurbitaceae,
along with the Brassicaceae and Asteraceae, can be considered to be families of extraordinary
importance to humans, and follow after cereals and legumes in their economic significance for the
human economy (Lebeda et al., 2007).

Cucurbit downy mildew, caused by P. cubensis (Berk. and Curt.) Rostov (Figures 18.1 and 18.2) is
one of the most important diseases affecting field and glasshouse cucumbers, and other cucurbits
around the world (Lebeda and Widrlechner, 2003). Although cucurbit downy mildew most often
occurs in tropical, subtropical and warm, temperate areas of the world (Lebeda and Widrlechner,
2003), it is also known to occur in cooler regions, such as Sweden (Forsberg, 1986) and Finland
(Tahvonen, 1985), to where inoculum is transported by air flows. Overwintering as oospores is known
and was observed in natural conditions in Japan (Hiura and Kawada, 1933), China (Chen et al., 1959;
Zhang et al., 2006), India (Bains et al., 1977), Italy (D’Ercole, 1975) and Austria (Bedlan, 1989). P.
cubensis attacks a broad spectrum of cucurbitaceous plants, especially cucumbers (Cucumis sativus),
muskmelons (Cucumis melo), watermelons (Citrullus lanatus) and cucurbits (Cucurbita pepo, C.
maxima). Palti and Cohen (1980) reported about 40 host species; however, more recently at least 60
species have been reported to be affected (Lebeda 1992a, 1999; Lebeda and Widrlechner, 2003, 2004).

Interactions and host-parasite specificity between Cucurbitaceae and P. cubensis are very
heterogeneous and complex. The existence of P. cubensis pathogenicity variation on the level of
pathotypes and races has been confirmed in different countries around the world (Lebeda and
Widrlechner, 2003; Lebeda et al., 2006). The differentiation of pathotypes is described in the final part
of this chapter (Tables 18.1-3).

30
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Race-specificity was observed in interactions between C. melo and P. cubensis, and between
Cucurbita spp. and P. cubensis (Lebeda, 1991, 1999; Lebeda and Gadasová, 2002; Lebeda and
Widrlechner, 2003, 2004); however, until now no differential set for race determination has been
developed (Lebeda et al., 2006). Recently, some previously unknown and highly virulent isolates were
described from Israel (Cohen et al., 2003) and the Czech Republic (Lebeda and Urban, 2004a, 2004b).

Disease control through the growing of resistant cucumber (C. sativus L.) cultivars has not, as yet,
been effective (Lebeda, 1992b; Lebeda and Prášil, 1994; Lebeda and Widrlechner, 2003; Lebeda and
Urban, 2004a, 2004b). Despite research progress in this field (Lebeda, 1999; Lebeda and Doležal,
1995; Lebeda et al., 1996), no effective source of resistance of C. sativus has been found. Sitterly
(1972) only reported field resistance of some cultivars to P. cubensis. On the contrary, effective
sources of resistance based on race specificity are known in the case of C. melo and Cucurbita spp.
(Lebeda, 1991; Lebeda and Křístková, 1993, 2000; Lebeda and Widrlechner, 2004). However,
according to the terminology of McDonald and Linde (2002), P. cubensis belongs to the group of “the
highest risk pathogens” with high evolutionary potential (Lebeda and Urban, 2004a; Lebeda et al.,
2006; Urban and Lebeda, 2006) and, thus, the use of resistance genes has to be combined with other
practices of an integrated management system (antifungal compounds and plant defence inducers,
balanced fertiliser input, forecasting systems and diagnostics etc.) to minimize the risk of the pathogen
attack.

MATERIAL AND METHODS

Pathogen isolation, cultivation and maintenance

In order to isolate the pathogen, leaf samples infected by P. cubensis are placed in plastic pots (110 ×
85 × 45 mm) on wet filter paper. Depending on the degree of infection and destruction of the plant
tissue, it is possible to maintain these samples for 2-3 days at 10-15°C. Leaves are placed with the
adaxial surface down as conidiosporangiophores are especially produced on the abaxial surface. The
highest level of sporulation is usually achieved in fresh chlorotic lesions.

The primary inoculum is prepared from a lesion, surrounded by intact tissue, with well-developed
conidiosporangiophores with spores (Figure 18.1). The lesion is cut out with sterile scissors and
shaken by tweezers in a beaker (25 ml) with distilled water (approximately 5 ml); sterilised water is
more suitable. The plant tissue is then removed from the water, and the inoculum is atomised over the
abaxial surface of a leaf of a highly susceptible genotype (e.g., C. sativus ‘Marketer 430’ or C. sativus
‘Stela F1’). The leaf is then placed with the adaxial surface down on a moistened filter paper in a Petri
dish (Lebeda, 1986).

For genetic studies, it is more appropriate to use monospore isolates, which are prepared by isolation
and cultivation of a conidiospore as follows: A conidiospore suspension is prepared from a primary
isolate and diluted in distilled water (in a ratio of approximately 1:5 to 1:10). A suspension drop is
then placed on a slide with water agar (2-3 mm thick) and isolation is carried out using a monospore
insulator (Fassatiová, 1979). The agar column with a conidiospore is than transferred onto a leaf disk
(20 mm in diameter), placed with the adaxial surface down on a moistened filter paper in a Petri dish.
A modification of this method was described by Michelmore and Ingram (1982). It is also possible to
isolate a monospore with the help of a micropipette (Koch and Blok, 1985).

Pure cultures (isolates) are cultivated on leaf disks or whole leaves of a susceptible genotype placed in
Petri dishes. After ~ 10-14 days (optimum 10 days), re-inoculation onto fresh plant material must be
carried out. Longer time periods are not recommended because of destruction of plant tissues, loss of
isolate vitality or possible infection by saprophytic microflora (Lebeda, 1986). P. cubensis isolates can
be stored in Petri dishes packed in aluminium foil at – 80°C. The spores are vital for about six months;
after this period, it is necessary to renew the cultures through fresh inoculations (Lebeda and Urban,
2004a).

286 31
Preparation of plant material for screening

In our experience, true leaves (6-8 week old plants, 3-6 true leaf stage) are the most suitable for
assessing resistance. Plants should be grown in a glasshouse under optimal conditions (25°C/15°C
day/night, daily watering and weekly fertilisation), and should not be treated with chemicals
(fungicides, etc.) as these could significantly influence the infection of the pathogen isolates. Leaves
used in the tests have to be fully turgescent and without any signs of infections or pests. On the other
hand, it is also possible to use a primary intact leaf for the infection tests (Thomas, 1982).

Inoculum preparation, inoculation and incubation

The inoculum is multiplied on leaf disks (20 mm in diameter) or whole leaves of a highly susceptible
cultivar (depending on the original host of the P. cubensis isolate), which are placed with the adaxial
surface down on moistened filter paper in Petri dishes. The optimal conditions for inoculum
incubation are as follows: Usually, 7-9 days after inoculation, the abaxial surfaces of the leaves/disks
are covered with conidiosporangiophores and spores of the pathogen (Figure 18.2). The inoculum is
prepared by shaking disks/leaf pieces with well-developed spores in distilled water. The remaining
plant tissue should then be removed from the water, and the spore suspension diluted to 105-106
spores/ml. This concentration is optimal for maximum infection pressure (Lebeda, 1986). However,
lower concentrations (5 × 103) are also possible (Thomas, 1982).

The inoculum is applied onto leaf disks that were placed with the adaxial surface down on the
moistened filter paper in Petri dishes. Alternatively, water agar can be used instead of the moistened
filter paper. The inoculum is atomised over the surface of the disks with a glass sprayer
(approximately 2-3 ml per Petri dish). The leaf surface should be uniformly covered by the spore
suspension and the whole surface of the disks should touch the filter paper, otherwise only limited
infection could occur (Lebeda, 1986).

Infection by P. cubensis is strongly influenced by environmental conditions. For that reason, it is

necessary to ensure stable microclimate conditions during in vitro incubation. In field conditions,
incubation of P. cubensis takes 4-12 days, and the shortest incubation occurs at 25°C (day) and 15°C
(night). The production of spores requires high air humidity and a dark period of approximately six
hours, and is inhibited by water drops on the surface of the leaves (Cohen, 1981). In in vitro
conditions, inoculated disks are incubated in the growth chamber with stable conditions, i.e., a 12 hour
photoperiod, 18°C/15°C during light/dark cycles. During the first 16-24 hours after inoculation, it is
necessary to keep the inoculated leaves in the dark by covering the Petri dishes with black plastic foil.
The pathogen usually produces conidiosporangiophores with spores 7-9 days after in vitro inoculation.
The final evaluation is carried out 14 days after inoculation (Lebeda, 1986).

Disease assessment

The assessment of resistance/tolerance is made between 6 and 14 days after inoculation at two day
intervals. The level of tolerance of a particular host plant with known genotype and accession to a
pathogen isolate can be quantified both qualitatively and semi-quantitatively.

For a qualitative evaluation, three degrees of infection are used to determine the level of tolerance
according to Lebeda and Widrlechner (2003) (Figure 18.3):
– resistance: no visible symptoms of sporulation or very sparse sporulation (≤10% of leaf disks
covered by sporangiophores with spores;
± incomplete resistance: 10.1-35% of leaf disks covered by sporangiophores with spores;
+ susceptibility: > 35.1% of leaf disks covered by sporangiophores with spores.

For a semi-quantitative evaluation, a 0-4 scale is used (Lebeda, 1986; Lebeda and Widrlechner, 2003)
(Figure 18.4):

32
287
0 without sporulation;
1 ≤ 25% of the disk area covered by sporangiophores with spores;
2 25.1-50% of the disk area covered by sporangiophores with spores;
3 50.1-75% of the disk area covered by sporangiophores with spores;
4 > 75.1% of the disk area covered by sporangiophores with spores.

In both scales, the intensity of sporulation (i.e., the total degree of infection, DI) is expressed as a
percentage of the maximum scores according to Towsend and Heuberger (1943):

P = Σ(n × v) × 100/x × N

Where: P = the total degree of infection (DI); n = number of disks in every category of infection; v =
the category of infection (0-4); x = the range of the scale (in this case = 4); N = the total number of
evaluated disks. At least five disks in three replicates (i.e., 3 plants/accession) are tested in the case of
homogeneous plant material. Highly susceptible cultivars such as C. sativus ‘Marketer 430’ or C.
sativus ‘Stela F1’ are used as a control in the case of P. cubensis isolates from C. sativus.

Determination of pathogenic variability

The first differential set of cucurbitaceous plants showing varying degrees of resistance to P. cubensis
pathotypes was published in 1987 (Thomas et al., 1987). This set included six differential genotypes
from three cucurbitaceous genera (Cucumis, Cucurbita, Citrullus); however, they were not defined
either at the species (Cucurbita spp.) or genotype (Cucumis, Citrullus) levels. For this reason, a new
differential set was developed, and includes 12 cucurbitaceous genotypes from six genera, which are
sufficiently characterised at the levels of species, subspecies and genotypes/accessions (Table 18.1)
(Lebeda and Widrlechner, 2003). P. cubensis pathotypes (Table 18.2) are described by using unique
(tetrad) numerical codes following the proposed differential set of Cucurbitaceae (Table 18.1)
according to Lebeda and Widrlechner (2003) as shown in Table 18.3.

CONCLUSIONS

The methods described in this chapter have broad applications in basic research, screening of plant
germplasm and resistance breeding programmes of cucurbits (Lebeda et al., 2007). The method using
leaf disks is especially advantageous as it allows screening of resistance of a huge amount of plant
material in a relatively limited space and in precisely defined conditions, as well as the
characterisation of a large number of P. cubensis isolates from the viewpoint of pathotypes and races
(Lebeda et al., 2006). Data at the population level can contribute to elucidating temporal and spatial
pathogen variation and dynamics, as well as to clarifying host-pathogen interactions. It is also
important to consider the practical application of these data in resistance breeding, studies of fungicide
effectiveness and disease management (Urban and Lebeda, 2006).

Acknowledgements

This study was supported by the Ministry of Education, Youth and Sports of the Czech Republic,
project MSM 6198959215, and and by the project QH 71229 (NAZV).

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Bedlan G (1989) Erstmaliger Nachweis von Oosporen von Pseudoperonospora cubensis (Berk. et
Curt.) Rost. an Gewächshausgurken in Österreich. Pflanzenschutzberichte 50: 119-120
Chen CP, Sung CC, Ho CC (1959) A brief report of the discovery of oospores of downy mildew of
cucumber (Pseudoperonospora cubensis (Berk. et Curt.) Rostov.). Zhibing Zhishi 3: 144-145.

288 33
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Press, London, UK, pp 341-354.

Cohen Y, Meron I, Mor N, Zuriel S (2003) A new pathotype of Pseudoperonospora cubensis causing
downy mildew in cucurbits in Israel. Phytoparasitica 31: 458-466.

D’Ercole N (1975) La peronospora del cetriolo in coltura protetta. Inftore Fitopatol 25: 11-13.

Fassatiová O (1979) Plísně a vláknité houby v technické mikrobiologii (Moulds and Filamentous Fungi
in Technical Microbiology). SNTL, Prague, Czech Republic, pp 25-26.

Forsberg AS (1986) Downy mildew-Pseudoperonospora cubensis in Swedish cucumber fields.

Växtskyddsnotiser 50: 17-19.

Hiura M, Kawada S (1933) On the overwintering of Peronoplasmopara cubensis. Jap J Bot 6: 507-513.

Koch MF, Blok I (1985) Inheritance of virulence in Bremia lactucae to match several resistance factors
in lettuce. Neth J Plant Path 91: 15-26.

Lebeda A (1986) Pseudoperonospora cubensis. In: Lebeda A (ed.) Metody testování rezistence zelenin
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Sempra, VŠÚZ Olomouc, Czech Republic, pp 81-85.

Lebeda A (1991) Resistance in muskmelons to Czechoslovak isolates of Pseudoperonospora cubensis

from cucumbers. Sci Hort 45: 255-260.

Lebeda A (1992a) Screening of wild Cucumis species against downy mildew (Pseudoperonospora
cubensis) isolates from cucumbers. Phytoparasitica 20: 203-210.

Lebeda A (1992b) Susceptibility of accessions of Cucumis sativus to Pseudoperonospora cubensis. Tests

of Agrochem and Cult No 13 (Ann Appl Biol 120 Suppl.): 102–103.

Lebeda A (1999) Pseudoperonospora cubensis on Cucumis spp. and Cucurbita spp. – resistance
breeding aspects. Acta Hort 492: 363-370.

Lebeda A, Doležal K (1995) Peroxidase isozyme polymorphism as a potential marker for detection of
field resistance in Cucumis sativus to cucumber downy mildew (Pseudoperonospora cubensis (Berk. et
Curt.) Rostov.). J. Plant Dis. Prot.ec 102: 467-471.

Lebeda A, Gadasová V (2002) Pathogenic variation of Pseudoperonospora cubensis in the Czech

Republic and some other European countries. Acta Hort 588: 137-141.

Lebeda A, Křístková E (1993) Resistance in Cucurbita pepo and Cucurbita moschata varieties to
cucurbit downy mildew. Plant Varieties Seeds 6: 109-114.

Lebeda A, Křístková E (2000) Interactions between morphotypes Cucurbita pepo and obligate biotrophs
(Pseudoperonospora cubensis, Erysiphe cichoracearum and Sphaerotheca fuliginea). Acta Hort 510:
219-225.

Lebeda A, Křístková E, Kubaláková M (1996) Interspecific hybridization of Cucumis sativus × Cucumis

melo as a potential way to transfer resistance to Pseudoperonospora cubensis. In: Gómez-Guillamón
ML, Soria C, Cuartero J, Torés JA, Fernández-Muňoz R (eds.) Cucurbits Towards 2000. Proceedings of
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Lebeda A, Prášil J (1994) Susceptibility of Cucumis sativus cultivars to Pseudoperonospora cubensis.

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Genetics and Breeding, Palacký University in Olomouc, 2004, Olomouc, Czech Republic, pp 267-273.

Lebeda A, Urban J (2004b) Distribution, harmfulness and pathogenic variability of cucurbit downy
mildew in the Czech Republic. Acta Fytotechnica et Zootechnica 7: 170-173.

Lebeda A, Widrlechner MP (2003) A set of Cucurbitaceae taxa for differentiation of

Pseudoperonospora cubensis pathotypes. J. Plant Dis. Prot.ect 110: 337-349.

Lebeda A, Widrlechner MP (2004) Response of wild and weedy Cucurbita L. to pathotypes of

Pseudoperonospora cubensis (Berk. & Curt.) Rostov. (cucurbit downy mildew). In: Spencer-Phillips
PTN, Jeger M (eds.) Advances in Downy Mildew Research, Vol. 2. Kluwer Academic Publishers,
Dordrecht, Germany, pp 203-210.

Lebeda A, Widrlechner MP, Staub J, Ezura H, Zalapa J, Křístková E (2007) Cucurbits (Cucurbitaceae;
Cucumis spp., Cucurbita spp., Citrullus spp.), Chapter 8. In: Singh RJ (ed.) Genetic Resources,
Chromosome Engineering, and Crop Improvement Series, Vol. 3 – Vegetable Crops. CRC Press,
Francis & Taylor, Boca Raton, Florida, USA, pp 271-376.

Lebeda A, Widrlechner MP, Urban J (2006) Individual and population aspects of interactions between
Cucurbits and Pseudoperonospora cubensis: Pathotypes and races. In: Holmes GJ (ed.) Proceedings of
Cucurbitaceae 2006. Universal Press, Raleigh, North Carolina, USA, pp 453-467.

McDonald BA, Linde C (2002) Pathogen population genetics, evolutionary potential, and durable
resistance. Annu Rev Phytopathol 40: 349-37.

Michelmore RW, Ingram DS (1982) Secondary homothallism in Bremia lactucae. Trans Br Mycol
Soc 77: 131-137.

Palti J, Cohen Y (1980) Downy mildew of cucurbits (Pseudoperonospora cubensis): The fungus and
its hosts, distribution, epidemiology and control. Phytoparasitica 8: 109-147.

Sitterly WR (1972) Breeding for disease resistance in cucurbits. Annu Rev Phytopathol 10: 471-487.

Tahvonen R (1985) Downy mildew of cucurbits found for the first time in Finland. Växtskyddsnotiser
49: 42-44.

Thomas CE (1982) Resistance to downy mildew in Cucumis melo plant introductions and American
cultivars Plant Dis 66: 500-502.

Thomas CE, Inaba T, Cohen Y (1987) Physiological specialization in Pseudoperonospora cubensis.

Phytopathology 77: 1621-1624.

Towsend GR, Heuberger W (1943) Methods for estimating losses caused by diseases in fungicide
experiments. Plant Dis Rep 27: 340-343.

Urban J, Lebeda A (2006) Fungicide resistance in cucurbit downy mildew – methodological,

biological and population aspects Ann Appl Biol 149: 63-75.

Zhang, Yan-ju, Qin, Zhi-wei, Zhou, Xiu-yan (2006) Study on the over-wintering of cucumber downy
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Table 18.1. Differential set of cucurbit taxa for determination of pathogenic variability in
Pseudoperonospora cubensis*

No. Taxon Accession number Cultivar name Country of

Donor EVIGEZ 1 origin
1 Cucumis sativus H39-0121 ‘Marketer 430’ USA
2 C. melo subsp. melo PI 292008 H40-1117 ‘Ananas’ Israel
‘Yoqne’am’
3 C. melo subsp. agrestis var. CUM H40-0625 ‘Baj-Gua’ Japan
conomon 238/1974
4 C. melo subsp. agrestis var. PI 200819 H40-0611 Myanmar
acidulus
5 Cucurbita pepo subsp. pepo 2 PI 171622 H42-0117 ‘Dolmalik’ Turkey
6 C. pepo subsp. ovifera var. PI 614687 H42-0130 USA
texana
7 C. pepo var. fraternal 3 PI 532355 H42-0136 Mexico
8 Cucurbita maxima H42-0137 ‘Goliáš’ Czechoslovakia
9 Citrullus lanatus H37-0008 ‘Malali’ Israel
10 Benincasa hispida BEN 485 H15-0001 USA
11 Luffa cylindrica H63-0010 N/A
12 Lagenaria siceraria H63-0009 N/A
1
EVIGEZ: Czech genebank number; 2 Taxonomy of Cucurbita species is adjusted according to the
recent correspondence with J. H. Wiersema (ARC, Beltsville, USA); 3 Originally described as
Cucurbita fraterna (Lebeda and Gadasová, 2002). * Adapted from Lebeda and Widrlechner (2003).

Table 18.2. Examples of reaction patterns of some Pseudoperonospora cubensis isolates originating in
Europe and their degree of pathogenicity*

P. cubensis/isolate (PC)a
b
No. Differential genotype 3/00 13/00 1/88 3/98 1/98 4/00 12/00 1/97
1 Cucumis sativus +c + + + + + + +
2 C. melo subsp. melo + -d + - + - + +
3 C. melo subsp. agrestis - - - - - + - +
var. conomon
4 C. melo subsp. agrestis - + - - - - + +
var. acidulus
5 Cucurbita pepo subsp. - - - - - - - -
pepo
6 C. pepo subsp. ovifera var. - + + + + + + +
texana
7 C. pepo var. fraterna - - - - - + - -
8 C. maxima - + - + + + + +
9 Citrullus lanatus - - - - - - - +
10 Benincasa hispida - - + + + + + +
11 Luffa cylindrica - - - - - - + -
12 Lagenaria siceraria - - + + + + + +
a
PC codes represent the isolate number/year of collection; b 3/00 isolated in France, all others
in the Czech Republic; c resistant response (with no visible symptoms of sporulation or very
sparse sporulation convering < 10% of infected area; d susceptible response with intensity of
sporulation > 35% of infected area (Lebeda and Křístková, 1993). * Modified according to
Lebeda and Gadasová (2002).

291
36
Table 18.3. Unique (tetrad) numerical codes for Pseudoperonospora cubensis pathotypes (Table 18.2)
following the proposed differential set of Cucurbitaceae (Table 18.1)*

Genotype Cucumis spp. Cucurbita spp. Other cucurbits

Differential number 1 2 3 4 5 6 7 8 9 10 11 12

Isolate Scoring value 1 2 4 8 1 2 4 8 1 2 4 8 Identifier

PC 3/00 1 2 0 0 0 0 0 0 0 0 0 0 3.0.0
PC 13/00 1 0 0 8 0 2 0 8 0 0 0 0 9.10.0
PC 1/88 1 2 0 0 0 2 0 0 0 2 0 8 3.2.10
PC 3/98 1 0 0 0 0 2 0 8 0 2 0 8 1.10.10
PC 1/98 1 2 0 0 0 2 0 8 0 2 0 8 3.10.10
PC 4/00 1 0 4 0 0 2 4 8 0 2 0 8 5.14.10
PC 12/00 1 2 0 8 0 2 0 8 0 2 4 8 11.10.14
PC 1/97 1 2 4 8 0 2 0 8 1 2 0 8 15.10.11

* According to Lebeda and Widrlechner (2003)

292 37
Figure 18.1. Conidiosporangiophores and spores of Pseudoperonospora cubensis.

Figure 18.2. Abaxial leaf surface of Cucumis sativus cv. ‘Marketer 430’ with sporulating
.Pseudoperonospora cubensis

43

293
 A

Figure 18.3. Different responses in leaf disks to inoculation by Pseudoperonospora

cubensis (nine days after inoculation). (A) Resistant response of Cucurbita pepo subsp.
pepo cv. ‘Dolmalik’; (B) Susceptible response of C. pepo subsp. texana (reduced
sporulation of P. cubensis); (C) Susceptible response of Cucumis sativus cv. ‘Marketer
430’ (profuse sporulation of P. cubensis).

DI 0 DI 1 DI 2 DI 3 DI 4

Figure 18.4. Detail of leaf disks with different degree of infection (DI 0-4) nine days
after inoculation with Pseudoperonospora cubensis.

44

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 Chapter 19

Screening for resistance to cucurbit powdery mildews (Golovinomyces

cichoracearum, Podosphaera xanthii)

A. Lebeda* and B. Sedláková

Palacký University in Olomouc, Faculty of Science, Department of Botany,

Czech Republic.

*Correspondence: [email protected]

Abstract

In this chapter is summarized the basic knowledge about cucurbit powdery mildew (CPM) which is
the major cause of losses in cucurbit production worldwide. The disease is caused by two obligate
biotrophic ectoparasites, Golovinomyces cichoracearum s.l. (Gc) (syn. Erysiphe cichoracearum s.l.)
and Podosphaera xanthii (Px) (syn. Sphaerotheca fuliginea). They can be distinguished easily under
light microscopy according to the morphology of conidia and their germ tubes. Powdery mildew
species differ in host range, ecological requirements, and geographic distribution. Broad pathogenic
variation is represented by the existence of different pathotypes and races. Fungicide resistance of
CPMs is a serious problem worldwide, therefore suitable sources of resistance and resistance breeding
are considered as very important for all cucurbit crops. There are briefly described the basic methods
of pathogen determination, isolation, cultivation and maintenance, including preparation of plant
material for screening. The methods of adult leaf disks inoculation, disease assessment and calculation
of degree of infection (DI) are described, as well as determination of pathogenic variability
(pathotypes and races) by both species of cucurbit powdery mildew.

INTRODUCTION

Cucurbit powdery mildews (CPMs) cause a foliar fungal disease and are among the most intensively
studied powdery mildews because of their economic impact (Cohen et al., 2004; McCreight, 2006),
not only in Europe but also worldwide (Jahn et al., 2002). CPMs occur in all areas where cucurbits are
cultivated, both in field and greenhouse crops. In field conditions, CPMs are less problematic than in
the greenhouse. CPMs primarily infect leaf blades that can be totally destroyed during heavy infection.
Reduction or destruction of assimilatory leaf surface can markedly influence yield and the quality of
fruits (Skalický, 1961; Sitterly, 1978; Jahn et al., 2002).

During recent years, the taxonomical division of the order Erysiphales has been revised (Braun et al.,
2002) and two ascomycete fungi are considered to be the causal agents of CPM infection:
Golovinomyces cichoracearum (DC.) V. P. Gelyuta (Gc) (formely E. cichoracearum resp. E. orontii)
(Figure 19.1) and Podosphaera xanthii (Castag.) U. Braun and N. Shish. (Px) (formely Sphaerotheca
fuliginea or S. fusca) (Shishkoff, 2000) (Figure 19.2). The two species differ in their host range
(Braun, 1995), ecological requirements (Sitterly, 1978; Lebeda, 1983), response to certain fungicides
(McGrath, 2001; Sedláková and Lebeda, 2008) and pathogenicity (Bardin et al., 1997, 1999; Lebeda
et al., 2007a). The identification of both species is relatively easy by microscopic examination of the
morphological characters of conidia (Lebeda, 1983) (Figures 19.1 and 19.2). The occurrence of
cleistothecia (chasmothecia) is recorded quite rarely (Figures 19.3 and 19.4).

In general, Px occurs more frequently in warmer regions and on protected crops (greenhouses, plastic
tunnels). Gc is more common in temperate and cooler regions then in warmer areas. This fact was
confirmed in the 1970s and 1980s during studies on the distribution of CPMs in the former

40
295
Czechoslovakia (Lebeda, 1983) and specified by Zlochová (1990) for the territory of Slovakia. It was
also verified in recent observations (Lebeda, 1983; Křístková et al., 2002; Lebeda and Sedláková,
2004), and a potential tendency of Px spread to northern areas of the Czech Republic has been
indicated (Křístková et al., 2002; Lebeda and Sedláková, 2004). It has been shown that the CPM
species spectrum in the Czech Republic is markedly different from that in some western and southern
European countries, and many other parts of the world (Křístková et al., 2009), Px being the common
or even the predominant CPM species (Bertrand et al., 1992; McGrath, 1994; Vakalounakis et al.,
1994; Cohen et al., 2004). The genetic background of heterothalism of both CPM species was found to
be monogenic (McGrath et al., 1996). The low frequency of occurrence of sexual stages (teleomorphs,
Figure 19.4) is probably due to unequal distribution of sexually compatible mycelia (Bertrand, 1991;
McGrath et al., 1996).

The pathogenicity variation among Px and Gc has been described at the level of pathotypes and races
(Bardin et al., 1997, 1999). Pathotypes basically express pathogenicity variation at the host range level
(Table 19.1); on the other hand, races represent the level of virulence on a set of selected genotypes of
one host species (recently Cucumis melo, Table 19.2) with different resistance factors. Both species
are characterised by broad pathogenic variation represented by the existence of different pathotypes
and races (Bertrand et al., 1992; Vakalounakis and Klironomou, 1995; Jahn et al., 2002). 12 races of
Px (Cohen et al., 2004) and two races of Gc have been identified on melons, but recent results suggest
that even more pathotypes and races exist (Lebeda and Sedláková, 2004, 2006; Lebeda et al., 2004;
McCreight, 2006; Lebeda et al., 2008).

Jahn et al. (2002) published a comprehensive review of sources and genetic control of resistance to
powdery mildew in cucurbits. It is clear that only relatively little progress has been made towards
breeding Cucumis sativus resistant to CPM. The main reason for this is the absence of good resistance
resources (Lebeda et al., 2007). As opposed to this, in watermelon (C. melo) there are many sources of
race-specific resistance available (Lebeda, 1991), including commercially cultivated cultivars (Lebeda
et al., 2007b). Broad variation of resistance against powdery mildews is known in gourds (Cucurbita
spp.). We found important differences in field resistance of C. pepo and in some other species (Lebeda
and Křístková, 1994, 1996; Lebeda et al., 1999; Jahn et al., 2002).

The application of fungicides is the principal tool for managing powdery mildews (Hollomon and
Wheeler, 2002). Fungicide resistance of CPMs is a serious problem worldwide, as many reports show
that strains are becoming resistant to eight groups of fungicides (e.g., fenarimol, benomyl, triforine
etc.) (Epinat et al., 1993; McGrath, 1996, 2001; and McGrath and Shishkoff, 2001, 2003). Resistance
to some fungicides (e.g., triadimefon) can be expected to develop rapidly (McGrath and Shishkoff,
2001). The occurrence of fungicide resistance of CPM on cucurbit crops was also recently reported in
the Czech Republic (Sedláková and Lebeda, 2003, 2004a, 2004b, 2008). Further research should focus
on obtaining more comprehensive data about the geographical distribution, spatial and temporal
variability, and shift of fungicide resistance, including the relationship with pathogenicity variation of
CPM (Křístková et al., 2004; Lebeda and Sedláková, 2006; Lebeda et al., 2007a).

MATERIAL AND METHODS

Pathogen isolation, cultivation and maintenance

Fresh and heavily infected leaves of host plants (e.g., Cucurbita pepo L., C. maxima Duchesne,
Cucumis sativus L. and C. melo L.) must be collected in the field or from under cover. Before
isolation, it is necessary to carry out microscopic examination of the sample to identify the pathogen.
Mixed cultures are not suitable for this work. The identification of Px and Gc is carried out by
microscopic examination of the morphological characters of fresh or dry conidia in a 3% KOH
solution (Lebeda, 1983). Conidia of pure cultures are transferred by tapping on the primary leaves of
the highly susceptible cucumber (C. sativus) cultivar ‘Stela F1’ or other susceptible host
genotype/species. By doing this, it is possible to obtain a pathogen culture (population of conidia) that
is considered to be homogeneous.

296 41
An exact description for obtaining monosporic isolates of Gc is given in Schnathorst (1958). Leaves
with pathogen cultures are softly atomised with distilled water with a glass sprayer. Conidia are then
transferred by tapping them onto the surface of water agar in Petri dishes. Single conidia are isolated
(under the microscope) and transferred with a glass needle in a drop of water to the surface of the leaf,
which is placed on a piece of filter paper in a Petri dish. Incubation is at room temperature.

Because powdery mildew cannot be cultivated on synthetic media, it is necessary to maintain the
culture on the primary leaves of susceptible host seedlings. Isolates are cultured in plastic boxes (24°C
/18°C day/night with a 12 hour photoperiod) and transferred at approximately two week intervals
(Lebeda, 1986). Sitterly (1978) also mentioned that it is possible to store CPM on dry leaves in paper
bags for longer periods of time (up to one year) at room temperature. Based on the research of Bardin
and Nicot (1999) and our own preliminary experiments (unpublished data), it appears that conidia of
Px can withstand long-term storage in a deep freezer (– 40°C) after thorough drying. Pérez-García et
al. (2006) recommend cold storage of Px conidia desiccated with silica gel at – 80°C. This is probably
possible due to oil droplets present in the conidia.

Preparation of plant material for screening

Resistance screening of cucurbits against powdery mildews is mostly performed with adult plants.
Plants at the seedling stage (cotyledons) are not recommended for screening because expression of
resistance mostly appears in later developmental stages (Angelov and Petkova, 1979; Lebeda, 1984).
Plants at the stage of 3-6 true leaves are the most suitable for screening (Lebeda, 1986) and should be
cultivated in greenhouses or growth chambers at 22-26 °C. It is recommended that plants have buttress
(wire, string) for climbing. During the collection of leaves for resistance screening, the plants must be
fully turgescent and not treated with chemicals (fungicide application can considerably influence the
final response to powdery mildew). Throughout cultivation, it is necessary to keep plants free of
spontaneous infection with powdery mildews, which can frequently occur during the summer months.
From this viewpoint, the winter months are the most suitable time to perform these experiments as the
risk of a spontaneous inoculum transfer and infection from outside the greenhouse or growth chamber
is very low at this time.

Inoculum preparation, inoculation and incubation

The fungal inoculum is prepared and multiplied on fully developed cotyledons or the first primary
leaves of a highly susceptible cucumber cultivar (e.g., ‘Stella F1’) or another host species. Plants are
grown in plastic pots filled with Perlite with two or three seedlings per pot (Figures 19.3 and 19.5).
After the inoculation of cotyledons or primary leaves, the pots are put in transparent plastic boxes with
a detachable cover (Figure 19.5). The inside of the box must be moderately ventilated to prevent the
formation of water drops on the walls of the box and/or on the leaf surface. High air humidity may
reduce sporulation and cause limited dispersion of conidia during inoculation. A sufficient amount of
fresh conidia appears on the leaves 9-12 days after inoculation (Figure 19.6).

As mentioned above, it is best to use leaves from adult plants at the 3-6 leaf stage for inoculation. The
disks (diameter: 15 mm; Figure 19.7) are cut with a cork borer from the leaves, and placed with their
abaxial side on moistened filter paper in the Petri dishes (Lebeda, 1984, 1986). Another method has
been proposed in which the leaf disks are put onto polystyrene pills floating in distilled water in the
Petri dishes (Nagy, 1972). In this case, the Petri dishes must be opened every two days following
inoculation so that the evaporated water can be gradually added. Leaf disks may also be cultivated on
water agar supplemented with benzimidazole (40 mg/l) (Király et al., 1974). Inoculation of leaf disks
is performed by mechanical transfer of conidia (tapping, dusting). After inoculation, the surface of the
leaf disks should be covered with a white coat of conidia (Lebeda, 1986). Sitterly (1978) proposed that
the inoculum be prepared as a water suspension of conidia. However, we do not recommend this
method because the conidia germinate badly in water drops, and mycelium does not develop well on
the moist leaf surface.

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Opinions about the optimal conditions for powdery mildew infection differ (Nagy, 1976; Sitterly,
1978; Bashi and Aust, 1980; Zlochová, 1990). However, in our experience, the most suitable
temperature for incubation is 18-22°C with a photoperiod of 12 hours (Lebeda, 1986). During the
incubation, it is necessary to keep the temperature constant, because it is known that, in cucumbers,
the expression of resistance versus susceptibility may depend on the temperature (Munger, 1979). For
optimal infection and good sporulation, the humidity inside the Petri dishes must be low (~ 60 - 70%).

Methods of disease assessment

The assessment of infection intensity versus resistance is first carried out on the basis of the degree of
coverage of disks with mycelium. Eventually, it is possible to combine this criterion with sporulation
intensity. Disks are evaluated 14 days after inoculation using the following scale (Lebeda, 1984, 1986)
(Figure 19.7):

0 = without symptoms of infection;

1 = ≤ 25% of disk surface covered with mycelium;
2 = > 25 - ≤ 50% of disk surface covered with mycelium;
3 = > 50 - ≤ 75% of disk surface covered with mycelium;
4 = > 75% of disk surface covered with mycelium.

Each plant genotype is represented by five-leaf disks (diameter: 15 mm) in at least three replicates
(one replicate per plant) totalling at least 15 disks per genotype.

The intensity of sporulation, also called the degree of infection (DI), is expressed as a percentage of
the maximum scores according to Towsend and Heuberger (1943):

P = Σ(n × v) × 100/x × N

Where: P = the total degree of infection (DI); n = the number of disks in every category of infection; v
= the category of infection (0-4); x = the range of the scale (in this case = 4) and N = the total number
of evaluated disks.

Genotypes with low or no sporulation (i.e., DI = 0 - 1) are considered to be resistant (R); genotypes
with a DI of 2 - 4 are scored as susceptible (S).

Determination of pathogenic variability

To determine pathogenic variability (pathotypes and races), isolates are also screened using a leaf-disk
method (Lebeda, 1986; Bertrand et al., 1992; see above). In this case, a set of six differential cucurbit
taxa (C. melo Védrantais and PMR 45, C. sativus Marketer, C. pepo Diamant F1, C. maxima Goliáš
[Czech origin] and Citrullus lanatus Sugar Baby) (Bertrand et al., 1992) is used for determination of
pathotypes as shown in Table 19.1. The races are identified by using a set of 11 differential genotypes
of C. melo (Iran H, Védrantais, Solartur, PMR 45, WMR 29, Edisto 47, PI 414723, PMR 5, PI 124112,
MR-1, Nantais Oblong) as shown in Table 19.2 (Bardin et al., 1999). Recently is in progress the
development of a new system of determination and denomination of pathotypes and races of both
cucurbit powdery mildews species (Lebeda et al., 2008).

CONCLUSIONS

Cucurbit powdery mildew is one of the most devastating diseases of cucurbits and is caused by two
species (Golovinomyces cichoracearum and Podosphaera xanthii) which are distributed worldwide.
However, till now there is limited success in resistance breeding of the most cucurbit crops because of
enormous pathogenic variation of both CPM species and rather limited sources of efficient and durable
resistance. Detailed and broad international research of CPMs pathogenic variability and sources of
resistance, including their genetic background, can yield new knowledge in this area. For this purpose

298 43
are required unified and efficient methods of resistance screening by using well defined pathotypes
and races (Lebeda et al., 2008). The methods described in this chapter have a broad potential for
applications both in screening of Cucurbitaceae germplasm and in cucurbits breeding programmes
(Lebeda et al., 2007b). The methodology is also useful for basic research of host-pathogen
interactions, pre-breeding and mass-screening selection. The described methods can be also applied to
the research of pathogenic variability (determination of pathotypes and races) of both powdery mildew
species, as well as in their population studies (Lebeda et al., 2008).

Acknowledgements

This study was supported by the Ministry of Education, Youth and Sports of the Czech Republic,
project MSM 6198959215, and by the project QH 71229 (NAZV).

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 Table 19.1. Response of different genotypes of Cucurbitaceae to pathotypes of Golovinomyces cichoracearum and Podosphaera xanthii

France Czech Republic

Differential genotype/abbreviation Gc (4)1 Px (3) Gc (10) Px (6)
A2 AB1B2 AC ACD A AB1C AB1B2C AB1B2CC AB1B2CCm AB1CC AB1CCm
m D m D
Cucumis sativus ‘Markete 430’ A S S S S S S S S S S S
Cucumis melo ‘Védrantais’ B1 R S R R R S S S S S S
Cucumis melo ‘PMR 45’ B2 R S R R R R S S S R R
Cucurbita pepo ‘Diamant F1’ C R R S S R S S S S S S
Cucurbita maxima ‘Goliáš’* Cm - - - - - - - S S S S
Citrullus lanatus ‘Sugar Baby’ D R R R S R R R R S R S
1
Pathogen species (No. of identified pathotypes): Gc = Golovinomyces cichoracearum, Px = Podosphaera xanthii. 2Designation of pathotypes based on the
host response reaction: R = resistant, S = susceptible, - not tested; *proposed by Lebeda and Sedláková (2004). Sources: for France: Bertrand (1991), Bertrand
et al. (1992); for the Czech Republic: Lebeda and Sedláková (2004, 2006), Lebeda et al. (2007a).

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 Table 19.2. Examples of reaction patterns of Golovinomyces cichoracearum and Podosphaera xanthii for determining of pathogen races

304
Race Cucumis melo genotypes*
IrH Véd P45 W29 E47 PI41 P5 PI12 MR1 TpM Ans Nobl
Golovinomyces cichoracearum
0 S R R R R R R R R - - R
Pitrat et al. (1998)
1 S S S S S R R R R - - R
f R S S R R R R R R - - S Lebeda and Sedláková
k S S S R R S S R R - - R (2004, 2006), Lebeda et
S S S S S S S S S S - - S al. (2007a)
Podosphaera xanthii
0 S R R R R R R R R R R R
1 S S R R R R R R R S S S Pitrat et al. (1998)
2US S S S het S S R R R S S S
2F S S S R R R R R R S S S
2S S S S het R S R R R S - - McCreight (2006)
2Z S - S R R R R R - - - - Alvarez et al. (2000)
3 - S S - R R S R R S S S
Pitrat et al. (1998)
4 S S S S R R R R R S S S
5 S S S S S R R R R S S S
6 - S S S S R R R - - - - Bertrand (2002)
N1 - - R R R S R - - - - -
N2 - - S R R S R - - - - - Hosoya et al. (2000)
N3 - - R R S S R - - - - -
N4 - - R R S R R - - - - -
R1 R S S R R S S R R - - S
Lebeda and Sedláková
G S S S S S S S R R - - S
(2004, 2006), Lebeda et
H S S S R S S S S S - - S
al. (2007a)
F S S S S S S S S S - - S

Reaction: R = resistant, S = susceptible, het = heterogeneous, - = not tested.

2

*C. melo genotypes: IrH (‘Iran H’), Véd (‘Védrantais’), P45 (PMR 45), W29 (WMR 29), E47 (‘Edisto 47’), PI41 (PI 414 723), P5 (PMR 5), PI12 (PI 124
112), MR1 (MR-1), TpM (‘Top Mark’), Ans (‘Ananas’), Nobl (‘Nantais Oblong’). Sources: Pitrat et al. (1998), Bardin et al. (1999), Jahn et al. (2002),
McCreight (2006), Lebeda et al. (2008).
 Figure 19.2. Conidia of Podosphaera xanthii.

Figure 19.1. Conidia of

Golovinomyces cichoracearum.

Figure 19.3. Sporulation of cucurbit powdery mildew on cotyledons of Cucumis sativus,

susceptible cv. ‘Stela F1’ (formation of cleistothecia on the necrotic tissue).

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 Figure 19.4. Cleistothecium with asci of Golovinomyces cichoracearum.

Figure 19.5. Maintenance of cucurbit powdery mildew isolates.

306
Figure 19.6. Sporulation of cucurbit powdery mildew (Golovinomyces cichoracearum)
on cotyledons of susceptible Cucumis sativus cv. ‘Stela F1’.

A B C

D E

Figure 19.7. Detail of leaf disks with different degrees of infection (DI) 14 days after
inoculation with Golovinomyces cichoracearum. (A) DI = 0: no symptoms of pathogen
development; (B) DI = 1: mild development of mycelium without sporulation; (C) DI =
2: well-developed mycelium with mild sporulation; (D) DI = 3: intensive sporulation and
well-developed mycelium covering <50% of the leaf disk area; (E) DI = 4: intensive
sporulation and well-developed mycelium covering 50-100% of the leaf disk area.
Source: Lebeda (1983).

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4
 Chapter 20

Screening onions and related species for resistance to Anthracnose

(Colletotrichum gloeosporioides)

G. Galván

Plant Production Department,

Facultad de Agronomía de la Universidad de la República,
Montevideo,
Uruguay.

Correspondence: [email protected]

Abstract

Anthracnose (Colletotrichum gloeosporioides) is an airborne disease which causes significant yield

losses in onion (Allium cepa var. cepa) and shallot (A. cepa var. ascalonicum) in tropical regions.
There is scant variation in the response to the pathogen within A. cepa, but high resistance was found
in accessions of A. fistulosum, A. galanthum, A. roylei and other onion-related species. Their
introgression recently became feasible, and their resistances to temperate diseases are currently being
exploited. Screening for resistance to anthracnose is facilitated by in vitro multiplication of the plant
material and the use of massive experimental inoculation under conditions highly conducive to the
disease (27°C, 95% relative humidity) in a growing chamber. These elements provide economic,
quick, repeatable and reliable screening. Protocols for the in vitro introduction of Allium plant
material, for its multiplication and its acclimatisation are provided. On average, the number of
explants doubles every three to four weeks. An aggressive strain should be selected, and the inoculum
produced as a suspension of conidia, which is mass sprayed on the populations to be screened. High
relative humidity (100%) should be ensured by covering the plants with plastic bags during the
incubation period (48 to 96 hours after inoculation). The disease is scored after two weeks. A high
level of resistance was found in accessions of A. galanthum and A. fistulosum, and partial resistance in
A. roylei. A genetic analysis of the resistance in A. roylei to a Brazilian isolate, revealed that it is
dominantly inherited and most probably determined by more than one gene.

INTRODUCTION

Anthracnose (C. gloeosporioides Penz; teleomorph: Glomerella cingulata Spould & Schrenk) is an
airborne disease which causes significant yield losses in onion (A. cepa var. cepa) and shallot (A. cepa
var. ascalonicum) in tropical regions. It is widespread in Indonesia, India, Brazil and other countries,
and in extreme cases can affect 100% of the crop (Chawda and Rajasab, 1992; Wietsma et al., 1998;
Pedrosa et al., 2004). Anthracnose causes abnormal twisting and elongation of the pseudostem,
slender and distorted leaves, and soft rotting of the bulbs. Leaf lesions, scattered with minute, slightly
raised acervuli (fruiting bodies) carrying pink masses of conidia, develop (Chawda and Rajasab, 1992;
Boff, 1996; Weeraratne, 2002).

Temperature and wetness period are major factors contributing to infection and disease development.
The optimum temperature for spore germination is 26°C, while maximum mycelium growth occurs at
23-27°C, and spore production is optimal at 27°C (Suhardi, 1994; Carneiro and Amorim, 1999). A leaf
wetness period of two hours can be enough to cause infection (Carneiro and Amorim, 1999), although
Suhardi (1991) found that the longer the wetness period, the higher the disease severity. The control of
onion and shallot anthracnose is based on chemicals, and cultural practices that tend to reduce the

309
levels of initial inoculum and to cause unfavourable conditions for disease development (Suhardi,
1993; Haddad et al., 2003; Pedrosa et al., 2004).

Breeding resistant cultivars may be an environmentally sound method of disease control; however,
there is very limited variation in the response within A. cepa. The shallot cultivar ‘Sumenep’ showed
reduced disease severity in Indonesia (Suhardi, 1993), but being sterile, it is not useful for breeding.

In Brazil, variation in the reaction of onion cultivars to anthracnose was found. The local cultivar
‘Barreiro’ was identified first, and its derivative ‘Pira Ouro’ was selected for resistance (Melo and
Costa, 1983). Recently, after screening 15 isolates under controlled conditions, cultivars developed
locally such as ‘Vale Ouro IPA 11’ and ‘Roxinha de Belem’ differed from the susceptible cultivars
‘Texas EG 502’ and ‘Brownsville’ (Assunção et al., 1999). In the same way, Pedrosa et al. (2004)
screened a set of Brazilian onion cultivars and Uruguayan germplasm. Local germplasm was more
resistant than introduced susceptible cultivars, and differed in the frequency of initial infection (4.85 to
3.64 lesions/leaf) and in the rate of disease development (Gompertz rg: 0.13 to 0.20), but not in the
maximum severity nine days after inoculation, nor in the latent period (13.3 to 14.5 days). This scant
variation in the local germplasm, in a highly conducive screening test, was also found for other onion
diseases (Galván et al., 2004), and is an important tool for small household farming, and therefore a
valuable source of resistance.

Higher levels of resistance to anthracnose were found in accessions of A. fistulosum, A. galanthum, A.

roylei and other onion-related species (Galván et al., 1997). Introgression of resistances and other
useful characters from onion-related species has been viable since the late 1980s, with successful
crosses with A. roylei and A. galanthum. The generation of tri-hybrid populations by the bridge cross
A. cepa × (A. roylei × A. fistulosum) allows the introduction of A. fistulosum features into the genetic
basis of the crop (Kik, 2002).

The exploitation of resistances to temperate diseases from A. cepa-allied species is ongoing. A. roylei
resistance to Peronospora destructor was found to be controlled by one gene, located in the distal
portion of the linkage group assigned to chromosome 2 (Kofoet et al., 1990; Kik, 2002). Resistance to
Botrytis squamosa was found in A. roylei (de Vries et al., 1992) and A. fistulosum (Currah and
Maude, 1984), the former being exploited by backcrossing (Alan et al., 2003). Similar introgression
schemes could be implemented to breed for high resistance to C. gloeosporioides.

The development and availability of accurate screening to identify the response of specific genotypes
become an important issue for the use of molecular techniques, such as the development of molecular
markers via bulk segregant analysis, or via linkage disequilibrium. Indeed, the screening stage forms a
bottle-neck for many pathosystems and, therefore, deserves attention. As a general goal, the adopted
screening method should be economical, quick and repeatable.

In vitro multiplication of Allium species is a useful technique when screening inter-specific

populations and other valuable plant material (de Melo et al., 2003). This technique allows: (1) the
maintenance of specific genotypes and populations over long periods, and (2) the availability of a
large enough number of replications for the proper characterisation of specific genotypes.

Screening for resistance to anthracnose is facilitated by in vitro multiplication of the plant material and
the use of mass experimental inoculation under conditions highly conducive to disease development in
a growing chamber. These elements provide a reliable screening procedure.

310
MATERIAL AND METHODS

Inoculum production

C. gloeosporioides isolates differ in their virulence, when comparing isolates from different onion or
shallot cultivation areas (Suhardi, 1994; Assunção et al., 1999; Pedrosa et al., 2004) and continents
(Galván et al., 1997). There is no indication of isolate-specific responses, but rather differences in the
presence of pathogenic factors among isolates, thus leading to differences in virulence. Suhardi (1991)
also found differences between two isolates with regard to their temperature requirements for in vitro
spore germination and mycelium growth, suggesting ecological adaptation to local conditions.

An aggressive strain, isolated from the target cropping area, should be selected for screening. So far,
no advantage has been gained by mixing isolates in the resistance tests.

The pathogen is in vitro multiplied on potato dextrose agar (PDA) at 27°C in darkness. The inoculum
is produced as a suspension of conidia by gently rubbing 10 day old colonies (usually fully sporulated)
and filtering with a cheese-cloth. The suspension is then adjusted to 1.0-1.2 × 106 spores/ml and a
surfactant such as Tween-80 is added (10 drops/l). The inoculation is performed with a hand atomizer.

Plant material

Onion plants are produced from seeds in pots in a greenhouse, and shallot cultivars are vegetatively
propagated as usually done for commercial purposes. The test is carried out with 60 to 90 day old
plants (three to four true expanded leaves). In this chapter, the response of A. fistulosum, A. roylei and
A. galanthum to C. gloeosporioides are presented and discussed, including results on genetic analysis
of an F2 A. cepa × A. roylei segregant population screened at Wageningen University and Research
Centre (The Netherlands) and results previously reported in Galván et al. (1997). In addition, the
response of tri-hybrid A. cepa × (A. roylei × A. fistulosum) clones is reported and discussed
(Galván, 1996). The parental species were propagated from seeds, and the F1 and F2 genotypes were
vegetatively cloned in a greenhouse.

When testing the response of genetically valuable populations (e.g., inter-specific progenies), in vitro
propagation allows the maintenance of the genotypes, and increases the number of replications (de
Melo et al., 2003). The introduction is performed by direct plant regeneration, using pieces of the
basal stem as initial explants (Kahane et al., 1992). Plants in vegetative growth would be preferable
for obtaining explants; however, non-dormant bulbs could be used as an alternative (dormant bulbs
and flowering stems give a lower rate of success).

The in vitro culture protocols presented here are, in essence, the same as those described by de Melo et
al. (2003). First, the plants (or bulbs) are washed with tap water. The outer sheaths, the roots and
senescent parts of the basal plate are removed, and the false stem is cut to 15-25 mm in length from
the basal plate. This piece is surface disinfected under aseptic conditions (flux chamber). This
involves: (1) 20 minutes of immersion in 96% ethanol, followed by (2) 90 minutes of immersion in 15
g/l sodium hypochlorite (pH6) plus 10 drops/l of Tween-80 or a similar surfactant, under frequent or
permanent shaking; and (3) rinsing with sterile water three times to remove the hypochlorite.

Next, the outer sheaths of the disinfected basal stems are again removed, and the stem or bulb is cut
shorter just above the basal plate containing the sprouting points (5-10 mm). This basal plate is
divided in a radial pattern to obtain the final explants measuring 3 to 8 mm in all dimensions, each one
potentially containing sprouting points. Vegetatively growing plants yield two to six explants
(dependent on the initial diameter of the basal plate), whereas bulbs may yield 10 to 20 explants
(Kahane et al., 1992).

311
Initial explants are in vitro established using MS growing medium (Murashige and Skoog, 1962),
including vitamins, 40 g/l of sucrose, 2 mg/l of benzyl-amino-purine (BAP) and 0.2 mg/l of
naphthalene acetic acid (NAA), adjusted to pH5.7. 125 mg/l of cefotaxime is added as an antibiotic,
during cooling down. Both the initial establishment and the sub-culture are incubated at 20-25°C and a
photoperiod of 16 hours using fluorescent light (70 μmol/m2/s). During sub-culturing, the leaves and
the roots of the plants are trimmed. For sub-culture and multiplication, the growing medium is half-
concentrated MS with vitamins and 40 g/l of sucrose adjusted to pH5.7. On average, the number of
plantlets doubles every three to four weeks, by naturally generated lateral stems or by splitting,
although the rate depends on the genetic background of the material.

Plantlets with well developed roots are chosen for acclimatisation. The temperature should be in the
range of 15-25°C. The relative humidity should be kept close to 100% during the first days to prevent
desiccation by covering the pots with a plastic film and, if necessary, a shadowing net to prevent
excessive temperature and sun burning. After a few days, the plastic is progressively removed,
allowing the plants to gradually adapt to the conditions of the greenhouse.

At least three to four replications per genotype should be included in the screening, up to a maximum
of 10 whenever possible. All genotypes and/or accessions should also be included in a non-inoculated
control treatment, sprayed with pure water.

Mass screening test

The screening is carried out under controlled conditions in a growing chamber or phytotron. The
temperature is set to 26°C, relative humidity is 95-100%, and the plants are exposed to a photoperiod
of 12 hours (78 μmol/m2/s1) (Figure 20.1). The pots are transferred to the phytotron a few days before
inoculation.

The suspension of conidia is mass sprayed onto the accessions being screened. Pedrosa et al. (2004)
reported that spraying was faster, simpler, and provided higher infection and lower variability than
inoculation by deposition of mycelial disks on the leaves. Figures 20.2A and B show the distribution
of C. gloeosporioides conidia on the leaves, 24 hours after atomization, aligned along the edges of the
dried drops. In order to obtain uniform distribution of conidia, the atomizer should be adjusted to
produce very tiny drops. Excessive spraying should be avoided as this rinses the inoculum.

Immediately after inoculation and during the first two days (incubation period), high relative humidity
(100%) should be ensured by covering the plants with plastic bags (Figure 20.1A). Afterwards, high
relative humidity (95-100%) should be achieved by including a fog producer, spraying water on the
plants and keeping the floor wet. The effect of different durations of the “incubation” treatment
studied in Galván (1996) is presented and discussed in this chapter.

The disease is scored one and/or two weeks after inoculation by considering the characteristic
anthracnose spots, and avoiding other reactions of the plants by comparison with the control treatment.
A simple, ordinal scale is preferred to record the observed genetic variation. Working with A. cepa-
related species, a logarithmic-based scale was appropriated (Galván et al., 1997): 0: no symptoms; 1:
< 5% of the leaf area affected, one or a few small spots; 2: 5-20% of the leaf area affected; 3: > 20%
of the leaf area affected.

RESULTS AND DISCUSSION

Screening procedure

Three different “incubation” periods were tested, in which plants remained covered with plastic bags
for one, two and four days after inoculation (Galván, 1996). A minimum duration of 48 hours was
necessary to cause maximum disease severity (Figure 20.3).

312
This result is in agreement with Suhardi (1994), who reported that disease severity increases with the
duration of the wetness period in the range of 6 to 45 hours, but not with Carneiro and Amorim
(1999), who found that disease severity was not correlated with wetness duration. Although a few
hours wetness period could be enough to produce infections, at least 48 hours is suggested in order to
ensure highest disease expression.

Germination of Colletotrichum conidia comprises the development of a germ tube, followed by the
formation of appressoria (Bailey et al., 1992). Subsequently, the cuticle is penetrated. Preliminary
microscopic observations, 24 hours after inoculation (Figures 20.2C and D), showed the germinated
conidia and appressoria, but no clear fungal growth through the epidermis yet. These observations
suggest that, if the incubation period to increase leaf wetness is too short, the efficiency of detecting
susceptible genotypes may be affected.

To discriminate between susceptible and resistant responses, scoring disease expression 14 days after
inoculation was better than 7 days after inoculation (Figure 20.3). Pedrosa et al. (2004) found that
scoring 9 days after inoculation gave the best results when distinguishing the reaction among Brazilian
onion cultivars. However, for testing Allium species related to onion, the largest differences between
susceptible and resistant germplasm were found two weeks after inoculation, when the susceptible
controls were significantly diseased.

Resistance

A high level of resistance was found in accessions of A. galanthum and A. fistulosum (Figure 20.3).
These species were highly resistant to a Brazilian isolate from Santa Catarina, and partially resistant to
an Indonesian and a Nigerian isolate (Galván et al., 1997). Partial resistance in these species is
characterised by a much lower number of infections, which mainly occur in older leaves, than in a
susceptible control. In partially resistant material, the infections lead to spots of limited size with
suppressed or delayed sporulation (Figure 20.4). Although A. cepa-type infections were observed, the
typical reaction was tiny white spots (Galván et al., 1997).

A. roylei was resistant to the Brazilian isolate, but was susceptible to the Indonesian and Nigerian
isolates. The pattern of reaction was similar to that of A. galanthum and A. fistulosum, but with a lower
level of resistance (Galván et al., 1997).

After infection with Colletotrichum diseases, two phases are recognised: (1) a short hemi-biotrophic
phase, followed by (2) a visible and destructive necrotrophic phase (Bailey et al., 1992). Considering
the first phase, two types of Colletotrichum pathosystems are distinguished: (1) intracellular hemi-
biotrophic species with formation of haustoria, and (2) the subcuticular intramural species. In both
systems, there is contact between the pathogen and the host living cells, which makes it possible for
resistance responses to be triggered before the collapse of the host tissue. These phases have not been
described for A. cepa anthracnose.

The mode of resistance found in A. fistulosum, A. galanthum, A. roylei and other Allium species
possibly involves mechanisms operating during the early infection process, which might be combined
to other mechanisms at a later infection stage delaying the progress of the disease (Galván et al.,
1997).

The screening of an F2 inter-specific population of A. cepa × A. roylei with the Brazilian isolate was
carried out in order to analyse the genetic basis of the resistance in A. roylei (Figure 20.5). The
experiment was repeated twice and gave rise to different results, probably as a result of the screening
conditions. Seven days after inoculation, A. cepa was already very affected, while most of the A. roylei
replications, F1 and F2 clones had few or no symptoms. 14 days after inoculation, disease expression
increased overall, but the difference between the parents remained significant. The F1 clones and most
of the F2 clones were partially resistant, a response similar to that in A. roylei parent, revealing a

313
dominant inheritance pattern. The occurrence of significant differences between F1 plants, suggests the
presence of more than one gene controlling A. roylei resistance (Galván et al., 1997).

Tri-hybrid clones A. cepa × (A. roylei × A. fistulosum) (known as CC × RF, Wageningen Accession
No. 89446) were screened in the aforementioned experiments (Galván, 1996). Out of 10 clones, seven
and eight in each experiment fell into the resistant or partially resistant classes. Clones from the
backcross of A. cepa (CC × [CC × RF], No. 91074) were also screened, choosing parents with no
previous selection for resistance. In each experiment, eight out of 15 and one out of six clones, were
still characterised as resistant or partially resistant. These results suggest a combined effect of A.
fistulosum and A. roylei resistances, and the potential of the try-hybrid approach for the introgression
of A. fistulosum, as suggested by Kik (2002). Further research, combining efficient screening with
molecular tools, would provide insight into the genetic control of these resistance sources.

CONCLUSIONS

Successful and efficient screening for resistance to onion and shallot anthracnose can be achieved
under conditions that are highly conducive for disease development. The screening can be facilitated
by the use of in vitro propagation of Allium plant material, allowing clonal replication of genetically
valuable populations.
Under these conditions, the establishment of a reliable screening test, playing a key role in speeding up
the selection procedures, and applying molecular tools oriented to understanding the genetic basis of
the resistance, is attainable.

Acknowledgements

Thanks are due to C. Kik and W. Wietsma, who introduced the author to research on onion
anthracnose, and the potential of onion-related species for breeding; and to K. Burger-Meijer and
O. Scholten who made possible the use of in vitro culture for screening tests at Wageningen
University and Research Centre (The Netherlands). Grateful thanks are due to A. Castro for critically
reading the manuscript (Universidad de la República, Uruguay).

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 A

Figure 20.1. Overview of the screening tests in Allium spp. under controlled conditions.
(A) Incubation period, in which the plants are covered with a plastic bag to ensure
100% relative humidity. (B) Thereafter, fog is produced with a humidifier.

A B

a
co
C D

Figure 20.2. Microscopic observations of onion leaf pieces 24 hours after spray
inoculation with a suspension of Colletotrichum gloeosporioides conidia. (A) 200×
magnification; (B) 300×; (C) 400×, above the epidermis; (D) 400×, epidermal layer.
Arrows point at an appresorium (a) and a conidium (co).

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 7 DAI 14 DAI

Allium cepa Allium cepa

20 20
24 hours 24 hours

No. of clones
No. of clones

15 48 hours 15 48 hours
96 hours 96 hours
10 10

5 5

0 0
0-1 1-2 2-3 0-1 1-2 2-3

Allium roylei Allium roylei

8 No. of clones 8
No. of clones

6 6
4 4
2 2
0 0
0-1 1-2 2-3 0-1 1-2 2-3

Allium fistulosum Allium fistulosum

4 4
No. of clones

No. of clones

2 2

0 0
0-1 1-2 2-3 0-1 1-2 2-3
Disease severity classes Disease severity classes

Figure 20.3. Distribution over disease severity classes of genotypes of A. cepa (cvs.
‘Albeno’ and ‘Marathon’), A. roylei (Wageningen Accession No. 95001) and A. fistulosum
(No. 84036), after 24, 48 and 96 hours of “incubation” treatment, 7 and 14 days after
inoculation (DAI). A Brazilian isolate of Colletotrichum gloeosporioides from Santa
Catarina State was used. The disease severity classes in the x-axis comprise the lower
limit, but not the upper one (Galván et al., 1997).

14

318
 A B

Figure 20.4. (A) Reaction to anthracnose caused by Colletotrichum gloeosporioides on

a susceptible onion (Allium cepa). (B) Partial resistance of A. fistulosum, with small to
tiny white spots developing and little or no sporulation.

Allium cepa Allium roylei

60 5
No. of clones

Experiment 1
No. of clones

50 4
40 Experiment 2
3
30
2
20
1
10
0 0
0-1 1-2 2-3 0-1 1-2 2-3

F1 clones F2 clones
5 40
No. of clones

No. of clones

4 30
3
20
2
1 10
0 0
0-1 1-2 2-3 0-1 1-2 2-3
Disease severity classes Disease severity classes

Figure 20.5. Distribution over disease severity classes of an individual observation of A.

cepa plants (cvs. ‘Albeno’ and ‘Marathon’), and clonal medians of A. roylei
(Wageningen Accession No. 84037), F1 A. cepa × A. roylei clones (No. 86282), and F2 A.
cepa × A. roylei clones (No. 89163), in two experiments represented by light and dark
bars, respectively, 14 days after inoculation. A Brazilian isolate of Colletotrichum
gloeosporioides from Santa Catarina State was used. The disease severity classes in the
x-axis comprise the lower limit, but not the upper one (Galván et al., 1997).

15

319
16
 CONTRIBUTORS TO DRAFING AND REVIEW

Afza, R. Joint FAO/IAEA Programme,

Plant Breeding Unit, Agency’s Laboratories,
Seibersdorf,
Austria

Albino, J.de Cássia Centro de Energia Nuclear na Agricultura (CENA),

Universidade de São Paulo,
C.P.96, Piracicaba, São Paulo,
Brazil

Albuquerque, F.C. de Empresa Brasileira de Pesquisa Agropecuária (EMBRAPA),

Amazônia Oriental, C.P.48, Belém, Pará,
Brazil

Amusa, N. A. Institute of Agricultural Research and Training, Obafemi

Awolowo University,
PMB 5029 Moor Plantation,
Ibadan, Nigeria

Ando, A. Centro de Energia Nuclear na Agricultura (CENA),

Universidade de São Paulo,
C.P.96, Piracicaba,
São Paulo,
Brazil

Bandyopadhyay, R. International Institute of Tropical Agriculture,

Oyo Road, PMB 5320,
Ibadan,
Nigeria

Barbosa, M.L. Embrapa Genetic Resources and Biotechnology,

70770-900 Brasilia, DF,
Brazil

Bermúdez, R.S. Bioplants Centre. Carretera de Morón Km 9,

Ciego de Avila, C.P.69450,
Cuba

Borrás-Hidalgo, O. Centre for Genetic Engineering and Biotechnology (CIGB),

P.O. Box 6162,
10600 Havana,
Cuba

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321
Cedeño, M. Instituto Nacional Autónomo de Investigaciones
Agropecuarias (INIAP),
Estación Experimental Tropical Pichilingue, Km 5 vía
Quevedo-El Empalme,
Apartado postal 024, Los Ríos,
Ecuador

Degi, K. Institute of Radiation Breeding,

National Institute of Agrobiological Sciences,
Hitachiohmiya, Ibaraki, 319-2293,
Japan

Dostálová, R. Agritec Plant Research,

Zemědělská 16, 787 01 Šumperk,
Czech Republic

Erzurum, K. Ankara University

Agricultural Faculty Department of Plant Protection,
Ankara,
Turkey

Galván, G. Plant Production Department,

Facultad de Agronomía de la Universidad de la República,
Av. Garzón 780, 12900 Montevideo,
Uruguay

Garzón, I. Instituto Nacional Autónomo de Investigaciones

Agropecuarias (INIAP),
Estación Experimental Tropical Pichilingue,
Km 5 vía Quevedo-El Empalme,
Apartado postal 024, Los Ríos,
Ecuador

Gemma, H. Graduate School of Life and Environmental Sciences,

University of Tsukuba.
Tsukuba, Ibaraki 305-8572,
Japan

Grasso, G. Joint FAO/IAEA Programme,

Agriculture and Biotechnology Laboratory,
International Atomic Energy Agency, Seibersdorf,
Austria

Hase, Y. Department of Ion-Beam Applied Biology,

Japan Atomic Energy Agency,
Watanukii 1233, Takasaki,
Gunma 370-1292,
Japan

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Kantoglu, Y. Division of Agriculture,
Saraykoy Nuclear Research and Training Center,
Turkish Atomic Energy Authority,
Ankara,
Turkey

Kunter, B. Division of Agriculture,

Saraykoy Nuclear Research and Training Center,
Turkish Atomic Energy Authority,
Ankara,
Turkey

Lebeda, A. Palacký University in Olomouc,

Faculty of Science, Department of Botany,
Šlechtitelů 11,
783 71 Olomouc-Holice,
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Plant Breeding Unit, Agency’s Laboratories, Seibersdorf,
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70770-900 Brasilia, DF,
Brazil

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Faculty of Science, Department of Botany,
Šlechtitelů 11, 783 71 Olomouc-Holice,
Czech Republic

Miranda, M. UNEP, Convention on Biological Diversity,

413 Saint-Jacques Street, Suite 800,
Montreal QC H2Y 1N9,
Canada;
e-mail: [email protected]

Morpurgo, R. Joint FAO/IAEA Programme,

Plant Breeding Unit, Agency’s Laboratories,
Seibersdorf,
Austria

Nagatomi, S. Institute of Radiation Breeding,

National Institute of Agrobiological Sciences,
Hitachiohmiya, Ibaraki, 319-2293,
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Novák, F.J. Joint FAO/IAEA Programme,
Plant Breeding Unit, Agency’s Laboratories,
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Oyo Road, PMB 5320, Ibadan,
Nigeria

Okole, B. Council for Scientific and Industrial Research (CSIR)

Biosciences Unit, P.O. Box 395,
Pretoria 0001,
South Africa

Pande, S. International Crops Research Institute for the Semi-Arid

Tropics, Patancheru 502 324,
Andhra Pradesh,
India

Pathak, M. International Crops Research Institute for the Semi-Arid

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Andhra Pradesh, India

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Ankara, Turkey

Petrželová, Irena Palacký University in Olomouc, Faculty of Science,

Department of Botany, Šlechtitelů 11,
783 71 Olomouc-Holice,
Czech Republic

Poltronieri, Marli Costa Empresa Brasileira de Pesquisa Agropecuária (EMBRAPA),

Amazônia Oriental,
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Brazil

Rao, Joginedi Narayana International Crops Research Institute for the Semi-Arid
Tropics,
Patancheru 502 324,
Andhra Pradesh,
India

Reyes-Borja, W.O. Graduate School of Life and Environmental Sciences,

University of Tsukuba,
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Sağel, Zafer Ankara University Agricultural Faculty Department of Plant
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Japan

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Turkish Atomic Energy Authority,
Ankara,
Turkey

Sedláková, B. Palacký University in Olomouc, Faculty of Science,

Department of Botany,
Šlechtitelů 11, 783 71 Olomouc-Holice,
Czech Republic

Sekozawa, Y. Graduate School of Life and Environmental Sciences,

University of Tsukuba,
Tsukuba, Ibaraki 305-8572,
Japan

Sharma, M. International Crops Research Institute for the Semi-Arid

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Patancheru 502 324, Andhra Pradesh,
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Agropecuarias (INIAP),
Estación Experimental Tropical Pichilingue,
Km 5 vía Quevedo-El Empalme,
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Ecuador

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70770-900 Brasilia, DF,
Brazil

Spencer, M.M. Joint FAO/IAEA Division of Nuclear Techniques in Food

and Agriculture
International Atomic Energy Agency,
Vienna International Centre, PO Box 100,
1400 Vienna,
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Stevenson, P. Natural Resources Institute, University of Greenwich,
Kent ME4 4TB,
UK & Royal Botanic Gardens, Kew,
Richmond, Surrey TW9 3AB,
United Kingdom

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University of Tsukuba,
Tsukuba,
Ibaraki 305-8572,
Japan

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221-10, Yamaguchi,
Nogi, Aomori, 030-0142
Japan

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Zemědělská 16,
787 01 Šumperk,
Czech Republic

Tanaka, Atsushi Department of Ion-Beam Applied Biology,

Japan Atomic Energy Agency,
Watanukii 1233, Takasaki,
Gunma 370-1292,
Japan

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70770-900 Brasilia, DF,
Brazil

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Humid Forest Ecoregional Center,
BP 2008, Messa,
Yaounde,
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Agriculture and Biotechnology Laboratory,
International Atomic Energy Agency,
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Agropecuarias (INIAP),
Estación Experimental Tropical Pichilingue,
Km 5 vía Quevedo-El Empalme,
Apartado postal 024, Los Ríos,
Ecuador

23

327
 No. 21, July 2006

Where to order IAEA publications

In the following countries IAEA publications may be purchased from the sources listed below, or from
major local booksellers. Payment may be made in local currency or with UNESCO coupons.
Australia
DA Information Services, 648 Whitehorse Road, Mitcham Victoria 3132
Telephone: +61 3 9210 7777 • Fax: +61 3 9210 7788
Email: [email protected] • Web site: http://www.dadirect.com.au

Belgium
Jean de Lannoy, avenue du Roi 202, B-1190 Brussels
Telephone: +32 2 538 43 08 • Fax: +32 2 538 08 41
Email: [email protected] • Web site: http://www.jean-de-lannoy.be

Canada
Bernan Associates, 4611-F Assembly Drive, Lanham, MD 20706-4391, USA
Telephone: 1-800-865-3457 • Fax: 1-800-865-3450
Email: [email protected] • Web site: http://www.bernan.com

Renouf Publishing Company Ltd., 1-5369 Canotek Rd., Ottawa, Ontario, K1J 9J3
Telephone: +613 745 2665 • Fax: +613 745 7660
Email: [email protected] • Web site: http://www.renoufbooks.com

China
IAEA Publications in Chinese: China Nuclear Energy Industry Corporation, Translation Section, P.O. Box 2103, Beijing

Czech Republic
Suweco CZ, S.R.O. Klecakova 347, 180 21 Praha 9
Telephone: +420 26603 5364 • Fax: +420 28482 1646
Email: [email protected] • Web site: http://www.suweco.cz

Finland
Akateeminen Kirjakauppa, PL 128 (Keskuskatu 1), FIN-00101 Helsinki
Telephone: +358 9 121 41 • Fax: +358 9 121 4450
Email: [email protected] • Web site: http://www.akateeminen.com

France
Form-Edit, 5, rue Janssen, P.O. Box 25, F-75921 Paris Cedex 19
Telephone: +33 1 42 01 49 49 • Fax: +33 1 42 01 90 90 • Email: [email protected]

Lavoisier SAS, 14 rue de Provigny, 94236 Cachan Cedex

Telephone: + 33 1 47 40 67 00 • Fax +33 1 47 40 67 02
Email: [email protected] • Web site: http://www.lavoisier.fr

Germany
UNO-Verlag, Vertriebs- und Verlags GmbH, August-Bebel-Allee 6, D-53175 Bonn
Telephone: +49 02 28 949 02-0 • Fax: +49 02 28 949 02-22
Email: [email protected] • Web site: http://www.uno-verlag.de

Hungary
Librotrade Ltd., Book Import, P.O. Box 126, H-1656 Budapest
Telephone: +36 1 257 7777 • Fax: +36 1 257 7472 • Email: [email protected]

India
Allied Publishers Group, 1st Floor, Dubash House, 15, J. N. Heredia Marg, Ballard Estate, Mumbai 400 001,
Telephone: +91 22 22617926/27 • Fax: +91 22 22617928
Email: [email protected] • Web site: http://www.alliedpublishers.com

Bookwell, 24/4800, Ansari Road, Darya Ganj, New Delhi 110002

Telephone: +91 11 23268786, +91 11 23257264 • Fax: +91 11 23281315
Email: [email protected] • Web site: http://www.bookwellindia.com

Italy
Libreria Scientifica Dott. Lucio di Biasio “AEIOU”, Via Coronelli 6, I-20146 Milan
Telephone: +39 02 48 95 45 52 or 48 95 45 62 • Fax: +39 02 48 95 45 48

Japan
Maruzen Company, Ltd., 13-6 Nihonbashi, 3 chome, Chuo-ku, Tokyo 103-0027
Telephone: +81 3 3275 8582 • Fax: +81 3 3275 9072
Email: [email protected] • Web site: http://www.maruzen.co.jp
Korea, Republic of
KINS Inc., Information Business Dept. Samho Bldg. 2nd Floor, 275-1 Yang Jae-dong SeoCho-G, Seoul 137-130
Telephone: +02 589 1740 • Fax: +02 589 1746
Email: [email protected] • Web site: http://www.kins.co.kr

Netherlands
Martinus Nijhoff International, Koraalrood 50, P.O. Box 1853, 2700 CZ Zoetermeer
Telephone: +31 793 684 400 • Fax: +31 793 615 698 • Email: [email protected] • Web site: http://www.nijhoff.nl

Swets and Zeitlinger b.v., P.O. Box 830, 2160 SZ Lisse

Telephone: +31 252 435 111 • Fax: +31 252 415 888 • Email: [email protected] • Web site: http://www.swets.nl

New Zealand
DA Information Services, 648 Whitehorse Road, MITCHAM 3132, Australia
Telephone: +61 3 9210 7777 • Fax: +61 3 9210 7788
Email: [email protected] • Web site: http://www.dadirect.com.au

Slovenia
Cankarjeva Zalozba d.d., Kopitarjeva 2, SI-1512 Ljubljana
Telephone: +386 1 432 31 44 • Fax: +386 1 230 14 35
Email: [email protected] • Web site: http://www.cankarjeva-z.si/uvoz

Spain
Díaz de Santos, S.A., c/ Juan Bravo, 3A, E-28006 Madrid
Telephone: +34 91 781 94 80 • Fax: +34 91 575 55 63 • Email: [email protected]
[email protected] • [email protected] • [email protected]
Web site: http://www.diazdesantos.es

United Kingdom
The Stationery Office Ltd, International Sales Agency, PO Box 29, Norwich, NR3 1 GN
Telephone (orders): +44 870 600 5552 • (enquiries): +44 207 873 8372 • Fax: +44 207 873 8203
Email (orders): [email protected] • (enquiries): [email protected] • Web site: http://www.tso.co.uk

On-line orders:
DELTA Int. Book Wholesalers Ltd., 39 Alexandra Road, Addlestone, Surrey, KT15 2PQ
Email: [email protected] • Web site: http://www.profbooks.com

Books on the Environment:

Earthprint Ltd., P.O. Box 119, Stevenage SG1 4TP
Telephone: +44 1438748111 • Fax: +44 1438748844
Email: [email protected] • Web site: http://www.earthprint.com

United Nations (UN)

Dept. I004, Room DC2-0853, First Avenue at 46th Street, New York, N.Y. 10017, USA
Telephone: +800 253-9646 or +212 963-8302 • Fax: +212 963-3489
Email: [email protected] • Web site: http://www.un.org

United States of America

Bernan Associates, 4611-F Assembly Drive, Lanham, MD 20706-4391
Telephone: 1-800-865-3457 • Fax: 1-800-865-3450
Email: [email protected] • Web site: http://www.bernan.com

Renouf Publishing Company Ltd., 812 Proctor Ave., Ogdensburg, NY, 13669
Telephone: +888 551 7470 (toll-free) • Fax: +888 568 8546 (toll-free)
Email: [email protected] • Web site: http://www.renoufbooks.com

Orders and requests for information may also be addressed directly to:
Sales and Promotion Unit, International Atomic Energy Agency
Vienna International Centre, PO Box 100, 1400 Vienna, Austria
Telephone: +43 1 2600 22529 (or 22530) • Fax: +43 1 2600 29302
Email: [email protected] • Web site: http://www.iaea.org/books
10-16971
 spine:
19.48 mm

Mass Screening Techniques for Selecting Crops Resistant to Disease

Mass Screening Techniques
for Selecting Crops
Resistant to Disease
This publication presents an overview of mass screening
techniques for the selection of disease resistant crops. Various
aspects are considered in choosing the most suitable selection
technique for specific plant–pathogen interactions. The careful
selection of the infectious agent is very much dependent on
its origin, method of preparation, content of active substances,
and ease of use. The publication also covers radiation induced
mutations, which in addition to in vitro and in vivo screening
methods, provide a secure and rapid means of developing
resistant genotypes for fruit trees, legumes, vegetables and
tuber crops, with greater emphasis on banana — dessert and
plantain — for its high nutritive value.

INTERNATIONAL ATOMIC ENERGY AGENCY

VIENNA
ISBN 978-92-0-105110-3

Atoms for Peace

P1458 Cover.indd 1 2010-05-18 15:24:49

Atoms for Peace