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 CAMBRIDGE MONOGRAPHS IN
EXPERIMENTAL BIOLOGY
No. 19

EDITORS:

P. W. BRIAN
J. W. S. PRINGLE

DEFENCE MECHANISMS OF PLANTS

 T H E SERIES
1 v. B. WIGGLESWORTH. The Physiology of Insect Metamorphosis
2 G. H. BE ALE. The Genetics of Paramecium aurelia
3 G. v. T. MATTHEWS. Bird Navigation. 2nd edition
4 A. D. LEES. The Physiology of Diapause in Arthropods
5 E. B. EDNEY. The Water Relations of Terrestrial Arthropods
6 LILIAN E. HAWKER, The Physiology of Reproduction in Fungi
7 R. A. BEATTY. Parthenogenesis and Polyploidy in Mammalian
Development
8 G. HOYLE. Comparative Physiology of the Nervous Control of
Muscular Contraction
9 j . w. s. PRINGLE. Insect Flight
10 D. B. CARLISLE and SIR FRANCIS KNOWLES. Endocrine Control

in Crustaceans
11 DAVID D. DAVIES. Intermediary Metabolism in Plants
12 W.H.THORPE. Bird-song
13 JANET E. HARKER. The Physiology of Diurnal Rhythms
14 j . E. TREHERNE. The Neurochemistry of Arthropods
15 R. N. ROBERTSON. Protons, Electrons, Phosphorylation and
Active Transport
16 GEORGE SALT. The Cellular Defence Reactions of Insects
17 D. w. T. CROMPTON. An Ecological Approach to Acanthocephalan
Physiology
18 G. H. SATCHELL. Circulation in Fishes
DEFENCE MECHANISMS
OF
PLANTS

BY

Emeritus Professor B. J. Deverall

Faculty of Agriculture, Food and Natural Resources
University of Sydney

CAMBRIDGE UNIVERSITY PRESS

CAMBRIDGE
LONDON ' NEW YORK • MELBOURNE
 CAMBRIDGE UNIVERSITY PRESS
Cambridge, New York, Melbourne, Madrid, Cape Town, Singapore, Sao Paulo, Delhi

Cambridge University Press

The Edinburgh Building, Cambridge CB2 8RU, UK

Published in the United States of America by Cambridge University Press, New York

www.cambridge.org
Information on this title: www.cambridge.org/9780521112857

© Cambridge University Press 1977

This publication is in copyright. Subject to statutory exception

and to the provisions of relevant collective licensing agreements,
no reproduction of any part may take place without the written
permission of Cambridge University Press.

First published 1977

Reprinted 1979
This digitally printed version 2009

A catalogue record for this publication is available from the British Library

Library of Congress Cataloguing in Publication data

Deverall, Brian J.
Defence mechanisms of plants.
(Cambridge monographs in experimental biology; no. 19)
Bibliography: p.
Includes index.
1. Plants - Disease and pest resistance. 2. Host-
parasite relationships. I. Title. II. Series.
SB750.D48 581.2'32 76-12917

ISBN 978-0-521-21335-6 hardback

ISBN 978-0-521-11285-7 paperback
 Contents

Preface page vii

1 INTRODUCTION TO THE HOST-PARASITE
INTERACTION I
Infection type, susceptibility and virulence
Biotrophy and cellular compatibility
Genetic determination of the host-parasite interaction

2 DISCRIMINATORY EVENTS BEFORE AND DURING

PENETRATION INTO PLANTS 7
Effect of roots on parasites in the soil
Interactions with parasites on aerial part of plants
Relationships between virulence and penetration into
plants
Conclusion

3 CYTOLOGICAL CHANGES IN HOST AND PARASITE

AFTER INFECTION l8
The cellular location of parasites in host tissues
Cessation of growth of parasites in resistant plants
The association of hypersensitivity with resistance
Other cellular reactions observed to be associated with
resistance
Conclusion

4 CROSS-PROTECTION AND INDUCED RESISTANCE 30

Modes of action of cross-protection
Direct action of the protectant against the pathogen
Induced changes in the host plant
The nature of the induced change in the host plant
Prevention of hypersensitivity by heat-killed bacterial
cells
Conclusion
5 PHYTOALEXINS AND THEIR INDUCED FORMATION
AND BIOSYNTHESIS 43
Phytoalexins of the Leguminosae
Phytoalexins of the Solanaceae
Phytoalexins in other families
Induction and sites of phytoalexin formation
The biosynthesis of phytoalexins
Conclusion
6 ROLE OF PHYTOALEXINS IN DEFENCE MECHANISMS 62
Relationships between phytoalexin accumulation and
resistance
The significance of phytoalexin metabolism by fungi in
disease development
Phytoalexins and bacterial diseases of plants
Conclusion
7 MEDIATION OF HOST-PARASITE SPECIFICITY 75
Host-specific toxins
Common antigens
Specific cross-protection factors
An RNA as recognition product of an avirulent rust
Elicitation or suppression of phytoalexin formation
Conclusion
References 89
Index 106
 Preface

This book is concerned with the dynamic mechanisms involved

in the defence of plant cells against attack by parasitic bacteria
and fungi. Thus I scarcely discuss those plant features such as
bark and cuticle which play an obvious role in defence, but which
are essentially static contributors. Circumvent these barriers and
the ability of apparently undifferentiated parenchyma to defend
itself is revealed. Furthermore, this ability is dependent upon
particular genes in plant and parasite which interact after infec-
tion. My interest is with the processes by which plant cells perceive
the approach of an intruder and occasionally permit, but com-
monly discourage, its further progress. How do the genes of host
and parasite communicate to determine the outcome of attemp-
ted parasitism? Is there a universal defence mechanism in all
plants, and, if so, what is it? What contribution does the much
studied process of phytoalexin formation make to the defence
of plants?
Research on the physiology of host-parasite relationships has
been prolific in recent years and a number of multi-author
treatises are being published on different aspects of this work.
Hopefully, this monograph will make a useful contribution by
presenting a shorter and personal view of those parts of this
research which bear directly upon the processes of resistance in
plants. My envisaged readership comprises research workers in
the subject, and University teachers and their advanced students
in plant pathology, botany and plant biochemistry.
I wish to thank Professor A. H. Ellingboe for his suggestions
and comments on some of the chapters, Elizabeth Froggatt for
typing the manuscript, Stella McLeod for assistance with the
References and my family for their tolerance and encourage-
ment.
University of Sydney B.J.DEVERALL
February igj6
 CHAPTER I

Introduction to the Host-Parasite

Interaction
Plants at all stages of their life-cycles are exposed to many
potentially parasitic micro-organisms. Seeds germinate in soils
which contain numerous resting parasites awaiting the arrival of
roots to stimulate them into activity. Aerial parts of plants are
inoculated by fungal spores and bacterial cells carried in air
currents and rain-splash droplets. Under favourable conditions
of moisture and temperature, plant tissues are thus subjected to
attempted infection on numerous occasions. However, these
attempts often fail, and most plants remain healthy. Successful
establishment of a parasite depends upon a special genetical and
physiological relationship so that the cells of the host accept the
parasite.
This book is concerned with the processes whereby plants
succeed in remaining healthy despite their constant exposure to
potential parasites. It leads to a consideration of one of the most
interesting problems in biology and biochemistry, namely the
molecular basis of the high degree of specialization which is
often observed in relationships between parasites and hosts. As
will become apparent, there are reasons to believe that the basis
of much specialized parasitism rests in the ability of parasites to
confound a recognition system linked to other reactions in host
cells. Through this linked system, the host normally notices and
then fails to accept the intrusion of an alien organism.

INFECTION TYPE, SUSCEPTIBILITY AND VIRULENCE

Before starting to analyse these processes, it is essential to
re-emphasize that a disease is the product of the interaction of
two organisms, host and parasite. This product can depend
upon fine differences in the properties of the two organisms, as
is well recognized in research on rust diseases. Thus under
T A B L E I. The alternative attributes of parasite and host and the result
of their interaction

Attributes of Attributes of Resulting

parasites host plants infection type
Virulence Susceptibility High
Lower virulence Lower resistance Intermediate
Avirulence Resistance Low

standard and ideal conditions for infection, an isolate of a wheat

rust fungus will fail to develop on one cultivar of wheat Triticum
aestivum, but will produce large uredia on another and an
intermediate condition on a third cultivar, comprising, for
example, small uredia surrounded by an area of necrosis or
chlorosis in the host. These different products are termed infec-
tion types, and they are determined by the genetic constitutions of
the rust isolate and wheat cultivar under particular environmen-
tal conditions. A common convention is to qualify infection type
by high where there is substantial rust development and by low
where there is little or no development (Loegering, 1966). A low
infection type implies that the cultivar was resistant to the rust
isolate and that the isolate was avirulent on that cultivar. High
infection type implies the product of an interaction between a
susceptible host and a virulent rust isolate. Thus, resistance or
susceptibility as properties of a plant are defined with respect to
the response of that plant to infection by a particular isolate of
parasite. Similarly, virulence or avirulence as properties of a
parasite are defined with respect to success or failure to colonize
a particular host plant. Intermediate infection types result from
interactions between parasites and hosts possessing lower
degrees of virulence and resistance. The interaction between
these alternative attributes of parasites and hosts is shown in
Table 1.
The concept of infection type will be used in considering all
host-parasite interactions in this book, and will not be restricted
to the rust diseases.
 BIOTROPHY AND CELLULAR COMPATIBILITY
Many virulent parasites such as most rust fungi grow in an
apparently harmonious relationship with their susceptible hosts
because they cause no visible adverse reaction in the host cells
which they penetrate. It is generally assumed that these parasites
derive their nutrients from the living host cells, and they are
thus said to be biotrophic in their parasitism. By contrast, soft-rot
parasites such as the bacterium Erwinia in tubers of the potato
Solarium tuberosum or leaf-spot parasites such as Botrytis fabae in
the leaves of the broad bean Viciafaba kill adjacent plant cells by
chemical secretions. These highly successful parasites presum-
ably derive their nutrients from cells which they have killed and
thus are said to be necrotrophic in their parasitism.
The rust fungi and B. fabae are extremely different types of
parasite in their relationships with host cells, but it is important
to appreciate that some parasites, such as Colletotrichum lin-
demuthianum in bean Phaseolus vulgaris, are often biotrophic for
part of their development and necrotrophic for the remaining
part. Analyses of the features essential to the host-parasite
interaction must consider these possibly changing relationships.
Very special capacities of the parasite might be anticipated
during its biotrophic phase when the integrity and function of
living host cells are maintained despite the intrusion of hyphae
or haustoria into protoplasts. During this phase, the relation-
ships between the cells of host and parasite can be termed
compatible. Compatibility will be used in this book to describe
harmonious relationships between parasite and host, and incom-
patibility to describe interactions which cause deleterious
changes in cells of host and/or parasite.
The use of the concept of compatibililty is thus more
restricted here compared with its use by Day (1974) to refer to
any host-parasite interaction which gives rise to a high infection
type. This restriction might facilitate an analysis of the nature of
the compatibility which enables cells of a biotroph and host to
live together harmoniously. Quite a different sequence of
physiological events might be envisaged to underlie the success,
as a parasite, of the virulent necrotroph B. fabae in giving rise to
a high infection type despite the incompatibility of broad bean
cells to the fungus. The concept of compatibility in the
host-parasite cellular relationships should therefore be kept
distinct from the concept of final infection type.
 GENETIC DETERMINATION OF THE
HOST-PARASITE INTERACTION
No attempt is made here to give a detailed review of the genetics
of the host-parasite interaction, and the reader is referred to the
book bearing that title by Day (1974) and to reviews by Ellingboe
(1976), Johnson (1976a, b) and Day (1976) for more complete
discussions of this subject. However, as part of a general intro-
duction to the nature of the host-parasite interaction, it is well to
recall the widely held generalizations pertaining to the genetic
control of the interaction. These arise from results of analyses
done by Flor on the inheritance of resistance and virulence in
flax rust disease. Different genes conferred resistance in cul-
tivars of flax Linum usitatissimum to different races of the rust,
and different genes conferred avirulence in rust races to differ-
ent flax cultivars. The analyses were the basis for the gene-for-
gene hypothesis of Flor (1956) which implies that infection type
is determined by complementary single genes for resistance in
the host and avirulence in the parasite.

TABLE 2. Dependence of infection type upon genes in host and parasite

Parasite alleles for avirulence

Host alleles ^ R Low High

for [
resistance J r High High

The gene-for-gene hypothesis is considered to apply to many

host-parasite interactions, and its basis is illustrated in the
simplest form in Table 2 where infection type depends upon the
alleles at single gene loci in parasite and host. A low infection
type results from the genetic interaction between the dominant
alleles for avirulence in the parasite and for resistance in the
host. Absence of the dominant allele in either partner results in
a high infection type where virulence and susceptibility are
expressed. Thus expression of avirulence and resistance is a
more particular phenomenon, requiring precise matching of
genetic information in both partners.
TABLE 3. Dependence of infection type upon complementary genes in
host and parasite

Alleles at gene loci for avirulence

in the parasite
Ai A2 Ai a,2 ai A2 ai CL2

Alleles at Ri R2 Low Low Low High

gene loci ri R2 Low High Low High
for resistance Ri T2 Low Low High High
in the host XI T2 High High High High

The complementary role of alleles at specific gene loci in

control of infection type is illustrated in Table 3, where two gene
loci are depicted in both host and parasite. Here it can be seen
that low infection type results from the matching of specific
complementary dominant alleles in host and parasite. Thus
avirulence and resistance are expressed only when avirulence
gene Ai matches resistance gene Ri or when A2 matches R2.
Virulence and susceptibility are expressed if avirulence gene Ai
is matched with recessive allele ri or dominant allele R2 in the
host. Thus Table 3 emphasizes more strongly than Table 2 that
expression of avirulence and resistance is the result of a highly
specific genetic interaction.
Many analyses show that resistance in crop plants is inherited
as single dominant genes, but relatively few corresponding
analyses of inheritance of virulence in parasites have been
performed. Putative genes for avirulence are often assigned to
races of parasites based on the gene-for-gene hypothesis and
knowledge of corresponding genes for resistance in the host.
However, exceptions to the major generalizations discussed
above are known where susceptibility and virulence are the
dominant characters in host and parasite; for example, the
relationship between oats Avena sativa and the fungus Helmin-
thosporium victoriae which will be considered in a later chapter.
Polygenic control of resistance towards particular parasites has
been claimed in a number of important crop plants, but this
phenomenon requires critical genetic analysis under controlled
conditions of environment and, in at least two cases, has not
withstood this test (see Ellingboe, 1976; Johnson, 1976ft).
The genetic control of parasitism in natural vegetation is not
 well known, and we can only speculate about how resistance and
avirulence evolved before man sought resistance genes for
incorporation into his crop plants. The selective advantage of
resistance to plants seems self-evident, but the role of avirulence
in the development of populations of parasites is more difficult
to conceive except as a means of separating evolving populations
within species.
The most important implications for physiological and
biochemical analyses of expression of resistance and avirulence
to arise from genetic studies are that infection type is under
control of genes in host and parasite, and that the expression of
these interdependent properties is often determined by highly
specific interactions between particular complementary genes in
both partners. Ellingboe (1976) and Johnson (19766) have
pointed out that the most specific interactions are usually for
expression of resistance and avirulence. The simplest mechan-
ism mediating this expression would be based upon confronta-
tion of primary products of the genes for resistance and avirul-
ence following upon infection. Evidence bearing upon these
hypothetical products will be discussed later, but firstly it is
useful to consider what is known about stages in parasitism when
resistance is expressed. This will be done by assessing the fate of
potential parasites at the stages of attempted entry and then
early growth into plants. Attempts to understand natural pro-
cesses of defence in plants should accommodate reports that
plants can be cross-protected against normally virulent parasites
by previous infection by other organisms. The extent will be
sought to which cross-protection is achieved by activation of a
process of induced resistance in plants. Any analysis of the
process of expression of resistance must assess the contribution
of the many anti-microbial chemical compounds in plants, and
especially of those which form after infection. Finally, however,
it is essential to return to the initial question concerning the
nature of recognition between parasites and plants, and to
consider the molecular means by which genes in parasite and
host interact to determine specificity.
 CHAPTER 2

Discriminatory Events before and during

Penetration into Plants
Many micro-organisms are dispersed in air currents or in splash
droplets caused by rain, and thereby arrive on leaves and stems.
Other fungi and bacteria move in soil water before encountering
roots or persist as resting stages until roots grow into their
vicinity. Plants can then influence micro-organisms around their
surfaces by physical and chemical means, thus starting an
interaction which must be followed by entry of the parasite into
the plant by a specialized route, if parasitism is to have a chance
of success. This chapter is concerned with the few attempts that
have been made to assess quantitatively the contributions to the
success or failure of parasitism of these primary interactions
between potential parasites and hosts.

EFFECT OF ROOTS ON PARASITES IN THE SOIL

The principal effect of roots on organisms in the soil is a general
stimulation of germination and growth, and this is particularly
important for parasites most of which remain dormant until
contacted by their living substrates. Fungal parasites lie dormant
in soil as a number of different types of resting body such as
sclerotia, oospores, chlamydospores, basidiospores and hyphal
fragments. Their dormancy is considered to be of two types,
constitutive or exogenous (Sussman, 1966). Constitutive dor-
mancy is thought to be maintained by internal factors in the
fungus, and is particularly important in basidiospores. Experi-
mentally, constitutive dormancy can be broken, in at least a small
proportion of spores, by temperature shocks, treatment with
certain chemicals, and proximity to other micro-organisms and
some plant roots in culture. Presumably environmental fluctua-
tions in soils cause some of these spores to germinate in favour-
able seasons. It is also likely that emanations from roots induce
 breaking of dormancy of basidiopsores of mycorrhizal fungi
(Fries, 1966). Exogenous dormancy is conferred by external
factors in the soil. Among the emanations from other soil
inhabitants which prevent germination of fungi are ethylene
(Smith, 1973; Smith & Cook, 1974) and possibly antibiotics, many
of which were discovered as products of soil fungi and soil
Actinomycetes in culture, although their role in natural soils
remains uncertain. The effects of some of the fungistatic factors
in soil are overcome by stimulatory substances which diffuse
from plant roots. Many substances exude from the zone of
elongation of roots in particular (Schroth & Hildebrand, 1964)
and these include mineral salts, sugars, amino acids, organic
acids, nucleotides and vitamins (Rovira, 1965).
Reports of stimulatory emanations from plant roots are plen-
tiful, but toxic factors such as the cyanogenic glucoside linama-
rin in Linum (Trione, 1950), and compounds inhibitory to
nematodes in Asparagus (Rohde & Jenkins, 1958) and Tagetes
(Winoto Suatmadji, 1969) are also known to occur in roots.
These factors or their anti-microbial products may diffuse in the
soil. The role of inhibitors in the pre-penetration interactions
between roots and parasitic micro-organisms is not well estab-
lished, as discussed below, but Wallace (1973) considers that such
factors may influence parasitic nematodes before they reach the
roots of some plants, although he states that resistance to
nematodes usually occurs during or after penetration.
There is as yet little evidence that susceptible roots are excep-
tionally stimulatory to parasitic fungi in the soil or that resistant
roots are repressive. As Schroth & Hildebrand (1964) emphasize,
the soil environment is complex, and stimulants and inhibitors
diffusing from plant roots are likely to affect the activities of
many soil inhabitants, including saprophytic antagonists, with
unpredictable consequences for the success or failure of a
parasitic organism. Some laboratory experiments, which pre-
cluded these complexities, indicated that susceptible roots stimu-
late the formation of infection structures and that resistant roots
suppress the germination of spores in a selective way, but the
specificity of these effects has been discounted in subsequent
work. Thus Flentje (1957) and Kerr & Flentje (1957) showed that
the formation of appressoria of Pelliculariafilamentosawas stimu-
lated by exudates from susceptible roots, but Flentje, Dodman &
Kerr (1963) and de Silva & Wood (1964) concluded that there
8
was little difference between hosts and non-hosts in this
phenomenon. Buxton (1957) found that washings from resistant
cultivars of the pea Pisum sativum inhibited the germination of
conidia of Fusarium oxysporum f. sp. pisi, and then revealed a
similar phenomenon with washings from resistant cultivars of
the banana Musa sp. and conidia of F. oxysporum f. sp. cubense
(Buxton, 1962). However, specificity in the effects of exudates
from pea roots on the germination of micro-conidia or
chlamydospores of F. oxysporum f. sp. pisi was not found in the
work of Kommedahl (1966). Furthermore, sterile exudates from
resistant and susceptible cultivars were equally stimulatory to
different physiologic races of the parasite in vitro and also when
allowed to diffuse through porous blocks into soil containing
chlamydospores (Whalley & Taylor, 1973). There is therefore
little reason to believe that susceptible roots selectively encour-
age germination and chemotropic growth of virulent parasites.
The attraction of zoospores of several Phytophthora spp.,
Aphanomyces euteiches, Olpidium spp. and Pythium aphanider-
matum to the zones of elongation of different roots has been
established, as discussed by Hickman & Ho (1966). Zoospores
swim towards, attach to and encyst on the roots. Water-soluble
extracts of roots were attractive to zoospores of P. aphanider-
matum (Royle & Hickman, 1964a, b) as also were many of the
individual compounds known to exude from roots. The only
substance in this work which caused chemotaxis, trapping and
encystment of zoospores was glutamic acid in the presence of
ammonium bases. However, glutamic acid was considered
unlikely to act alone in attracting zoospores to roots. Troutman
& Wills (1964) found that zoospores of Phytophthora parasitica
were attracted to negative electrodes, and suggested that elec-
trostatic adhesion might occur to the zone of elongation of roots.
Contrary to most research on the response of zoospores to roots,
Zentmyer (1961) saw that some zoospores were specifically
attracted to host roots and not to non-host roots. P. cinnamomi
moved towards roots of susceptible cultivars of the avocado
Persea americana within a few minutes, and encysted and germi-
nated within an hour. Zoospores of this fungus moved less
readily to roots of resistant cultivars and were not attracted to
roots of the tomato Lycopersicon esculentum, tobacco Nicotiana
tabacum and Citrus sp. Similarly, zoospores of P. citrophthorawere
attracted to roots of citrus but not to those of avocado. In
further work, Khew & Zentmyer (1973) found that zoospores of
five species of Phytophthora including P. cinnamomi and P. cit-
rophthora responded in a similar chemotactic way to many com-
pounds known to exude from plant roots. Later Khew & Zent-
myer (1974) showed that zoospores of seven species including P.
parasitica responded electrotactically to positive, and not nega-
tive, electrodes, contrary to the report by Troutman & Wills
(1964). With the awareness of so many non-specific attractants to
roots, it is difficult to understand the nature of any specific taxis
to susceptible roots.
Most evidence, therefore, suggests the non-specific stimula-
tion of activity in soil micro-organisms by plant roots, but there
are a few reports, requiring substantiation, that some events at
this pre-penetration stage might determine resistance or suscep-
tibility of the host plant to soil-borne plant parasites. Verification
of reports of specific interactions should desirably be based
upon the more difficult experimentation in the complex envi-
ronment of natural soils, and not solely on the necessary testing
of phenomena in model situations in the laboratory.

INTERACTIONS WITH PARASITES ON AERIAL PARTS

OF PLANTS
The early investigations of Brown (1922) suggested that leaves
and petals act as sources of stimulants and inhibitors of fungal
development in overlying infection droplets. Water droplets
were shown not only to change in electrical conductivity during
incubation on plant parts but also to accumulate compounds
which affected spore germination of Botrytis cinerea. These
observations suggest that aerial parts of plants might influence
the behaviour of micro-organisms on surfaces in much the same
way as roots affect microbial activity. However, recent research
has revealed that some of these effects might be caused by other
organisms on the plant surface.
Bacteria multiply rapidly in droplets of water on leaf surfaces,
especially in the presence of fungal spores, and some of these
bacteria produce inhibitors of germination of fungi on the leaf
(Blakeman & Fraser, 1971). Irradiation of water droplets on
leaves of beetroot Beta vulgaris and Chrysanthemum sp. with
high-intensity ultra-violet light failed to decrease bacterial popu-
lations (Sztejnberg & Blakeman, 1973). Because similar bacteria
10
were eliminated in control droplets on glass slides, this suggests
that bacteria are sheltered from irradiation on the leaf, either
persisting in intercellular spaces or beneath folds on the cuticle.
Bacteria may therefore be endemic to leaf surfaces, and can
clearly multiply and influence potential parasites of plants.
Pollen grains can act as major sources of stimulants to the
germination of spores of many fungi, particularly necrotrophic
parasites such as Botrytis and Fusarium spp. The remarkable
effect of pollen was shown following experiments on infection of
fruit of the strawberry Fragaria ananassa by B. cinerea (Chou &
Preece, 1968). When anthers were removed before spraying the
fruit with conidia, no infection occurred, but in the presence of
anthers rapid rotting of the fruit took place. Pollen and pollen
extracts were then shown to promote germination of conidia
and infection of strawberry petals and broad bean leaves. Thus
emanations from pollen confer virulence on B. cinerea, normally
avirulent on all but ageing flowers and leaves. Wheat anthers
stimulate the infection of wheat spikelets by F. graminearum
(Strange 8c Smith, 1971). The fact that spikelets are resistant
before anthesis, or if the anthers are removed, suggests that the
substances which diffuse from the anthers to stimulate the
fungus are essential contributors to the virulence of the fungus
on this part of the wheat plant. Choline and betaine have been
identified as two major components of wheat anthers that stimu-
late germination of F. graminearum (Strange, Majer 8c Smith,
1974), but it is not yet confirmed that they are the virulence
factors in anthers. Anthers and fallen pollen grains may be
contributory factors to the development of outbreaks of certain
types of disease under wet conditions when inoculum is present.
Extensive experimentation on changes in the myco-flora of the
leaves of rye Secale cereale, as a result of exposure to pollen under
natural and artificial conditions, shows that competitive and
antagonistic fungi may be stimulated also (Fokkema, 1968, 1971).
As a consequence, parasitism is not necessarily promoted by
anthesis under field conditions, and the extent of the signifi-
cance of anthesis in disease development remains for wider
investigation.
Following a number of demonstrations of interactions
between parasitic and saprophytic fungi and bacteria on the leaf
surface, the ecology of the leaf surface is now recognized as an
important subject for research (Preece 8c Dickinson, 1971).
11 2-2
TABLE 4. Percentage of spores of avirulent and virulent Colletotri-
chum lindemuthianum giving rise to appressoria on resistant
and susceptible bean hypocotyls (from Skipp & Deverall, igy2)

Hours from Low infection High infection

inoculation type (%) type (%)
24 21 12
48 45 45
72 53 43

These ecological interactions can also be influenced by the

underlying cuticle, which is a complex material with many
components and an intricate surface configuration. Some cuti-
cles may permit movement of molecules from beneath, thus
affecting parasites on the surface. Other cuticles yield antifungal
substances upon extraction with organic solvents, and it is possi-
ble that these substances may interfere with attempted parasit-
ism either by diffusion into infection droplets or during penetra-
tion. However, Martin & Junifer (1970) in their comprehensive
account of plant cuticles were reluctant to conclude that cuticles
had much direct inhibitory or stimulatory effect on potential
parasites. They considered that resistance might be conferred
indirectly by physical effects on infection droplets. The chemical
composition of the cuticular surface may deter wetting and
minimize moisture retention, so affecting the establishment of
fungal parasites, most of which require persistence of moisture
films for several hours during germination and penetration.
The physical nature of the cuticular surface or chemical
emanations from particular sites may influence location of
points of entry for parasites. Thus Dickinson (1964) showed that
germ-tubes of a rust fungus can detect and grow across ridges
on membranes, and suggested that they may do the same on leaf
surfaces as they locate stomata. A few attempts have been made
to assess the importance of phenomena of these types in resis-
tance. Appressoria of Colletotrichum lindemuthianum commonly
form on those parts of the cuticle immediately above vertical
walls in underlying epidermal cells, but they do this in a similar
way on both resistant and susceptible cultivars of bean (Skipp &
Deverall, 1972), thus discounting any special role in determining
infection type (Table 4).
12
TABLE 5. Relationship between zoospore association with stomata
and resistance of hop cultivars to Pseudoperonospora humuli as
measured by yield of sporangia {from Royle & Thomas,

Hop cultivars in Zoospores on stomata Sporangia per leaf

order of resistance after 16 hours (%) disc after 7 days

Ringwood Special 96.8 62600

Eastwell Golding 56.7 60600
1/63/37 76.3 18400
7k 491 78.4 4300
2L 118 9'-5 4000

Zoospores of Pseudoperonospora humuli are capable of detecting

and settling on positive 'Perspex' replicas of stomata on the
leaves of the hop Humulus lupulus, although relatively slowly.
Furthermore, Royle & Thomas (1973) showed that the zoospores
had the ability to move very rapidly towards open stomata on the
leaf in the light, apparently in response to a chemical stimulus
associated with photosynthesis. However, this chemotaxis to
stomata occurred similarly on both resistant and susceptible
cultivars of hop (Royle & Thomas, 1971), again discounting any
special role in regulating virulence and avirulence (Table 5).
Clearly there are many direct and indirect interactions
between parasites and aerial parts of plants, but there is not
much evidence to support the idea that the fate of avirulent
parasites is often determined by inability to establish infections
on resistant cultivars. However, relatively little is yet known of
the fate in nature of the many parasitic micro-organisms which
must alight on a wide variety of leaves and stems under favour-
able conditions for infection. Many may fail to attempt penetra-
tion because they are affected by anti-microbial compounds or
are disoriented by the physical contours on the cuticle'. Exuda-
tions from pollen can contribute to the susceptibility of plants to
some parasites, but again more research is needed to reveal the
general significance of this phenomenon to the ecology of the
leaf surface and to parasitism.
 RELATIONSHIPS BETWEEN VIRULENCE AND
PENETRATION INTO PLANTS
Entrance into plants via stomata is the most important or only
route for many bacteria and most downy mildew and rust fungi.
The possibility that failure to gain stomatal entrance might be an
important basis of avirulence has been considered by a number
of workers, particularly with rusts. Allen (1923) surveyed a
number of wheat cultivars resistant to Puccinia graminis f. sp.
tritici, and obtained clear evidence that one cultivar imposed an
impediment to entry of infection hyphae via stomatal apertures.
However, the same restriction was imposed on races virulent
and avirulent on that cultivar. Brown 8c Shipton (1964) meas-
ured the frequency of penetration of stomatal apertures of
many wheat cultivars by races of P. graminis f. sp. tritici. They
found remarkable differences in penetration on different cul-
tivars, but these were not related to the final infection type which
developed, as shown in Table 6. Perhaps the most remarkable
observations were that less than 2 % of the stomata contacted by
appressoria were penetrated in some cultivars, yet the rust was
able to grow relatively freely once successful entrance was
achieved. These results suggest that the combination by plant
breeding of other endogenous factors for resistance, with the
impediments imposed by some stomata to entrance, could make
major contributions to reduction of rust development in wheat.
The evidence of these studies with rust fungi is against the
idea that size of rust pustule is controlled by difficulties in
entering stomata. Effects of these difficulties on number of rust
colonies, as distinct from colony size and host reaction which
comprise infection type, do not appear to have been reported.
Assessment of ease of penetration of cuticles by the many
fungi which enter by this route is technically difficult, but some
pertinent experiments have been done with isolated cuticles and
with artificial membranes supplemented with extracts of cuti-
cles. Maheshwari, Allen & Hildebrand (1967) obtained cuticle
from Antirrhinum leaves and found that it supported normal
germination and development of appressoria and infection pegs
of several different rusts irrespective of their usual hosts. Yang
& Ellingboe (1972) performed a quantitative study of the
behaviour of powdery mildew fungi on surfaces of host and
non-host cereals, mainly wheat and barley Hordeum vulgare. The
14
TABLE 6. Relationship between host infection type and percentage of
successful penetration from appressoria on wheat varieties inoculated
with strain 2i-Anz-2 o/Puccinia graminis fsp. tritici (from Brown
& Shipton, ig64)

Variety Infection type Penetrations (%)

Steinwedel 3 19.26
Khapstein 1451 2 I
747
Einkorn 292 ;i 17.17
Khapli 12 ;i 15-59
Bobbin 3 9-23
Eureka 3 6.63
Emmer II ;i 6.36
Gabo 2-3 485
Celebration 3 4-77
Little Club 4 4.48
Mentana 3 4.01
Kanred 0 2.80
Gaza 3 1.85
C.I. 12632 1 1.83
Kenya 117A 2 J
-35
II-52-73 2 0.60
Mengavi 1 o-33

fungi produced appressoria freely on the non-host cereals and

also on host cereals, even in the presence of specific genes for
resistance to the fungal races tested. Similar results were
obtained using isolated cuticles, suggesting that there was little
involvement of surface contours or chemical composition in
resistance or susceptibility. Mere thickness of cuticle does not
necessarily present a barrier to infection; for example, the shrub
Euonymus japonicus has an exceptionally heavy cuticular mem-
brane and yet is highly susceptible to a powdery mildew fungus
which penetrates via the cuticle (Roberts, Martin & Peries, 1961).
However, the age of cuticles of the poplar Populus tremuloides
had a marked effect on the ability of the fungus Colletotrichum
gloeosporioides to penetrate and initiate infections (Marks, Berbee
& Riker, 1965), thus showing that some cuticles can present a
barrier to penetration. Most investigations based on quantitative
comparisons of behaviour on resistant and susceptible plants
support the conclusion of Martin (1964) that the contribution of
cuticle to resistance is slight compared with the contributions of
endogenous interactions after penetration. However, relatively
few comprehensive studies have been reported.
 Penetration of cuticles must be followed by penetration of the
epidermal wall by most fungi which enter through intact plant
surfaces, except Venturia spp. and Diplocarpon rosae which grow
between the cuticle and the epidermis. Observation of penetra-
tion points in epidermal walls, by electron microscopy, such as
that of McKeen (1974) studying Botrytis cinerea, suggests that this
is a neat process accomplished by localized enzyme action by the
fungus. Many fungal enzymes are known which can affect
different components of walls of plant cells (Wood, 1967). It
seems likely that these enzymes are attached to or secreted by
tips of infection hyphae during initial penetration of the epider-
mal wall. An intriguing hypothesis was advanced by Albershiem,
Jones & English (1969) to explain avirulence and resistance
based on interactions between fungal enzymes and components
of epidermal walls. The idea was inspired by (1) their observa-
tion that when grown on cell walls as a sole carbon source
parasitic fungi produce a sequence of different hydrolytic
enzymes and (2) their discoveries that cell walls contain many
different polysaccharides. Thus, a sequence of interactions was
postulated whereby a wall might be resistant if it released a sugar
suppressive to synthesis of a critical enzyme, or if it failed to
release a needed sugar. Knowledge of the existence of processes
in micro-organisms which can regulate synthesis of enzymes by
substrate induction and catabolite repression permits many
ingenious explanations of the mechanisms of resistance in the
cell wall. The hypothesis requires evidence that avirulent para-
sites stop growing while attempting to penetrate resistant cell
walls. Especially contradictory are the results of an examination
by Skipp & Deverall (1972) of the stage in the infection process
when avirulent races of Colletotrichum lindemuthianum fail in
their attempted parasitism of resistant bean cultivars, because
this interaction was the model used for development of the
hypothesis. Some difficulty was experienced in deciding, by use
of light microscopy, whether the fungus stopped before or after
penetration of the wall, because necrosis of the underlying cells
accompanied expression of resistance and the necrotic cells had
granular and brown cytoplasm. However, treatment of the
tissue with dilute alkali cleared the browning and granulation
from the dead cells and revealed that over 80% of these cells
contained short infection hyphae. A parallel study, using elec-
tron microscopy, by Mercer, Wood & Greenwood (1974) con-
16
firmed that hyphae were present inside the dead protoplasts of
some hypersensitive cells. Other comparisons of behaviour of
fungi in cells of resistant and susceptible cultivars of several
plant species have confirmed that hyphae usually grow equally
well through the first cell walls penetrated - for example, the
development of Phytophthora infestans on cut surfaces of potato
tubers. Hyphae from zoospores penetrated resistant cell walls
within a few hours from inoculation (Tomiyama, 1967) and
hyphal growth was noted to stop inside resistant cells several
hours later. Another series of observations concerned the stage
of the infection process when four different genes for resistance
in wheat to Erysiphe graminis f. sp. tritici expressed themselves.
Working with nearly synchronously developing inocula under
controlled environmental conditions, Slesinski 8c Ellingboe
(1969) found that none of the genes had any effect in the first 12
hours when appressoria formed, nor during the next few hours
when primary haustoria formed inside epidermal cells. Hyphal
growth stopped at later stages, as described in Chapter 3, again
indicating that critical events occurred after penetration of cell
walls.

CONCLUSION
Failure of parasitism may occur in some host-parasite interac-
tions during attempted penetration of cuticle and epidermal
walls as with Colletotrichum gloeosporioides in the poplar. These
penetrations are very difficult to observe and to measure quan-
titatively. However, in most other studies of early stages of
infection, failure of parasitism occurs at a later stage, at least
after entry into the first cell penetrated. The best-known
defence mechanisms of plants operate, therefore, after penetra-
tion into the cells, but it must be repeated that little is known of
the fate of many potential parasites in nature. Clearly, many
interactions are possible during the earliest stages of attempted
parasitism as indicated in this chapter. Further research might
reveal these interactions to be more important in the general
defence of plants than present evidence indicates.
 CHAPTER 3

Cytological Changes in Host and

Parasite after Infection
It must be clear from the previous chapter that the fate of most
potential parasites is decided after they have entered their host
plants. It is important, therefore, to know the location of para-
sites within and between cells, the stages during attempted
infection when parasitism succeeds or fails, and the associated
responses of host cells as revealed by light and electron micro-
scopy. Based on this knowledge, the significance of the numer-
ous physiological and biochemical changes in infected plants to
the processes of resistance and susceptibility can be assessed.

THE CELLULAR LOCATION OF PARASITES IN HOST TISSUES

There have been a limited number of quantitative studies on
related cytological changes in host and parasite during the
critical stages when resistance is expressed or parasitism is
established. Before considering these studies which are confined
to a few much analysed host-parasite relationships, it is valuable
to review the different ecological niches presented by plants and
the ways in which they are exploited by specialized micro-
organisms. In doing so, it is well to emphasize the types of
physiological interaction which might be anticipated between
the cells of parasite and host, and to appreciate the rarity of
direct contact of the protoplasts of the two organisms. The
parasitic protoplast is usually separated by its cell wall from the
host, and in many cases a host cell wall also separates the two
protoplasts.
Perhaps one of the most unusual niches is that between the
cuticle and the outer epidermal wall of leaves, which is exploited
by the mycelium of Venturia inaequalis and Diplocarpon rosae in
the apple Mains sylvestris and Rosa spp., respectively. Successful
growth of mycelium in this site is followed some time later by the
deterioration and discoloration of the underlying epidermal
18
cells, which are not directly contacted by the fungus (Nusbaum
& Keitt, 1938; Preece, 1963). Envisaged physiological interactions
with the host deciding the fate of parasitism might be with
components of cuticle and epidermal wall or, more remotely,
with the epidermal cells beneath.
The outer surface of many plants provides a site for the
growth of powdery mildew fungi in the genus Erysiphe. Contact
with epidermal cells is by means of haustoria, which are put
through the cuticle and epidermal wall. The protoplasmic mem-
branes of these cells are invaginated by the haustoria which
become large and develop finger-like lobes. The host protoplasts
are always separated from the fungal protoplast, not only by a
fungal wall but also by a sheath which may be a product of the
fungus and host or of the host alone (Bracker & Littlefield,
1973). Compatibility of haustoria and host protoplasts must be a
prerequisite for the development of powdery mildews, which
are biotrophic parasites. Not only can many possible inter-
changes be envisaged between the haustorium and the host cell,
but also there is evidence of a metabolic interaction between host
and parasite in the first hours of mildew development, before
the host is penetrated. Germinating conidia of Erysiphe, but not
of other organisms, affect the physiology of underlying wheat
leaves and cause stomatal closure (Martin, Stuckey, Safir &
Ellingboe, 1975). Some volatile or diffusible fungal metabolite
must pass into host cells at the very earliest stages of attempted
parasitism.
Many parasitic organisms exploit the intercellular spaces
between mesophyll cells of leaves as a habitat for growth. Thus,
most rust fungi grow from substomatal cavities by means of
intercellular hyphae which push haustoria into contacted
mesophyll cells. Suggestions that the young haustorium lacked a
fungal wall (Ehrlich & Ehrlich, 1963) and that direct contact
between rust and host protoplast occurred have been discounted
by recent examinations of the fine structure of the parasite
during cell penetration (Heath & Heath, 1971; Bracker &
Littlefield, 1973). Compatibility between the walled haustorium,
the surrounding sheath and the host plasma membrane again
seems essential for successful parasitism. Interactions are known
to occur between intercellular hyphae and host cells before wall
penetration and are quite common between young haustoria
and protoplasts.
Intercellular spaces in leaf mesophyll are the major sites for
the multiplication of many parasitic bacteria in the genera
Pseudomonas and Xanthomonas. Substantial population growth
occurs within hours of inoculation (Ercolani & Crosse, 1966)
when major differences between the multiplication rates of
avirulent and virulent organisms become apparent. Host cells
are not entered during this important period, and exchanges
with the bacterial cells must occur by diffusion of substances
through walls. Even when very low concentrations of avirulent
bacteria are introduced into intercellular spaces, host cells die
within a few hours (Turner & Novacky, 1974), whereas virulent
bacteria maintain a compatible relationship with host cells for
much longer periods.
Some parasitic fungi undergo most of their development
inside living host cells. Thus, Colletotrichum lindemuthianum
penetrates through the cuticle and produces large hyphae inside
successive epidermal and cortical cells of susceptible bean stems,
petioles and veins (Skipp & Deverall, 1972). Only the hyphal wall
separates the fungal protoplast from the host plasma mem-
brane, which is pushed aside as the hyphae grow through the
cells (Mercer, Wood & Greenwood, 1974). Harmony between
host and parasite must depend on the compatibility of the host
protoplast with the fungal wall and with substances secreted
through it.
Direct contact of host and parasite protoplasts occurs in the
relationships between plants in the Cruciferae and the plasmo-
dial parasites Olpidium brassicae (Lesemann & Fuchs, 1970) and
Plasmodiophora brassicae (Williams & McNabola, 1970). Very soon
after penetration from encysted zoospores of P. brassicae, the
parasitic amoebal cell in compatible host cells becomes bounded
by a multi-layer of membranes. Williams, Aist & Bhattacharya
(1973) have speculated that the fate of parasitism might be
decided within moments of introduction of the amoeba into the
cell.
Successful development of vascular parasites such as Fusarium
oxysporum and Verticillium spp. depends upon their ability to
penetrate and grow through root tissues until they reach the
lumen of xylem vessels. They colonize these non-living vessels by
mycelial growth and liberation of conidia which are carried in
the transpiration stream through perforation plates into adja-
cent vessels. Interactions with plant tissues affecting the success
of parasitism could occur at any stage before entry into xylem
20
vessels and, thereafter, with components of vessel walls and
possibly with the live parenchyma cells adjacent to vessels
(Beckman, 1971; Talboys, 1972).
Those organisms, which are necrotrophic for most phases of
their parasitic development, probably kill host cells by toxic
secretions and thus rarely contact living cells. Fungal and bacter-
ial parasites which cause soft-rot diseases in parenchyma of
tubers and fruit are wound entrants and then producers of
enzymes which separate and kill host cells (Tribe, 1955;
Garibaldi & Bateman, 1970; Stephens & Wood, 1975). In return,
metabolites may diffuse from responding live host cells through
the dead tissue to affect the parasites. Similar secretions might
also emanate from host cells ahead of the necrotrophic leaf-spot
fungi which destroy cell walls and protoplasts in their immediate
vicinity. Thus, much less immediate interactions between the
necrotrophic parasites and their hosts might be anticipated, in
comparison with the many parasites which are biotrophic for
much of their lives.

CESSATION OF GROWTH OF PARASITES IN

RESISTANT PLANTS
Answers to the questions of where and when parasites stop
growing in resistant plants have been provided in varying detail
for different types of parasite. Greatest precision has been
achieved by EUingboe and his colleagues, in their analysis of the
fate of the powdery mildew fungus Erysiphe graminis f. sp. tritici
in wheat cultivars bearing different single genes for resistance.
As discussed by EUingboe (1968, 1972) this work was organized
so that the effects of genes conferring resistance or susceptibility
in the plant and avirulence or virulence in the fungi could be
discerned under controlled environmental conditions. These
conditions were not only controlled for the growth and infection
of the wheat but also for the production of the conidia used as
inocula. Thus, successive experiments could be conducted to
relate physiological changes to observed morphological events.
Furthermore, within any experiment, the development of all
conidia was almost synchronous. The results showed that the
different genes controlling the host-fungus interaction had no
effect in the first 12 hours, when conidia germinated and pro-
duced appressoria (Slesinski & EUingboe, 1969; Stuckey &
21
Ellingboe, 1974). Primary haustoria were produced similarly in
the different interactions. Some of the genes for resistance
caused the parasite to stop growing within the next 18 hours, but
other genes acted less effectively or at much later stages. Cessa-
tion of hyphal growth required the presence of complementary
genes for avirulence in the fungus and resistance in the host, as
predicted from the gene-for-gene hypothesis (Slesinski & Elling-
boe, 1970). Thus the parasite-host genotype P^IPmq. proved to
be the most effective, causing the collapse of about 95 % of the
secondary hyphae elongating from appressoria 22 hours from
inoculation, when host cytoplasm discoloured around the haus-
toria. Next in effectiveness was the Pi/Pmi genotype, which
caused about 80 % of secondary hyphae to cease growth 26 hours
from inoculation, but without associated discoloration in penet-
rated host cells. Less effective was the P^a/Pm^a genotype which
allowed at least 30 % of secondary hyphae to continue to elon-
gate after 26 hours. The P2JPma genotype was effective only
after several days had passed, when host cells died and only a
limited crop of conidiophores was produced. These elegant
studies demonstrate that different genotypes for low infection
type act in different ways and at different stages of the
host-parasite interaction. Molecular explanations of the differ-
ent phenomena are not yet available, but the cytological studies
have been followed by analyses of the ways in which different
parasite and host genes affect the transfer of 35S from the plant
to the mildew mycelium (Slesinski & Ellingboe, 1971; Hsu &
Ellingboe, 1972).
It is more difficult to follow the progress of parasites which
penetrate deeply in plant tissues, unlike the members of the
genus Erysiphe. Particularly difficult problems are presented by
the rust fungi because it is necessary not only to record the
growth of intercellular hyphae but also to note the stages of
hyphal growth when contact is made, via haustoria, with cells
within leaves and stems. The possibility exists that haustorial
penetration might occur more frequently in some host-parasite
interactions than in others, and that it might be unrelated to the
spread of intercellular hyphae. The earliest investigators (Ward,
1905; Stakman, 1915) observed that a very common result of the
contact of rust hyphae with cells of resistant hosts was the rapid
death of the cells and cessation of further growth of the hyphae.
The rapid death of resistant cells was termed hypersensitivity by
22
 Stakman. Although Stakman made no implications concerning
the sequence and nature of physiological factors which resulted
in the failure of further hyphal growth, it is clear that any
analysis of the time course of events in resistant plants must
record the hypersensitivity of host cells, in addition to the
penetration of these cells from intercellular hyphae. Hyper-
sensitivity of plant cells has been recognized to be commonly
associated with resistance of plants to many fungi (Miiller, 1959)
and bacteria (Klement & Goodman, 1967). The processes
involved in the initiation of hypersensitivity and in the limitation
of potential parasites are matters of lively debate and investiga-
tion, as discussed in later parts of this book.
The time in the infection process when the development of
rust hyphae stops in resistant plants has been estimated in
several ways. Ogle & Brown (1971) cleared and stained whole
leaves of cereals and measured areas of mycelial colonies of
Puccinia graminis f. sp. tritici by microscopic examination
through surfaces. They concluded that rust growth stopped
within 48 hours of inoculation in highly resistant wheat cultivars
and in other cereals. They also observed that the area of these
limited colonies was exceeded by an area of necrotic host cells.
Skipp & Samborski (1974) followed the progress of one race of
this rust in isogenic lines of wheat, differing only in the presence
or absence of the Sr6 gene governing resistance to this race at
20 °C. In general, they confirmed that most rust hyphae grew
little more after the second day in the presence of the gene for
resistance. However, they observed that the first haustoria were
produced in a similar way in resistant and susceptible lines 16 to
20 hours after inoculation. Differences between the lines became
apparent several hours later, when some hypersensitivity occur-
red in resistant mesophyll cells. Differences in hyphal growth
became noticeable 36 hours after inoculation and substantial 24
hours later. Although growth continued freely in the susceptible
line, growth was restricted in resistant tissue where necrosis was
visible. The problems presented for an analysis of the sequences
of the physiological processes that control these events are
emphasized by their observations that infection sites behaved
differently, even in a resistant leaf. For example, epidermal cells
never underwent hypersensitivity although penetrated by haus-
toria; some mesophyll cells became necrotic rapidly, possibly
without haustorial penetration; and other mesophyll cells did
23
 not become necrotic until several neighbouring cells contained
haustoria.
Another way of attempting to decide when the process of
resistance is complete, following rust infections, is through man-
ipulation of the sensitivity of expression of the Sr6 gene to
temperature. This gene confers low infection type when plants
are grown and incubated at 20 °C but high infection types at
26 °C. Antonelli & Daly (1966) found that transfers of plants
from one of these temperatures to the other, at any stage within
three days from inoculation, caused the development of the
infection type normally characteristic of the final temperature.
This suggested that metabolic processes regulating the infection
type are not effective until three days after inoculation. Trans-
fers at stages after this period caused the development of
intermediate infection types. When Skipp & Samborski (1974)
did similar experiments and observed histological changes dur-
ing the three-day period, they noted that host cells responded
within 10 to 20 hours to a change in temperature. Thus, cellular
necrosis and accompanying metabolic changes can occur at very
early stages in the infection of resistant leaves. The results of
these experiments and observations show that rust hyphae are
not killed in necrotic tissue. Although haustoria and subtending
hyphae die, growth can resume from a more remote mycelium
when appropriate changes in temperature are made. Skipp &
Samborski (1974) also deduced that the temperature depen-
dence of the expression of the Sr6 gene probably resided in the
wheat plant, because they found that the temperature at which
plants were grown before inoculation influenced the sensitivity
of cells to the rust. Some cells in plants grown at 20 °C became
necrotic when inoculated and incubated at 25 °C, unlike those in
plants kept at 25 °C throughout. Knowledge of these visible
cellular reactions should aid interpretation of experiments on
metabolic changes, as resistance conferred by the Sr6 gene is
expressed. The relationship between hypersensitivity and rust
development will be considered again later.
Although not the natural sites for infection by fungi, the
interiors of legume pods and the fruit of the pepper Capsicum
frutescens have often been used to study physiological interac-
tions with different fungi, for the reasons explained in Chapter
5. It is therefore useful to note here the types of cytological
change which take place in pepper fruit cavities following infec-
24
tion by some virulent and avirulent parasites (Jones, Graham &
Ward, 1974, 19750, b). Introduction of zoospores of the virulent
fungus Phytophthora capsici caused rapid responses in the under-
lying cells which produced many ribosomes and showed nuclear
changes within four hours. Chloroplasts were beginning to
degenerate at this time, and two hours later the cytoplasms of
these cells were disorganized as the hyphae penetrated deeper
into the tissue. Newly approached cells also became ribosome-
dense before degenerating after penetration. Thus, this virulent
parasite seemed to reactivate cells before killing them and grow-
ing on apparently unchecked. Greater incompatibility with pep-
per cells was shown by P. infestans which killed the first cells
penetrated within four hours. The fungus spread into two or
three layers of cells which died after penetration. Adjacent
uninvaded cells became ribosome-dense, and the fungus was
limited within 36 hours. Hyphal death and disintegration were
observed to follow host cell death. The soft-fruit parasite
Monilinia fructicola was highly incompatible with pepper because
it only succeeded in penetrating a few cells, yet caused wide-
spread host necrosis ahead of the hyphae. This study shows that
two types of cellular response could be distinguished, namely
rapid necrosis and slower death often preceded by an apparent
reactivation of the ribosomal apparatus for synthesis.
Phytophthora capsici, unlike the other fungi studied, was able to
colonize pepper fruit despite its incompatible relationship with
penetrated cells.

THE ASSOCIATION OF HYPERSENSITIVITY

WITH RESISTANCE

A recurrent theme in many investigations on resistance of plants

to rust parasites and many other parasites is the close association
between hypersensitivity and resistance. Thus it is quite likely
that resistance results from the hypersensitive death of the host
cell, perhaps because the haustorium within the host cell is
starved when the protoplast dies, or even killed by toxic sub-
stances in the dead cell. However, the idea that hypersensitivity
is a cause of resistance has been challenged in several ways in
recent years.
One way results from extensive analyses of the growth of
black stem rust Puccinia graminis f. sp. tritici in a range of wheat
3 25 DDM
 cultivars by Brown, Shipton & White (1966) and by Ogle &
Brown (1971). The essence of the argument was that three
categories of host reaction could be recognized on the basis of
rust growth and host necrosis, namely resistant, susceptible and
intermediate. In the resistant reaction, mentioned in the previ-
ous section, the small rust colonies were exceeded by areas of
necrotic tissue. In the susceptible reaction, rust colonies became
very large and host necrosis was negligible. These two observa-
tions were consistent with the idea that necrosis caused limita-
tion of rust growth. However, in the intermediate reactions,
moderate colony areas exceeded very variable areas of host
necrosis; this implied that the necrosis had not prevented rust
growth and was the basis for the suggestion that resistance was
not caused by death of the host cells.
It would be most interesting if some of these intermediate
reactions could be examined for the sequence of interactions
between hyphae, haustoria and host cells in the first stages of
infection, as done by Skipp & Samborksi (1974) for the resistant
reaction mediated by the Sr6 gene. A low sensitivity of cells to
haustorial penetration in some intermediate reactions might
have resulted in late necrosis and permitted considerable hyphal
growth compared with the resistant types. One of the inter-
mediate reactions was in the cultivar Einkhorn, which had also
been regarded as distinctive by Stakman (1915) because rust
growth was limited despite the absence of any obvious cellular
reaction. Clearly, in this cultivar, a process quite unrelated to
cellular necrosis limits rust development. The observations on
the remaining intermediate reactions indicate nicely the need
for further histological investigations. They also draw attention
to the deficiencies in knowledge of fungal growth and host
response in plant cultivars intermediate in reaction to other
parasites, particularly in cultivars with so-called * field' resis-
tance.
The role of hypersensitivity in resistance of wheat to Puccinia
graminis f. sp. tritici has also been brought into question by
observations on host cell death, as measured by fluorescence
microscopy, in plants possessing the Sr6 gene (Mayama, Daly,
Rehfeld & Daly, 1975). The number of fluorescent sites per unit
area of leaf increased in the same way irrespective of whether a
low infection type was being produced at 20 °C, or a high
infection type at 26 °C. Thus, substantial uredial formation
26
 occurred at 26 °C despite the occurrence of the same pattern of
cell death in response to haustorial penetration as at 20 °C.
Assuming that the technique of detecting fluorescent cells
records cells that died in response to infection, this study implies
either that cell death per se has no effect on rust development or
that some rust-limiting process linked to cell death does not
function at 26 °C.
Another way in which hypersensitivity as a cause of resistance
is questioned, arises from experiments performed by Kiraly,
Barna & Ersek (1972). They used chemical agents to stop fungal
growth in susceptible plants, and then observed responses in
host cells around the infection hyphae. For example, chloram-
phenicol prevented the growth of a compatible race of
Phytophthora infestans in potato tubers which, in turn, caused
hypersensitivity of the tuber cells. Similarly, necrosis was
induced in wheat leaves inoculated with a compatible race of
Puccinia graminis, and in bean leaves inoculated with Uromyces
phaseoli after treatment with fungal inhibitors. The implications
of this work are that fungal growth in resistant plants is stopped
by an unknown factor, and that the inhibited fungus then
releases a toxic substance which kills the host cells. The latter
possibility was supported by evidence that a killed mycelium of
Phytophthora infestans released a fluid in vitro which induced
hypersensitivity when introduced into potato tissue. These
experiments lead to the conclusion that hypersensitivity can be a
consequence and not a cause of resistance.
The apparent force of these arguments is much decreased by
observations that, in a number of host-parasite interactions,
fungal hyphae continue to grow, albeit increasingly more slowly,
in plant cells after these cells have undergone hypersensitivity.
For example, Tomiyama (1955) observed the growth of incom-
patible hyphae of Phytophthora infestans inside potato cells for
several hours after the cells had become necrotic. Growth even-
tually stopped before the hyphae passed into a second cell.
These observations are supported by a study of the ultrastruc-
ture of the interaction between this fungus and potato leaves
(Shimony & Friend, 1975). Penetrated and adjacent epidermal
and mesophyll cells of resistant leaves died within 9 to 12 hours
of inoculation, but the hyphae were not killed until several hours
later. Skipp & Deverall (1972) measured hyphal lengths in
'marked' cells in excised bean tissue mounted on microscope
27 3-2
slides through an 18-hour period, when the cells were beginning
to undergo hypersensitivity to an incompatible race of Colleto-
trichum lindemuthianum. Growth rates of hyphae inside resistant
cells before symptoms were detected ranged up to 9 /jm/hour,
but were lower in cells where cytoplasm was becoming granular
as part of the necrotic reaction. However, slow hyphal growth
was detected in cells which had become pale brown in colour.
This was supported by other observations using whole
hypocotyls which indicated that some hyphae remained alive
and grew in brown, apparently dead, cells, although they did not
grow out of these cells. This suggests that a progressive inhibi-
tion of fungal growth follows hypersensitivity of the host cell.
This traditional view of the sequence of events in hypersensitive
cells is supported by the report by Maclean, Sargent, Tommerup
& Ingram (1974) that the membranes and cytoplasm of resistant
cells of the lettuce Lactuca sativa, viewed beneath the electron
microscope, became severely disrupted four hours after penet-
ration by the downy mildew Bremia lactucae. At this time, fungal
cytoplasm appeared normal. The fungus continued to grow in
the cell for another 12 hours before dying. The details of
observed changes in hypersensitive cells do not, therefore,
support the conclusions, based on the experiments performed
by Kiraly et al. (1972), that hypersensitivity is a consequence of
resistance having been expressed earlier by unknown factors.

OTHER CELLULAR REACTIONS OBSERVED TO BE

ASSOCIATED WITH RESISTANCE
Hypersensitivity is not the only visible cellular process associated
with resistance. Reports have shown that other reactions can
occur, and a good example is that of the enclosure of rust
haustoria by a particularly dense sheath. Electron microscopy on
sections of cells of the cowpea Vigna sinensis resistant to the
cowpea rust Uromyces phaseoli f. sp. vignae reveal at least two fates
of incompatible haustoria, namely collapse in hypersensitive
cells and encapsulation by very thick sheaths in otherwise
unaffected cells (Heath & Heath, 1971). Similar studies by Heath
(1974) show that a diversity of cellular responses to cowpea rust
can occur in cowpea cultivars and other plant species, some
apparently deterring haustorial formation and others affecting
the haustorium in the penetrated cell. In addition to hyper-
28
sensitivity and encapsulation, two other phenomena visible by
electron microscopy were deposition of osmiophilic material on
the inside of a cell wall opposite to an intercellular hypha, and
poor adhesion of haustorial mother-cells and plant cell walls. As
a result of this survey, Heath (1974) envisaged that parasitism by
a rust can be regulated by a series of * switching-points', and that
the result at each determines whether the fungus can progress
to the next. The greater the compatibility between parasite and
host, the further the rust progressed so that in a susceptible
plant, no signs of deleterious reactions to the rust could be
discerned.

CONCLUSION
Recent research reveals that growth of parasites can stop at a
number of stages inside the tissues of resistant plants, and
different types of processes are likely to be involved at these
different stages. Most studies have concerned the more complete
forms of resistance where infection stops within one or two days
before the parasite has progressed far. Although there are some
contrary indications, processes accompanying hypersensitive
host cell death often seem to be related to the expression of
rapid resistance to incompatible races, but other imperfections
in the host-parasite interface can occur. Relatively little informa-
tion is available on the progress of parasites inside plants which
are intermediate in reaction type or which possess so-called
* field' resistance. However, one excellent example was provided
of the delayed expression of a gene-for-gene interaction causing
incompatibility and low infection type after a period of compati-
ble growth of powdery mildew in wheat. This suggests that
genetically determined incompatibility can occur at any stage in
the ontogeny of the interaction between host and parasite.

2
9
 CHAPTER 4

Cross-Protection and Induced Resistance

Cross-protection is the phenomenon whereby a plant is pro-

tected from infection by earlier or simultaneous exposure to
another organism. This chapter will be concerned with protec-
tion against fungal and bacterial parasites as a result of infection
by related and unrelated fungi and bacteria and of local lesion
formation by viruses. It will not be concerned with the interfer-
ence of the development of viruses by related or unrelated
viruses (Matthews, 1970).
For a long time people have been interested in the idea that
plants, like animals, can acquire physiological immunity to
pathogens. Research, mainly in Europe in the late part of the
nineteenth century and in the early decades of this century, was
inspired by observations which suggested that some perennial
plants were less severely affected by a disease following earlier
infection. For example, the varied development of powdery
mildew infections on the foliage of oak trees Quercus spp. in
successive seasons had intrigued early investigators. Experi-
ments had also been performed which seemed to show that
herbaceous plants became more resistant to infection after the
plants or the soil bearing them were sprayed with the extracts of
fungal cultures. Numerous observations and claims of this type
were reviewed by Chester (1933) who criticized the lack of
sufficient replication for proper appraisal of the experiments
and the frequent inadequacy of controls. Sources of error which
had rarely been eliminated before drawing conclusions were
losses of virulence in the parasites during experiments, differ-
ences in environmental conditions on successive occasions for
assessing resistance, and natural increases in resistance as plants
aged during the periods of supposed acquisition of resistance.
From all of this work, there remain possibilities, requiring
substantiation, that trees or new branches are less susceptible
after infection of earlier growth and that antibiotics released by
3°
 protecting organisms might be responsible for some of the
effects.
The early work with the most convincing outcome followed
that of Bernard on the interactions between germinating
orchid seed and fungi, leading to the development of functional
seedlings with mycorrhizal roots. Some fungi destroyed the
seed, some did not survive attempted infection of the seed and
others entered into association with the roots of the seedling to
form mycorrhiza. Using excised orchid embryos, Bernard (1909)
found that one of these types of fungi penetrated several layers
of cells before it stopped growing and disintegrated. Attempts to
re-infect the embryo with a normally destructive fungus were
then unsuccessful, suggesting acquisition of resistance in the
embryo. Further experiments gave an indication of the involve-
ment of antifungal compounds arising from the orchid tissue.
Bernard (1911) plated a piece of surface sterilized tuber of the
orchid Loroglossum hircinum a short distance from the pathogen
Rhizoctonia repens on solid culture medium. After beginning to
grow in all directions, the fungus became inhibited as it
approached the orchid tissue but before it reached the tuber.
Although these tuber fragments were fungistatic, it was not
possible to detect any activity in extracts of crushed fresh tubers.
Fragments heated at 55 °C for 35 minutes were also inactive.
Nobecourt (1923) confirmed these results and found that tuber
fragments which had been frozen and thawed or exposed to
chloroform vapour also did not inhibit the growth of the fungus.
He thought it unlikely that such diverse treatments as exposure
to heat, cold and chloroform would destroy a pre-existing
fungistat in the tissue. The fact that all three treatments killed
the tissue led Nobecourt to suggest that they prevented the
tissues from synthesizing the fungistat in response to substances
diffusing from the fungus. Magrou (1924) disputed this
interpretation, and showed that sterilized pieces of tuber incu-
bated alone on medium for two weeks exuded a fungistat into
the medium. Although it was clear therefore that orchid tissue
liberated an antifungal material under some circumstances, it
was uncertain whether Magrou had detected the same material
as Nobecourt and Bernard. If it was the same material, there was
no indication of its quantity in the two types of experiment.
Furthermore, considerable doubt existed about the nature of
the stimulus required for its formation and/or liberation; cutting
31
the orchid tissue might have been the essential stimulus. These
important questions were unresolved in 1933 and remained so
until the work of Gaiimann and his associates, which led to the
discovery of a number of antifungal compounds produced by
different orchid tubers in response to infection, as discussed in
Chapter 5.
The next important and extensive investigation on acquired
resistance was that of Miiller & Borger (1941) on the interactions
of potato varieties with different fungi, particularly with races of
Phytophthora infestans. An essential basis for these experiments
was Miiller's earlier work on the selection of potato varieties
resistant to P. infestans, which also led to the detection and
collection of virulent and avirulent races of this fungus.
Miiller & Borger showed that treatment of cut surfaces of
potato tubers with an avirulent race prevented the development
of a virulent race inoculated a day later. Cut tubers, aged for
several days in humid chambers in the absence of the avirulent
race, retained their susceptibility to the virulent race. The pro-
tection was total when one or two days elapsed between succes-
sive inoculations but was recognizable only by weaker mycelial
growth when the intervals between inoculations were as short as
one hour. The protection was also localized, being confined to
the area of the tuber surface treated with the avirulent race, and
was apparently non-specific, being effective against an isolate of
Fusarium normally pathogenic towards potato tubers. Thus, by
means of these experiments, localized non-specific protection
was demonstrated in potato tubers.
There are now several recent reports of systemic cross-
protection (e.g. Kuc, Shockley & Kearney, 1975) to set beside the
many reports of localized and non-specific protection against
both fungal and bacterial pathogens. Thus, for example, avirul-
ent rust fungi will protect against virulent rusts on leaves and
stems of several plants (Littlefield, 1969; Kochman & Brown,
1975). Avirulent races of Pseudomonas spp. will protect against
virulent races in intercellular spaces of plants (Averre & Kel-
man, 1964). Avirulent isolates of Fusarium oxysporum protected
against virulent isolates in experiments with seedlings in test
tubes, under conditions which would seem greatly to have
favoured the development of a general parenchymatous infec-
tion by the pathogen, even if not a characteristic vascular infec-
tion (Davis, 1967). Selected reports will be referred to in the
32
remaining parts of this chapter where they make a contribution
to understanding the different possible modes of action of
protection.

MODES OF ACTION OF CROSS-PROTECTION

There are two distinct ways in which protection may be brought
about. Firstly, the protectant organism could act directly on the
normally pathogenic organism by physical impedance or chemi-
cal antagonism. Secondly, the protectant could activate a
physiological change in the host plant so that it becomes resis-
tant. Such induced resistance would be of greatest consequence
to the main theme of this book, but it is first desirable to consider
the evidence that induced resistance can be distinguished from
direct interference.

DIRECT ACTION OF THE PROTECTANT

AGAINST THE PATHOGEN
One of the most likely ways in which a protectant organism can
be effective is by blocking the stomatal sites of entry for the
normal pathogen. The most important stomatal entrants for
which cross-protection has been shown are the rust fungi. After
demonstrating localized protection of wheat leaves against Puc-
cinia recondita by treatment with the oat rust P. coronata f. sp.
avenae, Johnston & Huffman (1958) suggested that the stomata
may have been blocked by the protectant. However, in the
course of experiments in which he achieved marked localized
and non-specific protection against the flax rust Melampsora lini,
Littlefield (1969) calculated that insufficient stomatal apertures
would have been blocked by the low concentrations of uredo-
spores which were effective in causing protection. Evidence that
cross-protection between rusts is sometimes caused by blocking
sites of entry was provided by Kochman & Brown (1975) who
observed some occlusion of stomata on oat leaves by the appres-
soria of wheat rusts used as protectant in their experiments.
Johnston & Huffman (1958) also suggested that self-inhibitors
released by the uredospores of oat rust may have been effective
in preventing infection by wheat rust. It is well known that many
uredospores release inhibitors of germination into surrounding
fluids (Allen, 1955). Yarwood (1954) also showed that volatile
33
emanations from rusted bean and Antirrhinum leaves inhibited
germination of uredospores and impaired development of other
rust infections. Yarwood (1956) compared the efficacy of uredo-
spores with fungicides as protectants against rusts. Thus he
showed that 1-3 mg dry weight of uredospores of bean rust per
dm2 of the leaf of the sunflower Helianthus annuus gave 50%
control of uredial production by the sunflower rust Puccinia
helianthi. Conversely, 4 mg of uredospores of sunflower rust
gave similar control of bean rust. The effect was considered to be
caused by self-inhibitors. However, unless self-inhibition is
much stronger between uredospores of different species than
between those of the same species, it is surprising that there was
no self-limiting effect of increasing the dosage of a pathogenic
rust alone to 10 mg/dm2. This inoculum gave rise to so many
uredia that it was impossible to count them. Thus there is reason
to doubt that inhibition of spore germination by spore products
was the cause of protection in the earlier experiments. Several
self-inhibitors have now been characterized (Macko, Staples,
Allen & Renwick, 1971; Macko, Staples, Renwick & Pirone,
1972); bean rust, sunflower rust and some strains of wheat rust
uredospores have been shown to be sensitive, and flax rust
uredospores insensitive, to methyl-3,4-dimethoxycinnamate
released from uredospores of the sunflower and Antirrhinum
rusts. It should be possible now to assess the extent to which
self-inhibitors could cause cross-protection between rusts.
Littlefield (1969) observed that germination of flax rust was
unimpaired by the protecting rust on flax leaves, thus showing
clearly that self-inhibitors were not involved in his experiments.
Direct chemical antagonism by a protective organism is likely
to be involved in the prevention of crown-gall development
Agrobacterium tumefaciens by treatment of susceptible plants with
the non-pathogenic A. radiobacter. This treatment is the basis of
a practical control of crown-gall disease in South Australia
(Kerr, 1972; New & Kerr, 1972). The mode of action is thought
to be through the secretion of a specific antibiotic, a bacteriocin,
by the protectant bacterium. Kerr & Htay (1974) showed that
many strains of A. tumefaciens were prevented from causing
crown-gall when they were inoculated into wounds in tomato
stems in 1:1 mixtures with strain 84 of A. radiobacter. Growth of
all these strains of A. tumefaciens was inhibited on nutrient agar
plates around spots where strain 84 had previously been allowed
34
to grow for two days before being killed with chloroform. Thus,
it seems likely that protection in tomato is caused by a similar
inhibition of growth of A. tumefaciens. Diminished growth of one
pathogenic strain in tomato was demonstrated, but it is desirable
to show that the bacteriocin is produced in vivo by the protectant
strain. An alternative hypothesis to explain protection has been
suggested, namely competition for sites for bacterial attachment
or action within host tissues (Lippincott & Lippincott, 1969).
Both direct chemical antagonism by bacteriocins, and exclu-
sion from infection sites, were considered unlikely explanations
by Garrett & Crosse (1975) for the similar suppressive effect of
plum strains of Pseudomonas morsprunorum and some other
Pseudomonas spp. on the development of leaf-scar infections
and cankers in the cherry Prunus avium caused by the cherry
strains of this organism (Crosse & Garrett, 1970). There was
no relationship between the suppressive action of Pseudomonas
spp. in cherry and their antagonistic effects on the cherry strain
by lysogeny or bacteriocin production in vitro. Furthermore,
related Pseudomonas spp. which might be expected to compete
for infection sites in cherry had no effect on canker develop-
ment by the cherry strain. The common feature of the strains
and species of Pseudomonas which suppressed canker develop-
ment was their ability to induce hypersensitivity in non-
host plants. Thus, Garrett & Crosse (1975) suggested that
avirulent strains inhibited virulent strains as a result of induction
of hypersensitivity in the host, in agreement with conclusions
by Averre & Kelman (1964) about a similar phenomenon
revealed in studies with P. solanacearum.

INDUCED CHANGES IN THE HOST PLANT

There are several types of evidence that cross-protection can
result from a change in the physiology of the host plant. The
first arises from the studies of Miiller & Borger (1941) on
cross-protection in tubers against potato blight. When a thin
layer of tuber tissue was excised beneath a surface treated a day
earlier with an avirulent race of Phytophthora infestans, the under-
lying tissue retained its resistance to a virulent race. This elimi-
nated physical hindrance as a cause of the protection but left the
possibility that an antibiotic had diffused from the avirulent
race. However, the occurrence of direct chemical antagonism
35
between the avirulent race and the virulent race was eliminated
by the results of an experiment in which a mixture of the races
was inoculated into a potato cultivar susceptible to both races. A
large crop of sporangia was produced, and a dilution series of
these sporangia was inoculated onto differential cultivars of
potato. By this means, both races of P. infestans were recognized
in equal amounts among the sporangia. The avirulent race
therefore exerted its action on the virulent race only in resistant
host tissue, implying that it activated a host process to become
effective against the normally virulent race.
A second type of evidence comes from direct observations of
the behaviour of protectant and pathogenic spores and germ-
tubes during the infection process. Littlefield (1969) working
with flax rust and Skipp & Deverall (1973) with bean anthracnose
saw no effect of avirulent spores on normal germination and
penetration by virulent spores. Their conclusion was that pro-
tection was achieved after entry of both types of fungus into the
host plant, and that there was no direct interference between the
fungi.
Striking evidence of a change in host physiology leading to
resistance to a fungal pathogen is provided by work with tobacco
and a strain of tobacco mosaic virus (TMV) which caused local
lesion formation. Local lesion formation on one side of a tobacco
leaf made the opposite side of the leaf much more resistant to
the fungal pathogen Thielaviopsis basicola (Hecht & Bateman,
1964). Strains of virus which did not cause local lesions had no
effect on fungal development. This is not the only report of
remote cross-protection in tobacco, because field observations by
Pont (1959) prompted Cruickshank & Mandryk (i960) to investi-
gate the decrease of foliar infection by Peronospora tabacina as a
result of earlier stem infection by the same fungus. When
conidia were injected into the stub of a cut petiole near the base
of tobacco stems, the upper foliage became completely resistant
to sporulation of the downy mildew sprayed onto the leaves
under optimal test conditions four weeks later. This remarkable
result is put into better perspective when it is realized that the
stem-infected plants became prematurely senescent compared
with the water-injected controls. The leaves on the protected
plants showed signs of senescence, and the plants matured and
flowered two weeks before the controls. Thus it is not clear
whether the resistance of the leaves was caused indirectly by
36
their premature senescence. These varied phenomena in
tobacco are open to several other interpretations such as com-
petition between infection sites and the formation of systemi-
cally mobile antifungal substances. The value of tobacco as a
worthwhile subject for deeper investigation of induced resis-
tance is increased still further by the discoveries of Lozano &
Sequeira (1970) of its response to heat-killed bacteria to be
discussed later.
Remote cross-protection, as distinct from the often demon-
strated localized form, has also been claimed in recent research
with anthracnose diseases of bean and cucurbits and with bacter-
ial diseases of pome fruit. Avirulent races of Colletotrichum
lindemuthianum caused sites 5 mm away on etiolated bean
hypocotyls to become resistant to virulent races (Elliston, Kuc &
Williams, 1971). This implies that some factor is translocated in
etiolated tissue to induce a change at a distance of numerous
cell-widths, although it must be noted that Skipp & Deverall
(1973) were unable to achieve similar results on green
hypocotyls. More remarkable, is the demonstration that infec-
tion of the first leaf of the cucumber Cucumis sativus with
Colletotrichum lagenarium renders the younger foliage resistant
to infection by the same fungus when this is applied again one or
more weeks later (Kuc, Shockley & Kearney, 1975). Direct inter-
ference of one inoculum with the next is clearly impossible and
induction of premature senescence of the plant seems unlikely
in the time involved and from the appearance of the plants.
Thus, the activation of an uncharacterized process of resistance
throughout the plant is implied. Also largely unexplained is the
means by which application of preparations containing DNA
from the bacterium Erwinia amylovora, to roots or cut shoots of
seedlings of the pear Pyrus communis, protects these seedlings
from subsequent infection by the bacterium (Mclntyre, Kuc &
Williams, 1975).

THE NATURE OF THE INDUCED CHANGE IN THE HOST PLANT

A simple explanation of the types of change in host plants which
increase their resistance would be the diffusion of anti-microbial
compounds from the host tissues at the sites of inoculation with
the protectant organism. In fact, the original concept of
phytoalexins was that they were produced when cells became
37
necrotic in hypersensitive responses to avirulent fungi (Miiller,
1958). This idea had arisen during earlier work on cross-
protection in potato (Miiller & Borger, 1941); a hypothetical
antifungal principle was conceived to diffuse out from hyper-
sensitive cells to prevent development of virulent races of the
potato blight fungus. The formation of phytoalexins in and
around necrotic cells in a number of plant families has now been
demonstrated clearly, as discussed in Chapter 5. However, there
is no evidence that any of the known phytoalexins diffuse from
sites of formation, although there is a demonstration of
phytoalexin formation in live cells next to dead cells, as discus-
sed in Chapter 5. There is no reason as yet, therefore, to assume
that cross-protection is caused by diffusion of phytoalexins over
the distance of even several cell widths.
As a matter of speculation, it is quite possible that phytoalex-
ins with appropriate solubility might be able to diffuse through
necrotic cells, where barriers to permeability and movement of
molecules have been destroyed. Furthermore, systemic fungi-
cides must be able to move for considerable distances in healthy
plant tissue. It is possible that some of the known phytoalexins
may be able to move through the same routes taken by systemic
fungicides, but this has not been investigated. What is not
possible at present is to give any evidence that the known
examples of cross-protection are caused by the movement of
phytoalexins. The remote cross-protection in cucumber discus-
sed in the previous section seems most unlikely to be mediated
by phytoalexins because of the failure to detect such compounds
in the Cucurbitaceae, as described in Chapter 5.
There are at present stronger indications of other types of
translocated change, involving the diffusion of unknown sub-
stances from cells affected by the protecting organism to nearby
cells, thus causing a change in sensitivity of these cells to nor-
mally virulent (compatible) fungi. This was the conclusion of
Skipp & Deverall (1973) arising from observations and experi-
ments on cross-protection in the anthracnose disease of bean.
Following the observation that the protection was caused after
the entrance of the avirulent and virulent races into the plant, as
mentioned earlier in this chapter, it was found that the avirulent
fungus would also exert its effect when it was added one day
after the virulent fungus. By this means, it was possible to see
that the virulent hyphae grew into the host cells before the
38
avirulent germ-tubes caused nearby cells to undergo hyper-
sensitivity. Once the latter had happened, normally compatible
cells containing virulent hyphae also underwent hypersensitiv-
ity. This occurrence, even at a distance of several cells from the
avirulent appressorium, suggested a change in the sensitivity of
cells to normally compatible hyphae. Further evidence for a
change in the sensitivity of bean cells around those which had
undergone hypersensitivity to avirulent germ-tubes came from
the use of heat treatments. Four days after inoculation of
hypocotyls with the avirulent race, when penetrated cells were
necrotic, the hypocotyls were given a heat shock of 50 °C for 30
seconds. As a result, all neighbouring cells became necrotic
whereas those at a distance from the penetrated cells remained
healthy. Thus the physiology of bean cells around hypersensi-
tive cells had changed and they had become abnormally sensitive
to either the presence of compatible hyphae or to heat. Similar
observations of changed sensitivity of tobacco cells to heat
around local lesions caused by a strain of TMV were made by
Ross & Israel (1970).
The report by King, Hampton & Diachun (1964) that infection
of leaves of the red clover Trifolium pratense by bean yellow
mosaic virus affected their susceptibility to the powdery mildew
Erysiphe polygoni is also relevant. Virus infection altered metabol-
ism of the leaves so that they underwent hypersensitivity to the
normally compatible powdery mildew. The visible responses of
the host cells to the virus were the same as those reported by
Smith (1938) for varieties of red clover genetically resistant to E.
polygoni.
Thus there are a number of reasons why a factor, emanating
from hypersensitive cells and able to change the sensitivity of
susceptible cells so that they become resistant, should be sought.
Some of the results of experiments described in the last chapter,
concerning specific cross-protection factors in bean and an RNA
from rust fungi in hypersensitive wheat leaves, may aid in this
search.
The apparent implication of the remote cross-protection in
cucumber is that two separate processes require investigation, at
least in this phenomenon. One concerns the nature and action
of a factor which passes from the inoculated leaf. The second
concerns the nature of the change induced in the upper foliage.
Is the acquired resistance associated with hypersensitivity of the
39
cells to attempted infection, or is some other type of change in
host physiology involved?

PREVENTION OF HYPERSENSITIVITY BY HEAT-KILLED

BACTERIAL CELLS
There remains the need to consider the questions posed by the
series of discoveries following the demonstration by Lozano &
Sequeira (1970) that heat-killed cells of an avirulent race of
Pseudomonas solanacearum prevented hypersensitivity of tobacco
leaves to normal cells of the same race. The areas of tobacco leaf
injected with a minimum population of 3.5Xio7 cells/ml of an
avirulent race normally become completely necrotic 24 hours
later. This necrosis was prevented when the same areas were
infiltrated with an equal concentration of heat-killed cells 18
hours before injection with live cells. Lower concentrations of
heat-killed cells than live cells, and shorter intervals between
infiltration and injection, were much less effective. Not only did
the heat-killed cells prevent the necrosis of the leaf, but they also
caused a more -rapid decline in populations of the avirulent race
than normally occurs as hypersensitivity develops. It was also
noted that the preventive effect of the heat-killed cells was light
dependent. Substantial prevention of necrosis was also caused
by heat-killed cells of other races of P. solanacearum, and of P.
lachrymans and Xanthomonas axonopodis, but not of Escherichia
coli. These findings, along with further, more interesting dis-
coveries by Lozano and Sequeira, should be considered with that
of Loebenstein & Lovrekovich (1966) that heat-killed cells of
Pseudomonas syringae interfered with local lesion formation by
TMV in tobacco. Firstly, longer periods of time between infiltra-
tion with heat-killed cells and injection permitted the protective
effect to spread not only into neighbouring areas of the treated
leaf but later (within two days) to leaves immediately above this
leaf. The preventive effect in the more remote leaves was seen by
their response to injection as giving small necrotic spots rather
than total necrotic collapse. Secondly, the heat-killed cells also
prevented symptoms caused when virulent cells were injected
into the treated area of leaf 24 hours later. Furthermore, the
populations of these virulent cells declined instead of increasing
rapidly as in untreated leaves.
Attempts have been made to separate the factor from heat-
40
 killed bacterial cells which prevents the development of hyper-
sensitivity. Sequeira, Aist & Ainslie (1972) obtained several active
crude extracts from the cells. A crude extract obtained by brief
sonication of cells was completely effective, at a protein content
of 0.1 mg/ml, when introduced into leaves seven hours before
injection with the avirulent race. High molecular weight com-
pounds separated from this extract by ethanol precipitation and
fractionation on a column of Sephadex G-200 were active at a
protein content of 2.4 mg/ml, even after heating at 95 °C for 10
minutes. The likely proteinaceous nature of the factor is shown
by the destruction of activity by proteolytic enzymes, but first
attempts to characterize and isolate the active protein were
unsuccessful (Wacek & Sequeira, 1973).
This research, being mainly concerned with prevention of
hypersensitivity, differs in its major theme from most of the
phenomena described in this chapter. However, it is appropriate
that it should be considered here because it is relevant to the
debate concerning direct interference of one organism, albeit a
dead one, with a pathogen, and it adds to the reports of
systemically induced changes in response to infection in tobacco.
Direct interference by blocking special sites in intercellular
spaces is a possible mode of action when the need to infiltrate the
same number of dead cells as living cells is considered, but this
explanation is rendered improbable because of the delay of 18
hours before these dead cells can fully affect the reaction of the
leaf. An induced metabolic change in the host thus seems the
likely explanation of the phenomenon, and this is supported by
the changes in response which occur in leaves remote from the
site of treatment.

CONCLUSION
Although most of the earliest subjects for research in the study
of cross-protection have not been re-investigated by modern
methods, there are now clearly numerous convincing reports of
localized protection against many important pathogens, and
intriguing new claims of systemic protection in a few diseases.
The major challenges for the future concern their exploitation
in new methods of disease control and comprehension of their
mode of action. In most cases, protection seems to be brought
about by a change in host physiology and this give rise to the
4 41 DDM
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Title: Successful Baking for Flavor and Texture: Tested Recipes

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BAKING FOR FLAVOR AND TEXTURE: TESTED RECIPES ***
 SUCCESSFUL
Baking
FOR FLAVOR AND TEXTURE

Tested Recipes

BY Martha Lee Anderson

6th EDITION

CHURCH & DWIGHT COMPANY, INC.

70 PINE STREET, NEW YORK, N. Y.

COPYRIGHT, 1937 BY CHURCH & DWIGHT CO. INC., N. Y.

Helpful Kitchen Hints—pages 34-38
2
 INTRODUCTION

You may use these recipes with confidence. You will find that we
have carefully selected a most pleasing variety of old favorites and
new, delicious tempters. Many are pictured in color, so you can see
what your results will be. All have been critically tested ... for your
protection.

For the first time, you may observe the rich flavor and delicate
texture of baked products leavened and lightened with nature’s fruit
juices and nature’s acids, combined with baking soda. The crumb,
soft as velvet, the moisture thoroughly retained and the color, rich
and inviting, are outstanding results of this natural leavening. You
don’t need to wait for milk to sour, either; but ... the secret of
successful baking is in these pages ... read them.

We invite you to practice this art women regard so highly ...

successful baking.

Research Test Kitchen

Church & Dwight Co., Inc.
 4

A Secret for successful baking

Successful baking is another way of keeping a family happy. For who
isn’t filled with the joy of living when tempted by the penetrating
aroma of Gingerbread, rich and spicy ... or a piece of luscious
velvety Chocolate Cake, full of flavor? What adds more zest to a
meal than a surprise plate of hot breads ... fragrant Cinnamon Buns,
maybe Lemon Clover Rolls, delicate Soda Biscuits or Old Fashioned
Corn Bread?

The secret for making these successfully is as “old as the hills” but
as new as the morrow. Baking soda! Yes, grandmother used it in her
prized recipes and the modern homemaker finds it making her
baking day a success.

Arm & Hammer Brand and Cow Brand Baking Soda are refined
bicarbonate of soda whose standard of purity is that set up by the
United States Pharmacopoeia. For over 90 years this mild, healthful,
alkaline substance has been creating baking history. All these years
homemakers have depended on baking soda to make their baked
products deliciously moist and delicately light and tender.

“How,” asks the inexperienced homemaker, “does baking soda make

cakes, cookies and quick breads light and tender every time?”

Baking soda has stored in it a tremendous quantity of carbon dioxide

gas, the same gas found in soda water and ginger ale. This is
released when it comes in contact with any acid material such as the
many mild acids naturally found in cooking ingredients.

Among those ingredients are chocolate, cocoa, brown sugar, tomato

juice, sour milk, buttermilk, apple sauce, spices, cottage cheese,
molasses, vinegar, citrus fruit juices and many more. These acid
ingredients are familiar to everyone. One or more of them, you will
notice, is used almost every time you bake.

The baking soda gently but surely reacts with these natural acids,
freeing millions of tiny carbon dioxide bubbles which are held
enmeshed in the batters and doughs. As this gas expands during the
baking, the product becomes light and tender. Thus it is that baking
soda uses nature’s own unrivaled acids to leaven and lighten baked
products.

5
 Success Assured
Leavening nature’s way is surprisingly easy. The acid content of
citrus fruit juices or vinegar may be used to develop the
unsurpassed flavor and texture associated with baking soda
products. The following amounts,

1⅓ tablespoons vinegar (4 teaspoons)

1½ tablespoons lemon juice (4½ teaspoons)
¾ cup orange juice (12 tablespoons)

may be used with ½ teaspoon baking soda. Many of the recipes in

this booklet are especially designed for this natural combination of
baking soda and acid juices. Sometimes the acid is added last as in
the “Lemon Loaf Cake” on page 11, while in the “Lemon Clover
Rolls,” page 28, it is combined with the liquid, then added to the
mixture. In any case, you will be pleased with the results.

IMPORTANT! You don’t need natural sour milk or buttermilk to

prepare your old time favorite delicacies. It is the acid normally
found in these ingredients which reacts with baking soda for
leavening. If sour milk or buttermilk with its natural acid is not
available, you may provide the necessary acid by using citrus fruit
juices or vinegar with sweet milk. It is surprisingly simple to change
sweet milk to milk that contains as much acid as natural sour milk or
buttermilk when it is at its best for baking.

For example, when vinegar is used to provide this acid, place 1⅓

tablespoons vinegar (white vinegar makes a whiter product) in a
standard measuring cup, then fill to the one-cup mark with sweet
milk. Mix well. The resulting liquid can be used in place of sour milk
or buttermilk in any baking soda recipe. Use 1½ tablespoons lemon
juice or ¾ cup orange juice in a similar manner.

In many of these recipes, designed in our Research Test Kitchen,

one or more acid ingredient is used to create perfect leavening with
baking soda.

Follow these recipes accurately and carefully, then enjoy the finer
flavor and even texture produced when baking with baking soda.

6
 How to Bake

FLOUR. Preferably use the kind of flour specified in the recipe. If

you substitute cake or pastry flour for all-purpose flour, use 2
additional tablespoonfuls of flour for each cup required; to substitute
all-purpose flour for cake or pastry flour, remove 2 tablespoonfuls of
flour from each cup.

FATS. Solid fats can be used interchangeably. Melted fats or oils

should not be used in recipes specifying creaming of the shortening.

LIQUID. The use of citrus fruit juices, lemon and orange, is the
most recent accompaniment with sweet milk and baking soda for
leavening. With the health-giving qualities, this new use for fruit
juices in baking is widely accepted.

Sweet milk may be used in place of sour milk if clabbered

artificially. To sour or clabber sweet milk quickly, place 1½
tablespoonfuls of lemon juice or 1⅓ tablespoonfuls of
vinegar (white vinegar makes a whiter product) in a
standard measuring cup, then fill to the one-cup mark with
sweet milk. Mix well. The resulting liquid will contain as
much acid as natural sour milk or buttermilk when it is at its
best for baking, and may be used exactly as natural sour
milk or buttermilk in any baking soda recipe.
MEASURING. Always use level measurements. Use standard
measuring equipment: a ½ pint cup marked in quarters and thirds;
a set of standard measuring spoons consisting of a tablespoon,
teaspoon, ½ teaspoon and ¼ teaspoon.

MIXING. There is no such thing as “luck” in baking. Success

depends on good ingredients correctly combined. Follow the
directions carefully as set down in the following recipes.

BAKING. Keep the oven at the temperature specified in the recipe.

You have mastered an important part of baking, if you keep your
oven under control. Oven regulators and thermometers safeguard
baking.

CARE AFTER BAKING. Let cakes stand in pan on cooling rack for 3
to 5 minutes after baking; then turn out on rack and finish cooling
before frosting. Cookies should also be cooled on a rack.
ALWAYS SIFT FLOUR
USE STANDARD MEASURING EQUIPMENT
FOLLOW RECIPES CAREFULLY
AFTER BAKING LET CAKES AND COOKIES COOL

7
 Facts regarding plain white flour

In Arm & Hammer or Cow Brand Baking Soda recipes, certain types
of flour are used or specified simply to indicate that such a flour
gives the most desirable characteristics to that particular baked
product, but it does not mean that another type of flour cannot be
substituted, nor that an inferior product will result if a correct
substitution is made.

BREAD FLOUR. This is used to a large extent by commercial

bakers and generally is made from hard wheats: it contains a high
percentage of a protein product known as gluten. The gluten in
this flour is hard, capable of taking up and retaining a large
quantity of water. This type of flour is admirably adapted for bread
making, since the strong gluten gives an excellent skeleton to the
loaf. Such a flour is seldom used in the home today, except by
those who make large quantities of home-made white bread.
Usually, the gluten is present in this flour to the extent of 11 to
12%.

GENERAL PURPOSE OR FAMILY FLOUR. This flour is intended

to fill all needs and, consequently, is made by blending flours from
soft and hard wheats. It contains a moderate amount of medium
hard gluten, and is used in baking hot breads, such as muffins and
scones. However, it can be used for pastries as well. When
employed in place of pastry or cake flour, two level tablespoonfuls
less per cup should be used. General-purpose flours range in
gluten content from 10 to 11% and, in this respect, are about half
way between bread and true pastry or cake flours.

PASTRY OR CAKE FLOUR. Such flours not only have the lowest
gluten content, but a weak soft gluten as well, and are very
satisfactory for making all pastries except such items as fruit
cakes. These are preferably made with all-purpose flour, to
support the fruit and maintain a desirable structure. Pastry flours
contain 9 to 10% gluten and are made from various types of soft
wheat. Special cake flours belong to the pastry flour class, but are
finer in texture. They are slightly lower in gluten content (8 to
9%), and the gluten is even softer. Pastry flour gives baked
products a tender thin crust and a delicate crumb. Pastry flour can
neither absorb nor retain moisture like bread and all-purpose
flours and, therefore, sour milk or buttermilk is splendidly adapted
for use with this flour, since both are capable of retaining
moisture.

If all-purpose or family flour is specified in a recipe, and only

pastry is available, increase the pastry flour slightly (two
tablespoonfuls for every cup of family flour specified). When
pastry flour is used in biscuit recipes, the dough is rather soft and
inclined to be somewhat difficult to roll. Instead of further
increasing the pastry flour to stiffen the dough, better results are
realized by using the dough for dropped biscuits.

Flour, baking soda and many other similar materials should be

stored in a dry cool place, free from odors.

8
 9

Cakes

ORANGE LOAF
2 cups pastry flour
½ teaspoon Arm & Hammer or Cow Brand Baking Soda
¼ teaspoon salt
⅓ cup butter, or other shortening
1 cup sugar
2 eggs
Grated rind of 1 orange
¾ cup orange juice, strained

1. Sift, then measure the flour. Sift three times with the baking soda
and salt.

2. Cream the butter until light and lemon colored. Add sugar
gradually, beating after each addition.

3. Slowly add the eggs which have been beaten until they are almost
as stiff as whipped cream.

4. Add the orange rind. Alternately add the dry ingredients and
orange juice, beating until smooth after each addition.
5. Turn into a greased paper lined loaf pan. Bake.

6. When cool frost with Coconut Orange Frosting.

Amount: 6 × 10 inch pan Temperature: 350° F. Time:

50 minutes
See page 8

MARBLE CAKE
2½ cups pastry flour
1 teaspoon Arm & Hammer or Cow Brand Baking Soda
¼ teaspoon salt
½ cup butter, or other shortening
1 cup sugar
2 eggs
2 tablespoons lemon juice
¾ cup sweet milk
½ teaspoon vanilla
1 teaspoon cinnamon
½ teaspoon cloves
¼ teaspoon nutmeg
¼ teaspoon Arm & Hammer or Cow Brand Baking Soda
1 tablespoon molasses

1. Sift, then measure the flour. Sift three times with the 1 teaspoon
baking soda and salt.

2. Cream the butter until light and lemon colored. Add sugar
gradually, beating after each addition.

3. Slowly add the eggs which have been beaten until they are almost
as stiff as whipped cream.
4. Combine the lemon juice and milk. Alternately add the dry
ingredients and the liquid, a small amount at a time, beating until
smooth after each addition.

5. Divide batter in two equal parts.

6. To part one, add the vanilla.

7. To the other, add the well mixed ¼ teaspoon baking soda and
spices, then the molasses. Blend well.

8. Place batter in greased loaf pan by spoonfuls, alternating the light

and dark batters, thus giving a marbled effect. Bake.

9. Frost with Butter Frosting.

Amount: 9 × 5 inch pan Temperature: 350° F. Time: 45-

50 minutes See page 8

10

DATE NUT LAYER CAKE

2⅓ cups all-purpose flour
¾ teaspoon Arm & Hammer or Cow Brand Baking Soda
½ teaspoon salt
½ cup butter, or other shortening
1 cup sugar
2 eggs
1 cup buttermilk
1 cup dates, very finely cut
1 cup nutmeats, coarsely chopped

1. Sift, then measure the flour. Sift three times with baking soda and
salt. All-purpose flour is used to prevent settling of dates to the
bottom of the cake.
2. Cream the butter until light and lemon colored. Add sugar
gradually, beating after each addition.

3. Slowly add the eggs which have been beaten until they are almost
as stiff as whipped cream.

4. Alternately add the dry ingredients and the liquid, beating until
smooth after each addition.

5. Quickly fold in the dates and nuts which have been floured with 1
tablespoon of the dry ingredients.

6. Turn into greased layer cake pans. Bake.

7. Frost with Maple Cream Frosting.

Amount: 2—9 inch pans Temperature: 350° F. Time: 30-

35 minutes

SOUR MILK CHOCOLATE CAKE

2 cups pastry flour
1 teaspoon Arm & Hammer or Cow Brand Baking Soda
¼ teaspoon salt
½ cup butter, or other shortening
1 cup sugar
2 eggs
2 squares (2 ounces) unsweetened chocolate
1 cup sour milk
1 teaspoon vanilla

1. Sift, then measure flour. Sift three times with baking soda and
salt.
2. Cream the butter until light and lemon colored. Add sugar
gradually, beating after each addition.

3. Slowly add the eggs which have been beaten until they are almost
as stiff as whipped cream.

4. Gradually add the chocolate which has been melted and cooled.

5. Stir the vanilla into the milk. Alternately add the dry ingredients
and the liquid, a small amount at a time, beating until smooth
after each addition.

6. Turn into a greased loaf pan. Bake.

7. Frost with Soft Chocolate Icing.

Amount: 8 × 8 inch pan Temperature: 325° F. Time: 60 minutes

11

DESSERT GINGERBREAD
1½ cups all-purpose flour
1 teaspoon Arm & Hammer or Cow Brand Baking Soda
¼ teaspoon salt
1 teaspoon ginger
⅓ cup shortening
½ cup sugar
1 egg
½ cup molasses
¾ cup boiling water

1. Sift, then measure the flour. Sift three times with the baking soda,
salt and ginger.

2. Cream the shortening until it is light and fluffy. Add the sugar
gradually, beating after each addition.
3. Next, add the unbeaten egg, beating briskly.

4. Add the molasses. Then add the dry ingredients, beating until
smooth. Stir in boiling water.

5. Turn into greased loaf pan. Bake.

Amount: 8 × 8 inch pan Temperature: 350° F. Time: 30-

40 minutes See page 8

LEMON LOAF CAKE

2 cups pastry flour
½ teaspoon Arm & Hammer or Cow Brand Baking Soda
¼ teaspoon salt
½ cup butter, or other shortening
1 cup sugar
2 eggs
½ cup sweet milk
1½ tablespoons lemon juice

1. Sift, then measure flour. Sift three times with baking soda and
salt.

2. Cream the butter until light and lemon colored. Add sugar
gradually.

3. Slowly add the eggs which have been beaten until they are almost
as stiff as whipped cream.

4. Alternately add the dry ingredients and the liquid, beating until
smooth after each addition. Add lemon juice, blending in well.

5. Turn into greased loaf pan. Bake.

6. Cover with Lemon Filling and top with ½ recipe of Fluffy Frosting.

Amount: 8 × 8 inch pan Temperature 350° F. Time: 45 minutes

See page 17

THANKSGIVING STEAMED PUDDING

3 cups all-purpose flour
1 teaspoon Arm & Hammer or Cow Brand Baking Soda
1½ teaspoons salt
½ teaspoon cloves
½ teaspoon mace
½ teaspoon allspice
½ teaspoon cinnamon
1 cup suet, finely ground
1 cup molasses
1 cup sweet milk
1½ cups seedless raisins, chopped

1. Sift, then measure the flour. Sift three times with the baking soda,
salt and spices.

2. Combine suet, molasses and milk.

3. To the suet mixture, add the dry ingredients, beating until

smooth. Add raisins.

4. Turn into a well greased pan or mold. Cover. Steam 3 hours.

5. Serve with Hard Sauce or Foamy Sauce.

Amount: 12 servings

12
 DARK FRUIT CAKE
5 cups all-purpose flour
1 teaspoon Arm & Hammer or Cow Brand Baking Soda
½ teaspoon salt
½ teaspoon cloves
½ teaspoon cinnamon
½ teaspoon mace
1 pound butter, or other shortening
1 pound sifted brown sugar
8 eggs
½ pound each candied cherries, citron, orange and lemon peel,
finely sliced
1 pound almonds, blanched and shredded
1 pound seedless raisins
1 pound currants
½ cup water
1 cup honey
½ cup molasses

1. Sift, then measure the flour. Sift three times with baking soda, salt
and spices.

2. Cream the butter until light and lemon colored. Add sugar
gradually, beating after each addition.

3. Slowly add the eggs which have been beaten until they are almost
as stiff as whipped cream.

4. Add the fruits and nuts, then add water, honey and molasses.

5. Add dry ingredients, beating until smooth after each addition.

6. Turn into 2 paper-lined tube pans. Bake.

Amount: 10 pounds Temperature: 250° F. Time: 3½ hours

 BAKED PRUNE PUDDING
1½ cups all-purpose flour
½ teaspoon Arm & Hammer or Cow Brand Baking Soda
½ teaspoon salt
½ teaspoon cinnamon
¼ cup butter, or other shortening
¾ cup sugar
1 egg
½ cup juice from prunes
1 cup stewed prunes, drained and finely chopped
½ cup nutmeats, coarsely cut

1. Sift, then measure flour. Sift three times with baking soda, salt
and cinnamon.

2. Cream the butter until it is light and lemon colored. Add sugar
gradually, beating after each addition.

3. Briskly stir in the well beaten egg.

4. Alternately add the dry ingredients and prune juice, a small

amount at a time, beating until smooth after each addition.

5. Last, carefully stir in the prunes and nutmeats.

6. Turn into a greased tube pan. Bake.

7. Serve with whipped cream.

Amount: 2 qt. tube pan Temperature: 375° F. Time: 1 hour

13

FAVORITE SPICE CAKE

 2½ cups pastry flour
1 teaspoon Arm & Hammer or Cow Brand Baking Soda
¼ teaspoon salt
2 teaspoons cinnamon
½ teaspoon cloves
¼ teaspoon nutmeg
½ cup butter, or other shortening
1 cup sifted brown sugar, firmly packed
2 eggs
¾ cup sweet milk
2 tablespoons vinegar

1. Sift, then measure flour. Sift again with baking soda, salt and
spices.

2. Cream butter until light and lemon colored. Add sugar gradually,
beating after each addition.

3. Slowly add the eggs which have been beaten until they are almost
as thick as whipped cream.

4. Combine vinegar and milk. Alternately add the dry and the liquid
ingredients, beating until smooth after each addition.

5. Turn into a greased cake pan. Bake in a moderate oven.

6. Frost with Butter Frosting.

Amount: 8 × 8 inch pan Temperature: 350° F. Time: 40-

45 minutes

RED DEVIL’S CAKE

2 cups pastry flour
1¼ teaspoons Arm & Hammer Cow Brand Baking Soda
 ¼ teaspoon salt
½ cup butter, or other shortening
1 cup sugar
2 eggs
2 squares (2 ounces) unsweetened chocolate
1 teaspoon vanilla
¾ cup sour milk or buttermilk
⅓ cup boiling water

1. Sift, then measure the flour. Sift three times with the baking soda
and salt.

2. Cream the butter until light and lemon colored. Add sugar
gradually, beating after each addition until light and fluffy.

3. Slowly add the eggs which have been beaten until they are almost
as stiff as whipped cream. Gradually add the chocolate which has
been melted and cooled.

4. Stir the vanilla into the milk. Alternately add the dry ingredients
and the milk, beating until smooth after each addition. Add the
boiling water and beat in well.

5. Turn into a greased cake pan. Bake.

6. Frost with Quick Butterscotch Icing. Let cake stand two hours
before cutting to allow the red color to develop.

Amount: 2—8 inch layers Temperature: 350° F. Time: 25-

30 minutes
See page 17

14

HONEY DIAMONDS
2 cups pastry flour
 ½ teaspoon Arm & Hammer or Cow Brand Baking Soda
¼ teaspoon salt
½ teaspoon cinnamon
¼ cup butter, or other shortening
1 cup sifted brown sugar, firmly packed
⅓ cup honey
2 eggs
½ cup sweet milk
Nutmeats

1. Sift, then measure the flour. Sift three times with the baking soda,
salt and cinnamon.

2. Cream the butter until light and lemon colored. Add sugar
gradually, beating after each addition.

3. Combine honey and eggs which have been beaten until they are
almost as stiff as whipped cream. Add to the butter-sugar mixture.
Blend well.

4. Alternately add the dry ingredients and milk, beating after each
addition.

5. Turn into a greased shallow cake pan. Bake.

6. Frost with Butter Icing. Garnish with nutmeats. Cut in diamond

shaped pieces.

Amount: 9 × 9 inch pan Temperature: 350° F. Time: 45-

50 minutes See page 8

FRUIT CUP CAKES

2 cups pastry flour
1 teaspoon Arm & Hammer or Cow Brand Baking Soda
 ½ teaspoon salt
1 teaspoon cinnamon
½ teaspoon allspice
½ cup butter, or other shortening
1 cup sifted brown sugar, firmly packed
2 eggs
1⅓ tablespoons vinegar
⅔ cup sweet milk
1 cup dates, finely cut
1 cup nutmeats, coarsely cut
½ cup citron, sliced

1. Sift, then measure the flour. Sift three times with baking soda, salt
and spices.

2. Cream the butter until light and lemon colored. Add sugar
gradually, beating after each addition.

3. Slowly add the eggs which have been beaten until they are almost
as thick as whipped cream.

4. Combine vinegar and milk. Alternately add the dry ingredients and
the liquid, a small amount at a time, beating until smooth after
each addition.

5. Lastly add fruit and nuts.

6. Fill greased muffin tins ⅔ full.

Amount: 3 dozen small cakes Temperature: 375° F. Time: 20-

25 minutes
See page 17

15

APPLE SAUCE CAKE

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