African Journal of Biotechnology Vol. 9 (7), pp.

991-995, 15 February, 2010

Available online at http://www.academicjournals.org/AJB
ISSN 1684–5315 © 2010 Academic Journals

Full Length Research Paper

Fungal contaminants observed during

micropropagation of Lilium candidum L. and the effect
of chemotherapeutic substances applied after
sterilization
Filiz Altan1, Betül Bürün1* and Nurettin ahin2
1
Mu la University, Faculty of Science and Arts, Department of Biology, Mu la, Turkey.
2
Mu la University, Faculty of Education, Department of Biology Education, Mu la, Turkey.
Accepted 16 July, 2009

Lilium candidum L. is a species which grows in the South West Anatolia region of Turkey. It is a
bulbous plant with beautifully scented flowers and is used in the floral industry. The bulbs are
produced by using traditional propagation and in vitro techniques. Micropropagation is a rapid
propagation technique, but the greatest problem is contamination with fungi and bacteria. Antibiotic
and fungicide treatments were done after sterilization for micropropagation of L. candidum. Fungal
contaminants formed during the culture were determined. Bulb scales were used as explants (5 - 10 mm
width) and were cultured in photoperiodic conditions (16 h light, 8 h dark) or complete darkness. Bulb
scales rinsed in water were surface sterilized, then solutions containing chemotherapeutic substances
(Benomyl, Nystatin, Streptomycin, Penicillin) in different combinations were applied for 30 min and
-3 -3
subsequently were cultured in MS medium with supplement 0.1 mg dm NAA + 0.01 mg dm BA. During
the experiment, fungal contaminants were observed in full treatments. Determined contaminants were
identified according to their morphological and cultural characteristics by cultivation and were
comprised of: Fusarium, Penicillium, Alternaria, Rhizopus, Cylindrocarpon and Aspergillus species.
The most effective treatment against fungal contaminations was achieved by utilizing a Benomyl (100
-3 -3
mg dm ) + Nystatin (100 mg dm ) treatment combination.

Key words: Contaminating microorganisms, Lilium candidum, bulb scale, in vitro culture, treatment, antibiotic,
fungicide.

INTRODUCTION

Lilium are bulbous plants with many different species that area of biotechnology for the commercial production of
have a special importance as cut flowers among orna- ornamental plants (Bürün et al., 2001).
mental plants (Uzun, 1984) and are economically impor- Even though it is possible to produce a large number of
tant due to their attractive flowers (Nhut, 1998). Tradi- plants by micropropagation, the greatest problem in this
tional production is carried out with bulbils, bulblets and technique is contamination. A wide range of micro-
bulbs. It is also possible to produce plants by tissue rganisms (filamentous fungi, yeasts, bacteria, viruses and
culture techniques. With the micropropagation technique, viroids) and micro-arthropods (mites and thrips) have
a large number of plants can be propagated in a very been identified as contaminants in plant tissue cultures.
short time. Micropropagation has been widely used as an Contaminants may be introduced with the explant, during
manipulations in the laboratory, by micro-arthropod
vectors (Tanprasert and Reed, 1997; Leifert and
Cassells, 2001) or endophytic bacteria (Reed et al., 1995;
*Corresponding author. E-mail: [email protected]. Tel.: Pereira et al., 2003). Fungus may arrive with an explant,
002522825619. Fax: 002522238656.
or airborne, or enter a culture (Babao lu et al., 2001).
Abbreviations: MS, Murashige and Skoog; BA, benzyladenine; Frequently encountered bacterial and fungal contamina-
NAA, naphthalene acetic acid. tions especially in laboratories of commercial micro-
992 Afr. J. Biotechnol.

propagation pose a considerable problem (Reed et al., Medium was prepared and used in the following mixture: 30 g
1998). Studies on the effect of antibiotics and fungicides dm-3 sugar, 8 g dm-3 agar was added to MS medium (Murashige-
Skoog, 1962) containing 0.1 mg dm-3 NAA + 0.01 mg dm-3 BA plant
on these kinds of contaminants have been carried out growth regulator were prepared according to Franklin and Dixon
(George, 1993). (1994) and pH adjusted to 5.7 before autoclaving for 15 min. The
Shields et al. (1984) analysed the effects of a number sterilization of fungus identification media was made in the same
of fungicides against in vitro fungal contaminants and way by autoclaving.
their toxicity in tobacco cultures. They recommend 2
-3
fungicides, carbendazim and fenbendazole (30 g cm ).
-3 -3 Isolation and identification of filamentous microfungi
In addition imizalil (20 g cm ) and captofol (100 g cm )
were alternative fungicides to prevent fungal contami- During micropropagation, fungal contaminants were transferred into
nation and a mixture of propiconazole plus carbendazim tubes containing potato dextrose agar (PDA, Difco 0013) and were
was effective to control fungal contaminants (George, kept at +4oC for identification.
1993). The Fungi were later inoculated on PDA (Difco 0013), malt ex-
tract agar (Difco 0112) and yeast and mould agar (Oxoid CM920).
Wittenbach and Bukavoc (1980) reported that benomyl
-3 Plates were incubated in the dark at laboratory temperature (25°C)
(100 g cm ) reduced the contamination of cherry fruits for 5-15 d and the microscopic fungi were identified using the diag-
without affecting fruit growth unfavourably (George, nostic keys of Haseneko lu (1991) and Barnett and Hunter (1998).
1993). For identification purposes, slide cultures were prepared on malt
It has been stated that not single but combinatorial use extract agar and stained with lactophenol-blue.
of antibiotics has shown synergistic effects in both control
of microorganizms and reduction of plant damage. Be- Surface sterilization of plant material and treatments of
cause most antibiotics have a narrow target spectrum for chemotherapeutic substances
bacteria, it is typical that they be used in an ordered or
step-by-step fashion. It has been determined that 10 g Lilium bulb scales were rinsed in tap water. They were dipped in
-3 -3 96% ethanol for 2 min, then in 2.25% Na–hypochlorite solution, with
cm Rifampin + 1 g dm Benomyl has been an important
effective combination in the control of fungal and bacterial one drop of 0.1% Tween 80 for 20 min and rinsed 4 times in sterile
distilled water.
contaminants in Camellia cultures (Haldeman et al., Lilium bulb scales were treated with solutions containing anti-
1987). biotics and fungicides in different combinations for 30 min after
Reed et al. (1998) had observed internal bacterial con- surface sterilization of bulb scales. These combinations are as
tamination in hazelnut shoot cultures and contaminants follows:
were evident at culture establishment, or became appa-
Treatment 1. Streptomycin 200 mg dm-3 + Penicillin 200 mg dm-3
rent after several subcultures. They had treated plant
Treatment 2. Streptomycin 400mg dm-3 + Penicillin 400 mg dm-3
material with antibiotics timentin + streptomycin or genta- Treatment 3. Benomyl 50 mg dm-3 + Nystatin 50 mg dm-3
mycin + streptomycin and determined that the antibiotics Treatment 4. Benomyl 100 mg dm-3 + Nystatin 100 mg dm-3
combination was more effective, where no single Treatment 5. Streptomycin 300 mg dm-3 + Benomyl 75 mg dm-3
antibiotic was effective for all bacterial isolates from Treatment 6. Streptomycin 600 mg dm-3 + Benomyl 150 mg dm-3
hazelnut shoot cultures.
Kubota and Tadokoro (1999) examined photoauto- The test of phytotoxicity
trophic (sugar-free) micropropagation for many different
plant species. One of the advantages of photoautotrophic In the trials, it was analyzed that superface had or not phytotoxicity
micropropagation is the low risk of contamination and on the plant of secondary metabolites that were reproduced during
addition of AgNO3 in the medium suppressed growth of the development of fungal contaminants that were isolated. For that
nonpathogenic contaminants without reducing fresh and reason, contaminating products were sown in a medium (Sabo-
raund Dextrose Broth) and they were applied on Lolium perenne
dry weight, and number of leaves of tomato plantlets. seeds, as Lolium normally germinate in a very short time. The
In this study, the identification of fungal contamination treated seed were tested for germination and phytotoxicity in the
emerging during micropropagation of Lilium candidum isolates was assessed.
and the effects of antibiotic and fungicide treatments after
sterilization was determined.
The conditions of culture room

Culture room temperature was 23oC, with a photoperiod of 16 h

MATERIALS AND METHODS light (1600 lux), 8 h dark and total darkness. Development of the
bulblets from explants was observed in the culture room.
Plant material and nutrition medium for micropropagation

In the micropropagation of species Lilium, bulb scales and leaves RESULTS

have been successfully used as explants (Mansuro lu and Gürel,
2001). The L. candidum bulbs were collected from Dalyan - Mu la.
Each bulb-scale was cut into 5-10 mm pieces and each part Fungal contaminants were identified during micropro-
excised was placed into a culture tube. For each treatment, 40 pagation of L. candidum. The fungal contaminants found
explants (40 culture tubes) were used. are listed in Table 1 according to fungicide and antibiotic
 Altan et al. 993

Table 1. The list of fungal species that were determined in accordance with their applications.

a c
Treatment and Determined fungal contaminations
b
environmental conditions 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16
Photoperiodic conditions
Control x x
T1 x x x
T2 x x
T3 x
T4 x x
T5 x x
T6 x x
Dark Conditions
Control
T1 x x x
T2 x x x x
T3 x x x
T4 x x x x x
T5 x x x x x x x
T6 x x x x x
a
: Treatment
Control
T1: Streptomycin (200 mg dm-3) + Penicillin (200 mg dm-3 )
T2:Streptomycin (400 mg dm-3) + Penicillin (400 mg dm-3)
T3: Benomyl (50 mg dm-3) + Nystatin (50 mg dm-3)
T4: Benomyl (100 mg dm-3) + Nystatin (100 mg dm-3)
T5: Streptomycin (300 mg dm-3) + Benomyl (75 mg dm-3)
T6: Streptomycin (600 mg dm-3) + Benomyl (150 mg dm-3)
b
: Environmental conditions
Photoperiodic conditions (16 hours day, 8 hours darkness).
Totally dark conditions
c
: Fungal contaminants
1: Alternaria sp.; 2: Aspergillus sp.; 3: Aspergillus niger ; 4.Cylindrocarpon sp.; 5. Fusarium sp.; 6: Penicillium sp.1;
7: Penicillium sp. 2; 8: Penicillium sp. 3; 9: Penicillium sp. 4; 10: Rhizopus sp.; 11: Rhizopus stolonifer; 12: Mycellia sterilia
Unidentified isolates:
13: Colony type 1; 14: Colony type 2; 15: Colony type 3; 16: Colony type 4.

treatments. In the cultured photoperiodic condition (16 h rate of contamination as Colony type 1, in Treatment 1),
light, 8 h dark) a total of 29 contamined cultures were and Colony type 4 (68.9% of total contamination, in all of
identified (10.43% of cultured explants) and the fungal the treatments). The contami-nations in controls were
agent, Aspergillus niger, Penicillium sp. 4, Alternaria sp. identified as Fusarium sp. and formed 6.89% of total
and Fusarium sp. were identified. In experiments using contamination. The most contami-nants were observed in
-3
antibiotic/fungicide treatments, Treatment 1, Treatment 6 Treatment 1 (Streptomycin 200 mg dm + Penicillin 200
-3 -3
and control, the contaminants were observed as Asper- mg dm ), Treatment 2 (Strepto-mycin 400 mg dm +
-3 -
gillus niger and formed 10.34% of total contamination. Penicillin 400 mg dm ), Treatment 3 (Benomyl 50 mg dm
3 -3
The fungus that caused contamination in Treatment 2 + Nystatin 50 mg dm ) and Treatment 5 (Streptomycin
-3 -3
was identified as Penicillium sp. 4 and formed 3.44% of 300 mg dm + Benomyl 75 mg dm ).
total contamination. The fungus that caused contami- In dark-grown culture, a total of 46 contaminated tubes
-3
nation in Treatment 4 (Benomyl 100 mg dm + Nystatin were observed (16.60% contaminations in this experi-
-3
100 mg dm ) was identified as Alternaria sp. and formed ment) including Penicillium sp (1, 2, 3, 4), Aspergillus sp.
the same rate of contamination as Penicillium. The rest of and A. niger, Rhizopus sp., R. stolonifer and Cylindro-
contaminations were unknown and these unidentified carpon sp. Penicillium sp. 1 (2.17% of total contami-
isolates were classified under four colony types in both nation) and Penicillium sp. 2 (2.17% of total contami-
-3
the photoperiodic growth condition and darkness growth nation) were observed only in streptomycin 300 mg dm
-3
condition (but Colony type 3 was noticed only in the + Benomyl 75 mg dm treatments (in treatment).
darkness growth condition). Colony type 1 (3.44% of total Penicillium sp. 3 (2.17% of total contamination) were ob-
-3
contamination, in Treatment 5), Colony type 2 (the same served only in Treatment 6 (Streptomycin 600 mg dm +
994 Afr. J. Biotechnol.

-3
Benomyl 150 mg dm ). The fungi that causes contamina- morphological changes. In this study, phytotoxicity has
-3
tion in Treatment 4 (Benomyl 100 mg dm + Nystatin 100 not been determined by any specific phytotoxicity test.
-3 -3
mg dm ), Treatment 6 (Streptomycin 600 mg dm + Fungi are widespread plant pathogens and they are
-3
Benomyl 150 mg dm ) and both of Streptomycin + saprophytic soil living beings. Many fungal species have
Penicillin treatments (Treatments 1, 2) were identified as relations with plant tissues and they generally cause
Penicillium sp. 4 and formed 17.39% of total contamination in plant tissues. The main offenders are
contamination. Contaminants in Treatment 1 (Strepto- species of Aspergillus, Candida, Microsporum and
-3 -3
mycin 200 mg dm + Penicillin 200 mg dm ), Treatment Phialophora (George, 1993). In this study Aspergillus has
-3 -3
4 (Benomyl 100 mg dm + Nystatin 100 mg dm ) and been encountered frequently in a similar way (Table 1).
-3
Treatment 5 (Streptomycin 300 mg dm + Benomyl 75 In a study concerning the effect of benomyl on shoot
-3
mg dm ) were identified as Aspergillus niger and and root development on explants of Asparagalus
comprised 17.39% of total contamination. Contaminants officinalis grown in culture media, Yang (1976) reported
-3
in Treatment 2 (Streptomycin 400 mg dm + Penicillin that benomyl as a systemic fungicide had the same effect
-3 -3
400 mg dm ), Treatment 3 (Benomyl 50 mg dm + as cytokinin. Different doses (10, 25, 50, 100 and 250 mg
-3 -3
Nystatin 50 mg dm ), Treatment 5 (Streptomycin 300 mg dm ) of benomyl were added in culture medium. At low
-3 -3 -1
dm + Benomyl 75 mg dm ) and Treatment 6 (Strepto- doses (10 and 50 µg g ) benomyl addition encouraged
-3 -3
mycin 600 mg dm + Benomyl 150 mg dm ) were effective development, but at high levels (100 and 250 µg
-1
identified as Aspergillus sp. and formed the same rate of g ) benomyl addition affected both root and shoot
contamination as A. niger. Contaminations in Treatments formation unfavourably and it also caused an abnormally
1, 4 were identified as Rhizopus stolonifer and formed short and thick shoot development. In the present study
4.34% of total contamination. Treatments 4 and 6 had benomyl is one of the fungicides which was used to treat
contaminants identified as Rhizopus sp. and formed Lilium bulb-scale explants and when used with nystatin,
6.52% of total contamination. The cause of contamination has been observed to be effective.
-3
in Treatment 5 (Streptomycin 300 mg dm + Benomyl 75 Fusarium oxysporum and C. radicola fungi are primary
-3
mg dm ) was identified as Cylindrocarpon sp. and pathogens for lily. They show symptoms of illness in
formed 4.34% of total contamination. 2.17% of total Lilium scales like tip rot and stem lesions. With the loss of
contamination formed mycelia-sterilia in Treatment 2. The bulbs in Asiatic Lilies, basal root illnesses are a serious
rest of contaminations were unknown and they were problem. F. oxysporum together with C. radicola or Phy-
classified under 4 types, in the same manner as in the thium caused infection in basal root. After treatment with
o
photoperiodic condition. Colony type 1 (2.17% of total hot water at 39 C for 2 h. Bulbs of lily, treated with in
contamination, in Treatment 4), Colony type 2 (2.17% of benomyl for 30 min, has caused increase in yields in in
total contamination, in Treatment 3), Colony type 3 (in vivo (Lawson and Hsu, 1996). Also in our study we
Treatment 5 and the same rate contamination with observed these species. In the in vitro culture of benomyl
Colony type 1 and 2), Colony type 4 (the cause of conta- treatment explants, Fusarium had not been observed.
mination in Treatments 2, 3, 4, 5 and 6 and formed In order to free plant tissues from pathogens, there are
17.39% of total contamination) were determined. some recommendations to use hot water treatments.
o
Langens et al. (1988) determined that hot water at 40 C
DISCUSSION for Lilium decreased contamination, but that hot water at
o
45 C decreased regeneration capacity.
The contamination in plant tissue cultures can originate Kritzenger and Van Vuuren (1998), in their study about
from several sources. These can suddenly occur when elimination of contamination in rhizomes of Zandedeschia
there is ineffective surface sterilization and microorga- aethiopica, indicated that it was possible to eliminate
nisms that were concealed within explants or introduced fungal and bacterial contaminations in rhizomes using
during subculturing or via contamination which occurs fungicide pretreatment and antibiotic or combinations of
simultaneously in cultures after a long period of growth antibiotics. According to the study results, fungicides
(Cassells, 1990; George, 1993). (Captab 500 WP and Dithane M-45) showed an increa-
All of the types of contaminants including by fungi, singly antibacterial effect and antibiotics (ABM1 and
bacteria, viruses, yeasts and mollicutes and rickettsias Imipenem) proved to be effective against contaminants.
have been causing considerable economical losses in In the culture of bulb-scale explants for L. candidum
plant tissue culture laboratories (George, 1993; Leifert, micropropagation, contamination has been identified and
2000). For that reason it is of great importance that an studied. After surface sterilization with Na-hypochlorite,
effective sterilization process is developed. antibiotic and fungicide combinations and treatments, it
The use of antibiotics for controlling plant contaminants was stated that observed contamination were fungi such
is limited because of its impairing effect on plastids and as Fusarium, Penicillium, Aspergillus, Alternaria, Rhizo-
mitochondria and chlorophyll formation (George, 1993). pus and Cylindrocarpon genus. In dark growth conditions,
Reed et al. (1998) in their study phytotoxicity of anti- more microorganism genera were observed in L. candi-
biotics observed visually browning, chlorosis and dum micropropagation cultures.
 Altan et al. 995

REFERENCES Leifert C (2000). Quality Assurance systems for plant cell and tissue
culture; The problem of latent persistence of bacterial pathogens and
Babao lu M, Yorgancılar M, Akbudak MA (2001). Doku kültürü: Temel Agrobacterium-based transformation vector systems. Acta Hortic. p.
laboratuar teknikleri (Plant tissue culture: Basic laboratory 530: International Symposium on Methods and Markers for Quality
techniques).- In: Babao lu M, Gürel E, Özcan S (eds), Bitki assurance in Micropropagation.
Biyoteknolojisi: Doku Kültürü ve Uygulamaları (Biotechnology of Leifert C, Cassells AC (2001). Microbial hazards in plant tissue and cell
Plant: Plant Tissue Culture and Application). Selçuk Üniversitesi cultures. In vitro: Cell Dev. Biol. Plant, 37(2): 133-138.
Vakfı Yayınları-Konya- pp.1-35. Mansuro lu S, Gürel E (2001). Mikroço altım (Micropropagation). In:
Barnett HL, Hunter BB (1998). Illustrated Genera of Imperfect Fungi, 4th Babao lu M, Gürel E, Özcan S (eds), Bitki Biyoteknolojisi : Doku
Ed. American Phytopathological Society. Kültürü ve Uygulamaları (Plant Biotechnology: Tissue culture and
Bürün B, Altan F, Turasay B (2001). [The using in vitro techniques on applications). Selçuk Üniversitesi Vakfı Yayınları, Konya, pp. 262-
propagation plants commercially]. XII. Biyoteknoloji Kongresi 281.
(Biotechnology Congress), Ayvalık-Balıkesir, 4-7 Eylül (September) Murashige T, Skoog F (1962). A revised medium for rapid growth and
2001. bioassays with tobacco tissue culture. Physiol. Plant, 15: 473-497.
Cassells AC (1990). Problem in tissue culture: Culture contamination. Nhut DT (1998). Micropropagation of lily (Lilium longiflorum). Plant Cell
In: Debergh PC, Zimmerman RH (eds), Micropropagation- Rep. 12(17): 913-916.
Techonology and Application. pp. 31-45. Pereira JES, Mattos MLT, Fortes GRD (2003). Identification and
Franklin CI, Dixon RA (1994). Initiation and maintenance of callus and antibiotic control of endophytic bacteria contaminants in
cell suspension cultures. In: Dixon RA, Gonzales RA (eds), Plant Cell micropropagated potato explants. Pesquisa Agropecuaria Brasileira
Culture A. Practical Approach, Second edition, New York: Oxford 38(7): 827-834.
University Pres, pp. 1-23. Reed BM, Buckley PM, Dewilde TN (1995). Detection and eradication of
George EF (1993). Plant Propagation by Tissue Culture, Part 1, endophytic bacteria from micropropagated mint plants. In vitro: Cell
Techonology, England: Exegetics Ltd., pp. 121-145. Dev. Biol. Plant, 31(1): 53-57.
Haldeman JH, Thomas RL, McKamy DL (1987). Use of benomyl and Reed BM, Mentzer J, Tanprasert P, Yu X (1998). Internal bacterial
rifampicin for in vitro shoot tip culture of Camellia sinensis and C. contamination of micropropagated hazelnut : identification and
japonica. Hort. Sci. 22: 306-307. antibiotic treatment. Plant Cell Tissue Organ Cult. 52(1-2): 67-70.
Haseneko lu I (1991). Toprak Mikrofungusları, Atatürk Univ. Yayınları, Shields R, Robinson SJ, Anslow PA (1984). Use of fungicides in plant
7 Cilt, Sayı 689, Erzurum. tissue culture. Plant Cell Rep. 3: 33-36.
Kritzenger EM, Jansen Van Vuuren R (1998). Elimination of external Tanprasert P, Reed BM (1997). Detection and identification of bacterial
and internal contaminants in rhizomes of Zantedeschia aethiopica contaminants from strawberry runner explants. In vitro: Cell Dev. Biol.
with commercial fungucides and antibiotics. Plant Cell,Tissue Organ Plant, 33(3): 221-226.
Cult. 52: 61-65. Uzun G (1984). Zambak Yeti tiricili i. Yalova- TAV Yayınları, Vol. 7.
Kubota C, Tadokoro N (1999). Control of microbial contamination for Yang HJ (1976). Effect of benomyl on Asparagus officinalis L. shoot
large-scale photoautotrophic micropropagation. In Vitro: Cell. Dev. and root development in culture media. Hort. Sci. 11(5): 473-474.
Biol. Plant, 35(4): 296-298.
Langens-Gerrits Albers M, Jan de Klerk G (1998). Hot water teatment
before tissue culture reduces initial contamination in Lilium and Acer.
Plant Cell Tissue Organ Cult. 52: 75-77.
Lawson RH, Hsu HT (1996). Lily diseases and their control. Acta Hortic.
p. 414.