Microbiol. Res. (1995) 150, 71- 75 Microbiological Research

© Gustav Fischer Verlag Jena

Biological control of Pythium mamillatum causing damping-off of cucumber seedlings by a soil bacterium, Bacillus mycoides

Bernard Pault, Romond Charles2, Tej Bhatnagar3

1 Laboratoire de Mycologie, U.ER. Sciences de la vie, B.P. 138, 21004 Dijon, Cedex, France 2 Laboratoire de Bacteriologie, Faculte de Pharmacie, B.P. 83, 59006, Lille, France 3 Tej Bhatnagar, Chemin du vergon, 13510 Eguilles, France

Accepted: November 5, 1994

Abstract

Pythium mamillatum Meurs was isolated from soil samples and was found to be pathogenic to cucumber seedlings, causing severe damping-off disease. However, in vitro and in vivo studies reveal that this fungus was completely inhibited by a soil bacteria Bacillus mycoides strain SU-23. The details of the parasitic fungus, the antagonistic bacteria and the biological control of the disease are described in this article.

Keywords: Pythium - Bacillus - antagonism - damping­off - biological control

Introduction

The members of the genus Pythium, with more than 100 species, are distributed throughout the world and are well known for their parasitic nature on a wide variety of plants. Most of these infect mainly juvenile or succulent tissues, thus restricting their parasitic behaviour to seedlings or to the feeder roots or root tips of older plants, and to watery fruits or stem tissues (Hendrix and Campbell 1973). The most important diseases caused by this genus due to the attack of juvenile tissues are "seed rots", and "dam­ping-offs". The latter can be observed mostly in nursery bed, greenhouse flats and row crops, where symptoms are developped suddenly resulting in the toppling off of large number of seedlings. Another important disease caused by the species of Pythium are the "feeder root necrosis" which are associated

Corresponding author: B. Paul

with "decline" diseases reported on a wide variety of plants, including perennial plants. A common symp­tom in peaches, turf, citrus, apples, pines and other perennials is the gradual to sudden deterioration of the established plants and poor survival of replants. Due to the infection by the pythiaceous pathogen, the declining plants lack a healthy root system (Hendrix et al. 1966) and extensive damages are caused to the host plants.

During the recent years, efforts have been made towards the biological control of soil borne diseases. Dennis and Webster (1971 a, b, c) have shown the antagonistic properties of the fungus Trichoderma. Bolton (1978) showed that the addition of Tricho­derma viride and a Streptomyces species gave a degree of protection to pelargonium against the pathogen Pythium splendens. Locke et al. (1979) used a seed dressing of Trichoderma spp. to control the Pythium induced seed rot of peas, and they showed that Trichoderma established itself in the rhizosphere of the treated -peas. Fajola and Alasoadura (1975) reported the antagonistic properties of Trichoderma against Pythium aphanidermatum, the damping-off agent of tobacco in Nigeria. Hoch and Abawi (1979) discovered that Corticium sp. was an effective biolo­gical control agent against Pythium ulti11jium induced damping-off of table beets. Pseudomonas has been used extensively as a biological control agent against wilts caused by Fusarium (Lemanceau and Alabou­vette 1993; Sivamani and Gnanamanickam 1988; Ziegle and Correl1988). This bacterium has also been used as an antagonist against the proliferation of Pythium. Loper_ (1988) demonstrated the role of fluorescent siderophore produced by Pseudomonas

MicrobioJ. Res. 150 (1995) 1 71

fluorescens in the biological control of Pythium ultimum. Howell and Stipanovic (1980) showed the role of an antibiotic: Pyolucteorin, produced by Pseudomonas fluorescens in the control of damping­off of cotton seedlings. Paul (1992) obtained the control of damping-off of pine seedlings caused by Pythium also by the use of Pseudomonas fluorescens. Antagonistic effects of actinomycetes have also been described against several species of Pythium (Knauss, 1976; Reddi 1972; Kusakari and Ueyama 1974; Tims 1932). Hadar et al. (1983) demonstrated the antago­nistic effects of Enterobacter cloacae on cucumber and pea rots caused by Pythium sp. Soil bacteria belonging to the genus Bacillus have also been known to suppress soil borne pathogens such as Phytophtho­ra cactorum (Gupta and Utkhede 1986, 1987), Bacil­lus cereus is known to be an antagonist towards Phytophthora megasperma f. sp. medicaginis, the cau­sal agent of the damping-off of alfalfa seedlings (Handelsman et al. 1990); Bacillus cereus is also known to suppress the cottony leak of cucumber's pathogen Pythium aphanidermatum (Smith et al. 1993).

Pythium mamilla tum is a soil fungus , characterized by its spiny oogonia and globose, ovoid or broadly ellipsoidal, non-proliferating sporangia (Plaats-Nite­rink 1981). It was originally described as causing damping-off of Beta vulgaris seedlings (Meurs 1928). This fungus is now known to be pathogenic to a wide variety of plants (Plaats-Niterink 1981). In this study we have found the fungus to be very pathogenic to cucumber seedlings and this is the first report of its biological control obtained by the soil bacterium Bacillus mycoides (SU-23).

Materials and methods

Bacillus mycoides strain SU-23 was isolated from a soil sample collected in Alma-Ata (Kazakstan). This was grown on nutrient agar slants and in Trypcase­soy broth. Pythium mamillatum (F -60) was isolated from a soil sample collected in Compiegne (France). This was cultured on solid media like potato carrot agar (PCA) or corn meal agar (CMA), and on boiled hemp-seed halves in water. All the cultures of bacte­ria and fungi were maintained at 25 °C. Seeds of cucumber were surface sterilized as described by Dodd and Stewart (1992) and were put in 3 plastic pots, each containing 30 g of compost soil and were sown with the treated seeds. These were humified with sterilized distilled water (SDW). All the three pots were kept at 25 °C for germination. Pot 1 served as "control" and was humidified with SDW only. Upon the first sign of germination (72 hours) the pots

72 Microbial. Res. 150 (1995) 1

1

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Fig. 1. Cucumber seedlings. a: pot 1 with cucumber seeds only - healthy seedling; b: pot 2 with cucumber seeds + Pythium mamilla tum - damped-otT seedling; c: pot 3 with cucumber seeds + Pythium mamillatum + Bacillus mycoides - healthy seedling.

11m IlItIIl/llUln

b

Fig. 2. Cucumber seedlings. a: healthy seedling without pathogen; b: damped-off seedling caused by Pythium mamillatum; c: healthy seedling resulting from antagonism between Bacilllus mycoides and Pythium mamillatum.

2 and 3 were innoculated with the fungus Pythium mamillatum: 7 days old, bacteria free fungal colonies, actively growing on hemp-seed halves in SDW were placed in the vicinity of the germinating cucumber grains. The pot 2 was humidified by SDW, while the pot 3 was humidified with 20 ml24 hours old Bacillus mycoides (SU-23) bacterial broth. All the pots were maintained at 25 °C for 7 days. The pots were regularly humified with SDW. This experiment was repeated three times. The fungus was identified with the help of keys provided by Middleton (1943), Waterhouse (1967), Plaats-Niterink (1981), and Dick (1990). The bacterial sample was identified by the microbiological department of the faculty of Phar­macy, University of Lille II.

4

Fig.3-5. Pythium mamillatum; Fig 3: sporangia. Fig. 4: sporangia and oogonia. Fig. 5: spiny oogonium attached to a sporangium.

Figures 3 - 5 bar = 25 Jlm.

Microbiol. Res. 150 (1995) 1 73

Results

The fungus (F -60): The fungus grows luxuriantly on boiled hemp seed halves in water, and has a daily growth rate of20 mm at 25 DC on PCA. Main hyphae are upto 7 - 8 /lm wide. Sporangia are globose, ovoidal, ellipsoidal to somewhat cylindrical, intercalary, sometimes termi­nal, measuring 13 - 28 /lm diam (av. 19.4 /lm). Oogo­nia intercalary or terminal, globose to slightly ovoi­dal, provided with blunt to conical and at times curved spines of 2 - 6 J.1m in length and 1 - 3 /lm in breadth, measuring 13 - 24 /lm in diameter (av. 18.3/lm). Antheridia mostly monoclinous, usually one, infrequently two per oogonium, antheridial cells clavate making apical contact with the female game­tangia. Oospores globose, plerotic, one per oogo­nium, measuring 12 - 22 (av. 16.4) /lm diam., provi­ded with a moderately thin wall.

The Bacterium (SU-23): The antagonist bacterium (SU-23) was isolated from a soil sample taken in Alma-Ata (Kazakstan). It is a readily recognizable species: Bacillus mycoides because of its distinctive rhizoid colonies covering the agar plates within 48 hours. The bacterial cells are rod shaped measu­ring 3 - 5 /lm in length and 1-1.5 /lm in width. They are Gram-positive bacteria, producing an ellipsoidal central spore.

All the three repeats of the above described experiment yielded the following results: Pot 1: Normal growth of the cucumber seedlings. Pot 2: Severe damping-off of the cucumber seedling. Pot 3: Better growth of the cucumber seedlings as compared to pot 1.

The damped-off material, when transferred to PCA or CMA plates yielded growth of Pythium mamilla tum.

The above results show that the fungus, Pythium mamillatum, is a pathogen of cucumber, causing post-emergence damping-off of its seedlings (pot 2); that the bacterium SU-23 is an antagonist to the fungus, as it completely suppresses its expression as a damping-off agent (pot 3), and is in some way also responsible for enhancing the growth of the seedling which in this case is healthier than pot 1.

Conclusion

Pythium mamilla tum is a well known fungus causing damping-off of Beta vulgaris (Meurs 1928), of conifer seedlings (Vaartaja 1967), causing poor ratooning of sugar-cane (Chu et al. 1966), and after artifical infection with this fungus severe damage was obser-

74 Microbiol. Res. 150 (1995) 1

ved in seedlings of lucerne, cauliflower, and gherkin (Schultz 1950). In this study it was found to be very pathogenic to cucumber seedlings bringing about damping-off symptoms within 7 days. The study also reveals that flooding the fungus with Bacillus mycoi­des causes not only a complete suppression of the parasite, but also better growth of the seedlings. Thus, biological control of the fungus by means of the bacterium Bacillus mycoides (SU-23) can be intended.

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