5.2.59
IN VITRO STUDY OF THE MODE OF ACTION OF A NEW BIOCONTROL AGENT, MICROSPHAEROPSIS SP.

M BENYAGOUB1, N BENHAMOU2 and O CARISSE1

1Agriculture and Agri-Food Canada, 430 Boul. Goum, St-Jean, Québec, Canada; 2Dept of Plant Science, Laval University, Québec, Canada

Background and objectives
Recently, Microsphaeropsis sp. was described as a potential biocontrol agent against the apple scab agent, Venturia inaequalis [1]. In an attempt to better understand its mode of action, the interaction between this fungus and some phytopathogenic fungi was studied by using ultrastructural and cytochemical investigations. In addition, the culture filtrate of the antagonist was screened for the detection of antifungal molecules.

Material and methods
Mycelial samples from the interaction region between Microsphaeropsis sp. and its hosts were collected, fixed in glutaraldehyde, rinsed in cacodylate buffer and post-fixed in osmium tetroxide. They were then dehydrated in ethanol and embedded in resin. Labelling with the gold-complexed exoglucanase for the detection of cellulose was performed by incubating the ultrathin sections a drop of the enzyme-gold complex. After a washing, sections were contrasted with uranyl acetate and lead citrate. For the indirect labeling of chitin, sections were incubated on a drop of wheat germ agglutinin (WGA) and finally incubated on a drop of the ovomucoid-gold complex. Sections were contrasted as described above. Concerning the detection of antifungal molecules, one litre of culture filtrate of Microsphaeropsis sp was extracted with chloroform and analyzed by thin layer chromatography (TLC). The antifungal molecules were isolated from the crude extracts by preparative TLC and flash chromatography.

Results and conclusion
Cytological observations revealed that interactions between Microsphaeropsis sp., V. inaequalis and Rhizoctonia solani involved a sequence of events: (i) attachment and local penetration into the host hyphae; (ii) induction of host structural response; (iii) alteration of host cytoplasm; and (iv) active multiplication of the antagonist in the pathogen. The detection of cellulose on the cell wall of V. inaequalis refutes the classification of ascomycetes as only a glucan-chitin group. At an advanced state of parasitism, the labelling pattern of chitin and cellulose in V. inaequalis, which clearly showed that the level of integrity of these compounds was affected, suggested the production of chitinolytic and cellulolytic enzymes by the antagonist. The chitinolytic activity was also detected against R. solani. Wall appositions formed in response to antagonist attack contain both cellulose and chitin in V. inaequalis and only chitin in R. solani.

Chronological events associated with the degradation of Botrytis cinerea by Microsphaeropsis sp. were also studied using ultrastructural and cytochemical investigations. The first cytological changes were observed 48 h after the contact between the two colonies, and were characterized by punctuated invaginations of the B. cinerea cell wall and plasmalemma. These reactions were accompanied by disorganization of the cytoplasm. The first evidence of penetration of B. cinerea was recorded 96 h after contact. The evidence of chitinolytic degradation of host cell wall was confirmed by the random and reduced presence of gold particles over the host cell wall. These results suggest that Microsphaeropsis sp. antagonized B. cinerea first and foremost by antibiosis, leading to cell death, followed by degradation of the cell by means of chitinolytic enzymes. Against Pythium ultimum and Sclerotinia scleotiorum, cytochemical investigations revealed that the antagonist displayed the antibiosis as a sole mode of action. In an attempt to identify which molecules are involved in antibiosis, at least two compounds with antifungal activity were revealed by a bioassay on TLC plates. The synthesis of these antimicrobial molecules by Microsphaeropsis sp. would explain its strong activity as an antagonist against several phytopathogenic fungi.

References
1. Philion V, Carisse O, Paulitz T, 1997. European Journal of Plant Pathology 103:441-452.