Screening Of Biocontrol Bacteria Against Botrytis Cinerea In Strawberry And Research On Its Antibacterial Mechanism

Botrytis cinerea in strawberry is one of the major constraints which made the yield and quality reduced. The traditional chemical control has led to the formation of serious resistance and environmental problems. The microbial control of Botrytis cinerea in strawberry with safety and low toxicity is an important and effective way. In this research, we hope to get the antagonistic strain by screening the bacteria isolated from rhizosphere soil of strawberry, strawberry plants and other plants and study identification of the strains, fermentation optimization, and its antagonistic reaction mechanism.The main results are as follows:1.Six strong antagonistic strains were isolated from the rhizosphere soil of strawberry, in them, strain SL₆ isolated from the leaf of strawberry was found to have the best control efficacy, the inhibition zone diameter was 15 mm and the inhibition rate could reach 97.6% with the fermentation filtrate, revealing potential in the aspect of antagonistic action. Based on the morphological,physiological,biochemical characteristics and 16S rDNA sequence analysis,through its 16S rDNA phylogenetic tree constructed with MEGA4, SL₆ was identified as Bacillus subtilis.2. Study on inhibition mechanism of strain SL₆ demonstrated that metabolites produced by antagonistic strain SL₆ could do great damage to the mycelia and had inhibitory effects on the growth of Botrytis cinerea. Microscopic observation indicated that the mycelium inhibited by strain SL₆ mostly twisted, inflated, deformed, the mycelia wall broke, the protoplasm leaked from the wound and even hyphae lysed. The bacterial metabolites in the culture filtrate could be precipitated by 70% ammonium sulfate and its inhibition activity was similar to the damage of mycelium by strain SL₆, while the supernate couldn't inhibit the pathogen growth, thereby indicating that the antagonistic substance was protein. The optimal culture condition of SL₆ was shaking culture at 37℃with initial pH 7.0 in 100mL PSB liquid medium for 3 days.3. The bacterial metabolites in the culture filtrate could be precipitated by 70% ammonium sulfate. The crude protein was obtained by the dialysis of the precipitate and purified by using ammonium sulfate precipitation following DEAE Sepharose Fast Flow chromatography. There were three bands with similar molecular weight in SDS-PAGE and PAGE of the active substances which had high protease activity. By mass spectrometry, three proteins were identified respectively as 30S ribosomal protein, nuclease subunit and uncharacterized protein. The crude protein was rather stable, its antibacterial activity remained above 60% after treated for 30 min at 100℃, and its pH ranged from 2.0 to 9.0.4. The fermentation filtrate of SL₆ showed effective control to Botrytis cinerea in strawberry in field experiments. After three treatments, the effect of fermentation broth was 86.6% and effectively inhibited the spread of disease. It could arrive to the conclusion that it could play a stable role in the long-term natural environment.Studies had shown that biological agents, which could prevent or reduce gray mold of strawberry flowers from infection, can effectively control gray mold damage, and endophytic bacteria can play the role. The endophytic bacteria of strawberry could promote the plant growth by producing indole acetic acid or dissolving organic phosphorus. But their roles in biological control have not yet been reported at domestic and overseas reports. This highly effective strain was selected from strawberry leaves, whose fermentation liquid could play a very good role in the field test. Studying the biological control mechanisms and their associated proteins which were expected to be applied to production had important theoretical and practical significance.