Study On Antagonistic Bacteria For Biocontrol Of The Fusarium Wilt

Fusarium wilt is a destructive soil-borne disease of the crops. It has been paid more attention to biocontrol of the disease by many countries. To obtain satisfactory biocontrol agents for the successful management the present study was undertaken in antagonistic bacteria screening, mechanisms of the disease inhibition, biosafety, conditions for fermentation, colonization, extraction of the inhibition substance against the pathogen.The potential biocontrol bacteria against Fusarium oxysporum f. sp. radicis lycopersici causing tomato wilt were screened from the soil samples of different ecosystems (Zhejiang, Xinjiang, Sichuang and Fujian Provinces) by plate inhibition method combining with pot growing method. One hundred-sixty-nine isolates of Bacillus spp. were obtaind and fifteen of them showed strong antagonistic effect against the pathogen. JK-2 identified as Brevibacillus brevis exhibited 83.82% and 74.70% of control effectiveness in pot and field experiment respectively. JK-2 strongly inhibited mycelium growth and spore germination of F. oxysporum f. sp. radicis lycopersici. The results of observation under scanning electron microscope illustrated that JK-2 could dissolve the mycelium, produce bubbles in the cell plasma, destroy the growth points and cause the leak of cytoplasmic substances of the pathogen. The interaction of JK-2 and F. oxysporum f. sp. radicis lycopersici on wounded tomato roots was studied. It would be supposed that the mode of action of JK-2 was antibiosis. Single-factor design and multiply-factor design were used for optimizing culture conditions of JK-2. The results showed that the optimal cultural media for antibiotics production was composed of 1.0% starch, 0.5% beef extract, 0.3% peptone, 1.0% sucrose, 0.5% yeast extract and 0.5% CaCl₂ with initial pH 7. JK-2 was nopathogenic and its 50% lethal dosage for white mouse was LD₅₀>2000mg/kg. The bacterial and actinomycetic community in the soil was not significantly different 30 days after inoculation of JK-2. It indicated that JK-2 is safety used as a biocontrol agent in field. The crude antibiotic extract of JK-2 was not sensitive to trypsin, acid, alkaline and ultraviolet rays but it denaturalized to some extent under 100℃. One antagonistic peptide with MW of 4.1kD and one polysaccharide were extracted and purified from the crude antibiotic substances. This is the first report on the antifungal polysaccharide secreted by Brevibacillus brevis. To monitor the colonization of JK-2 in the environment, a green fluorescent protein (GFP) expression and erythromycin resistant plasmid PCM20 was used and transformed into the JK-2 strain by electroration. The results showed that the JK-2-gfp maintained a population of 10⁴-10⁶cfu/g in the rhizosphere of tomato 60 days after watering roots inoculation .Endophytic bacteria in healthy and Fusarium-infected banana plants collected from Zhangzhou, Fujian province were surveyed for their distribution patterns and antagonistic activities against Fusarium wilt pathogen. The results showed that endophytic bacteria population in healthy banana plants was significantly higher than that in diseased ones. Except for rhizome, the top of pseudostem and leafstalk, the endophytic bacterial populations of other parts of healthy plants were significantly higher than those of the diseased plants. The population distribution of different endophytic bacterial species were different between the two types of banana plants. There was no Bacillus megaterium isolated from healthy banana plants and no Acinetobacter baumannii and Microbacterium chocolatum found in diseased ones. Four hundred and eleven bacterial strains were isolated and screened for antagonists against Fusarium oxysporum f. sp. cubertse in vitro. The isolation frequency of the endophytic antagonistic bacteria was about 8.52 % of the total bacteria obtained. Eight isolates exhibiting strongly antagonism against the pathogen were identified at species or biotype level by FAME-GC. They belong to Pseudomonas aeruginosa, Zoogloea ramigera, P. putida biotype and P. aeruginosa. H4-3-1 exhibited strong antagonism against Fusarium oxysporum f. sp. radicis lycopersici, F. oxysporum f. sp. cubense, F. oxysporum f. sp. niveum, F. oxysporum f. sp. vasinfectum, Botrytis cinerea, Sclerotinia sclerotiorura et al. It strongly inhibited mycelium growth of F. oxysporum f. sp. radicis lycopersici. The results of observation under scanning electron microscope showed that the terminal mycelia were distorted and swollen and cell wall was ruptured after treated with H4-3-1 filtrate culture. It promoted the growth of tomato significantly in the pots. The optimal cultural period of H4-3-1 for antibiotics production was 24h at 30℃in nutrient agar in 250-ml shake flask at 170 r/min. The aeration ratio was 25 ml, and the inoculation volume was 1%. Antagonistic crude protein extract of H4-3-1 was obtained by 70% ammonium sulphate precipitation. Antifungal substance of H4-3-1 was stable to heat, trypsin, acid, alkaline and ultraviolet rays. Isolate H4-3-1 was proved to be able to colonize in banana, tomato, eggplant, watermelon, melon and cucumber by watering roots or dipping seeds inoculation with the culture of rifampicin mutant strain. The results indicated that the colonization of H4-3-1 was not host specialization.