| Cucumber Fusarium wilt disease is one of the most serious diseases which is more epidemic under conditions of protected field cultivation at present. Since a lot of synthetic chemicals were used in disease prevention and control, it has already caused serious environmental pollution and economic loss. It seems urgently necessary to explore biopesticide that will provide effective control while minimizing negative consequences for human health and the enviroment. Ubiquitous in soils and plant rhizosphere, the antagonistic strains of Bacillus subtilis have shown great application and development value in the field of biocontrol and have the ability of high stress resistance and antifungal activities. In order to control cucumber Fusarium wilt disease efficiently, screening strong antagonistic strain of Bacillus subtilis and studying the mechanism and control efficiency in the field would not only be of great value theoretically, but practically as well.The antagonistic strain B29 was isolated from 10 soil samples of plant rhizo- sphere against Fusarium oxysporum, which was identified as Bacillus subtilis on the basis of its morphological, biochemical and physiological characteristics, and BIOFOSUN microbial identification and analysis.The culture medium and fermentation conditions were optimized. The results showed that optimal medium was composed of 1% glucose, 0.6% beef extract and 0.3% yeast extract. The optimal fermentation conditions were culture temperature 30℃, initial p H7.0~7.5, flasks containing 50 m L in 250 m L flask, inoculation age 15~18h and culture time 32 h. The fermentation of Bacillus subtilis B29 was studied with 20 L and 2000 L fermentor. In stable pilot-scale process, the agent production of Bacillus subtilis B29 reached 3.5×109cfu/m L.The indoor control experiment showed the EC50 of Bacillus subtilis B29 against Fusarium oxysporum was 9.17×105 cfu/ m L. The control efficiencies of 100(3.5×107cfu/m L), 250(1.4×107cfu/m L) and 500(7×106cfu/m L) dilution times of Bacillus subtilis agent to cucumber Fusarium wilt were 70.3~88.2%, 62.3~85.9% and 54.7~80.6% respectively in 4 field plots in 2 seasons in 1 year. The Fusarium wilt of cucumber was suppressed by using B29 to an average of 84.9% in field trials during 2 years, and the yield was increased by 12.57%.Toxicology, ecotoxicology and the safety to several major crops of Bacillus subtilis agent were studied. The acute toxicity of Bacillus subtilis strain B29 to big mouse through its mouth and skin was examined and the LD50 was beyond 5000 mg/kg. It showed no stimulation on rabbit eyes, less stimulation on rabbit skin and less allergic reaction on guinea pig skin. It was also low toxic to partridges, bees, zebra fish and silkworms. The safety of strain B29 to 9 major crops was investigated and no inhibition on their seedling rate, growth and development was found.The fermentation conditions of antifungal substance produced from strain B29 were optimized. The optimal fermentation conditions were shown with initial p H7.5, 75 m L/250 m L and at 30 ℃ for 120 h. The antifungal substance was precipitated by(NH4)2SO4 from 30% to 70%. The crude antifungal substance of strain B29 was stable to heat to some extent, partially tolerant to proteinase K, and more sensitive to trypsinase and pepsin. An antifungal protein was isolated from a culture of Bacillus subtilis B29. The isolation procedure comprised ion exchange chromatography on DEAE-52 cellulose and chromatography on Bio-Gel® P-100. The protein was absorbed on DEAE-cellulose and Bio-Gel® P-100. The purified antifungal fraction was designated as B29 I, with a molecular mass of 42.3 k Da by SDS-PAGE, p I value 5.69 by IEF-PAGE, and 97.81% purity as detected by HPLC. The PMF of antifungal protein B29 I was obtained by MALDI-TOF mass spectrometry, and it was an unreported protein as searched in the Mascot database. The amino acid of the antifungal protein B29 I was sequenced. The result showed that N-terminal amino acid sequences were GRIWHN and the three peptide segments amino acid sequences were KTHVLEDEFK, KGYQTGDFGAYLH, and RTYEVAEESPVLGL respectively. B29 I exhibited inhibitory activity on mycelial growth in Fusarium oxysporum, Rhizoctonia solani, Fusarium moniliforme and Sclerotinia sclerotiorum. The IC50 values of its antifungal activity toward Fusarium oxysporum and Rhizoctonia solani were 45 μM and 112 μM, respectively. B29 I also demonstrated an inhibitory effect on conidial spore germination of Fusarium oxysporum, suppression of germ-tube elongation, and induced distortion, tumescence, and rupture of a portion of the germinated spores.In order to investigate the colonization of Bacillus subtilis B29 in cucumber rhizosphere, the strain was marked by using the method of antibiotic(rifampicin) resistance or EYFP gene. The results showed that strain B29 colonized in the root, basal stem and rhizosphere soil of cucumber seedling after soaking seed or watering root treatment or combination treatment in greenhouse or in field. It was better for colonization by the treatment first soaking seed then watering root. The strong ability of Bacillus subtilis B29 to colonize helped it to compete site and nutrients with pathogens and then to inhibit pathogens effectively.The changes of defense enzymes induced by Bacillus subtilis B29 in cucumber roots were studied. Results showed that the activities of defense enzymes(PAL, PPO, POD) were increased to some extent after inoculation with Bacillus subtilis B29. Especially after challenging inoculation with Fusarium oxsporum, the changes of defense enzymes were increased much more than single inoculation. It suggested that challenging inoculation was good for induced systemic resistance. It could come to a conclusion that induced systemic resistance was one of biocontrol mechanisms combined with the control efficiencies of Bacillus subtilis B29 to cucumber Fusarium wilt disease. |
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