Investigation On The Biocontrol Mechanism Of Bacillus Subtilis Strain TR21 Against Banana Fusarium Wilt

In order to expedite investigation of biocontrol agents against banana fusarium wilt and its mechanism, the biochemical and molecular identification, biological characteristics, enzyme-producing abilities, antagonistic properties and its component of a banana fusarium wilt biocontrol Bacillus strain TR21 had been accomplished. And the biocontrol effect, colonization of the strain TR21 and the related enzymes and genes of disease resistance of banana seedlings were investigated. Insight into mechanisms involved in TR21 control pathogen would lead to further research and application of Bacillus strain TR21. The results were as follows:1,Based on the API 50 CH system tests, the strain TR21 were as similarity as 90.3% with B. subtilis. Combined with 16S rDNA, gyrA and gyrB gene sequence analysis, the strain TR21 was confirmed as B. subtilis.2,The determination results of biological characteristics showed that the growth pH range of TR21 strain was pH 5.0～9.0, and the optimum pH was pH 6.0, glucose was the optimum carbon source, and yeast extract was the optimum nitrogen source, it got the stationary growth phase by cultured in NB medium in 14 hours. The strain TR21 could produce extracellular protease in NB medium, and couldn't produce protease and chitinase in Musa AAA tissues extract medium. The strain TR21 antagonized a broad spectrum of plant pathogenic fungi. The extracted extracellular crude protein and lipopeptide from fermentation broth filtrate of strain TR21 had antagonistic activity against Fusarium oxysporum f. sp. cubense.3,Pot planted banana seedlings were inoculated by injuring root and dropping leaf axil methods, the results showed that the control effects of fermentation solution of strain TR21 were (81.10±2.80)% and (79.32±4.49)% for 40 d after inoculating the pathogen, respectively. Inoculated by injuring root and drenching root methods, the population of marked strain showed the downward trend generally in banana plant, root surface and rhizosphere soil during 1 to 15 d after inoculation. Inoculated by dropping leaf axil method, the marked strain could only colonize in leaf, the population showed a trend "first increasing then decreasing". The TR21 strain could colonize and transfer in banana tissues when inoculated by three methods, and it also displayed good colonization in the rhizosphere soil and root surface. It suggested that induced systemic resistance was the main antagonistic mechanism of the strain TR21 against Fusarium wilt pathogen.4,After being inoculated with different fractions of TR21 fermented liquor by drenching root method to reveal the disease control mechanism. The results showed that the PPO, POD and PAL activities of banana roots inoculated with TR21 fermented liquor, cells suspension, a cell-free fermented supernatant fluid and NB liquid medium were always higher than those just inoculated with sterile water control, but the roots inoculated with TR21 fermented liquor or cells suspension had higher activities of PPO, POD and PAL than those inoculated with a cell-free fermented supernatant fluid and NB liquid medium control. Therefore, the main bioactive fraction in the strain TR21 fermented liquor was cell. Banana seedlings were inoculated with TR21 cells suspension by drenching root method, and the related enzymes activities of disease resistance in leaves were determinated. The results showed that the leaves had higher activities of PPO, POD and PAL than those inoculated with sterile water control. It suggested that induced systemic resistance was one of antagonistic mechanism of the strain TR21 against banana fusarium wilt.5-. The effect of strain TR21 on expression of four defense-related genes in roots of Musa AAA Cavendish subgroup cv. Brazil was investigated by semi-quantitative RT-PCR method. The results showed that the expression of PAL,POD,PR-3,PR-1 was up-regulated after being inoculated with strain TR21, but PAL,POD had higher increase amplitude. The expression of PAL,POD reached the maxium in roots at 12 h after inoculation, it was similar with colonization of strain TR21 in roots of Musa AAA Cavendish subgroup cv. Brazil. It suggested that induced systemic resistance was main antagonistic mechanism of the strain TR21 against banana fusarium wilt.