Induced Resistant Physiological And Biochemical Characteristics Of Banana By Bacillus Subtilis Tr21

Bacillus subtilis strain TR21can effectively control banana wilt disease in fields by spraying on banana leaf axils. In order to discover the mechanism, the colonization ability of TR21on banana leaves and the changes of banana disease resistant physiology and resistance related genes expression induced by TR21colonization were studied. First, green fluorescent protein (GFP) gene tagged TR21and its colonization on the Brazil leaves were researched. Secondly, the signaling messenger hydrogen peroxide (H2O2) dynamic accumulation in leaves caused by TR21inoculation on the roots were tested in two banana varieties (Musa spp. AAA cv. Brazil and Musa spp. AAA cv. Nongke No.1). Then, the dynamic changes of the root phenolic compounds and lignin contents in two genotypes of susceptible banana(Musa spp. AAA cv. Brazil and Musa spp. ABB cv. Guangfen No.1) were tested after inoculated by TR21on leaves or by Fusarium oxysporum f. sp. cubense race4strain FOC009on roots. Finally, the real-time quantitative PCR technique were used to analysis the expression of6resistance related genes, such as CAT (encoding Catalase), POD(encoding Peroxidase),PAL(encoding Phenylalanine ammonialyase), PAE(encoding Pectin acetylesterase), PR-1(encoding Pathogenesis Related Protein1) and PR-3(encoding Pathogenesis Related Protein3) of Brazil banana by inoculated by TR21on leaves, or FOC009on roots, or challenge inoculation of TR21and FOC009. The results as follows:1.By protoplast transformation method, plasmid pHT315-gfp was successfully imported into B. subtilis TR21strain, and the engineering strain TR21-gfp can steadily secrete green fluorescent protein. The colonization process of TR21-gfp strain on Brazil leaves were observed using Fluorescent microscope. After spraying3h, TR21-gfp mainly gathered on the surface of epidermal cells, but5h later, some entered the cortical cells through the intercellular space of epidermal cell, and into the mesophyll cells at7h. It was difficult to find any bacteria on the surface of epidermal cells after6d, but in petiole cells, TR21-gfp could be observe. It was showed that TR21can colonized in banana leaf and petiole.2. By DAB staining and quantitative determination of H2O2content, H2O2dynamic changes of banana leaves caused by TR21root inoculation on Brazil banana and Nongke No.1were checked. The results showed that either susceptible (Brazil banana) or resistant (Nongke No.1.) banana could be induced burst of reactive oxygen species in leaves by TR21inoculation on roots. The hydrogen peroxide content of TR21treatmen reached peak at12h after inoculation, but the content peak of chitosan treatment was8h. The concentration of inoculated TR21had dose effect on the hydrogen peroxide content,108cfu.mL-1TR21induced the highest hydrogen peroxide content. And plant disease susceptibility also influence on hydrogen peroxide production,10cfu.mL-1TR21could induced more hydrogen peroxide in resistance banana cultivar Nongke No.1than Brazil.3. Root inoculation with F. oxysporum f. sp. cubense race4strain FOC009were handled as a positive control, seedlings treated with water as a negative control, the root total soluble phenolic acids (RTSPA), cell wall bound phenolic acids (CWBPA), lignin content of seedling roots were tested after spraying TR21on leaves at0h,12h,24h,36h,48h,60h and72h. The results showed that in two susceptible banana cultivars Brazil and Guangfen No.l, followed the time expansion, RTSPA, CWBPA and lignin content was increased, at and after48h, RTSPA, CWBPA and lignin contents in FOC009and TR21treatments were significantly higher than water control in different banana cultivars. Except for RTSPA in Guangfen No.1, the RTSPA, CWBPA and lignin contents of FOC009treatment were significantly higher than TR21treatment in two susceptible cultivars. It was concluded that spraying leaf axils by TR21could induced the susceptible banana cultivars accumulating phenolic compounds and lignin on roots, and this were similar with the action induced by F. oxysporum, but the level induced by TR21were lower than pathogen.4. Adopting double standard curve method, bananas Actin gene as an internal reference, Real-time fluorescent quantitative PCR technique was used to study Brazil six kinds of disease-related gene expression caused by foliar spray inoculated TR21, roots inoculated with pathogen FOC009and first foliar spray inoculation TR213d after root challenge inoculation pathogen FOC009. The results show that, compare to water control, TR21treatment can quickly induce CAT and POD expression up-regulation, after12h, TR21treatment has significant difference with control, but can not induce expression of PAE. After48h TR21inoculation alone can cause PAL, PR-1and PR-3gene expression up-regulation. Challenge and pathogen FOC009treatments group can induce the expression up-regulation of all six genes, but the induced up-regulation ratio of genes in challenge group were significantly higher than the pathogen alone, which means that the endogenous bacteria TR21could induce the banana into priming state, if the priming banana suffer from diseases, the disease resistant response will become more faster and stronger.