Investigation On The Biocontrol Mechanism Of Bacillus Subtilis Strain R31Against Banana Fusarium Wilt

Bacillus subtilis strain R31was isolated from Dendrobium leaf, its biological characteristics, capability to control banana Fusarium wilt in field and fermentation technology has been studied. This thesis focus the emphasis on its biocontrol mechanism. Firstly, pot experiment was conducted to test the control effect of R31to Panama disease of Musa spp. AAA cv. Brazil cased by Fusarium oxysporum f. sp. cubense Race4. Secondly, the qualitative research on the antimicrobial substances generated by R31was performed, such as whether R31had the ability to produce volatile antagonistic substance, the antagonistic activity of fermentation supernatant of R31, whether R31could produce lipopeptide and its antagonistic effect, whether R31could produce antagonistic protein and its effect. Thirdly, the colonization ability of R31in the banana root and rhizosphere was determined by antibiotic marked R31. Then, the biofilm forming ability of R31and its influencing factor was determinated. Finally, R31was marked by green fluorescent protein and its colonization in banana root was tested. The results were as follows:(1) Pot experiment of banana Fusarium Wilt control by R31:Pot planted banana seedlings were inoculated by R31and F. oxysproum f. sp. cubense Race4strain FOC009, the results showed that the control effects of fermentation solution of R31were74.19%.(2) Qualitative research of antagonistic substances produced by R31:R31could not produce volatile substance; R31could grow in the culture medium that chitin as sole carbon and nitrogen source, but chitinase activity of fermented solution and broken cells had not been detected by the method of DNS; Protease activity was only detected when R31was fermented in corn soybean meal medium, although the NB and banana tissue extract liquid culture medium were tested at the same time.(3)The antifungi ability of R31fermentation supernatant:suppression of R31fermentation supernatant cultured in NB medium for24h,48h and72h on FOC009was compared, and it was found that the highest inhibition rate,16.87%, was by48h fermentation supernatant. Temperature and pH had significant influence on the antifungi ability of supernatant.(4) The antagonistic protein of R31:The antagonistic protein produced by R31were precipitated by different concentrations of saturated ammonium sulfate. It was found that proteins precipitated by different concentrations of ammonium sulfate all had inhibition effect on FOC009, but the highest inhibition rate was31.32%, which was expressed by90%ammonium sulfate. After treated with boiling water and proteinase K, the inhibition of antagonistic proteins did not significantly weakened.(5)The lipopeptid of R31:lipopeptid produced by R31was precipitated by acidand its inhibitory rate on FOC009was32.04%. After purification, the inhibition rate of methanol dissolving component was34.47%. The crude lipid peptides component had hemolytic activity, oil discharge capacity and droplet collapsed capacity suggest surfactin was one component of crude lipid peptides. Primers were designed according to surfactin synthesis essential gene sfp, the sfp gene sequence of R31was cloned to prove that R31had the ability to produce surfactant.(6) Colonization ability of strain R31on banana root:R31was marked by antibiotic, and pot planted banana seedlings were inoculated by R31to determine its colonization ability in Brazil banana and dwarf banana rhizosphere. Each seedlings inoculated with50ml1.78×10cfu/ml bacteria liquid,60d after inoculation, the marked strains in banana rhizosphere soil, root surface, root and corm were4.14×106cfu/g (dry),1.09×107cfu/g (fresh weight, FW),104cfu/g(FW),4.66×104cfu/g(FW) and1.23×103cfu/g(FW), respectively. Similarly,40d after inoculation R31*in Guangfen No.1, the number of R31*on rhizosphere soil and root surface were9.87×105cfu/g (dry) and7.9×104cfu/g(FW), respectively. It was found that R31*could colonize the rhizosphere soil and root surface of banana successfully, but the colonization quantity is less than rhizosphere in root and corm. And the population of R31*in rhizosphere soil and root surface have a relationship with inoculation concentration and daily maximum temperature.(7) Biofilm formation of R31:R31had the capability to form biofilm, and the biofilm were influenced by fungi toxin produced by FOC009.(8) Green fluorescence label of R31:R31were marked by green fluorescent protein vector PHT315-gfp, and gained visible fluorescent marker strains. The antagonistic stability and fluorescence stability of marked strain were tested, results showed that antagonistic activity of fluorescence labeled strains did not disappear. The banana root was green after inoculated by R31-gfp when checked in microscope.