| Marinus Bacillus strain TC-1is a potential biocontrol agent against a variety of plantpathogens.Biocontrol mechanism of strain TC-1against banana Fusarium oxysporum andeucalyptus Ralstonia solanacearum was observed through the pot experiment. And themechanism of antimicrobial protein was further discussed after been purified andidentificatied. The results were as follows:1、Biocontrol mechanism of strain TC-1against banana Fusarium oxysporum wasobserved based on colonization、control effect and growth-promoting and inducedresistance. The results showed that:(1) strain TC-1could colonize on the the roots, stems,leaves and the rhizosphere ofbanana and the organisms could maintain with8.7×10~4、5.7×10~3、1.0×10~3and2.0×10~5cfu/g after120d, showing good adaptability and stability of the environment.(2) strain TC-1also significantly reduced Fusarium oxysporum in the pot experiment.The banana seedlings increased significantly (P<0.05),treated by strain TC-1, with the plantheight increased by27%after treated90days. When the Disease Severity Index (DSI) forleaf symptoms was38.3, the preventive effect of TC-1reached35.3%,and the treatmenteffect to29.4%; In contrast with the DSI for rhizome discoloration by60.7, both thepreventive effect and the treatment effect of TC-1reached10%.(3) there were no significant differences(P>0.05) on the five kinds of defense enzymeactivities in unvaccinated bananas between banana seedlings treated with Marine bacteriaTC-1and seedlings with untreated. The five kinds of defense enzymes activities weresignificantly induced (P<0.05) in the banana seedlings inoculated Fusarium oxysporumafter treated with TC-1. The activities of POD and PPO in leaf were respectively increased248%and12%at6day after treatment (DAT);the activities of PAL and SOD wererespectively increased16%and10%at9DAT; Simulta-neously, the activity of CAT wererespectively increased363%at12DAT,compared with untreated plant. It was concludedthat strain TC-1can played an important role on banana resistance to infection Bananavascular wilt by inducing the activities of banana plant CAT, POD PPO, PAL and SODimprove. 2、The crude protein was extracted by adding ammonium sulfate to culture filtratewhich was heated in water bath at100℃after20min with a saturation of50%-60%. Theisolation and purification were operated by sephadex G-50molecular sievechromatography column.Two single bands in SDS-polyacrylamide gel electrophoresiswere obtained which molecular weight were50kD and38kD respectively. Antimicrobialproteins were identified by the whole mass spectrum analysis,including nine antimicrobialproteins respectively: a member of the following regulons, AprE, nprE, Translationelongation factor Tu (Fragment), YhfE, Dps, MrgA, Putative uncharacterized protein,Endoxylanase glycosyl hydrolase family11protein.And the relative molecular mass of theproteins were alkali protease and neutral protease.3、The antifungal mechanism of antimicrobial protein was studied by studying theeffect of TC-1of conidium germination, morphology of mycelia and spores, cellmembrane and cell wall of Fusarium oxysporium. The results are as follows:(1) The effect of antimicrobial protein on the conidium germination of bananaFusarium oxysporum. The antimicrobial protein was diluted gradiently to deal with sporesuspension of banana Fusarium oxysporum, it was determinated that the EC50of inhibitingspore germination was11.97μg/ml. inhibition of spore germination was70%as theconcentration of untimicrobial protein being14μg/ml.(2) The effect of antimicrobial protein on the morphology of mycelia and spores. Themisshapen mycelia and vesicles existing in top or middle of mycelia treated byantimicrobial protein were observed under optical microscope. Under scanning electronmicroscope, the surface of deformed mycelia became rough and the appearance of swollenvesicle exhibited sags and crests and had some debris.(3) The leakage of cell membrane of mycelia treated by antimicrobial protein wasdetected by electrical conductivity.The antimicrobial protein was added in the sporesuspension of banana Fusarium oxysporum which density was1.0×106cfu/ml, making itsend concentration of14μg/ml. The conductivities of them which were diluted by100times were measured. The results showed that, the conductivity was significantly increasedtreated by antimicrobial protein.It was concluded that antimicrobial protein coulddestroyed the cell membrane permeability to inhibit pathogen growth.(4) The leakage of cell wall of mycelia treated by antimicrobial protein was detectedby alkaline phosphatase changes. The antimicrobial protein was added in the sporesuspension of banana Fusarium oxysporum which density was1.0×106cfu/ml, making itsend concentration of14μg/ml. the influence of antimicrobial protein on the cell wall was measured through the medium of the content of alkaline phosphatase. The results showedthat, alkaline phosphatase activity increased apparently treated by antimicrobial protein. Itwas concluded that antimicrobial protein could destroyed the cell wall permeability toinhibit pathogen growth. |
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