6株贝莱斯芽胞杆菌对土传病原菌的抑制活性及其作用机理

Inhibitory Activity and Mechanism of Bacillus velezensi Strains against Soil-borne Pathogens

【目的】明确6株贝莱斯芽胞杆菌(Bacillus velezensi)对7种土传病原菌的抑制活性,探究其拮抗机理,为土传病害的生物防治提供菌种资源和理论依据。【方法】采用平板对峙法测定拮抗菌株对7种土传病原菌的抑菌活性,用显微镜观察拮抗菌对病原物菌丝形态的影响;采用叶片离体接种法测定其防治效果,鉴别性培养基测定拮抗菌株产生的胞外酶,并用PCR扩增技术检测拮抗菌携带抗生素相关基因(mycB、fenB、ituA、sfp、bamC、erisA、spaS、bacA、yndJ和qk)。【结果】所有测试菌株(NN01、NN02、NN04、NN05、NN88和NN95)对核盘菌(Sclerotinia sclerotiorum)、灰葡萄孢菌(Botrytis cinerea)、立枯丝核菌(Rhizoctonia solani)、尖孢镰刀菌古巴专化型(Fusarium oxysporum f.sp.cubense)、齐整小核菌(Scleritium rolfsii)和烟草疫霉(Phytophthora nicotianae)等土传病原菌的菌丝生长均具有明显的抑制作用,其中对齐整小核菌抑制作用最明显;NN01、NN02、NN04和NN88对尖孢镰刀菌菌丝有较好的抑制效果,菌丝生长抑制率为40.56%~56.30%;抑菌带边缘菌丝的形态发生明显改变,原生质浓缩或外泄、菌丝破裂和颜色加深;所有拮抗菌株均能较好抑制桑白绢病和莴苣菌核病病斑的发展,防治效果分别为53.40%~71.32%和43.57%~65.68%,高于或与枯草芽胞杆菌对照防治效果相当;所有拮抗菌都能产纤维素酶和蛋白酶,除了NN95基因组不携带fenB,其余所有菌株都携带mycB、ituA、fenB、bacA和yndJ等脂肽类抗生素相关基因。【结论】所有测定的贝莱斯芽胞杆菌对6种土传病原菌均具有抑菌活性,能产纤维素酶和蛋白酶,携带mycB、ituA、fenB、bacA和yndJ等5种抗生素相关基因,具有防治土传病害的潜力。

【Objective】Inhibitory activities and antagonistic mechanisms of Bacillus velezensi(Bv)strains against certain soil-borne pathogens were studied in search for new venues of biological disease control.【Method】Antagonism of 6 Bv strains,NN01,NN02,NN04,NN05,NN88,and NN95,on 7 soil-borne pathogens,Fusarium oxysporum f.sp.cubense,Sclerotinia sclerotiorum,Scleritium rolfsii,Botrytis cinerea,Rhizoctonia solani,and Phytophthora nicotianae,were studied using a plate confrontation test.Morphological responses of the pathogen mycelia to the Bv strains were observed under an optic microscope,and the control efficacy verified in vitro on mulberry and lettuce leaves.Activities of the extracellular enzymes(e.g.,cellulase,protease,andβ-1,3-glucanase)produced by the Bv strains were identified with differential media,and 10 antibiotic-related genes(i.e.,mycB,fenB,ituA,sfp,bamC,Erisa,spaS,bacA,yndJ,and Qk)in the bacteria detected by PCR amplification with specific primers.【Result】To varying extents the 6 Bv strains inhibited the mycelia growth of the 6 target pathogens.The strongest effect was shown on S.rolfsii.NN01,NN02,NN04,and NN88 displayed inhibition rates ranging from 40.56%to 56.30%on F.oxysporum.The appearance of mycelia on edge of inhibition rings changed significantly with broken,leaking intracellular substances,and darked color.In vitro the Bv strains significantly inhibited disease development on the plant leaves by S.sclerotiorum with a control effect of 53.40-71.32%and 43.57-65.68%by S.rolfsii,which were higher than or equal to the inhibition by B.subtilis.All 6 Bv strains secreted proteases and cellulases with the presence of 5 lipopeptide antibiotic-related genes(i.e.,mycB,fenB,ituA,bacA,and yndJ),except no fenB found in NN95.【Conclusion】All 6 Bv strains had varying inhibitory effects against the 7 soil-borne pathogens.They showed extracellular protease and cellulase activities,and almost all of them carried 5 lipopeptide antibiotic-related genes.These Bv strains could potentially be applied as biocontrol agents for control of diseases caused by the soil-borne pathogens.