桑树内生枯草芽孢杆菌的分离鉴定及其对桑椹菌核病的生防机理

Isolation and identification of an endophytic Bacillus subtilis from mulberry and preliminary exploration of its biocontrol mechanisms against mulberry fruit sclerotiniose

【目的】从菌核病抗性桑树品种中筛选具有生防潜力的内生拮抗菌,为绿色防控桑椹菌核病提供优质菌种及有效策略。【方法】通过植物组织培养法及平板对峙培养法分离、筛选桑椹菌核病拮抗菌,根据形态学特征、生理生化特征和基于16S rRNA基因序列的系统发育分析对其进行菌种鉴定;分析拮抗菌抑菌谱及离体防效,评估其应用潜力;通过观察拮抗菌发酵上清液对病原菌菌丝生长的影响,检测拮抗菌作用后病原菌糖原和活性氧积累量变化,并测定病原菌基因表达变化,初步探究拮抗菌的抑菌机理。【结果】从健康桑枝中分离筛选出一株对桑椹菌核病菌(Sclerotinia sclerotiorum)PZ-2抑制作用强且稳定的内生细菌C1R32,其形态学和生理生化特征与芽孢杆菌一致,基于16SrRNA基因序列的系统发育分析显示该菌株与枯草芽孢杆菌聚在同一最小分支,将其鉴定为枯草芽孢杆菌(Bacillus subtilis)。B.subtilis C1R32对核盘菌在内的多种植物病原菌具有抑制作用;桑椹菌核病离体防效实验结果表明,B.subtilis C1R32菌悬液和发酵上清液处理组的防效分别为52.94%和46.43%;抑菌机理初探结果显示,B.subtilis C1R32发酵上清液能使S.sclerotiorum PZ-2菌丝膨大畸变,细胞壁破裂,胞质流出;该菌可通过减少S.sclerotiorum PZ-2糖原积累、促进活性氧迸发,影响其抗氧化相关基因的表达抑制病原菌。【结论】本研究从桑椹菌核病抗性品种中分离获得一株内生枯草芽孢杆菌,并初步探究其拮抗机理,为桑椹菌核病生物防治提供了潜在菌种资源。

[Objective]To provide candidate strains and effective strategies for the control of mulberry fruit sclerotiniose,we screened out the endophytic bacteria with biocontrol potential for mulberry fruit sclerotiniose from a resistant mulberry cultivar.[Methods]The endophytic bacteria antagonistic to mulberry fruit sclerotiniose were isolated from mulberry plants by the tissue culture and confrontation culture methods.The antagonistic strain was identified based on morphological features,physiological and biochemical characteristics,and the phylogenetic relationship based on 16S rRNA gene sequences.The antimicrobial spectrum and control efficiency to detached mulberry fruits were determined to evaluate the application potential of the antagonistic strain.Furthermore,we observed the inhibitory effect of the fermentation supernatant of the strain on the mycelial growth of the pathogen,measured the variations in glycogen and reactive oxygen species accumulation of the pathogen treated with the antagonistic strain,and determined the expression of pathogen-related genes after treatment with the antagonistic strain to decipher the antagonistic mechanism of this strain.[Results]An endophytic bacterial strain C1R32 with strong and stable antagonistic activity on Sclerotinia sclerotiorum PZ-2(the pathogen of mulberry fruit sclerotiniose)was isolated from a healthy mulberry branch.C1R32 showed similar morphological features and physiological and biochemical characteristics with Bacillus.The phylogenetic analysis based on 16S rRNA gene sequences revealed that C1R32 was located in the same clade with B.subtilis.Therefore,strain C1R32 was identified as B.subtilis.B.subtilis C1R32 had antagonistic activities against a variety of phytopathogens including S.sclerotiorum.The suspension and fermentation supernatant of B.subtilis C1R32 showed the control effects of 52.94%and 46.43%,respectively,on sclerotiniose of detached mulberry fruits.The cell-free fermentation supernatant of B.subtilis C1R32 caused the hypha swelling and distorting,cell wall breaking,and cytoplasm leakage of S.sclerotiorum PZ-2.Moreover,B.subtilis C1R32 inhibited S.sclerotiorum PZ-2 by reducing glycogen accumulation,promoting reactive oxygen species burst,and influencing the expression of genes associated with antioxidant activity.[Conclusion]We isolated an endophytic B.subtilis strain capable of controlling mulberry fruit sclerotiniose from a resistant mulberry cultivar and preliminarily explored its antagonistic mechanism,providing potential strain resources for the biocontrol of mulberry fruit sclerotiniose.