一株桑树内生拮抗菌的分离、鉴定及发酵条件优化

Screening, identification and optimization of fermentation conditions of an antagonistic endophytic bacterium from mulberry

【目的】利用植物内生拮抗菌防治植物病害是一种有效的生物防治手段。本研究从健康桑树中分离筛选桑椹菌核病拮抗性内生细菌,为桑椹菌核病生物防治提供优良菌种。【方法】釆用组织分离培养法及抑菌圈法分离、筛选桑椹菌核病拮抗性内生细菌;根据菌体形态、培养特征、生理生化特性及基于16S rDNA序列的系统发育分析,对抑菌活性显著且稳定的菌株进行菌种鉴定;进而利用菌丝生长速率法检测活性发酵液的抑菌谱与热稳定性,并通过单因素及正交试验优化该菌株产生抑菌活性物质的发酵条件。【结果】从健康桑树中共分离获得55株内生细菌,其中XP-27菌株对核盘菌PZ-2的抑菌活性稳定且拮抗效果明显;XP-27菌株形态、培养特征、生理生化特性与芽孢杆菌属相符,基于16S rDNA序列的系统发育分析结果显示该菌株与多株甲基营养型芽孢杆菌(Bacillus methylotrophicus)的亲缘关系最近,且处于系统发育树的同一分枝,故将XP-27菌株鉴定为甲基营养型芽孢杆菌,命名为B.methylotrophicusXP-27;抑菌谱与热稳定性实验结果表明XP-27菌株对灰霉菌SWU5、腐霉菌SWU3等10种常见植物病原菌具不同程度的抑制作用,且其发酵滤液热稳定性强;发酵条件优化结果表明该菌株最佳培养基配方与培养条件为:牛肉膏1.00%,淀粉1.50%,K_2HPO_4 0.05%,MgSO_4·7H_2O 0.10%,初始pH值为8.0,培养温度为30°C,接种量为7%,发酵时间为120 h。【结论】筛选获得的桑树内生细菌B. methylotrophicus XP-27对桑椹菌核病病原菌核盘菌PZ-2具有显著拮抗作用,可作为开发桑椹菌核病生防制剂的候选菌株。

[Objective]Application of antagonistic endophyte is a potential strategy for biological control of plant diseases.In this study,the antagonistic endophytic bacteria were isolated and screened from healthy mulberry,to provide candidate strains for the biological control of mulberry fruit sclerotiniose.[Methods]Tissue isolation method was used to isolate the endophytic bacteria from healthy mulberry tissues.The endophytic bacterium with strong and stable antagonistic activity on the pathogens of mulberry fruit sclerotiniose was screened by inhibition zone method.The antagonistic bacterium was identified through morphological features,cultural,physiological and biochemical characteristics and 16S rDNA phylogenetic analysis.Antimicrobial spectrum and thermal stability of cell-free fermentation supernatant were assayed by mycelial growth rate.The fermentation conditions and medium composition were optimized through single factor and orthogonal experiment.[Results]In total 55 endophytic bacteria were isolated from healthy mulberry.Among them,strain XP-27 was found to have strong and stable antagonistic activity against Sclerotinia sclerotiorum PZ-2(the pathogen of mulberry fruit sclerotiniose). Morphological features,cultural,physiological and biochemical characteristics indicated that XP-27belongs to genus of Bacillus.Based on 16S rDNA phylogenetic analysis,XP-27was close to the Bacillus methylotrophicus and within the same clade with several strains of Bacillus methylotrophicus.Therefore,strain XP-27was identified as Bacillus methylotrophicus,and named to be B.methylotrophicus XP-27.Antimicrobial spectrum assayed results showed that strain XP-27 had variously antagonistic activity on 10 plant pathogens and had excellent heat stability. The results of fermentation optimization showed that the optimal medium was composed of 1.00% beef extract, 1.50% starch,0.05% K2HPO4,0.10% MgSO4·7H2O and the optimal culture conditions were inoculation size of 7% for 120h at 30℃ with initial pH 8.0.[Conclusion]The above results indicated that mulberry endophytic bacterium B.methylotrophicus XP-27has strong antagonistic activity on S.sclerotiorum PZ-2,being a candidate strain for developing the biological control agent against mulberry fruit sclerotiniose.