杜仲黑斑病菌(Pestalotiopsis trachicarpicola)拮抗细菌的分离和鉴定

Isolation and identification of antagonistic bacterial strain against Pestalotiopsis trachicarpicola

摘要:

【背景】杜仲黑斑病菌(Pestalotiopsis trachicarpicola)是引起杜仲黑斑病的新病原，当前尚未见生防菌对其产生拮抗作用的报道。【目的】从杜仲健康叶片上分离筛选出对P. trachicarpicola DZHBB-1拮抗效果最好的生防芽孢杆菌(Bacillus sp.)，利用16S rRNA基因结合蛋白质编码基因，实现对该菌株的快速准确鉴定。【方法】采用稀释分离法从健康杜仲叶片上分离菌株，通过平板对峙实验和生长速率法进行筛选，联合16S rRNA、gyrA、gyrB基因序列构建系统发育树分析，结合形态学特征及生理生化特性鉴定目标菌株，盆栽试验验证其对杜仲黑斑病的防治效果。【结果】总共分离得到62株芽孢杆菌，初筛出14株对病原菌有较好生防效果的菌株，复筛结果证明菌株J1拮抗效果最好且稳定，抑制率达到66.67%，16S rRNA、gyrA、gyrB基因序列系统发育树分析结果综合显示，菌株J1为解淀粉芽孢杆菌(Bacillus amyloliquefaciens)。盆栽试验结果显示，该菌能有效防治杜仲黑斑病的发生，防治效果均超过57%。【结论】该菌株具有防治P. trachicarpicola引起相关植物病害的生防潜力，16S rRNA、gyrA、gyrB基因联合鉴定芽孢杆菌，可为快速准确地鉴定芽孢杆菌及其近缘种提供借鉴方法。

Abstract:

[Background] Pestalotiopsis trachicarpicola is a new pathogen causing black spot disease of Eucommia ulmoides. There is no report that biocontrol bacteria had antagonistic activity on it. [Objective] The best Bacillus sp. antagonistic activity against P. trachicarpicola DZHBB-1 was isolated and selected from healthy leaves of E. ulmoides, the rapid and accurate identification of this strain was achieved by using the 16S rRNA gene binding protein-encoding gene. [Methods] The strains were isolated from healthy E. ulmoides leaves by dilution separation method, screened by sputum experiments and growth rate methods, combined with 16S rRNA, gyrA and gyrB gene sequences to construct phylogenetic tree analysis. The target strains were identified by its morphological characteristics, physiological and biochemical properties. The pot experiment was carried out to verify its control effect on black spot of E. ulmoides. [Results] A total of 62 strains of Bacillus were isolated and 14 strains with good biocontrol effect on pathogens were screened out. The results of rescreening showed that the strain J1 had the best antagonistic activity and was stable, the inhibition rate reached 66.67%. Phylogenetic tree analysis of 16S rRNA, gyrA and gyrB gene sequences showed that strain J1 was B. amyloliquefaciens. In the pot experiment, the strain could effectively prevent the occurrence of black spot disease of E. ulmoides and the control effect exceeded 57%. [Conclusion] This strain has the potential to prevent and cure the related plant diseases caused by P. trachicarpicola. 16S rRNA, gyrA and gyrB genes are combined to identify Bacillus sp., which can provide a reference method for the rapid and accurate identification of Bacillus sp. and its related species.