桑树内生拮抗菌的分离鉴定及其对桑断枝烂叶病的生防初探

Isolation and identification of an antagonistic endophytic bacterium from mulberry for biocontrol against Boeremia exigua

摘要:

[目的] 本研究从健康桑树茎中分离筛选对桑断枝烂叶病菌具显著拮抗作用的内生细菌，为该病生物防治奠定研究基础。[方法] 采用组织培养法分离桑树内生菌，抑菌圈法和平板对峙法筛选抑菌活性稳定的内生拮抗菌；根据形态学、生理生化特征检测和基于16S rDNA、gyrA和gyrB基因的系统发育分析对拮抗菌进行菌种鉴定；利用抑菌圈法测定拮抗菌株活性发酵液热稳定性，菌丝生长速率法检测活性发酵液抑菌谱；并通过观察拮抗菌对桑断枝烂叶病菌Boeremia exigua GXH1菌株生长及菌丝形态的影响，扩增抑菌活性物质合成关键基因，以及采用酸沉淀法提取拮抗菌株脂肽类化合物并进行高效液相色谱串联质谱分析（LC-MS），初步探究可能的抑菌机制。[结果] 从健康桑树茎中共分离获得17株桑树内生细菌，并从中筛选获得一株对桑断枝烂叶病菌B.exigua GXH1有稳定拮抗作用的桑树内生细菌NPJ13菌株。该菌株形态学、生理生化特征与芽孢杆菌属一致，基于16S rDNA、gyrA和gyrB基因序列的系统发育分析结果显示该菌株与贝莱斯芽孢杆菌（Bacillus velezensis）的亲缘关系最近，且处于系统发育树的最小分枝，故将NPJ13菌株鉴定为贝莱斯芽孢杆菌，命名为B.velezensis NPJ13。NPJ13菌株对灰霉病菌SWU5、核地杖菌SXSG-5、核盘菌PZ-2及烟草疫霉SWU20等12种病原真菌具有不同程度的拮抗作用，其活性发酵液具有较好的热稳定性。NPJ13菌株会导致桑断枝烂叶病菌GXH1菌丝发生扭曲、膨大、透明度增加、断裂等畸变现象；基因检测结果显示NPJ13菌株基因组中具有PKSI、NRPS、Sfp、ItuD、Srfc等5种抑菌活性物质合成关键基因，LC-MS检测结果表明菌株NPJ13脂肽类粗提物中含有表面活性素和伊枯草菌素。[结论] 本研究分离筛选获得一株对桑断枝烂叶病菌具有显著拮抗作用的桑树内生细菌B.velezensis NPJ13菌株，为桑断枝烂叶病的生物防治提供了候选菌株。

Abstract:

[Objective] Antagonistic endophytic bacteria were isolated and screened from the healthy stems of mulberry using Boeremia exigua (the pathogen of mulberry snags rotten leaves disease) as target, for the biological control of mulberry snags rotten leaves disease.[Methods] Endophytic bacteria were isolated from mulberry by tissue isolation method, and the antagonistic bacteria with stable antifungal activity were screened out by the combination of inhibition zone method and confrontation culture method. The antagonistic bacterium was identified through morphological features, cultural, physiological and biochemical characteristics and phylogenetic analysis based on 16S rDNA, gyrA and gyrB sequence. The thermal stability and antimicrobial spectrum of cell-free fermentation supernatant were assayed by inhibition zone method and mycelial growth rate method, respectively. Finally, the antimicrobial mechanism was explored through observation the affection of the antagonistic bacteria on B. exigua GXH1, amplification the key genes that were involved in the biosynthesis of antimicrobial active substances, and the lipopeptide compounds produced by antagonistic bacteria were extracted by acid precipitation and analyzed by LC-MS.[Results] In total 17 endophytic bacteria were isolated from healthy mulberry. Among them, strain NPJ13 exhibited strong and stable antagonistic activity against B. exigua GXH1. Morphological features, cultural, physiological and biochemical characteristics analysis results indicated that NPJ13 belongs to genus of Bacillus. Phylogenetic analysis results based on 16S rDNA, gyrA and gyrB genes revealed that NPJ13 strain and several strains of Bacillus velezensis were in the same minimal clade. Therefore, strain NPJ13 was identified as Bacillus velezensis and named B. velezensis NPJ13. The results of thermal stability test showed that strain NPJ13 had superior heat stability. Antimicrobial spectrum showed that strain NPJ13 had different degrees of antagonism against 12 kinds of pathogenic fungi, such as Botrytis cinerea SWU5, Scleromitrula shiraiana SXSG-5, Sclerotinia sclerotiorum PZ-2, Phytophthora nicotianae SWU20 and so on. The hyphaes of B. exigua GXH1 treated by B. velezensis NPJ13 would become distorted, intumescent, broken and more transparent. The results of functional genes detection showed that the genome of B. velezensis NPJ13 contains 5 antimicrobial metabolite synthesis related genes, which are PKSI, NRPS, Sfp, ItuD, Srfc. Surfactins and iturins were detected from the lipopeptide compounds of strain NPJ13 by LC-MS.[Conclusion] The above results indicated that mulberry endophytic bacterium B. velezensis NPJ13 has strong antagonistic activity on B. exigua GXH1, being a candidate strain for developing the biological control agent against mulberry snags rotten leaves disease.