一株花椒根腐病拮抗菌的分离鉴定及全基因组序列分析

Isolation, identification, and whole genome analysis of a strain against Zanthoxylum bungeanum root rot

摘要:

【背景】花椒根腐病的防治一直是生产中难以解决的问题，优良生防菌的筛选是微生物菌剂研发的重要方向。【目的】解析花椒根腐病拮抗菌T-1的遗传信息，深入挖掘其拮抗基因簇资源，揭示该菌的拮抗机制。【方法】采用平板对峙法、形态观察、生理生化测定结合分子生物学等方法进行拮抗菌的分离鉴定，同时对菌株进行全基因组测序，并对其序列进行分析及比较基因组学分析。【结果】分离获得的菌株经鉴定为贝莱斯芽孢杆菌，编号T-1，该菌对花椒根腐病的抑制率可达72%，可使菌丝前端的生长严重受阻，抑菌谱检测和花椒根片的离体拮抗试验结果表明，拮抗菌T-1具有较广的抑菌活性且离体状态下对花椒根片具有一定的拮抗作用。其全基因组序列数据提交到NCBI的SRA数据库中获得登录号为SRX11086663，基因组总长为3 886 726 bp，GC含量为46.42%，全基因组中有4 015个编码基因，占总基因组的89.74%，比较基因组学分析结果显示，菌株T-1与贝莱斯芽孢杆菌模式菌株FZB42相似性高，拮抗基因簇预测结果发现B. velezensisT-1基因组序列中有12个编码次级代谢产物基因合成簇，其中8个与已知功能基因簇高度相似，分别为butirosin A/butirosin B、macrolactin H、backland、泛革素(fengycin)、difficidin、bacillibactin、溶杆菌素(bacilysin)和表面活性素(surfactant)，4个基因簇功能未知。【结论】本研究明确了拮抗菌T-1全基因组的遗传信息，获得了拮抗基因相关的基因簇，为后期研究该菌株在抑菌方面的分子机理提供了参考。

关键词:贝莱斯芽孢杆菌;全基因组序列;拮抗基因;次级代谢产物

Abstract:

[Background] It remains a challenge to prevent and control Zanthoxylum bungeanum root rot in production, and the screening of biocontrol bacteria for the development of microbial agents seems to be a promising solution. [Objective] To analyze the genetic information of the antagonistic strain T-1, explore the root rot-antagonizing gene clusters, and reveal the antagonistic mechanism. [Methods] The methods of plate confrontation, morphological observation, physiological and biochemical index determination, and molecular biology were used to isolate and identify the antagonistic bacteria. The whole genome of the strain was sequenced, followed by sequence analysis and comparative genomics analysis. [Results] The strain was identified as Bacillus velezensis and numbered T-1. It inhibited 72% of the Fusarium solani, the pathogen of Z. bungeanum root rot, and hindered the growth of the front end of the mycelia. The results of in vitro antagonism experiments showed that T-1 had a wide range of antibacterial activities and had certain antagonistic effect on Z. bungeanum root pieces in vitro. Its whole-genome sequence data were submitted to SRA of NCBI to yield the accession number of SRX11086663. The genome was 3 886 726 bp, with GC content of 46.42% and 4 015 coding genes (89.74% of the genome). Comparative genomics analysis suggested that it had a high homology with the model strain B. velezensis FZB42, and the antagonistic gene cluster prediction indicated 12 gene clusters encoding the secondary metabolites in T-1 genome. Eight of them had functions known (butirosin A/butirosin B, macrolactin H, backland, fengycin, difficidin, bacillibactin, bacilysin, and surfactant), and the rest four had functions unknown. [Conclusion] This paper dissects the whole genome of B. velezensis T-1 and clarifies the gene clusters related to antagonism, which can serve as a reference for further research on the molecular antibacterial mechanism of this strain.

Key words:Bacillus velezensis;whole genome sequence;antagonist genes;secondary metabolites