一株盐湖生防芽孢杆菌YH7-4对大豆根腐病的防效测定及全基因组测序分析

Biocontrol effect assessment and whole genome sequencing of Bacillus sp. YH7-4 from salt lake against soybean root rot

摘要:

层出镰孢菌(Fusarium proliferatum)是大豆根腐病的一种重要病原菌，前期从运城盐湖中分离到一株耐盐生防芽孢杆菌YH7-4。目的 探究菌株YH7-4对大豆生长的影响及对根腐病的防治效果，并从全基因组层面解析其抑菌机理、挖掘抗菌基因。方法 采用平板对峙培养检测YH7-4的抑菌效果；利用盆栽接种试验测定YH7-4对大豆幼苗的安全性和对根腐病的防效等；利用Illumina和PacBio平台相结合的测序技术对菌株YH7-4进行全基因组测序，并进行代谢系统分析、毒力基因预测、转运蛋白分析、与生防作用相关基因分析、比较基因组学分析以及生化测定等。结果 菌株YH7-4对大豆根腐病层出镰孢菌、大豆疫霉、炭疽菌、拟茎点种腐病菌等病原菌的抑菌率均达到75.00%以上。盆栽试验结果显示，YH7-4菌悬液在OD₆₀₀为0.8时对大豆幼苗的根长和干重有显著促进作用，浓度过高则失去促进作用；其对层出镰孢菌引起的大豆根腐病的防治效果达到56.02%。全基因组测序结果显示，其基因组全长为3 945 352 bp，G+C含量为46.51%，预测到3 756个编码基因。将预测得到的基因序列分别与NR、Swiss-Prot、Pfam、COG、GO、KEGG等数据库比对，分别有3 753、3 537、3 358、3 082、1 756和2 845个基因得到注释。其中，有130个蛋白质结构域属于CAZy家族；12个次级代谢产物基因簇，包括：8个已知抗生素合成基因簇(surfactin、macrolactin H、bacillaene、fengycin、difficidin、bacillibactin、bacilysin和butirosin A/butirosin B)和4个未知功能基因簇。此外，还发现2个与铁离子转运相关的基因、1个与诱导抗性相关的基因以及15个与生物膜形成相关的基因。比较基因组分析结果显示，YH7-4属于贝莱斯芽孢杆菌(Bacillus velezensis)，直系同源核心基因簇有2 898个。生化特征结果表明，YH7-4具有产生淀粉酶、蛋白酶、果胶酶、纤维素酶等特性。结论 从运城盐湖中分离到的耐盐贝莱斯芽孢杆菌YH7-4对大豆根腐病具有较好的防治效果，且含有与生防特性相关的基因以及产生多种抗菌物质的基因，为植物真菌病害的防治提供了应用材料。本研究将盐湖中分离到的细菌菌液应用于植物根部，对大豆生长产生了一定影响，为进一步解析贝莱斯芽孢杆菌YH7-4的抑菌机制提供了理论基础。

Abstract:

Fusarium proliferatum is a critical pathogenic fungus causing soybean root rot. A halotolerant biocontrol strain Bacillus sp. YH7-4 was isolated from the Yuncheng Salt Lake.Objective To investigate strain YH7-4 in terms of the effect on soybean growth and the control efficacy against soybean root rot. Additionally, we sought to elucidate the antifungal mechanisms of this strain and identify antimicrobial genes through whole genome sequencing.Methods The plate dual-culture method was adopted to assess the antifungal activity of strain YH7-4. Pot experiments were conducted to evaluate the safety of the strain to soybean seedlings and the control efficacy against root rot. Illumina and PacBio platforms were used for whole genome sequencing of YH7-4. Subsequent analyses included metabolic system assessment, virulence factor prediction, transporter analysis, identification of genes related to biocontrol functions, comparative genomics, and biochemical assays.Results Strain YH7-4 demonstrated the inhibition rates exceeding 75.00% against several plant pathogens, including F. proliferatum, Phytophthora sojae, Colletotrichum truncatum, and Phomopsis longicolla. Pot experiments showed that at the OD₆₀₀ value of 0.8, YH7-4 suspension significantly increased the root length and dry weight of soybean seedlings, while excessively high concentrations abolished this effect. The control efficacy of YH7-4 against F. proliferatum-induced soybean root rot reached 56.02%. Whole genome sequencing revealed a genome of 3 945 352 bp with the G+C content of 46.51% and 3 756 predicted coding genes. These genes were annotated against databases including NR, Swiss-Prot, Pfam, COG, GO, and KEGG, with 3 753, 3 537, 3 358, 3 082, 1 756, and 2 845 sequences successfully annotated, respectively. Among the proteins encoded by these genes, 130 proteins belonged to the CAZy family. Twelve secondary metabolite biosynthetic gene clusters were identified, including eight known biosynthetic gene clusters for antibiotics (surfactin, macrolactin H, bacillaene, fengycin, difficidin, bacillibactin, bacilysin, and butirosin A/butirosin B) and four gene clusters with unknown functions. Additionally, two siderophore-related genes, one gene encoding 2,3-butanediol (associated with induced systemic resistance), and 15 genes involved in biofilm formation were identified. Comparative genomics analysis indicated that YH7-4 was a strain of Bacillus velezensis and shared 2 898 orthologous core gene clusters. Biochemical characterization showed that YH7-4 had the ability to produce amylase, protease, pectinase, and cellulase.Conclusion The halotolerant strain B. velezensis YH7-4 isolated from the Yuncheng Salt Lake shows excellent control efficacy against soybean root rot. Its genome harbors genes linked to biocontrol traits and antimicrobial substance production, which makes this strain a promising candidate for managing soybean root rot and other plant fungal diseases. This study applies salt lake-derived bacteria to plant roots, demonstrating their influence on soybean growth while providing a theoretical basis for further elucidating the antifungal mechanisms of B. velezensis YH7-4.