一株具有促生作用的生防细菌YN-2A的分离、鉴定及全基因组测序分析

A biocontrol bacterium YN-2A with growth-promoting effect: isolation, identification, and genome sequencing

摘要:

【背景】根际土壤中分布着丰富的微生物类群，其中不少微生物具有拮抗病原菌的能力，以此保护植株免受病害侵袭。【目的】分离并鉴定出一株具有促生作用的生防细菌，从基因组学与生物信息学角度探究生防菌的抑菌机制。【方法】采用平板稀释涂布法筛选出具有拮抗黄瓜炭疽病作用的细菌。通过16S rRNA基因和基因组序列比对来确定生防细菌的生物学分类。以500倍生防菌稀释液(5×10⁸cfu/mL)为材料，测定其对番茄种子、幼苗的促生效果。通过平板对峙试验测定生防细菌的广谱抑菌性。利用三代(PacBio、Nanopore)结合二代测序技术解析菌株的基因组信息、基因功能注释等。【结果】从鹅掌柴根际土壤分离出一株生防细菌YN-2A，结合16S rRNA基因与核酸序列数据库(nucleotide sequence database, NT)比对结果，鉴定出菌株YN-2A为贝莱斯芽孢杆菌(Bacillus velezensis)。菌株YN-2A是一株好氧型的革兰氏阳性菌，具有产生生物膜的能力。菌株YN-2A的500倍稀释液(5×10⁸ cfu/mL)能够有效促进番茄种子发芽率和幼苗的生长，同时提高叶片的叶绿素荧光参数。在72、96和120 h的发芽率分别比CK组提高1.45倍、1.42倍和1.09倍；在幼苗形态指标上，株高提升5.90 cm。光化学猝灭指数(photochemical quenching, qP)在荧光参数中提升最为显著，处理后番茄叶片qP指数同CK组相比提高1.27倍。平板对峙试验中菌株YN-2A能够拮抗多种真菌病原。全基因组测序结果表明，菌株YN-2A全基因组长度为4 046 066 bp，G+C含量为46%，包含4 090个编码基因。将全基因组测序数据提交至NCBI获得GenBank登录号为CP139086。基本功能注释中大部分基因注释到氨基酸与碳水化合物代谢途径。另外，在病原与宿主互作(pathogen host interactions, PHI)数据库中毒力降低(reduced virulence)分类涉及基因数量高达905个，并有69个基因注释到病原菌失去致病力(loss of pathogenicity)分类。初步推断生防细菌YN-2A因其含有大量抗病、降低毒力的基因来发挥抑菌作用。【结论】分离并鉴定出一株贝莱斯芽孢杆菌(B. velezensis) YN-2A，该菌株对番茄的种子和幼苗具有良好的促生效果，同时该菌株具备广谱抑菌性。通过测序技术，获得了菌株YN-2A的全基因组信息，为深入挖掘菌株YN-2A的抑菌机制提供参考信息。

关键词:生防细菌;拮抗作用;全基因组测序;基因功能注释

Abstract:

[Background] There are numerous microorganisms in the rhizosphere soil, many of which can antagonize pathogens and thus protect plants from diseases. [Objective] To isolate and identify a biocontrol bacterial strain capable of promoting plant growth and explore the antifungal mechanism from genomics and bioinformatics. [Methods] The dilution-plate coating method was employed to isolate a bacterial strain with antagonistic effects against Colletotrichum orbiculare. The strain was identified based on 16S rRNA gene sequencing and genome sequence alignment. The effects of the strain suspension (5×10⁸ CFU/mL) on tomato seeds and seedlings were determined. The antifungal spectrum of the strain was determined by the plate confrontation test. Furthermore, the third-generation (PacBio, Nanopore) sequencing was combined with the second-generation sequencing to analyze the genomic information and gene function annotation of the strain. [Results] A strain of biocontrol bacteria isolated from the rhizosphere soil of Schefflera heptaphylla was named YN-2A. Based on the alignment results of 16S rRNA gene and nucleotide sequence database (NT), the strain YN-2A was identified as Bacillus velezensis. Strain YN-2A was an aerobic Gram-positive bacterial strain with the ability to produce biofilms. Strain YN-2A suspension (5×10⁸ CFU/mL) promoted tomato seed germination and seedling growth and increased the leaf chlorophyll fluorescence parameters. Specifically, the suspension increased the germination rate at the time points of 72, 96 and 120 h by 1.45 times, 1.42 times and 1.09 times, respectively, and it increased the plant height of tomato seedlings by 5.90 cm. Among the fluorescence parameters, photochemical quenching (qP) exhibited the most significant increase of 1.27 times in the strain treatment group compared with the control group. The results of the plate confrontation test showed that strain YN-2A can antagonize a variety of fungal pathogens. The results of whole genome sequencing showed that the whole genome length of strain YN-2A was 4 046 066 bp, with the G+C content of 46% and 4 090 coding genes. The whole genome sequencing data was submitted to NCBI to obtain the GenBank accession number CP139086. Most genes were annotated to the amino acid and carbohydrate metabolic pathways. Particularly, 905 genes were annotated to reduced virulence in the pathogen host interactions (PHI) database, and 69 genes were annotated to the loss of pathogenicity database. It is thus hypothesized that the antifungal effect of YN-2A is attributed to the disease-resistant and virulence-reducing genes. [Conclusion] A biocontrol strain B.velezensis YN-2A was isolated and identified. The strain promoted tomato seed germination and seedling growth and demonstrated a broad antifungal spectrum. The whole genome information of strain YN-2A was obtained by sequencing, which provided reference information for deciphering the antifungal mechanism of strain YN-2A.

Key words:biocontrol bacteria;antagonistic effect;whole genome sequencing;gene function annotation