根际来源弗吉尼亚链霉菌JL7001对番茄青枯病的防治作用及全基因组序列分析

Biocontrol effect of rhizosphere-derived Streptomyces virginiae JL7001 against tomato bacterial wilt and whole genome sequence analysis

摘要:

背景 番茄青枯病由青枯菌引起，传播迅速且难以治理，已成为番茄生产中的主要毁灭性病害。随着化学防控受限，开发安全高效的生防菌资源愈发关键。目的 从不同药用植物的根际中筛选出对青枯菌(Ralstonia solanacearum)有拮抗作用的放线菌，探究其对青枯菌的抗菌活性及对番茄青枯病的防治效果，分析目的菌株的基因组序列信息，深入探索其功能基因及次级代谢产物相关基因资源，为后续防治病害微生物菌剂的研发奠定理论基础。方法 利用共培养法筛选出目标菌株后对其进行菌种鉴定及发酵粗提物萃取。通过微量稀释法测定目标菌株粗提物对青枯菌的抗菌活性，并通过盆栽试验测定其发酵液对番茄青枯病的防治效果。采用Oxford Nanopore Technologies (ONT)测序技术对其全基因组进行测序，并对测序数据进行系统分析。结果 根据基因组测序结果，将从黄精根际分离得到的目标菌株JL7001鉴定为弗吉尼亚链霉菌(Streptomyces virginiae)，对青枯菌的抑菌圈直径达29.93 mm。利用乙酸乙酯萃取发酵液的粗提物在1 mg/mL时对青枯菌的抑制率为83.3%。盆栽防病试验表明，发酵液对番茄青枯病的防治效果为53.1%。菌株JL7001基因组全长为8 611 226 bp，G+C含量为72.28%，编码7 692个编码基因。在NR、GO、KEGG、eggNOG、Pfam、Swiss-Prot、CAZy、转运蛋白分类数据库(transporter classification database, TCDB)、综合抗生素耐药性数据库(comprehensive antibiotic research database, CARD)、病原与宿主互作(pathogen host interaction, PHI)、毒力因子数据库(virulence factor database, VFDB^[35])中注释到的基因分别为7 613、5 220、4 809、5 627、5 981、3 627、323、1 596、2 228、358、1 188个。鉴定了32个次级代谢产物生物合成基因簇(biosynthetic gene clusters, BGCs)，其中14个为潜在新型BGCs，并检测到多种与抗菌活性相关的BGCs (如ε-聚赖氨酸、链丝菌素和拟无枝酸霉素等)。结论 本研究从黄精根际土壤分离获得一株弗吉尼亚链霉菌S. virginiae JL7001，该菌株对青枯菌具有显著拮抗活性且表现出良好的番茄青枯病防控效果，其基因组中含有多种抑菌代谢物基因簇，具有作为生防菌剂开发的潜在应用价值。

Abstract:

Background Tomato bacterial wilt, caused by Ralstonia solanacearum, spreads rapidly and is difficult to control, which make it one of the most destructive diseases in tomato production. With increasing restrictions on chemical control, the development of safe and effective microbial resources for biocontrol has become increasingly important.Objective To screen actinomycetes with antagonistic activity against R. solanacearum from the rhizosphere of various medicinal plants, evaluate their antibacterial activity and biocontrol efficacy against tomato bacterial wilt, and analyze the genome of the target strain to explore its functional genes and biosynthetic gene clusters (BGCs) for secondary metabolites, thus providing a theoretical basis for developing microbial agents for disease control.Methods A co-culture method was used for target strain screening, followed by strain identification and extraction of crude metabolites from fermentation broth. Antibacterial activity of the crude extract against R. solanacearum was determined via a microdilution assay, and the biocontrol efficacy of fermentation broth against tomato bacterial wilt was evaluated through pot experiments. Whole genome sequencing was performed via Oxford Nanopore Technologies (ONT), and the sequencing data were systematically analyzed.Results Genome sequencing identified strain JL7001 isolated from the rhizosphere of Polygonatum sibiricum as Streptomyces virginiae. The strain produced an inhibition zone with the diameter of 29.93 mm against R. solanacearum. The ethyl acetate extract (1 mg/mL) of the fermentation broth exhibited an inhibition rate of 83.3% against R. solanacearum. Pot experiments showed that the fermentation broth provided the control efficacy of 53.1% against tomato bacterial wilt. The JL7001 genome was 8 611 226 bp long with the G+C content of 72.28%, encoding 7 692 protein-coding genes. Genes annotated in NR, GO, KEGG, eggNOG, Pfam, Swiss-Prot, CAZy, TCDB, CARD, PHI, and VFDB databases numbered 7 613, 5 220, 4 809, 5 627, 5 981, 3 627, 323, 1 596, 2 228, 358, and 1 188, respectively. In addition, 32 BGCs were identified, including 14 putative novel BGCs, and several BGCs associated with antibacterial activity were detected, such as those responsible for the biosynthesis of ε-poly-l-lysine, streptothricins, and amycomicin.Conclusion A S. virginiae strain JL7001 was isolated from the rhizosphere soil of P. sibiricum, exhibiting significant antagonistic activity against R. solanacearum and effective biocontrol performance against tomato bacterial wilt. The genome of JL7001 harbors multiple BGCs related to antibacterial metabolites, which indicates the potential application of the strain as a biocontrol agent.