Abstract: To screen strains with antagonistic activity against Phytophthora nicotianae and to develop microbial resources for the biological control of tobacco black shank，antagonistic strains were isolated using dilution plating and dual‐culture assays. Dominant antagonistic strains were identified based on morphological characteristics，16S rRNA gene，and the sequences of 31 housekeeping genes.Their antimicrobial capacity was further evaluated via broad‐spectrum inhibition assays and determination of antibacterial activity in sterile fermentation filtrate.The control efficacy of sterile fermentation filtrates against P.nicotianae was assessed by measuring lesion length on detached leaves.Whole‐genome sequencing of the antagonistic strain was performed using next‐generation sequencing technology，and the genomic data were analyzed with KEGG database and antiSMASH software.Active antimicrobial compounds were identified by ultra‐performance liquid chromatography coupled with high‐resolution mass spectrometry（UPLC‐MS）. Results indicated that a strain of Bacillus cereus YC5 with antagonistic activity against P.nicotianae was successfully isolated and screened.Dual‐culture assays demonstrated that strain YC5 exhibited a 74.22% inhibition rate against P.nicotianae，along with antifungal activity against multiple plant pathogenic fungi including the causal agents of tobacco Fusarium wilt and wheat crown rot.In vitro leaf tests confirmed that the sterile fermentation filtrate of YC5 significantly suppressed lesion expansion，demonstrating remarkable antifungal efficacy.Genomic analysis revealed that YC5 possessed a genome size of 5 859 938 bp with 34.779% GC content.Among the 5 824 predicted genes，3 813 were annotated in the KEGG database.antiSMASH predicted 14 secondary metabolite biosynthetic gene clusters，and UPLC‐MS identified various upregulated antimicrobial compounds.These findings demonstrated that YC5 exerted its biocontrol activity primarily through the production of diverse antimicrobial substances，which effectively suppressed the growth of P.nicotianae and other phytopathogens，thereby providing protective effects for plants.In summary，strain YC5 has the ability to synthesize multiple antibacterial compounds，and has antagonistic effects on Phytophthora nicotianae and various plant pathogenic fungi.

Key words: Tobacco black shank, Phytophthora nicotianae, Antagonistic strain, Biological control, Bacillus cereus, Genome sequencing, Metabolnomics

摘要： 为筛选具有烟草疫霉拮抗活性的菌株，开发烟草黑胫病生防菌菌种资源，采用稀释涂布法、对峙培养法筛选拮抗菌株，结合形态学、16S rRNA基因和31个看家基因序列鉴定优势拮抗菌株，通过广谱抑菌鉴定、无菌发酵滤液抑菌活性测定探究拮抗菌株的抑菌能力，通过离体叶片病斑长度研究拮抗菌株无菌发酵滤液对烟草黑胫病的防治效果，借助二代测序技术对拮抗菌株进行基因组测序，结合KEGG、antiSMASH对拮抗菌株的基因组数据进行分析，利用超高效液相色谱-质谱联用技术（UPLC-MS）鉴定有效抗菌成分。结果表明，分离筛选出一株具有烟草疫霉拮抗活性的蜡样芽孢杆菌（Bacillus cereus）YC5。平板对峙发现，YC5对烟草疫霉的抑制率达到74.22%，且对烟草枯萎病病原菌、小麦茎基腐病病原菌等多种植物病原真菌具有抑菌能力。离体叶片防效结果表明，YC5无菌发酵滤液能显著抑制病斑长度，表现出优异的抗真菌活性。基因组数据分析结果表明，YC5基因组长度为5 859 938 bp，平均GC含量为34.779%，得到5 824个预测基因，其中有3 813个基因被注释到KEGG数据库，antiSMASH预测到14个次级代谢产物合成基因簇，在发酵液上调表达物中筛选鉴定出多种抗菌物质，表明YC5可通过分泌抗菌物质抑制烟草疫霉及多种病原菌生长，从而为植物提供保护。综上，菌株YC5具有合成多种抗菌化合物的能力，对烟草疫霉及多种植物病原真菌具有拮抗作用。

关键词: 烟草黑胫病, 烟草疫霉, 拮抗菌株, 生物防治, 蜡样芽胞杆菌, 基因组测序, 代谢组学