枯草芽胞杆菌HMB19198菌株抑菌物质的鉴定及其对番茄灰霉病的防治

 设施番茄灰霉病发生频繁,易产生抗药性,利用微生物杀菌剂防治番茄灰霉病是切实可行且对环境友好的措施之一。本研究通过抑菌活性测定和田间棚室试验,获得一株有效防治番茄灰霉病的生防细菌HMB19198,通过16S rDNA联合gyrA、gyrB、rpoB和rpoC等看家基因序列比对将菌株HMB19198鉴定为枯草芽胞杆菌(Bacillus subtilis)。对峙培养结果表明,菌株HMB19198的脂肽提取物对灰霉菌表现较强的抑菌活性,通过色谱分离技术结合质谱分析,证明菌株HMB19198主要产生脂肽类抗生素丰原素(fengycin)和表面活性素surfactin,其中fengycin包含fengycin A(C15-C18)和fengycin C(C19-C20)两种同系物。抑菌活性测定表明,fengycin对灰霉菌表现较强的抑菌活性,显微观察发现,fengycin处理后造成灰霉菌菌丝畸形。通过对菌株HMB19198全基因组序列进行分析,获得fengycin合成酶基因簇,从基因水平证明菌株HMB19198可以产生fengycin。

Identification of antifungal compounds of Bacillus subtilis HMB19198 and their biocontrol efficacy against tomato gray mold

Gray mold (Botrytis cinerea) is an important disease of tomato and causes huge economic loss to tomato production. Microbial fungicide is proved as an alternative and environmentally friendly method to suppress the gray mold. In this study, bacterial strain HMB19198, a potential biocontrol agent for tomato gray mold was obtained based on the inhibitory test against B. cinerea in vitro and suppressing ability against gray mold in the greenhouse. Using 16S rDNA sequences, strain HMB19198 was preliminarily identified as Bacillus genus and further identified as B. subtilis species based on the phylogenetic tree constructed using multiple genes sequences of gyrA, gyrB, rpoB, and rpoC. The lipopeptide extract of strain HMB19198 showed strong inhibitory effect against the growth of B. cinerea in vitro as well as in vivo. The active compounds were isolated from the lipopeptide extract using high-performance liquid chromatography (HPLC) and identified by UPLC-Triple TOF-MS/MS. Results showed the lipopeptide extract contained fengycin and surfactin, and only the fengycin showed strong inhibitory effect against B. cinerea and caused abnormality of the hypha. The fengycin produced by strain HMB19198 contained isoforms of fengycin A (C15-C18) and fengycin C (C19-C20). Bioinformatic analysis further revealed that the genome sequence of strain HMB19198 carries a gene cluster for the synthesis of fengycin.