Abstract

Botrytis cinerea is the causal agent of gray mold disease affecting Paeoniaceae plants. The present study focuses on the potential of Bacillus amyloliquefaciens Mdgb15 as a promising biological control agent toward B. cinerea. The strain Mdgb15 showed the highest inhibition rate (55.56%) among all the strains tested against B. cinerea growth and was effective in controlling tree peony gray mold in detached leaves. A conventional PCR with three specific primer pairs detected lipopetides substance synthesis genes of Mdgb15. The results revealed that Mdgb15 had srfAA, fenA and ituD genes, which resulted in the production of surfactin, fengycin and iturin. An accurate qRT-PCR protocol with specifically designed primers was developed to characterize the expression of lipopeptide synthesis gene of Mdgb15. The qRT-PCR analysis revealed that the expression of the ituD and fenA genes in Mdgb15 was significantly up-regulated during the coculture with B. cinerea. Subsequently, the lipopeptides were extracted by acid precipitation of culture filtrate of Mdgb15 and were separated by Sephadex Gel chromatography. Three separated fractions exhibited strong antifungal activity against B. cinerea, and were identified as bacillomycin D by MALDI-TOF-MS analysis. This study has demonstrated that B. amyloliquefaciens Mdgb15 is a future potential new biocontrol agent for controlling tree peony gray mold disease. It suggests that the antifungal mechanisms of Mdgb15 may be at least partially associated with its ability to secret bacillomycin D.

中文翻译：

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内生解淀粉芽孢杆菌 Mdgb15 是一种针对灰霉病引起的牡丹灰霉病的潜在生物防治剂

摘要

灰葡萄孢是影响芍药科植物的灰霉病的病原体。本研究的重点是解淀粉芽孢杆菌Mdgb15 作为一种有前景的灰霉病生物防治剂的潜力。在所有测试的菌株中，菌株Mdgb15对灰霉病的生长抑制率最高（55.56%），可有效控制牡丹离体灰霉病。采用3对特异性引物进行常规PCR检测Mdgb15脂肽物质合成基因。结果表明，Mdgb15具有srfAA、fenA和ituD基因，从而产生surfactin、fengycin和iturin。开发了具有专门设计的引物的准确 qRT-PCR 方案来表征 Mdgb15 脂肽合成基因的表达。qRT-PCR分析表明， Mdgb15中ituD和fenA基因的表达在与B. cinerea共培养期间显着上调。随后，通过酸沉淀Mdgb15的培养滤液提取脂肽，并通过Sephadex凝胶色谱分离。三个分离的级分对灰霉病菌表现出很强的抗真菌活性，并通过 MALDI-TOF-MS 分析鉴定为杆菌霉素 D。本研究表明解淀粉芽孢杆菌Mdgb15是未来潜在的防治牡丹灰霉病的新型生防剂。这表明 Mdgb15 的抗真菌机制可能至少部分与其分泌芽孢杆菌素 D 的能力有关。