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2014 MR 枯草芽孢杆菌NCD-2产生的丰原素在生物防治棉花幼苗枯萎病中起重要作用

已有 295 次阅读 2024-5-15 08:48 |个人分类:生物农药|系统分类:科研笔记

原文链接：Fengycin produced by Bacillus subtilis NCD-2 plays a major role in biocontrol of cotton seedling damping-off disease - ScienceDirect

Abstract

Bacillus subtilis strain NCD-2 is strongly antagonistic toward phytopathogenic fungi, and functions as an excellent biocontrol agent for cotton soil-borne diseases. The aims of this study were to characterize the main active antifungal compound from strain NCD-2 and clarify its role in suppressing cotton damping-off disease. Strain NCD-2 and lipopeptide extract prepared from an NCD-2 culture strongly inhibited the growth of Rhizoctonia solani in vitro. The lipopeptides of strain NCD-2 were separated by fast protein liquid chromatography (FPLC) and the antifungal compound was identified as a cluster of fengycin homologs analyzed by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry. A fengycin-deficient mutant was obtained by in-frame deletion of the fengycin synthetase gene in B. subtilis NCD-2. Compared with the wild-type strain, this mutant showed decreased abilities to inhibit the growth of R. solani in vitro and to suppress cotton damping-off disease in vivo. Studies showed that the population of fengycin-deficient mutant was almost same as that of the wild-type NCD-2 strain in the cotton rhizosphere. However, the population of R. solani in the cotton rhizosphere colonized by the fengycin-deficient mutant was twice that in the cotton rhizosphere colonized by the NCD-2 wild-type strain. This study demonstrated that fengycin-type lipopeptides are the main antifungal active compounds produced by B. subtilis NCD-2. These compounds play a major role in restricting the population of R. solani in the cotton rhizosphere and in suppressing cotton damping-off disease.

摘要

枯草芽孢杆菌菌株NCD-2对植物病原真菌具有较强的拮抗作用，是棉花土传病害的优良生防剂。本研究的目的是鉴定NCD-2菌株的主要活性抗真菌化合物，并阐明其在抑制棉花枯萎病中的作用。菌株NCD-2和从NCD-2培养物制备的脂肽提取物在体外强烈抑制了立枯丝核菌的生长。用快速蛋白液相色谱法（FPLC）分离菌株NCD-2的脂肽，并用基质辅助激光解吸电离飞行时间质谱法鉴定该抗真菌化合物为一簇丰原素同源物。通过对枯草芽孢杆菌NCD-2中的丰霉素合成酶基因进行框内缺失，获得丰原素缺陷突变体。与野生型菌株相比，该突变体在体外抑制立枯丝核菌生长和在体内抑制棉花枯萎病的能力降低。研究表明，丰霉素缺乏突变体与野生型NCD-2菌株在棉花根际的群体基本相同。然而，丰霉素缺乏突变体定殖的棉花根际中的立枯丝核菌种群是NCD-2野生型菌株定殖的棉根际中的两倍。本研究表明，丰原素型脂肽是枯草芽孢杆菌NCD-2产生的主要抗真菌活性化合物。这些化合物在限制棉花根际立枯丝核菌种群和抑制棉花抗枯萎病方面发挥着重要作用。

图1 来自枯草芽孢杆菌菌株NCD-2的脂肽的FPLC分析（A），以及活性组分对立枯丝核菌生长的抑制活性（B）。LP-I、LP-II和LP-III是FPLC分析的三个主要组分。对照（CK）为150 μL 50%甲醇。

表2 棉花根际立枯丝核菌和细菌的种群动态

图2 枯草芽孢杆菌NCD-2级分LP-II的MALDI–TOF-MS分析[M+H]+。

图3 枯草芽孢杆菌NCD-2中fenC基因缺失的琼脂糖凝胶电泳分析。M： 1-kb DNA标记；泳道1和2，引物FenC-P1/FenC-P2和FenC-P3/FenC-P4以NCD-2 DNA为模板扩增的PCR片段；泳道3，引物FenC-P1/FenC-P4以NCD△fen-DNA为模板扩增的PCR片段；泳道4，引物FenC-P1/FenC-P4以NCD-2野生型DNA为模板扩增的PCR片段。与从NCD-2野生型扩增的扩增子相比，来自NCD-fen的扩增子小500bp，证实部分fenC缺失。在用丰原素缺乏突变体处理的土壤中，保护作用显著降低（图7）。基于这些结果，我们得出结论，菌株NCD-2中的丰原素产物有助于其控制棉花幼苗枯萎病的能力。