一株西瓜枯萎病生防菌Bacillus methylotrophicus的筛选、鉴定及抑菌物质分析

Screening, identification, and antifungal substances of an antagonistic strain Bacillus methylotrophicus against Fusarium oxysporum f. sp. niveum

摘要:

【背景】西瓜专化型尖孢镰刀菌（Fusarium oxysporum f.sp.niveum，Fon）是西瓜枯萎病的致病菌，严重威胁连作西瓜的生产。【目的】筛选拮抗西瓜枯萎病菌的菌株并推测其无菌上清液（cell-free supernatant，CFS）的抑菌成分，为其在农业生产中的应用提供理论依据。【方法】利用平板对峙法筛选拮抗Fon的菌株，通过盆栽试验验证其抑制西瓜枯萎病的能力，并通过扫描电镜、共聚焦显微镜观察和抗氧化酶活性测定等方法研究Fon暴露在拮抗菌株次级代谢物下孢子形态的变化、膜的完整性和造成的氧化损伤，通过二代Illumina Hiseq联合三代PacBio Sequel测序平台进行全基因组测序、LC-MS非靶代谢组学、超高液相色谱系统和高分辨率质谱（UHPLC-ESI-MS/MS），明确拮抗菌株次级代谢物中的抑菌功能物质。【结果】从健康西瓜根际土筛选到拮抗Fon的一株优良菌株J4，盆栽试验表明其对西瓜枯萎病的防治效果为72.6%。经全基因组学鉴定为Bacillus methylotrophicus J4，该菌株可产蛋白酶、淀粉酶、葡聚糖酶和纤维素酶并有分泌铁载体的能力，其衰亡期无菌上清液对Fon菌丝生长抑制率为71.87%，菌株J4的CFS使Fon孢子表面凹陷，破坏其细胞膜的完整性，造成细胞死亡。菌株J4诱导Fon菌丝体活性氧（reactive oxygen species，ROS）的积累；菌株J4的CFS引起Fon菌丝超氧化物歧化酶（superoxide dismutase，SOD）、过氧化氢酶（catalase，CAT）、过氧化物酶（peroxidase，POD）、谷胱甘肽还原酶（glutathione reductase，GR）活性，以及可溶性蛋白和丙二醛（malondialdehyde，MDA）含量的显著升高。该菌株基因组中含有13个抑菌功能的次级代谢产物合成基因簇，菌株J4的CFS中含有3种多肽化合物，并且这3种脂肽具有抑制Fon菌丝生长的能力，推测菌株J4上清液中的抑菌功能物质是伊枯草菌素A （iturin A）、丰原素（fengycin）和表面活性素（surfactin）。【结论】菌株J4对西瓜枯萎病有较强的防效，在西瓜枯萎病生物防治中具有广泛的应用前景。

Abstract:

[Background] Fusarium oxysporum f. sp. niveum (Fon) is the causal agent of watermelon wilt, seriously threatening the production of watermelon in continuous cropping. [Objective] To screen out the antagonistic strain against Fon and identify the antifungal components in the cell-free supernatant (CFS), providing a theoretical basis for application of the strain in agricultural production. [Methods] Plate confrontation was employed to screen the antagonistic strain against Fon. Pot experiments were conducted to verify the control effect of the antagonistic strain on watermelon wilt. Scanning electron microscopy and confocal microscopy were employed to observe the conidial morphology and membrane integrity of Fon. The activities of antioxidant enzymes were determined to evaluate the oxidative damage. Whole genome sequencing through second-generation Illumina Hiseq combined with third-generation PacBio Sequel sequencing platform, LC-MS-based non-target metabolomics, and ultra-performance liquid chromatography-electrospray ionization-tandem mass spectrometry (UHPLC-ESI-MS/MS) were employed to identify the antifungal substances in the secondary metabolites of the strain. [Results] An antagonistic strain J4 was screened out from the rhizosphere soil of healthy watermelon. The pot experiments showed that its control effect against watermelon wilt was 72.6%. The strain was identified as Bacillus methylotrophicus J4 by whole genome sequencing, and it could produce protease, amylase, glucanase, cellulose, and siderophores. The CFS of J4 in the decline stage presented the inhibition rate of 71.87% on the growth of Fon. Moreover, it led to the collapsing of Fon spore surface, destroyed the integrity of the cell membrane, and caused cell death. Strain J4 induced the accumulation of reactive oxygen species (ROS) in the mycelia of Fon. The CFS of strain J4 significantly increased the activities of superoxide dismutase, catalase, peroxidase, and glutathione reductase and the content of soluble protein and malondialdehyde in the mycelia of Fon. The genome of strain J4 carried 13 gene clusters for the synthesis of secondary metabolites with antimicrobial functions. The CFS of strain J4 contained three peptides, which were proved to inhibit the growth of Fon. It was hypothesized that the antifungal functional substances in the CFS of strain J4 were iturin A, fengycin, and surfactin. [Conclusion] Strain J4 had a strong control effect on watermelon wilt and could be used to control watermelon wilt in the future.