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

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.