ABSTRACT: Isolation of Bacillus velezensis XZ3-6 With Antagonistic Activity Against Apple Canker and Transcriptome Analysis Uncover Its Biocontrol Mechanism
Project description:Lecanicillium fungicola, the causative agent of dry bubble disease on Agaricus bisporus results in significant crop losses for mushroom growers worldwide. Dry bubble disease is treated through strict hygiene control methods and the application of chemical fungicides but an increase in fungicide resistant L. fungicola strains has resulted in a need to develop alternative biocontrol treatment methods. The aim of the work presented here was to evaluate the response of L. fungicola to a Bacillus velezensis isolate to assess its potential as a novel biocontrol agent. The bacterial species in Serenade, a commercially available biocontrol treatment was also included in this analysis. Exposure of 48 hr L. fungicola cultures to 25% v/v 96h B. velezensis culture filtrate resulted in a 45% reduction in biomass (P < 0.0002) and a disruption in hyphal structure and morphology. Characterisation of the proteomic response of L. fungicola following exposure to B. velezensis culture filtrate revealed an increase in the abundance of a variety of proteins associated with stress response (Norsolorinic acid reductase (+8 fold), isocitrate lyase (+7 fold) and MMS19 nucleotide excision repair protein (+4 fold). There was also a decrease in the abundance of proteins associated with transcription (40S ribosomal protein S30 (-33 fold), 60S ribosomal protein L5 (-45 foldThe results presented here indicate that B. velezensis culture filtrate is capable of inhibiting the growth of L. fungicola and inducing a stress response, thus indicating its potential to control this important pathogen of mushrooms.
Project description:Extracellular vesicles (EVs) are increasingly recognized as an important mechanism for cell-cell interactions. Their role in fungi is still poorly understood and they have been isolated from only a handful of species. Here, we isolated and characterized EVs from Aureobasidium pullulans, a biotechnologically important black yeast-like fungus that is increasingly used for biocontrol of phytopathogenic fungi and bacteria. After optimization of the isolation protocol, characterization of EVs from A. pullulans by transmission electron microscopy (TEM) revealed a typical cup-shaped morphology and different subpopulations of EVs. These results were confirmed by nanoparticle tracking analysis (NTA), which revealed that A. pullulans produced 6.1 × 10^8 nanoparticles per milliliter of culture medium. Proteomic analysis of EVs detected 642 proteins. A small fraction of them had signal peptides for secretion and transmembrane domains. Proteins characteristic of different synthesis pathways were found, suggesting that EVs are synthesized by multiple pathways in A. pullulans. Enrichment analysis using Gene Ontology showed that most of the proteins found in the EVs were associated with primary metabolism. When sequencing the small RNA fraction of A. pullulans EVs, we found two hypothetical novel mil-RNAs. Finally, we tested the biocontrol potential of EVs from A. pullulans. The EVs did not inhibit the germination of spores of three important phytopathogenic fungi – Botrytis cinerea, Colletotrichum acutatum, and Penicillium expansum. However, exposure of grown cultures of C. acutatum and P. expansum to A. pullulans EVs resulted in visible changes in morphology of colonies. These preliminary results suggest that EVs may be part of the antagonistic activity of A. pullulans, which is so far only partially understood. Thus, the first isolation and characterization of EVs from A. pullulans provides a starting point for further studies of EVs in the biotechnologically important traits of the biocontrol black fungus A. pullulans in particular and in the biological role of fungal EVs in general.
Project description:Cladobotryum mycophilum, the causative agent of cobweb disease on Agaricus bisporus results in significant crop losses for mushroom growers worldwide. Cobweb disease is treated through strict hygiene control methods and the application of chemical fungicides but an increase in fungicide resistant Cladobotryum strains has resulted in a need to develop alternative biocontrol treatment methods. The aim of the work presented here was to evaluate the response of C. mycophilum to a Bacillus velezensis isolate to assess its potential as a novel biocontrol agent. Exposure of 48 hr C. mycophilum cultures to 25% v/v 96h B. velezensis culture filtrate resulted in a 57% reduction in biomass (P < 0.0002), a disruption in hyphal structure and morphology, and the appearance of aurofusarin in culture medium. Proteomic analysis of B. velezensis culture filtrate revealed the presence of peptidase 8 (subtilisin), peptide deformylase and probable cytosol aminopeptidase which are known to induce cell disruption. Characterisation of the proteomic response of C. mycophilum following exposure to B. velezensis culture filtrate revealed an increase in the abundance of a variety of proteins associated with stress response (ISWI chromatin-remodelling complex ATPase ISW2 (+24 fold), carboxypeptidase Y precursor (+3 fold) and calmodulin (+2 fold). There was also a decrease in the abundance of proteins associated with transcription (40S ribosomal protein S30 (-26 fold), 40S ribosomal protein S21 (-3 fold) and carbohydrate metabolism, (L-xylulose reductase (-10 fold). The results presented here indicate that B. velezensis culture filtrate is capable of inhibiting the growth of C. mycophilum and inducing a stress response, thus indicating its potential to control this important pathogen of mushrooms.
Project description:Fusarium spp with biocontrol activity against pathogenic Fusarium
| PRJNA361522 | ENA
Project description:Comparative transcriptome analysis reveals the biocontrol mechanism of Bacillus velezensis F against Fusarium oxysporum f. sp. niveum
| PRJNA503589 | ENA
Project description:Tree pathogens influence the biocontrol activity of Bacillus velezensis BY6
Project description:We analyzed differential gene expression in wt and a snf2 mutant (W8) cells; the pucherimin biosynthesis genes were among the top diff. regulated genes (reduced expression in the mutant). ABSTRACT: Metschnikowia pulcherrima synthesizes the pigment pulcherrimin, from cyclodileucine (cyclo(Leu-Leu)) as a precursor, and exhibits strong antifungal activity against notorious plant pathogenic fungi. This yeast therefore has great potential for biocontrol applications against fungal diseases; particularly in the phyllosphere where this species is frequently found. To elucidate the molecular basis of the antifungal activity of M. pulcherrima, we compared a wildtype strain with a spontaneously occurring, pigmentless, weakly antagonistic mutant derivative. Whole genome sequencing of the wildtype and mutant strains identified a point mutation that creates a premature stop codon in the transcriptional regulator SNF2 in the mutant strain. Complementation of the mutant strain with the wildtype SNF2 gene restored pigmentation and recovered the strong antifungal activity. Mass spectrometry (UPLC HR HESI-MS) proved the presence of the pulcherrimin precursors cyclo(Leu-Leu) and pulcherriminic acid and identified new precursor and degradation products of pulcherriminic acid and/or pulcherrimin. All of these compounds were identified in the wildtype and complemented strain, but were undetectable in the pigmentless snf2 mutant strain. These results thus identify Snf2 as a regulator of antifungal activity and pulcherriminic acid biosynthesis in M. pulcherrima and provide a starting point for deciphering the molecular functions underlying the antagonistic activity of this yeast.