ABSTRACT: Isolation of Bacillus velezensis XZ3-6 With Antagonistic Activity Against Apple Canker and Transcriptome Analysis Uncover Its Biocontrol Mechanism
Project description:Southern corn leaf blight (SCLB), caused by Bipolaris maydis, poses a serious threat to maize production worldwide. In our previous study, Bacillus velezensis BV3 was isolated and demonstrated strong antagonistic activity against maize leaf spot pathogens and effective disease control in greenhouse experiments.
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.