Comparative genomics and secondary metabolite analysis to gain novel insights into the mycoparasite

Abstract

The main aims of this study were to gain genome level novel insights into the functional basis of the mycoparasite Coniothyrium minitans, including the identification of novel genes/gene clusters associated with the biocontrol process. For this research, a set of seven C. minitans isolates were selected, representing geographical diversity, alongside two isolates of the fungal crop pathogen Sclerotinia sclerotiorum. Two interaction types were utilised, 1. hyphal-hyphal and 2. spore-sclerotia, to decipher patterns and variations of mycoparasitism. Hyphal-hyphal interactions revealed that both C. minitans isolates were able to colonise and invade regions containing S. sclerotiorum, however, this did not lead to complete loss of viability of the host fungus hyphae or sclerotia. Spore-sclerotial interactions displayed distinct differences between the C. minitans isolates; with isolate Conio all sclerotia colonised were non-viable, whilst with isolate DPM01 despite colonisation all sclerotia were viable. These assays revealed that overall, isolate Conio had stronger mycoparasitic capabilities and S. sclerotiorum isolate DSS03 was less susceptible to both C. minitans isolates, particularly in hyphal-hyphal interactions. A high-quality reference genome (e.g., 34 scaffolds) has been established for C. minitans isolate Conio, alongside resequenced genome assemblies for an additional six C. minitans isolates representing diverse geographic locations. Comparative analysis highlighted new insights into genome-level variations in orthologous protein clusters among the isolates, and the overall conservation of secretory and effector-like proteins, CAZymes and GPCRs. Phylogenomic analysis based on single copy orthologs (~800 to ~1100) revealed a lack of clear geographic association; further analysis including sister-species of C. minitans and alongside Trichoderma species, including mycoparasites, presented that the C. minitans clade was genetically highly conserved. Four biosynthetic gene clusters were identified in this study, potentially associated with mycoparasitism. The complete ETP-type gene cluster has been annotated for all C. minitans isolates including isolate ZS-1. Additionally, this study is the first to present an annotated gene cluster of macrosphelide A in C. minitans, alongside two novel gene clusters, one of which is putatively annotated as involved in the biosynthesis of macrophorin A and is the first report of these gene clusters in C. minitans. The quantification of macrosphelide A carried out with the seven C. minitans isolates using LC-MS/MS analysis enabled categorisation as high producers including isolates Conio, DPM01 and DPM05, and low producers including isolates DPM02, DPM03, DPM04 and DPM06. This is the first report of the quantification of macrosphelide A using a range of geographically distinctive C. minitans isolates. Additionally, this study is the first to confirm the production of an ETP-type compound by C. minitans. Current results suggest that the compound is gliovirin and confirmed that it is not gliotoxin as previously hypothesised. The new knowledge and resources developed in this study will contribute to an advancement in the understanding of the functional basis of mycoparasitism by C. minitans providing a platform for further investigations into the production of secondary metabolites and their association with mycoparasitism by this fungus.