Bioprospecting for fungal-based biocontrol agents

The research objective of this project was to investigate how a virus infecting the fungus can improve its effectiveness as a pesticide. This mycovirus-induced hypervirulence was investigated using microbiological and genomic techniques to characterise the molecular interactions between the virus and the fungus host so that an improved mycopesticide can be deployed commercially. Prior to this work, it was reported that a newly proposed virus family called the Polymycoviridae can confer mild hypervirulence to their fungal host. In this work, attempts to cure a B. bassiana isolate (ATHUM 4946) which harbours a polymycovirus-3 (BbPmV-3) were successful and I built and confirmed two isogenic lines of virus-free and virus-infected. Furthermore, BbPmV-3 has six genomic dsRNA segments and its complete sequence is reported here. Phylogenetic analysis of RNA-dependent RNA polymerase (RdRP) protein sequences revealed that BbPmV-3 is closely related to BbPmV-2 but not BbPmV-1. Consequently, examining the effects of BbPmV-3 and BbPmV-1 on their respective hosts revealed similar phenotypic effects including increased pigmentation, sporulation, and radial growth. However, this polymycovirus-mediated effect on growth is dependent on the carbon and nitrogen sources available to the host fungus. When sucrose is replaced by lactose, trehalose, glucose, or glycerol both BbPmV-3 and BbPmV-1 increase growth of ATHUM 4946 and EABb 92/11-Dm respectively, whereas these effects were reversed on maltose and fructose. Similarly, both BbPmV-3 and BbPmV-1 decrease growth of ATHUM and EABb 92/11-Dm when sodium nitrate is replaced by sodium nitrite, potassium nitrate, or ammonium nitrate. To this extent, this hypervirulent effect was tested on Tenebrio molitor, where a virus-infected EABb 92/11-Dm line demonstrated increased mortality rate when compared to the commercial B. bassiana ATCC 74040, B. bassiana GHA, and the virus-free isogenic line. Furthermore, gene expression data from five timepoints of the two isogenic lines were used in a candidate pathway approach, investigating key pathways known to affect resistance to stresses and carbon uptake. Secretion of organic acid during growth can change the pH of the growth medium creating a toxic environment causing stress and death. Consequently, genes involved in stress tolerance such as heat shock proteins, trehalose and mannitol biosynthesis, and calcium homeostasis were upregulated in virus-infected isolate. Likewise, genes involved in carbon uptake such as BbAGT1 and BbJen1 transporters were upregulated in virus-infected isolate. Equally, here we demonstrate that BbPmV-1 drives the up-regulation of nirA gene which is linked to nitrate uptake and/or assimilation and secondary metabolites such as Tenellin, Beauvericin and Bassianolide. These results reveal a symbiotic relationship between BbPmV-1 and its fungal host. To conclude, these data present a crucial first step in characterising how mycopesticides can be improved to deliver better and safer pest management