Investigations on Mycoviruses found in Dothistroma septosporum and Beauveria bassiana and their Effects on Fungal Pathogenicity

Abstract

Mycoviruses specifically infect and replicate in fungi, and they are frequently cryptic in nature and usually associated with hypovirulence (reduced virulence) or, more unusually, hypervirulence. The research aim of the project is to gain further understanding of the molecular biology of viruses infecting the entomopathogenic fungus Beauveria bassiana and the phytopathogenic fungus Dothistroma septosporum, together with any effects on pathogenicity. D. septosporum is a foliar pathogen of pine that causes Dothistroma needle blight (DNB), the incidence of which has rapidly increased over the last few decades, particularly in the northern hemisphere. Screening of over 50 D. septosporum isolates for virus infection revealed one isolate D752.1 harboring a tetrapartite mycovirus with a double stranded (ds) RNA genome. The sequence of these dsRNA elements was determined using random cDNA PCR amplification combined with a genome walking strategy and RNA ligase-mediated rapid amplification of cDNA ends (RLM RACE). Following sequence analysis, the mycovirus was designated as Dothistroma septosporum chrysovirus (DsCV-1) and was found to belong to the family Chrysoviridae. Subsequently, attempts were made to construct virus-free and virus-infected isogenic lines, using matric stress, the protein synthesis inhibitor cycloheximide and the potent antiviral drug ribavirin; the latter was proven successful in eradicating the virus. Subsequently, small-scale field trial testing was developed in collaboration with Forest Research UK to investigate the virulence of D. septosporum isolates on Pinus saplings. The trials included virus-infected and virus-free isogenic lines of D752.1 and other isolates D622, D584, D233, D1216, D700 whose infection status was determined using RT-PCR. The DsCV-1-free D752.1 isolate was revealed to be more infectious, cause significantly more extended needle damage in the form of red bands and have significantly higher fungal burden as compared to the DsCV-1-infected D752.1 isolate, four other DsCV-1-infected D. septosporum isolates from Scotland and one DsCV-1-free D. septosporum isolate from Southern England, as shown by image analysis and qPCR. B. bassiana is an entomopathogenic fungus with a worldwide distribution and a wide arthropod host range and is a popular biocontrol agent against insect pests. The effects of virus infection on the growth and pathogenicity of B. bassiana were investigated through a comparative study of virus-infected and virus-free isogenic lines of the fungus. Radial expansion experiments conducted on Czapek-Dox complete and minimal media revealed that the virus-infected (VI) B. bassiana isolate EABb 92/11-Dm grows faster as compared to the virus-free (VF) isogenic EABb 92/11-Dm, virus-free non-isogenic KVL-03-144 and KVL-03-122, and virus-free Botanigard and Naturalis which are commercially used as biocontrol agents. Subsequently, the pathogenicity of the isogenic lines EABb 92/11-Dm VI and VF was studied in a controlled environment using the greater wax moth Galleria mellonella infection model. Larvae infected with EABb 92/11-Dm VF had a significantly higher survival rate as compared to EABb 92/11-Dm VI. In summary, these experiments yielded promising results, as virus infection was shown to promote hypovirulence and hypervirulence respectively in D. septosporum and B. bassiana. Virus infection could therefore be utilised in the context of biological control in the future.