Characterization of dsRNA Mycoviruses Infecting Plant Pathogenic Fungi

Abstract

Mycoviruses are ubiquitous and their infection has been reported from almost all major fungal taxonomic groups. Numerous mycoviruses have been identified and characterized so far, which revealed neo-lifestyles of mycoviruses. This study reports an extensive screening survey on Pakistani filamentous fungal isolates for the identification of viral infections. A total of 125 fungal samples were screened, 6 isolates were found positive for double-stranded (ds) RNA elements when analyzed by a classical dsRNA isolation method with an infection frequency of 4.8%. All six isolates (1NL, NFN, SP1, HK, E, L3) were subjected to Next-generation sequencing for virome analysis. Obtained contigs sequences were completed by 3′ RACE-PCR strategy and subsequent sequencing by a classical Sanger sequencing method. A total of 28 mycoviruses were identified in the infected isolates, belonging to the families Chrysoviridae, Polymycoviridae, Partitiviridae, Totiviridae, Narnaviridae, Mitoviridae, Botourmiaviridae, Tombusviridae and a proposed member of Hadakavirus genera. Based on the International Committee on Taxonomy of Viruses (ICTV) demarcation criteria, the majority of the identified mycoviruses are considered novel. Viruses from three isolates (1NL, NFN, SP1) were completely characterized and sequences have been submitted in the NCBI database. Isolate SP1 was found to harbor co-infection of 11 viruses which is among the largest number of viruses infecting single fungal isolate. Isolates 1NL and NFN showed unique molecular features. Following genomic characterization, the biological assessment was also conducted in which curing mycoviral infection was attempted. HadV1-1NL infecting 1NL was easily cured in aged mycelia however viruses of isolate SP1 and NFN showed persistent infections. Artificial transfection of C. parasitica (model fungus) using virus particles Abstract xxv showed that some mycoviruses can infect and maintain a stable infection in experimental host, whilst others can′t cope with host silencing machinery.