Assessment of mycoviral diversity in Pakistani fungal isolates revealed infection by 11 novel viruses of a single strain of Fusarium mangiferae isolate SP1

Abstract

An extensive screening survey was conducted on Pakistani filamentous fungal isolates for the identification of viral infections. A total of 396 fungal samples were screened, of which 36 isolates were found double-stranded (ds) RNA positive with an overall frequency of 9% when analysed by a classical dsRNA isolation method. One of 36 dsRNA-positive strains, strain SP1 of a plant pathogenic fungus Fusarium mangiferae, was subjected to virome analysis. Next-generation sequencing and subsequent completion of the entire genome sequencing by a classical Sanger sequencing method showed the SP1 strain to be co-infected by 11 distinct viruses, at least seven of which should be described as new taxa at the species level according to the ICTV (International Committee on Taxonomy of Viruses) species demarcation criteria. The newly identified F. mangiferae viruses (FmVs) include two partitivirids, one betapartitivirus (FmPV1) and one gammapartitivirus (FmPV2); six mitovirids, three unuamitovirus (FmMV2, FmMV4, FmMV6), one duamitovirus (FmMV5), and two unclassified mitovirids (FmMV1, FmMV3); and three botourmiavirids, two magoulivirus (FmBOV1, FmBOV3) and one scleroulivirus (FmBOV2). The number of coinfecting viruses is among the largest ones of fungal coinfections. Their molecular features are thoroughly described here. This represents the first large virus survey in the Indian sub-continent.

Mycovirus Hunting Revealed the Presence of Diverse Viruses in a Single Isolate of the Phytopathogenic Fungus Diplodia seriata From Pakistan

“…The fungal strain L3 ( D. seriata ) was collected and identified during a mycoviral screening survey conducted in 2018 from a diseased leaf of the Lokath plant ( Khan et al., 2021b ). Some mycoviruses identified as a result of this screening survey have already been published ( Khan et al., 2021a ; Khan et al., 2021b ; Khan et al., 2022 ).…”

“…The fungal strain L3 ( D. seriata ) was collected and identified during a mycoviral screening survey conducted in 2018 from a diseased leaf of the Lokath plant ( Khan et al., 2021b ). Some mycoviruses identified as a result of this screening survey have already been published ( Khan et al., 2021a ; Khan et al., 2021b ; Khan et al., 2022 ). For isolating purified cultures of L3, surface sterilization of leaves was performed with 1% sodium hypochlorite solution and further washing was done using autoclaved sterile distilled water.…”

“…Next-Generation Sequencing of Viral RNA Total RNA fractions were separately obtained from three dsRNApositive fungal isolates, L3 (D. seriata, a phytopathogen), SP1 (F. mangiferae, a phytopathogen) and HK (Geotrichum candidum, a soil-inhabitant) and were pooled together as already described by Khan et al (Khan et al, 2021a;Khan et al, 2021b). Ribosomal RNA depletion, cDNA library construction and RNA sequencing were performed by Macrogen Inc. (Tokyo, Japan) as already described by Khan et al (Khan et al, 2021b).…”

“…Next-Generation Sequencing of Viral RNA Total RNA fractions were separately obtained from three dsRNApositive fungal isolates, L3 (D. seriata, a phytopathogen), SP1 (F. mangiferae, a phytopathogen) and HK (Geotrichum candidum, a soil-inhabitant) and were pooled together as already described by Khan et al (Khan et al, 2021a;Khan et al, 2021b). Ribosomal RNA depletion, cDNA library construction and RNA sequencing were performed by Macrogen Inc. (Tokyo, Japan) as already described by Khan et al (Khan et al, 2021b). The assembly of RNA sequencing reads was performed using the Read Mapping algorithm with a default parameter in the CLC Genomics Workbench (version 11, CLC Bio-Qiagen) assembled 42,770 contig sequences were subjected to local BLAST searches to check sequence similarities with deposited virus reference sequences (RefSeq) in the National Center for Biotechnology Information (NCBI) database (data not shown).…”

“…We also have screened many different Pakistani fungal strains from different sources for mycovirus hunting and have reported molecular characterization of some of the discovered novel mycoviruses ( Khan et al., 2021a ; Khan et al., 2022 ). The tested fungal strains included the SP1 strain of Fusarium mangiferae that was found to be co-infected with 11 mycoviruses ( Khan et al., 2021b ).…”