Macrophomina phaseolina is an important necrotrophic phytopathogenic fungus and cause extensive damage in many oilseed crops. Twelve M.phaseolina isolates with diverse biological phenotypes were selected for a high-throughput sequencing-based metatranscriptomic and bioinformatics analysis to identify viruses infecting M.phaseolina. The analysis identified 40 partial or nearly complete viral genome segments, 31 of which were novel viruses. Among these viral sequences, 43% of the viral genomes were double-stranded RNA (dsRNA), 47% were positive single-stranded RNA (ssRNA+), and the remaining 10% were negative sense-stranded RNA (ssRNA−). The 40 viruses showed affinity to 13 distinct viral lineages, including Bunyavirales (four viruses), Totiviridae (three viruses), Chrysoviridae (five viruses), Partitiviridae (four viruses), Hypoviridae (one virus), Endornaviridae (two viruses), Tombusviridae (three viruses), Narnaviridae (one virus), Potyviridae (one virus), Bromoviridae (one virus), Virgaviridae (six viruses), ‘Fusagraviridae’ (five viruses), and Ourmiavirus (four viruses). Two viruses are closely related to two families, Potyviridae and Bromoviridae, which previously contained no mycovirus species. Moreover, nine novel viruses associated with M.phaseolina were identified in the family Totiviridae, Endornaviridae, and Partitiviridae. Coinfection with multiple viruses is prevalent in M.phaseolina, with each isolate harboring different numbers of viruses, ranging from three to eighteen. Furthermore, the effects of the viruses on the fungal host were analyzed according to the biological characteristics of each isolate. The results suggested that M.phaseolina hypovirus 2, M.phaseolina fusagravirus virus 1-5 (MpFV1-5), M.phaseolina endornavirus 1-2 (MpEV1-2), M.phaseolina ourmia-like virus 1-3 (MpOLV1-3), M.phaseolina mitovirus 4 (MpMV4), and M.phaseolina mycobunyavirus 1-4 (MpMBV1-4) were only detected in hypovirulent isolates. Those viruses associated with hypovirulence might be used as biological control agents as an environmentally friendly alternative to chemical fungicides. These findings considerably expand our understanding of mycoviruses in M.phaseolina and unvailed the presence of a huge difference among viruses in isolates from different hosts in distant geographical regions. Together, the present study provides new knowledge about viral evolution and fungus-virus coevolution.

Production of pycnidia and pycnidiospores by Macrophomina phaseolina is not often seen in vitro. The objective of this study is to develop a simple and effective technique to obtain pycnidiospores of M. phaseolina isolates in vitro and to evaluate germination rates and pathogenicity of pycnidiospores. We found M. phaseolina isolates can produce pycnidia on oatmeal agar (OMA) under ultraviolet light with 365 nm wavelength (UV). For evaluating the effect of OMA and UV on growth of M. phaseolina, combinations of two agar media and three lighting conditions were tested. The results confirm that all six M. phaseolina isolates produced pycnidia only on OMA under UV. The pycnidiospores produced on OMA under UV had germination rates higher than 90%. In pathogenicity tests, inoculation with the pycnidiospores showed symptoms later than inoculation with hypha-colonized toothpicks. Significant differences in the pathogenicity is detected between isolates Mp2014003 and Mp2014024 when inoculation is done with the pycnidiospores (P < 0.001), but not when hypha-colonized toothpicks are used as inoculum (P = 0.091). This study provides a new method for obtaining pycnidiospores of M. phaseolina for future investigations.

Fusarium oxysporum is a cosmopolitan plant pathogen causing Fusarium wilt and Fusarium root rot in many economically important crops. There is still limited information about mycoviruses that infect F. oxysporum. Here, a novel mitovirus tentatively named Fusarium oxysporum mitovirus 1 (FoMV1) was identified from F. oxysporum strain B2-10. The genome of FoMV1 is 2,453 nt in length with a predicted AU content of 71.6%, and contains one large open reading frame (ORF) using the fungal mitochondrial genetic code. The ORF encodes RNA-dependent RNA polymerases (RdRp) of 723 amino acids with a molecular mass of 84.98 kDa. The RdRp domain of FoMV1 shares 29.01–68.43% sequence similarity to the members of the family Mitoviridae. Phylogenetic analysis further suggested that FoMV1 is a new member of a distinct species in the genus Mitovirus.

Macrophomina phaseolina is a widely distributed soilborne phytopathogenic fungus that causes destructive diseases such as charcoal rot and stem canker, posing serious threats to crop yield and quality. In recent years, mycoviruses have gained attention as potential biological control agents. In this study, a novel double-stranded RNA (dsRNA) virus was identified from M. phaseolina strain 22C-8, isolated from sesame (Sesamum indicum L.) charcoal rot samples in Fuyang, Anhui Province, China. The viral genome comprised four dsRNA segments, each encoding a single open reading frame (ORF) predicted to encode RNA-dependent RNA polymerase (RdRp), coat protein (CP), and two hypothetical proteins. Phylogenetic analysis classified the virus as a new member of the genus Betachrysovirus in the family Chrysoviridae, and it was designated Macrophomina phaseolina chrysovirus 2 (MpChrV2). Pathogenicity assays in sesame seedlings revealed that MpChrV2 infection significantly reduced the virulence of M. phaseolina strain 22C-8. In contrast, virus-free derivatives (22C-8-VF18), obtained via protoplast regeneration, caused more severe symptoms and exhibited enhanced growth rates, indicating that MpChrV2 alters fungal physiology and pathogenicity. These findings suggest that MpChrV2 possesses a typical hypovirulence phenotype and holds promise as a biocontrol agent for sesame charcoal rot.

Fusarium oxysporum is a widespread plant pathogen that causes Fusarium wilt and Fusarium root rot in many economically significant crops. Here, a novel dsRNA virus tentatively named Fusarium oxysporum Virus 1 (FoV1) was identified from F. oxysporum strain 3S-18. The genome of FoV1 spans a length of 2,944 nucleotides (nt) and encompasses two distinct open reading frames (ORF1-2), which do not overlap with each other. The larger ORF2 encodes RNA-dependent RNA polymerases (RdRp) of 590 amino acids with a molecular mass of 67.52 kDa. The ORF1 encodes a putative nucleocapsid protein consisting of 134 amino acids with a molecular mass of 34.25 kDa. The RdRp domain of FoV1 shares 60.00–84.24% sequence similarity to the non-segmented dsRNA virus. Phylogenetic analysis further suggested that FoV1 is a new member of the unclassified monopartite dsRNA virus.