White root rot, caused by the ascomycete Rosellinia necatrix, is a devastating disease worldwide, particularly in fruit trees in Japan. Here we report on the biological and molecular properties of a novel bipartite double-stranded RNA (dsRNA) virus encompassing dsRNA-1 (8,931 bp) and dsRNA-2 (7,180 bp), which was isolated from a field strain of R. necatrix, W779. Besides the strictly conserved 5 (24 nt) and 3 (8 nt) terminal sequences, both segments show high levels of sequence similarity in the long 5 untranslated region of approximately 1.6 kbp. dsRNA-1 and -2 each possess two open reading frames (ORFs) named ORF1 to -4. Although the protein encoded by 3-proximal ORF2 on dsRNA-1 shows sequence identities of 22 to 32% with RNA-dependent RNA polymerases from members of the families Totiviridae and Chrysoviridae, the remaining three virus-encoded proteins lack sequence similarities with any reported mycovirus proteins. Phylogenetic analysis showed that the W779 virus belongs to a separate clade distinct from those of other known mycoviruses. Purified virions ϳ50 nm in diameter consisted of dsRNA-1 and -2 and a single major capsid protein of 135 kDa, which was shown by peptide mass fingerprinting to be encoded by dsRNA-1 ORF1. We developed a transfection protocol using purified virions to show that the virus was responsible for reduction of virulence and mycelial growth in several host strains. These combined results indicate that the W779 virus is a novel bipartite dsRNA virus with potential for biological control (virocontrol), named Rosellinia necatrix megabirnavirus 1 (RnMBV1), that possibly belongs to a new virus family.

The transmission of viral infections between plant and fungal hosts has been suspected to occur, based on phylogenetic and other findings, but has not been directly observed in nature. Here, we report the discovery of a natural infection of the phytopathogenic fungus by a plant virus, cucumber mosaic virus (CMV). The CMV-infected strain was obtained from a potato plant growing in Inner Mongolia Province of China, and CMV infection was stable when this fungal strain was cultured in the laboratory. CMV was horizontally transmitted through hyphal anastomosis but not vertically through basidiospores. By inoculation via protoplast transfection with virions, a reference isolate of CMV replicated in and another phytopathogenic fungus, suggesting that some fungi can serve as alternative hosts to CMV. Importantly, in fungal inoculation experiments under laboratory conditions, could acquire CMV from an infected plant, as well as transmit the virus to an uninfected plant. This study presents evidence of the transfer of a virus between plant and fungus, and it further expands our understanding of plant-fungus interactions and the spread of plant viruses.

Mycoviruses are wide spread throughout almost all groups of fungi but only a small number of mycoviruses can attenuate the growth and virulence of their fungal hosts. Alternaria alternata is an ascomycete fungus that causes leaf spot diseases on various crop plants. In this study, we identified a novel ssRNA mycovirus infecting an A. alternata f. sp. mali strain isolated from an apple orchard in China. Sequence analyses revealed that this virus is related to hypoviruses, in particular to Wuhan insect virus 14, an unclassified hypovirus identified from insect meta-transcriptomics, as well as other hypoviruses belonging to the genus Hypovirus , and therefore this virus is designed as Alternaria alternata hypovirus 1 (AaHV1). The genome of AaHV1 contains a single large open-reading frame encoding a putative polyprotein (∼479 kDa) with a cysteine proteinase-like and replication-associated domains. Curing AaHV1 from the fungal host strain indicated that the virus is responsible for the slow growth and reduced virulence of the host. AaHV1 defective RNA (D-RNA) with internal deletions emerging during fungal subcultures but the presence of D-RNA does not affect AaHV1 accumulation and pathogenicities. Moreover, AaHV1 could replicate and confer hypovirulence in Botryosphaeria dothidea , a fungal pathogen of apple white rot disease. This finding could facilitate better understanding of A. alternata pathogenicity and is relevant for development of biocontrol methods of fungal diseases.

Rosellinia necatrix megabirnavirus 1 (RnMBV1) with a bipartite dsRNA genome (dsRNA1 and dsRNA2) confers hypovirulence to its natural host, the white root rot fungus, and is thus regarded as a potential virocontrol (biocontrol) agent. Each segment has two large ORFs: ORF1 and partially overlapping ORF2 on dsRNA1 encode the major capsid protein (CP) and RNAdependent RNA polymerase (RdRp), whilst ORF3 and ORF4 on dsRNA2 encode polypeptides with unknown functions. Here, we report the biological and molecular characterization of this virus in the chestnut blight fungus, Cryphonectria parasitica, a filamentous fungus that has been used as a model for mycovirus research. Transfection with purified RnMBV1 particles into an RNAsilencing-defective strain (Ddcl-2) of C. parasitica and subsequent anastomosis with the WT strain (EP155) resulted in stable persistent infection in both host strains. However, accumulation levels in the two strains were different, being~20-fold higher in Ddcl-2 than in EP155. Intriguingly, whilst RnMBV1 reduced both virulence and growth rate in Ddcl-2, it attenuated virulence without affecting significantly other traits in EP155. Western blot analysis using antiserum against recombinant proteins encoded by either ORF1 or ORF2 demonstrated the presence of a 250 kDa protein in purified virion preparations, suggesting that RdRp is expressed as a CP fusion product via a "1 frameshift. Antiserum against the ORF3-encoded protein allowed the detection of 150, 30 and 23 kDa polypeptides specifically in RnMBV1-infected mycelia. Some properties of an RnMBV1 mutant with genome rearrangements, which occurred after transfection of Ddcl-2 and EP155, were also presented. This study provides an additional example of C. parasitica serving as a versatile, heterologous fungus for exploring virus-host interactions and virus gene expression strategies.