Mycoviruses are widespread in all major groups of plant pathogenic fungi. They are transmitted intracellularly during cell division, sporogenesis, and cell fusion, but apparently lack an extracellular route for infection. Their natural host ranges are limited to individuals within the same or closely related vegetative compatibility groups. Recent advances, however, allowed the establishment of experimental host ranges for a few mycoviruses. Although the majority of known mycoviruses have dsRNA genomes that are packaged in isometric particles, an increasing number of usually unencapsidated mycoviruses with positive-strand RNA genomes have been reported. We discuss selected mycoviruses that cause debilitating diseases and/or reduce the virulence of their phytopathogenic fungal hosts. Such fungal-virus systems are valuable for the development of novel biocontol strategies and for gaining an insight into the molecular basis of fungal virulence. The availability of viral and host genome sequences and of transformation and transfection protocols for some plant pathogenic fungi will contribute to progress in fungal virology.

Viruses of plant pathogenic fungi.

Mycoviruses (fungal viruses) are reviewed with emphasis on plant pathogenic fungi. Based on the presence of virus-like particles and unencapsidated dsRNAs, mycoviruses are common in all major fungal groups. Over 80 mycovirus species have been officially recognized from ten virus families, but a paucity of nucleic acid sequence data makes assignment of many reported mycoviruses difficult. Although most of the particle types recognized to date are isometric, a variety of morphologies have been found and, additionally, many apparently unencapsidated dsRNAs have been reported. Until recently, most characterized mycoviruses have dsRNA genomes, but ssRNA mycoviruses now constitute about one-third of the total. Two hypotheses for the origin of mycoviruses of plant pathogens are discussed: the first that they are of unknown but ancient origin and have coevolved along with their hosts, the second that they have relatively recently moved from a fungal plant host into the fungus. Although mycoviruses are typically readily transmitted through asexual spores, transmission through sexual spores varies with the host fungus. Evidence for natural horizontal transmission has been found. Typically, mycoviruses are apparently symptomless (cryptic) but beneficial effects on the host fungus have been reported. Of more practical interest to plant pathologists are those viruses that confer a hypovirulent phenotype, and the scope for using such viruses as biocontrol agents is reviewed. New tools are being developed based on host genome studies that will help to address the intellectual challenge of understanding the fungal-virus interactions and the practical challenge of manipulating this relationship to develop novel biocontrol agents for important plant pathogens.

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Mycoviruses of filamentous fungi and their relevance to plant pathology

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 approximately 50 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.

A novel bipartite double-stranded RNA Mycovirus from the white root rot Fungus Rosellinia necatrix: molecular and biological characterization, taxonomic considerations, and potential for biological control.

Many bands were detected on an electrophoretic profile of double-stranded (ds) RNA preparation from a single strain of Fusarium poae isolated from wheat. When the purified dsRNA sample was deep-sequenced by a next-generation sequencer, sixteen virus-like assembled contigs with predicted amino acid sequences showing homologies to respective viral RNA-dependent RNA polymerases (RdRps) were found by BLAST analysis. Fourteen out of sixteen sequences showed homologies to RdRps of known mycoviruses, that is, four mitoviruses, two narnaviruses, two partitiviruses, an alternavirus, a fusarivirus, a hypovirus, a victorivirus, and two unclassified mycoviruses, Sclerotinia sclerotiorum dsRNA mycovirus-L and Aspergillus foetidus slow virus 2, respectively. The other two putative viral RdRp sequences showed homologies to those of members of negative-stranded RNA viruses, the Ophiovirus and the Phlebovirus respectively, which mycoviruses had been not ever assigned to. Based on genome structure and phylogenetic analysis, both viruses were thought to be members of novel respective negative-stranded RNA virus groups. The presences of all sixteen viral RdRp sequences identified by BLAST analysis were confirmed by sequencing RT-PCR products generated from the starting dsRNA material using primers designed from the de novo assembled sequences of respective putative mycoviruses. Since the single strain of F. poae was considered to be multiply infected with mycoviruses from novel taxonomical groups in addition to many common mycoviruses, the RNA virome of the strain was found to be highly diverse.

Multiple virus infection in a single strain of Fusarium poae shown by deep sequencing

Botryosphaeria dothidea is an important pathogenic fungus causing fruit rot, leaf and stem ring spots and dieback, stem canker, stem death or stool mortality, and decline of pear trees. Seven double-stranded RNAs (dsRNAs; dsRNAs 1 to 7 with sizes of 3,654, 2,773, 2,597, 2,574, 1,823, 1,623, and 511 bp, respectively) were identified in an isolate of B. dothidea exhibiting attenuated growth and virulence and a sectoring phenotype. Characterization of the dsRNAs revealed that they belong to two dsRNA mycoviruses. The four largest dsRNAs (dsRNAs 1 to 4) are the genomic components of a novel member of the family Chrysoviridae (tentatively designated Botryosphaeria dothidea chrysovirus 1 [BdCV1]), a view supported by the morphology of the virions and phylogenetic analysis of the putative RNA-dependent RNA polymerases (RdRps). Two other dsRNAs (dsRNAs 5 and 6) are the genomic components of a novel member of the family Partitiviridae (tentatively designated Botryosphaeria dothidea partitivirus 1 [BdPV1]), which is placed in a clade distinct from other established partitivirus genera on the basis of the phylogenetic analysis of its RdRp. The smallest dsRNA, dsRNA7, seems to be a noncoding satellite RNA of BdPV1 on the basis of the conservation of its terminal sequences in BdPV1 genomic segments and its cosegregation with BdPV1 after horizontal transmission. This is the first report of a chrysovirus and a partitivirus infecting B. dothidea and of a chrysovirus associated with the hypovirulence of a phytopathogenic fungus.

IMPORTANCE Our studies identified and characterized two novel mycoviruses, Botryosphaeria dothidea chrysovirus 1 (BdCV1) and Botryosphaeria dothidea partitivirus 1 (BdPV1), associated with the hypovirulence of an important fungus pathogenic to fruit trees. This is the first report of a chrysovirus and a partitivirus infecting B. dothidea and of a chrysovirus associated with the hypovirulence of a phytopathogenic fungus. BdCV1 appears to be a good candidate for the biological control of the serious disease induced by B. dothidea. Additionally, BdPV1 is placed in a clade distinct from the established genera. The BdCV1 capsid has two major structural proteins, and the capsid is distinct from that made up by a single polypeptide of the typical chrysoviruses. BdPV1 is the second partitivirus in which the putative capsid protein shares no significant identity with any mycovirus protein. A small accompanying dsRNA that is presumed to be a noncoding satellite RNA of BdPV1 is the first of its kind reported for a partitivirus.

Hypovirulence of the Phytopathogenic Fungus Botryosphaeria dothidea: Association with a Coinfecting Chrysovirus and a Partitivirus

The W8 isolate of the phytopathogenic fungus, Rosellinia necatrix that causes white root rot, contained three segments of double-stranded (ds) RNA, namely L1, L2 and M. Purified viral particles of about 25 nm in diameter contained an RNA segment with almost the same mobility as M-dsRNA, but the band was sensitive to S1 nuclease. Molecular analysis revealed that M-dsRNA consisted of two (RNA 1 and RNA 2) similarly sized species of 2299 and 2279 bp excluding an interrupted poly (A or U) tail of 16-51 bp. The predicted largest open reading frame in RNA 1 and RNA 2 was similar to those of RNA dependent RNA polymerase (RdRp) and coat protein (CP), respectively, encoded by the family Partitiviridae. The non-coding regions (NCR) of the two segments were similar (approximately 70% base identity) at the 5' end, but different at the 3' end. The NCR at the 3' end contained adenosine-uracil rich elements (AREs) in both segments. Northern analyses revealed RNA 1 and RNA 2 in mycelial and viral particle fractions. We coined the name Rosellinia necatrix partitivirus 1-W8 (RnPV1-W8) for M-dsRNA based on viral particle morphology and sequence information.

M. Miyanishi

Papers

Molecular characterization of a partitivirus from the plant pathogenic ascomycete Rosellinia necatrix