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

Abstract

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.

FIG. 1.

Mycelial growth of virus-carrying field strain W779 and virus-cured isogenic strain W1015 of R. necatrix. (A) Colony morphology of R. necatrix strains W779 and W1015. The virus-carrying W779 and virus-cured W1015 fungal strains were grown on PDA for 5 days in the dark and photographed. (B) Mycelial growth on apple rootstocks. Twigs of Japanese pear (1.5 cm long) were placed for 3 weeks on 5-day-old PDA cultures of fungal strains W779 and W1015. Japanese pear twigs covered with mycelia of each fungal strain were fixed on stems of apple rootstocks (M. prunifolia var. ringo) that had been precultured in a soil medium for horticulture (Kenbyou, Yae Agricultural Co., Ltd., Nagasaki, Japan). Inoculated apple plants were cultured in the soil for an additional 2 weeks. Mycelial expansion is shown by white arrows, while inoculation sites are denoted by arrowheads. Levels of mycelial growth on apple rootstocks are equivalent to virulence levels.

FIG. 2.

Composition of virus particles isolated from strain W779. (A) Electron micrograph of virus particles. Virus particles purified by differential and sucrose gradient density centrifugation were negatively strained with 2% uranyl acetate and observed in a Hitachi model H-7000 transmission electron microscope. The scale bar indicates 100 nm. (B) RNA components of W779 particles. Nucleic acids were extracted from virus particles by the SDS-phenol method, electrophoresed in a 1% agarose gel, and stained with ethidium bromide (lane VP). dsRNAs were isolated from mycelia of the same W779 culture as used for virus purification (lane mycelia) and a virus-free W1015 culture (VF) and applied to an agarose gel in parallel. M refers to the 1-kb DNA ladder (GeneRuler; Fermentas). (C) Protein components of particles from strain W779. Purified particle preparations were denatured by boiling for 3 min in Laemmli's sample buffer containing 2% SDS and 0.05% β-mercaptoethanol and electrophoresed in a 10% polyacrylamide gel (lane VP) (43). A fraction obtained from virus-free strain W1015 by the same method as for virus particles was also analyzed (lane VF). Proteins were stained by Coomassie brilliant blue. Prestained protein size standards are from Bio-Rad (Precision Plus Protein Standards) (lane M). The black and gray arrowheads denote the major and minor protein bands, respectively.

FIG. 3.

Molecular characteristics of W779 dsRNA-1 and dsRNA-2. (A) Northern blot analysis of W779 dsRNA-1 and dsRNA-2. Total RNA and dsRNA prepared as stated in Materials and Methods were electrophoresed under denaturing conditions, blotted onto nylon membranes, and probed by DIG-labeled DNA fragments (probes 1 to 3). The positions of probes 1 to 3 are shown in panel B. (B) Schematic representation of the genetic organization of the strain W779 dsRNA-1 and dsRNA-2 segments. dsRNA-1 and -2 are 8,931 and 7,180 nt in length, respectively. dsRNA-1 has a 1,636-nt-long 5′ UTR, two ORFs (ORF1 and ORF2), and a 57-nt-long 3′ UTR, while dsRNA-2 has a 1,656-nt-long 5′ UTR, two ORFs (ORF3 and ORF4), and a 406-nt-long 3′ UTR. ORF1 to -4 are composed of 1,240, 1,111, 1,427, and 227 codons, respectively. Open boxes drawn with solid lines denote ORFs, while that drawn with dotted lines indicates a possible extension of ORF2 by frameshifting. Numbers above solid lines refer to map positions of initiation and termination codons of the respective ORFs.

FIG. 4.

Characteristics of the terminal sequence domains of dsRNA-1 and dsRNA-2. (A) Conserved terminal sequences of dsRNA-1 and dsRNA-2. The terminal sequences of the ORF-containing strands of dsRNA-1 and -2, obtained by sequencing the RACE clones, are shown. The 5′ 24-mer and 3′ octamer (lines above the sequences) are shared by the segments. Identical nucleotides are denoted by asterisks. Sequence similarities are found in the entire 5′ UTRs (see Fig. S3 at http://www.rib.okayama-u.ac.jp/pmi/2003/html/JV09suppl.html). (B) Possible secondary structures of the 3′-terminal sequences of W779 dsRNA-1 and dsRNA-2. The 3′-terminal 90-nt sequences of each of the plus-sense strands of dsRNA-1 and -2 were analyzed with the Mfold program. The default settings were utilized for the analyses. Secondary structures are depicted in a modified style from the ones provided by the program. The stop codon of ORF2 is indicated. (C) Possible inverted repeats formed by the terminal sequences of W779 dsRNA-1 and dsRNA-2. Both dsRNA-1 and -2 potentially form inverted repeats between the conserved terminal sequences. Nucleotides that differ between the inverted repeat structures of dsRNA-1 and -2 are shown by asterisks.

FIG. 5.

Molecular evolutionary analysis of the W779 dsRNA virus. Multiple alignment of sequences of the RdRp motifs (I to VIII) encoded by W779 dsRNA-1 and other mycoviruses. The alignment was prepared by the program CLUSTAL_X and modified manually based on those reported by Bruenn (7), Jiang and Ghabrial (32), and Hillman et al. (26). Abbreviated virus names: LeV-HKB, Lentinula edodes mycovirus HKB; PgV1, Phlebiopsis gigantea mycovirus dsRNA-1; PcV, Penicillium chrysogenum virus; HvV145S, Helminthosporium victoriae 145S virus; ACD-CV, Amasya cherry disease-associated chrysovirus; CCRS-CV, cherry chlorotic rusty spot-associated chrysovirus; FoV1, Fusarium oxysporum chrysovirus 1; AbV1, Agaricus bisporus virus 1; ScV-L-A, Saccharomyces cerevisiae virus L-A; ScV-L-BC, Saccharomyces cerevisiae virus L-BC; UmV-H1, Ustilago maydis virus H1; HmV-17, Helicobasidium mompa no. 17 dsRNA virus; GaRV1, Gremmeniella abetina RNA virus L1; BfTV1, Botryotinia fuckeliana totivirus 1; MoV1, Magnaporthe oryzae virus 1; Hv190SV, Helminthosporium victoriae 190S virus; FpV1, Fusarium poae virus 1 (AF047013); RnPV1, Rosellinia necatrix partitivirus 1-W8 (NC_007537); AhV, Atkinsonella hypoxylon virus (L39125); FsV1, Fusarium solani virus 1 (D55668); PsV-S, Penicillium stoloniferum virus S (NC_005976); CHV1, Cryphonectria hypovirus 1-EP713 (M57938); CHV2, Cryphonectria hypovirus 2-NB58 (L29010). See Table 1 for the accession numbers, except for members of the families Partitiviridae and Hypoviridae. LeV-HKB and PgV1 are partially characterized, and their entire genome sequences are not available.

FIG. 6.

Phylogenetic analysis of the W779 virus. A multiple alignment of RdRps from 24 related viruses representing the established dsRNA mycovirus families and genera and virus-like elements (Fig. 5) was used to construct a dendrogram. The neighbor-joining tree was constructed by using CLUSTAL_X in which hypoviruses with ssRNA genomes were included as an outgroup. Numbers at the nodes denote bootstrap values out of 1,000 replicates.

FIG. 7.

Effects of W779 virus introduction on the phenotypes of mycelially incompatible fungal strains. (A) Colony morphology of transfected and untransfected fungal strains. Purified virus particles were introduced into spheroplasts derived from W97 and W370T1 (vegetatively incompatible with W779 and W1015). Regenerated isolates were grown on PDA for 6 days at 25°C in the dark. Representative colonies transfected with virus particles [virus (+)] are shown. Untransfected strains W97 and W370T1 were cultured in parallel [virus (−)]. Mean colony sizes and standard deviations of five cultures are shown in graphic form below the colony photograph. (B) Virulence assay on apple rootstocks. Nursery rootstocks (M. prunifolia var. ringo) were planted in plastic containers (five rootstocks per container), cultured for approximately 1.5 months, and subsequently inoculated with fungal mycelia of each of the following fungal strains: virus-free W370T1 [virus (−)], virus-infected W370T1 [virus (+)], virus-free W97 [virus (−)], and virus-infected W97 [virus (+)]. Ten plants in two containers were inoculated with each strain. Phenotypes of apple plants 4 weeks following inoculation with virus-infected and uninfected W370T1 are shown. Virus-free W370T1 induced lethal-type symptoms in all of the plants in the two left pots, while virus-infected W370T1 is attenuated in virulence in the right two pots. The graph below the photograph shows the number of apple plants destroyed out of 10 scored at 4 weeks postinoculation.