Double-stranded RNA mycovirus from Fusarium graminearum

Abstract

Double-stranded RNA (dsRNA) viruses in some fungi are associated with hypovirulence and have been used or proposed as biological control agents. We isolated 7.5-kb dsRNAs from 13 of 286 field strains of Fusarium graminearum isolated from maize in Korea. One of these strains, DK21, was examined in more detail. This strain had pronounced morphological changes, including reduction in mycelial growth, increased pigmentation, reduced virulence towards wheat, and decreased (60-fold) production of trichothecene mycotoxins. The presence or absence of the 7.5-kb dsRNA was correlated with the changes in pathogenicity and morphology. The dsRNA could be transferred to virus-free strains by hyphal fusion, and the recipient strain acquired the virus-associated phenotype of the donor strain. The dsRNA was transmitted to approximately 50% of the conidia, and only colonies resulting from conidia carrying the mycovirus had the virus-associated phenotype. Partial nucleotide sequences of the purified dsRNA identify an RNA-dependent RNA polymerase sequence and an ATP-dependent helicase that are closely related to those of Cryphonectria hypovirus and Barley yellow mosaic virus. Collectively, these results suggest that this dsRNA isolated from F. graminearum encodes traits for hypovirulence.

FIG. 1.

dsRNA molecules purified from F. graminearum isolates and separated on a 1% agarose gel. Lanes M1 and M2, 1-kb ladder (New England BioLabs) and λ DNA digested with HindIII, respectively; lanes 1 to 6, isolates YWD3, YWD6, DK21 YDP16, YWD5, and JB53, respectively. Numbers on the left and right indicate sizes in kilobases.

FIG. 2.

Cultural morphology of isolate DK21 and pathogenicity test. A dsRNA-containing single-conidial isolate (A) grew slowly and was deep red. A dsRNA-free isolate (B) grew fast and was pink. Through anastomosis, hygromycin-resistant colonies (C to F) that showed different cultural appearances on CM plates were selected as candidates for dsRNA transmission. The slow-growing colonies (C, D, and E) were hygromycin resistant and contained dsRNA, and the fast-growing one (F) was dsRNA free. For pathogenicity tests, conidial suspensions were inoculated onto wheat plants. Plants infected with dsRNA-free spores showed more severe symptoms than those infected with dsRNA-containing spores. CK, water-sprayed control plants.

FIG. 3.

Conserved sequence motifs in the RNA-dependent RNA polymerases (RdRp) (A) and helicases (B) of positive-strand RNA viruses, related dsRNA viruses, and the 7.5-kb dsRNA of strain DK21. The tentative superpositions of the conserved motifs correspond to the motifs described by others (21, 30, 48). The number of amino acid residues between the motifs and the distances from the protein termini are shown (question marks show that numbers are unknown). In the consensus line above the sequences, “&” designates a bulky hydrophobic residue (either aliphatic or aromatic). GenBank accession numbers are as follows: CHV1-EP713 (Cryphonectria hypovirus 1), AAA67458; LRV2-1 (Leishmania RNA virus 1-1), AAB50024; ScV-L-A (Saccharomyces cerevisiae virus L-A), AAA50321; DaV (Diaporthe ambigua RNA virus 1), AAF22958; FusoV (Mycovirus FusoV from Fusarium solani), BAA09520; HvV190S (Helminthosporium victoriae virus 190S), AAB94791; RsV (Rhizoctonia solani virus), AF133290; CHV1-713 (Cryphonectria hypovirus EP713), AAA67458; CHV2-NB58 (Cryphenectria hypovirus 2-NB58), AAA20137; CHV3-GH2 (Cryphonectria hypovirus 3-GH2), AF13604; and BaYMV (Barley yellow mosaic virus), CAA49412.

FIG. 4.

Northern blot analysis. Total RNA was extracted from dsRNA-free and -containing derivatives of DK21 and several colonies obtained from anastomosis. The extracts were treated with DNase and blotted to a nylon membrane. Gels were probed with a dsRNA-specific probe (A) and with ethidium bromide (B). The arrowhead indicates the position of the dsRNA. Lanes: 1, λ/HindIII; 2, prebiotinylated DNA marker; 3, dsRNA-free derivative; 4, dsRNA-containing derivative; 5 and 6, dsRNA-free derivatives obtained from hyphal anastomosis T-DK F2 and T-DK F4, respectively; 7 to 9, dsRNA-containing derivatives obtained from hyphal anastomosis T-DK B2, T-DK C5, and T-DK D1, respectively. Numbers on the left indicate sizes in kilobases.