APPLIED AND ENVIRONMENTAL MICROBIOLOGY, May 2002, p. 2529–2534 Vol. 68, No. 50099-2240/02/$04.00�0 DOI: 10.1128/AEM.68.5.2529–2534.2002Copyright © 2002, American Society for Microbiology. All Rights Reserved.

Double-Stranded RNA Mycovirus from Fusarium graminearumYeon-Mee Chu,1 Jae-Jin Jeon,1 Sang-Jin Yea,1 Yong-Ho Kim,2 Sung-Hwan Yun,2 Yin-Won Lee,1

and Kook-Hyung Kim1*School of Agricultural Biotechnology, Seoul National University, Suwon 441-744,1 and Division of Life Sciences,

Soonchunhyang University, Asan 336-745,2 Korea

Received 13 November 2001/Accepted 6 February 2002

Double-stranded RNA (dsRNA) viruses in some fungi are associated with hypovirulence and have been usedor proposed as biological control agents. We isolated 7.5-kb dsRNAs from 13 of 286 field strains of Fusariumgraminearum isolated from maize in Korea. One of these strains, DK21, was examined in more detail. Thisstrain had pronounced morphological changes, including reduction in mycelial growth, increased pigmenta-tion, reduced virulence towards wheat, and decreased (60-fold) production of trichothecene mycotoxins. Thepresence 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 thevirus-associated phenotype of the donor strain. The dsRNA was transmitted to approximately 50% of theconidia, 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 andan ATP-dependent helicase that are closely related to those of Cryphonectria hypovirus and Barley yellow mosaicvirus. Collectively, these results suggest that this dsRNA isolated from F. graminearum encodes traits forhypovirulence.

Double-stranded RNA (dsRNA) mycoviruses have been de-scribed for a wide variety of fungi and plant-pathogenic fungi(7, 9, 27, 42, 44, 55, 58). These dsRNA mycoviruses are clas-sified into three families based on number of genome seg-ments, capsid structure, and nucleotide sequences, with somedsRNA mycoviruses remaining unclassified (8, 20, 24, 31, 39,45). The isometric dsRNA mycoviruses are classified into twofamilies, Totiviridae and Partitiviridae, consisting of viruses thatare enclosed in nonenveloped isometric particles of 25 to 50nm in diameter and typically cause latent infections in theirhost fungi (20). Totiviruses have a nonsegmented genome,while partitiviruses have segmented genomes. Hypoviruses(family Hypoviridae) are the unusual exception in that they canresult in considerable morphological and physiological changes,including cytological alterations, changes in colony morphologyand growth rate, and persistently attenuate novel virulence-re-lated phenotypes (hypovirulence) (2, 7, 13, 25, 28, 34, 42). Hypo-viruses lack conventional virions, and their dsRNAs are enclosedin host-encoded vesicles (17).

Among Fusarium species, dsRNA mycoviruses have beenreported to be present in F. poae and F. solani f. sp. robiniae(14, 39, 40). dsRNA elements of the same electrophoreticmobility isolated from vegetatively compatible strains of F.poae were similar or identical in many cases. Similar-sizeddsRNA elements of vegetatively incompatible strains of thefungus could contain both homologous and nonhomologousdsRNAs (14). Morphological alterations or signs of degener-ation of F. poae, however, were not observed in any of thedsRNA-containing isolates.

We have isolated Fusarium graminearum Schwabe [tele-morph: Gibberella zeae (Schwein.) Petch] and occasionallyfound colonies with unusual morphology, e.g., slow growth,ameboid mycelia, and increased pigmentation, similar to thatdescribed earlier for some F. graminearum isolates from China(60). Phenotypic characteristics of F. graminearum have notbeen reliably attributed to the presence of virus. In this study,we screened isolates of F. graminearum recovered from Koreanmaize for dsRNA mycoviruses. Our objectives in this studywere (i) to determine if dsRNA mycoviruses were present infield isolates of F. graminearum, (ii) to determine if thedsRNAs result in discernible changes in fungal phenotype,and (iii) to conduct sequence analysis of the identifieddsRNA. Results of the experiments in this work showed forthe first time that the presence of the dsRNA mycovirusresults in changes in morphological and pathogenicity phe-notypes of F. graminearum. Partial nucleotide sequenceanalysis also showed that the dsRNA contained sequencesthat had identities with RNA-dependent RNA polymerasesof plus-strand RNA viruses.

MATERIALS AND METHODS

Maize samples. Samples of maize seeds, approximately 500 g each, werecollected from 40 fields in eight corn-producing regions in the Gangwon provinceof Korea during November 1999.

Isolation of fungi. One hundred seeds of each sample were soaked in 2%NaOCl for 2 min, rinsed in sterile distilled water, transferred to potato dextroseagar (PDA) (Difco Laboratories, Detroit, Mich.), and incubated for 4 to 7 daysat 25°C. Fusarium isolates, with carmine red pigmentation, were transferred tohomemade PDA (20% potato extract, 2% dextrose, 1.5% agar), carnation leafagar (18), or both, incubated under fluorescent lamps (cool white type; 5,000 lx)at 25°C, and identified to the species level by the procedures of Nelson et al. (38).We recovered 809 Fusarium isolates from the maize seed. Isolates were stored asspore suspensions in 15% glycerol at �80°C and subcultured on PDA as needed.We screened 286 F. graminearum isolates for the presence of dsRNA. dsRNAvirus-containing strain DK21 has been deposited in the Korean Collection of

* Corresponding author. Mailing address: School of AgriculturalBiotechnology, Seoul National University, Suwon 441-744, Korea.Phone: 82-31-290-2441. Fax: 82-31-294-5881. E-mail: kookkim@snu.ac.kr.

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Type Cultures at the Genetic Resources Center, Korea Research Institute ofBioscience and Biotechnology, Daejon, Korea, under accession number 26916.

Culture conditions, dsRNA extraction, and enzymatic digestions. To deter-mine if dsRNA mycoviruses were present in field isolates of F. graminearum,each isolate was grown in 100 ml of complete medium (CM) broth (16) for 4 to5 days at 25°C in a orbital shaker (100 rpm). Mycelia were collected by filtrationthrough four layers of cheesecloth, followed by washing with distilled water.Mycelia were dried by blotting with paper towels and stored frozen at �70°Cuntil extracted (54). Extraction of nucleic acids and purification of dsRNA bycellulose chromatography were performed as previously described (54). Thequality and relative concentration of dsRNA were checked by electrophoresis on1% agarose gels at 4°C and visualized following ethidium bromide staining andillumination with 350-nm UV light. RNase A digestion under high- and low-ionic-strength buffer conditions and DNase digestion of extracted nucleic acidswere carried out as described by Vilches and Castillo (57).

Mycelial growth and sporulation. Mycelial growth of dsRNA-containing iso-lates was measured on CM after incubation for 3 days at 25°C (33). Conidia weregrown in carboxymethyl cellulose (CMC) broth (15 g of CMC, 1 g of yeastextract, 0.5 g of MgSO4, 1 g of NH4NO3, and 1 g of KH2PO4 per liter) for 9 daysat 25°C in a rotary shaker (100 rpm). One thousand conidia in 100 �l wereinoculated into 5 ml of CMC broth in a test tube (1.7 by 17 cm). Sporulation wasestimated after incubation for 8 days as described by Ann and Lee (3). Twentyindependent experiments were performed to obtain means and standard devia-tions.

Characteristics of dsRNA obtained from F. graminearum strain DK21. Tofurther characterize the molecular nature and the role(s) of the dsRNAs in theirfungal hosts, one F. graminearum strain, DK21, containing a 7.5-kb dsRNA andwith altered fungal morphology, was arbitrarily selected and used for the follow-ing assays.

(i) Transmission of dsRNA. To determine if the dsRNAs were transmittedthrough the conidia, we collected conidia from F. graminearum DK21 and platedthem on 2% water agar. After incubation for 8 h at 25°C, colonies originatingfrom a germinated conidium, as determined microscopically, were transferred tofresh CM, and the presence of the dsRNA was determined.

(ii) Pathogenicity. To determine if the dsRNAs affect virulence of the hostfungus, conidial suspensions of dsRNA-containing and dsRNA-free isolates wereinoculated onto wheat plants. Conidia were grown in CMC broth for 9 days at25°C. Conidia were harvested, filtered through cheesecloth, and diluted withsterile water if necessary. Fifty-milliliter portions of spore suspension (105 or 103

conidia/ml) were prepared and sprayed onto two sheaves each of the OlgruMiland GumgangMil wheat varieties; each sheaf contained at least 50 heads. Afterbeing sprayed, the plants were placed in a plastic bag for 3 days to maintain highrelative humidity. Approximately 2 weeks after inoculation, plants were har-vested and disease severity was rated on a scale of 0 to 5, as follows: 0, no disease;1, weak head blight with less than one-third of a head infected; 2, less than halfof a head infected; 3, more than half of, but not the entire, head infected but few(�10%) completely infected heads observed; 4, more than half of, but not theentire, head infected and more than 10% of heads completely infected; and 5,entire head usually infected. The mean of the disease severity from three inde-pendent experiments was used as the virulence value.

(iii) Mycotoxin analyses. dsRNA-containing and dsRNA-free F. graminearumsubcultures of DK21 were grown on sterile rice for 2 weeks at 25°C as previouslydescribed (51) to see whether the presence of the dsRNAs affected mycotoxinproduction by F. graminearum. The mycelial mass and substrate were dispersedonto a screen-bottom tray and allowed to air dry in a ventilated hood (25°C) for5 days. Ten grams of dried substrate was ground in a blender and extracted asdescribed by Tanaka et al. (51). Briefly, each ground sample (10 g) was extractedwith 100 ml of acetonitrile-water (3:1, vol/vol) in a 500-ml Erlenmeyer flask for30 min on a rotary shaker (150 rpm), and the extract was filtered throughWhatman (Maidstone, Kent, England) no. 1 filter paper. A 50-ml portion of thefiltrate was defatted with the same volume of n-hexane. The aqueous phase wasmixed with 100 ml of ethanol and evaporated to dryness. The residue wasdissolved in 3 ml of methanol and applied directly to a Florisil column (2 by 20cm) containing 10 g of Florisil (60/100 mesh; Fisher Scientific Co., Pittsburgh,Pa.). The column was washed with 100 ml of n-hexane and eluted with 100 ml ofchloroform-methanol (9:1, vol/vol). The eluate was concentrated to drynessunder reduced pressure, and the residue was redissolved in 2 ml of methanol.Mycotoxins were analyzed with a Shimadzu (Kyoto, Japan) QP-5000 gas chro-matograph-mass spectrometer as previously described (43, 49).

(iv) Preparation of fungal protoplasts, transformation, and anastomosis. Theplasmid pUCHI (53) was used for transformation. Preparation and inoculationof fungal protoplasts were performed as previously described (53). Fungal trans-formation was performed with the virus-free F. graminearum strain DK. One

milliliter of the transformed mixture was mixed with 10 ml of regenerationmedium (1 g of yeast extract, 1 g of casein enzymatic hydrolysate, 342 g ofsucrose, and 18 g of agar per liter), and poured into a petri dish. After incubationat 25°C overnight, the plate was overlaid with 10 ml of 1% agar containinghygromycin B (150 ppm). The resulting dsRNA mycovirus-free hygromycin B-resistant transformant, T-DK, was used for studies of hyphal anastomosis.

To transfer the dsRNA from DK21 to the hygromycin B-resistant transfor-mant, T-DK, mycelial plugs of T-DK were placed around colonies of 13-day-oldDK21 on CM and allowed to incubate for at least 4 days after the colonies of thetwo strains made contact. Mycelial plugs from the edges of the T-DK colonieswere transferred to a CM plate containing hygromycin (100 ppm). Hygromycin-resistant colonies were selected as candidates for dsRNA transmission. dsRNAwas extracted from each colony and visualized on a 1% agarose gel.

(v) Cloning and sequence analysis. Five micrograms of gel-purified dsRNAwas heat denatured at 99°C for 5 min and primed with random hexamers. First-and second-strand cDNA synthesis reactions and cDNA cloning procedures wereperformed as described by Gubler and Hoffman (23) and Hillman et al. (26).dsRNA-specific, 25-base oligonucleotide primers (8F, 5�-AAAAGTGTCCTTGACCAAAA-3�; 8R, 5�-AAGCCTTTCAAGCAGTTGTG-3�; 11F, 5�-AGTTTCGCCAGGTCTTTCCA-3�; and 11R, 5�-CTATAGTCCTTTAGATAAGC-3�)were designed based on the sequences of the initial cDNA clones and used forfirst-strand cDNA synthesis. About 250 ng of the first-strand cDNA was ampli-fied by PCR in a final volume of 100 �l containing 10 mM Tris-HCl (pH 8.8), 50mM KCl, 1.5 mM MgCl2, a 125 �M concentration of each deoxynucleosidetriphosphate, 50 pmol of designed and random primers, and 2.5 U of Taq DNApolymerase (TaKaRa Shuzo Co. Ltd., Shiga, Japan). Amplification was carriedout using 1 cycle of 94°C for 1 min, 42°C for 1 min, and 72°C for 5 min followedby 35 cycles of 94°C for 1 min, 60°C for 1 min, and 72°C for 1 min. After the cycleswere completed, the reaction mixtures were held at 72°C for 10 min and then at4°C. PCR products were purified and ligated into the TOPO cloning vector(Invitrogen, Carlsbad, Calif.) or pT7 Blue-2 T-vector (Novagen, Darmstadt,Germany). Recombinant plasmids containing cDNA inserts were sequenced bythe dideoxynucleotide chain termination method by using the ABI Prism Ter-minator Cycle Sequencing Ready Reaction Kit and ABI Prism 377 GeneticAnalyzer (Perkin-Elmer, Foster City, Calif.), located at the National Instrumen-tation Center for Environmental Management of Seoul National University,according to the manufacturer’s instructions. All clones were sequenced in bothdirections, and at least three different DNA clones representing each amplifiedDNA fragment were sequenced. Polyprotein-related sequences were initiallyidentified using the BLAST search program of GenBank (61). Multiple-sequencealignment was performed with the CLUSTAL W program (52).

(vi) Northern blotting. dsRNAs were resolved on a 1% agarose gel in TAEbuffer (40 mM Tris-acetate, 1 mM EDTA, pH 8.0), and denatured by soaking thegel in 50 mM NaOH for 30 min at room temperature. Gels were neutralized with1.5 M Tris-HCl (pH 7.5) containing 0.5 M NaCl for 20 min. The RNAs weretransferred to a nylon membrane (Sigma, St. Louis, Mo.) by capillary blottingwith 2� SSC (0.3 M NaCl, 30 mM sodium citrate, pH 7.0) for 10 min andcross-linked to the membrane by UV irradiation for 1 min. dsRNA-specificprobes were prepared with the NEBlot Phototope kit, and viral bands weredetected by chemiluminescence (Phototope-Star Detection kit; New EnglandBioLabs, Boston, Mass.).

Nucleotide sequence accession numbers. The partial sequences of the dsRNAisolated from DK21 have been deposited in GenBank under accession numbersAF443212 (pDK-2) and AF443213 (pDK-1).

RESULTS

Natural occurrence of 7.5-kb dsRNA in F. graminearum iso-lated from corn. We found that 13 of 286 isolates of F. gra-minearum from maize contained dsRNA. CF-11 cellulose col-umn chromatography and enzyme digestions using RNase Aand DNase confirmed the dsRNA nature of the genome. Thepurified viral genome was resistant to DNase and RNase A ata high salt concentration (0.3 M NaCl) (data not shown).

Morphological differences between a dsRNA-free strain anda dsRNA-containing strain. We detected significant differ-ences in mycelial growth rates (Table 1). A 7.5-kb dsRNA waspresent in all of the F. graminearum isolates with altered phe-notypes (Fig. 1). dsRNA-free derivatives of strain DK21 cov-ered the entire plate after 3 days. In contrast, the dsRNA-

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containing subcultures took more than 9 days to do so. We didnot observe a significant difference, however, in the productionof conidia. dsRNA-free and dsRNA-containing subculturesproduced 135 and 133 conidia per ml of CMC broth, respec-tively. In addition to the 7.5-kb dsRNA, smaller dsRNAs, of 5.5to 6 kb, were also found in some isolates (Fig. 1). The presenceof these smaller RNAs, however, did not change the alteredphenotypes of the F. graminearum isolates.

Effects of dsRNAs on pathogenicity and mycotoxin produc-tion. Head blight symptoms were observed on all wheat plantsinoculated with both the dsRNA-free and dsRNA-containingisolates. The disease severity of the dsRNA-containing single-conidial isolates, however, was significantly less than that of thedsRNA-free isolates. Disease severities of dsRNA-free isolateson inoculated wheat sheaves were 2.3 � 1.01 and 4.7 � 0.36,whereas those of dsRNA-containing isolates were 1.7 � 0.71and 2.9 � 0.80, when inoculated with suspensions of 103 and105 conidia per ml, respectively. No symptoms were associatedwith the control inoculation. Deoxynivalenol (DON) was de-tected at significantly lower levels in dsRNA-containing deriv-atives than in dsRNA-free isolates. The relative levels of DONin the single-conidial isolate of dsRNA-free and dsRNA-con-taining strains were 76 to 84 ppm and 3 to 7 ppm, respectively.

Transmission of dsRNAs to conidia. Single-conidial deriva-tives of strain DK21 could be placed into two groups. The

growth rates of isolates in one group were higher than those ofisolates in the other group and resembled those of the dsRNA-free derivatives. Mycelial growth and pigmentation also weredifferent (Fig. 2; Table 1). dsRNA profiles of 55 single-conidialisolates were tested. The 28 isolates that lacked dsRNA all hadthe wild-type dsRNA-free phenotype.

Transmission of dsRNA following hyphal anastomosis. Werecovered several colonies containing dsRNA when we at-tempted to transmit dsRNA from DK21 to T-DK. ColoniesT-DK B2, T-DK C5, T-DK C7, T-DK D1 to D3, and T-DK F1to F6, which were hygromycin resistant, were further studied.T-DK B2, T-DK C5, T-DK C7, and T-DK D1 to D3 hadphenotypes similar to those of strains known to carry thedsRNA, while the phenotypes of T-DK F1 to F6 resembledthose of other dsRNA-free isolates. Isolates T-DK B2, T-DKC5, T-DK C7, and T-DK D1 to D3 contained dsRNA, butT-DK F1 to F6 did not and are simply subcultures of T-DK(Fig. 2). When the dsRNA was introduced into strain T-DK, it

FIG. 1. dsRNA molecules purified from F. graminearum isolatesand separated on a 1% agarose gel. Lanes M1 and M2, 1-kb ladder(New England BioLabs) and � DNA digested with HindIII, respec-tively; lanes 1 to 6, isolates YWD3, YWD6, DK21 YDP16, YWD5, andJB53, respectively. Numbers on the left and right indicate sizes inkilobases.

FIG. 2. Cultural morphology of isolate DK21 and pathogenicitytest. A dsRNA-containing single-conidial isolate (A) grew slowly andwas deep red. A dsRNA-free isolate (B) grew fast and was pink.Through anastomosis, hygromycin-resistant colonies (C to F) thatshowed different cultural appearances on CM plates were selected ascandidates for dsRNA transmission. The slow-growing colonies (C, D,and E) were hygromycin resistant and contained dsRNA, and thefast-growing one (F) was dsRNA free. For pathogenicity tests, conidialsuspensions were inoculated onto wheat plants. Plants infected withdsRNA-free spores showed more severe symptoms than those infectedwith dsRNA-containing spores. CK, water-sprayed control plants.

TABLE 1. Characteristics of dsRNA-containing and dsRNA-freeisolates of DK21

Isolatea dsRNA Morphologyb Colony diam(cm)c

dsRNA-containing DK21 � Dark red, irregular 0.86 � 0.25d

dsRNA-free DK21 � Pink, circular 2.34 � 0.13T-DK B2 � Dark red, irregular 1.01 � 0.33d

T-DK C5 � Dark red, irregular 0.98 � 0.51d

T-DK D1 � Dark red, irregular 0.88 � 0.08d

T-DK F1 � Pink, circular 2.38 � 0.04

a F. graminearum isolates T-DK B2, T-DK C5, T-DK D1, and T-DK F1 wereobtained following anastomosis between dsRNA-free T-DK and dsRNA-con-taining DK21.

b dsRNA-containing isolates produced dark red pigment, and their colonyshape was ameboid. dsRNA-free isolates were pink and had a circular colonyshape.

c Values are the means and standard deviations compiled from 20 independentexperiments. Colony diameter was determined after 3 days of incubation on CM.

d The value is significantly different by Student’s t test (P 0.05) from that forthe dsRNA virus-free isolate.

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altered the colony morphology. This result suggests that theunusual cultural morphology of DK21 was due to the dsRNAmycovirus infection.

Partial sequence analysis of 7.5-kb dsRNA. Two cDNA se-quences, one of 825 bp and the other of 1,080 bp in length(pDK-1 and pDK-2; GenBank accession no. AF443213 andAF443212, respectively), were obtained from overlappingcDNA clones, i.e., pDK0112-12 (511 bp), pDK1229-1 (157 bp),and pDK1026-8 (283 bp) for the 825-bp fragment andpDK1026-11 (545 bp) and pDK1229-4 (570 bp) for the1,080-bp fragment. The sequences from the dsRNAs haveidentity to RNA-dependent RNA polymerase (RdRp) andATP-dependent helicase sequences of several viruses, includ-ing Cryphonectria hypovirus and Barley yellow mosaic virus (Fig.

3). The tentative superposition of the conserved motifs ofRdRp is designated as described by Koonin (30), and theconserved helicase motif is shown as suggested by Gorbalenyaet al. (21) and Smart et al. (48).

These two DNA sequences were used as probes for North-ern blot analyses to confirm that they originated from the7.5-kb dsRNA genome. Each probe specifically hybridized tothe 7.5-kb dsRNA in dsRNA-containing isolates T-DK B2,T-DK C5, and T-DK D3 (Fig. 4). No signal was observed witheither probe in the lanes with virus-free isolates T-DK F1 andT-DK F3. Thus, the sequences that we obtained are from the7.5-kb dsRNA mycovirus genome. Smaller dsRNAs (5.5 to 6kb) observed in some F. graminearum isolates were detected inNorthern blots using both the pDK-1 and pDK-2 probes (datanot shown), indicating that these smaller dsRNAs contain se-quences similar or identical to the 7.5-kb dsRNA.

DISCUSSION

We found that a 7.5-kb dsRNA mycovirus isolated from F.graminearum strain DK21 is associated with altered fungalmorphology. Loss of dsRNA through conidial passage resultedin faster mycelial growth and less pigmentation, whereas itsacquisition through hyphal anastomosis restored the abnormaldsRNA-containing DK21 phenotypes.

Since the 7.5-kb dsRNA-containing F. graminearum isolatesgrew slowly compared to dsRNA-free isolates, we expectedinfected strains to have drastically reduced symptoms on inoc-ulated wheat plants. Although the dsRNA-containing isolateshad significantly reduced virulence, some symptom develop-ment still occurred. Since the dsRNA of F. graminearum iso-lates that contain the 7.5-kb dsRNA was transferred to onlyabout half of the conidia, only about half of the spores in the

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

FIG. 4. Northern blot analysis. Total RNA was extracted fromdsRNA-free and -containing derivatives of DK21 and several coloniesobtained from anastomosis. The extracts were treated with DNase andblotted to a nylon membrane. Gels were probed with a dsRNA-specificprobe (A) and with ethidium bromide (B). The arrowhead indicatesthe position of the dsRNA. Lanes: 1, �/HindIII; 2, prebiotinylatedDNA marker; 3, dsRNA-free derivative; 4, dsRNA-containing deriv-ative; 5 and 6, dsRNA-free derivatives obtained from hyphal anasto-mosis T-DK F2 and T-DK F4, respectively; 7 to 9, dsRNA-containingderivatives obtained from hyphal anastomosis T-DK B2, T-DK C5, andT-DK D1, respectively. Numbers on the left indicate sizes in kilobases.

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suspension prepared from dsRNA-containing isolates proba-bly have dsRNA. Isolates carrying the dsRNA also producemuch less mycotoxin (DON) than do the dsRNA-free isolates.Trichothecene production has a role in virulence; therefore,dsRNA-containing F. graminearum isolates caused muchslower disease development on infected wheat plants. Devel-opment of disease, however, is a complicated phenomenon, soadditional studies are required to confirm these results.

A hypovirulence phenotype associated with dsRNA mycovi-ruses has been reported for several plant pathogenic fungi,including Ophiostoma novo-ulmi and Cryphonectria parasitica(1, 35, 41, 56). O. novo-ulmi, the causal agent of Dutch elmdisease, may contain 12 unencapsidated mitochondrialdsRNAs that result in dramatic changes in the fungal host,including slow growth, formation of colonies with abnormal,irregular margins, and a reduction in the number of viableasexual spores (6, 13, 46, 47). Extensive studies of the biolog-ical role of dsRNA have been conducted with C. parasitica, thechestnut blight fungus. dsRNA-containing isolates of C. para-sitica have reduced levels of virulence (hypovirulence), sup-pressed sporulation, altered colony morphology, reduced pig-mentation, increased oxalate accumulation, and alteredcellulase and laccase activities (2, 12, 25). The dsRNA could becytoplasmically transmitted from one strain of C. parasitica toanother during hyphal fusion (anastomosis). Furthermore, ap-plication of a compatible hypovirulent strain to an existingcanker resulted in the conversion of the resident virulent strainto the hypovirulence phenotype and consequent healing of thecanker. This observation led to the development of severalprograms that employed the artificial introduction of hypoviru-lent strains as a practical means of controlling chestnut blight(22). These characteristics and transmission of dsRNA fromone strain to another during hyphal anastomosis provided thebasis for potential biological control of many fungal diseases(1, 11). However, it should be noted that there are manyvegetative compatibility groups reported (5, 37) for F. gra-minearum, which thus serve as a major constraint for dsRNAtransmission. Studies on the population structure of the vege-tative compatibility groups and disease development will fur-ther extend the possible implication of this dsRNA for biolog-ical control.

The pDK-1 and pDK-2 DNA fragments possess conservedRdRp motifs (III and IV), and motifs IV to VI of the helicasegenes, respectively (32). Although overall sequence similarityamong RNA viruses is poor, the RdRp and helicase genes arerelatively highly conserved. Thus, sequence similarities of theRdRp and helicase genes often are used for phylogenetic anal-yses of RNA viruses (30, 32). Although the deduced aminoacid sequence of the 7.5-kb dsRNA RdRp gene contains moresequence upstream of motif III, we could not identify anyconserved motifs in this region (data not shown). Motifs IV toVI of RdRp are regarded as core motifs, are conserved acrossall RdRp classes, and may have a role in binding nucleosidetriphosphate substrates (30). As the alignment of motifs I to IIIis more tentative, it is not surprising that there is no clearconserved motif upstream of motif III in the 7.5-kb dsRNA.RNA viruses that belong to superfamilies I and II containseven conserved motifs within their helicase genes (30, 32).pDK-2 contained motifs IV to VI of the helicase gene at the Nterminus. Although we have not sequenced the regions con-

taining all seven motifs, the degree of sequence similarity leftvirtually no doubt that the 7.5-kb dsRNA possesses helicasesequences. Experiments to more accurately identify the phylo-genic relationships with other RNA viruses and to prove thepolymerase activity of this region are under way.

F. graminearum grows rapidly on PDA, with dense arialmycelia and pigmentation ranging from carmine red to tan inthe center and white at the margins, and it is an importantplant pathogen that causes head and seedling blight of smallgrains such as wheat and barley, stalk and ear rot of corn, andstem rot of carnation (15, 29, 50). Head blight and ear rotreduce the grain yield, and harvested grain often is contami-nated with mycotoxins that have a trichothecene structure,including DON, nivalenol, and zearalenone (36). Direct eco-nomic losses that can result from the pathogen, including lowercrop yields and poor grain quality, have become common (59),as has reduced animal performance when the product is usedfor feed (10). There also is concern about the public healthimplications of exposure to Fusarium mycotoxins, such as feedrefusal, vomiting, and skin necrosis (4, 10, 19). If the 7.5-kbdsRNA in strain DK21 is transferable to dsRNA-free isolatesthrough hyphal fusion with a high incidence, and if the viru-lence level and the mycotoxin production are reduced due tothe dsRNA as described above, biological control of diseasescaused by F. graminearum could be achieved.

In summary, we have identified a new fungal dsRNA myco-virus that results in changes in the morphological and patho-genicity phenotypes of the fungal host. In addition to theOphiostoma and Cryphonectria hypoviruses, this is only thethird such example of a virus with these biologically and eco-nomically important characteristics.

ACKNOWLEDGMENTS

This research was supported in part by grant 2000-2-22100-004-3from the Korea Science & Engineering Foundation and by grants(M101KG010001-01K070104910 and M101KG010001-01K070102810)from the Crop Functional Genomics Center of the 21st Century Fron-tier Research Program funded by the Ministry of Science and Tech-nology of the Republic of Korea. Y.-M.C. and J.-J.J. were supported bygraduate fellowships from the Ministry of Education through the BrainKorea 21 Project.

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