Fusarium graminearum is an important plant pathogen that causes head and seedling blight and scab disease of small grains such as wheat, barley and corn in Korea. F. graminearum infections were associated with direct economic losses due to lower yields and poor grain quality. Double-stranded RNA (dsRNA) mycoviruses in some fungi are associated with hypovirulence and have been used or proposed as biological control agents. In case of F. graminearum, Fusarium graminearum virus-DK21 (FgV-DK21) which isolated from Korea has been studied that related to a hypovirus that attenuates virulence (hypovirulence) expressing phenotypic traits such as reduced mycelial growth, reduced sporulation, less toxin production but increased pigmentation. To search the more isolates of F. graminearum related to hypovirulence, we isolated the F. graminearum containing the dsRNAs by cellulose CF11 chromatography. Among these, we selected 2 isolates such as DK3 and 98-8-60 isolates which the presence of dsRNA did not affect colony morphology and pigmentatation and did associated with morphological changes including reduction in mycelial growth and increased pigmentation, respectively. The dsRNA of these isolates were characterized the full length sequence and transmissibility of progeny conidia and ascospores. The isolate DK3 has two different dsRNA viruses, which are referred to as FgV-3 and FgV-4. FgV-3 consists of one dsRNA fragment and FgV-4 is divided to two dsRNA fragments. The another isolate 98-8-60 has one dsRNA virus containing seven dsRNA fragments, which are refered to as FgV-2. Phylogenetic analysis of the deduced amino acid sequences of RdRp regions revealed that RdRp sequences from isolate 98-8-60 is closely related to family Chrysoviridae. DsRNA1 of isolate DK3 was closely related to the family of Totiviriridae and Chrysoviridae but distantly related within the cluster, whereas dsRNA2 of isolate DK3 formed a distinct clade with the family Partitiviridae. To investigate the transformation technology for transmission of hypovirus using FgV-DK21, we developed the efficient fungal promoter for virus gene overexpression in fugal host cell. EF1 alpha promoter was adapted to fungal transformation with pCB1004 which DNA based vector containing Hygromycin gene as selection marker. To identify protein of FgV-DK21 related to hypovirulence, each ORF protein for four ORFs was cloned. All clones were transformed to virus-free F. graminearum strain by polyethylene glycol (PEG)-uptake method. As a result, transformants by ORF2 and ORF4 overexpression showed irregular morphology and slow growth rate. This result indicate the ORF2 and 4 of FgV-DK21 are pathogenecity determinants related to hypovirulence. Finally, to test the application of biological control using FgV-DK21 hypovirus to field, we tried the transmission of hypovirulence-associated dsRNA between mycelia of F. graminearum. However, transmission beteween mycelia in field is prevented by vegetative incompatibility barrier in filamentous fungi strains. Vegetative incompatibility can be overcome through the use of protoplast fusion. To determine whether FgV-DK21 can overcome vegetative incompatibility barrier, protoplasts was prepared from virus-free recipients, strain DK3 (lineage7), strain 88-1 (lineage6), F. culmorum (F39;outgroup), F. oxysporum f. sp. lycopersici (outgroup) and F. meridionale (F34; lineage2) that were transformed with a hygromycin B resistant gene, and virus-infected donor, strain DK21, were fused by the PEG-mediated method. Colonies regenerated from donor were excluded by growing fused protoplasts on a medium with hygromycin B. The morphology of colonies arising from virus-infected strains was severely altered from their virus-free strains such as reduction in growth rate and change in pigmentation. We confirmed infection with FgV-DK21 by the presence of genomic dsRNA and by northern blot analysis. We also examined the protoplast fusion method on Fusarium sp., Cryphonectria parasitica and other filamentous fungi to elucidate potential hosts of FgV-DK21. Altogether, these results indicate that protoplast fusion can be used to introduce FgV-DK21 dsRNA in to various filamentous fungi.