A HYPOVIRULENT ISOLATE OF CRYPHONECTRIA-PARASITICA WITH MULTIPLE, GENETICALLY UNIQUE DSRNA SEGMENTS

Many different dsRNA-containing isolates of Cryphonectria (Endothia) p arasitica, the chestnut blight fungus, have been identified in surveys conducted in the central Appalachians. Isolate C-18, collected in 198 9 from a virulent canker in southern West Virginia, was found to conta in 11 segments of dsRNA ranging from 1 to 5 kb in size. Virulence expe riments, using Golden Delicious apples, excised dormant American chest nut stems, and American chestnut sprouts, determined that the dsRNA as sociated with isolate C-18 significantly reduced virulence and altered cultural morphology when isogenic dsRNA-containing and dsRNA-free ase xual progeny were compared. The 11 dsRNA segments were transmitted int o other isolates of C. parasitica via hyphal anastomosis and into coni dia in an all-or-none fashion. Complementary DNA clones from a library representing C-18 dsRNA did not hybridize to other dsRNAs associated with hypovirulent isolates D2 (Pennsylvania), Ep-713 (Europe), NB58-88 (New Jersey), to another multisegmented dsRNA from West Virginia (9B- 2-1), or to wound tumor virus. None of the cDNA clones tested hybridiz ed to more than one dsRNA segment by Northern blot analysis. Eight of the 11 dsRNA segments were demonstrated to be genetically unique. Usin g a phosphate buffer extraction protocol, icosahedral particles approx imately 60 nm in diameter were purified from the mycelial tissue of is olate C-18. The dsRNA virus associated with hypovirulence of isolate C -18 is therefore fundamentally different from other hypovirulence-asso ciated viruses characterized to date.