Journal of General Virology (1992), 73, 681-686. Printed in Great Britain 681 A North American hypovirulent isolate of the chestnut blight fungus with European isolate-related dsRNA Bradley I. Hillman,* Yanan Tian, Peter J. Bedker and Matthew P. Brown Department of Plant Pathology, Rutgers University, New Brunswick, New Jersey 08903, U.S.A. We have synthesized and mapped a cDNA library representing the one major dsRNA element associated with hypovirulence in strain NB58 of the chestnut blight fungus, Cryphonectria (=Endothia) parasitica, which was isolated from recovering chestnut trees in New Jersey, U.S.A. The linear dsRNA has a size of approximately 12- 5 kbp and is polyadenylated at the 3' terminus of one strand. Molecular hybridization experiments indicate that there is sequence similarity between the NB58 dsRNA and dsRNAs from Euro- pean isolates of C. parasitica, but not among dsRNAs of NB58 and those associated with other North American isolates. Hybridization experiments with mapped cDNA clones representing different regions of the 12.5 kbp dsRNA indicate that the termini and the 3'-proximal two-thirds (relative to the plus strand) are more conserved among NB58 and the European isolates than the rest of the 5'-proximal one-third. Nucleotide sequence analysis of the termini of NB58 dsRNA suggests common organizational features between it and the dsRNA from French-derived strain EP713. Introduction The chestnut blight fungus, Cryphonectria (=Endothia) parasitica, was responsible for considerable damage to European chestnut forests and virtually eliminated the American chestnut in the first half of this century (for reviews see Van Alfen, 1982; Anagnostakis, 1987). The term hypovirulence generally refers to infections of the fungus by cytoplasmic agents that can be transmitted via hyphal anastomosis, often resulting in altered pheno- types as well as reduced fungal virulence. Because of the unrestrained nature of this pandemic and the aesthetic and economic importance of the chestnut tree, a number of debilitated, hypovirulent C. parasitica isolates of European origin were released in the 1970s in various parts of northeastern U.S.A., including New Jersey, in attempts at biological control of the fungus by transmis- sion of the hypovirulence-inducing agents. Unfortu- nately, records identifying exactly which strains were released are incomplete, and the investigators involved are not available (S. L. Anagnostakis, personal commun- ication). Previous studies regarding the interrelatedness of dsRNAs from C. parasitica indicated that dsRNAs from European strains are related to one another, and dsRNAs from several North American strains are related to one another, but that dsRNAs from European strains are not closely related to those from North American strains (L'Hostis et al., 1985 ; Paul et al., 1988). The apparent absence of a protein coat surrounding the dsRNA molecules and the inability of researchers to infect a dsRNA-free strain of C. parasitica with any purified form of a hypovirulence-inducing agent have greatly hindered their study from a virological perspec- tive. However, the weight of evidence clearly indicates that many such agents are sufficient to cause hypoviru- lence in the fungal host and are of viral origin (Day et al., 1977; Anagnostakis & Day, 1979; Fulbright, 1984; Hansen et al., 1985; Hiremath et al., 1986; Rae et al., 1989; Choi et al., 1991a, b; Shapira et al., 1991a, b). In the French-derived strain EP713, the easily isolated dsRNA form of the genome is encapsulated within lipid vesicles that appear to be devoid of a major structural protein component, but demonstrate RNA-dependent RNA polymerase activity (Dodds, 1980; Hansen et al., 1985). Besides dsRNA, the cytoplasmic fraction contains virus-specific ssRNA which is polyadenylated and hybridizes to probes representing only one strand of the dsRNA (Hiremath et al., 1986; Tartaglia et al., 1986). This poly(A)-containing strand is referred to as the plus strand, and utilizes two open reading frames (ORFs) to express at least four polypeptides (Choi et al., 1991b; Shapira et al., 1991b). We are interested in comparing hypovirulence-asso- ciated viruses of C. parasitica with the aim of understand- ing the mechanisms of hypovirulence itself and differen- tiating those from the hypovirulence-associated changes in fungal morphology (Hillman et al., 1990). Strain NB58, isolated from recovering chestnut trees in New 0001-0481 © 1992 SGM