DIVERSITY OF CRYPHONECTRIA-PARASITICA HYPOVIRULENCE-ASSOCIATED DOUBLE-STRANDED RNAS WITHIN A CHESTNUT POPULATION IN NEW-JERSEY

The effects on colony morphology, sporulation, laccase production, and virulence of hypovirulence-associated double-stranded (ds)RNAs of the chestnut blight fungus, Cryphonectria parasitica, from a population of chestnut trees in eastern New Jersey were examined. Size and number of dsRNAs were determined by gel electrophoresis, and their genetic variability was determined by polymerase chain reaction (PCR) and nucleotide sequencing. The New Jersey hypovirulent isolates were similar in cultural phenotype and electrophoretic characteristics of their dsRNAs. PCR amplification primed by oligonucleotides specific for a conserved region of a New Jersey-derived Cryphonectria hypovirus dsRNA (CHV2-NB58) for which the entire nucleotide sequence is known resulted in amplification of a single band of predicted size from all but one of the New Jersey dsRNAs. Neither the European-derived dsRNA, CHV1-EP713, nor a Rhode Island dsRNA amplified with these primers. The sequences of 400 nucleotides of PCR-amplified products primed by two oligonucleotides (200 residues from each primer) were determined. Alignments of these sequences with CHV1-EP713 and CHV2-NB58 and phylogenetic trees drawn from these alignments indicated that all New Jersey isolates were closely related and easily distinguishable from CHV1-EP713. In the region sequenced, none of the dsRNAs from fungal isolates from different trees were identical; however, CHV2-NB58 dsRNA isolated in 1992 was identical to the original 1988 CHV2-NB58 dsRNA from the same tree. This suggests that there is significant drift among dsRNA sequences even within a small C. parasitica population but that there is stability within a particular fungal thallus.