Genetic variation of Cryphonectria hypoviruses (CHV1) in Europe, assessed using restriction fragment length polymorphism (RFLP) markers.

A total of 72 hypovirus-infected isolates of the chestnut blight fungus Cryphonectria parasitica were sampled from nine European countries between 1975 and 1997. The double-stranded RNA of the Cryphonectria hypoviruses (CHV1) was isolated and reverse transcription (RT)-PCR products were obtained for two different regions of the viral genome (ORF A and ORF B) using primer sequences of the type species CHV1-EP713. Both PCR products of each viral isolate were digested with four restriction endonucleases recognizing sequences of four nucleotides. The restriction fragment length polymorphism (RFLP) analysis revealed 41 genetically distinct RFLP types of CHV1 with 10 types occurring more than once. Identical RFLP types were detected nine times among viruses collected in the same location. Cluster analysis based on the RFLP banding patterns separated the viral isolates into five CHV1 clusters or subtypes. Most viral isolates (64 out of 72) grouped into one large cluster which comprised all viruses from Italy (including CHV1-EP747), Switzerland, Crotia, Bosnia, Hungary, Greece, and the French island Corsica, as well as five out of 11 isolates from continental France. Two additional subtypes of CHV1 were found in France (one related to CHV1-EP713) and one each in Spain and Germany. The Swiss samples collected over a period of 20 years showed that very little RFLP variation has evolved during this time. The results of this study are consistent with the hypothesis of multiple introductions of CHV1 into Europe.