Importance of Cryphonectria parasitica stromata production and intermediate-pigmented isolates in spread of Cryphonectria hypovirus 1 on grafted American chestnut trees

Large, surviving American chestnut trees, grafted in 1980, were inoculated in 1982 and 1983 with four 'white' European (French and Italian) hypovirulent strains of Cryphonectria parasitica infected with Cryphonectria hypovirus 1 (CHV1). Spread of Italian CHV1-Euro7, indicated by nucleotide sequence analysis of CHV1 isolates, and a high level of blight control, occurred on these trees for over 20 years. However, the means by which CHV1 spreads and the possible role of stromata production in that spread are unknown. In this study, 249 C. parasitica isolates were recovered from stromata excised from natural cankers on the grafted trees and plated on an agar medium; 5.2% of the stromata yielded white phenotype isolates, 9.2% yielded intermediate-pigmented isolates (30-70% pigmentation) and the remainder were normal-pigmented isolates. For comparison, cankers artificially established on blight-free, forest-clear-cut American chestnut trees, following inoculation with three Italian white hypovirulent strains, were evaluated in a similar manner. Of 241 C. parasitica isolates recovered from stromata, 66.4% had a white colony phenotype, 19.1% had an intermediate-pigmented phenotype and the remainder were normal-pigmented isolates. For single conidia collected from stromata and plated, nearly equal frequencies of only white and intermediate-pigmented colony phenotypes were obtained. Following dsRNA extraction and electrophoresis, 21 of 33 intermediate-pigmented isolates were positive for CHV1. Some normal pigmented isolates also were positive for CHV1. Single-sporing a CHV1-positve, normal-pigmented, natural-canker, stroma isolate (Str 1) from the grafts resulted in several deeply red-orange pigmented (JR) isolates as well as some white isolates. The dsRNA in the JR isolate was extracted and cDNAs made by reverse transcriptase-polymerase chain reaction (RT-PCR) for part of a region (p29) in ORF A. Nucleotide sequencing indicated 100% identity to CHV1 present in the inoculated Italian white strain, Ep 47. The results indicate that stromata production on the grafted trees may contribute to CHV1 spread, and the presence of CHV1 in intermediate-pigmented isolates and some normal pigmented isolates indicates these isolates, often overlooked, may be important in CHV1 spread and the high level of blight control on the grafted trees.