Nucleotide sequence identification of Cryphonectria hypovirus 1 infecting Cryphonectria parasitica on grafted American chestnut trees 12–18 years after inoculation with a hypovirulent strain mixture
Natural blight cankers on grafted American chestnut Castanea dentata trees were inoculated with one French-derived hypovirulent Cryphonectria parasitica strain, with a predominantly orange-pigmented colony phenotype and three Italian hypovirulent C. parasitica strains, with a predominantly white colony phenotype, in 1982 and 1983. Cryphonectria hypovirus 1 (CHV1) dsRNA was extracted from the four inoculated strains and from 11 predominantly white or orange pigmented C. parasitica isolates recovered from blight-controlled cankers on the grafted trees during 1994–2000; cDNAs were made by reverse transcriptase-polymerase chain reaction for two hypovirus regions: (i) an 844-bp region in the helicase domain of open reading frame B (ORF B), designated OB and (ii) an 894-bp region that included part of the 5′ non-coding region and part (p29) of ORF A (5′OA). Nucleotide sequence analysis indicated that hypovirus isolates from the grafted trees and Italian inoculated strains had high homologies (identities) to each other and high homologies for OB (98.7–99.9%) and 5′OA (99.2–99.6%) to reference hypovirus, CHV1-Euro7; homologies to reference hypovirus, CHV1-EP713, were low (≤89.8%). In contrast, the hypovirus from the predominantly pigmented, French-derived inoculated strain had high homologies for OB (99.1%) and 5′OA (99.7%) to reference CHV1-EP713, and low homologies (87.7–89.7%) to the predominantly pigmented grafted-tree isolates. These results provide strong evidence that hypoviruses present in the two predominantly pigmented isolates and nine predominantly white isolates of C. parasitica from the grafted trees were of Italian inoculated hypovirulent strain origin. For the Phe(25) through Gln(73) phenotype-determinant region in p29 of 5′OA, hypovirus isolate amino acid sequences were dependent on whether they were CHV1-Euro7 or CHV1-EP713 hypoviruses, but predominantly white and predominantly pigmented phenotypes within CHV1-Euro7 or within CHV1-EP713 had identical amino acid sequences. Grafted-tree hypovirus isolates infecting the predominantly pigmented C. parasitica isolates may originate from chance transmission and replication of a CHV1-Euro7 hypovirus variant or variant mixture, possibly present in one or more of the Italian inoculated hypovirulent strains, such as Ep 49.
Document Type: Research Article
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Publication date: February 1, 2004