A mixture of hypovirulent strains of Cryphonectria parasitica, including four white (European) strains infected with Cryphonectria hypovirus 1 (CHV1), was used in 1982 and 1983 to inoculate natural blight cankers located within a zone ranging from the ground to 183 cm on grafted American chestnut trees. These four white strains belonged to three vegetative compatibility (vc) types. Using pigmented, single-spore colonies from white isolates, 48 vc types were identi®ed among 110 white isolates recovered in 1996, 1998, and 1999 from cankers located outside the inoculated zone. Twenty-®ve of the 48 white vc types consisted of two or more isolates. The 25 major white vc types were vegetatively incompatible with all four of the original white hypovirulent strains, providing evidence for spread of CHV1 but not for spread of the original inoculated strains. Forty-®ve vc types represent the minimum number of`new' vc types into which CHV1 had spread. The ratio of white vc types to white isolates tested (S/N) and Shannon diversity index were 0.436 and 3.64, respectively. The spatial pattern of white vc types on the grafts was found to be non-random (p 0.019). White single-spore colonies of white isolates were placed into four cultural morphology (CM) groups. The two largest groups contained 37 (CM group 3) and 33 (CM group 1) isolates. Single-spore colonies from the original, white inoculated strain, Ep 49, were classi®ed into CM groups 3 and 1, and colonies of Ep 51 W were classi®ed into CM group 1.

Summary 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.

Summary American chestnut trees, grafted in 1980 from large survivors, were inoculated in 1982 and 1983 with four white (European) hypovirulent strains of Cryphonectria parasitica, infected with C. hypovirus 1 (CHV1); this hypovirus has been shown to be capable of moving rapidly within the mycelium of a vegetative compatibility (vc) type of C. parasitica in blight cankers. Using a 49‐cell lattice plot, 17.8×17.8 cm, the spatial patterns and frequencies of white and pigmented isolates and white and pigmented vc types were investigated within superficial cankers on the grafts located outside the hypovirulent‐strain‐inoculated zone. Four of six cankers assayed contained white isolates, and three of the four had random spatial patterns of white isolates, based on join‐count statistics. Vc tests, using pigmented isolates and pigmented single‐spore colonies of white isolates, indicated that the majority of white and pigmented isolates recovered from each of two cankers assayed were in one vc type. White and pigmented lattice‐plot cells of the same vc type were frequently in contact with each other, indicating incomplete movement of CHV1 within a vc type. Nine and 10 vc types were found in the two cankers; it is hypothesized that small, white vc type areas in each canker may be a source of CHV1 transmission to the major vc types. Based on join‐count statistics, the spatial pattern of the single, major vc type in one canker was non‐random (aggregated), whereas the other canker had a random major vc type pattern. White and pigmented in vitro variants (sectors) of C. parasitica, that resemble white and pigmented in vivo variants in spatial contact and vc compatibility, were intermediate hypovirulent and virulent on forest American chestnuts, and dsRNA positive and negative, respectively. Incomplete movement of CHV1 within a vc type could be a major cause of the prevalence of pigmented isolates in superficial cankers on chestnut trees.

Large, surviving American chestnut trees, grafted in 1980, were inoculated in 1982 and 1983 with four ÔwhiteÕ 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.