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The dissemination of European (Euro-7) and North American (GH2) hypovirulent (H) isolates of Cryphonectria parasitica to American chestnut saplings was investigated. Nine 0.01 ha plots were established in a clearcut. Each of 24 trees per plot received 4 punch- and 1 branch wounds in October, 1985; January, April, and July, 1986. Control plots received a virulent (V) strain of the fungus. H inocula were placed in three plots per treatment, respectively, at 2–4 month intervals. Cankers were sampled over a 2.5-year period. Isolate morphology, growth rate and sporulation were evaluated. Three hundred forty-three of the 3,456 punch wounds and 112 of 856 branch wounds developed cankers. July wounds were most likely to become infected. Isolates from 28 atypical cankers were examined for hypovirus double-stranded RNA (dsRNA). Four isolates and 7, respectively, had the Euro-7 or GH2 hypovirus. The large numbers of vegetative compatibility types (up to 38) of the pathogen, or the lack of hypovirus-containing conidia may have curtailed dissemination. A second study examined the relationship between time of year of wounding and callus formation, and the susceptibility of wounds to colonization by C. parasitica. Stems were wounded in May, August, November, 1987, and in early March, 1988. Each of 54 trees received 4 punch wounds at each date. Wound closure by callus tissue and/or C. parasitica infections were evaluated at 2–4-month intervals. Eighty-three percent of May wounds closed after 15 months. Closure declined with each subsequent wounding. Thirty-nine of 69 cankers developed in wounds without callus ridges. Twenty-one infections occurred in partially callused wounds and 8 cankers developed in closed wounds. May wounds exhibited the most rapid wound closure but also were most likely to develop cankers. The in vivo developmental stage of V cankers at which they may be receptive to conversion also was examined. Two sexually compatible V isolates were used to establish perithecial cankers. Wounds in other groups of trees received a single V isolate in July to produce pycnidial cankers, or in August to produce nonsporulating cankers. Cankers were exposed to a vegetatively compatible H strain on bark patches in November. Control patches contained sterile agar. Inoculum was removed in December and the stems were harvested in April. Canker total surface area and sporulation were determined. Isolate growth rate, sporulation and pigmentation were evaluated on PDA. Perithecial, pycnidial and prestromal cankers had average surface areas of 173.5 and 154.7 cm2, and 16.3 cm2, respectively. Two of 18 perithecial cankers and 5 of 18 pycnidial cankers were partially colonized by Euro-7. None of the prestromal or control cankers contained dsRNA. Total canker surface area appeared critical to colonization by H inoculum.