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Transgenic hypovirulent (HV) strains of Cryphonectria parasitica [Murrill] M.E. Barr engineered to contain an infectious cDNA copy of Cryphonectria hypovirus 1 (Hypoviridae; CHV1) may provide an efficient mechanism to introduce hypoviruses into genetically heterogeneous populations of the chestnut blight fungus. However, elucidating the factors that affect the infection of chestnut and spread of C. parasitica is needed to fully utilize the potential of transgenic HV strains for the biological control of chestnut blight. In one study, three experiments were conducted at a forested site near Bartow, West Virginia. The seasonality of HV and virulent canker development was measured in the first experiment by inoculating healthy chestnut trees at 8-wk intervals (May, July, September) with cytoplasmic HV (EP146/tpTE7), transgenic HV (EP146/pXHE7), or virulent (EP146) inoculum. The cytoplasmic and transgenic HV isolates were significantly less able to infect and grow in chestnut bark when compared to the virulent isolate. Inoculations made with virulent conidia or mycelium resulted in more infections and larger cankers after 1-yr than those made with conidia or mycelium of the two HV isolates. Virulent inoculations initiated in May incited the greatest amount of disease (i.e., infection and canker growth) after 1-yr followed by September and July inoculations. A similar seasonal pattern of infection was observed for cytoplasmic HV inocula, but when infection occurred, the growth of cytoplasmic HV cankers was not affected by month of inoculation. The frequency of infection and canker growth for the transgenic HV isolate exhibited little seasonal variation as described above except inoculations were made with either 300 or 3,000 conidia or ascospores per wound. Inoculum concentration had no effect on the incidence of infection or canker growth for either the cytoplasmic or transgenic HV isolate after seven months. In contrast, inoculations using 3,000 virulent conidia significantly increased the frequency of infection compared to those made with 300 virulent conidia. The third experiment examined the effect(s) of inoculum formulation, delivery substrate, and wound-size the development of virulent and transgenic HV cankers. Inoculations using virulent conidia in agar (potato dextrose agar [PDA] or 2.5% water agar) and a wound diameter of nine mm resulted in the greatest incidence of infection after seven months when compared to inoculations using virulent ascospores or an agar suspension containing mycelia and conidia. Transgenic HV inoculations with a nine mm wound and a mycelium-conidia agar suspension produced the largest number of cankers compared to transgenic HV inoculations initiated in the first two experiments and were as effective as virulent inoculations with nine mm wounds. Because the fungal propagule responsible for fertilizing the female thallus of C. parasitica in the sexual cycle has yet to be identified, a second study was conducted to determine whether ascospores, conidia, or mycelial fragments function as spermatia. Brown-pigmented cytoplasmic (EP146/tpTE7) and transgenic HV (EP146/pXHE7) and virulent (EP146) isolates were used for in situ spermatization of a virulent orange-pigmented isolate (EP155) pre-established as cankers on healthy chestnuts at the forested site described above. The formation of brown and orange ascospores (∼1:1) in new perithecia was the marker for successful spermatization. Over a two-year period, cytoplasmic and transgenic HV and virulent conidia of C. parasitica were the only propagules to successfully spermatized virulent cankers. When virulent or cytoplasmic HV conidia were used as spermatia, ascospore progeny were hypovirus-free. However, ascospores resulting from crosses involving transgenic HV conidia were hypovirus-infected and resistant to hygromycin, thereby indicating the successful inheritance of the CHV1 transgene in these progeny.