Long-term field study of transgenic hypovirulent strains of Cryphonectria parasitica in a forest setting

Transgenic hypovirulent strains of Cryphonectria parasitica, the chestnut blight fungus, engineered to contain a chromosomally integrated full-length infectious cDNA copy of virulence-attenuating hypoviruses, differ from natural hypovirulent strains in the ability to transmit hypoviruses to ascospore progeny and with 100% efficiency through asexual spores. We report the results of a long-term field study that examined whether these properties result in enhanced hypovirulence establishment, dissemination and persistence under field conditions. Informed by previous field results using a severe hypovirus, this study that employed 144 American chestnut trees was designed to provide improved inoculum formulation and delivery and to include the use of a mild hypovirus isolate (less debilitating) CHV-1/Euro7 in an attempt to increase dissemination. Isogenic transgenic hypovirulent (TG), non-transgenic cytoplasmic hypovirulent (CH) or virus-free virulent (V) treatment strains were applied to artificially initiated and natural C.parasitica cankers three times each year for 7years. Reservoirs of treatment inoculum also were initiated and refreshed annually for the first 6years of the study. Sampling of 111,000 individual ascospores from 4,500 perithecia confirmed hypovirus-containing spermatia successfully transmitted TG hypoviruses to ascospore progeny under field conditions. Surprisingly, TG ascospore progeny were recovered 3years after the last annual application of treatment inoculum. Repeated sampling of over 440 cankers revealed dissemination of both CH and TG hypovirulent strains. However, no significance differences in establishment or dissemination were observed for the two hypovirulent strains. The results are discussed in terms of the contribution of ascospore progeny to infection, competition by endemic virulent C.parasitica, size of inoculated trees and the biological control potential of TG hypovirulent strains.