Multi‐seasonal field release and spermatization trials of transgenic hypovirulent strains of Cryphonectria parasitica containing cDNA copies of hypovirus CHV1‐EP713

Three indigenous field strains of the chestnut blight fungus, Cryphonectria parasitica collected in 1996 from a test site in the Meshomasic State Forest, Connecticut, USA, were engineered to contain a chromosomally integrated full‐length infectious cDNA copy of the severe, virulence‐attenuating hypovirus CHV1‐EP713. These transgenic hypovirulent strains were introduced into the test site after it had been clear‐cut, as sprayed conidia in multiple applications over 4 years, beginning in 1997. Evidence was obtained for cytoplasmic transmission of cDNA‐derived hypovirus RNA and for recovery of transgenic strains from canker tissue of the spray‐treated trees. However, there was no indication of spread of transgenic strains or cDNA‐derived viral RNA to the control plot or outside the treatment plot. A single transgenic C. parasitica isolate was identified from a total of 156 isolates recovered from 1330 insects representing five insect orders. Only 2.6% of the 1082 C. parasitica bark isolates recovered during the course of this study were vegetatively compatible with the untransformed input strains. Transgenic ascospore progeny were produced by 40% of the perithecia collected in 1999 and by only 7.3% of the perithecia collected in 2000. A concurrent field release study performed in the Monongahela National Forest in West Virginia in 1998 and 1999 with transgenic hypovirulent strains constructed with the same CHV1‐EP713 cDNA‐containing plasmid identified seasonal application dates and delivery methods that resulted in very high levels of transgenic ascospore production. Results of the combined field release studies are discussed in terms of requirements for improved formulations and delivery methods and the importance of balancing ecological fitness and virulence attenuation.