The biological control potential of Cryphonectria parasitica strains containing an infectious cDNA copy of hypovirus CHV1-Euro7

Abstract

Transmissible hypovirulence has not become established in most areas of North America as it has in Europe where it has been associated with increased biological control of chestnut blight. Transgenic strains of Cryphonectria parasitica provide several mechanisms that may improve biological control. This study was designed to evaluate whether transgenic C. parasitica strains containing a cDNA transgene encoding the viral genome of CHV1-Euro7 show greater potential to biologically control blight than their cytoplasmically infected counterparts. Three treatments were employed that compared: transgenic hypovirulent strains (TG), cytoplasmic hypovirulent strains (CH), and virulent strains (V). Two types of cankers were initiated in each treatment to generate inoculum. To produce ascospore inoculum, naturally occurring and artificially established cankers were spermatized by painting cankers in June, July, and September with a conidial mixture that contained both mating types (MAT-1 and MAT-2) of the appropriate treatment strain (TG, CH, or V). To produce conidial inoculum, cankers were scratch-initiated (SI) on separate trees in June 2004 by scratching the surface of the bark and painting the wounded area with a mycelial-agar slurry of the appropriate treatment strain (TG, CH, or V). Non-treated trees also were left to monitor natural canker formation. In October, 2004, tree condition and natural canker establishment were assessed for all trees. Most trees were asymptomatic after the first treatment season and the incidence of natural infection remained relatively low. There were 27 natural cankers in TG plots, 13 cankers in CH plots, and 20 cankers in V plots. Cankers also were sampled to determine the hypovirus infection status of the thallus. Although the purpose of the spermatization treatment was to produce ascospores, many treated cankers also acquired hypovirus from the treatment inoculum. Cankers occurring below SI cankers also acquired hypovirus. Ascospore production was assessed by collecting bark discs in October 2004 and serially diluting ascospore contents from perithecia in the winter of 2004-2005. Pigmentation and morphology then were recorded for ascospores from all three plot treatments (TG, V, and CH). Hypovirulent ascospore (HVA) isolates were only collected from TG plots and at less than expected Mendelian ratios. Pigmentation segregated as expected in V and CH plots. The transgenic MAT-1 treatment strain effectively spermatized and hence produced HVAs on both initiated cankers and treated natural infections. The transgenic MAT-2 treatment strain did not produce morphologically distinct HVA isolates. To assess the increased conversion capability of HVAs, 18 HVA isolates were paired with 17 vegetative compatibility (v-c) types isolated from the study site; pairings then were examined for hypovirus transmission. Collectively, HVA isolates consistently (i.e. 4-5 conversions out of 5 replications) converted 12 of the v-c types; three additional v-c types were able to be converted at…