Molecular Monitoring of Wild-Type and Genetically Engineered Colletotrichum coccodes Biocontrol Strains In Planta

Abstract

Two strains of Colletotrichum coccodes, the wild type (DAOM 183088) and T-20a, engineered with the necrosis- and ethylene-inducing peptide (NEP1) gene for hypervirulence on velvetleaf (Abutilon theophrasti, Medik.), were monitored in planta for the first 2 weeks after infection. Real-time quantitative polymerase chain reaction (QPCR) was used to assess the extent of colonization of both strains on velvetleaf using SYBR Green chemistry. Quantification of both strains was successful as soon as the conidia were sprayed on the leaves and up to 14 days after infection. The increase in fungal DNA amounts corroborated with the appearance of necrotic lesions on velvetleaf leaves infected with the wild-type strain. The wild-type C. coccodes was more efficient at infecting velvetleaf than the transgenic T-20a strain. In addition, detection of host DNA allowed us to quantitatively monitor the decrease in plant DNA amounts in response to wild-type strain infection. Expression of the NEP1 transgene by conventional retro-transcription (RT)-PCR was absent from T-20a growing on either V8 agar or in planta, suggesting that the gene may be silenced. The application of QPCR to monitor fungal growth was proven to detect the target organisms in planta prior to the appearance of symptoms.