RESUMO
This study provides a novel qRT-PCR protocol for specific detection and proof of viability of Phytophthora in environmental samples based on differential accumulation of cox II transcripts. Chemical and physical treatments were tested for their ability to induce in vitro the accumulation of cytochrome oxidase genes encoding subunits II (cox II) transcripts in Phytophthora cambivora. Glucose 170 mM, KNO3 0.25 mM and K3 PO3 0.5 and 0.8 mM induced the transcription of cox II in P. cambivora living mycelium while no transcription was observed in mycelium previously killed with 0.5% (p/v) RidomilGold(®) R WG. Living chestnut tissue was artificially infected with P. cambivora and treated with inducers. In vivo experiments confirmed the ability of glucose to induce the accumulation of P. cambivora cox II transcripts. Based on these results, pretreatment of environmental samples with glucose prior to nucleic acid extraction increased the accumulation of specific cox II transcripts, and therefore the sensitivity of qRT-PCR assay for detection of P. cambivora in living tissues. Furthermore, differential accumulation of transcripts between treated and untreated samples represents an unequivocal proof of inoculum viability.
