Detection of Phaeomoniella chlamydospora in soil using species-specific PCR

摘要

Phaeomoniella chlamydospora is a fungal pathogen of woody grapevine tissue that causes "Petri disease". As one potential source of inoculum is infested soil, a SDS/phenol/chloroform DNA extraction method and PCR assay utilising species-specific primers were evaluated for the ability to detect P. chlamydospora in grapevine nursery soil. Using a nested PCR approach, the assay was able to detect 10~2 conidia/ml when a spore suspension was added to sterilised soil samples and 50 fg when P. chlamydosporagenomic DNA was added directly to the reaction. In this process, preamplification of a 600 bp region of the ribosomal DNA was followed by amplification with primers Pch1 and Pch 2 to produce a 360 bp species-specific product. This highly sensitive diagnostic tool will be used in future studies to determine if pathogen propagules are present in soils collected from New Zealand grapevine nurseries.