A protocol for molecular detection of Phaeomoniella chlamydospora in grapevine wood
PETRI DISEASE IS A SERIOUS DECLINE AND dieback disease of young grapevines. The principal causal organism, Phaeomoniella chlamydospora, is distributed mainly by infected propagation material. Pathogen detection and accurate diagnosis are currently based on fungal isolation in artificial growth media. The fungus is extremely slow-growing, however, and cultures are often overgrown by co-isolated fungi before it can be identified. To avoid this problem, we have developed and validated an efficient and cost-effective protocol for the molecular detection of Pa. chlamydospora in grapevine wood. This novel molecular technique, using a species-specific PCR, detected as little as 1 pg of genomic Pa. chlamydospora DNA. The protocol was validated with grafted grapevines from different nurseries, including grapevines that were first treated with hot water. The basal end of the rootstock was analysed for Pa. chlamydospora by means of both isolations in artificial medium and molecular detection. The latter was found to be considerably more sensitive than isolations, and detected Pa. chlamydospora in samples that recorded both positive and negative in isolations. The identity of PCR products obtained from a subset of samples that tested positive only for Pa. chlamydospora, based on molecular detection, was confirmed to be Pa. chlamydospora-specific through restriction digestion with AatII. The pathogen was not isolated from samples treated in hot water. However, as expected, Pa. chlamydospora DNA was detected in samples exposed to hot water at rates similar to those detected in material not immersed in hot water.