A real-time PCR assay for the detection of European Stone Fruit Yellows Phytoplasma (ESFYP) in plant propagation material.

A severe outbreak of European Stone Fruit Yellows has been reported recently in apricot orchards located in the province of Trento, where partial or total tree dieback caused major economic losses to growers. In order to prevent the disease spreading, the presence of ESFYP-vector, the psyllid cacopsylla pruni, was monitored toghetre with wild reservoirs of the phytoplasma. Five experimental orchards were planted using ESFYP-free material (cv. Bergeron and Goldrich grafted on "Wavit" or "Myrobalan 29C") to perform epidemiological studies. A multiplex real-time PCR procedure (TaqMan) was set up using 2 primers/probe combinations for simultaneous detection of ESFYP and host DNA, in order to avoid false negatives due to PCR inhibition . Real-time PCR assays were performed on: propagation material, groups of C. pruni (2 insects per group) and wild individuals of several Prunus spp. collected in areas close to the experimental orchards and individual samples from apricots showing ESFYP-like symptoms. The results obtained indicate that the primers/prove combination used in the real-time procedure allows reliable and specific detection of ESFYP. The pathogen was detected in 93% of the apricot trees showing ESFYP-symptoms and in about 33% of the insect groups and in several wild species collected in different locations. No phytoplasmas were found in healthy plants or in propagation material. This result suggest that new infection of trees is presumably due to ESFYP transmission by vectors rather than by contamination of propagation material. Further research is in progress to check the presence of ESFYP-sources in wild plants close to experimental orchards and to monitor pathogen's dissemination.