Pseudomonas syringaepv.actinidiaedetection in kiwifruit plant tissue and bleeding sap

The rapid spreading of the disease during last few years highlighted the need of a quick, sensitive and reliable method forPseudomonas syringaepv.actinidiae(Psa) detection, to find possible inoculum sources and limit the pathogen spreading. APCRmethod, using new primers designed on the gene encoding a putative outer membrane proteinP1, was developed to detect Psa in symptomatic and asymptomatic tissue; a nested‐PCRwas also applied. Bleeding sap samples, collected in early spring from orchards with symptomatic and asymptomatic trees, were used both forPCRassays and for pathogen isolation and identification. ThePCRand nestedPCRmethods were able to detect Psa presence at very low concentration from plant and pollen extracts;RFLPanalyses withBclI on PCR and nested PCR amplicons confirmed the assay specificity, while the digestion withBfmI andAluI allowed to discriminate Psa strains isolated before 2008 from those isolated after 2008. Furthermore, the PCR and nested PCR on crude bleeding sap samples detected the presence of the pathogen in 3 and 5 of the 15 assayed samples, respectively. Direct isolation from the same samples and bacterial identification confirmed the results of molecular analysis.