GENE SEQUENCE ANALYSIS FOR THE MOLECULAR DETECTION OF PSEUDOMONAS SYRINGAE pv. ACTINIDIAE: DEVELOPING DIAGNOSTIC PROTOCOLS

Bacterial canker of kiwifruit caused by Pseudomonas syringae pv. actinidiae (Psa) is seriously damaging Actinidia deliciosa and A. chinensis in central Italy. Since this severe outbreak of the disease may reflect on the trade of kiwifruit pollen and fruits, standardized protocols are needed for the extraction of the bacterium from different matrices and for its detection and identification. Current PCR detection of Psa is aspecific, as the amplified product has the same size as that of P syringae pv. theae. To improve the specificity of molecular detection of Psa, a gene-sequence analysis was done to identify new specific DNA markers. This enabled us to develop a duplex-PCR that distinguishes Psa from P. syringae pv. theae and from other genetically related P. syringae pathovars. This method was also successfully applied to detect Psa directly in infected kiwifruit matrices such as leaves, wood, flowers and in experimentally contaminated pollen and fruits. We propose two protocols for Psa extraction and detection from pollen and fruits. These protocols can be used for epidemiological studies, to establish whether symptomless fruits or pollen can harbour Psa, and can help diagnostic laboratories in the analysis of these type of materials.