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Abstract
The aim of this study was to characterise the performance of new molecular methods for the detection and identification of Pseudomonas syringae pv. actinidiae (Psa) and to provide validation data in comparison to the assays mentioned in official diagnostic protocols and being currently used. Eleven molecular tests for the Psa detection were compared in an inter-laboratory comparison where each laboratory had to analyse the same panel of samples consisting of thirteen Psa-spiked kiwifruit wood extracts. Laboratories had to perform also isolation from the wood extracts. Data from this interlaboratory test performance study (TPS) was statistically analysed to assess the performance of each method. In order to provide complete validation data, both for detection and identification, this TPS was supplemented by a further study of identification from pure culture of phylogenetically closely related Pseudomonas spp., Psa, and bacterial strains associated with kiwifruit. The results of both these studies showed that simplex-PCRs gave good results, whereas duplex-PCR and real-time PCR were the most reliable tools for detection and identification of Psa. Nested and multiplex-PCR gave false-positive results. The use of the most reliable detection test is suggested for routine analyses, but when Psa-free status needs to be accurately assessed, it is recommended that at least two detection tests are used. This work provides a wide comparison of the available diagnostic methods, giving new information for a possible revision of the official diagnostic protocols (e.g. European and Mediterranean Plant Protection Organization (EPPO) protocol PM7/120 for the detection of Psa).
Highlights
Bacterial canker of kiwifruit caused by Pseudomonas syringae pv. actinidiae (Psa) was first described in Japan (Takikawa et al 1989) and subsequently in Italy and in Korea (Koh et al 1984; Scortichini 1994)
Values followed by the same letter in a column are not significantly different (p = 0.05) according to Fisher’s exact test d For each criterion, we present data derived from the scenario H1 described in the Materials and methods section for the interpretation of indeterminate results
Because no full proof control strategy has been developed for Psa, special attention need to be paid to disease monitoring and to the certification of the sanitary status of the propagation material and other kiwifruit plant material
Summary
Bacterial canker of kiwifruit caused by Pseudomonas syringae pv. actinidiae (Psa) was first described in Japan (Takikawa et al 1989) and subsequently in Italy and in Korea (Koh et al 1984; Scortichini 1994). Bacterial canker of kiwifruit caused by Pseudomonas syringae pv. Actinidiae (Psa) was first described in Japan (Takikawa et al 1989) and subsequently in Italy and in Korea (Koh et al 1984; Scortichini 1994). Whereas the disease caused severe economic losses in Japan and in Korea, in Italy remained sporadic and with a low incidence for 20 years. In 2007/2008 economic losses started to be observed in Italy, and in 2010/2012 in all the main areas of kiwifruit cultivation in the world (https://gd.eppo.int/taxon/PSDMAK/reporting). Four different Psa population (named biovars) have been previously described and characterised by different virulence (Chapman et al 2012). The biovar 1 ( named Psa1) include strains associated with the first epidemics of bacterial canker in Japan and in Italy.
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