A reliable method for Pseudomonas syringae pv. actinidiae detection in asymptomatic kiwifruit plants three years after pathogen inoculation in micropropagated shoots and buds.

According to recent statistics, in Italy a significant spread of the bacterial canker of actinidia (BCA) is recorded with peaks of 70% and 100% and lower percentages ranging around 50%. The control of the BCA spreading is a priority especially considering the hazard represented by the endophytic bacterial presence in the nursery material. In the Italian MD (02/07/2011), art. 10 relates to the kiwifruit production from controlled mother plant in accordance with specifications set out in Annex II. In the EU legislation, Actinidia spp. is still considered an ornamental plant and only from 2013 with the EU Decision 756 (05/12/2012) on “Measures to prevent the introduction and spread in the EU of P. syringae pv. actinidiae (Psa)” the plant passport is mandatory like the other traditional fruit species. To evaluate the risk that Psa could be associated with micropropagated tissue and transferred from asymptomatic mother plants to new individuals, the survival of Psa was studied in micropropagated shoots of A. deliciosa cv. Hayward inoculated more than three years ago with different concentrations of a virulent Psa gfp-expressing/rifampicin-resistant strain (Psa::gfp-Rifres), through microbiological and molecular analysis at each in vitro transfer for a total of seven generations. After the multiplication and rooting phase, the plantlets were transferred to the greenhouse for re-establishment. A similar trial was carried out using micropropagated buds, instead of shoots, as starting materials for Psa::gfp-Rifres inoculation. In the three years after the inoculation, the Psa::gfp-Rifres reisolation on selective media, whose identity was confirmed by PCR analysis and by the ability to induce symptoms in actinidia and HR in tobacco, was verified in both the micropropagated materials and in the plant analyzed in toto. In particular at low inoculum dose, Psa remains latent without inducing disease symptoms. A reliable and efficient method for the detection of Psa, which showed to be latent for such a long period of time in asymptomatic nursery material, is proposed.