Experimental confirmation that Xylella fastidiosa subsp. pauca, ST53, does not colonise grapes

Xylella fastidiosa is able to colonise a very large number of plant species, but considering each subspecies/phylogenetic clade the number of associated susceptible hosts is significantly reduced. Although strains genetically related most likely share similar host range, using phylogenetic relationships to infer information regarding the potential host range of new strains is still problematic, and pathogenicity tests remain the only means to assess the capability of a given strain to infect or not a specific plant species. Based on the current European legislative provisions on X. fastidiosa, information on the host range of the strain(s) causing an outbreak have regulatory consequences. In this context, we have made efforts to prove experimentally the capability of X. fastidiosa subsp. pauca, ST53, one of the most virulent European genotypes, to infect grapes, using 23 grape varieties (Vitis vinifera) and four rootstocks. Upon needle inoculation, plants were monitored for 18 months, using standand diagnostic methods (qPCR and isolation), supported by vector-transmission tests and observation of thin sections of the inoculated stems, stained using the LIVE/DEAD BacLight kit. qPCR assays on samples collected at the inoculation points (i.p.) and from the distal portions, 6 and 12 months post-inoculation, yielded positive reactions in more than 90% of the i.p., whereas in half of the cultivars scattered amplifications (the majority yielding Cq values > 30) occurred in some replicates at 15-20 cm from the i.p., but none of the apical portions tested positive. Isolations made 18 months after the inoculation, either from mature leaf petioles and stems portions harbouring the inoculation points, failed to recover actively growing colonies. At the same time, microscope observation of the thin sections showed only the presence of aggregates of dead Xylella-cells at the i.p. Transmission tests performed using specimens of Philaenus spumarius caged on the inoculated grapes produced negative results for both insects and recipient plants. The overall results showed that the bacterium was successfully delivered into the stem of the grapes and bacterial residual could be qPCR-detected even one year after the inoculation, but none of the inoculated cultivars sustained active bacterial multiplication and colonisation.