Occurrence of plasmids pXF64-Hb_ESVL and pUCLA-ESVL associated with infections caused by Xylella fastidiosa subsp. multiplex ST6 in the demarcated area of Alicante, Spain

An outbreak of Xylella fastidiosa (Xf) subsp. multiplex ST6 was first identified in June 2017 in Alicante province (Valencian Community, Spain) affecting mainly almond trees, but also other species including ornamentals, cultivated Prunus spp. and landscape plants. The current demarcated area (DA) in Alicante covers 134,581 hectares, and affects more than 70 municipalities. Genetic analysis may provide evidence of biological, ecological and host-range diversity among Xf strains of the same subspecies and STs and help to elucidate the pathway of introduction and spread in an affected area. Pairwise comparisons of the chromosomal genomes of two ST6-sequenced strains isolated from almond trees in Alicante, ESVL and IVIA5901, showed an average nucleotide identity higher than 99.9%. Interestingly, the two strains differ for the presence of the plasmids pXF64-Hb_ESVL and pUCLA-ESVL detected only in the ESVL strain. The aim of this study was to determine the incidence and distribution of plasmid-bearing strains of Xf subsp. multiplex ST6 in Alicante. PCR tests were performed on 20 strains isolated from different hosts, and on approximately 100 DNA samples from infected almond trees collected in eight municipalities within the DA of Alicante. PCR results on the cultured isolates showed the occurrence of ST6-strains harbouring the two plasmids, or only the plasmid pUCLA-ESVL or none of them. Interestingly, in some of the DNA samples from infected almonds, only the plasmid pXF64-Hb_ESVL could be detected. More specifically, 4% of ST6-infected samples harboured the plasmid pXF64-Hb_ESVL, 12% only pUCLA-ESVL, 16% both plasmids, and 44% neither of the two plasmids. In five of the eight municipalities, we found samples harbouring the two plasmids, and only in the samples from the municipality of Benifato the PCR tests failed to amplify the targeted plasmids. Future work will increase the number of samples to cover the entire DA to better understand and relate the presence of plasmids to the epidemiology of this disease in combination with the use of multilocus sequence typing and genome sequencing of more Xf subsp. multiplex ST6 strains