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Xylella fastidiosa (Xf) is a vascular phytopathogenic bacterium native to the Americas, known for causing severe diseases and significant economic losses in important crops. In Europe, Xf is considered a quarantine pathogen. Since its first detection in 2013 in Italy, mandatory surveys to search for Xf across Europe revealed the presence of three Xf subspecies (fastidiosa, multiplex and pauca) in different outbreaks occurring in France, Spain, and Portugal. Xf subspecies sandyi and morus have not been associated to epidemic outbreaks in Europe; however, subspecies sandyi has been detected in intercepted plants imported from Costa Rica and Honduras. By combining data from the Oxford Nanopore Technologies and Illumina sequencing platforms, we obtained complete genomes for Xf strains CO33 and CFBP8478. Both strains belong to the subspecies sandyi and were isolated from coffee plants intercepted in Europe that were imported from Costa Rica. These two genome resources increase the scarce number of Xf genomes available belonging to the subspecies sandyi which is crucial for comparative genomic analysis and population studies of this important plant pathogenic bacterium.

The Gram-negative bacterium Xylella fastidiosa (Xf) was originally found in the Americas, but has now been identified in more than 20 countries across America, Asia, and Europe. This plant pathogen is currently listed as a priority pest in Europe due to its socio-economic and ecological impacts. Within the three Xf subspecies fastidiosa, multiplex and pauca, subsp. multiplex displays a notably wider range of host plants than the other two subspecies. Comparative genomics may allow determination of how Xf subsp. multiplex adapts to new and diverse hosts and environments, so it is important that more genomes of this subspecies are defined. Twelve complete closed genomes sequences of Xf subsp. multiplex were obtained using a hybrid assembly approach combining Illumina and Oxford Nanopore technologies. The combined use of Canu and Unicycler assemblers enabled identification and closure of several plasmid sequences with high similarity to other plasmids described in strains of Xf subsp. fastidiosa and subsp. pauca. The analysis also revealed prophage sequences and contigs outside the chromosomes, annotated as phages. These new genomes, in conjunction with those existing in GenBank, will facilitate exploration of the evolutionary dynamics of Xf subsp. multiplex, its host adaptation mechanisms, and the potential emergence of novel strains of this important plant pathogen.

In the original publication [1], the information contained in Table 1 concerning the Sequence Type (ST) and host of some of the Xylella fastidiosa strains was not correct. Xylella fastidiosa subsp. multiplex strains RH1 and LM10 belong to ST 7 instead of ST 6. Xylella fastidiosa subspecies multiplex Fillmore belongs to ST 81 instead of ST 6. Regarding to Xylella fastidiosa, subsp. multiplex ST 6 IVIA6586-2 was isolated from Helicrysum italicum, and IVIA6629 was isolated from Rhamnus alaternus instead of Prunus dulcis, as it is said in the original table. This mistake does not affect the results or information provided in the main body of the manuscript.

Xylella fastidiosa is a major transboundary plant pest, causing severe socioeconomic impacts. Development of preventive strategies and methods for surveillance, early detection, monitoring, and accurate diagnosis of X. fastidiosa and its vectors, are keys to preventing the effects of this plant pathogen, and assist timely eradication or optimisation of containment measures. This review focuses on approaches for early detection of X. fastidiosa in the Mediterranean Basin, including development of climatic suitability risk maps to determine areas of potential establishment, and epidemiological models to assist in outbreak management through optimized surveillance and targeted responses. The usefulness of airborne hyperspectral and thermal images from remote sensing to discriminate X. fastidiosa infections from other biotic and abioticinduced spectral signatures is also discussed. The most commonly used methods for identifying X. fastidiosa in infected plants and vectors, and the molecular approaches available to genetically characterize X. fastidiosa strains, are described. Each of these approaches has trade-offs, but stepwise or simultaneous combinations of these methods may help to contain X. fastidiosa epidemics in the Mediterranean Basin.

The plant pathogenic bacterium Xylella fastidiosa (Xf) is a major threat to agriculture and the environment worldwide. Recent devastating outbreaks in Europe highlight the potential of this pathogen to cause emergent diseases. Xf subsp. multiplex ESVL and IVIA5901 strains that belong to sequence type (ST) 6 were isolated from almond orchards within the outbreak area in Alicante province (Spain). Both strains share more than 99% of the chromosomal sequences), but the ESVL strain harbours two plasmids (pXF64-Hb_ESVL and pUCLA-ESVL). Here, virulence phenotypes and genome content were compared between both strains, using three strains from USA as a reference for the phenotypic analyses. Experiments in microfluidic chambers, used as simulation of xylem vessels, showed that twitching motility was absent in the IVIA5901 strain, while the ESVL strain had reduced twitching motility. Both Spanish strains had less biofilm formation, cell aggregation and lower virulence in tobacco compared with USA reference strains. Genome analysis of the two plasmids from ESVL revealed 51 unique coding sequences (CDS) that were absent in the chromosome of IVIA5901. Comparison of the chromosomes of both strains showed some unique CDS and SNPs in each strain with potential deleterious mutations. Genomic differences found in genes previously associated with adhesion and motility might explain the differences in the phenotypic traits studied. Although additional studies are necessary to infer the potential role of Xf plasmids, our results indicate that the presence of plasmids should be considered in the study of the mechanisms of pathogenicity and adaptation in Xf to new environments.

Xylella fastidiosa (Xf) is a vascular plant pathogenic bacterium native to the Americas that is causing significant epidemics and economic losses in olive and almonds in Europe, where it is a quarantine pathogen. Since its first detection in 2013 in Italy, mandatory surveys across Europe revealed the presence of the bacterium also in France, Spain, and Portugal. Combining ONT and Illumina sequencing data, we assembled high-quality complete genomes of seven Xf subsp. fastidiosa strains isolated from different plants in Spain, the USA, and Mexico. Comparative genomic analyses discovered differences in plasmid content among strains, including plasmids that had been overlooked previously when using Illumina sequencing platform alone. Interestingly, in strain CFBP8073, intercepted in France from plants imported from Mexico, three plasmids were identified, including two (plasmid pXF-P1.CFBP8073 and pXF-P2.CFBP8073) not previously described in X. fastidiosa, and one (pXF5823.CFBP8073) almost identical to a plasmid described in a Xf strain from citrus. Plasmids found in the Spanish strains here were similar to those described previously in other strains from the same subspecies and ST1 isolated in the Balearic Islands and the USA. The genome resources from this work will assist in further studies on the role of plasmids in the epidemiology, ecology, and evolution of this plant pathogen.

The bacterium Xylella fastidiosa (Xf) is a plant pathogen that can block the flow of water and nutrients through the xylem. Xf symptoms may be confounded with generic water stress responses. Here, we assessed changes in biochemical, biophysical and photosynthetic traits, inferred using biophysical models, in Xf-affected almond orchards under rainfed and irrigated conditions on the Island of Majorca (Balearic Islands, Spain). Recent research has demonstrated the early detection of Xf-infections by monitoring spectral changes associated with pigments, canopy structural traits, fluorescence emission and transpiration. Nevertheless, there is still a need to make further progress in monitoring physiological processes (e.g., photosynthesis rate) to be able to efficiently detect when Xf-infection causes subtle spectral changes in photosynthesis. This paper explores the ability of parsimonious machine learning (ML) algorithms to detect Xf-infected trees operationally, when considering a proxy of photosynthetic capacity, namely the maximum carboxylation rate (Vcmax), along with carbon-based constituents (CBC, including lignin), and leaf biochemical traits and tree-crown temperature (Tc) as an indicator of transpiration rates. The ML framework proposed here reduced the uncertainties associated with the extraction of reflectance spectra and temperature from individual tree crowns using high-resolution hyperspectral and thermal images. We showed that the relative importance of Vcmax and leaf biochemical constituents (e.g., CBC) in the ML model for the detection of Xf at early stages of development were intrinsically associated with the water and nutritional conditions of almond trees. Overall, the functional traits that were most consistently altered by Xf-infection were Vcmax, pigments, CBC, and Tc, and, particularly in rainfed-trees, anthocyanins, and Tc. The parsimonious ML model for Xf detection yielded accuracies exceeding 90% (kappa = 0.80). This study brings progress in

Xylella fastidiosa (Xf) is a phytopathogenic bacterium with a repertoire of self-replicating genetic elements, including plasmids, pathogenicity islands, and prophages. These elements provide potential avenues for horizontal gene transfer both within and between species and have the ability to confer new virulence traits, including the ability to colonize new host plants. However, they can also serve as a 'footprint' to type plasmid-bearing strains. Genome sequencing of several strains of Xf subsp. fastidiosa sequence type (ST) 1 from Mallorca Island, Spain, revealed the presence of a 38 kb plasmid (pXFAS_5235). In this study, we developed a PCR-based typing approach using primers targeting the traC gene to determine the presence of pXFAS_5235 plasmid or other plasmids carrying this gene in a world-wide collection of 65 strains X. fastidiosa from different subspecies and STs or in 226 plant samples naturally infected by the bacterium obtained from the different outbreaks of Xf in Spain. The traC gene was amplified only in the plant samples obtained from Mallorca Island infected by Xf subsp. fastidiosa ST1 and from all Spanish strains belonging to this ST. Maximum-likelihood phylogenetic tree of traC revealed a close relatedness among Spanish and Californian strains carrying similar plasmids. Our results confirm previous studies, which suggested that a single introduction event of Xf subsp. fastidiosa ST1 occurred in the Balearic Islands. Further studies on the presence and role of plasmids in Xf strains belonging to the same or different subspecies and STs can provide important information in studies of epidemiology, ecology, and evolution of this plant pathogen.

The detection of the diseases caused by the plant pathogenic bacterium Xylella fastidiosa (Xf) is difficulted by the occurrence of asymptomatic infections, its long incubation period and non-specific and diagnostic symptoms. Early detection of infected plants based on the assessment of changes in physiological plant traits due to bacterial infection might be an important tool for pathogen detection, disease management and breeding for resistance to Xf in woody crops. Therefore, the objectives of this study were to evaluate the susceptibility to four Spanish Xf strains and the response to infection of three olive cultivars widely grown in Spain by measuring plant physiological traits with different leaf-level sensors and Xf-specific molecular diagnostic assays. Leaf measurements and petiole samplings were taken at the inoculation point, and at 5, 10, 15 and 20 cm above the inoculation points at 1, 3, 6, 12 and 18 months post-inoculation. Additionally, sampling of wood tissue was performed at 8 month post inoculation at the same plant sections of leaf sampling. Molecular diagnosis based on two qPCR tests was used to determine the presence and colonization level of the bacteria into the olive xylem tissues. The Dualex Scientific leaf clip sensor and two spectrometers (PolyPen RP400 and DLP NIRscan nano) covering the range of the electromagnetic spectrum from 340 and 1700 nm were used for leaf measurements to detect physiological changes. The Spanish strain XYL1961 from Xf subsp. pauca ST80 showed the highest colonization in the different olive cultivars as compared to strains from Xf subsp. multiplex from ST81 and ST6 from Spain and the Italian strain ‘DeDonno’ from Xf subsp. pauca ST53. On the other hand, from a total of 70 vegetation indices calculated related with chlorophyll, carotenoid and flavonoids content and structural related indexes a set was selected to discriminate between olive plants infected by Xf from those non-inoculated even in the absence of visible

Xylella fastidiosa (Xf) is a vascular plant pathogen native to the Americas. In 2013, it was first reported in Europe, implicated in a massive die-off of olive trees in Apulia, Italy. This finding prompted mandatory surveys across Europe, successively revealing that the bacterium was already established in some distant areas of the western Mediterranean. To date, the Balearic Islands (Spain) hold the major known genetic diversity of Xf in Europe. Since October 2016, four sequence types (ST) belonging to the subspecies fastidiosa (ST1), multiplex (ST7, ST81), and pauca (ST80) have been identified infecting 28 host species, including grapevines, almond, olive, and fig trees. ST1 causes Pierce’s disease (PD) and together with ST81 are responsible for almond leaf scorch disease (ALSD) in California, from where they were introduced into Mallorca in around 1993, very likely via infected almond scions brought for grafting. To date, almond leaf scorch disease affects over 81% of almond trees and Pierce’s disease is widespread in vineyards across Mallorca, although producing on average little economic impact. In this perspective, we present and analyze a large Xf-hosts database accumulated over four years of field surveys, laboratory sample analyses, and research to understand the underlying causes of Xf emergence and spread among crops and wild plants in the Balearic Islands. The impact of Xf on the landscape is discussed.

Plant pathogens pose increasing threats to global food security, causing yield losses that exceed 30% in food-deficit regions. Xylella fastidiosa (Xf) represents the major transboundary plant pest and one of the world’s most damaging pathogens in terms of socioeconomic impact. Spectral screening methods are critical to detect non-visual symptoms of early infection and prevent spread. However, the subtle pathogen-induced physiological alterations that are spectrally detectable are entangled with the dynamics of abiotic stresses. Here, using airborne spectroscopy and thermal scanning of areas covering more than one million trees of different species, infections and water stress levels, we reveal the existence of divergent pathogen- and host-specific spectral pathways that can disentangle biotic-induced symptoms. We demonstrate that uncoupling this biotic–abiotic spectral dynamics diminishes the uncertainty in the Xf detection to below 6% across different hosts. Assessing these deviating pathways against another harmful vascular pathogen that produces analogous symptoms, Verticillium dahliae, the divergent routes remained pathogen- and host-specific, revealing detection accuracies exceeding 92% across pathosystems. These urgently needed hyperspectral methods advance early detection of devastating pathogens to reduce the billions in crop losses worldwide.

Xylella fastidiosa (Xf) strains from Mallorca belong to subspecies fastidiosa Sequence Type (ST) 1. Isolates from Xf subsp. fastidiosa cause Pierce’s Disease on grapes and leaf scorch disease on almonds in California. Xf colonization and disease development in host plants have been shown to be related to the size of cell aggregates, bacterial motility, and biofilm formation, which are mediated by Type I and Type IV pili, among other traits. In this study several Xf subsp. fastidiosa strains from Spain were phenotypically characterized and compared with strains from the USA. The four Spanish Xf strains used included IVIA5235, IVIA5770, XYL461, R2XF4358 that were isolated from cherry tree, grapevine and Rhamnus alaternus. The Californian strains included Temecula 1 (reference strain), WM1-1 and CFBP7970 that were isolated from grapevines; M23, CFBP8073, CFBP7969, CFBP8068, CFBP8082 isolated from almonds, Coffea canephora, Vitis rotundifolia, Ulmus sp., and Ambrosia artemifolia, respectively. To study bacterial behavior and phenotypic characteristics several experiments were performed to determine adhesion force to substrate, biofilm formation, movement, cell–cell aggregation, twitching motility and patterns of bacterial growth. Taking together our results showed that Spanish strains do not have twitching motility as M23, CFBP7969, CFBP8068, CFBP8082 strains, whereas the remaining strains have. Standardized area under the growth progress curve showed that IVIA5770 and IVIA5235 have similar growth rate and planktonic growth than M23, but lower biofilm formation as compared to the remaining strains. Future research will be performed to determine the relationship between those phenotypic traits and virulence level on host plants of agronomic interests.

Xylella fastidiosa (Xf) has the capacity of uptaking extracellular DNA from the environment and incorporate it into its genome by homologous recombination, process known as Natural Competence. The process of genetic acquisition and recombination of extracellular DNA could be related to the acquisition of new traits such as antibiotic resistance and virulence factors, giving rise to the origin of new pathotypes. We took a step forward in comparative genomics analysis of Spanish populations of Xf by determining the complete genome of several isolates from Alicante, Mallorca and Ibiza and established potential recombination events that may have occurred with isolates from other places in the world. For this purpose, we used combined Illumina and Oxford Nanopore sequencing platforms and combined assembly approaches to obtain complete circularized genomes. Our results showed that olive and almond isolates belonging to subspecies pauca ST80 from Ibiza (Balearic Islands), the only place where this ST has been detected, showed an unusual level of recombination, being so far the isolates showing the highest number of recombination events among all the Xf isolates sequenced to date. On the order hand, we have confirmed the high degree of homology among genomes from Xf strains from Mallorca and California belonging to subspecies fastidiosa ST1 and subspecies multiplex ST81 isolated from almond and grapes, and we have also identified specific long-size genomic rearrangements in isolates of Xf subspecies multiplex ST6 from Alicante, compared to their homologous strains from the Americas. Research is now underway to determine the source and the functionality of these recombinant genes and their role in pathogenicity.

The recent introductions of the bacterium Xylella fastidiosa (Xf) into Europe are linked to the international plant trade. However, both how and when these entries occurred remains poorly understood. Here, we show how almond scorch leaf disease, which affects ~79% of almond trees in Majorca (Spain) and was previously attributed to fungal pathogens, was in fact triggered by the introduction of Xf around 1993 and subsequently spread to grapevines (Pierceʼs disease). We reconstructed the progression of almond leaf scorch disease by using broad phylogenetic evidence supported by epidemiological data. Bayesian phylogenetic inference predicted that both Xf subspecies found in Majorca, fastidiosa ST1 (95% highest posterior density, HPD: 1990–1997) and multiplex ST81 (95% HPD: 1991–1998), shared their most recent common ancestors with Californian Xf populations associated with almonds and grapevines. Consistent with this chronology, Xf-DNA infections were identified in tree rings dating to 1998. Our findings uncover a previously unknown scenario in Europe and reveal how Pierce’s disease reached the continent.

Pathogen introductions have led to numerous disease outbreaks in naive regions of the globe. The plant pathogen Xylella fastidiosa has been associated with various recent epidemics in Europe affecting agricultural crops, such as almond, grapevine, and olive, but also endemic species occurring in natural forest landscapes and ornamental plants. We compared whole-genome sequences of X. fastidiosa subspecies multiplex from America and strains associated with recent outbreaks in southern Europe to infer their likely origins and paths of introduction within and between the two continents. Phylogenetic analyses indicated multiple introductions of X. fastidiosa subspecies multiplex into Italy, Spain, and France, most of which emerged from a clade with limited genetic diversity with a likely origin in California, USA. The limited genetic diversity observed in X. fastidiosa subspecies multiplex strains originating from California is likely due to the clade itself being an introduction from X. fastidiosa subspecies multiplex populations in the southeastern United States, where this subspecies is most likely endemic. Despite the genetic diversity found in some areas in Europe, there was no clear evidence of recombination occurring among introduced X. fastidiosa strains in Europe. Sequence type taxonomy, based on multilocus sequence typing (MLST), was shown, at least in one case, to not lead to monophyletic clades of this pathogen; whole-genome sequence data were more informative in resolving the history of introductions than MLST data. Although additional data are necessary to carefully tease out the paths of these recent dispersal events, our results indicate that whole-genome sequence data should be considered when developing management strategies for X. fastidiosa outbreaks.

Xylella fastidiosa is an economically important plant pathogenic bacterium of global importance associated, since 2013, with a devastating epidemic in olive trees in Italy. Since then, several outbreaks of this pathogen have been reported in other European member countries including Spain, France, and Portugal. In Spain, the three major subspecies (subsp. fastidiosa, multiplex, and pauca) of the bacterium have been detected in the Balearic Islands, but only subspecies multiplex in the mainland (Alicante). We present the first complete genome sequences of two Spanish strains: X. fastidiosa subsp. fastidiosa IVIA5235 from Mallorca and X. fastidiosa subsp. multiplex IVIA5901 from Alicante, using Oxford Nanopore and Illumina sequence reads, and two hybrid approaches for genome assembly. These completed genomes will provide a resource to better understand the biology of these X. fastidiosa strains.

Identification of sequence types (ST) of Xylella fastidiosa based on direct MultiLocus Sequence Typing (MLST) of plant DNA samples is partly efficient. In order to improve the sensitivity of X. fastidiosa identification, we developed a direct nested-MLST assay on plant extracted DNA. This method was performed based on a largely used scheme targeting seven housekeeping gene (HKG) loci (cysG, gltT, holC, leuA, malF, nuoL, petC). Samples analyzed included 49 plant species and two insect species (Philaenus spumarius, Neophilaenus campestris) that were collected in 2017 (106 plant samples in France), in 2018 (162 plant samples in France, 40 plant samples and 26 insect samples in Spain), and in 2019 (30 plant samples in Spain). With the nested approach, a significant higher number of samples were amplified. The threshold was improved by 100 to 1000 times compared to conventional PCR. Using nested-MLST assay, plants that were not yet considered hosts tested positive and revealed novel alleles in France, whereas for Spanish samples it was possible to assign the subspecies or ST to samples considered as new hosts in Europe. Direct typing by nested-MLST from plant material has an increased sensitivity and may be useful for epidemiological purposes.

A growing number of recent and current studies on Xylella fastidiosa outbreaks in Europe emphasise genomic sequencing of bacterial isolates as a mean to infer their potential origin, biology, recombination events and possible determinants of host specificity. In the present work we have undertaken a re-assessment of publicly available X. fastidiosa genomic sequences from a comparative standpoint. With limitations due to the fact that most of the available sequences were left at the draft stage, and that only a few are complete, we undertook to compare: i) genomic organisation (synteny); ii) number, origin and genomic position of insertion elements (IS, islands, phage-related elements, plasmids); iii) individual gene complements within the species and subspecies pan-genomes; and iv) exclusive or enriched genes associated with subspecies or host of isolation. Our preliminary results suggest that X. fastidiosa genomes show a great plasticity in their organisation, possibly aided by the widespread presence of insertion elements, and that the traditional subspecies classification, although useful from an operational standpoint, is probably insufficient to explain the observed genomic variability.

Outbreaks in Europe associated with Xylella fastidiosa(Xf) subspecies multiplex are the most frequent, accounting for the larger number of susceptible plant species (more than 70%), with some host overlap among the different regions. Diverse Sequence Types (ST) have been detected across distinct geographical regions: ST6, ST7 and ST79 in Corsica and PACA region (France), ST7 and ST81 in the Balearic Islands (Spain), ST6 in the province of Alicante and Madrid (mainland Spain), ST7 in the Douro Littoral region (Portugal), and ST87 in the region of Tuscany (northern Italy). In recent years, genetic analysis and pathogenicity tests have provided evidence of biological, ecological and host range diversity among strains of the same subspecies and STs. Draft genomes of 12 Spanish isolates of Xf subsp. multiplex (ST6and ST81) were used for comparative genomic studies with currently available genomes of the same subspecies from France and Italy. Phylogenetic analysis based on core genomes, accessory genomes and single nucleotide polymorphisms indicate that: (i) European outbreaks associated with strains of Xf subsp. multiplex most likely result from distinct independent introductions; (ii) ST6-strains recovered from Spain and France, although sharing the same ST, fell in distinct phylogenetic subgroups; (iii) ST81 strains from the Balearic Islands and ST6 strains from Alicante differentiated in distinct phylogenetic groups (i.e. ST81 isolates are closer to ST6 isolates from California and France than ST6 isolates from Alicante, which in turn are closer to ST7 isolates from the USA and France); (iv) a low number of SNPsare detected among the strains recovered in Alicante, suggesting a recent introduction in the area. Additionally, whereas some recombination events were found among ST81 isolates from Mallorca and ST6 isolates from France with isolates of Xf subsp. fastidiosa ST1 from Mallorca, no evidence of recombination among ST6 isolates from Alicante with other European Xf

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 plas

An outbreak of Xylella fastidiosa subsp. multiplex sequence type ST6 was discovered in 2017 in mainland Spain affecting almond trees. Two cultured almond strains, “ESVL” and “IVIA5901,” were subjected to high throughput sequencing and the draft genomes assembled. Phylogenetic analysis conclusively indicated they belong to the subspecies multiplex, and pairwise comparisons of the chromosomal genomes showed an average nucleotide identity higher than 99%. Interestingly, the two strains differ for the presence of the plasmids pXF64-Hb_ESVL and pUCLA-ESVL detected only in the ESVL strain. The availability of these draft genomes contribute to extend the European genomic sequence dataset, a first step toward setting new research to elucidate the pathway of introduction and spread of the numerous strains of this subspecies so far detected in Europe.