Complete Circularized Genome Resources of Seven Strains of Xylella fastidiosa subsp. fastidiosa Using Hybrid Assembly Reveals Unknown Plasmids.

Xylella fastidiosa 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 Oxford Nanopore Technologies and Illumina sequencing data, we assembled high-quality complete genomes of seven X. fastidiosa subsp. fastidiosa strains isolated from different plants in Spain, the United States, and Mexico. Comparative genomic analyses discovered differences in plasmid content among strains, including plasmids that had been overlooked previously when using the Illumina sequencing platform alone. Interestingly, in strain CFBP8073, intercepted in France from plants imported from Mexico, three plasmids were identified, including two (plasmids 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 X. fastidiosa 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 United States. 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.

Xylella fastidiosa Infection Reshapes Microbial Composition and Network Associations in the Xylem of Almond Trees.

Xylella fastidiosa represents a major threat to important crops worldwide including almond, citrus, grapevine, and olives. Nowadays, there are no efficient control measures for X. fastidiosa, and the use of preventive measures and host resistance represent the most practical disease management strategies. Research on vessel-associated microorganisms is gaining special interest as an innate natural defense of plants to cope against infection by xylem-inhabiting pathogens. The objective of this research has been to characterize, by next-generation sequencing (NGS) analysis, the microbial communities residing in the xylem sap of almond trees affected by almond leaf scorch disease (ALSD) in a recent X. fastidiosa outbreak occurring in Alicante province, Spain. We also determined community composition changes and network associations occurring between xylem-inhabiting microbial communities and X. fastidiosa. For that, a total of 91 trees with or without ALSD symptoms were selected from a total of eight representative orchards located in five municipalities within the X. fastidiosa-demarcated area. X. fastidiosa infection in each tree was verified by quantitative polymerase chain reaction (qPCR) analysis, with 54% of the trees being tested X. fastidiosa-positive. Globally, Xylella (27.4%), Sphingomonas (13.9%), and Hymenobacter (12.7%) were the most abundant bacterial genera, whereas Diplodia (30.18%), a member of the family Didymellaceae (10.7%), and Aureobasidium (9.9%) were the most predominant fungal taxa. Furthermore, principal coordinate analysis (PCoA) of Bray-Curtis and weighted UniFrac distances differentiated almond xylem bacterial communities mainly according to X. fastidiosa infection, in contrast to fungal community structure that was not closely related to the presence of the pathogen. Similar results were obtained when X. fastidiosa reads were removed from the bacterial data set although the effect was less pronounced. Co-occurrence network analysis revealed negative associations among four amplicon sequence variants (ASVs) assigned to X. fastidiosa with different bacterial ASVs belonging to 1174-901-12, Abditibacterium, Sphingomonas, Methylobacterium-Methylorubrum, Modestobacter, Xylophilus, and a non-identified member of the family Solirubrobacteraceae. Determination of the close-fitting associations between xylem-inhabiting microorganisms and X. fastidiosa may help to reveal specific microbial players associated with the suppression of ALSD under high X. fastidiosa inoculum pressure. These identified microorganisms would be good candidates to be tested in planta, to produce almond plants more resilient to X. fastidiosa infection when inoculated by endotherapy, contributing to suppress ALSD.

Complete Circularized Genome Data of Two Spanish strains of Xylella fastidiosa (IVIA5235 and IVIA5901) Using Hybrid Assembly Approaches.

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.

Emergence of a Plant Pathogen in Europe Associated with Multiple Intercontinental Introductions.

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.IMPORTANCEXylella fastidiosa is an economically important plant-pathogenic bacterium that has emerged as a pathogen of global importance associated with a devastating epidemic in olive trees in Italy associated with X. fastidiosa subspecies pauca and other outbreaks in Europe, such as X. fastidiosa subspecies fastidiosa and X. fastidiosa subspecies multiplex in Spain and X. fastidiosa subspecies multiplex in France. We present evidence of multiple introductions of X. fastidiosa subspecies multiplex, likely from the United States, into Spain, Italy, and France. These introductions illustrate the risks associated with the commercial trade of plant material at global scales and the need to develop effective policy to limit the likelihood of pathogen pollution into naive regions. Our study demonstrates the need to utilize whole-genome sequence data to study X. fastidiosa introductions at outbreak stages, since a limited number of genetic markers does not provide sufficient phylogenetic resolution to determine dispersal paths or relationships among strains that are of biological and quarantine relevance.

Draft Genome Resources of Two Strains ("ESVL" and "IVIA5901") of Xylella fastidiosa Associated with Almond Leaf Scorch Disease in Alicante, Spain.

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.

Draft Genome Sequence of Xylella fastidiosa subsp. fastidiosa Strain IVIA5235, Isolated from Prunus avium in Mallorca Island, Spain.

We report the complete annotated genome sequence of the plant-pathogenic bacterium Xylella fastidiosa subsp. fastidiosa strain IVIA5235. This strain was recovered from a cherry tree in Mallorca, Spain.