The plant-pathogenic bacterium Xylella fastidiosa 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. X. fastidiosa subsp. multiplex ESVL and IVIA5901 strains that belong to sequence type 6 were isolated from almond orchards within the outbreak area in Alicante province (Spain). Both strains share more than 99% of the chromosomal sequences (average nucleotide identity), but the ESVL strain harbors two plasmids (pXF64-Hb_ESVL and pUCLA-ESVL). Here, virulence phenotypes and genome content were compared between both strains, using three strains from the United States as a reference for the phenotypic analyses. Experiments in microfluidic chambers, used as a simulation of xylem vessels, showed that twitching motility was absent in the IVIA5901 strain, whereas the ESVL strain had reduced twitching motility. In general, both Spanish strains had less biofilm formation, less cell aggregation, and lower virulence in tobacco compared with U.S. reference strains. Genome analysis of the two plasmids from ESVL revealed 51 unique coding sequences that were absent in the chromosome of IVIA5901. Comparison of the chromosomes of both strains showed some unique coding sequences and single-nucleotide polymorphisms 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 X. fastidiosa plasmids, our results indicate that the presence of plasmids should be considered in the study of the mechanisms of pathogenicity and adaptation in X. fastidiosa to new environments. [ ] Copyright © 2023 The Author(s). This is an open access article distributed under the CC BY-NC-ND 4.0 International license .

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.

Strains of the bacterial pathogen Xylella fastidiosa subspecies multiplex (Xfm) and pauca (Xfp) isolated from symptomatic almond and olive plants in Spain and Italy were used in this study. Because of the risk of host jump and considering the importance of southern highbush blueberry production in Spain, we tested a small set of these strains for their potential to infect and cause disease symptoms in blueberries under greenhouse experiments. Xfm IVIA5901 (isolated from almonds in Alicante, Spain) caused symptoms similar to those caused by Xfm AlmaEm3 (isolated from blueberries in Georgia, U.S.A., and used as a reference strain capable of inducing severe symptoms in blueberry). Nevertheless, bacterial populations of Xfm IVIA5901 in planta were significantly lower than those of Xfm AlmaEm3. Xfm ESVL (isolated from almonds, Alicante, Spain) and Xfp XYL1961/18 (isolated from olives, Ibiza Island, Spain) caused limited symptoms, while Xfm XYL466/19 (isolated from wild olives, Mallorca Island, Spain) and Xfm XF3348 (isolated from almonds, Mallorca Island, Spain) and Xfp De Donno (isolated from olives, Puglia, Italy, and representative of the devastating olive quick decline syndrome) did not cause symptoms nor colonize blueberries. This study suggests that certain strains already found in Europe could infect blueberry if conditions conducive for a host jump in this region are met, such as proximity of blueberries to other infected hosts and presence of insect vectors that feed on these crops. Surveys on the presence of X. fastidiosa in blueberries in Spain and other European countries are needed to anticipate possible issues.

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 have 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 with 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. [ ] Copyright © 2024 The Author(s). This is an open access article distributed under the CC BY-NC-ND 4.0 International license .

Xylella fastidiosa (Xf) is a quarantine plant pathogen in the European Union, recognized as a high-priority pest due to its devastating cultural and economic impact on crops, ornamental plants, and landscape vegetation. The development and implementation of reliable, sensitive, and specific diagnostic methods for Xf detection are critical to ensure the production and trade of healthy plant material and to facilitate effective control measures, primarily aimed at eradication. Despite the availability of numerous detection protocols, their diagnostic parameters remain not precisely defined, and no universally accepted gold standard protocol exists. This study compared the global accuracy and performance of six molecular assays using almond samples collected from naturally infected almond trees in the Alicante Demarcated Area, Spain. Additionally, the study evaluated the influence of plant sample type (leaf petioles versus woody chips) on diagnostic accuracy. Harper-qPCR and Li-qPCR assays demonstrated the highest sensitivity, with detection limits as low as 2.8 to 3 fg of Xf DNA. Droplet digital PCR (ddPCR) exhibited excellent sensitivity for woody chip samples, while Li-qPCR showed superior specificity across both tissue types. In contrast, recombinase polymerase amplification displayed lower detection limits and reproducibility compared with qPCR-based methods. Bayesian latent class models indicated that combining Harper-qPCR and Li-qPCR for petioles, or Harper-qPCR and ddPCR for wood samples, optimized diagnostic reliability by reducing false negatives, which is critical in buffer zones under eradication while maintaining high specificity. These findings emphasize the need for tailoring diagnostic protocols to the epidemiological context, balancing sensitivity and specificity to optimize surveillance schemes for Xf and to support effective phytosanitary management strategies. [ ] Copyright © 2026 The Author(s). This is an open access article distributed under the CC BY-NC-ND 4.0 International license .

Emergence of Xylella fastidiosa (Xf) in Europe threatens agriculture and natural ecosystems. Its adaptability to diverse environments raises concerns regarding potential host shifts and increased virulence. Effective disease management depends on accurate and timely detection. Additionally, understanding its interaction with local host cultivars is essential for developing risk mitigation measures. This study evaluated the pathogenicity and the extension of stem colonization of five Xf strains from different subspecies, isolated from almond and olive trees in Spain and Italy, inoculated on three widely cultivated Spanish olive cultivars. All strains successfully infected and colonized olive plants, although detection frequencies decreased over time, and none of the strains induced disease symptoms. Overall, strains XYL1961/18 and De Donno from subspecies pauca showed higher colonization frequencies and bacterial loads compared with strains from subspecies multiplex. Furthermore, physiological responses were assessed using leaf spectral data retrieved with handheld proximal sensors, enabling the calculation of 74 vegetation indices to detect physiological alterations linked to infection status in asymptomatic plants. Of the 74 indices, between 17 and 31, mainly related to pigment composition (carotenoids, flavonoids, and xanthophylls), were selected as reliable predictors (>90% accuracy) of infection status across strains and olive cultivars. This study highlights the importance of evaluating the extent of stem colonization and interactions among local olive cultivars and Xf strains from different subspecies. We showed that proximal sensing may offer a promising noninvasive tool for early detection and monitoring of Xf infection, supporting timely management, thereby reducing the risk of new outbreaks. These methodologies may also assist in breeding programs aimed at developing Xf-resistant olive cultivars.