A Survey of Xylella fastidiosa in the U.S. State of Virginia Reveals Wide Distribution of Both Subspecies fastidiosa and multiplex in Grapevine.

Global travel and trade in combination with climate change are expanding the geographic distribution of plant pathogens. The bacterium Xylella fastidiosa is a prime example. Native to the Americas, it has spread to Europe, Asia, and the Middle East. To assess the risk that pathogen introductions pose to crops in newly invaded areas, it is key to survey their diversity, host range, and disease incidence in relation to climatic conditions where they are already present. We performed a survey of X. fastidiosa in grapevine in Virginia using a combination of quantitative PCR, multilocus sequencing, and metagenomics. We also analyzed samples from deciduous trees with leaf scorch symptoms. X. fastidiosa subspecies fastidiosa was identified in grapevines in all regions of the state, even in Northern Virginia, where the temperature was below -9°C for 10 days per year on average in the years preceding sampling. Unexpectedly, we also found for the first time grapevine samples infected with X. fastidiosa subspecies multiplex (Xfm). The Xfm lineage found in grapevines had been previously isolated from blueberries in the Southeastern United States and was distinct from that found in deciduous trees in Virginia. The obtained results will be important for risk assessment of X. fastidiosa introductions in other parts of the world.

New Host Plant Species of Grapevine Virus A Identified with Vector-Mediated Infections.

Grapevine virus A (GVA) is an economically important virus and a member of the genus Vitivirus (family Betaflexiviridae) that causes a range of symptoms with qualitative and quantitative effects on grape production. Wild and domesticated species of Vitis, including hybrids used as rootstocks, are considered important natural hosts of GVA. Mechanical transmission to some herbaceous plant species, graft transmission, and vector transmission from grape to grape by various mealybugs and soft scale insects have been reported. Under laboratory and greenhouse conditions, this study demonstrates the transmission of GVA from grapes to alternative hosts by the vine mealybug (Planococcus ficus). Results of ELISA, end-point one-step RT-PCR, and real-time RT-PCR, and in some cases electron microscopy and genome sequencing, confirmed successful transmission to three new plant species commonly found in Croatian vineyards: velvetleaf (Abutilon theophrasti), redroot pigweed (Amaranthus retroflexus), and field poppy (Papaver rhoeas), along with Chenopodium murale and the previously known host Nicotiana benthamiana, with variable infection rates. Depending on the host species, symptoms in the form of leaf reddening, yellow spots, reduced growth of lateral shoots, systemic vein clearing, foliar deformation and rugosity, and dwarfism were observed in GVA-infected plants, whereas no symptoms were observed in infected plants of A. theophrasti. Reverse transmission from these new hosts to grapevines by Pl. ficus was not successful. These results confirm four new GVA host species and open new research venues.

The Multifaceted Role of Homologous Recombination in a Fastidious Bacterial Plant Pathogen.

Homologous recombination plays a key function in the evolution of bacterial genomes. Within Xylella fastidiosa, an emerging plant pathogen with increasing host and geographic ranges, it has been suggested that homologous recombination facilitates host switching, speciation, and the development of virulence. We used 340 whole-genome sequences to study the relationship between inter- and intrasubspecific homologous recombination, random mutation, and natural selection across individual X. fastidiosa genes. Individual gene orthologs were identified and aligned, and a maximum likelihood (ML) gene tree was generated. Each gene alignment and tree pair were then used to calculate gene-wide and branch-specific r/m values (relative effect of recombination to mutation), gene-wide and branch-site nonsynonymous over synonymous substitution rates (dN/dS values; episodic selection), and branch length (as a proxy for mutation rate). The relationships between these variables were evaluated at the global level (i.e., for all genes among and within a subspecies), among specific functional classes (i.e., COGs), and between pangenome components (i.e., accessory versus core genes). Our analysis showed that r/m varied widely among genes as well as across X. fastidiosa subspecies. While r/m and dN/dS values were positively correlated in some instances (e.g., core genes in X. fastidiosa subsp. fastidiosa and both core and accessory genes in X. fastidiosa subsp. multiplex), low correlation coefficients suggested no clear biological significance. Overall, our results indicate that, in addition to its adaptive role in certain genes, homologous recombination acts as a homogenizing and a neutral force across phylogenetic clades, gene functional groups, and pangenome components. IMPORTANCE There is ample evidence that homologous recombination occurs frequently in the economically important plant pathogen Xylella fastidiosa. Homologous recombination has been known to occur among sympatric subspecies and is associated with host-switching events and virulence-linked genes. As a consequence, is it generally assumed that recombinant events in X. fastidiosa are adaptive. This mindset influences expectations of how homologous recombination acts as an evolutionary force as well as how management strategies for X. fastidiosa diseases are determined. Yet, homologous recombination plays roles beyond that of a source for diversification and adaptation. Homologous recombination can act as a DNA repair mechanism, as a means to facilitate nucleotide compositional change, as a homogenization mechanism within populations, or even as a neutral force. Here, we provide a first assessment of long-held beliefs regarding the general role of recombination in adaptation for X. fastidiosa. We evaluate gene-specific variations in homologous recombination rate across three X. fastidiosa subspecies and its relationship to other evolutionary forces (e.g., natural selection, mutation, etc.). These data were used to assess the role of homologous recombination in X. fastidiosa evolution.

Progression of Xylella fastidiosa Infection in Grapevines Under Field Conditions.

The pathogen Xylella fastidiosa subsp. fastidiosa has circulated through California's vineyards since its introduction from Central America in the 1800s. This pathogen is responsible for a bacterial disease called Pierce's disease (PD) of grapevine. With no known cure, PD has had devastating effects on some vineyards. Important factors that impact disease severity and persistence include: the presence of insect vectors, grapevine cultivar, management, ecology, and winter temperatures. Removal of infected vines is critical for reducing pathogen spread but relies on accurate and rapid pathogen detection. In this study, we foster a greater understanding of disease symptom emergence by way of a 3-year field inoculation project in Napa Valley. Although PD emergence and symptom progression have been studied in greenhouse and experimental plots, there is a large knowledge gap in quantifying disease progression under commercial conditions. After inoculating 80 mature Vitis vinifera vines in April 2017, we measured bacterial populations and six symptom types at four locations within each plant throughout the subsequent three growing seasons. The main foci of the project were understanding X. fastidiosa movement through the plants, infection, overwinter curing, and symptom development. We observed greater winter recovery than expected, and shriveled grape clusters proved to be a more reliable early indication of infection than other more commonly used symptoms. Although there were differences among wine grape cultivars, this work suggests that disease progression in the field may not fit the paradigm of predominant leaf scorch and low recovery rates as neatly as has been previously believed.

The XadA Trimeric Autotransporter Adhesins in Xylella fastidiosa Differentially Contribute to Cell Aggregation, Biofilm Formation, Insect Transmission and Virulence to Plants.

Surface adhesion strategies are widely employed by bacterial pathogens during establishment and systemic spread in their host. A variety of cell-surface appendages such as pili, fimbriae, and afimbrial adhesins are involved in these processes. The phytopathogen Xylella fastidiosa employs several of these structures for efficient colonization of its insect and plant hosts. Among the adhesins encoded in the X. fastidiosa genome, three afimbrial adhesins, XadA1, Hsf/XadA2, and XadA3, are predicted to be trimeric autotransporters with a C-terminal YadA-anchor membrane domain. We analyzed the individual contributions of XadA1, XadA2, and XadA3 to various cellular behaviors both in vitro and in vivo. Using isogenic X. fastidiosa mutants, we found that cell-cell aggregation and biofilm formation were severely impaired in the absence of XadA3. No significant reduction of cell-surface attachment was found with any mutant under flow conditions. Acquisition by insect vectors and transmission to grapevines were reduced in the XadA3 deletion mutant. While the XadA3 mutant was hypervirulent in grapevines, XadA1 or XadA2 deletion mutants conferred lower disease severity than the wild-type strain. This insight of the importance of these adhesive proteins and their individual contributions to different aspects of X. fastidiosa biology should guide new approaches to reduce pathogen transmission and disease development. [Formula: see text] Copyright © 2022 The Author(s). This is an open access article distributed under the CC BY-NC-ND 4.0 International license.

Phylogenetics of Historical Host Switches in a Bacterial Plant Pathogen.

Xylella fastidiosa is an insect-transmitted bacterial plant pathogen found across the Americas and, more recently, worldwide. X. fastidiosa infects plants of at least 563 species belonging to 82 botanical families. While the species X. fastidiosa infects many plants, particular strains have increased plant specificity. Understanding the molecular underpinnings of plant host specificity in X. fastidiosa is vital for predicting host shifts and epidemics. While there may exist multiple genetic determinants of host range in X. fastidiosa, the drivers of the unique relationships between X. fastidiosa and its hosts should be elucidated. Our objective with this study was to predict the ancestral plant hosts of this pathogen using phylogenetic and genomic methods based on a large data set of pathogen whole-genome data from agricultural hosts. We used genomic data to construct maximum-likelihood (ML) phylogenetic trees of subsets of the core and pan-genomes. With those trees, we ran ML ancestral state reconstructions of plant host at two taxonomic scales (genus and multiorder clades). Both the core and pan-genomes were informative in terms of predicting ancestral host state, giving new insight into the history of the plant hosts of X. fastidiosa. Subsequently, gene gain and loss in the pan-genome were found to be significantly correlated with plant host through genes that had statistically significant associations with particular hosts. IMPORTANCE Xylella fastidiosa is a globally important bacterial plant pathogen with many hosts; however, the underpinnings of host specificity are not known. This paper contains important findings about the usage of phylogenetics to understand the history of host specificity in this bacterial species, as well as convergent evolution in the pan-genome. There are strong signals of historical host range that give us insights into the history of this pathogen and its various invasions. The data from this paper are relevant in making decisions for quarantine and eradication, as they show the historical trends of host switching, which can help us predict likely future host shifts. We also demonstrate that using multilocus sequence type (MLST) genes in this system, which is still a commonly used process for policymaking, does not reconstruct the same phylogenetic topology as whole-genome data.

Xylella fastidiosa Outbreak in Israel: Population Genetics, Host Range, and Temporal and Spatial Distribution Analysis.

Diseases caused by the insect-transmitted bacterium Xylella fastidiosa have been reported in the Americas since the 19th century, causing diseases such as Pierce's disease of grapevine, almond leaf scorch (ALS), and citrus variegated chlorosis. In the last decade X. fastidiosa was reported from different parts of the world, most notably from southern Italy, infecting olives. In 2017, X. fastidiosa was reported to be associated with ALS symptoms in Israel. Here, we investigated the causal agent of ALS in Israel, its genetic diversity, and host range, and we characterized the temporal and spatial distribution of the disease. X. fastidiosa subsp. fastidiosa sequence type 1 was isolated from symptomatic almond trees and was used to infect almond and grapevine by mechanical inoculation. The pathogen, however, did not infect olive, peach, cherry, plum, nectarine, clementine, and grapefruit plants. Genomic analysis of local isolates revealed that the local population is derived from a single introduction and that they are closely related to X. fastidiosa strains from grapevines in California. Distribution analyses revealed that ALS did not expand from 2017 to 2019; however, since 2020, newly symptomatic trees appeared in the tested orchards. Symptomatic trees were located primarily in clusters, and symptoms tended to spread within rows. Our study confirms that X. fastidiosa is the causal agent of ALS in Israel and describes its genetic and host range characteristics. Although there is no clear evidence yet for the identity of the vectors in Israel, ALS spread continues to threat the almond and grapevine industries in Israel.

Allopatric Plant Pathogen Population Divergence following Disease Emergence.

Within the landscape of globally distributed pathogens, populations differentiate via both adaptive and nonadaptive forces. Individual populations are likely to show unique trends of genetic diversity, host-pathogen interaction, and ecological adaptation. In plant pathogens, allopatric divergence may occur particularly rapidly within simplified agricultural monoculture landscapes. As such, the study of plant pathogen populations in monocultures can highlight the distinct evolutionary mechanisms that lead to local genetic differentiation. Xylella fastidiosa is a plant pathogen known to infect and damage multiple monocultures worldwide. One subspecies, Xylella fastidiosa subsp. fastidiosa, was first introduced to the United States ∼150 years ago, where it was found to infect and cause disease in grapevines (Pierce's disease of grapevines, or PD). Here, we studied PD-causing subsp. fastidiosa populations, with an emphasis on those found in the United States. Our study shows that following their establishment in the United States, PD-causing strains likely split into populations on the East and West Coasts. This diversification has occurred via both changes in gene content (gene gain/loss events) and variations in nucleotide sequence (mutation and recombination). In addition, we reinforce the notion that PD-causing populations within the United States acted as the source for subsequent subsp. fastidiosa outbreaks in Europe and Asia.IMPORTANCE Compared to natural environments, the reduced diversity of monoculture agricultural landscapes can lead bacterial plant pathogens to quickly adapt to local biological and ecological conditions. Because of this, accidental introductions of microbial pathogens into naive regions represents a significant economic and environmental threat. Xylella fastidiosa is a plant pathogen with an expanding host and geographic range due to multiple intra- and intercontinental introductions. X. fastidiosa subsp. fastidiosa infects and causes disease in grapevines (Pierce's disease of grapevines [PD]). This study focused on PD-causing X. fastidiosa populations, particularly those found in the United States but also invasions into Taiwan and Spain. The analysis shows that PD-causing X. fastidiosa has diversified via multiple cooccurring evolutionary forces acting at an intra- and interpopulation level. This analysis enables a better understanding of the mechanisms leading to the local adaptation of X. fastidiosa and how a plant pathogen diverges allopatrically after multiple and sequential introduction events.

Emerging plant disease epidemics: Biological research is key but not enough.

The effective management of plant diseases is of fundamental importance for food production, forestry, and other plant-derived products, as well as for the sustainability of natural environments. When considering the impact of a plant pathogen, the financial costs incurred by an outbreak usually receive the most focus, but there are other much less understood consequences for the affected society, culture, and environment due to disease. This poorly studied layer of complexity is particularly relevant for emerging outbreaks, of which often only limited knowledge is available to devise management strategies, but decisions and actions must be made quickly. The recent outbreak of a bacterial plant pathogen in Europe illustrates how understanding not only the biology of an emerging pathogen but also the cultural context is critical for effectively communicating and engaging with stakeholders and policy makers in order to implement successful disease control strategies.

Inference of Bacterial Pathogen Instantaneous Population Growth Dynamics.

Although bacterial host colonization is a dynamic process that requires population growth, studies often focus on comparing bacterial populations at a given time point. However, this may not reflect the dynamics of the colonization process. Time-course assays provide important insights into the dynamics of host colonization but are laborious and may still lack resolution for immediate processes affecting populations. An alternative way to address this issue, using widely accessible tools (such as quantitative PCR [qPCR]), is to take advantage of the relationship between bacterial chromosomal replication and cell division to determine population growth status at the sampling time. Conceptually, the ratio between the number of copies at the origin of replication and that at the terminus of replication should be correlated with the measured bacterial growth rate. This peak-to-trough ratio (PTR) to estimate instantaneous population growth status was tested with the slow-growing plant-pathogenic bacterium Xylella fastidiosa. We found no correlation between PTR and the measured growth rate when using genome-level data but overall sequencing depth of coverage trends matched theoretical expectations. On the other hand, the population growth status of X. fastidiosa was predicted by PTR when using qPCR data, which was improved by the pretreatment of cells with a photoreactive DNA-binding dye. Our results suggest that PTR could be used to determine X. fastidiosa growth status both in planta and in insect vectors. We expect PTR will perform better with fast-growing bacterial pathogens, potentially becoming a powerful tool for easily and quickly assessing population growth status.

Impacts of local population history and ecology on the evolution of a globally dispersed pathogen.

BACKGROUND: Pathogens with a global distribution face diverse biotic and abiotic conditions across populations. Moreover, the ecological and evolutionary history of each population is unique. Xylella fastidiosa is a xylem-dwelling bacterium infecting multiple plant hosts, often with detrimental effects. As a group, X. fastidiosa is divided into distinct subspecies with allopatric historical distributions and patterns of multiple introductions from numerous source populations. The capacity of X. fastidiosa to successfully colonize and cause disease in naïve plant hosts varies among subspecies, and potentially, among populations. Within Central America (i.e. Costa Rica) two X. fastidiosa subspecies coexist: the native subsp. fastidiosa and the introduced subsp. pauca. Using whole genome sequences, the patterns of gene gain/loss, genomic introgression, and genetic diversity were characterized within Costa Rica and contrasted to other X. fastidiosa populations. RESULTS: Within Costa Rica, accessory and core genome analyses showed a highly malleable genome with numerous intra- and inter-subspecific gain/loss events. Likewise, variable levels of inter-subspecific introgression were found within and between both coexisting subspecies; nonetheless, the direction of donor/recipient subspecies to the recombinant segments varied. Some strains appeared to recombine more frequently than others; however, no group of genes or gene functions were overrepresented within recombinant segments. Finally, the patterns of genetic diversity of subsp. fastidiosa in Costa Rica were consistent with those of other native populations (i.e. subsp. pauca in Brazil). CONCLUSIONS: Overall, this study shows the importance of characterizing local evolutionary and ecological history in the context of world-wide pathogen distribution.

Population structure and adaptation of a bacterial pathogen in California grapevines.

Xylella fastidiosa subsp. fastidiosa causes Pierce's disease of grapevine (PD) and has been present in California for over a century. A singly introduced genotype spread across the state causing large outbreaks and damaging the grapevine industry. This study presents 122 X. fastidiosa subsp. fastidiosa genomes from symptomatic grapevines, and explores pathogen genetic diversity associated with PD in California. A total of 5218 single-nucleotide polymorphisms (SNPs) were found in the dataset. Strong population genetic structure was found; isolates split into five genetic clusters divided into two lineages. The core/soft-core genome constituted 41.2% of the total genome, emphasizing the high genetic variability of X. fastidiosa genomes. An ecological niche model was performed to estimate the environmental niche of the pathogen within California and to identify key climatic factors involved in dispersal. A landscape genomic approach was undertaken aiming to link local adaptation to climatic factors. A total of 18 non-synonymous polymorphisms found to be under selective pressures were correlated with at least one environmental variable highlighting the role of temperature, precipitation and elevation on X. fastidiosa adaptation to grapevines in California. Finally, the contribution to virulence of three of the genes under positive selective pressure and of one recombinant gene was studied by reverse genetics.

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.

Genomic Diversity and Recombination among Xylella fastidiosa Subspecies.

Xylella fastidiosa is an economically important bacterial plant pathogen. With insights gained from 72 genomes, this study investigated differences among the three main subspecies, which have allopatric origins: X. fastidiosa subsp. fastidiosa, multiplex, and pauca The origin of recombinogenic X. fastidiosa subsp. morus and sandyi was also assessed. The evolutionary rate of the 622 genes of the species core genome was estimated at the scale of an X. fastidiosa subsp. pauca subclade (7.62 × 10-7 substitutions per site per year), which was subsequently used to estimate divergence time for the subspecies and introduction events. The study characterized genes present in the accessory genome of each of the three subspecies and investigated the core genome to detect genes potentially under positive selection. Recombination is recognized to be the major driver of diversity in X. fastidiosa, potentially facilitating shifts to novel plant hosts. The relative effect of recombination in comparison to point mutation was calculated (r/m = 2.259). Evidence of recombination was uncovered in the core genome alignment; X. fastidiosa subsp. fastidiosa in the United States was less prone to recombination, with an average of 3.22 of the 622 core genes identified as recombining regions, whereas a specific clade of X. fastidiosa subsp. multiplex was found to have on average 9.60 recombining genes, 93.2% of which originated from X. fastidiosa subsp. fastidiosa Interestingly, for X. fastidiosa subsp. morus, which was initially thought to be the outcome of genome-wide recombination between X. fastidiosa subsp. fastidiosa and X. fastidiosa subsp. multiplex, intersubspecies homologous recombination levels reached 15.30% in the core genome. Finally, there is evidence of X. fastidiosa subsp. pauca strains from citrus containing genetic elements acquired from strains infecting coffee plants as well as genetic elements from both X. fastidiosa subsp. fastidiosa and X. fastidiosa subsp. multiplex In summary, our data provide new insights into the evolution and epidemiology of this plant pathogen.IMPORTANCEXylella fastidiosa is an important vector-borne plant pathogen. We used a set of 72 genomes that constitutes the largest assembled data set for this bacterial species so far to investigate genetic relationships and the impact of recombination on phylogenetic clades and to compare genome content at the subspecies level, and we used a molecular dating approach to infer the evolutionary rate of X. fastidiosa The results demonstrate that recombination is important in shaping the genomes of X. fastidiosa and that each of the main subspecies is under different selective pressures. We hope insights from this study will improve our understanding of X. fastidiosa evolution and biology.

Understanding How an Invasive Vector Drives Pierce's Disease Epidemics: Seasonality and Vine-to-Vine Spread.

For vector-borne plant pathogens, disease epidemics may be attributable to multiple mechanisms, including introduction of a novel vector whose epidemiological role differs from that of native vectors. In such cases, understanding an exotic vector's ability to drive an epidemic is central to mitigating its impact. We studied how the invasive glassy-winged sharpshooter (Homalodisca vitripennis Germar) can drive Pierce's disease outbreaks in vineyards, focusing on its potential to promote vine-to-vine (i.e., secondary) spread of Xylella fastidiosa relative to potential constraints stemming from seasonality in the pathosystem. First, we developed a general vector-borne disease model to understand the consequences for disease dynamics of (i) seasonal acquisition efficiency and (ii) seasonal host recovery from infection. Results of the modeling indicate that these two sources of seasonality could constrain disease incidence, particularly when working in concert. Next, we established a field cage experiment to determine whether H. vitripennis promotes vine-to-vine spread, and looked for evidence of seasonality in spread. Broadly, results from the experiment supported assumptions of the model; there was modest to significant increase in the frequency of pathogen spread over the first season, and those new infections that occurred later in the season were more likely to recover during winter. Ultimately, by the end of the second season, there was not evidence of significant secondary spread, likely due to a combination of seasonal constraints and low transmission efficiency by H. vitripennis. Collectively, these results suggest that, although H. vitripennis may be able to promote vine-to-vine spread in certain contexts, it may not be the key factor explaining its impact. Rather, the ability of H. vitripennis to drive epidemics is likely to be more directly related to its potential to reach higher population densities than native vectors.

Transmission Efficiency of Xylella fastidiosa subsp. pauca Sequence Types by Sharpshooter Vectors after In Vitro Acquisition.

Xylella fastidiosa subsp. pauca is genetically diverse and has many vector species. However, there is limited information on vector specificity and efficiency for different sequence types (STs) within the pathogen subspecies. Both STs of X. fastidiosa and vectors differ in their associations with plants; therefore, assessment of vector competence should include the standardized vector acquisition ability of bacteria from artificial diets. This work aimed to adapt and validate an in vitro acquisition system for strains of X. fastidiosa that cause citrus variegated chlorosis, and to compare the transmission efficiency of STs of subsp. pauca by different species of sharpshooter vector. First, acquisition and transmission of ST13 by Bucephalogonia xanthophis and Macugonalia leucomelas was tested using an artificial diet with bacteria grown on minimum defined medium (X. fastidiosa medium) with or without 1% galacturonic acid (GA). Subsequently, four sharpshooter species (B. xanthophis, M. leucomelas, M. cavifrons, and Sibovia sagata) were compared as vectors of ST13 acquired from artificial diets, and four STs of subsp. pauca (11, 13, 65, and 70) were tested for acquisition and transmission by M. leucomelas. The artificial system allowed efficient acquisition and transmission of ST13 to plants, with no differences between the media tested. ST13 was transmitted more efficiently by B. xanthophis and M. leucomelas when compared with M. cavifrons and S. sagata. Different STs influenced acquisition and transmission rates by M. leucomelas. The differences in vector competence, despite the standardized acquisition system, suggest that ST-vector foregut or vector-plant interactions may influence bacterial acquisition, retention and inoculation by the insect.

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.

Screening of some Croatian autochthonous grapevine varieties reveals a multitude of viruses, including novel ones.

Next-generation sequencing of total RNA samples from four Croatian autochthonous grapevine varieties revealed the presence of a novel virus in two grapevine accessions. The complete genome sequence of a novel virus, tentatively named "grapevine badnavirus 1" (GBV-1), was reconstructed from a de novo-assembled contig. GBV-1 has a genome of 7,145 nucleotides containing three ORFs with sequence similarity to other badnaviruses. In addition, several other viruses and viroids, including grapevine virus G, grapevine virus K/D, grapevine virus T, grapevine Roditis leaf discoloration-associated virus, grapevine yellow speckle viroids 1 and 2, and hop stunt viroid were detected and identified for the first time in Croatian grapevines in the course of this study.

Natural Competence Rates Are Variable Among Xylella fastidiosa Strains and Homologous Recombination Occurs In Vitro Between Subspecies fastidiosa and multiplex.

Xylella fastidiosa, an etiological agent of emerging crop diseases around the world, is naturally competent for the uptake of DNA from the environment that is incorporated into its genome by homologous recombination. Homologous recombination between subspecies of X. fastidiosa was inferred by in silico studies and was hypothesized to cause disease emergence. However, no experimental data are available on the degree to which X. fastidiosa strains are capable of competence and whether recombination can be experimentally demonstrated between subspecies. Here, using X. fastidiosa strains from different subspecies, natural competence in 11 of 13 strains was confirmed with plasmids containing antibiotic markers flanked by homologous regions and, in three of five strains, with dead bacterial cells used as source of donor DNA. Recombination frequency differed among strains and was correlated to growth rate and twitching motility. Moreover, intersubspecific recombination occurred readily between strains of subsp. fastidiosa and multiplex, as demonstrated by movement of antibiotic resistance and green fluorescent protein from donor to recipient cells and confirmed by DNA sequencing of the flanking arms of recombinant strains. Results demonstrate that natural competence is widespread among X. fastidiosa strains and could have an impact in pathogen adaptation and disease development.

Fasting alters aphid probing behaviour but does not universally increase the transmission rate of non-circulative viruses.

A fasting period prior to non-circulative virus acquisition has been shown to increase the rate of transmission by aphids. However, this effect has only been studied for a few virus-vector combinations, and there are contradictory results in the literature as to the role of fasting on virus acquisition. We analysed the influence of fasting on the transmission of three non-circulative viruses, Cucumber mosaic virus, Zucchini yellow mosaic virus and Cauliflower mosaic virus, by two aphid vector species: Myzus persicae Sulzer (Hemiptera: Aphididae) and Aphis gossypii Glover (Hemiptera: Aphididae). All variables tested, including the virus species and isolate, and the species of aphid, influenced the effect of a fasting period on virus transmission efficiency. Furthermore, when aphids were subjected to an overnight feeding period on a sucrose solution, the fasting effect disappeared and the probing behaviour of these aphids was markedly different to plant-reared aphids. The electrical penetration graph (EPG) technique revealed that fasting altered the probing behaviour of M. persicae and A. gossypii, with fasted aphids beginning to feed sooner and having a significantly longer first intracellular puncture, measured as a potential drop. Significantly longer sub-phase II-3 of the potential drop and more archlets during this sub-phase were also observed for fasted aphids of both species. However, these behavioural changes were not predictive of increasing virus transmission following a fasting period. The impacts of pre-acquisition fasting on aphid probing behaviour and on the mechanisms of non-circulative virus transmission are discussed.