Seasonal Abundance and Infectivity of Philaenus spumarius (Hemiptera: Aphrophoridae), a Vector of Xylella fastidiosa in California Vineyards.

The meadow spittlebug, Philaenus spumarius (Linnaeus) (Hemiptera: Aphrophoridae), is a vector of the plant pathogen Xylella fastidiosa; however, its role in recent outbreaks of Pierce's disease of grapevine (PD) in California is unclear. While the phenology and ecology of P. spumarius can help determine its contributions to PD epidemics, both remain poorly described in the North Coast vineyards of California. We assessed the phenology of P. spumarius in the region. Spittlemasses were first observed in February or March, while the emergence of adult spittlebugs did not occur until April or May depending on the year. Analysis of sweep and trap data from 2016 to 2018 revealed significant effects of survey month, vineyard site, and year on adult abundance in sweep and trap surveys. Spittlebug adults were present in the vineyards from April until December, with the greatest number of adults by sweep net in May or June, whereas adults on traps peaked between July and November. Analysis of natural infectivity in groups of field-collected spittlebug adults showed significant difference in transmission rates among months. Spittlebugs successfully transmitted Xylella fastidiosa (Wells) (Xanthomonadales: Xanthomonadaceae) to potted grapevines between July and December. The greatest risk of X. fastidiosa transmission by P. spumarius was in December (60%) followed by October (30%). However, the infectivity patterns of the meadow spittlebug did not align with the historical paradigm of California North Coast PD. We discuss alternative hypotheses in which P. spumarius could play a role in the epidemiology of this disease.

Updated pest categorisation of Xylella fastidiosa.

Following a request from the European Commission, the EFSA Plant Health Panel updated its pest categorisation of Xylella fastidiosa, previously delivered as part of the pest risk assessment published in 2015. X. fastidiosa is a Gram-negative bacterium, responsible for various plant diseases, including Pierce's disease, phony peach disease, citrus variegated chlorosis, olive quick decline syndrome, almond leaf scorch and various other leaf scorch diseases. The pathogen is endemic in the Americas and is present in Iran. In the EU, it is reported in southern Apulia in Italy, on the island of Corsica and in the Provence-Alpes-Côte d'Azur region in France, as well as in the Autonomous region of Madrid, the province of Alicante and the Balearic Islands in Spain. The reported status is 'transient, under eradication', except for the Balearic Islands, Corsica and southern of Apulia, where the status is 'present with a restricted distribution, under containment'. The pathogen is regulated under Council Directive 2000/29/EC and through emergency measures under http://eur-lex.europa.eu/legal-content/EN/TXT/?uri=CELEX:32015D0789 (as amended http://eur-lex.europa.eu/legal-content/EN/TXT/?uri=CELEX:32017D2352). The pest could enter the EU via host plants for planting and via infectious insect vectors. The host range includes hundreds of host species listed in the EFSA host plant database. In the EU, host plants are widely distributed and climatic conditions are favourable for its establishment. X. fastidiosa can spread by movement of host plants for planting and infectious insect vectors. X. fastidiosa is known to cause severe direct damage to major crops including almonds, citrus, grapevines, olives, stone fruits and also forest trees, landscape and ornamental trees, with high impacts. The criteria assessed by the Panel for consideration as a potential Union quarantine pest are met (the pathogen is present in the EU, but it has a restricted distribution and is under official control). X. fastidiosa is not considered as a regulated non-quarantine pest (RNQP) as the pathogen may spread also via insect vector transmission.

Paradigms: examples from the bacterium Xylella fastidiosa.

The history of advances in research on Xylella fastidiosa provides excellent examples of how paradigms both advance and limit our scientific understanding of plant pathogens and the plant diseases they cause. I describe this from a personal perspective, having been directly involved with many persons who made paradigm-changing discoveries, beginning with the discovery that a bacterium, not a virus, causes Pierce's disease of grape and other plant diseases in numerous plant species, including important crop and forest species.

Xylem structure of four grape varieties and 12 alternative hosts to the xylem-limited bacterium Xylella fastidious.

BACKGROUND AND AIMS: The bacterium Xylella fastidiosa (Xf), responsible for Pierce's disease (PD) of grapevine, colonizes the xylem conduits of vines, ultimately killing the plant. However, Vitis vinifera grapevine varieties differ in their susceptibility to Xf and numerous other plant species tolerate Xf populations without showing symptoms. The aim of this study was to examine the xylem structure of grapevines with different susceptibilities to Xf infection, as well as the xylem structure of non-grape plant species that support or limit movement of Xf to determine if anatomical differences might explain some of the differences in susceptibility to Xf. METHODS: Air and paint were introduced into leaves and stems to examine the connectivity between stem and leaves and the length distribution of their vessels. Leaf petiole and stem anatomies were studied to determine the basis for the free or restricted movement of Xf into the plant. KEY RESULTS: There were no obvious differences in stem or petiole vascular anatomy among the grape varieties examined, nor among the other plant species that would explain differences in resistance to Xf. Among grape varieties, the more tolerant 'Sylvaner' had smaller stem vessel diameters and 20 % more parenchyma rays than the other three varieties. Alternative hosts supporting Xf movement had slightly longer open xylem conduits within leaves, and more connection between stem and leaves, when compared with alternative hosts that limit Xf movement. CONCLUSIONS: Stem--leaf connectivity via open xylem conduits and vessel length is not responsible for differences in PD tolerance among grape varieties, or for limiting bacterial movement in the tolerant plant species. However, it was found that tolerant host plants had narrower vessels and more parenchyma rays, possibly restricting bacterial movement at the level of the vessels. The implications of xylem structure and connectivity for the means and regulation of bacterial movement are discussed.

Effect of Benzothiadiazole on Transmission of X-Disease Phytoplasma by the Vector Colladonus montanus to Arabidopsis thaliana, a New Experimental Host Plant.

Colladonus montanus (Van Duzee), a leafhopper vector of X-disease phytoplasma (Xp), efficiently transmitted the pathogen to Arabidopsis thaliana Columbia wild type. During transmission trials, the phytoplasma was inoculated into 22-, 34-, and 40-day-old plants. Phytoplasma infections were confirmed by polymerase chain reaction (PCR) using primers specific for Xp Symptoms in Xp-positive A. thaliana were overall stunting and reduced or no fruit (silques). All symptom-free plants were PCR negative. Leafhopper nymphs free of Xp that fed on diseased A. thaliana acquired and transmitted Xp to celery plants, a diagnostic host, causing typical X-disease symptoms. Foliar spray applications of the plant resistance elicitor benzothiadiazole (BTH) to A. thaliana 1 week before phytoplasma inoculation significantly reduced phytoplasma infection, ranging from an infection rate of 73.7% for untreated plants to 50 and 35% for plants treated with 1.2 and 4.8 mM BTH, respectively. Vector leafhoppers survival was significantly reduced on BTH-treated A. thaliana compared with leafhoppers on nontreated plants, suggesting that systemic acquired resistance in this plant may have some detrimental effect on the leafhopper C. montanus.

Xylella fastidiosa cultivation on a minimal solid defined medium.

A simple defined solid medium containing citrate and succinate, three amino acids (L-glutamine, L-asparagine, and L-cysteine), hemin chloride, potato starch, gellan gum (GelRite), and mineral salts supported the growth of grape strains of Xylella fastidiosa, the bacterial pathogen that causes Pierce's disease of grape. Isolation efficiency from infected grape plant samples, determined by the number of colony forming units recovered, on the defined medium was slightly less ( approximately 10-fold) or indistinguishable from two standard rich media used for culturing X. fastidiosa, PWG and PD3, respectively. The bacterium also grew on media with citrate and L-glutamine as the only carbon and nitrogen sources. Potato starch was not essential for bacterial growth, but no growth was observed on media without hemin chloride. Agar inhibited bacterial growth when used as the gelling agent.

Cell-cell signaling controls Xylella fastidiosa interactions with both insects and plants.

Xylella fastidiosa, which causes Pierce's disease of grapevine and other important plant diseases, is a xylem-limited bacterium that depends on insect vectors for transmission. Although many studies have addressed disease symptom development and transmission of the pathogen by vectors, little is known about the bacterial mechanisms driving these processes. Recently available X. fastidiosa genomic sequences and molecular tools have provided new routes for investigation. Here, we show that a diffusible signal molecule is required for biofilm formation in the vector and for vector transmission to plants. We constructed strains of X. fastidiosa mutated in the rpfF gene and determined that they are unable to produce the signal activity. In addition, rpfF mutants are more virulent than the wild type when mechanically inoculated into plants. This signal therefore directs interaction of X. fastidiosa with both its insect vector and plant host. Interestingly, rpfF mutants can still form in planta biofilms, which differ architecturally from biofilms in insects, suggesting that biofilm architecture, rather than a passive response to the environment, is actively determined by X. fastidiosa gene expression. This article reports a cell-cell signaling requirement for vector transmission. Identification of the genes regulated by rpfF should elucidate bacterial factors involved in transmission and biofilm formation in the insect.

The biology of xylem fluid-feeding insect vectors of Xylella fastidiosa and their relation to disease epidemiology.

Xylophagous leafhopppers are common and abundant insects of tropical and subtropical environments and play important ecological roles in these ecosystems. The feeding biology of these insects is unique in terms of their high feeding rates and a digestive physiology that allows them to assimilate amino acids, organic acids, and sugars at approximately 99% efficiency. For those species well studied, fluctuations in plant xylem chemistry and tension appear to determine the diurnal and seasonal use of their host plants. Relatively few species of xylem fluid-feeding leafhoppers are considered important pests in commercial agriculture, as they transmit the bacterial plant pathogen Xylella fastidiosa. X. fastidiosa induces diseases of grapevines, citrus, coffee, almond, alfalfa, stone fruits, landscape ornamentals, and native hardwoods for which there is no cure. Two Xylella diseases, citrus variegated chlorosis (CVC) and Pierce's disease (PD) of grapevines, have emerged as important issues within the past decade. In Brazil, CVC became important in the early 1990s and has now expanded throughout many citrus-growing areas of South America and threatens to spread to North America. The recent establishment of the exotic glassy-winged sharpshooter (Homalodisca coagulata) in California now threatens much of the United States' wine grape, table grape, and almond production. The spread of H. coagulata throughout southern California and the spread of CVC northward from Argentina through Brazil exemplifies the biological risks from exotic species. The occurrence and epidemiology of leafhopper-vectored Xylella diseases are discussed.

Use of a green fluorescent strain for analysis of Xylella fastidiosa colonization of Vitis vinifera.

Xylella fastidiosa causes Pierce's disease of grapevine as well as several other major agricultural diseases but is a benign endophyte in most host plants. X. fastidiosa colonizes the xylem vessels of host plants and is transmitted by xylem sap-feeding insect vectors. To understand better the pattern of host colonization and its relationship to disease, we engineered X. fastidiosa to express a green fluorescent protein (Gfp) constitutively and performed confocal laser-scanning microscopic analysis of colonization in a susceptible host, Vitis vinifera. In symptomatic leaves, the fraction of vessels colonized by X. fastidiosa was fivefold higher than in nearby asymptomatic leaves. The fraction of vessels completely blocked by X. fastidiosa colonies increased 40-fold in symptomatic leaves and was the feature of colonization most dramatically linked to symptoms. Therefore, the extent of vessel blockage by bacterial colonization is highly likely to be a crucial variable in symptom expression. Intriguingly, a high proportion (>80%) of colonized vessels were not blocked in infected leaves and instead had small colonies or solitary cells, suggesting that vessel blockage is not a colonization strategy employed by the pathogen but, rather, a by-product of endophytic colonization. We present evidence for X. fastidiosa movement through bordered pits to neighboring vessels and propose that vessel-to-vessel movement is a key colonization strategy whose failure results in vessel plugging and disease.

Biological traits of Xylella fastidiosa strains from grapes and almonds.

Xylella fastidiosa is a xylem-limited bacterium that causes various diseases, among them Pierce's disease of grapevine (PD) and almond leaf scorch (ALS). PD and ALS have long been considered to be caused by the same strain of this pathogen, but recent genetic studies have revealed differences among X. fastidiosa isolated from these host plants. We tested the hypothesis that ALS is caused by PD and ALS strains in the field and found that both groups of X. fastidiosa caused ALS and overwintered within almonds after mechanical inoculation. Under greenhouse conditions, all isolates caused ALS and all isolates from grapes caused PD. However, isolates belonging to almond genetic groupings did not cause PD in inoculated grapes but systemically infected grapes with lower frequency and populations than those belonging to grape strains. Isolates able to cause both PD and ALS developed 10-fold-higher concentrations of X. fastidiosa in grapes than in almonds. In the laboratory, isolates from grapes overwintered with higher efficiency in grapes than in almonds and isolates from almonds overwintered better in almonds than in grapes. We assigned strains from almonds into groups I and II on the basis of their genetic characteristics, growth on PD3 solid medium, and bacterial populations within inoculated grapevines. Our results show that genetically distinct strains from grapes and almonds differ in population behavior and pathogenicity in grapes and in the ability to grow on two different media.

Transmission of Xylella fastidiosa to grapevines by Homalodisca coagulata (Hemiptera: Cicadellidae).

Pierce's disease (PD) of grapevines is caused by a xylem-limited bacterium Xylella fastidiosa (Wells, Raju, Hung, Weisburg, Mandelco-Paul, and Brenner) that is transmitted to plants by xylem sap-feeding insects. The introduction of the sharpshooter leafhopper Homalodisca coagulata (Say) into California has initiated new PD epidemics in southern California. In laboratory experiments, the major characteristics of H. coagulata's transmission of X. fastidiosa to grapevines were the same as reported for other vectors: short or absent latent period; nymphs transmitted but lost infectivity after molting and regained infectivity after feeding on infected plants; and infectivity persisted in adults. Adult H. coagulata acquired and inoculated X. fastidiosa in <1 h of access time on a plant. Inoculation rates increased with access time, but acquisition efficiency (20% per individual) did not increase significantly beyond 6-h access. Estimated inoculation efficiency per individual per day was 19.6, 17.9, and 10.3% for experiments where plant access was 1, 2, and 4 d, respectively. Freshly molted adults and nymphs acquired and transmitted X. fastidiosa more efficiently than did older, field-collected insects. H. coagulata transmitted X. fastidiosa to 2-yr-old woody tissues of grapevines as efficiently as to green shoots. H. coagulata transmitted X. fastidiosa 3.5 mo after acquisition, demonstrating persistence of infectivity in adults. About half (14/29) of the H. coagulata from which we failed to culture X. fostidiosa from homogenized heads (with a detection threshold of 265 CFU/head) transmitted the pathogen to grape, and 17 of 24 from which we cultured X. fastidiosa transmitted.

Effects of Date of Inoculation on the Within-Plant Movement of Xylella fastidiosa and Persistence of Pierce's Disease Within Field Grapevines.

ABSTRACT The effects of date of inoculation on the development of Pierce's disease (PD) were evaluated in California grapevines during 1997 through 2000 at four locations. Some vines that had been inoculated either by using blue-green sharpshooters (Graphocephala atropunctata) as vectors or mechanically by needle puncture with the PD causal bacterium Xylella fastidiosa became infected during each month and at each location where infection was attempted. Vines inoculated on the earliest inoculation dates (April to May) developed more extensive and severe PD symptoms, and only 54% of these vines recovered from PD after the following winter, compared with vines that had been inoculated during June through August, of which 88% recovered from PD after the following winter. For the 1999 inoculations, the number of vines infected at a central California site (Parlier) was higher than the number of vines infected at a north coastal site (Hopland). For the best-fitting regression equation, percent recovery of vines infected with X. fastidiosa increased significantly with date of inoculation (r(2) = 0.737) at all sites excluding Hopland. The Hopland site had the highest percentage of vines that recovered from PD (100%). At most sites, only early infection (April and May) resulted in chronic disease unless the vines were inoculated at the bases instead of the distal tips of canes. Vines inoculated early in the growing season (April and May) have less chance to recover from Pierce's disease than vines inoculated later (July and August).

Draft sequencing and comparative genomics of Xylella fastidiosa strains reveal novel biological insights.

Draft sequencing is a rapid and efficient method for determining the near-complete sequence of microbial genomes. Here we report a comparative analysis of one complete and two draft genome sequences of the phytopathogenic bacterium, Xylella fastidiosa, which causes serious disease in plants, including citrus, almond, and oleander. We present highlights of an in silico analysis based on a comparison of reconstructions of core biological subsystems. Cellular pathway reconstructions have been used to identify a small number of genes, which are likely to reside within the draft genomes but are not captured in the draft assembly. These represented only a small fraction of all genes and were predominantly large and small ribosomal subunit protein components. By using this approach, some of the inherent limitations of draft sequence can be significantly reduced. Despite the incomplete nature of the draft genomes, it is possible to identify several phage-related genes, which appear to be absent from the draft genomes and not the result of insufficient sequence sampling. This region may therefore identify potential host-specific functions. Based on this first functional reconstruction of a phytopathogenic microbe, we spotlight an unusual respiration machinery as a potential target for biological control. We also predicted and developed a new defined growth medium for Xylella.

Whole-genome comparative analysis of three phytopathogenic Xylella fastidiosa strains.

Xylella fastidiosa (Xf) causes wilt disease in plants and is responsible for major economic and crop losses globally. Owing to the public importance of this phytopathogen we embarked on a comparative analysis of the complete genome of Xf pv citrus and the partial genomes of two recently sequenced strains of this species: Xf pv almond and Xf pv oleander, which cause leaf scorch in almond and oleander plants, respectively. We report a reanalysis of the previously sequenced Xf 9a5c (CVC, citrus) strain and the two "gapped" Xf genomes revealing ORFs encoding critical functions in pathogenicity and conjugative transfer. Second, a detailed whole-genome functional comparison was based on the three sequenced Xf strains, identifying the unique genes present in each strain, in addition to those shared between strains. Third, an "in silico" cellular reconstruction of these organisms was made, based on a comparison of their core functional subsystems that led to a characterization of their conjugative transfer machinery, identification of potential differences in their adhesion mechanisms, and highlighting of the absence of a classical quorum-sensing mechanism. This study demonstrates the effectiveness of comparative analysis strategies in the interpretation of genomes that are closely related.

Temperature-Dependent Growth and Survival of Xylella fastidiosa in Vitro and in Potted Grapevines.

Xylella fastidiosa is a xylem-inhabiting bacterium that causes Pierce's disease (PD) of grapevine. Growth rates of X. fastidiosa in a rich liquid medium were determined by culturing methods at various temperatures. The slope of the regression line between the points of 18 and 28°C was similar to that reported for Escherichia coli between 12 and 30°C and for Erwinia amylovora between 9 and 18°C. For three PD strains, two almond strains, and an oleander strain, X. fastidiosa grew fastest at 28°C but did not grow at 12°C. Grape seedlings kept at 5, 10, 17, or 25°C for 18 days, beginning 2 weeks postinoculation at 25°C, had 230-fold lower populations of X. fastidiosa when kept at 5°C, but populations did not change significantly over time at the other temperatures. In planta populations of X. fastidiosa decreased 3 days after placing the seedlings at 5 and 37°C, and subsequent samples yielded no culturable bacteria at 37°C. Based on in vitro and in planta studies, it appears that temperatures between 25 and 32°C may be critical for the epidemiology of Pierce's disease because of its rapid growth rate at these temperatures, whereas temperatures below 12 to 17°C and above 34°C may affect the survival of X. fastidiosa in plants.