Host plant identification in the generalist xylem feeder Philaenus spumarius through gut content analysis

The meadow spittlebug, Philaenus spumarius L. (Hemiptera: Aphrophoridae), is the main vector of the phytopathogenic bacterium Xylella fastidiosa in Europe, where the ST53 strain induces the olive quick decline syndrome, causing severe economic damage in southern Italy. The wide range of plant species infected by X. fastidiosa, and the wide host range of P. spumarius suggest that a large number of wild and cultivated plants may become infected by the pathogen following unintentional introduction events. Therefore, it is necessary to detail the host plant preference of the vector, in order to include preferred plants in the field, in pathogen-targeted diagnostic efforts. This would allow the identification of main sources of X. fastidiosa acquisition by P. spumarius; such plant species represent an important target for rational disease management. Here, we investigated the host plants of P. spumarius in north-western Italy, a region where X. fastidiosa is still not present but is regarded as a primary threat. We designed a new molecular diagnostic tool targeting chloroplast DNA, to characterize the gut content of single P. spumarius adults. The newly set up, nested PCR/sequencing-based identification protocol was proven to be useful for retrieving sequences from the last two different host plants used by P. spumarius, even if limited persistence of intact chloroplast DNA was reported in the spittlebug gut. We propose this protocol as a new tool for supporting research on xylem feeder biology that could be particularly useful for highly polyphagous species such as P. spumarius. Furthermore, the method could help monitor X. fastidiosa invasion, and contribute to the study of vector ecology and pathogen epidemiology.     

Xylella fastidiosa Does Not Occur in Lebanon

Xylella fastidiosa has been reported as responsible for a devastating disease on olive trees in Apulia region (south‐eastern Italy), characterized by a quick decline syndrome. In Lebanon, the pathogen was recently associated with leaf scorch symptoms on oleander, and reports on leaf scorch and dieback of olive trees branches by technicians and farmers have shown an increasing trend in the main agricultural areas. To assess the occurrence and distribution of the pathogen in Lebanon, samples of twigs from olive trees (82), olive seedlings (26), grapevine (30), oleander (32) and ornamentals imported from Italy (48) were analysed by isolation on four agarized media, serological techniques (ELISA and DTBIA) using Xylella fastidiosa‐specific antibodies and by PCR, using three specific sets of primers. Results unequivocally demonstrated that all the collected samples were free from the pathogen. As well, both detection protocols and attempts at isolating the pathogen on agarized media demonstrated that oleander samples gathered from American University campus in Beirut, where X. fastidiosa was previously reported, were not infected. Nevertheless, continuous monitoring and rigorous control measures of propagative materials are necessary to prevent the introduction of Xylella fastidiosa in Lebanon.     

Transmission of Xylella fastidiosa by naturally infected Philaenus spumarius (Hemiptera,Aphrophoridae) to different host plants

The recent establishment of Xylella fastidiosa subspecies pauca in the southern Italian region of Apulia threatens agricultural crops and the environment. Olive is an important and widespread ancient crop in Italy and, so far, the most impacted host. The meadow spittlebug Philaenus spumarius (Hemiptera, Aphrophoridae) has been identified as a vector of X. fastidiosa in southern Italy; this species is one of the most common potential vectors in Europe. To generate disease management strategies, data on X. fastidiosa transmission by P. spumarius are necessary. Therefore, we carried out transmission experiments by using field‐collected spittlebugs in 2014 and 2015 (5 and 11 collection dates, respectively), and transferring groups of insects immediately on to recipient plants. Various host plant species were tested: olive, oleander, sweet orange, grapevine and the stone fruit rootstock GF677 (Prunus persica × Prunus amygdalus). Xylella fastidiosa was detected in all the host plants after insect plant access except for grapevine; infections to sweet orange and stone fruit were not systemic. In 2015, estimates of insect X. fastidiosa infectivity were obtained; the number of PCR‐positive P. spumarius on each plant was positively correlated with the plant infection status. The proportion of P. spumarius infected with X. fastidiosa ranged from 25% to 71% during the entire survey period. The number of X. fastidiosa cells detected in P. spumarius heads ranged from 3.5 × 10 to 4.0 × 10² (CFU equivalents), which is lower than that reported for leafhopper vectors in the Americas. These data show that field‐collected P. spumarius have high rates of X. fastidiosa infection and are competent vectors.     

Modelling the spread and control of <Emphasis Type="Italic">Xylella fastidiosa</Emphasis> in the early stages of invasion in Apulia,Italy

Xylella fastidiosa is an important plant pathogen that attacks several plants of economic importance. Once restricted to the Americas, the bacterium, which causes olive quick decline syndrome, was discovered near Lecce, Italy in 2013. Since the initial outbreak, it has invaded 23,000 ha of olives in the Apulian Region, southern Italy, and is of great concern throughout Mediterranean basin. Therefore, predicting its spread and estimating the efficacy of control are of utmost importance. As data on this invasive infectious disease are poor, we have developed a spatially-explicit simulation model for X. fastidiosa to provide guidance for predicting spread in the early stages of invasion and inform management strategies. The model qualitatively and quantitatively predicts the patterns of spread. We model control zones currently employed in Apulia, showing that increasing buffer widths decrease infection risk beyond the control zone, but this may not halt the spread completely due to stochastic long-distance jumps caused by vector dispersal. Therefore, management practices should aim to reduce vector long-distance dispersal. We find optimal control scenarios that minimise control effort while reducing X. fastidiosa spread maximally—suggesting that increasing buffer zone widths should be favoured over surveillance efforts as control budgets increase. Our model highlights the importance of non-olive hosts which increase the spread rate of the disease and may lead to an order of magnitude increase in risk. Many aspects of X. fastidiosa disease invasion remain uncertain and hinder forecasting; we recommend future studies investigating quantification of the infection growth rate, and short and long distance dispersal.     

Vessel occlusion in three cultivars of Olea europaea naturally exposed to Xylella fastidiosa in open field

Xylella fastidiosa is a Gram‐negative, xylem‐limited, bacterium which is responsible, in Italy, for the olive quick decline syndrome (OQDS). The disease is caused by the subspecies pauca and emerged a few years ago in the Apulia province of Lecce, in the Salento peninsula, on Olea europaea plants. X. fastidiosa can infect different plant species and is well known in California as the causal agent of Pierce's disease on grape. Infections of susceptible hosts with X. fastidiosa are known to result in xylem vessel occlusions, water movement impairment, and accordingly to induce the typical desiccation symptoms. In this study, we investigated xylem vessel occlusions in healthy and naturally infected O. europaea plants grown in open field by analysing three olive cultivars widespread in the region that show different degree of susceptibility to the disease: the susceptible cultivars “Ogliarola salentina” and “Cellina di Nardò,” and the tolerant cultivar “Leccino.” Our results show that occlusions were caused by tyloses and gums/pectin gels, and not by bacterial cell aggregates. Our data also indicate that occlusions are not responsible for the symptomatology of the OQDS and, as observed in Leccino plants, they are not a marker of tolerance/resistance to the disease.     

Genetic Analysis of a Novel Xylella fastidiosa Subspecies Found in the Southwestern United States

Xylella fastidiosa, the causal agent of several scorch diseases, is associated with leaf scorch symptoms in Chitalpa tashkentensis, a common ornamental landscape plant used throughout the southwestern United States. For a number of years, many chitalpa trees in southern New Mexico and Arizona exhibited leaf scorch symptoms, and the results from a regional survey show that chitalpa trees from New Mexico, Arizona, and California are frequently infected with X. fastidiosa. Phylogenetic analysis of multiple loci was used to compare the X. fastidiosa infecting chitalpa strains from New Mexico, Arizona, and trees imported into New Mexico nurseries with previously reported X. fastidiosa strains. Loci analyzed included the 16S ribosome, 16S-23S ribosomal intergenic spacer region, gyrase-B, simple sequence repeat sequences, X. fastidiosa-specific sequences, and the virulence-associated protein (VapD). This analysis indicates that the X. fastidiosa isolates associated with infected chitalpa trees in the Southwest are a highly related group that is distinct from the four previously defined taxons X. fastidiosa subsp. fastidiosa (piercei), X. fastidiosa subsp. multiplex, X. fastidiosa subsp. sandyi, and X. fastidiosa subsp. pauca. Therefore, the classification proposed for this new subspecies is X. fastidiosa subsp. tashke.Xylella fastidiosa is a gram-negative bacterium that multiplies within the xylem and causes serious disease problems in many diverse plant species. X. fastidiosa is considered a “new world” pathogen and is mainly found within North, Central, and South America (30). In many native plant species this bacterium exists as an apparently benign endophyte, while in other instances proliferation of X. fastidiosa within the xylem leads to disease typified by symptoms, including leaf scorch, chlorosis, stunting, branch dieback, inedible fruit, and eventually the death of the plant (4, 15). X. fastidiosa is transmitted by xylem-feeding insect vectors such as sharpshooters, leafhoppers, and spittle bugs (35). Diseases caused by X. fastidiosa include Pierce''s disease in grapes (7), citrus variegated chlorosis (CVC) (6), coffee leaf scorch (18), pecan leaf scorch (36), phony peach (41), plum leaf scald (32), and almond leaf scorch (25). X. fastidiosa has also been shown to be the causative agent of diseases found in landscape plants such as oleander leaf scorch (31), mulberry leaf scorch (14), and oak leaf scorch (3). In addition to the examples above proven through the completion of Koch''s postulates, X. fastidiosa is known to be associated with leaf scorch type diseases in several other ornamental landscape species including crape myrtle, olive, day lily, and southern magnolia (12).Chitalpa (Chitalpa tashkentensis Elias and Wisura) is an ornamental landscape plant that was developed for arid landscapes such as California, Arizona, Texas, and New Mexico. Chitalpa, originally bred in Russia and introduced into the United States in 1977, is an intergenic hybrid between desert willow (Chilopsis linearis Cav.) and Catalpa bignonioides Walt. (28). In the past, chitalpa trees across the Southwest were observed to display leaf scorch symptoms of unknown origin. X. fastidiosa was detected in many chitalpa trees that displayed leaf scorch symptoms in southern New Mexico (34). The first known occurrence of Pierce''s disease in New Mexico was reported in 2007, and the strains of X. fastidiosa found in infected New Mexico grapes were very similar to those present in chitalpa trees from the same area (33). The common use of chitalpa as a landscape plant in the Southwest coupled with the recent discovery that it can harbor X. fastidiosa strains similar to those associated with Pierce''s disease in New Mexico prompted a survey of chitalpa trees across the Southwest. The results of this survey show that chitalpa trees from New Mexico and Arizona are frequently infected with X. fastidiosa. Chitalpa plants imported into New Mexico nurseries from California were also found to contain similar strains of X. fastidiosa. A multilocus phylogenetic analysis was performed to further characterize these strains of X. fastidiosa. This analysis revealed that the X. fastidiosa isolates infecting chitalpa plants in New Mexico, Arizona, and imported into nurseries from California are highly related to each other and are distinct from the previously described subspecies fastidiosa (38).     

Presence of Cicadomorpha in olive orchards of Greece with special reference to Xylella fastidiosa vectors

Insects of the infraorder Cicadomorpha cause serious economic impact on agriculture because of pathogens’ transmission. The vector-borne bacterium Xylella fastidiosa, which is associated with olive quick decline syndrome, is exclusively transmitted by insects of this infraorder and more specific by sharpshooters and spittlebugs. Efficient control practices of the insect vectors are necessary for the control of the disease in cases of outbreaks. Therefore, the knowledge of the vectors' species as well as the robust understanding of their biology and ecology is of crucial importance. In the present study, extensive sampling was conducted in 28 olive orchards of Greece during the years 2017 and 2018 aiming to investigate which species of this infraorder are present, with special interest to those which are considered as vectors of the bacterium. During the surveys, 4,350 Cicadomorpha insects were collected. Forty-two, five and one species of the families Cicadellidae, Aphrophoridae and Cercopidae, respectively, were identified. The results of the surveys revealed that the main potential vectors of X. fastidiosa in Greek olive orchards are the spittlebugs Philaenus spumarius and Neophilaenus campestris. Those species have wide distribution in Greece and are present for a long period during the year, and they inhabit both the ground vegetation and the olive trees' canopy. A representative portion of the captured spittlebugs was analysed with molecular methods, and the analysis confirmed the absence of X. fastidiosa in the tested individuals.     

A Multigene Phylogenetic Study of Clonal Diversity and Divergence in North American Strains of the Plant Pathogen Xylella fastidiosa

Xylella fastidiosa is a pathogen that causes leaf scorch and related diseases in over 100 plant species, including Pierce's disease in grapevines (PD), phony peach disease (PP), plum leaf scald (PLS), and leaf scorch in almond (ALS), oak (OAK), and oleander (OLS). We used a high-resolution DNA sequence approach to investigate the evolutionary relationships, geographic variation, and divergence times among the X. fastidiosa isolates causing these diseases in North America. Using a large data set of 10 coding loci and 26 isolates, the phylogeny of X. fastidiosa defined three major clades. Two of these clades correspond to the recently identified X. fastidiosa subspecies piercei (PD and some ALS isolates) and X. fastidiosa subsp. multiplex (OAK, PP, PLS, and some ALS isolates). The third clade grouped all of the OLS isolates into a genetically distinct group, named X. fastidiosa subsp. sandyi. These well-differentiated clades indicate that, historically, X. fastidiosa has been a clonal organism. Based on their synonymous-site divergence (~3%), these three clades probably originated more than 15,000 years ago, long before the introduction of the nonnative plants that characterize most infections. The sister clades of X. fastidiosa subsp. sandyi and X. fastidiosa subsp. piercei have synonymous-site evolutionary rates 2.9 times faster than X. fastidiosa subsp. multiplex, possibly due to generation time differences. Within X. fastidiosa subsp. multiplex, a low level (~0.1%) of genetic differentiation indicates the recent divergence of ALS isolates from the PP, PLS, and OAK isolates due to host plant adaptation and/or allopatry. The low level of variation within the X. fastidiosa subsp. piercei and X. fastidiosa subsp. sandyi clades, despite their antiquity, suggests strong selection, possibly driven by host plant adaptation.     

Multilocus Simple Sequence Repeat Markers for Differentiating Strains and Evaluating Genetic Diversity of Xylella fastidiosa

A genome-wide search was performed to identify simple sequence repeat (SSR) loci among the available sequence databases from four strains of Xylella fastidiosa (strains causing Pierce's disease, citrus variegated chlorosis, almond leaf scorch, and oleander leaf scorch). Thirty-four SSR loci were selected for SSR primer design and were validated in PCR experiments. These multilocus SSR primers, distributed across the X. fastidiosa genome, clearly differentiated and clustered X. fastidiosa strains collected from grape, almond, citrus, and oleander. They are well suited for differentiating strains and studying X. fastidiosa epidemiology and population genetics.     

Specific Detection and Identification of <Emphasis Type="Italic">Xylella fastidiosa</Emphasis> Strains Causing Oleander Leaf Scorch Using Polymerase Chain Reaction

A pair of PCR primers, QH-OLS05/QH-OLS08 specific for strains of Xylella fastidiosa causing oleander leaf scorch was developed. The primers were designed according to a DNA sequence of a randomly amplified polymorphic DNA (RAPD)-polymerase chain reaction (PCR) product unique to oleander strains. The PCR assay using primer pair QH-OLS05/QH-OLS08 allowed quick and simple detection and identification of oleander strains in cultured bacterium and infected plant samples. The assay also can be applied to insect samples. Specific detection and identification of oleander strains of X. fastidiosa by PCR is useful for epidemiologic and etiologic studies of oleander leaf scorch by identifying what plants and insect vectors harbor or carry this particular strain of X. fastidiosa, especially in locations where mixed natural infections by oleander and other strains of X. fastidiosa occur.     

Two different Xylella fastidiosa strains circulating in Italy: phylogenetic and evolutionary analyses

Xylella fastidiosa, a bacterial species infecting a broad range plants, includes five subspecies, fastidiosa, multiplex, pauca, mulberry and sandyi. In Europe, Xylella was isolated in olive trees in southern Italy (Apulia region) during the year 2013. The aim of the present study was to apply phylogenetic and evolutionary analysis to trace the possible origin and way of the entrance of Xylella fastidiosa in Italy. All the genomes available for Xylella fastidiosa spp were downloaded from NCBI. A phylogeographic analysis was performed using BEAST. X. fastidiosa strains belonging to X. fastidiosa subsp. pauca and subsp. sandyi have been reported to infect olive trees and coffee plants, respectively. The phylogeographic analysis also revealed and confirmed these two different ways of provenience X. fastidiosa subsp. pauca from Costa Rica and X. fastidiosa subsp sandyi from California Phylogeny have been an important tool to validate and support the recent hypothesis for X. fastidiosa pauca provenience.     

Specific Detection and Identification of American Mulberry-Infecting and Italian Olive-Associated Strains of Xylella fastidiosa by Polymerase Chain Reaction

Xylella fastidiosa causes bacterial leaf scorch in many landscape trees including elm, oak, sycamore and mulberry, but methods for specific identification of a particular tree host species-limited strain or differentiation of tree-specific strains are lacking. It is also unknown whether a particular landscape tree-infecting X. fastidiosa strain is capable of infecting multiple landscape tree species in an urban environment. We developed two PCR primers specific for mulberry-infecting strains of X. fastidiosa based on the nucleotide sequence of a unique open reading frame identified only in mulberry-infecting strains among all the North and South American strains of X. fastidiosa sequenced to date. PCR using the primers allowed for detection and identification of mulberry-infecting X. fastidiosa strains in cultures and in samples collected from naturally infected mulberry trees. In addition, no mixed infections with or non-specific detections of the mulberry-infecting strains of X. fastidiosa were found in naturally X. fastidiosa-infected oak, elm and sycamore trees growing in the same region where naturally infected mulberry trees were grown. This genotype-specific PCR assay will be valuable for disease diagnosis, studies of strain-specific infections in insects and plant hosts, and management of diseases caused by X. fastidiosa. Unexpectedly but interestingly, the unique open reading frame conserved in the mulberry-infecting strains in the U. S. was also identified in the recently sequenced olive-associated strain CoDiRO isolated in Italy. When the primer set was tested against naturally infected olive plant samples collected in Italy, it allowed for detection of olive-associated strains of X. fastidiosa in Italy. This PCR assay, therefore, will also be useful for detection and identification of the Italian group of X. fastidiosa strains to aid understanding of the occurrence, evolution and biology of this new group of X. fastidiosa strains.     

An Evolutionary Perspective of Pierce's Disease of Grapevine,Citrus Variegated Chlorosis,and Mulberry Leaf Scorch Diseases

Xylella fastidiosa causes diseases on a growing list of economically important plants. An understanding of how xylellae diseases originated and evolved is important for disease prevention and management. In this study, we evaluated the phylogenetic relationships of X. fastidiosa strains from citrus, grapevine, and mulberry through the analyses of random amplified polymorphic DNAs (RAPDs) and conserved 16S rDNA genes. RAPD analysis emphasized the vigorous genome-wide divergence of X. fastidiosa and detected three clonal groups of strains that cause Pierce's disease (PD) of grapevine, citrus variegated chlorosis (CVC), and mulberry leaf scorch (MLS). Analysis of 16S rDNA sequences also identified the PD and CVC groups, but with a less stable evolutionary tree. MLS strains were included in the PD group by the 16S rDNA analysis. The Asiatic origins of the major commercial grape and citrus cultivars suggest the recent evolution of both PD and CVC disease in North and South America, respectively, since X. fastidiosa is a New World organism. In order to prevent the development of new diseases caused by X. fastidiosa, it is important to understand the diversity of X. fastidiosa strains, how strains of X. fastidiosa select their hosts, and their ecological roles in the native vegetation. Received: 7 February 2002 / Accepted: 7 March 2002     

Large-Scale Intersubspecific Recombination in the Plant-Pathogenic Bacterium Xylella fastidiosa Is Associated with the Host Shift to Mulberry

Homologous recombination plays an important role in the structuring of genetic variation of many bacteria; however, its importance in adaptive evolution is not well established. We investigated the association of intersubspecific homologous recombination (IHR) with the shift to a novel host (mulberry) by the plant-pathogenic bacterium Xylella fastidiosa. Mulberry leaf scorch was identified about 25 years ago in native red mulberry in the eastern United States and has spread to introduced white mulberry in California. Comparing a sequence of 8 genes (4,706 bp) from 21 mulberry-type isolates to published data (352 isolates representing all subspecies), we confirmed previous indications that the mulberry isolates define a group distinct from the 4 subspecies, and we propose naming the taxon X. fastidiosa subsp. morus. The ancestry of its gene sequences was mixed, with 4 derived from X. fastidiosa subsp. fastidiosa (introduced from Central America), 3 from X. fastidiosa subsp. multiplex (considered native to the United States), and 1 chimeric, demonstrating that this group originated by large-scale IHR. The very low within-type genetic variation (0.08% site polymorphism), plus the apparent inability of native X. fastidiosa subsp. multiplex to infect mulberry, suggests that this host shift was achieved after strong selection acted on genetic variants created by IHR. Sequence data indicate that a single ancestral IHR event gave rise not only to X. fastidiosa subsp. morus but also to the X. fastidiosa subsp. multiplex recombinant group which infects several hosts but is the only type naturally infecting blueberry, thus implicating this IHR in the invasion of at least two novel native hosts, mulberry and blueberry.     

Artificial diet delivery system for Philaenus spumarius,the European vector of Xylella fastidiosa

Artificial diets represent an essential tool for investigations on the intimate relationship between plant pathogens and their vectors. Previous research failed in devising an artificial diet delivery system for the meadow spittlebug Philaenus spumarius, to date considered the most important vector of the bacterium Xylella fastidiosa in Europe. Here, we describe a new delivery “tube system” by which we succeeded in artificial feeding of P. spumarius with holidic diets (one sucrose diet and two amino acids diets). Spittlebug probing and feeding behaviour on either the tube system or a traditional “flat system” realized out of a small Petri dish filled with diet and covered with stretched Parafilm^® were observed in real time by video‐EPG (Electrical Penetration Graph), in order to assess the occurrence of ingestion and excretion. Moreover, we evaluated P. spumarius survival on either the tube system filled with the two holidic diets that gave the best EPG results or an empty tube system serving as control. Contrary to the flat system, where just brief stylet insertions through the Parafilm^® were recorded, the spittlebug ingested the artificial diets when delivered with the tube system. Survival on the diets provided with the tube system was significantly greater than the control, with no differences between the diets tested. Furthermore, the tube system was suitable also for another spittlebug species shown to be a competent vector of X. fastidiosa, that is Neophilaenus campestris. The tool we devised opens new perspectives for investigations on X. fastidiosa/spittlebugs interactions, as well as for the functional analysis of mutant X. fastidiosa strains in respect to insect colonization and transmission.     

The Complex Biogeography of the Plant Pathogen Xylella fastidiosa: Genetic Evidence of Introductions and Subspecific Introgression in Central America

The bacterium Xylella fastidiosa is a plant pathogen with a history of economically damaging introductions of subspecies to regions where its other subspecies are native. Genetic evidence is presented demonstrating the introduction of two new taxa into Central America and their introgression into the native subspecies, X. fastidiosa subsp. fastidiosa. The data are from 10 genetic outliers detected by multilocus sequence typing (MLST) of isolates from Costa Rica. Six (five from oleander, one from coffee) defined a new sequence type (ST53) that carried alleles at six of the eight loci sequenced (five of the seven MLST loci) diagnostic of the South American subspecies Xylella fastidiosa subsp. pauca which causes two economically damaging plant diseases, citrus variegated chlorosis and coffee leaf scorch. The two remaining loci of ST53 carried alleles from what appears to be a new South American form of X. fastidiosa. Four isolates, classified as X. fastidiosa subsp. fastidiosa, showed a low level of introgression of non-native DNA. One grapevine isolate showed introgression of an allele from X. fastidiosa subsp. pauca while the other three (from citrus and coffee) showed introgression of an allele with similar ancestry to the alleles of unknown origin in ST53. The presence of X. fastidiosa subsp. pauca in Central America is troubling given its disease potential, and establishes another route for the introduction of this economically damaging subspecies into the US or elsewhere, a threat potentially compounded by the presence of a previously unknown form of X. fastidiosa.     

DC-EPG assisted comparison of European spittlebugs and sharpshooters feeding behaviour on grapevine

Xylem-feeding is apparently the only requirement making an insect a competent vector of the bacterium Xylella fastidiosa, an organism responsible for the devastation of the Southern Italian olive forest and nowadays considered one of the most feared threats to agriculture and landscape in Europe, including vineyards. Here, we used the direct current-electrical penetration graph (DC-EPG) technique to compare and describe the feeding behaviour on grapevine of four xylem-feeding species considered candidate vectors of X. fastidiosa widespread in Europe, namely two spittlebugs (the meadow spittlebug Philaenus spumarius and the spittlebug Neophilaenus campestris) and two sharpshooter leafhoppers (the rhododendron leafhopper Graphocephala fennahi and the green leafhopper Cicadella viridis). We created a standard for the analysis of EPG waveforms recorded with a DC-EPG device, describing feeding activities performed by these insects from stylet insertion into the plant to withdrawal. This standard, along with freely available software, has been developed to harmonize the calculation of feeding behavioural parameters in xylem-feeders. The most relevant differences between the two vector taxa were the probing frequency and the dynamics of xylem ingestion. Sharpshooters tended to perform significantly more probes than spittlebugs. In contrast, the latter spent longer times in low-frequency xylem ingestion, characterized by scattered contractions of the cibarial dilator muscle interspersed with periods of pump inactivity. Cicadella viridis was the species displaying the highest frequency of the electrical pattern found to be associated with X. fastidiosa inoculation in spittlebugs (Xe). Feeding behavioural data presented here represent an important step forward for deepening our knowledge of xylem-sap feeding insects' interaction with both the host plants and the bacterium they transmit.     

Photosynthetic Responses of a Temperate Liana to Xylella fastidiosa Infection and Water Stress

Xylella fastidiosa is a xylem‐limited bacterial plant pathogen that causes bacterial leaf scorch in its hosts. Our previous work showed that water stress enhances leaf scorch symptom severity and progression along the stem of a liana, Parthenocissus quinquefolia, infected by X. fastidiosa. This paper explores the photosynthetic gas exchange responses of P. quinquefolia, with the aim to elucidate mechanisms behind disease expression and its interaction with water stress. We used a 2 × 2‐complete factorial design, repeated over two growing seasons, with high and low soil moisture levels and infected and non‐infected plants. In both years, low soil moisture levels reduced leaf water potentials, net photosynthesis and stomatal conductance at all leaf positions, while X. fastidiosa‐infection reduced these parameters at basally located leaves only. Intercellular CO₂ concentrations were reduced in apical leaves, but increased at the most basal leaf location, implicating a non‐stomatal reduction of photosynthesis in leaves showing the greatest disease development. This result was supported by measured reductions in photosynthetic rates of basal leaves at high CO₂ concentrations, where stomatal limitation was eliminated. Repeated measurements over the summer of 2000 showed that the effects of water stress and infection were progressive over time, reaching their greatest extent in September. By reducing stomatal conductances at moderate levels of water stress, P. quinquefolia maintained relatively high leaf water potentials and delayed the onset of photosynthetic damage due to pathogen and drought‐induced water stress. In addition, chlorophyll fluorescence measurements showed that P. quinquefolia has an efficient means of dissipating excess light energy that protects the photosynthetic machinery of leaves from irreversible photoinhibitory damage that may occur during stress‐induced stomatal limitation of photosynthesis. However, severe stress induced by disease and drought eventually led to non‐stomatal decreases in photosynthesis associated with leaf senescence.     

Anterior Foregut Microbiota of the Glassy-Winged Sharpshooter Explored Using Deep 16S rRNA Gene Sequencing from Individual Insects

The glassy-winged sharpshooter (GWSS) is an invasive insect species that transmits Xylella fastidiosa, the bacterium causing Pierce''s disease of grapevine and other leaf scorch diseases. X. fastidiosa has been shown to colonize the anterior foregut (cibarium and precibarium) of sharpshooters, where it may interact with other naturally-occurring bacterial species. To evaluate such interactions, a comprehensive list of bacterial species associated with the sharpshooter cibarium and precibarium is needed. Here, a survey of microbiota associated with the GWSS anterior foregut was conducted. Ninety-six individual GWSS, 24 from each of 4 locations (Bakersfield, CA; Ojai, CA; Quincy, FL; and a laboratory colony), were characterized for bacteria in dissected sharpshooter cibaria and precibaria by amplification and sequencing of a portion of the 16S rRNA gene using Illumina MiSeq technology. An average of approximately 150,000 sequence reads were obtained per insect. The most common genus detected was Wolbachia; sequencing of the Wolbachia ftsZ gene placed this strain in supergroup B, one of two Wolbachia supergroups most commonly associated with arthropods. X. fastidiosa was detected in all 96 individuals examined. By multilocus sequence typing, both X. fastidiosa subspecies fastidiosa and subspecies sandyi were present in GWSS from California and the colony; only subspecies fastidiosa was detected in GWSS from Florida. In addition to Wolbachia and X. fastidiosa, 23 other bacterial genera were detected at or above an average incidence of 0.1%; these included plant-associated microbes (Methylobacterium, Sphingomonas, Agrobacterium, and Ralstonia) and soil- or water-associated microbes (Anoxybacillus, Novosphingobium, Caulobacter, and Luteimonas). Sequences belonging to species of the family Enterobacteriaceae also were detected but it was not possible to assign these to individual genera. Many of these species likely interact with X. fastidiosa in the cibarium and precibarium.     

Population Structure of the Bacterial Pathogen Xylella fastidiosa among Street Trees in Washington D.C.

Bacterial leaf scorch, associated with the bacterial pathogen Xylella fastidiosa, is a widely established and problematic disease of landscape ornamentals in Washington D.C. A multi-locus sequence typing analysis was performed using 10 housekeeping loci for X. fastidiosa strains in order to better understand the epidemiology of leaf scorch disease in this municipal environment. Samples were collected from 7 different tree species located throughout the District of Columbia, consisting of 101 samples of symptomatic and asymptomatic foliage from 84 different trees. Five strains of the bacteria were identified. Consistent with prior data, these strains were host specific, with only one strain associated with members of the red oak family, one strain associated with American elm, one strain associated with American sycamore, and two strains associated with mulberry. Strains found for asymptomatic foliage were the same as strains from the symptomatic foliage on individual trees. Cross transmission of the strains was not observed at sites with multiple species of infected trees within an approx. 25 m radius of one another. X. fastidiosa strain specificity observed for each genus of tree suggests a highly specialized host-pathogen relationship.