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.     

Natural infectivity of Xylella fastidiosa Wells et al. in sharpshooters (Hemiptera: Cicadellidae) from coffee plantations of Parana, Brazil

Xylella fastidiosa Wells et al., a gram-negative and xylem limited bacterium, causes significative economic on several crops, such as the leaf scorch in coffee. It is transmitted by xylem feeding insects and four sharpshooters species have been reported as vectors of X. fastidiosa in coffee. The objective of this study was to determine the natural infectivity of X. fastidiosa in five species of sharpshooters from coffee trees: Acrogonia citrina Marucci & Cavichioli, Bucephalogonia xanthophis (Berg), Dilobopterus costalimai Young, Oncometopia facialis (Signoret) and Sonesimia grossa (Signoret). Samples were collected from coffee plantations in five counties of the North and Northwest regions of the State of Parana, Brazil, from October 1998 through November 2001. A total of 806 samples containing three to five insects were examined for the presence of X. fastidiosa by using PCR and nested PCR tests. X. fastidiosa was present in samples of all five species of sharpshooters collected in the two coffee regions. The average level of natural infectivity potential was 30.4%. However, this natural infectivity ranged from 2.2% for O. facialis to 68.8% for A. citrina. Sharpshooters collected in the spring tended to have lower natural infectivity of X. fastidiosa as compared to those collected in other seasons. The results obtained showed the high potential of dissemination of X. fastidiosa by different insect vectors in coffee trees in Parana.     

Detection and differentiation of Xylella fastidiosa strains acquired and retained by glassy-winged sharpshooters (Hemiptera: Cicadellidae) using a mixture of strain-specific primer sets

Xylella fastidiosa Wells is a bacterial pathogen that causes a variety of plant diseases, including Pierce's disease (PD) of grapevine, almond leaf scorch, alfalfa dwarf, citrus variegated chlorosis, and oleander leaf scorch (OLS). Numerous strains of this pathogen have been genetically characterized, and several different strains occur in the United States. The dominant vector in southern California is the glassy-winged sharpshooter, Homalodisca coagulata (Say) (Hemiptera: Cicadellidae). The high mobility of this insect, and its use of large numbers of host plant species, provides this vector with ample exposure to multiple strains of X. fastidiosa during its lifetime. To learn more about the ability of this vector to acquire, retain, and transmit multiple strains of the pathogen, we developed a polymerase chain reaction (PCR)-based method to detect and differentiate strains of X. fastidiosa present in individual glassy-winged sharpshooter adults. Insects were sequentially exposed to plants infected with a PD strain in grapevine and an OLS strain in oleander. After sequential exposure, a few insects tested positive for both strains (7%); however, in most cases individuals tested positive for only one strain (29% PD, 41% OLS). In transmission studies, individual adults transmitted either the PD or OLS strain of the pathogen at a rate (39%) similar to that previously reported after exposure to a single strain, but no single individual transmitted both strains of the pathogen. PD and OLS strains of X. fastidiosa remained detectable in glassy-winged sharpshooter, even when insects were fed on a plant species that was not a host of the strain for 1 wk.     

Seasonal population dynamics of Draeculacephala minerva (Hemiptera: Cicadellidae) and transmission of Xylella fastidiosa

The grass sharpshooter, Draeculacephala minerva Ball (Hemiptera: Cicadellidae), is a very common and often abundant grass-feeding leafhopper in California. Its population dynamics and ability to transmit Xylella fastidiosa were monitored over a 2-yr period in California's San Joaquin Valley. Collections of individuals from natural populations in irrigated pastures and alfalfa, Medicago savita L. fields adjacent to X. fastidiosa-infected almond (Prunus spp.) orchards indicated the occurrence of three discrete generations per year that peaked during the summer. Population densities varied significantly among experimental field survey sites. Insects captured on intercepting mesh traps, yellow sticky cards, and UV-light traps indicated local movement of these insects into and surrounding X. fastidiosa-infected, almond orchards. Local movement and seasonal transmission of X. fastidiosa from infected almonds to Catharanthus roseus (L.) G. Don indicated that this insect may be partly responsible for the slow spread of almond leaf scorch now recently observed in California's San Joaquin Valley.     

Impact of duration versus frequency of probing by Homalodisca vitripennis (Hemiptera: Cicadellidae) on inoculation of Xylella fastidiosa

Successful infection of the plant pathogenic bacterium Xylella fastidiosa (Wells) from an infected plant to a new host involves three main steps: 1) acquisition of the bacterium by a vector; 2) inoculation of a noninfected host plant by the vector; and 3) establishment of sufficient titers of X. fastidiosa in the host plant to sustain a chronic infection. Understanding the basic biology of the transmission process is a key to limiting the spread of plant diseases induced by X. fasdidiosa and reducing agricultural losses, especially those experienced in California since the introduction of a new vector, Homalodisca vitripennis (Germar) (Hemiptera, Cicadellidae) (formerly H. coagulata Say), the glassy-winged sharpshooter. In this study, H. vitripennis adults that acquired X. fastidiosa were allowed access to chrysanthemum plant cuttings for 30, 60, 90, or 120 min. The numbers of X. fastidiosa acquired (i.e., cells present in the insect foregut) and the number inoculated to the plant cuttings were separately determined using quantitative real-time polymerase chain reaction (PCR). In addition, the number of times glassy-winged sharpshooter stylets probed plant cuttings and the amount of time glassy-winged sharpshooter spent actively ingesting were monitored using video surveillance. Linear regression did not indicate a relationship between the number of X. fastidiosa cells inoculated into the plant cutting and either the titer of pathogen present in the insect or amount of time spent ingesting per probe. However, the number of probes significantly influenced the number of X. fastidiosa cells inoculated. Due to the highly variable nature of transmission, our model could not account for all observed variation as indicated by low R2 values. However, our results suggest that the mechanism of transmission is dependent on probing behaviors more than ingestion duration.     

Phylogeny and biogeography of the sharpshooters (Hemiptera: Cicadellidae: Cicadellinae)

Sharpshooters (Cicadellinae), a large subfamily of the Cicadellidae, exhibit a global distribution and a broad array of ecological preferences. To explore the phylogenetic relationships and roles of global historical, biotic and biogeographic processes in the diversification of sharpshooters, we analysed DNA sequence data from three mitochondrial and two nuclear genes for 243 taxa representing all Cicadellinae tribes, generic groups, regional faunas and data of geographic distributions of sharpshooter species compiled from online databases and available literature. The maximum likelihood (ML) and Bayesian inference (BI) analyses strongly support the monophyletic clade including Cicadellinae and Phereurininae. Divergence time estimates and biogeographic analyses suggest that sharpshooters originated in the Neotropical region or were more widespread in Gondwana during the Early Cretaceous and diversified through a combination of ancient vicariance and dispersal following the evolution of angiosperm-dominated habitats. The earliest divergence during the Cretaceous gave rise to Oriental and New World lineages, the latter of which subsequently dispersed into the Old World and gave rise to the diverse endemic fauna of Madagascar. The Oriental lineage shows high diversity and endemism in tropical Asia and the Pacific, with striking distributional discontinuities in Wallacea. These results suggest that a combination of environmental and evolutionary factors including continental-scale vicariance, long-distance dispersal and diversification of terrestrial microhabitats and host plants may explain the diversity of the modern sharpshooter fauna.     

Hemipteran diversity (Cicadellidae and Clastopteridae) in three coffee production zones affected by Xylella fastidiosa (Wells et al.) in Costa Rica

A survey was conducted during 2002, 2003 and 2004 to determine the leafhopper species composition, abundance, richness, diversity, evenness, occurrence and flight activity among three coffee production zones of Costa Rica. Yellow sticky traps were used to qualify and quantify the number of aerial leafhoppers during the sampling period. A total of 82,500 individuals, belonging to 139 species within nine leafhopper subfamilies, were trapped. San Isidro de León Cortés site presented the highest diversity from the three surveyed sites. Twenty five species were frequently trapped at least in one of the studied zones, and only Coelidiana sp.1, Osbornellus sp.1, Scaphytopius sp.1 and Empoasca sp. were trapped throughout the sampling period. The flight activity of the taxa that contain the main vectors of Xylella fastidiosa Wells et al. showed differences among the sampling zones.     

Dispersion of Homalodisca coagulata (Hemiptera: Cicadellidae), a vector of Xylella fastidiosa, into vineyards in southern California

Recent epidemics of Pierce's disease of grapevine in California vectored by Homalodisca coagulata (Say), an invasive vector species, have characteristics that differ from epidemics involving native vectors. Among these differences are the longer distances and greater speed that the disease is spread by H. coagulata. In this investigation, we used yellow sticky traps to study the seasonal dispersion activity of H. coagulata in a southern California grape-growing area in which an epidemic of Pierce's disease has caused large losses. For 21 mo, we monitored adult H. coagulata at the edges of vineyards bordering citrus, an important crop host, natural coastal sage scrub vegetation, and natural riparian vegetation. We also monitored H. coagulata dispersion from 0 to 40 m into vineyards. Finally, we examined the vertical dispersion of H. coagulata adults into grapevines through a season. This investigation showed that H. coagulata is associated with citrus, from where it disperses deep into vineyards, and not just the vineyard edge as with Pierce's disease vectors that are native to California. Peak dispersion into vineyards occurred in the summer. Another period of H. coagulata activity occurred in the winter in vineyards bordering citrus. Through the period of peak flight activity, 97% of all H. coagulata adults trapped between 1 and 7 m were caught at an altitude of 5 m or lower, suggesting the potential of a barrier as a management tactic to keep H. coagulata out of vineyards.     

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.     

Genetic structure and biology of Xylella fastidiosa strains causing disease in citrus and coffee in Brazil

Xylella fastidiosa is a vector-borne, plant-pathogenic bacterium that causes disease in citrus (citrus variegated chlorosis [CVC]) and coffee (coffee leaf scorch [CLS]) plants in Brazil. CVC and CLS occur sympatrically and share leafhopper vectors; thus, determining whether X. fastidiosa isolates can be dispersed from one crop to another and cause disease is of epidemiological importance. We sought to clarify the genetic and biological relationships between CVC- and CLS-causing X. fastidiosa isolates. We used cross-inoculation bioassays and microsatellite and multilocus sequence typing (MLST) approaches to determine the host range and genetic structure of 26 CVC and 20 CLS isolates collected from different regions in Brazil. Our results show that citrus and coffee X. fastidiosa isolates are biologically distinct. Cross-inoculation tests showed that isolates causing CVC and CLS in the field were able to colonize citrus and coffee plants, respectively, but not the other host, indicating biological isolation between the strains. The microsatellite analysis separated most X. fastidiosa populations tested on the basis of the host plant from which they were isolated. However, recombination among isolates was detected and a lack of congruency among phylogenetic trees was observed for the loci used in the MLST scheme. Altogether, our study indicates that CVC and CLS are caused by two biologically distinct strains of X. fastidiosa that have diverged but are genetically homogenized by frequent recombination.     

Faunistic analysis of sharpshooters (Hemiptera: Auchenorrhyncha, Cicadellidae) in a 'Westin' sweet orange orchard

The purpose of this study was to analyze sharpshooter fauna in a five-year-old 'Westin' sweet orange orchard. Yellow sticky traps were placed on the edge of a forest, and on the periphery and inside the citrus stand. The traps were evaluated fortnightly, for three years. The most frequent species were Acrogonia citrina Marucci & Cavichioli, Bucephalogonia xanthophis (Berg), and Oncometopia facialis (Signoret). B. xanthophis occurred more in the forest edge, especially on spring and winter. A. citrina occurred most frequently in the forest edge, especially on spring. The highest incidence of O. facialis was inside the citrus stand, on spring and summer. Other cicadellids occurred more often in the forest edge, especially on summer. A. citrina, B. xanthophis, Dilobopterus costalimai Young, and O. facialis were predominant in all places studied. A. citrina, B. xanthophis and O. facialis were super dominant, super abundant, super frequent, and constant, except inside the stand, where B. xanthophis was dominant, very abundant, very frequent, and constant. D. costalimai and Homalodisca ignorata Melichar were dominant, very abundant, and very frequent in the forest edge and in the periphery of the stand, and D. costalimai was also predominant inside the stand. Scopogonalia subolivacea (St?l) was predominant in the forest edge and inside the stand, while Plesiommata corniculata Young was predominant in the periphery (both were dominant, very abundant, very frequent, and accessory).     

Spatial distribution of Oncometopiafacialis (Signoret) (Hemiptera: Cicadellidae) in citrus orchard

The spatial distribution of Oncometopia facialis (Signoret) was studied in sweet orange to elaborate sampling plans and decision-making procedure for the control of this sharpshooter in the field. The samplings were carried out fortnightly in a grid design disposition of 100 points (plants) in citrus orchard, with yellow sticky traps. The average number of O. facialis ranged from 0.35 to 1.17 insects/trap, with the I index varying from 0.89 to 1.82. The fit tests to negative binomial, Poisson distribution, I index and Morisita index indicated aggregated distribution of this insect. The b result of Taylor's power law was 1.6382 (t = 2.71; d.f. = 11; P < 0.05). Sampling plans were elaborated with a precision level of 10, 20 and 25% of error in the mean estimation.     

Gene disruption by homologous recombination in the Xylella fastidiosa citrus variegated chlorosis strain

Mutagenesis by homologous recombination was evaluated in Xylella fastidiosa by using the bga gene, coding for beta-galactosidase, as a model. Integration of replicative plasmids by homologous recombination between the cloned truncated copy of bga and the endogenous gene was produced by one or two crossover events leading to beta-galactosidase mutants. A promoterless chloramphenicol acetyltransferase gene was used to monitor the expression of the target gene and to select a cvaB mutant.     

Seasonal flight activity of two Homalodisca species (Homoptera: Cicadellidae) that spread Xylella fastidiosa in southern California.

Homalodisca coagulata (Say) and Homalodisca lacerta (Fowler) are vectors of a new bacterial disease of oleander in California known as oleander leaf scorch, induced by the bacterium Xylella fastidiosa. H. coagulata also has been implicated in the spread of the strain of X. fastidiosa that induces Pierce's disease of grapevines in California. We monitored the flight activity of H. coagulata and H. lacerta in oleander and citrus by using yellow sticky cards at three southern California locations where outbreaks of oleander leaf scorch have been documented, and where vector compliments are different. Areas sampled included a mesic coastal area (Irvine, CA) that supports predominantly H. coagulata and few H. lacerta, a dry inland location (Palm Desert, CA) that supports predominantly H. lacerta and few H. coagulata, and an intermediate area (Riverside, CA) supporting both Homalodisca species. From November 1996 to October 1999 peak catches of both Homalodisca species occurred during the midsummer at all locations. H. coagulata was trapped in greater numbers in citrus than in oleander at both the Riverside and the Irvine sites. Likewise, H. lacerta in Riverside was more associated with citrus than oleander, yet H. lacerta in Palm Desert was trapped in greater numbers in oleander than citrus.     

Influence of Xylella fastidiosa infection of citrus on host selection by leafhopper vectors

Infection of plants by pathogens can influence their attractiveness and suitability to insect vectors and other herbivores. Here we examined the effects of Citrus sinensis (L.) Osbeck (Rutaceae) infection by the bacterium Xylella fastidiosa, which causes citrus variegated chlorosis (CVC), on the feeding preferences of two sharpshooter vectors, Dilobopterus costalimai Young and Oncometopia facialis (Signoret) (Homoptera: Cicadellidae). Experiments were performed inside observation chambers, in which a healthy plant and an infected one (with or without CVC symptoms) were supplied to a group of 40 sharpshooters. The number of insects that selected each treatment was recorded at several time intervals in 48 h. In another experiment, the ingestion rate on healthy and infected (symptomatic or not) plants was evaluated by measuring the liquid excretion of sharpshooters that were confined on branches of each plant for 72 h. Both sharpshooter species preferred healthy plants to those with CVC symptoms. However, O. facialis did not discriminate between healthy citrus and symptomless infected plants. Feeding by D. costalimai was markedly reduced when confined on CVC‐symptomatic plants, but not on asymptomatic infected ones. The ingestion rate by O. facialis was not affected by the presence of CVC symptoms. The results suggest that citrus trees with early (asymptomatic) infections by X. fastidiosa may be more effective as inoculum sources for CVC spread by insect vectors than those with advanced symptoms.     

Correlations of cibarial muscle activities of Homalodisca spp. sharpshooters (Hemiptera: Cicadellidae) with EPG ingestion waveform and excretion

Fluid flow into and out of the stylets of xylem-ingesting sharpshooters (Hemiptera: Cicadellidae: Cicadellinae) is powered by muscles of the cibarial pump. Such fluid flow is crucial for transmission of Xylella fastidiosa, the Pierce’s Disease bacterium, yet has not been rigorously studied via electrical penetration graph (EPG) technology. We correlated EPG waveforms with electromyographically (EMG) recorded muscle potentials from the cibarial dilator muscles, which power the piston-like cibarial diaphragm. There was a 1:1 correspondence of each cycle of cibarial muscle contraction/relaxation with each plateau of EPG waveform C. Results definitively showed that the C waveform represents active ingestion, i.e. fluid flow is propelled by cibarial muscle contraction. Moreover, each C waveform episode represents muscular diaphragm uplift, probably combined with a “bounce” from cuticular elasticity, to provide the suction that pulls fluid into the stylets. Fine structure of the EPG ingestion waveform represents directionality of fluid flow, supporting the primary role of streaming potentials as the electrical origin of the C waveform. Rhythmic bouts of cibarial pumping were generally correlated with sustained production of excretory droplets. However, neither the onset nor cessation of ingestion was correlated with onset or cessation of excretion, respectively. Volume of excreta is an inexact measure of ingestion. Implications for using EPG to understand the mechanism of X. fastidiosa transmission are discussed.     

Diversity of endophytic bacterial populations and their interaction with Xylella fastidiosa in citrus plants

Citrus variegated chlorosis (CVC) is caused by Xylella fastidiosa, a phytopathogenic bacterium that can infect all Citrus sinensis cultivars. The endophytic bacterial communities of healthy, resistant, and CVC-affected citrus plants were studied by using cultivation as well as cultivation-independent techniques. The endophytic communities were assessed in surface-disinfected citrus branches by plating and denaturing gradient gel electrophoresis (DGGE). Dominant isolates were characterized by fatty-acid methyl ester analysis as Bacillus pumilus, Curtobacterium flaccumfaciens, Enterobacter cloacae, Methylobacterium spp. (including Methylobacterium extorquens, M. fujisawaense, M. mesophilicum, M. radiotolerans, and M. zatmanii), Nocardia sp., Pantoea agglomerans, and Xanthomonas campestris. We observed a relationship between CVC symptoms and the frequency of isolation of species of Methylobacterium, the genus that we most frequently isolated from symptomatic plants. In contrast, we isolated C. flaccumfaciens significantly more frequently from asymptomatic plants than from those with symptoms of CVC while P. agglomerans was frequently isolated from tangerine (Citrus reticulata) and sweet-orange (C. sinensis) plants, irrespective of whether the plants were symptomatic or asymptomatic or showed symptoms of CVC. DGGE analysis of 16S rRNA gene fragments amplified from total plant DNA resulted in several bands that matched those from the bacterial isolates, indicating that DGGE profiles can be used to detect some endophytic bacteria of citrus plants. However, some bands had no match with any isolate, suggesting the occurrence of other, nonculturable or as yet uncultured, endophytic bacteria. A specific band with a high G+C ratio was observed only in asymptomatic plants. The higher frequency of C. flaccumfaciens in asymptomatic plants suggests a role for this organism in the resistance of plants to CVC.     

Phenology and demography of Homalodisca coagulata (Hemiptera: Cicadellidae) in southern California citrus and implications for management

Populations of Homalodisca coagulata (Say) were sampled from citrus orchards in southern California, USA to characterize and quantify seasonal occurrences of nymphs and adults with the goal of identifying management opportunities through well-timed treatments and/or natural enemy releases. Higher densities of H. coagulata in 2001 contributed to a complete seasonal profile that began in early spring with the emergence of first instar nymphs and their progression through five nymphal instars lasting until mid-August. Adult emergence began in mid-June with peak adult densities attained from mid to late August followed by a gradual decline through autumn. A persistent and significant male bias was observed in the adult sex ratio from the time of first emergence through mid-October in oranges; the same trend was present in lemons, but with more variability. Adult densities gradually declined through the winter months into the following spring before rapidly increasing again in June as the 2002 spring generation of nymphs began emerging as adults. The seasonal timing of nymphs and adults in 2002 was nearly identical to that observed the previous year. Phenology data from both years were incorporated into a stochastic, temperature-dependent model that predicts the occurrences of H. coagulata stages through time. Applications of imidacloprid early in the spring generation of nymphs proved very effective at reducing nymphs and sustaining lower densities of adults through summer.     

Xylella fastidiosa Afimbrial Adhesins Mediate Cell Transmission to Plants by Leafhopper Vectors

The interactions between the economically important plant-pathogenic bacterium Xylella fastidiosa and its leafhopper vectors are poorly characterized. We used different approaches to determine how X. fastidiosa cells interact with the cuticular surface of the foreguts of vectors. We demonstrate that X. fastidiosa binds to different polysaccharides with various affinities and that these interactions are mediated by cell surface carbohydrate-binding proteins. In addition, competition assays showed that N-acetylglucosamine inhibits bacterial adhesion to vector foregut extracts and intact wings, demonstrating that attachment to leafhopper surfaces is affected in the presence of specific polysaccharides. In vitro experiments with several X. fastidiosa knockout mutants indicated that hemagglutinin-like proteins are associated with cell adhesion to polysaccharides. These results were confirmed with biological experiments in which hemagglutinin-like protein mutants were transmitted to plants by vectors at lower rates than that of the wild type. Furthermore, although these mutants were defective in adhesion to the cuticle of vectors, their growth rate once attached to leafhoppers was similar to that of the wild type, suggesting that these proteins are important for initial adhesion of X. fastidiosa to leafhoppers. We propose that X. fastidiosa colonization of leafhopper vectors is a complex, stepwise process similar to the formation of biofilms on surfaces.     

Specific PCR detection and identification of Xylella fastidiosa strains causing citrus variegated chlorosis

By cloning and sequencing specific randomly amplified polymorphic DNA (RAPD) products, we have developed pairs of PCR primers that can be used to detect Xylella fastidiosa in general, and X. fastidiosa that cause citrus variegated chlorosis (CVC) specifically. We also identified a CVC-specific region of the X. fastidiosa genome that contains a 28-nucleotide insertion, and single base changes that distinguish CVC and grape X. fastidiosa strains. When using RAPD products to develop specific PCR primers, we found it most efficient to screen for size differences among RAPD products rather than presence/absence of a specific RAPD band.