Quantitative comparison of stylet penetration behaviors of glassy-winged sharpshooter on selected hosts

New Zealand is threatened by invasion of the glassy-winged sharpshooter, Homalodisca vitripennis (Germar) (Hemiptera: Cicadellidae), an important vector of Xylella fastidiosa, a gram-negative bacterium that causes Pierce's disease in grape (Vitis spp.) and scorch diseases in many other horticultural crops. Therefore, an understanding of the host acceptability, feeding behavior, and potential vector efficiency of glassy-winged sharpshooter on New Zealand crops is important. We tested host plant acceptance and feeding behaviors of glassy-winged sharpshooter on three common horticultural crops grown in New Zealand (apple [Malus spp.], grape, and citrus [Citrus spp.]), and a native plant (Metrosideros excelsa [=tomentosa] Richard, pohutukawa), using the electrical penetration graph (EPG) technique. Probing (stylet penetration) behaviors varied among the host plants, primarily due to differences in waveform event durations. Apple and grape were the most accepted host plants, on which glassy-winged sharpshooter spent the majority of its time on the plant probing and readily located and accepted a xylem cell for ingestion. This resulted in long durations of sustained xylem fluid ingestion. In contrast, pohutukawa was the least accepted host. On this plant, glassy-winged sharpshooter spent less time probing and engaged in longer and more frequent testing/searching and xylem-testing activities, rejected xylem cells frequently, and spent less time with stylets resting, before accepting a xylem cell and ultimately performing the same amount of sustained ingestion. Citrus plants contaminated with sublethal insecticide residues were intermediate between these extremes, with some acceptance of xylem, but less ingestion, probably due to presumed partial paralysis of the cibarial muscles. Implications of the results in terms of host plant acceptance and the development of a stylet penetration index are discussed.     

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.     

Distribution and management of citrus in California: implications for management of glassy-winged sharpshooter

The epidemiology of Pierce's disease of grape (Vitis spp.) in California has changed over the past 10 yr due to the introduction of an exotic vector, Homalodisca vitripennis (Germar), the glassy-winged sharpshooter. Although this insect is highly polyphagous, citrus (Citrus spp.) is considered a preferred host and proximity to citrus has been implicated as a significant risk factor in recent epidemics of Pierce's disease in southern California. Consequently, a detailed knowledge of the distribution and management of citrus in relation to grape is needed to improve insect and disease management. Analysis of data on the area planted to these two commodities indicates that only five counties in California concomitantly grow >1,000 ha of grape and >1,000 ha of citrus: Riverside, Kern, Tulare, Fresno, and Madera counties. Comparison of the distribution of grape and citrus within each of these counties indicates that the percentage of grape that is in proximity to citrus is greatest for Riverside County, but the total area of grape that is in proximity to citrus is greater for Fresno, Kern, and Tulare counties. The use of carbamates, neonicotinoids, organophosphates, and pyrethroids as part of the citrus pest management program for control of key insect pests was compared among the same five counties plus Ventura County from 1995 to 2006. Ventura County was included in this analysis as this county grows >10,000 ha of citrus and has established glassy-winged sharpshooter populations. The use of these broad-spectrum insecticides was lowest in Riverside and Ventura counties compared with the other four counties. Analysis of historical trapping data at the county scale indicates a negative association of broad-spectrum insecticide use with glassy-winged sharpshooter abundance. These results are used to retrospectively analyze the Pierce's disease outbreaks in Kern and Riverside counties.     

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.     

Fate of a genetically modified bacterium in foregut of glassy-winged sharpshooter (Hemiptera: Cicadellidae)

Symbiotic control is a new strategy being investigated to prevent the spread of insect-transmitted pathogens by reducing vector competence. We are developing this strategy to reduce the spread of Xylella fastidiosa by Homalodisca vitripennis (Germar) [formerly Homalodisca coagulata (Say)] (Hemiptera: Cicadellidae), the glassy-winged sharpshooter. In this study, the fate of a transformed symbiotic bacterium, Alcaligenes xylosoxidans variety denitriicans (S1Axd), in the foregut of glassy-winged sharpshooter when fed on citrus (Citrus spp.) and grape (Vitris spp.) was assessed. TaqMan-based quantitative real-time polymerase chain reaction (PCR) was used to detect and quantify bacterial cells remaining in the foregut at 0, 2, 4, 9, and 12 d after acquisition. S1Axd titer dropped rapidly by 2 d after acquisition, but in spite of this, at end of the 12-d experimental period, 45 and 38% of the glassy-winged sharpshooters retained the transformed bacteria, when fed on grape and citrus, respectively.     

Context-dependent transmission of a generalist plant pathogen: host species and pathogen strain mediate insect vector competence

The specificity of pathogen–vector–host interactions is an important element of disease epidemiology. For generalist pathogens, different pathogen strains, vector species, or host species may all contribute to variability in disease incidence. One such pathogen is Xylella fastidiosa Wells et al., a xylem-limited bacterium that infects dozens of crop, ornamental, and native plants in the USA. This pathogen also has a diverse vector complex and multiple biologically distinct strains. We studied the implications of diversity in this pathogen–vector–host system, by quantifying variability in transmission efficiency of different X. fastidiosa strains (isolates from almond and grape genetic groups) for different host plants (grape, almond, and alfalfa) by two of the most important vectors in California: glassy-winged sharpshooter [ Homalodisca vitripennis (Germar)] and green sharpshooter ( Draeculacephala minerva Ball) (both Hemiptera: Cicadellidae). Transmission of isolates of the almond strain by H. vitripennis did not differ significantly, whereas transmission varied significantly among isolates from the grape strain (15–90%). Host plant species did not affect H. vitripennis transmission. Conversely, D. minerva efficiency was mediated by both host plant species and pathogen strain. No acquisition of an almond isolate occurred regardless of plant type (0/122), whereas acquisition of a grape isolate from alfalfa was 10-fold higher than from grape or almond plants. These results suggest that pathogen, vector, and host diversity impose contingencies on the transmission ecology of this complex disease system. Studies aimed at the development of management strategies for X. fastidiosa diseases should consider the complexity of these interactions as they relate to disease spread.     

Host selection by Homalodisca vitripennis: the interplay between feeding, egg maturation, egg load, and oviposition

For some phytophagous insects, egg maturation may be dependent on adult feeding. Accordingly, rates of egg maturation may be dependent on the quality and quantity of available food sources. In turn, oviposition behavior could be affected by diet quality via changes in egg load (number of mature eggs carried by a female). Experiments were conducted to determine whether adult feeding may affect oviposition behavior of the glassy-winged sharpshooter, Homalodisca vitripennis. No-choice tests demonstrated that eggs accumulated in glassy-winged sharpshooter abdomens as time since last oviposition increased largely as a function of feeding plant species. In choice tests, glassy-winged sharpshooter females were observed most frequently on the plant species that imparted the greatest egg maturation rate in no-choice tests. Direct tests of the effects of egg load on glassy-winged sharpshooter oviposition behavior found that females were more likely to deposit eggs as egg load increased. Similarly, acceptance of a low-ranked oviposition plant species by female glassy-winged sharpshooters increased with egg load and time since last oviposition. The results indicate that adult feeding affected glassy-winged sharpshooter egg maturation, plant species varied in quality for providing nutrients for egg maturation, and egg load affected oviposition behavior. Thus, the quantity and quality of available feeding plant species may affect glassy-winged sharpshooter egg maturation rates, which in turn may affect the plant species female glassy-winged sharpshooters select for oviposition.     

Estimation of feeding threshold for Homalodisca vitripennis (Hemiptera: Cicadellidae) and its application to prediction of overwintering mortality

The glassy-winged sharpshooter, Homalodisca vitripennis (Germar), vectors the bacterium Xylella fastidiosa that induces Pierce's disease of grape. This study determined the effect of temperature on the feeding activity of H. vitripennis adults and the resulting production of excreta. The Logan type I model described a nonlinear pattern that showed excreta production increased up to an optimal temperature (33.1°C), followed by an abrupt decline near an estimated upper threshold (36.4°C). A temperature threshold for feeding, at or below which adults cease feeding, was estimated to be 10°C using a linear regression model based on the percentage of adults producing excreta over a range of constant temperatures. A simulated winter-temperature experiment using fluctuating thermal cycles confirmed that a time period above the temperature threshold for feeding was a critical factor in determining adult survival. Using data from the simulated temperature study, a predictive model was constructed by quantifying the relationship between cumulative mortality and cooling degree-hours. In field validation experiments, the model accurately predicted the temporal pattern of overwintering mortality of H. vitripennis adults held under winter temperatures simulating conditions in Bakersfield and Riverside, California, in 2006-2007. Model prediction using winter temperature data from a Riverside weather station indicated that H. vitripennis adults would experience an average of 92% overwintering mortality before reproduction in the spring, but levels of mortality varied depending on winter temperatures. The potential for temperature-based indices to predict temporal and spatial dynamics of H. vitripennis overwintering is discussed.     

Temperature mediates vector transmission efficiency: inoculum supply and plant infection dynamics

Climate, particularly environmental temperature, frequently plays an important role in disease epidemiology. This study investigated the role of environmental temperature on transmission of the generalist plant pathogen Xylella fastidiosa by its leafhopper vectors. In this system temperature is known to influence both vector performance and feeding rate, yet the implications for pathogen transmission have not been documented. Experiments were conducted over a range of temperatures to document effects on transmission efficiency of the California native Graphocephala atropunctata (blue–green sharpshooter) and the invasive Homalodisca vitripennis (glassy-winged sharpshooter). Inoculation efficiency of H. vitripennis was positively related to temperature. Graphocephala atropunctata mortality and transmission responded non-linearly to temperature, with the highest rates of both at the highest temperature. The experiment also evaluated whether differences in inoculum supply contributed to plant infection level using quantitative PCR. Although total X. fastidiosa population within G. atropunctata was not related to plant infection, the number of infectious vectors was a strong predictor of plant infection level–suggesting that the number of inoculation events is important in the development of systemic infection of X. fastidiosa in grapevines. These results, along with existing evidence from the literature, point to wide-ranging impacts of climate on the epidemiology of X. fastidiosa diseases.     

Salivary enzymes are injected into xylem by the glassy-winged sharpshooter, a vector of Xylella fastidiosa

A few phytophagous hemipteran species such as the glassy-winged sharpshooter, Homalodisca vitripennis, (Germar), subsist entirely on xylem fluid. Although poorly understood, aspects of the insect's salivary physiology may facilitate both xylem-feeding and transmission of plant pathogens. Xylella fastidiosa is a xylem-limited bacterium that causes Pierce's disease of grape and other scorch diseases in many important crops. X. fastidiosa colonizes the anterior foregut (precibarium and cibarium) of H. vitripennis and other xylem-feeding vectors. Bacteria form a dense biofilm anchored in part by an exopolysaccharide (EPS) matrix that is reported to have a β-1,4-glucan backbone. Recently published evidence supports the following, salivation-egestion hypothesis for the inoculation of X. fastidiosa during vector feeding. The insect secretes saliva into the plant and then rapidly takes up a mixture of saliva and plant constituents. During turbulent fluid movements in the precibarium, the bacteria may become mechanically and enzymatically dislodged; the mixture is then egested back out through the stylets into plant cells, possibly including xylem vessels. The present study found that proteins extracted from dissected H. vitripennis salivary glands contain several enzyme activities capable of hydrolyzing glycosidic linkages in polysaccharides such as those found in EPS and plant cell walls, based on current information about the structures of those polysaccharides. One of these enzymes, a β-1,4-endoglucanase (EGase) was enriched in the salivary gland protein extract by subjecting the extract to a few, simple purification steps. The EGase-enriched extract was then used to generate a polyclonal antiserum that was used for immunohistochemical imaging of enzymes in sharpshooter salivary sheaths in grape. Results showed that enzyme-containing gelling saliva is injected into xylem vessels during sharpshooter feeding, in one case being carried by the transpiration stream away from the injection site. Thus, the present study provides support for the salivation-egestion hypothesis.     

Delivery of a Genetically Marked <Emphasis Type="Italic">Alcaligenes</Emphasis> sp. to the Glassy-Winged Sharpshooter for Use in a Paratransgenic Control Strategy

An artificial feeding system was designed for the glassy-winged sharpshooter (GWSS), Homalodisca coagulata Say (Hemiptera: Cicadellidae). The system, unlike previous systems, provided enough nutrients to GWSS to survive for 48 h. A system like this is a prerequisite to examining the potential use of paratransgenesis to interrupt transmission of Xylella fastidiosa, the bacterial pathogen causing Pierces disease of grape, by insect vectors. We developed a system for short-term feeding of GWSS that allows for the introduction of bacteria in liquid medium, and we have demonstrated the ability of Alcaligenes xylosoxidans denitrificans, expressing a red fluorescent protein (dsRed), to colonize the cibarial region of the GWSS foregut for up to 5 weeks post-exposure. Alcaligenes xylosoxidans denitrificans thus occupies the same region in the foregut as the pathogen, Xylella fastidiosa.     

Suitability of non-fertilized eggs of Homalodisca vitripennis for the egg parasitoid Gonatocerus morrilli

Gonatocerus morrilli (Howard) (Hymenoptera: Mymaridae) is an egg parasitoid used in California, USA to control glassy-winged sharpshooter (GWSS), Homalodisca vitripennis (Germar) (Hemiptera: Cicadellidae). Virgin GWSS females deposit non-fertilized eggs and mated females can exhaust sperm reserves for egg fertilization. However, nothing is known about Gonatocerus spp. performance when using non-fertilized GWSS eggs as hosts. Host age preference for oviposition and suitability of non-fertilized GWSS eggs as hosts for G. morrilli reproduction were investigated to determine whether non-fertilized eggs on sentinel plants could be used to monitor egg parasitoid populations. Gonatocerus morrilli parasitized all ages of GWSS eggs (1–8 days old) regardless if the host egg was fertilized or not. In choice tests (fertilized versus non-fertilized eggs), parasitoids failed to emerge as adults from non-fertilized eggs more often than from fertilized eggs. The results indicate that non-fertilized eggs were accepted by G. morrilli as suitable hosts for oviposition, but were less suitable for immature development compared to fertilized eggs.     

Estimating Xylella fastidiosa transmission parameters: decoupling sharpshooter number and feeding period

Compared to human- and wildlife-transmitted pathogens, less emphasis has been placed on developing models of plant pathogen transmission by insects. Here, we describe the transmission ecology of the bacterium Xylella fastidiosa Wells et al., the causal agent of Pierce's disease in grapevines, by its leafhopper vectors. First, we performed a meta-analysis of transmission studies of X. fastidiosa by its two most important vectors in the Western USA, the invasive glassy-winged sharpshooter, Homalodisca vitripennis Germar, and the native blue-green sharpshooter, Graphocephala atropunctata Signoret (both Hemiptera: Cicadellidae). The importance of vector number, pathogen acquisition period, and inoculation access period (IAP) for transmission differed between the two species. We fit these transmission datasets to two biologically derived transmission models, i.e., a binomial and a Poisson probability model. The Poisson model provided substantially better fit and produced estimates of H. vitripennis transmission efficiency that were dramatically lower than for G. atropunctata . We also conducted a separate pair of experiments that decoupled vector number from IAP. These experiments supported the results of the meta-analysis. Interestingly, high vector loads not only increased transmission rate, but also shortened X. fastidiosa incubation period in grapevines. This work provides quantitative estimates of transmission of an economically important pathogen that is analogous to risk models for arthropod-vectored human and wildlife diseases. In addition, this work suggests that heterogeneous vector loads may accelerate the disease cycle, increasing the potential for secondary spread in vineyards.     

Egg parasitoids (Hymenoptera: Mymaridae and Trichogrammatidae) of Kolla paulula (Walker) (Hemiptera: Cicadellidae) in Taiwan

Pseudoligosita nephotetticum (Mani) (Hymenoptera: Trichogrammatidae) and Gonatocerus (Cosmocomoidea) sp. (Hymenoptera: Mymaridae) have been identified for the first time as egg parasitoids of the sharpshooter leafhopper Kolla paulula (Walker) (Hemiptera: Cicadellidae: Cicadellinae: Cicadellini) in Taiwan. K. paulula has been recently identified as a candidate vector of the phytopathogenic bacterium Xylella fastidiosa, the causative agent of Pierce's disease of grapes and similar diseases of other affected plants in Taiwan, and thus has become of particular economic importance. Also provided is a summary of the known records of egg parasitoids (Mymaridae and Trichogrammatidae) of other leafhoppers from the tribe Cicadellini in the world, as well as taxonomic notes on P. nephotetticum.     

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.     

Production of Anagrus epos Girault (Hymenoptera: Mymaridae) on Homalodisca vitripennis (Germar) (Hemiptera: Cicadellidae) eggs

Anagrus epos Girault (Hymenoptera: Mymaridae) is a natural enemy candidate for a classical biological control program targeting the glassy-winged sharpshooter (GWSS), Homalodisca vitripennis (Germar) (Hemiptera: Cicadellidae), in California. Little is known about the biology or ecology of A. epos when it utilizes GWSS eggs as a host. Here, we report the results of laboratory studies that describe the host age preference for oviposition, longevity of A. epos adults provided with different food sources, and developmental rates at six different constant temperature regimes. Anagrus epos is a gregarious parasitoid in GWSS eggs with up to 14 adults emerging from each GWSS egg. In choice and no-choice tests for oviposition, A. epos females successfully parasitized all developmental ages of GWSS eggs (1–8 days old). In choice tests, parasitism rates were significantly higher in 1-, 3-, 4-, and 5-day-old GWSS eggs than in 2-, 6-, 7-, and 8-day-old eggs. If provided with honey and water, honey only, water only, or no food or water, A. epos females lived on average 8.2, 4.7, 2.6, and 1.6 days, respectively. Anagrus epos required 294.1 degree-days above a lower temperature threshold of 12.4 °C to develop from egg to adult (eclosion). Our results provide baseline information useful in the development of an efficient parasitoid mass rearing program for A. epos release and evaluation in California.     

Molecular characterization of Gonatocerus tuberculifemur (Ogloblin) (Hymenoptera: Mymaridae), a prospective Homalodisca vitripennis (Germar) (Hemiptera: Cicadellidae) biological control candidate agent from South America: divergent clades

We genetically characterized the prospective South American egg parasitoid candidate, Gonatocerus tuberculifemur, of the glassy-winged sharpshooter (GWSS), Homalodisca vitripennis, for a neoclassical biological control program in California. Two molecular methods, inter-simple sequence repeat-polymerase chain reaction DNA fingerprinting and a phylogeographic approach inferred from the mitochondrial cytochrome oxidase subunit I gene (COI), were utilized. Five geographic populations from South America were analyzed; in addition, a phylogenetic analysis was performed with several named and one unnamed Gonatocerus species using the COI gene. DNA fingerprinting demonstrated a fixed geographic banding pattern difference in the population from San Rafael, Mendoza Province, Argentina. The COI analysis uncovered haplotype or geographic structure in G. tuberculifemur. A neighbour-joining distance (NJ) and a single most parsimonious tree (MP) clustered the populations into two well-supported distinct clades with strong bootstrap values (97-99% and 92-99%, respectively) with populations from San Rafael clustering into clade 2 and the rest of the populations clustering into clade 1. No haplotype sharing was observed between individuals from the two clades. Phylogenetic analyses performed by NJ and MP methods with 15 Gonatocerus species confirmed species boundaries and again uncovered two distinct clades in G. tuberculifemur with strong bootstrap support (95-100% and 68-100%, respectively). However, the NJ tree supported the morphologically defined relationships better than the MP tree. The molecular evidence in the present study is suggestive of a species level divergence. Because G. tuberculifemur is under consideration as a potential biological control agent for GWSS in California, understanding cryptic variation in this species is critical.     

Biochemical monitoring of acetylcholinesterase sensitivity to organophosphorus insecticides in glassy-winged sharpshooter Homalodisca coagulata Say (Homoptera: Cicadellidae) and smoke-tree sharpshooter H. lacerta Fowler

The glassy-winged sharpshooter Homalodisca coagulata Say (Homoptera: Cicadellidae) is a new pest to California agriculture. It is the principal vector of several plant pathogenic diseases, particularly Pierce's Disease in grapevines, and oleander leaf scorch. A microplate-based assay is described that measures the sensitivity of acetylcholinesterase (AChE) activity to inhibition by organophosphorus (OP) insecticides in this important pest. The technique provides users with an accurate measure of the efficacy of OP binding to this target site, and is a valuable tool for monitoring field populations of the insect to determine whether the use of OP insecticides has selected for resistant individuals. The technique will also measure AChE sensitivity in the smoke-tree sharpshooter, H. lacerta Fowler. This species is native to California and is regarded only as a minor pest. Both inhibition and kinetic measurements for the AChE enzymes in these sharpshooters demonstrate the close phylogenetic relationships between the two species.     

Initial Genetic Analysis of Xylella fastidiosa in Texas

Xylella fastidiosa is the causative agent of Pierce’s Disease of grape. No published record of X. fastidiosa genetics in Texas exists despite growing financial risk to the U.S. grape industry, a Texas population of the glassy-winged sharpshooter insect vector (Homalodisca vitripennis) now spreading in California, and evidence that the bacterium is ubiquitous to southern states. Using sequences of conserved gyrB and mopB genes, we have established at least two strains in Texas, grape strain and ragweed strain, corresponding genetically with subsp. piercei and multiplex, respectively. The grape strain in Texas is found in Vitis vinifera varieties, hybrid vines, and wild Vitis near vineyards, whereas the ragweed strain in Texas is found in annuals, shrubs, and trees near vineyards or other areas. RFLP and QRT PCR techniques were used to differentiate grape and ragweed strains with greater efficiency than sequencing and are practical for screening numerous X. fastidiosa isolates for clade identity.