Glassy-winged Sharpshooter Research Articles

Effect of Host Plant Xylem Fluid on Growth, Aggregation, and Attachment of Xylella fastidiosa

In the Temecula Valley of California the proximity of citrus groves to vineyards influences the incidence and severity of Pierce's disease (PD) in grapes, a disease caused by the gram-negative bacterium, Xylella fastidiosa. Although the glassy-winged sharpshooter (GWSS), the major insect vector of the bacterium, feeds on and moves back and forth between citrus groves and vineyards, there are no visible symptoms of disease caused by X fastidiosa in citrus. Previous evidences suggested that while grapevines are susceptible to the PD strain of X. fastidiosa, citrus trees are resistant or tolerant but could be a reservoir to harbor the pathogen for the GWSS acquisition. We investigated the mechanisms of host plant resistance/susceptibility by examining the in vitro effect of xylem fluid from grapefruit, orange, lemon, and grape on the growth, aggregation, and attachment of a X. fastidiosa strain isolated from grape. Our results revealed that xylem fluid from grapefruit, orange, and lemon trees caused the bacterial cells to form aggregations of large whitish clumps, whereas the xylem fluid from grape vines created a visible thick biofilm. The densities of X. fastidiosa cells in grapefruit xylem fluid treatment were significantly higher at 6, 8, and 9 d postinoculation compared with those in grape xylem fluid treatment. The cell densities of X. fastidiosa cultures in orange or lemon xylem fluid were generally lower than those in grape xylem fluid treatment, whereas citrus xylem fluid significantly inhibited X. fastidiosa biofilm formation compared to grape xylem fluid.

Isolation and Evaluation of Beauveria bassiana (Deuteromycotina: Hyphomycetes) for the Suppression of the Glassy-winged Sharpshooter, Homalodisca coagulata (Homoptera: Cicadellidae)

The glassy-winged sharpshooter, Homalodisca coagulata (Say) (Homoptera: Cicadellidae), is a significant threat to California agriculture as a vector of the bacterium, Xylella fastidiosa Wells, causative agent of Pierce's disease. Control strategies target vector populations to prevent the spread of the bacterium. One of the potential means of controlling H. coagulata is the use of entomopathogenic fungi. To discover naturally-occurring fungal pathogens that are adapted to H. coagulata and its habitats, soil samples from organic citrus and conventional pomegranate orchards in Tulare and Riverside counties in southern California were screened for the presence of pathogens. Using a modified Galleria bait method that required small quantities of soil, 124 isolates of Beauveria bassiana (Balsamo) Vuillemin and four isolates of Metarhizium anisopliae (Metschnikoff) Sorokin (Deuteromycotina: Hyphomycetes) were recovered. Additionally, 22 isolates of B. bassiana were recovered by plating soil suspensions on selective growth media. Natural fungal infections were not detected in H. coagulata populations from periodic sampling in Kern, Riverside and Ventura counties in southern California. Some of the B. bassiana isolates recovered from soil and other insect hosts in southern California were evaluated against H. coagulata along with those isolated from H. coagulata in Texas and Mississippi. Growth of the selected isolates also was evaluated at 15, 23, 28 and 32°C. The Texas isolate and two California isolates of B. bassiana were virulent against H. coagulata and warrant further study.

Effect of Formulated Insecticides on Homalodisca vitripennis (Germar) (Hemiptera: Cicadellidae) and its Parasitoid Gonatocerus ashmeadi Girault (Hymenoptera: Mymaridae)

Eleven insecticides were tested using a bioassay of foliar insecticide residue to evaluate their impact on Homalodisca vitripennis nymphs and adults and on Gonatocerus ashmeadi parasitoid pupae and newly-emerged adults. Most compounds caused 100% mortality of H. vitripennis nymphs and adults within the first 24 h following exposure. Dimethoate was significantly less toxic to the nymphs than the other compounds. The insect growth regulators buprofezin and pyriproxifen caused high mortality levels at the end of the 72-h period of observation. They were efficacious against both the H. vitripennis nymphs and adults. Thiamethoxam, acetamiprid, cyfluthrin, fenpropathrin and endosulfan severely affected the immatures and newly-emerged adults of the parasitoid G. ashmeadi with total percent mortality exceeding 65%. Piriproxifen, methomyl, imidacloprid and carbaryl were moderately toxic to the parasitoid pupae and young adults (34–55% total mortality); whereas treatments using buprofezin or dimethoate allowed >89% survival of the parasitoids. These results may help predict the reliability of these compounds in the field and risks to natural enemies associated with their use. It is possible, however, to choose insecticides that are more selective in toxicity to the glassy-winged sharpshooter than to its natural enemies.

Dispersal of Homalodisca vitripennis (Homoptera: Cicacellidae) from a Point Release Site in Citrus

The glassy-winged sharpshooter, Homalodisca vitripennis (Germar), is an important vector of Xylella fastidiosa Wells et al., a bacterium that has caused substantial losses in the viticulture and ornamental industries in California. Area-wide management programs have been implemented to reduce vector populations and limit the spread of this disease. However, there is still a lack of information on the factors that influence this insect’s movement within and between host crops. In this study, we used mark-release-recapture (MRR) to examine the dispersal of H. vitripennis in a mature orange grove, Citrus sinensis Osbeck. Insects were doubly marked with chicken or rabbit immunoglobulin G (IgG) proteins and fluorescent dusts to enable monitoring over several weeks. Our objectives were to examine the reliability of IgG protein markers relative to fluorescent dusts, determine how sharpshooter movement differed in this landscape relative to a previous study, and develop a better understanding of the biotic and abiotic factors that could influence sharpshooter dispersal. Linear regressions of recapture data with a diffusion model provided significant fits to the data in five of six releases. Recapture data were fitted to a diffusion model, and based on parameters generated with the model, estimated dispersal distances for H. vitripennis at 72 h showed 50 and 99% remained within annuli of 31 and 150 m from the release site, respectively. Flight activity was greatest between 1000 and 1400 hours, and no flights occurred between 2200 and 0600 hours. Only temperature explained a significant amount of the variability in recapture of H. vitripennis, with sharpshooters rarely trapped below 18°C.

Dispersal of <I>Homalodisca vitripennis</I> (Homoptera: Cicacellidae) from a Point Release Site in Citrus

Abstract The glassy-winged sharpshooter, Homalodisca vitripennis (Germar), is an important vector of Xylella fastidiosa Wells et al., a bacterium that has caused substantial losses in the viticulture and ornamental industries in California. Area-wide management programs have been implemented to reduce vector populations and limit the spread of this disease. However, there is still a lack of information on the factors that influence this insect’s movement within and between host crops. In this study, we used mark-release-recapture (MRR) to examine the dispersal of H. vitripennis in a mature orange grove, Citrus sinensis Osbeck. Insects were doubly marked with chicken or rabbit immunoglobulin G (IgG) proteins and fluorescent dusts to enable monitoring over several weeks. Our objectives were to examine the reliability of IgG protein markers relative to fluorescent dusts, determine how sharpshooter movement differed in this landscape relative to a previous study, and develop a better understanding of the biot...

Spatial and Temporal Dynamics of Overwintering <I>Homalodisca coagulata</I> (Hemiptera: Cicadellidae)

A 4-yr landscape-scale study was conducted to investigate spatial and temporal dynamics of overwintering Homalodisca coagulata (Say) (Hemiptera: Cicadellidae) in the lower San Joaquin Valley, California. Spatial structures of H. coagulata distributions were characterized with Moran's I index, and spatial associations between H. coagulata and the surrounding environment were investigated with a geographic information system. H. coagulata was caught consistently with sticky traps throughout the winter, and trap catches formed a distinctive peak in December or January, indicating active flight of H. coagulata during the winter. In 2000-2001, the mean +/-SE trap count was 4.8 +/- 1.21 per trap per wk, and H. coagulata trap catches were spatially autocorrelated within approximately 1.3 km. Approximately 49% of H. coagulata were caught in citrus, 23% in stone fruit, and 11% in grape. After a control program began in spring 2001, the mean trap count was considerably lower (0.041 +/- 0.0004 per trap per wk), and no spatial autocorrelations were detected in 2001-2004. H. coagulata trap catch-crop associations also changed after initiation of the control program. Between 25 and 38% of H. coagulata trap catches were from citrus, between 8 and 20% were from stone fruit, and between 11 and 25% were from grape. Potential for winter-season spread and management of Xylella fastidiosa Wells et al., a pathogen causing Pierce's disease, are discussed.

A glassy-winged sharpshooter cell line supports replication of Rhopalosiphum padi virus ( Dicistroviridae)

Rhopalosiphum padi virus (RhPV) (family Dicistroviridae; genus Cripavirus) is an icosahedral aphid virus with a 10 kb positive-sense RNA genome. To study the molecular biology of RhPV, identification of a cell line that supports replication of the virus is essential. We screened nine cell lines derived from species within the Lepidoptera, Diptera and Hemiptera for susceptibility to RhPV following RNA transfection. We observed cytopathic effects (CPE) only in cell lines derived from hemipterans, specifically GWSS-Z10 cells derived from the glassy winged sharp shooter, Homalodisca coagulata and DMII-AM cells derived from the corn leaf hopper, Dalbulus maidis. Translation and appropriate processing of viral gene products, RNA replication and packaging of virus particles in the cytoplasm of GWSS-Z10 cells were examined by Western blot analysis, Northern blot hybridization and electron microscopy. Infectivity of the GWSS-Z10 cell derived-virus particles to the bird cherry-oat aphid, R. padi, was confirmed by RT-PCR and Western blot. The GWSS-Z10 cell line provides a valuable tool to investigate replication, structure and assembly of RhPV.

Molecular analysis of capsid protein of Homalodisca coagulata Virus-1, a new leafhopper-infecting virus from the glassy-winged sharpshooter, Homalodisca coagulata

A new virus that infects and causes increased mortality in leafhoppers was isolated from the glassy-winged sharpshooter, Homalodisca coagulata (Say) (Hemiptera: Cicadellidae). The virus, named Homalodisca coagulata virus -1, HoCV-1, was associated with increased mortality of cultured 5th instar H. coagulata. To identify the presence of H. coagulata viral pathogens, cDNA expression libraries were made from adult and nymphs. Analysis using reverse transcriptase PCR demonstrated that the virus was present in midgut tissues. As the viral capsid proteins are commonly used in classification of newly discovered viruses, the capsid proteins (CP) of the virus discovered in H. coagulata was examined. The order of the polyprotein subunits of HoCV-1 capsid proteins was determined to be CP2, CP4, CP3, and CP1. The CP4/CP3 (AFGL/GKPK) cleavage boundary site was clearly identified when the sequences were aligned. The putative CP3/CP1 (ADVQ/SAFA) cleavage site and the putative CP2/CP4 (VTMQ/EQSA) cleavage site of HoCV-1, respectively, were located in the same region as that of the other viruses. After alignment, the CP3/CP1 cleavage sites and CP2/CP4 cleavage sites of the viruses analyzed fell within 50 amino acids of one another. As with the cricket paralysis virus, HoCV-1 was found to be mainly comprised of β-sandwiches in CP1-3 with a jelly roll topological motif. CP4 of HoCV-1 appeared to be mainly α-helical in structure. CP1-4 domains are most homologous to insect picorna-like virus coat proteins as was demonstrated by the results of the BLASTP and PSI-BLAST tests, and is strongly supported by the structural modeling. While sequence homology between the cricket paralysis virus and HoCV-1 was low, the global structure of the proteins was conserved. Sequence identities were analyzed by in silico comparison to known genes in the public database, NCBI. Phylogenetic analysis performed using the optimized protein alignment generated a phylogram containing 5 clades. Clade 1 consisted of Drosophila C virus, Clade 2 consisted of cricket paralysis virus, Clade 3 of Triatoma virus, Plautia stali intestine virus, Himetobi P virus, black queen cell virus, and HoCV-1. Clade 4 encompassed acute bee paralysis virus and Kashmir bee virus, and Clade 5 consisted of Rhopalosiphum padi virus. Analysis of the capsid protein of this new leafhopper virus provided significant evidence that it is related to other ssRNA insect viruses within the Family, Dicistroviridae. The HoCV-1, capsid protein sequence has been deposited in GenBank, Accession number: DQ308403.

Development and Reproduction of the Egg Parasitoid, <I>Gonatocerus ashmeadi</I> Girault (Hymenoptera: Mymaridae), as a Function of Temperature

The development, fecundity, and life table parameters of Gonatocerus ashmeadi Girault, an egg parasitoid of the glassy-winged sharpshooter, Homalodisca coagulata (Say), were studied in the laboratory at six constant temperatures between 12 and 32°C. At 12°C, the parasitoid failed to develop beyond the third instar, and durations of the egg stage and the first and second instars were prolonged. Development from the egg stage to adult emergence varied from 27.1 d at 16°C to 9.5 d at 28°C. Temperature thresholds for development ranged from 3.8°C for first-instar larvae to 12.8°C for the pupal stage. The thermal constants were lowest for the second-instar larvae (26.7 DD) and highest for the pupae (75.4 DD). Nearly 207 DD were required above the lower temperature threshold of 8.5°C to complete development from egg to adult. The optimum temperature for egg to adult development was 29.2°C. Survival from egg to adult was 67.4% at 16°C and ranged from 83.4 to 86.7% between 20 and 32°C. At 16–32°C, the population had a type I survivorship pattern. At 16°C, longevity of adult females and males averaged 27.1 and 19.0 d, respectively, but declined to 6.4 and 6.9 d at 32°C. At 20–32°C, peak adult emergence occurred on the first day of emergence, but at 16°C, it was greatest on the second day. When exposed to temperatures ranging from 16 to 32°C, the female:male sex ratio was similar, ranging from 3.4 to 5.6. Lifetime fecundity was greatest at 24°C and lowest at 32°C, with the maximum net reproduction also occurring at 24°C. Greatest intrinsic and finite rates of increase, shortest population doubling time, and mean generation time occurred when G. ashmeadi was held at 28°C. The parameters defined in this study can influence geographical distribution and are important for the mass-rearing this wasp as biological control agent for the glassy-winged sharpshooter.

Susceptibility of Immature Stages of <I>Homalodisca coagulata</I> (Hemiptera: Cicadellidae) to Selected Insecticides

Susceptibility of immatures of the glassy-winged sharpshooter, Homalodisca coagulata (Say) (Hemiptera: Cicadellidae), to 10 insecticides that included chlorpyrifos, dimethoate, endosulfan, bifenthrin, cyfluthrin, esfenvalerate, fenpropathrin, acetamiprid, imidacloprid, and thiamethoxam was evaluated in the laboratory. All five instars were exposed to different doses of each foliar insecticide by the petri dish technique, whereas a systemic uptake method was used to assess the toxicity to imidacloprid and thiamethoxam. All test insecticides exhibited high toxicity to all immature stages of H. coagulata at concentrations below the field recommended rates of each insecticide. Although all five instars were susceptible to test insecticides, mortality was significantly higher in first instars than in the older immatures based on low LC50 values (ranging from 0.017 to 5.75 ng(AI)/ml) with susceptibility decreasing with each successive stage. Fifth instars were generally the least sensitive (LC50 values ranging from 0.325 to 216.63 ng (AI)/ml). These results show that mortality was directly related to age of the insect and suggest that chemical treatment at early stages is more effective than at late stages. Acetamiprid (neonicotinoid) and bifenthrin (pyrethroid) were the most toxic to all five instars, inducing most mortality within 24 h and showing lower LC50 values ranging from 0.017 to 0.686 ng/ml compared with other insecticides (LC50 values ranging from 0.191 to 216.63 ng(AI)/ml). Our data suggest that a diverse group of very effective insecticides are available to growers for controlling all stages of H. coagulata. Knowledge on toxicity of select insecticides to H. coagulata immatures may contribute to our understanding of resistance management in future for this pest by targeting specific life stages instead of the adult stage alone.

Susceptibility of Immature Stages of Homalodisca coagulata (Hemiptera: Cicadellidae) to Selected Insecticides

Susceptibility of immatures of the glassy-winged sharpshooter, Homalodisca coagulata (Say) (Hemiptera: Cicadellidae), to 10 insecticides that included chlorpyrifos, dimethoate, endosulfan, bifenthrin, cyfluthrin, esfenvalerate, fenpropathrin, acetamiprid, imidacloprid, and thiamethoxam was evaluated in the laboratory. All five instars were exposed to different doses of each foliar insecticide by the petri dish technique, whereas a systemic uptake method was used to assess the toxicity to imidacloprid and thiamethoxam. All test insecticides exhibited high toxicity to all immature stages of H. coagulata at concentrations below the field recommended rates of each insecticide. Although all five instars were susceptible to test insecticides, mortality was significantly higher in first instars than in the older immatures based on low LC50 values (ranging from 0.017 to 5.75 ng(AI)/ml) with susceptibility decreasing with each successive stage. Fifth instars were generally the least sensitive (LC50 values ranging from 0.325 to 216.63 ng(AI)/ml). These results show that mortality was directly related to age of the insect and suggest that chemical treatment at early stages is more effective than at late stages. Acetamiprid (neonicotinoid) and bifenthrin (pyrethroid) were the most toxic to all five instars, inducing most mortality within 24 h and showing lower LC50 values ranging from 0.017 to 0.686 ng/ml compared with other insecticides (LC50 values ranging from 0.191 to 216.63 ng(AI)/ml). Our data suggest that a diverse group of very effective insecticides are available to growers for controlling all stages of H. coagulata. Knowledge on toxicity of select insecticides to H. coagulata immatures may contribute to our understanding of resistance management in future for this pest by targeting specific life stages instead of the adult stage alone.

Studies on the fungi associated with the glassy-winged sharpshooter Homalodisca coagulata with emphasis on a new species Hirsutella homalodiscae nom. prov.

The glassy-winged sharpshooter (GWSS), Homalodisca coagulata (Say), is an efficient vector of the phytopathogenic bacterium Xyllela fastidiosa. Native to the Southeast US, it was introduced to California, where its presence has caused a sharp increase in X. fastidiosa-based disease. Here are described three fungi associated with GWSS in its native range, including a Hirsutella species that is the predominant mycopathogen detected in sampled GWSS populations. Also described are potentially novel species of Pseudogibellula and Sporothrix. Data garnered from examination of in␣situ and in vitro morphological characters were compared to published keys and type specimen descriptions to achieve morphology-based identification. ITS1-5.8S-ITS2 and partial sequences of the large subunit (LSU)-rDNA, actin, and β-tubulin genes of several GWSS Hirsutella isolates were sequenced and analyzed. Phylogenetic analysis of the LSU-rDNA and tubulin of the GWSS Hirsutella in combination with sequence data generated from other Hirsutella species defined the GWSS isolates to belong to subgroup II within this genus. The combination of phenotypic and genotypic data demonstrated GWSS Hirsutella to be a new species and as such, has been designated Hirsutella homalodiscae nom. prov. Species-specific PCR primers, designed from intron and exon regions of the actin and tubulin genes, were used in a multiplex reaction for the in situ identification of H. homalodiscae. A total of 246 mummified GWSS collected from sites in the southeastern US were subjected to multiplex PCR analysis. More than 70% of these samples tested positive for the presence of H. homalodiscae demonstrating it to be the major mycopathogen distributed throughout the native range of GWSS.

Evaluation of floral resources for enhancement of fitness of Gonatocerus ashmeadi, an egg parasitoid of the glassy-winged sharpshooter, Homalodisca vitripennis

The effect of five different food resources (Coccus hesperidum honeydew, honey-water, Lobularia maritima, Fagopyrum esculentum and water only) on the longevity, fecundity, and progeny fitness of female Gonatocerus ashmeadi, an egg parasitoid of the glassy-winged sharpshooter (Homalodisca vitripennis) was investigated in the laboratory. Female G. ashmeadi survived 208-405% longer (up to 11.7 days longer) when fed C hesperidum honeydew, honey-water, L. maritima and F. esculentum compared with water only. Similarly, all food treatments resulted in up to 378% more progeny (up to 80 more total progeny) than those females provisioned with water only. Mean percentage female progeny ranged from 70 to 81% and did not significantly differ between food treatments. The highest daily fecundity (2229 progeny) occurred on the first day of oviposition for each food treatment and generally declined as female G. ashmeadi aged. Females deposited 61-100% of offspring within the first 5 days, after which, daily fecundity usually declined to 0-6 offspring per day. For all experimental diets except L. maritima, the number of male progeny oviposited remained relatively constant over time (similar to 2 males per oviposition bout) but the proportion of males oviposited increased as female G. ashmeadi aged, as oviposition rates declined and fewer female eggs were subsequently laid. There was a strong positive correlation between hind tibia length (HTL) and total progeny produced by G. ashmeadi fed honey-water. There was no correlation between HTL and longevity of female G. ashmeadi fed honey-water. Mean HTL of G. ashmeadi progeny did not significantly differ between food treatments. Estimated progeny fecundity based on HTL, indicated that offspring produced by females provided with water only was 12-15 eggs or 9-12% lower than females that had access to food resources. This difference was not statistically significant. Female progeny that were oviposited by females 9-13 days of age with access to food resources were significantly larger and contained on average 14-17 more eggs than those female progeny resulting from eggs deposited by females 1-8 days of age. (c) 2006 Elsevier Inc. All rights reserved.

The effect of resource provisioning and sugar composition of foods on longevity of three Gonatocerus spp., egg parasitoids of Homalodisca vitripennis

The eVect of dietary supplements on the longevity of male and female Gonatocerus ashmeadi, G. triguttatus and G. fasciatus (Hymenoptera: Mymaridae), was determined in the laboratory. Treatments included: water only, 3:1 honey–water solution, Xoral and extraXoral nectars from Wve diVerent plants (excised stems from Fagopyrum esculentum, Lobularia maritima, Phacelia tanacetifolia, Anethum graveolens and Vicia faba), honeydew from Coccus hesperidum and Homalodisca vitripennis (formally H. coagulata), a commercially available food supplement (Eliminade) and citrus foliage. Additionally, the sugar composition of each food resource was determined using HPLC and whole Xower extracts. Honey–water and F. esculentum nectar signiWcantly increased longevity of male and female G. ashmeadi, G. triguttatus, and G. fasciatus up to 1860%, 1323% and 1459%, respectively, when compared with water. For both sexes and all three parasitoid species, survival on citrus foliage, H. vitripennis excrement, and P. tanacetifolia Xowers was equivalent to that on water only. The longevity of G. ashmeadi and G. triguttatus was up to 539% higher on Eliminade compared with water only, however there was no signiWcant eVect of Eliminade on survival of G. fasciatus. Coccus hesperidum honeydew increased survival times up to 665% for all mymarid species compared with citrus foliage alone. HPLC analysis indicated that food resources most beneWcial to Gonatocerus parasitoids possessed a high proportion of glucose (up to 44%) and fructose (up to 53%), suggesting that sucrose may not be as important for parasitoid survival. Citrus and P. tanacetifolia Xowers contained favorable proportions of glucose and fructose, but the inability of Gonatocerus spp. to beneWt from this may be related to Xower morphology which could prevent access to nectar.

First Report of a Proconiine Sharpshooter, <I>Anacuerna centrolinea</I> (Hemiptera: Cicadellidae), in Chile, with Notes on Its Biology, Host Plants, and Egg Parasitoids

The Þrst representative of the leafhopper tribe Proconiini (subfamily Cicadellinae), Anacuerna centrolinea (Melichar) is reported herein from the Tarapacaregion in northern Chile. This species was discovered at high elevation (4,000 m) in the course of a survey conducted in South America by the USDAÐARS for the neoclassical biological control program against the glassy-winged sharpshooter, Homalodisca coagulata (Say) in California. New data are given on the biology and host plants of A. centrolinea. Information also is provided on its egg parasitoid, Gonatocerus tuberculifemur (Ogloblin) (Hymenoptera: Mymaridae) which also was a Þrst record from Chile. This discovery encourages further exploration for leafhopper egg parasitoids in northern and central regions of Chile to identify new perspective biological control agents that are more adapted to Mediterranean climate (winters and wet summers), which are similar to California climate. In addition, it is possible that G. tuberculifemur may be a good candidate for the biological control of the recently discovered H. coagulata in Easter Island, Chile.

An American Hybrid Vineyard in the Texas Gulf Coast: Analysis within a Pierce’s Disease Hot Zone

The incidence of Pierce’s disease (PD) within an American hybrid vineyard in the Texas Gulf Coast was evaluated. Multiple viticultural parameters and <i>Xylella fastidiosa</i> levels were assessed for the varieties Black Spanish, Blanc du Bois, and Cynthiana in this region of intense disease pressure. Potential PD symptoms were observed uniformly in Cynthiana and sporadically in Blanc du Bois and Black Spanish postharvest. Consistent with these symptoms, Cynthiana showed significantly lower crop yield than either Blanc du Bois or Black Spanish and had significantly higher positive ELISA values for <i>X. fastidiosa</i> in summer petioles. Individual Cynthiana vines with higher bacterial levels as determined by ELISA exhibited correspondingly lower yields (<i>p</i> = 0.06). Indirect immunofluorescence microscopy of petiole xylem sap and PCR of petiole DNA confirmed positive <i>X. fastidiosa</i> ELISA readings in all three varieties. Cynthiana petioles tested by ELISA showed a dramatic increase in bacterial levels over the growing season. In contrast, bacterial levels in Black Spanish and Blanc du Bois remained lower all season despite high disease pressure. An evaluation of potential local plant reservoirs for <i>X. fastidiosa</i> demonstrated that at least six asymptomatic plant species tested positive by both ELISA and PCR: <i>Ilex vomitoria</i>, <i>Vitis mustangensis, Rubus trivialis</i>, <i>Platanus occidentalis</i>, <i>Lippia nodiflora,</i> and <i>Lonicera japonica</i>. In addition, June insect traps contained a significant number of <i>X. fastidiosa</i>-positive glassy-winged sharpshooters, <i>Homalodisca coagulata</i>, and several positive sharpshooters identified as <i>Oncometopia orbona,</i> suggesting the presence of multiple species of insect vector in this region.

Anatomy and Histology of Reproductive Organs of Female &lt;I&gt;Homalodisca coagulata&lt;/I&gt; (Hemiptera: Cicadellidae: Proconiini), with Special Emphasis on Categorization of Vitellogenic Oocytes

The anatomy and histology of female Homalodisca coagulata (Say) (Hemiptera: Cicadellida: Proconiini) reproductive organs is described using light microscopy. The reproductive organs of H. coagulata consist of one pair of ovaries, each with 10 telotrophic ovarioles, a pair of lateral oviducts, a common oviduct, a spermatheca, an enlarged genital duct, a complex bursa copulatrix, a vagina, two types of accessory glands, and a genital chamber. The reproductive organs follow the general pattern seen in cicadellids. The complex bursa copulatrix, important in copulation and sperm transfer, is described. A set of morphological criteria were selected, based on the stage of oocyte development, and used to evaluate and assign the rank of ovarian development for field-collected individuals and to assess the overall reproductive status of female insect populations. A principal component analysis of morphological and physiological characteristics suggests that the ovarian ranks reflect the reproductive status of the females. Understanding reproductive status and patterns is critical for determining the optimal time to implement control methods to suppress H. coagulata populations in southern California.

Patterns of &lt;I&gt;Xylella fastidiosa&lt;/I&gt; Colonization on the Precibarium of Sharpshooter Vectors Relative to Transmission to Plants

Xylellafastidiosa is a bacterial plant pathogen that causes many plant diseases, including PierceOs disease of grapevines. Sharpshooter leafhopper (Hemiptera: Cicadellidae: Cicadellinae) vectors transmit this bacterium to plants. Although the basic mechanism of pathogen transmission is not completely understood, previous studies implicated the foregut of infected insects as the source of bacterial inoculum because infective nymphs lose transmissibility after molting and no latent period is required for transmission. Scanning electron microscopy documented the spatial distribution of X. fastidiosa in the foreguts of the sharpshooter Graphocephala atropunctata (Signoret) after short (1-d) and long (4-d) acquisition access periods on infected plants and various inoculation access periods on grape test plants. After 1-d acquisition and 1-d inoculation periods, cells had only colonized portions of the precibarium between the stylets and the cibarial pump, often embedded in a matrix-like material. After long (15-d) periods after acquisition, we found bacterial cells attached polarly to the insectOs cuticle in a regular pattern throughout the precibarium but absent from speciÞc locations on both pharynges. Insects sampled for microscopy 2 wk after long acquisition access periods and that contained bacteria in their mouthparts transmitted X. fastidiosa to healthy grapevines independently of the length of the acquisition access period. In contrast, individuals free of bacteria on the precibarium did not transmit the pathogen. After successive short acquisition and inoculation periods, 5o f 12X. fastidiosa-positive insects did not transmit to plants. We also did transmission experiments with the sharpshooter Homalodisca coagulata; however, only one of 30 individuals had X. fastidiosa attached to its precibarium, and none transmitted it to plants. Our results suggest that sharpshooters introduce into plants X. fastidiosa cells that detach from the precibarial canal during feeding.

Glassy-Winged Sharpshooters Depend on Two Endosymbionts

The fruit of the vine does not suffice for the voracious glassy-winged sharpshooter, an insect pest and vector of diseases that feeds on grapevines, citrus, and other plant species. Remarkably, this insect also very much depends on not one, but two bacterial endosymbionts—one supplying amino acids and the other, vitamins—to sustain itself, according to Jonathan Eisen of the University of California, Davis, and his collaborators, who report their findings in the June PLoS Biology.

Detection and Differentiation of &lt;I&gt;Xylella fastidiosa&lt;/I&gt; 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.