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Lethal bronzing (LB) is a fatal palm disease caused by the phytoplasma ' Candidatus Phytoplasma aculeata'. This disease causes significant economic losses in palm industries and landscapes. The American palm cixiid, Haplaxius crudus , recently was identified as the vector of the phytoplasma. However, knowledge about LB phytoplasma transmission is limited due to the lack of a method to generate phytoplasma-infected insects in the laboratory. In this study, the acquisition and transmission of the LB phytoplasma by H. crudus were investigated. Successful acquisitions of the phytoplasma by H. crudus were observed at 2 days acquisition access period on LB-infected palm spear leaves. Analyses revealed increased phytoplasma infection rates of H. crudus with longer acquisition access periods and latent periods. A significantly higher phytoplasma infection rate was shown after various acquisition access periods and latent periods than the infection rate of the field-collected H. crudus population. Transmission of the phytoplasma from LB-infected spear leaves to sucrose media by H. crudus also was observed using digital PCR assays. These results further support the vector status of H. crudus and offer valuable information to understand LB phytoplasma transmission. Additionally, these results generate a critical baseline for future LB phytoplasma-vector research by providing a way to generate vectors with high phytoplasma infection rates in the laboratory setting.

The potato psyllid (Bactericera cockerelli Sulc) is an economically important insect pest of solanaceous crops such as potato, tomato, pepper, and tobacco. Historically, the potato psyllid's range included central United States, Mexico, and California; more recently, populations of this insect have been reported in Central America, the Pacific Northwest, and New Zealand. Like most phytophagous insects, potato psyllids require symbiotic bacteria to compensate for nutritional deficiencies in their diet. Potato psyllids harbor the primary symbiont, Candidatus Carsonella ruddii, and may also harbor many secondary symbionts such as Wolbachia sp., Sodalis sp., Pseudomonas sp., and others. These secondary symbionts can have an effect on reproduction, nutrition, immune response, and resistances to heat or pesticides. To identify regional differences in potato psyllid bacterial symbionts, 454 pyrosequencing was performed using generic 16S rRNA gene primers. Analysis was performed using the Qiime 1.6.0 software suite, ARB Silva, and R. Operational taxonomic units were then grouped at 97% identity. Representative sequences were classified to genus using the ARB SILVA database. Potato psyllids collected in California contained a less diverse microbial community than those collected in the central United States and Central America. The crop variety, collection year, and haplotype did not seem to affect the microbial community in potato psyllids. The primary difference between psyllids in different regions was the presence and overall bacterial community composition of Candidatus Carsonella ruddii and Wolbachia.

Accurate detection and quantification of Candidatus Liberibacter solanacearum (CLs), the putative causal agent of zebra chip disease of potato (Solanum tuberosum L.), in the potato psyllid, Bactericera cockerelli (Sulc), has become necessary to better understand the biology of the disease cycle. Studies on the transmission efficiency of potato psyllids have shown inconsistencies with field surveys. There have also been reports of laboratory colonies inexplicably losing and regaining CLs infection as detected by polymerase chain reaction (PCR). Until now, DNA primers were used to detect CLs in potato psyllid tissue using conventional polymerase chain reaction (PCR) and gel electrophoresis or by real-time quantitative PCR. In this study, CLs was detected using bacterial tag-encoded FLX amplicon pyrosequencing (bTEFAP) at levels identifiable by PCR, and low levels, including samples with only one cell of CLs. Potato psyllids with <300 pyrosequencing reads did not show positive using conventional PCR. These results indicate that the currently accepted PCR diagnostic technique produces false negatives due to detection limits higher than what is generally present in field collected psyllids, and also provides an explanation as to why laboratory colonies seem to lose and regain CLs infection.

The potato psyllid (Bactericera cockerelli, Sulc) is an invasive pest of solenaceous plants including potatoes (Solanum tuberosum L.)and tomatoes (Solanum lycopersicum L.). The insect transmits the phytopathogen Candidatus Liberibacter solanacearum, which has been identified as the causal agent of Zebra Chip in potatoes. The microbiome of the potato psyllid provides knowledge of the insect's bacterial makeup which enables researchers to develop targeted biological control strategies. In this study, the microbes associated with four B. cockerelli life stages were evaluated by 16S bTEFAP pyrosequencing. The sequences were compared with a 16S-rDNA database derived from NCBI's GenBank. Some bacteria identified are initial discoveries. Species of Wolbachia, Rhizobium, Gordonia, Mycobacterium, Xanthomonas and others were also detected and an assessment of the microbiome associated with B. cockerelli was established.

A series of oligonucleotide primer pairs covering the entire genome of Solenopsis invicta virus 1 (SINV-1) were used to probe the genome of its host, S. invicta, for integrated fragments of the viral genome. All of the oligonucleotide primer sets yielded amplicons of anticipated size from cDNA created from an RNA template from SINV-1. However, no corresponding amplification was observed when genomic DNA (from 32 colonies of S. invicta) was used as template for the PCR amplifications. Host DNA integrity was verified by amplification of an ant-specific gene, SiGSTS1. The representation of fire ant colonies included both social forms, monogyne and polygyne, and those infected and uninfected with SINV-1. Furthermore, no amplification was observed from genomic DNA from ant samples collected from Argentina or the US. Thus, it appears that SINV-1 genome integration, or a portion therein, has not likely occurred within the S. invicta host genome., (Published by Elsevier Inc.)

The glassy-winged sharpshooter, Homalodisca vitripennis (Germar), is an invasive pest that has spread across the southern and western United States. H. vitripennis is highly polyphagous and voracious, feeding on at least 100 plant species and consuming up to 100 times its weight in xylem fluid daily. The insect is a vector of the phytopathogen Xylella fastidiosa (Wells), which is the causative agent of Pierce's disease in grapevines. To evaluate the microbial flora associated with H. vitripennis, total DNA extracts from hemolymph, alimentary canal excretions, and whole insect bodies were subjected to 16S rDNA pyrosequencing using the bTEFAP methodology and the resulting sequences (370-520 bp in length) were compared with a curated high quality 16S database derived from GenBank http://www.ncbi.nlm.nih.gov. Species from the genera Wolbachia, Delftia (formerly Pseudomonas), Pectobacterium, Moraxella, Serratia, Bacillus, and many others were detected and a comprehensive picture of the microbiome associated with H. vitripennis was established. Some of the bacteria identified in this report are initial discoveries; providing a breadth of knowledge to the microbial flora of this insect pest can serve as a reservoir of information for developing biological control strategies., (© 2011 Entomological Society of America)

A red pigment is contained in the wing veins of the glassy-winged sharpshooter, Homalodisca vitripennis (Hemiptera: Cicadellidae). This insect is the main vector of the plant-pathogenic bacterium Xylella fastidiosa Wells (Xanthomonadales: Xanthomonadaceae), the causal agent of Pierce's disease of grapevines. Over the course of the H. vitripennis lifespan, the red pigment darkens and eventually becomes brown/black in color. These pigments are believed to be pheomelanin and eumelanin, respectively. The age of H. vitripennis can be determined by calculating the amount of red pigment found in the wings by analyzing high resolution wing photographs with image analysis software. In this study, a standard curve for the age determination of H. vitripennis was developed using laboratory-reared insects of known ages varying from 1 to 60 days. The impact of three environmental conditions on these readings was also investigated and found to have little effect on the age determination, and could be easily accounted for. Finally, field collected insects from several Central Texas vineyards were successfully analyzed for age determination suggesting that the annually reported influx of H. vitripennis was composed almost entirely of older insects.

Solenopsis invicta Buren (Hymenoptera: Formicidae), the red imported fire ant is native to South America but has invaded areas of the southeastern US, and parts of Southern California. The S. invicta virus-1 (SINV-1) is a positive sense, single-stranded RNA picorna-like virus that only affects Solenopsis species. The virus can infect all caste members and developmental stages. Infection of SINV-1 can result in colony collapse in less than 3 months under laboratory conditions. This study screened S. invicta colonies from Texas for the presence of SINV through Reverse Transcriptase PCR (RT-PCR). Positive samples were genetically characterized by direct sequencing and compared with known picorna-like viruses. SINV-1 was detected in ant colonies from Smith and Henderson TX counties. Amino acid sequence comparison of SINV-1 (TX5) ORF2 region showed homologies of 96% with SINV-1, 97% with SINV-1A, 17.6% with SINV-2, and 20.7% with SINV-3. In addition, SINV-1 (TX5) was compared to 18 other Dicistroviridae viruses. Ant-infecting viruses may provide new approaches to suppressing these important economic pests., ((c) 2010 Elsevier Inc. All rights reserved.)

The glassy-winged sharpshooter, Homalodisca vitripeninis Germar (Hemiptera: Cicadellidae), is a xylophagous insect that is an endemic pest of several economically important plants in Texas. H. vitripennis is the main vector of Xylella fastidiosa Wells (Xanthomonadales: Xanthomonadaceae), the bacterium that causes Pierce's disease of grapevine and can travel long distances putting much of Texas grape production at risk. Understanding the movement of H. vitripennis populations capable of transmitting X. fastidiosa into Pierce's-disease-free areas is critical for developing a management program for Pierce's disease. To that end, the USDA-APHIS has developed a program to sample vineyards across Texas to monitor populations of H. vitripennis. From this sampling, H vitripennis collected during 2005 and 2006 over the months of May, June, and July from eight vineyards in different regions of Texas were recovered from yellow sticky traps and tested for the presence of X. fastidiosa. The foregut contents were vacuum extracted and analyzed using RT-PCR to determine the percentage of H. vitripennis within each population that harbor X. fastidiosa and have the potential to transmit this pathogen. H. vitripennis from vineyards known to have Pierce's disease routinely tested positive for the presence of X. fastidiosa. While almost all H. vitripennis collected from vineyards with no history of Pierce's disease tested negative for the presence of the pathogen, three individual insects tested positive. Furthermore, all three insects were determined, by DNA sequencing, to be carrying a strain of X. fastidiosa homologous to known Pierce's disease strains, signifying them as a risk factor for new X. fastidiosa infections.

Evidence that symbiotic microorganisms can impact the development and fitness of insects has been shown in many species. Hemolymph-associated symbiotic bacteria have been identified in larvae of Solenopsis invicta Buren, the red imported fire ant; however, their association with adult red imported fire ants and the mode by which these organisms are transmitted from queens to offspring are not well known. In this study, Bacillus spp. bacteria were routinely recovered in the hemolymph of queen S. invicta. Genetic analysis of the 16S gene confirmed the most common bacteria isolated were Bacillus spp.; several Staphylococcus species were also collected. Ovaries from reproductive and nonreproductive queens, freshly laid eggs, first-instar larvae, and hemolymph were collected from queens and analyzed for the presence of specific Bacillus spp. bacteria. It was indicated that these bacteria may be transmitted vertically from queen to progeny.

Yellow sticky traps were placed in six vineyards in central Texas from 2003 to 2006 and in locations outside the vineyards in 2004-2006. In total, 72 collections on 55 dates were examined. Xylem fluid-feeding insects were removed and identified to species and then analyzed by polymerase chain reaction to determine the presence or absence of Xylella fastidiosa Wells et al. Of the 1318 insects removed, 13 species were found, dominated by Homalodisca vitripennis (Germar), Clastoptera xanthocepahala Germar, and Graphocephala versuta (Say). Insects testing positive for X. fastidiosa were analyzed further using fluorescence resonance energy transfer probes to determine the genotype of the bacterium, which fell into four groups: subspecies fastidiosa, multiplex, sandyi, and unknown subspecies. Vineyards known to be affected by Pierce's disease had more insects that were contaminated by the bacterium than those that were not as affected. X. fastidiosa subsp. fastidiosa, the causative agent of Pierce's disease, was found more commonly in insects collected from vineyards than from insects collected outside the vineyards. Conversely, the subspecies multiplex and sandyi, which are not known to cause disease in grape, were more commonly found in insects collected outside the vineyard. The percentage of individuals contaminated with the bacterium increased over the course of the growing season, and the data suggest that vector insects acquired X. fastidiosa subsp. fastidiosa from infected grapevines, a necessary precursor for vine to vine transmission to occur. Management options, including the use of systemic insecticides and plant roguing, would be effective for this type of transmission model.

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.

Pathotype differentiation within Xylella fastidiosa (Wells) is necessary to accurately identify the causal agents of several leaf scorch diseases in agronomic crops and ornamentals. Three strain groups, Pierce's disease (PD), almond leaf scorch (ALS), and oleander leaf scorch (OLS), have been described in California and inhabit multiple hosts with varied severity. An SYBR Green-based quantitative real-time PCR (QRT-PCR) protocol with a single primer set was developed for X. fastidiosa genotype differentiation utilizing melting temperature (T(m)) analysis. The goal of this work was to develop a rapid diagnostic tool that could separate X. fastidiosa strains from one another similar to the placement in previous reports. Placement of eight PD, six ALS, and six OLS strains into respective strain groups was consistent with T(m) curve analysis using a single primer set. These groupings were consistent with published data on the phylogenetic placement of the strains.

Homalodisca coagulata Say (Hemiptera: Cicadellidae) is a major agronomic pest because it transmits Xylella fastidiosa (Wells), the bacterium that causes Pierce's disease of grapevine. The ability to easily detect X. fastidiosa in populations of H. coagulata facilitates epidemiological studies and development of a monitoring program supporting disease management. Such a program depends on a detection protocol that is rapid, reproducible, and amenable to large sample sizes, while remaining sensitive enough to detect low amounts of pathogen DNA. In this study, we developed an improved method to speed DNA extraction by implementing a simple vacuum step that replaces labor- and time-intensive maceration of tissue samples and that is compatible with manufactured DNA extraction kits. Additionally, we have developed a SYBR Green-based real-time (RT)-polymerase chain reaction (PCR) system, which uses traditional PCR primers that are relatively inexpensive and effective. Using this extraction/RT-PCR system, we found no statistically significant differences in the detection of X. fastidiosa among samples that were either immediately extracted or stored dry or in mineral oil for 10 d at -4 degrees C. In further testing, we found no significant reduction in detection capabilities for X. fastidiosa-fed H. coagulata left in the sun on yellow sticky cards for up to 6 d. Therefore, we recommend a field-based detection system that includes recovery of H. coagulata from sticky traps for up to 6 d after trapping, subsequent freezing of samples for as long as 10 d before vacuum extraction is performed, and detection of the bacterium by SYBR Green-based RT-PCR.

Alcaligenes xylosoxidans subsp. denitrificans, originally isolated from the cibarial region of the foregut of the glassy-winged sharpshooter (Homalodisca coagulata), was transformed using the Himar1 transposition system to express EGFP. Seedlings of six potential host plants were inoculated with transformed bacteria and 2 weeks later samples were taken 5 cm away and analyzed by quantitative real-time PCR using primers designed to amplify the gene insert. The largest colony of 3,591,427 cells/2 cm of A. xylosoxidans subsp. denitrificans was found in Citrus limon, with almost all plants testing positive in both trials. The amount of colonization decreased in the other plants tested in the following order: orange (Citrus sinensis "sweet orange") > chrysanthemum (Chrysanthemum grandiflora cv. "White Diamond") > periwinkle (Vinca rosea) > crepe myrtle (Lagerstroemia indica) > grapevine (Vitis vinifera cv. Chardonnay). The bacterium's preference for citrus paralleled the host insect's preference for this same plant. Additional tests determined that A. xylosoxidans subsp. denitrificans thrives as a nonpathogenic, xylem-associated endophyte.

The recent spread of the plant pathogenic bacterium Xylclla fastidiosa Wells et al. by an invasive vector species, Homalodisca coagulata Say, in southern California has resulted in new epidemics of Pierce's disease of grapevine. Our goal is to develop an efficient method to detect low titers of X. fastidiosa in H. coagulata that is amenable to large sample sizes for epidemiological studies. Detection of the plant pathogenic bacterium X. fastidiosa in its insect vector is complicated by low titers of bacteria, difficulty in releasing it from the insect mouthparts and foregut, and the presence of substances in the insect that inhibit polymerase chain reaction (PCr). To select the optimal protocol for DNA extraction to be used with PCR, we compared three standard methods and 11 commercially available kits for relative efficiency of X. fastidiosa DNA extraction in the presence of insect tissue. All of the protocols tested were proficient at extracting DNA from pure bacterial culture (1 x 10(5) cells), and all but one protocol successfully extracted sufficient bacterial DNA in the presence of insect tissue. Three DNA extraction techniques, immunomagnetic separation, the DNeasy Tissue kit (Qiagen, Hercules, CA), and Genomic DNA Purification kit (Fermentus, Hanover, MD), were compared more closely using a dilution series of X. fastidiosa (5000-0 cells) with and without insect tissue present. The DNeasy Tissue kit was the best kit tested, allowing detection of 5 x 10(3) X. fastidiosa cells with an insect head background.

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 Pierce's 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.

Yellow vine (YV) of cucurbits, associated with a phloem-limited bacterium, causes rapid wilting and death in affected plants. In a previous study, experimental insecticide-treated plots had a lower incidence of YV than untreated plots, suggesting that insects were involved in the transmission of the bacterium. In the study reported here, we compared the incidence of YV and polymerase chain reaction (PCR) detection of the YV bacterium in noncovered squash plants (Cucurbita pepo var. melopepo) with plants covered with fine-mesh fabric secured in such a way that insects were excluded. Rows of squash were covered with row mesh cover that was stretched over hoops and anchored in the soil. The row cover was removed after 40 or 50 days, at which time all plants were sampled destructively by harvesting the crown and root. In the first experiment, 3% of the noncovered plants had foliar symptoms, 7% were positive with the use of Dienes' stain, and 25% were positive when analyzed by PCR with specific primers. No covered plants were positive by any detection method, and no plants in the second experiment had foliar symptoms or tested positive with Dienes' stain. However, 20% of noncovered and 0% of covered plants were PCR positive. These data support the hypothesis that insects were involved in the transmission of the bacterium.