Blake Bextine

Delivery of a Genetically Marked Alcaligenes 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 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.

Establishment of a Genetically Marked Insect-Derived Symbiont in Multiple Host Plants

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.

A SYBR Green-Based Real-Time Polymerase Chain Reaction Protocol and Novel DNA Extraction Technique to Detect Xylella fastidiosa in Homalodisca coagulata

Abstract 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°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.

Evaluation of methods for extracting Xylella fastidiosa DNA from the glassy-winged sharpshooter.

Abstract The recent spread of the plant pathogenic bacterium Xylella 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 × 105 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 (5,000–0 cells) with and without insect tissue present. The DNeasy Tissue kit was the best kit tested, allowing detection of 5 × 103 X. fastidiosa cells with an insect head background.

Comparison of Whole-Tissue and Xylem Fluid Collection Techniques to Detect Xylella fastidiosa in Grapevine and Oleander

Xylella fastidiosa is the xylem-limited bacterium that causes Pierces disease of grapevine and oleander leaf scorch. Detection of this pathogen prior to symptom development is critical for improved management of the disease. Enzyme-linked immunosorbent assay (ELISA) and polymerase chain reaction (PCR) currently are used for routine detection of the pathogen; however, both detection methods are limited by low titer or patchy distribution of the bacterium within a host plant. In the study reported here, we directly compared X. fastidiosa detection in whole-tissue samples with xylem fluid samples from grapevine and oleander. Collection of xylem fluid samples improved sensitivity of pathogen detection by ELISA (41.0%) compared with whole-tissue samples (20.5%) in asymptomatic grapevine. Additionally, pathogen detection in asymptomatic grapevine by PCR also was improved when xylem samples were tested (66.7%) compared with whole-tissue samples (23.1%). There were no differences in frequency of detection of X. fastidiosa in symptomatic grapevines by ELISA or PCR dependent upon sample collection method. Assays of xylem fluid samples did not improve detection of X. fastidiosa in symptomatic or asymptomatic oleander compared with assays of whole tissue. Finally, in a direct comparison of ELISA and PCR, we found no significant differences in frequencies of positive grapevine or oleander samples detected.

Novel Methods of Monitoring the Feeding Behavior of Homalodisca coagulata (Say) (Hemiptera; Cicadellidae)

Abstract The glassy-winged sharpshooter, Homalodisca coagulata (Say), is an important agricultural pest because it is an effective vector of Xylella fastidiosa, the pathogen that causes Pierce’s disease in grapevines. Knowledge of the feeding behavior of H. coagulata is important in understanding pathogen transmission, and this knowledge is important in developing innovative pathogen control strategies. Ingestion of fluid by sharpshooters was monitored as movement of fluid from reservoirs connected to short stems of plant tissue. We quantified the amount of fluid processed while the insects were freely moving on the plants stems offered for feeding. Females fed longer than males, and both ingested large amounts of plant fluid and both excreted large amounts of fluid. Excreta droplets were often actively flung from the body by flicking the abdomen. While actively ingesting, the abdomen made exaggerated movements that stopped during excretion. These movements only appeared after mouthparts penetrated the plant tissues. The abdominal movements were correlated with ingestion of plant fluids as monitored by fluid uptake from the reservoir.

Laboratory-based monitoring of an insect-transmitted plant pathogen system

winged sharpshooter, Homalodisca coagulata (Say), is challenging to study, not only because the bacterium is fastidious, but also because the insect does not survive well in captivity, and it will not feed from an artificial diet (i.e., parafilm sachet). We developed a simple and efficient transmission cycle for the study of X. fastidiosa transmission by H. coagulata, allowing collection of sufficient transmission data in 1 week. Specific numbers of cells can be detected both in the plant tissue and within the insect vector by realtime PCR (7). Real-time PCR is more sensitive than traditional PCR and is a single step process, thus reducing the possibility of error and speeding up detection time. A real-time PCR protocol for detecting the citrus variegated chlorosis strain of X. fastidiosa has already been established as a superior technique compared to traditional PCR (8). Real-time PCR was performed in a Rotor-Gene™ 3000 (Corbett Research, Mortlake, NSW, Australia) using iQ™ Supermix (Bio-Rad Laboratories, Hercules, CA, USA) in 20-µL reactions with 525 nM of each X.

Homalodisca coagulata (Hemiptera: Cicadellidae) Feeding Posture

Abstract The sharpshooter Homalodisca coagulata Say (Hemiptera: Cicadellidae) is a polyphagous insect that feeds primarily from the xylem vessels of host plants. This insect is an efficient vector of the xylem-limited, plant-pathogenic bacterium Xylella fastidiosa Wells, which is the causal agent of several important diseases in ornamental and agricultural crops. In wild populations, H. coagulata have been observed to almost exclusively orient with their anterior facing toward the roots (AFR), regardless of branch position. The orientation of H. coagulata seemed to be chosen before stylet insertion. Neither masking plant chemical and morphological surface cues nor placing test plants in the absence of light affected body posture. The introduction of 1.5-V direct current electricity to the plants resulted in altered body positioning. Removal of both front tarsi influenced body position; however, removal of only the right or left front tarsi had no effect on H. coagulata body position. Removal or masking of right, left, or both antennae resulted in altered body position.

Development of Quantitative Real-Time Polymerase Chain Reactionprotocols for rapid detection and differentiation of Xylella fastidiosasubsp. fastidiosa and Xylella fastidiosa subsp. multiplex

Xylella fastidiosa is a gram-negative, xylem-limited, plant pathogenic bacterium that is transmitted between hosts by the glassy-winged sharpshooter ( Homalodisca vitripennis) . Multiple subspecies of X. fastidiosa occur, exhibiting some degree of host specificity. X. fastidiosa subsp. fastidiosa is the causal agent of Pierce’s disease of grapevine. X. fastidiosa subsp. multiplex and X. fastidiosa subsp. sandyi are commonly found in North America but do not cause Pierce’s disease. Rapid diagnostics to determine presence of X. fastidiosa and differentiation of these subspecies is necessary for effective management and prevention of Pierce’s disease. In this study, three methods to distinguish X.fastidiosa subspecies using Quantitative Real-Time Polymerase Chain Reaction were compared. SYBR ® green, Eva Green ® , and Takara SYBR Green ® melt curve analysis of partial gyraseB amplicons, zot1 gene amplicons, and five tonB amplicons were evaluated for consistency and quality. Diagnostic protocols based on TaqMan ® and Molecular Beacon ® hybridization probes were developed with an emphasis placed on a X. fastidiosa subsp. multiplex insertion in the zot1 gene. We found SYBR ® Green and TaqMan ® based diagnostic protocols did not provide the necessary resolution for accurate and consistent differentiation of X. fastidiosa subspecies. Diagnostic protocols we developed utilizing the Molecular Beacon ® probe allow for highly specific and reliable differentiation of X. fastidiosa subspecies, even in cases where subspecies were mixed in solution. These new methods provide a more reliable protocol by which the subspecies of X. fastidiosa can be rapidly identified for the purposes of laboratory study and sample diagnostics.