David J. W. Morgan

Population Genetic Structure of Homalodisca coagulata (Homoptera: Cicadellidae), the Vector of the Bacterium Xylella fastidiosa Causing Pierce’s Disease in Grapevines

Abstract In the current study, inter-simple sequence repeat (ISSR) primers (p-13 and p-15) were used to estimate the population genetic structure of the sharpshooter Homalodisca coagulata (Say) (Homopera: Cicadellidae). Eighteen populations from throughout the United States and a population from Tahiti, French Polynesia, were analyzed. Populations were arbitrarily assigned to three regions: southeastern, southwestern, and western. Exact tests for population differentiation indicated highly significant differences in marker frequencies among the 18 populations with both primers. Analyses of molecular variance also indicated significant geographic structuring with both primers. A dendrogram based on Reynolds coancestry distance performed with p-15 clustered the U.S. populations into two main groups. The southeastern populations were grouped into one cluster and the southwestern and western populations into a second cluster. Within the western region, dendrograms produced with p-13 and p-15 showed in both cases that two populations (Edison and Bakersfield) clustered as outliers. The average divergence (D) among all populations was 0.099. Divergence values of 0.254, 0.103, and 0.102 were observed when comparing Bakersfield and the southeastern, southwestern, and western populations, respectively. Within the western region, D values for Bakersfield were 1.8- (p-13) and 2.4-fold (p-15) higher than the D of the western populations. The present results suggest that a subset of insects in California may have their origins in the southwestern region (Texas); furthermore, these results are suggestive of more than one founding event in California and/or biotypes or geographic races.

Spatiotemporal Colonization of Xylella fastidiosa in its Vector Supports the Role of Egestion in the Inoculation Mechanism of Foregut-Borne Plant Pathogens

The pathogen that causes Pierces disease of grapevine, Xylella fastidiosa, is the only known bacterial, arthropod-transmitted plant pathogen that does not circulate in the vectors hemolymph. Instead, bacteria are foregut-borne, persistent in adult vectors but semipersistent in immatures (i.e., bacteria colonize cuticular surfaces of the anterior foregut, are retained for hours to days, but are lost during molting). Yet, exactly how a sharpshooter vector inoculates bacteria from foregut acquisition sites is unknown. The present study used confocal laser-scanning microscopy to identify locations in undissected, anterior foreguts of the glassy-winged sharpshooter colonized by green fluorescent protein-expressing X. fastidiosa. Spatial and temporal distributions of colonizing X. fastidiosa were examined daily over acquisition access periods of 1 to 6 days for both contaminated field-collected and clean laboratory-reared Homalodisca vitripennis. Results provide the first direct, empirical evidence that established populations of X. fastidiosa can disappear from vector foreguts over time. When combined with existing knowledge on behavior, physiology, and functional anatomy of sharpshooter feeding, present results support the idea that the disappearance is caused by outward fluid flow (egestion) not inward flow (ingestion) (i.e., swallowing). Thus, results support the hypothesis that egestion is a critical part of the X. fastidiosa inoculation mechanism. Furthermore, results suggest a cyclical, spatiotemporal pattern of microbial colonization, disappearance, and recolonization in the precibarium. Colonization patterns also support two types of egestion, termed rinsing and discharging egestion herein. Finally, comparison of acquisition results for field-collected versus laboratory-reared sharpshooters suggest that there may be competitive binding for optimum acquisition sites in the foregut. Therefore, successful inoculation of X. fastidiosa may depend, in large part, on vector load in the precibarium.

Influences of Temperature on Homalodisca vitripennis (Hemiptera: Cicadellidae) Survival Under Various Feeding Conditions

ABSTRACT Survival of the glassy-winged sharpshooter, Homalodisca vitripennis (Germar) (Hemiptera: Cicadellidae), was studied under various constant temperatures and feeding conditions. When provided a host plant (Citrus limon L. Burm. f.) to feed on during a 21-d trial, 100% mortality occurred at 0.1, 3.2, and 40.1°C, whereas an average of 74–76% of adults survived in the 13.2–24.5°C range. When individually confined with moist cotton, adult longevity was greatest (16.3 d) at 13.3°C, but it was <3 d at -2.4 and 36.2°C. In a companion study comparing the presence versus absence of a host plant, the presence of a host plant was not a significant factor influencing survival at temperatures ≤7.8°C but was at temperatures ≥18.9°C. The relationship between temperature and survival was described by a nonlinear function that estimated the optimum temperature in each feeding regimen: no host plant or moist cotton (5.5°C), moist cotton (9.9°C), and accessible host plant (25.1°C). The model quantitatively predicted that H. vitripennis would survive longer periods at a wider temperature regimen when provided with a host plant than when provided with water alone (moist cotton) or when provided with neither plant host nor water. Our results suggest that continuous exposure to either low (<5°C) or high (>30°C) temperatures are detrimental for adult survival. Specifically, low temperatures caused early mortality because of inhibition of feeding activity and presumably this threshold lies between 7.8 and 13.2°C. Furthermore, this study clearly shows that temperature may influence the survival of H. vitripennis adults regardless of feeding regimens, and its implications for population dynamics are discussed.

Evaluation of Molecular Markers for Discriminating Gonatocerus morrilli (Hymenoptera: Mymaridae): a Biological Control Agent for Homalodisca vitripennis

Abstract We tested the utility of molecular markers for distinguishing between two closely related species, Gonatocerus morrilli (Howard) and Gonatocerus walkerjonesi S. Triapitsyn (Hymenoptera: Mymaridae), to evaluate whether postrelease G. morrilli specimens could be discriminated in the field. Initially, postrelease specimens from California collected in 2002 and 2003 were analyzed. Amplification size of the internal transcribed spacer (ITS) region 2 demonstrated that all of the specimens were of the G. walkerjonesi ITS2 genotype. Inter-simple sequence repeat-polymerase chain reaction DNA fingerprinting of specimens from the original G. morrilli“release” colony demonstrated that the DNA banding pattern was superimposable to that of G. walkerjonesi, confirming that the G. morrilli colony was contaminated. A new G. morrilli colony was initiated in spring 2005, and we continued to survey random postrelease specimens from the 2004–2006 collections. As expected, from 2004 and most of 2005, only the G. walkerjonesi ITS2 genotype was detected. In fall 2005 and in the spring and fall 2006, we detected the G. morrilli ITS2 genotype at sites where the new colony was previously released. Analyses with two newly developed “one-step” species-specific ITS2 diagnostic markers were in accordance with the results of the markers described above, demonstrating the usefulness of the former studies of natural enemy establishment in biological control programs.

Estimation of Feeding Threshold for Homalodisca vitripennis (Hemiptera: Cicadellidae) and Its Application to Prediction of Overwintering Mortality

ABSTRACT The glassy-winged sharpshooter, Homalodisca vitripennis (Germar), vectors the bacterium Xylella fastidiosa that induces Pierces 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.