|De leon, Jesus|
Submitted to: CDFA Pierce's Disease Control Program Research Symposium
Publication Type: Proceedings
Publication Acceptance Date: 11/7/2005
Publication Date: 12/1/2005
Citation: De Leon, J.H., Fournier, V., Hagler, J.R., Daane, K. 2005. Development of molecular diagnostic markers for glassy-winged and smoke-tree sharpshooters for use in predator gut content examinations. Proceedings of CDFA Pierce's Disease Control Program Research Symposium, December 5-7, 2005, San Diego, CA. p.293-297. Interpretive Summary: The purpose of the present study was to develop molecular diagnostic markers toward both the glassy-winged (GWSS), and smoke-tree (STSS) sharpshooters in order to begin to identify key predators of these sharpshooters. Both of these sharpshooters vector the bacterium, Xylella fastidiosa, the causative agent of Pierce's disease of grapevines. A total of six molecular diagnostic markers were developed toward these sharpshooters. All markers were highly specific toward their targets. Laboratory experiments demonstrated that both GWSS, and STSS eggs, nymphs, and adults were detected in the gut contents of several key predators. The results are important because they will allow us, for the first time, to begin to understand the ecology of the GWSS/STSS-predator complex inactions.
Technical Abstract: To aid in identifying key predators of Proconiini sharpshooter species present in California, we developed and tested molecular diagnostic markers for the glassy-winged sharpshooter Homalodisca coagulata (Say) and smoke-tree sharpshooter Homalodisca liturata (Ball) (Homoptera: Cicadellidae: Proconiini). Two different types of markers were compared: those targeting single-copy sequence characterized amplified regions (SCAR), and mitochondrial markers targeting the multi-copy cytochrome oxidase subunit genes I (COI) and II (COII). A total of six markers were developed: two SCAR, and four mitochondrial COI or COII markers. Specificity assays demonstrated that SCAR marker HcF5/HcR7 was H. coagulata-specific and HcF6/HcR9 was H. coagulata/H. liturata-specific. COI (HcCOI-F/R), and COII (HcCOII-F4/R4) markers were H. coagulata-specific, COII (G/S-COII-F/R) marker was H. coagulata/H. liturata-specific, and lastly, COII marker (Hl-COII-F/R) was H. liturata-specific. Sensitivity assays using genomic DNA showed the COI marker to be the most sensitive marker with a detection limit of 6 pg of DNA. This marker was 66-fold more sensitive than marker Hl-COII-F/R that showed a detection limit of 400 pg of DNA. In addition, the COI marker was 4.2-fold more sensitive than the COII marker. In predator gut assays, the COI and COII markers demonstrated significantly higher detection efficiency than the SCAR markers. Furthermore, the COI marker demonstrated slightly higher detection efficiency over the COII marker. Lastly, we described the inclusion of an internal control (28S amplification) for predation studies performing predator gut analyses utilizing PCR. This control was critical in order to monitor reactions for PCR failures, PCR inhibitors, and for the presence of DNA.