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Title: DEVELOPMENT AND UTILITY OF A ‘ONE-STEP’ SPECIES-SPECIFIC MOLECULAR DIAGNOSTIC MARKER FOR GONATOCERUS MORRILLI DESIGNED TOWARD THE INTERNAL TRANSCRIBED SPACER REGION 2 (ITS2) TO MONITOR ESTABLISHMENT IN CALIFORNIA

Author
item De Leon, Jesus
item MORGAN, DAVID - CDFA,RIVERSIDE,CA

Submitted to: CDFA Pierce's Disease Control Program Research Symposium
Publication Type: Proceedings
Publication Acceptance Date: 9/25/2006
Publication Date: 11/27/2006
Citation: De Leon, J.H., Morgan, D.J. 2006. Development and utility of a ‘one-step’ species-specific molecular diagnostic marker for gonatocerus morrilli designed toward the internal transcribed spacer region 2 (its2) to monitor establishment in california. CDFA Pierce's Disease Control Program Research Symposium.

Interpretive Summary: In the current study, we developed a ‘one-step’ species-specific molecular diagnostic marker (gmtx) for the imported natural enemy (Gonatocerus morrilli) from Texas (into California) directed toward a nuclear gene [ribosomal internal transcribed spacer region 2 (ITS2)]. G. morrilli is morphologically very similar to the native California species, G. walkerjonesi, and so molecular markers are needed to distinguish these two species in order to monitor the success of the G. morrilli biological control program in California against the invasive glassy-winged sharpshooter [Homalodisca vitripennis (=H. coagulata)]. We screened a total of 16 Gonatocerus species and showed that the molecular marker was highly specific toward G. morrilli as cross-reactivity was not seen with any other Gonatocerus species. We analyzed the release colony ‘TX/MX’ that was used before the summer of 2005 with the newly developed molecular marker (gmtx) and in accordance with previous data, we confirmed a colony contamination. The species of the colony (TX/MX) that was being used for releases was not what it was thought to be (G. morrilli), but rather, it was California’s own native species, G. walkerjonesi. After this, ARS-Weslaco sent G. morrilli to California in the summer of 2005 to restart the program. We analyzed post-released populations of G. morrilli with the new marker (gmtx) and detected G. morrilli in a location where it was previously released, in accordance with previous data using two other types of molecular markers. The current results confirm the utility of the newly developed molecular diagnostic marker (gmtx) as an excellent tool to aid in monitoring the success of the G. morrilli biological control program in California. We now have in our hands the molecular technology to evaluate the G. morrilli biological control program in California from start to finish, that is, monitor establishment, dispersal, and efficacy of natural enemies and improve mass rearing.

Technical Abstract: In addition to the ‘one-step’ species-specific molecular diagnostic ISSR-PCR DNA fingerprinting method, we developed an additional ‘one-step’ molecular diagnostic marker ‘gmtx’ toward Gonatocerus morrilli (Howard) designed toward the ribosomal internal transcribed spacer region 2 (ITS2) to aid in monitoring establishment in California. Gonatocerus morrilli, the imported natural enemy from Texas, is very closely related to G. walkerjonesi, the native California species. Specificity assays with this newly developed marker and a total of 16 Gonatocerus Nees species demonstrated that it was highly specific toward the species that it was designed for (G. morrilli), as cross-reactivity was not seen with any of the tested species, including all species belonging to the morrilli subgroup of the ater species group of Gonatocerus. Anaysis of the ‘release’ ‘TX/MX’ colony used before the summer of 2005 with this species-specific diagnostic marker confirmed previous results that the ‘release’ ‘TX/MX’ colony was not the imported G. morrilli, but the native species G. walkerjonesi, confirming a colony contamination. Analysis of post-released G. morrilli collections with this diagnostic marker detected G. morrilli in a site where it was previously released, in accordance with our recent finding using two other diagnostic markers used in combination, ITS2 fragment size and ISSR-PCR DNA fingerprinting. The current results confirm the utility of the newly developed species-specific ITS2 molecular diagnostic marker as an excellent tool to aid in monitoring the establishment of the imported natural enemy of the glassy-winged sharpshooter, G. morrilli. These results and molecular tools are critical to the biological control program in California. We now have in our hands the molecular technology to evaluate the G. morrilli biological control program in California from start to finish, that is, monitor establishment, dispersal, and efficacy of natural enemies and improve mass rearing.