2006 Annual Report 1.What major problem or issue is being resolved and how are you resolving it (summarize project aims and objectives)? How serious is the problem? Why does it matter? Invasive insects and weeds cost the United States over $122 billion per annum, impacting both agricultural and environmental areas. The glassy-winged sharpshooter (GWSS), Homalodisca coagulata, is a serious economic pest because it transmits a bacterium, Xylella fastidiosa, (Pierce's disease) that is lethal to grapevines and causes almond leaf scorch. GWSS recently invaded California and is threatening the $33 billion wine and table grape industry. Molecular techniques developed by scientists in the Beneficial Insects Research Unit (BIRU) were used to determine that Texas is the origin of the GWSS that invaded California. These techniques, along with others, have been used to determine if populations of morphologically indistinguishable natural enemies or egg parasitoids of GWSS from across the native range are separate species, each with a unique potential as biological control agents. For example, BIRU scientists have discovered that one of the primary GWSS egg parasitoids (Gonatocerus morrilli) was not one species but a complex of cryptic species. This led to the export and successful establishment of the unique Texas population of G. morrilli in California. BIRU scientists, in collaboration with other ARS scientists in Phoenix, AZ, have developed GWSS-specific molecular diagnostic assays to detect GWSS remains in the gut contents of native insect predators, which will help IPM specialists determine which species are most important to conserve in the citrus-grape agroecosystem. Intensive surveys in the native range of GWSS (Texas) for new biological control agents were conducted in wild mustang grapes and a fly parasitoid that attacks nymphal sharpshooters was discovered in native mustang grapes. Studies will be made on the chemical and behavioral ecology of herbivore-host plant-natural enemy tritrophic systems to elucidate the mechanisms that orchestrate these interactions. This information will be used to improve our understanding of the foraging ecology of crop pests, to enhance our ability to quantify their population abundance and distribution, and to devise conservation biological control strategies for their natural enemies. Research vineyards have been planted in Weslaco, TX, next to existing ARS citrus orchards to model the California agroecosystem where GWSS is invasive, which will allow us to integrate our laboratory and field research to evaluate management strategies. New approaches to manage invasive and exotic weeds are being developed. To improve augmentative biological control of waterhyacinth, a serious weed in the Southeastern U.S, pre-inoculation of established biological control agents with plant pathogenic fungi will be investigated. Plant pathogens will be investigated as bioherbicides to control pigweeds in the context of insect pests that feed on these weeds in subtropical sustainable cropping systems. In a collaborative research program with ARS scientists in Temple, TX, and Albany, CA, BIRU scientists will conduct field studies to determine the impact of biological control agents released for saltcedar on closely related athel pine. Athel pine is exotic, but valued as an ornamental and windbreak tree in Mexico. In addition, the influence of native herbivore insects on the establishment of saltcedar biological control agents will be investigated at field sites in south Texas. This research addresses goals identified under the ARS research area of Crop Production, Product Value, and Safety contained within National Program 304, Crop Protection and Quarantine. Objective 1 falls under the Biology of Pests and Natural Enemies; and Plant, Pest, and Natural Enemy Interactions and Ecology research components. Objective 2 contributes to the Plant, Pest, and Natural Enemy Interactions; and Postharvest, Pest Exclusion, and Quarantine Treatment research components; and Objective 3 contributes to the Biological Control of Weeds and Weed Management Systems research components. The research on GWSS directly benefits grape growers in California that are severely impacted by this invasive pest and lethal vine disease that it vectors. New pest management strategies will also benefit grape growers in the eastern U.S., where Pierce's disease is endemic. The weed research supports restoration of western rangelands infested with saltcedar and aquatic habitats with waterhyacinth. Specific pathogens tested in this program for pigweed could increase control of this weed in cotton and grain. 2.List by year the currently approved milestones (indicators of research progress) Year 1 (FY 2006) 1. Collaborate with the South American Biological Control Laboratory (SABCL) to determine the distribution and host range of South American sharpshooter parasitoids. 2. Complete colonization of GWSS. 3. Develop and initiate bioassays to detect presence of bioactive compounds in the GWSS-host plant-natural enemy tritrophic system. 4. Adopt/modify/develop techniques to collect, isolate, and analyze bioactive compounds. 5. Amplify and subclone GWSS mitochondrial COII genes from geographic populations for phylogenetic studies. 6. Amplify and subclone GWSS ITS2 regions from geographic populations for phylogenetic studies. 7. Complete developing and testing crude DNA extract procedure for GWSS predators. 8. Amplify and subclone GWSS mt-COII genes from sharpshooter species for predation studies. 9. Amplify and subclone G. morrilli mt-COI, II from geographic populations for phylogenetic studies. 10. Amplify and subclone G. ashmeadi ITS2 from geographic populations for phylogenetic studies. 11. DNA fingerprint parasitoid species with ISSR-PCR primer (A). 12. Amplify and subclone ITS2 region from parasitoid species for phylogenetic studies. 13. Complete study of vectoring of waterhyacinth pathogen cultures. 14. Complete study of pheromone and plant volatile lures on saltcedar beetle establishment. 15. Complete studies of effects of non-overexcretor pathogens on pigweeds in greenhouse. Year 2 (FY 2007) 1. Import candidate species that show habitat specificity, Mediterranean climatic requirements, and a narrow host range for quarantine host range for testing and risk assessment. 2. Continue bioassays to detect presence of bioactive compounds. Continue sampling and analysis studies. 3. Complete sequencing of GWSS mt-COII genes from geographic populations. 4. Complete sequencing ITS2 regions from geographic populations. 5. Complete testing of SCAR markers to detect all GWSS life stages. 6. Complete sequencing of GWSS mt-COII genes from sharpshooter species. 7. Complete sequencing of G. morrilli mt-COI II genes from geographic populations. 8. Complete sequencing ITS2 regions from G. ashmeadi populations. 9. DNA fingerprint parasitoid species with ISSR-PCR primer (B). 10. Complete sequencing ITS2 regions from parasitoid species. 11. Complete study of vectoring of fungal suspensions and impacts on waterhyacinth in tanks. 12. Complete study on nontarget effects of saltcedar beetles on athel in field. 13. Complete studies of new strains of pathogens on pigweeds and insect feeding in greenhouses. Year 3 (FY 2008) 1. Continue quarantine risk assessment of South American parasitoid species. 2. Collect and assay promising compounds. Continue sample collection, isolation, and analysis. 3. Complete processing GWSS mt-COII sequences, Sequencer software. 4. Complete processing GWSS ITS2 sequences, Sequencer software. 5. Complete retention time (0-36 h) experiment for one predator species to determine half-life of SCAR product. 6. Complete processing of G. ashmeadi ITS2 sequences, Sequencher software. 7. Complete processing G. morrilli mt-COI, II sequences, Sequencher software. 8. Complete processing G. morrilli ITS2 sequences, Sequencher software. 9. DNA fingerprint parasitoid species with ISSR-PCR primer (C). 10. Complete processing GWSS egg parasitoid ITS2 sequences, Sequencher software. 11. Complete field plot studies of waterhyacinth weevils and plant pathogens. 12. Complete multi-year study on field associations of saltcedar beetles and native insects. 13. Complete studies of overexcretor strain effects on pigweeds and insects in small field plots of pre-planted pigweed. Year 4 (FY 2009) 1. Finalize risk assessment and apply for release permit, if warranted. 2. Complete bioassays and ID of bioactive compounds. Initiate evaluation of attraction, monitoring, and/or trapping technologies. Seek CRADA partners, if appropriate. 3. Complete GWSS mt-COII sequence alignments. 4. Complete GWSS ITS2 sequence alignments. 5. Complete retention time (0-36 h) experiment for another predator species to determine half-life of SCAR product. 6. Complete design of GWSS-specific mt-COII primers. 7. Complete G. morrilli mt-COI, II sequence alignments. 8. Complete GWSS egg parasitoid ITS2 sequence alignments. 9. Pool ISSR-PCR reactions, construct marker matrix with POPGENE and TFPGA. 10. Complete G. ashmeadi ITS2 sequence alignments. 11. Complete open field release study of waterhyacinth weevils and plant pathogens. 12. Complete studies of leafhopper/planthopper effects on saltcedar beetles in bioassays. 13. Complete multi-year field plot sampling study of pigweeds in cotton crops. Year 5 (FY 2010) 1. Begin host range testing of second South American species. 2. Collaborate with researchers in California to evaluate establishment and impact of parasitoid. 3. Complete application of trapping and attraction technologies. 4. Complete analysis, bootstrap, draw trees (PAUP) for GWSS mt-COII sequences. 5. Complete analysis, bootstrap, draw trees (PAUP) for GWSS ITS2 sequences. 6. Complete analysis, graphs, and figures. 7. Complete retention time (0-36 h) experiment for one predator species to determine half-life of mt-COI or COII product. 8. Complete analysis, bootstrap, draw trees (PAUP) for G. morrilli mt-COI, II sequences. 9. Complete analysis, bootstrap, draw trees (PAUP) for G. ashmeadi ITS2 sequences. 10. Analysis genetic data, determine gene structure, polymorphic loci, gene diversity, GST, etc. 11. Complete analysis, bootstrap, draw trees, (PAUP) for Gonatocerus ITS2 Sequences. 12. Generate recommendations for use of waterhyacinth weevils and plant pathogens. 13. Generate recommendations for saltcedar beetle releases and test in new field sites. 14. Complete studies of large-scale application of bioherbicides to control pigweeds in cotton crops. 4a.List the single most significant research accomplishment during FY 2006. Discovery of a new species of egg prasitoid of the glassy-winged sharpshooter (GWSS): Scientists in the Beneficial Insects Research Unit, along with collaborators, discovered that one of the primary egg parasitoids of glassy-winged sharpshooter (GWSS) in California, (Gonatocerus morrilli), is actually a new species. The new species was described and named Gonatocerus walkerjonesi by a scientist at the University of California at Riverside. Diagnostic markers [inter-simple sequence repeat-polymerase chain reaction (ISSR-PCR) and internal transcribed spacer region 2 (ITS2)] were developed that distinguished the two very closely related species from California (G. walkerjonesi), and Texas and Florida (G. morrilli). It was recently determined that the GWSS that invaded California originated from Texas; it is crucial to choose the natural enemy from the origin of the GWSS. We now have the technology to monitor the success of the biological control program in California using G. morrilli as a control agent. This research falls under NP 304, Crop Protection and Quarantine, Component II, Biology of Pests and Natural Enemies. 4b.List other significant research accomplishment(s), if any. Saltcedar beetle ecotype safe for use near beneficial athel trees: As part of the national saltcedar biological control program, researchers at the Beneficial Insects Research Unit demonstrated that a saltcedar beetle ecotype from Crete defoliated saltcedar plants inside south Texas field cages, and laid fewer eggs on athel than on saltcedar, suggesting, preliminarily, that this beetle ecotype is safe for use near beneficial athel trees. This research falls under NP 304, Crop Protection and Quarantine, Biological Control of Weeds Research Component. Demonstrated the effect of phosphorous and nitrogen on waterhyacinth biocontrol agent performance: Phosphorous and nitrogen levels in water and plant tissue significantly affect the performance of a key biological control agent for the invasive aquatic weed, waterhyacinth. Positive interactions between the nitrogen and phosphorous content of waterhyacinth leaves and water nitrogen concentration in field populations, and a negative interaction between leaf damage by waterhyacinth weevils and plant nitrogen were discovered. Field sites with high water flow had reduced levels of damage. This accomplishment is consistent with the Project milestones to evaluate biological control of waterhyacinth, with the Weed Biological Control component of NP 304, and with ARS Strategic Plan Performance Measure 3.2.6 (Conduct biologically based integrated and areawide management of key invasive species). Development of a behavorial assay to evaluate response of GWSS: Scientists at the Kika de la Garza Subtropical Agricultural Research Center developed a behavioral assay to evaluate the response of GWSS to visual and olfactory stimuli associated with host plant foliage. This methodology was used to demonstrate, for the first time, that foliar odors are stimulatory and influence the host-plant searching behavior of both immature and adult GWSS. This research falls under NP 304, Crop Protection and Quarantine, Component II, Biology of Pests and Natural Enemies. Discovery of a fly parasitoid of nymphal sharpshooters: Surveys in the native range of glassy-winged sharpshooter GWSS (Texas) led to the discovery of a fly parasitioid of nymphal sharpshooters by scientists in the Beneficial Insects Research Unit and Ag Canada. The fly, Eurydorylas sp. (Pipunculidae), was collected from Oncometopia orbono, a closely related sharpshooter to GWSS. Intensive surveys are in progress to collect the Eurydorylas from GWSS. Nymphal parasitoids could complement the egg parasitoids already established in California. This research falls under NP 304, Crop Protection and Quarantine, Component II, Biology of Pests and Natural Enemies. 4c.List significant activities that support special target populations. None 4d.Progress report. Project 1: Determine the origin of the GWSS that invaded California and also determine the population genetic structure. DNA polymorphisms, for the first time, were detected in GWSS with four PCR-based DNA fingerprinting methods (ISSR-PCR, RAMP, SAMPL, and RAPD). The methods incorporating simple sequence repeats (SSR) were found to be the most sensitive and efficient methods with GWSS template. These methods were not only able to distinguish different sharpshooter species (H. coagulata, H. liturata, and H. insolita), but most importantly, they were able to detect geographic variation in GWSS. The development of molecular genetic markers for the GWSS is of great significance because it now allows us to genetically characterize the GWSS. For example, it allows us to. 1)determine genetic variation within and among populations,. 2)determine gene flow mechanisms,. 3)determine the population or geographic structure, and. 4)determine the origin of the GWSS that invaded California. In the population study of the GWSS, compound inter-simple sequence repeat (ISSR) primers containing CA-repeat motifs in their sequences were utilized to estimate the population genetic structure of GWSS from a total of 19 populations throughout the U.S. Results showed significant partitioning of gene diversity at three levels, among regions, among populations within regions, and among populations. The results estimated the population genetic structure of the GWSS, but most importantly, the study demonstrated that the GWSS that invaded California is of Texas origin, but more than one 'founding event' occurred in California. The data showed that GWSS populations in the U.S. were genetically distinct, clustering into two main groups or clades, a 'southeastern' and a 'southwestern and western' clade. Since the origin of the California GWSS infestation is Texas, the most effective natural enemies for release in California should be found in Texas. A separate study of egg parasitism in south Texas showed that an indigenous species parasitizes about 90% of GWSS egg masses throughout the season, perhaps at least partially explaining the low density of GWSS in its native range. Expected Benefits: Determination of the origin of the invasive California population of GWSS allows biological control scientists to focus the search in the origin to exploit the best adapted parasitoids. Project 2: Identify key predators of the GWSS [and the smoke-tree sharpshooter (STSS)]. Effective control of GWSS requires an area-wide, multi-tactic pest management program. A major component of such an approach is the exploitation of the pest's natural enemies, which, when utilized to their greatest potential, can increase the effectiveness of other control tactics. However, little is known about the predaceous enemies that feed on eggs, nymphs, or adult GWSS. Direct visual field observations of predation are difficult to obtain, and the field study of insect predation has often relied on indirect techniques for measurement and analysis. Jesus de León, ARS, Weslaco, in collaboration with J. Hagler, ARS, Phoenix, AZ; V. Fournier, Rutgers University; and K. Daane, University of California in Berkeley, developed, for the first time, GWSS-specific molecular diagnostic markers to aid in identifying key predators. The diagnostic markers [sequence characterized amplified region (SCAR) and mitochondrial COI and COII] were specific toward the GWSS (and STSS) and were able to identify GWSS remains at all life stages (eggs, nymphs, and adults) in predator gut contents. Preliminary field studies of predators in natural environments have shown good success using the newly developed GWSS-specific diagnostic markers, particularly the COI markers (V. Fournier, unpublished data). Expected benefits: The development of diagnostic markers for the GWSS will allow us to begin to understand the ecology of GWSS-predator interactions in natural environments. This information will be included in an area-wide pest management approach to aid in controlling this devastating pest. Project 3: Genetically characterize GWSS natural enemies or egg parasitoids in the genus Gonatocerus. Jesus de León and collaborators discovered that one of the primary egg parasitoids (Gonatocerus morrilli) of the GWSS from California is actually a new species (sp. n.). Serguei Triapitsyn (UC-Riverside) described the new species and named it Gonatocerus walkerjonesi. Diagnostic markers [inter-simple sequence repeat-polymerase chain reaction (ISSR-PCR) and internal transcribed spacer region 2 (ITS2)] were developed that distinguished the two very closely related species from California (G. walkerjonesi) and Texas and Florida, (G. morrilli). Since Jesus de León and collaborators recently determined that the GWSS that invaded California originated from Texas, it is crucial to choose the natural enemy from the origin of the GWSS. We now have the technology to monitor the success of the biological control program in California using G. morrilli as a control agent. Expected benefits: Accurate identification of natural enemies is critical to the success of classical biological control programs. Lack of proper identification procedures has affected several programs. Populations of G. morrilli from Texas and/or Mexico have been released in California since 2001 and it has, therefore, been difficult to distinguish between native (G. walkerjonesi), CA. and imported natural enemies (G. morrilli), TX, to determine their establishment. The genetic and hybridization results of the current study demonstrated that the native and imported natural enemies were actually different species. In addition, molecular diagnostic markers were developed from these studies that distinguished the two very closely related species. The Beneficial Insects Research Unit in Weslaco, sent G. morrilli from Texas to California in the summer of 2005 to restart the biological control program. The molecular diagnostic markers developed will be utilized to monitor the success of establishment and evaluate dispersal and efficacy of G. morrilli in California. Project 4: Post-release evaluation of the G. morrilli biological control program in California against the GWSS. Test the utility of the molecular diagnostic markers to monitor establishment of G. morrilli in California. The molecular markers distinguish the native California species (G. walkerjonesi) from the imported Texas species (G. morrilli). A four-year (2002-2005) survey was conducted on post-release populations collected in several counties in southern California. The results of this study indicated that the developed molecular diagnostic markers were highly successful in detecting and monitoring the establishment of G. morrilli in California on a small scale. In addition, the markers were used to monitor egg parasitoid colonies against contamination with unwanted species. Amplification of the ITS2 fragments of post-released G. morrilli populations detected only the native G. walkerjonesi ITS2 genotype from California. These results indicated that G. morrilli (Texas species) was not establishing in California. This raised a concern as to whether the G. morrilli biological control program was successful. In addition, there was a concern that the original 'release' colony may have been contaminated with an unwanted species. After testing the original G. morrilli colony that was used for release by ISSR-PCR DNA fingerprinting, it was determined that the colony was contaminated with California's own native species (G. walkerjonesi). The results demonstrated that what was being released was G. walkerjonesi and not G. morrilli, and therefore that is why only the California G. walkerjonesi ITS2 genotype was being detected. After determining that the original release colony was contaminated with California's own native species, the California Department of Food and Agriculture imported G. morrilli from TX (USDA, ARS Weslaco), the same area where GWSS found in California originated from. Importing G. morrilli from Texas for a classical biological control program in California was also important. This strategy can increase the likelihood of success because the parasitoid has co-evolved with its host (GWSS). In the fall of 2005, the G. morrilli (Texas) ITS2 genotype was detected in a location where it had been previously released in southern California, demonstrating the utility of the developed diagnostic markers. Intense surveying will continue for the next two years. Expected benefits: We now have the molecular technology to follow the biological control program of G. morrilli from start to finish, that is to determine the success of the biological control program in California. We can now evaluate establishment, dispersal, and efficacy of the natural enemy, and improve mass rearing by safeguarding for contamination of unwanted species. The application of molecular markers as diagnostic tools for enhancing the precision of classical biological control programs is clearly needed Past projects have often failed due to the inability to detect cryptic or different species, either prior to or after release. Project 5: Determine whether Gonatocerus ashmeadi, a primary egg parasitoid of the GWSS, exists in nature as a cryptic species complex or whether geographic variation or genetic differentiation can be detected. Current situation: The recognition of intraspecific variation can be as crucial for the success of biological control programs as is sound species determination. Populations of parasitoid from distinct geographical regions may differ in relevant biological characteristics of importance to biological control. Using ISSR-PCR DNA fingerprinting, Jesus de León and cooperators identified geographic variation and geographic-specific markers in certain populations of the GWSS egg parasitoid, G. ashmeadi. The results showed that all populations (Texas, Florida, Louisiana, California) of G. ashmeadi were the same species, a conclusion reached by other researchers. Though, and very importantly, the populations were highly differentiated. This observation is significant because studies have shown that recognition of intraspecific variation can be as crucial for the success of biological control programs as is sound species determination. Populations of parasitoid from distinct geographical regions may differ in relevant biological characteristics of importance to biological control. In other words, there may be a geographic population of G. ashmeadi that may be genetically better suited for the California environment. In addition, geographic-specific markers were identified in certain populations (Louisiana), and if releases were made from these individuals than it would become possible to monitor the success of the biological control program in California. Expected benefits: Biological control programs can benefit greatly by knowing that geographic populations of an egg parasitoid are highly differentiated, although there may be a geographic population that may be better suited to a specific environment of interest. In addition, identification of geographic-specific markers can aid greatly in monitoring the establishment of the same species in biological control programs. Nymphal parasitoids for GWSS will likely be discovered in the native range (Texas) since other closely related sharpshooter species in the same environment are known to be parasitized by Pipunculidae. All known Pipunculidae are nymphal parasitoids of leafhoppers and sharpshooters and are known to have narrow host ranges and high attack rates. In addition, they usually overwinter as immatures in their hosts. This biology may allow them to overwinter in large numbers and exert mortality on other life stages of GWSS, thus complementing the contributions of egg parasitoids, which are primarily only active in the summer months. We anticipate discovery and collection of Pipunculidae in 2007 and subsequent transfer of the parasitoids to the California Department of Food and Agriculture. We will also develop rearing methods and investigate the biology of pipunculids to aid the biological control program in California. Argentinean egg parasitoids collected from the closely related sharpshooter, Tajajosa rubromarginata, will be evaluated in Argentina and in U.S. quarantine facilities prior to release in California for GWSS. The host range of these parasitoids in regards to North American sharpshooters is not known, but assumed to be at the tribe level (Proconiini). In order to minimize the potential for non-target attack on native sharpshooters, parasitoids will be collected from climates in Argentina. The cool-Mediterranean regions of Argentina south and west of Buenos Aires (Mendoza and San Rafael) are the search areas for collection of egg parasitoids. Parasitoids from these areas will likely be better adapted to climates in California where GWSS is invasive and simultaneously poorly adapted to the climates of the southeastern U.S. and thus unable to establish where there are numerous native non-target sharpshooter species. The quarantine host range testing and risk analysis will be completed in 2008. This timeline could be shortened if researchers at the University of California at Riverside are funded by CDFA to collaborate on the project. 5.Describe the major accomplishments to date and their predicted or actual impact. This CRIS project will produce advances in the molecular biology, chemical ecology, and biological control of GWSS, as well as in procedures to augment waterhyacinth weevils and fungal plant pathogens, implementation of a biological control program of saltcedar, and application of a bio-herbicidal control option for pigweeds using fungal plant pathogens. All accomplishments made under this project are fully consistent with relevant milestones listed in the Project Plan, and with the relevant research components as defined in the National Program 304 Action Plan. Accomplishments under this project contribute to the achievement of ARS Strategic Plan Goal 3, Objective 2, Performance Measure 6, in that project accomplishments contribute substantially to attainment of the Agency goals to improve knowledge and understanding of the ecology, physiology, epidemiology, and molecular biology of emerging diseases and pests. This knowledge will be incorporated into pest risk assessments and management strategies to minimize chemical inputs and increase production. Accurate identification of natural enemies is critical to the success of classical biological control programs. Lack of proper identification procedures has affected several programs. Populations of G. morrilli from Texas and/or Mexico have been released in California since 2001 and it has, therefore, been difficult to distinguish between native (G. walkerjonesi) (California) and imported natural enemies (G. morrilli) (Texas) to determine their establishment. The genetic and hybridization results of the current study demonstrated that the native and imported natural enemies were actually different species. In addition, molecular diagnostic markers were developed from these studies that distinguished the two very closely related species. The USDA, ARS, Beneficial Insects Research Unit (Weslaco) sent G. morrilli from Texas (origin of the GWSS) to California in the summer of 2005 to restart the biological control program. The molecular diagnostic markers developed by BIRU scientists will be utilized to monitor the success of establishment and evaluate dispersal and efficacy of G. morrilli in California. A novel behavioral assay was developed to evaluate the response of GWSS to visual and olfactory stimuli associated with host-plant foliage. This methodology was used to provide evidence, for the first time, that GWSS is stimulated by exposure to host-plant odor and that chemical cues play an important role in GWSS host-searching behavior. Both immature and adult GWSS were shown to be responsive to host-plant odor. The results of this study also indicated that exposure to host-plant odor mediates GWSS response to visual cues. Information from this study has increased our basic knowledge of GWSS searching behavior and may contribute to developing management techniques (i.e., deficit-irrigation management) that could be used to influence the GWSS distribution at the scale of individual farms. The research completed in the first year of this project on biological control of waterhyacinth, saltcedar, and pigweeds provides a foundation that will lead to major accomplishments and impact in future years in controlling these harmful weeds. The use of vectored plant pathogens in augmentative releases of waterhyacinth weevils will increase biological control impacts, circumvent the need for large-scale mass-production and inoculation of waterhyacinth pathogens, and reduce the need for costly mechanical and chemical control. Research on saltcedar will answer the critical question of non-target effects on athel, an exotic, closely related, beneficial tree. The results will largely determine the acceptability of biological control of saltcedar with the saltcedar leaf beetle to Mexican and US-Mexico border stakeholders and customers, which will in turn determine the future of biological control releases in these areas. Procedures designed to maximize the success of saltcedar beetle establishment will also be generated and transferred under this project. The development of bioherbicidal control methods against pigweeds, when transferred, will benefit sustainable/organic crop producers in South Texas and elsewhere, and may reduce herbicide use in conventional crop production. 6.What science and/or technologies have been transferred and to whom? When is the science and/or technology likely to become available to the end-user (industry, farmer, other scientists)? What are the constraints, if known, to the adoption and durability of the technology products? Development of the diagnostic marker (especially the COI marker) technology for the GWSS was transferred to scientists; (ARS-Maricopa, AZ), (Rutgers University, NJ), and (UC-Berkeley, CA). The COI diagnostic marker is working well and is being used successfully to screen various predator species in the field. This information is leading to the identification of various key important predators. This will now allow us to begin to understand the ecology of GWSS-predator interactions in natural environments. This information will be included in an area-wide pest management approach to aid in controlling this devastating pest. Development of molecular diagnostic markers to distinguish two very closely related Gonatocerus species (G. morrilli and G. walkerjonesi) has been transferred to the California Department of Food and Agriculture (CDFA) and the University of California system (UC-Riverside). These markers allow us to distinguish the imported natural enemy, (G. morrilli), from the native species (G. walkerjonesi). We now have the molecular technology to follow the biological control program of G. morrilli from start to finish to determine the success of the biological control program in California. We can now evaluate establishment, dispersal, and efficacy of the natural enemy, and improve mass rearing by safe guarding for contamination of unwanted species. The research completed in the first year of this project on biological control of waterhyacinth, saltcedar, and pigweeds provides a foundation that will lead to major accomplishments and impact in future years in controlling these harmful weeds. The use of vectored plant pathogens in augmentative releases of waterhyacinth weevils will increase biological control impacts, circumvent the need for large-scale mass-production and inoculation of waterhyacinth pathogens, and reduce the need for costly mechanical and chemical control. Research on saltcedar will answer the critical question of non-target effects on athel, an exotic, closely related, beneficial tree. The results will largely determine the acceptability of biological control of saltcedar with the saltcedar leaf beetle to Mexican and US-Mexico border stakeholders and customers, which will in turn determine the future of biological control releases in these areas. Procedures designed to maximize the success of saltcedar beetle establishment will also be generated and transferred under this project. The development of bioherbicidal control methods against pigweeds, when transferred, will benefit sustainable/organic crop producers in South Texas and elsewhere, and may reduce herbicide use in conventional crop production. 7.List your most important publications in the popular press and presentations to organizations and articles written about your work. (NOTE: List your peer reviewed publications below). Fournier, V., Hagler, J., Daane, K., de León, J. H., Prabhaker, N., Costa, H. 2005. Identifying key predators of the various glassy-winged sharpshooter lifestages, pp. 314-317. In: Proceedings of the Pierce’s Disease Research Symposium, December 5-7, 2005, San Diego, CA. Complied by M. Athar Tariq, Stacie Oswalt, Peggy Blincoe, Amadou Ba, Terrance Lorick, and Tom Esser, Sacramento, CA. Moran P.J., Graham C.J. 2005. Vectoring of plant pathogenic fungi by waterhyacinth weevils (Neochetina spp.) and biological control of waterhyacinth (poster). Annual Meeting of the Entomological Society of America, December 15-18, 2005, Fort Lauderdale, Florida. Moran, P.J., Deloach, C.J. 2006. Biological control of saltcedar in Kingsville and other sites in south Texas (oral presentation). Second Meeting of the Southwestern Section of the Saltcedar Biological Control Consortium, March 2-3, 2006, Austin, Texas. Moran, P. J., Deloach, C. J. Biological control of saltcedar in Texas, New Mexico and the northwestern U. S. (oral and poster presentation in Spanish). Meetings with land managers from PRONATURA and CONANP to discuss arundo and saltcedar control, Cuatrocienegas, Coahuila, Mexico, May 24-25, 2006. Review Publications De Leon, J.H., Jones, W.A., Setamou, M., Morgan, D.J. 2006. Genetic and hybridization evidence confirms that a geographic population of Gonatocerus morrilli (Hymenoptera: Mymaridae) from California is a new species: egg parasitoids of the glassy-winged sharpshooter Homalodisca coagulata (Homoptera: Cicadellidae). Biological Control. 38:282-293. De Leon, J.H., Fournier, V., Hagler, J.R., Daane, K. 2006. Development of molecular diagnostic markers for sharpshooters Homalodisca coagulata and H. liturata (Homoptera: Cicadellidae: Proconiini) for use in predator gut content examinations. Entomologia Experimentalis et Applicata. 119:109-119. Fournier, V., Hagler, J.R., Daane, K.M., Groves, R.L., De Leon, J.H., Costa, H.S., Henneberry, T.J. 2006. Development and application of a glassy-winged and smoke-tree sharpshooter egg-specific predator gut content elisa. Biological Control. Biological Control 37: 108-118. De Leon, J.H., Morgan, D. 2005. Small scale post-release evaluation of a Gonatocerus morrilli program in California against the glassy-winged sharpshooter: Utility of developed molecular diagnostic tools. Proceedings of CDFA Pierce's Disease Control Program Research Symposium, December 5-7, 2005, San Diego, California. p. 306-309. De Leon, J.H., Hagler, J.R., Logarzo, G., Morgan, D.J. 2005. The utility of inter-simple sequence repeat-polymerase chain reaction (ISSR-PCR) to distinguish geographic populations of the smoke-tree sharpshooter Homalodisca liturata and egg parasitoid species of the genus Gonatocerus. Proceedings of CDFA Pierce's Disease Control Program Research Symposium, December 5-7, 2005, San Diego, California. p. 298-301. De Leon, J.H., Jones, W.A., Setamou, M., Morgan, D.J. 2005. Discovery of a cryptic species complex in Gonatocerus morrilli (Hymenoptera: Mymaridae), a primary egg parasitoid of the glassy-winged sharpshooter. Proceedings of CDFA Pierce's Disease Control Program Research Symposium, December 5-7, 2005, San Diego, California. p. 302-305. Moran, P.J., Greenberg, S.M. 2006. Winter cover crops and vinegar as weed control techniques in sustainable cotton production. In: Proceedings of the Beltwide Cotton Conferences, January 3-6, 2006, San Antonio, Texas. 2006 CDROM. p. 2188-2195. Patt, J.M., Setamou, M. 2005. Relationship between olfactory and visual stimuli during host plant recognition in immature and adult glassy-winged sharpshooter. Proceedings of the CDFA Pierce's Disease Control Program Research Symposium, December 5-7, 2005, San Diego, California. p. 122-123. Setamou, M., Goolsby, J., Patt, J.M. 2005. Effect of host plant fertilization on the developmental biology and feeding preference of the glassy-winged sharpshooter. Proceedings of CDFA Pierce's Disease Control Program Research Symposium, December 5-7, 2005, San Diego, California. p. 101-104. Goolsby, J., Setamou, M. 2005. Exploration for biological control agents in the native range of the glassy-winged sharpshooter. Proceedings of CDFA Pierce's Disease Control Program Research Symposium, December 5-7, 2005, San Diego, California. p. 318-320.