2011 Annual Report 1a.Objectives (from AD-416) 1. Chemical Ecology: Develop semiochemical-based control methods for citrus pests, particularly Asian citrus psyllid (ACP). 1a. Develop behavioral assays and antennogram methods for key citrus pests to identify conspecific odors that mediate mate location, recognition or aggregation and use these compounds for control and monitoring. 1b. Insect-Plant Interactions: Identification of host and nonhost compounds that affect host selection, location or repellency. 2. Biological Control: Develop new and improve existing biological control methods for ACP and sharpshooter species. 2a. Establish natural enemies of ACP in Florida. 2b. Identify new viral pathogens of ACP and sharpshooter species, and evaluate the potential of these as management strategies. 3. Host Plant Resistance: Develop host plant resistance in citrus to key pests. 3a. Identify sources of plant resistance to ACP in citrus germplasm. 3b. Develop novel control methods based on disrupting key processes in pest biology. 1b.Approach (from AD-416) Behavioral assays and antennogram (EAG/EAD) methods will be developed for key citrus pests to identify conspecific odors that mediate mate location, recognition or aggregation. Methods for monitoring and control using these compounds will be developed. Host and nonhost compounds will be identified that affect host selection, location or repellency through the use of gas chromatography (GC), GC-EAD, and GC-mass spectroscopy (GC-MS). To increase natural biological control of the Asian citrus psyllid, haplotypes of the parasitoid Tamarixia radiata will be characterized and released as deemed appropriate. Viral pathogens of Asian citrus psyllid and sharpshooter species will be identified by genomic methods and evaluated as management strategies. Existing citrus germplasm will be screened for host plant resistance to Asian citrus psyllids under greenhouse and field conditions. Genes and proteins involved in key biological processes such as salivary sheath formation will be studied to develop novel control strategies that block these processes. 3.Progress Report Databases of volatile and cuticular compounds from male and female Asian citrus psyllid (ACP) were generated; one compound was identified as a putative pheromone. Technical obstacles with electronic signal amplification from antennae of Asian citrus psyllid delayed identification of active compounds. Additional effort was made to develop behavioral tests to identify compounds for control and monitoring. A pheromone from the Diaprepes root weevil (DRW) was elucidated, synthesized and confirmed by gas chromatogram-linked electroantennography. Synthetic pheromone was obtained. Bioassays were developed to determine the response of male and female weevils to the pheromone. This work is expected to result in improved trapping and monitoring methods. A new device for deployment of citrus leafminer pheromone was fabricated for small scale and dooryard citrus. For commercial citrus, a tractor-mounted, computer-controlled applicator using global positioning system data was fabricated and tested in collaboration with International Fly Masters, Inc., Ft. Pierce, Florida. The applicator applied pheromone to over 250 acres per day within one percent of the desired rate resulting in continuous mating disruption for several weeks. The product containing 0.15 percent citrus leafminer (CLM) pheromone is now commercially available (SPLAT-CLM™, ISCA Technologies Inc., Riverside, California. An attract-and-kill product was developed (MalEx™, AlphaScents, Inc., Portland, OR); field trials reduced leafminer populations and damage. These two products offer the first semiochemical-based alternatives to traditional insecticides for control of a major global pest of citrus. A draft genome of the Asian citrus psyllid was completed and released as downloadable files. This dataset enables development of gene-based tools for research and development. Ribonucleic Acid-interference (RNAi) agents for managing psyllids and leafhoppers are being developed with private industry. Double-stranded ribonucleic acid molecules were toxic to psyllids and leafhoppers when delivered by root drench or injection into trees, grapevines or vegetables. These agents were detected in citrus trees for at least two months. These engineered molecules are highly target-specific tools for control of citrus pests. Salivary sheaths of Asian citrus psyllids were shown to be predominantly carbohydrate with a minor component comprised of biosynthetic enzymes that catalyze carbohydrate polymerization. This new insight into the process of psyllid feeding creates opportunities to disrupt sheath formation by psyllids and other insects that feed on plant phloem and xylem. Field and greenhouse trials tested formulations of bioactive antimicrobial molecules capable of moving through living bark and into the phloem of citrus trees. The goal is to develop an efficient strategy for infusion of engineered molecules to control Asian citrus psyllid and citrus greening. In greenhouse studies, antimicrobial molecules applied in such a manner were found in the phloem and reduced the abundance of the citrus greening bacterium. 4.Accomplishments 1. New environmentally friendly options for control of citrus pests based on insect pheromones. Agricultural Research Service entomologists at Fort Pierce, Florida in collaboration with the University of Florida and private industry are developing new products based on insect pheromones for control of major citrus pests and diseases, and new methods for their application in commercial citrus groves. The Agricultural Research Service collaborated with International Fly Masters, Inc. to design and fabricate a novel application device capable of delivering large (1 gram) dollops of high viscosity products using global positioning system telemetry to assure accurate placement and applicaton rate. The products use the pheromone of the citrus leafminer to confuse male moths so mating cannot occur (SPLAT-CLM™, ISCA Technologies, Riverside, CA) or to lure males to a fatal encounter with a droplet containing pesticide (MalEx™, AlphaScents Inc., Portland, OR). SPLAT-CLM is now available commercially; availability of MalEx is pending EPA registration. These products will contribute to reduced losses to leafminer damage and citrus canker disease as well as reduced dependence on traditional pesticides. 2. Discoveries about insect feeding open the door for new control strategies. Salivary sheath composition analysis may revolutionize our understanding of plant phloem feeding hemipterans, a group that includes major agricultural pests limiting food production worldwide. Agricultural Research Service molecular biologists and entomologists at Fort Pierce, FL, demonstrated that salivary sheaths are primarily composed of polymerized glucose associated with large amounts of free fructose, major components of phloem on which these insects feed. Thus, sheaths may form from regurgitated carbohydrates of plant origin polymerized by secreted salivary enzyme(s) and not solely secreted from salivary glands. Elucidation of the feeding mechanism is providing new opportunities for development of specific control strategies that have minimal if any collateral effects on beneficial organisms. 3. Novel application method for insect deterrents and antimicrobial compounds. New control molecules (proteins, double-stranded ribonucleic acid molecules, sheath inhibitors, antimicrobials) that have adverse effects on Asian citrus psyllids and/or citrus greening disease are often costly to manufacture. Application methods that minimize volume and maximize the amount of active ingredient that reaches the desired location are needed. Agricultural Research Service molecular biologists and entomologists at Fort Pierce, FL, developed and successfully demonstrated in greenhouse studies a citrus trunk application method that moves antimicrobial compounds across the bark and into the phloem. This resulted in a reduction of the citrus greening disease causative agent in the plant. The method is being optimized for commercial application. Review Publications Westbrook, C.J., Hall, D.G., Stover, E.W., Duan, Y., Lee, R.F. 2011. Colonization of citrus and citrus-related germplasm by Diaphorina citri (Hemiptera: Psyllidae). HortScience. 46(7):997-1005.