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:
Progress was achieved toward most objectives while others were modified during FY14. Semiochemical control of citrus leafminer has advanced to commercial launch of a new mating disruption product, DCEPT CLM™, ISCA Technologies, Inc. Disruption was maintained for more than 30 weeks with one application. The Citrus Research and Development Foundation subsidized the launch. Discovery of volatiles that influence Asian citrus psyllid (ACP) behavior has been difficult. Recordings confirmed response of ACP antennae to degradation products of citrus volatiles. The number of ACP stylet sheaths was greater on a wax matrix containing degraded volatiles compared with common citrus volatiles. A patent for a male-produced pheromone from Diaprepes root weevil was received; exclusive rights were licensed to Citrus Research and Development Foundation. A putative female pheromone is being sought. Cold stress increased ACP infestation of sour orange. Cold-treated trees had elevated nitrogenous nutrients and decreased polyphenols that may be insect deterrents (Objective 1b). The addition of citrus-scented lures to yellow sticky traps did not increase ACP capture. Fungal pathogen spores infected more than 50% of ACP on potted citrus (Objective 2). Adults transferred spores and infected more than 30% of immatures. Infected individuals died and released spores; dispenser tests are underway. Releases of parasitoids in urban areas (Objective 2), including new Tamarixia haplotypes from Vietnam, China and Pakistan, did not increase biological control of ACP. Population genetics studies of Tamarixia at release sites will determine haplotype frequency. Some accessions of Poncirus trifoliata and xCitroncirus screened for ACP oviposition and development (Objective 3) were susceptible; 12 Poncirus trifoliata accessions and six of 17 xCitroncirus hybrids were resistant. Development and survival were normal when first instars were placed on accessions resistant to oviposition. Electronic Penetration Graph recordings documented reduced feeding on resistant accessions. Within this objective, focus shifted to short-term solutions; delivery of non-transgenic RNA interference (RNAi) products has become the priority. Work on the virus will be done as time allows. Current research develops delivery systems for double-stranded RNA (dsRNA) products by root drench, foliar spray and trunk injection. Positive detection of dsRNA interference occurred at 2 weeks post-treatment for root drench and 4 weeks after trunk injection. dsRNA constructs were encapsulated under a Cooperative Research and Development Agreement (CRADA) for testing in the greenhouse and field. Peptides that bind ACP gut membranes were identified and will be screened for competitive inhibition of Candidatus Liberibacter asiaticus (CLas) movement within ACP (Objective 3). Down-regulation of an ACP gene by RNAi resulted in reduced ability to transmit CLas. Topical bactericides showed reduced CLas titer and improved tree quality. Natural products induced aberrant salivary sheath structure in artificial diets. A CRADA was established to commercialize therapeutic delivery of bactericides to huanglongbing infected citrus. An invention disclosure was submitted.
1. New product launched for mating disruption of citrus leafminer. Researchers at USDA-ARS in Fort Pierce, Florida partnered with private industry to launch a mating disruption product for control of the leafminer, Phyllocnistis citrella, and citrus canker disease. The new device (DCEPT CLM™), developed by ARS scientists and produced by ISCA Technologies Inc., was applied to 3,000 acres of grapefruit at three locations in 2014 through an innovative arrangement with the Commercial Product Delivery Committee of the Citrus Research and Development Foundation. Based on 2013 trials, it is expected that a single application will provide season-long disruption of leafminer mating and reduce incidence and severity of associated canker disease.
2. New inhibitors that block psyllid feeding process were discovered. USDA-ARS scientists at Fort Pierce, Florida have identified the major components of the cross-linking proteins that allow polymerization of Asian citrus psyllid (ACP) salivary sheath synthesis. As a result of work on salivary sheath composition, they identified natural products that create aberrant sheath formation and are ready to test these as potential new molecules for blocking the ability of ACP to feed.
3. Novel therapeutic application strategy developed for tree crops. Researchers at the USDA-ARS at Fort Pierce, Florida developed a novel application strategy to support highly efficient double-stranded RNA (dsRNA) uptake by plants. dsRNA constructs that target genes of sucking insects can be toxic to the insect if present in the vascular tissues on which the insect feeds. This application strategy is being developed for control of pest insects with emphasis on Asian citrus psyllid and other pests of perennial crops.
4. New assay developed for speedy testing of disease transmission by Asian citrus psyllid (ACP). Researchers at USDA-ARS, Fort Pierce, Florida developed a new ‘excised-leaf assay’ that can speed up disease transmission tests on ACP from 3 to 12 months (when using whole plants) to 2 to 3 weeks thereby saving time, materials and space, and it may enhance vector relation and epidemiological studies of the citrus greening pathogen.
5. Two new predators of Asian citrus psyllid (ACP) identified. USDA-ARS entomologists at Fort Pierce, Florida discovered that ACP eggs and nymphs were consumed by Western Flower Thrips (Frankliniella occidentalis) and a minute pirate bug (Orius insidiosus). Predation by thrips was facultative. The minute pirate bug may be useful in some settings for managing thrips but incompatible with psyllid rearing programs.
6. Heat treatments for killing Asian citrus psyllids (ACP) identified. Researching ACP tolerance to heat, entomologists with USDA-ARS in Fort Pierce, Florida identified lethal heat treatments for ACP in shipments of citrus leaves to culinary or medicinal markets. E.g., one lethal combination was 60°C for a minimum of 10 minutes.
7. Storing parasitoids prior to release for biological control of Asian citrus psyllid (ACP). USDA-ARS scientists at Fort Pierce, Florida showed that newly-emerged adult Tamarixia radiata, parasitoid of the ACP, can be stored at 20 to 30°C (14 hours photophase) in vials containing honey with more than 95% survival for 14 days. Adult parasitoids can be stored when necessary but should be released as soon as possible for maximal parasitism.
8. Environmental degradation of double-stranded RNA (dsRNA) in citrus trees and soil. USDA-ARS scientists at Fort Pierce, Florida in collaboration with scientist from Brazilian Agricultural Research Corporation, Brazil and the University of Florida, Apopka showed that dsRNA degrades after 24 hours in soil treated with a dsRNA product applied in water. Tree leaf samples from these same treatments had no detectable levels of dsRNA after 3 months. Rapid degradation of dsRNA's supports claims that dsRNA does not persist in the environment.
Reese, J., Christenson, M.K., Leng, N., Saha, S., Cantarel, B., Lindeberg, M., Tamborindeguy, C., MacCarthy, J., Weaver, D., Trease, A.J., Ready, S.V., Davis, V.M., Mccormick, C., Haudenschild, C., Han, S., Johnson, S.L., Shelby, K., Huang, H., Bextine, B.R., Shatters, R.G., Hall, D.G., Davis, P.H., Hunter, W.B. 2013. Characterization of the Asian citrus psyllid transcriptome. Journal of Genomics. 2:54-58.
Lapointe, S.L., Stelinski, L.L., Keathley, C.P., Mafra Neto, A. 2014. Coverage pattterns reduce cost of mating disruption for control of the leafminer Phyllocnistis citrella. Journal of Economic Entomology. 107:718-726.
Hall, D.G., Klein, E.M. 2014. Survival of adult Tamarixia radiata subjected to different short-term storage methods prior to field releases for biological control of Asian citrus psyllid. Florida Entomologist. 97:298-300.
Hall, D.G., Hentz, M.G. 2014. Asian citrus psyllid (Diaphorina citri) tolerance to heat. Annals of the Entomological Society of America. 107:641-649.
Monti, M., Mandrioli, M., Bextine, B., Hunter, W.B., Alma, A., Tedeschi, R. 2014. Maintenance of primary cell cultures of immunocytes from Cacopsylla sp. psyllids: a new in vitrio tool for the study of pest insects. In Vitro Cellular and Developmental Biology - Animals. https://doi.org/10.1007/s11626-014-9785-7.
Hall, D.G. 2014. Interference by western flower thrips in rearing Asian citrus psyllid: damage to host plants and facultative predation. Crop Protection Journal. 60:66-69.