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:
Significant progress was made during the past year. Under the first objective (Chemical Ecology), recordings from Asian citrus psyllid identified plant compounds that elicit antennal responses. Bioassays demonstrated adult psyllid movement towards blends. Validation trials for a mating disruption product, SPLAT CLM™, were established to document control of citrus leafminer and citrus canker. A patent for an attractant and a provisional patent for a pheromone from the Diaprepes root weevil were received. A second Diaprepes pheromone was discovered. Related to the second objective (Biological Control), the abundance and phenology of Hirsutella citriformis and activity against Asian citrus psyllids were assessed and a method of culturing the fungus was developed. Glassy winged sharpshooter cell cultures were established for propagation of a leafhopper-infecting and a hymenoptera-infecting single-stranded ribonucleic acid virus. The system demonstrated ability of ribonucleic acid-interference to slow virus replication, prolong cell survival and produce higher virus titers. Progress towards the third objective (Host Plant Resistance) advanced through traditional and novel methods. Resistance was identified in Citrus and Citrus-related genotypes to Asian citrus psyllid and citrus leafminer. Orange jasmine, a favored host of Asian citrus psyllid, was shown to be a reservoir of citrus greening disease. Urban plantings of jasmine were a minor source of inoculum suggesting possible resistance to greening disease in orange jasmine. Sequencing of Candidatus Liberibacter asiaticus, the bacterium associated with citrus greening, was developed using whole genome polymerase chain reaction (PCR) amplification. This illuminates CLas diversity and the influence of bacterial genetics on disease severity, acquisition and movement within the psyllid. Pure salivary sheaths were isolated using soluble membranes through which psyllids feed. Salivary sheaths were composed of a specific carbohydrate polymer and key protein crosslinks, targets for disruption. Specific decameric peptide mixtures influenced psyllid mortality and behavior in artificial diet and may be incorporated into psyllid control strategies. The combinatorial library screen to identify peptide sequences is ongoing. Psyllid mortality can be induced by ingestion of double-stranded ribonucleic acid sequences that match essential genes linked to down regulation of targeted gene(s). This can be incorporated into a psyllid control strategy using a Citrus Tristeza virus expression vector to produce infected citrus plants that synthesize these sequences. Survival of psyllids fed on leaves of these plants was reduced. Fifty psyllid genes were targeted representing 10 major gene ontology classes of biological function. Double-stranded ribonucleic acid was detected in citrus cuttings, seedlings and trees up to 2.5 months after injection. Dose-response bioassays determined lethal dose. Persistence of the constructs in inoculated seedlings was variable. Also, formulations of penetrants and antimicrobial compounds as basal bark applications reduced titer of the greening disease pathogen in citrus trees.
1. Discovery of pheromones from the Diaprepes root weevil. Agricultural Research Service entomologists at Fort Pierce, Florida in collaboration with ARS colleagues at Beltsville, Maryland and Gainesville, Florida received a patent and a provisional patent for kairomones and a pheromone for the Diaprepes root weevil, respectively. The work included identification of plant compounds that elicit antennal responses and discovery of a novel compound from the frass and gut of male weevils that is attractive to females. A second compound found in the frass of both sexes also appears to play a role in orientation by both sexes. Deciphering the behavioral repertoire of Diaprepes in response to blends of these odors should result in new lures, traps and control methods for this important pest of citrus.
2. Discovery of plant compounds attractive to Asian citrus psyllid. Agricultural Research Service entomologists at Fort Pierce, Florida in collaboration with colleagues at the University of Florida have identified compounds collected from citrus leaves that elicit large and consistent antennal responses from the Asian citrus psyllid and may result in the development of new or improved trapping and control methods. In two cases, common plant volatiles that do not elicit antennal responses were shown to decompose into highly active compounds. Behavioral trials are underway to determine the value of these compounds for new trapping, monitoring and control methods.
3. The Glassy-Winged Sharpshooter genome to be annotated. Agricultural Research Service entomologists at Fort Pierce, Florida established a working group to annotate the genome of the glassy-winged sharpshooter, Homalodisca vitripennis, in collaboration with the i5K project and the Baylor College of Medicine, Human Genome Center, Houston, TX. Genomic DNA from male, female, nymphs, and eggs, has been sent to Baylor for library construction. These results will advance the ability to understand the genetic basis of leafhopper biology and the development of new leafhopper control measures, e.g., Ribonucleic acid interference and biological controls.
4. A new draft of the Asian citrus psyllid genome is now available. Agricultural Research Service entomologists at Fort Pierce, FL established an international genome consortium and completed the first draft genome of the Asian citrus psyllid in 2011. The first draft used a single sequencing platform that provided limited assembly of fragments. The Los Alamos National Laboratory performed a second round of sequencing using the PacBio system resulting in an improved psyllid genome, DIACI 1.2. This expanded draft genome provides a foundation for further investigation of the functional genomics of psyllids and serves as a reference genome for sequencing of other psyllid species. Genomics enables elucidation of the genetic basis of key biological processes such as reproduction, pathogen transmission and insecticide resistance and provides critical information needed to develop methods for psyllid control and plant improvement to reduce losses to this pest and citrus greening disease.
5. Towards a biological control program for Asian citrus using an entomopathogenic fungus. Agricultural Research Service entomologists at Fort Pierce, Florida in collaboration with colleagues at the University of Florida and Purdue University assessed the abundance and phenology of the entomopathogen Hirsutella citriformis and its activity against the Asian citrus psyllid. Psyllids killed by the fungus were most abundant during the fall and winter months and least abundant during the spring, apparently due to seasonal differences in relative humidity. A method of culturing the fungus was developed. The research lays a foundation for developing an applied biological control program for the psyllid using the fungus. Several chemicals commonly used in citrus were found to significantly reduce infectivity of the fungus. Growers interested in capitalizing on the fungus should avoid using chemicals which inhibit the fungus.
6. Discovery of double-stranded RNA (dsRNA) molecules toxic to Asian citrus psyllids. Researchers at the USDA, ARS, USHRL in Fort Pierce, FL in collaboration with University of Florida scientists showed that psyllid mortality was significantly increased when they fed on citrus plants manipulated to produce dsRNA molecules that have a sequence identical to either of two psyllid genes. This offers the possibility of developing citrus resistant to psyllid feeding and therefore resistant to infection by the citrus greening disease bacterium (Candidatus Liberibacter species) through psyllid transmission, the primary source of infection in commercial groves.
Lapointe, S.L., Stelinski, L.L. 2011. A GPS-guided applicator for high viscosity semiochemical products and optimal coverage patterns for mating disruption of the leafminer Phyllocnistis citrella (Lepidoptera: Gracillariidae) in citrus. Entomologia Experimentalis et Applicata. 141:145-153.
Rodrigo, D., Aguirre, C., Wheeler, G.S., Lapointe, S.L., Rosskopf, E.N., Overholt, W. 2011. Differential performance of tropical soda apple and its biological control agent Gratiana boliviana (Coleoptera: Chrysomelidae) in open and shaded habitats. Environmental Entomology. 40:1437-1447.
Lapointe, S.L., Alessandro, R.T., Robbins, P.S., Khrimian, A., Svatos, A., Dickens, J.C., Otalora-Luna, F., Kaplan, F., Alborn, H.T., Teal, P.E. 2012. Identification and synthesis of a male-produced pheromone for the neotropical root weevil Diaprepes abbreviatus. Journal of Chemical Ecology. 38:408-417.
Rohrig, E.A., Hall, D.G., Stansly, P.A. 2012. Field releases in Florida of Diaphorencyrtus aligarhensis (Hymenoptera: Encyrtidae), an endoparasitoid of Diaphorina citri (Homoptera: Psyllidae) from mainland China. Florida Entomologist. 95(2):476-478.