Grant Information
| Knowledge Area | Subject of Investigation | Field of Science | Percent |
|---|---|---|---|
| 211 - Insects, Mites, and Other Arthropods Affecting Plants | 999 - Citrus, general/other | 1130 - Entomology and acarology | 100% |
This Small Business Innovation Research Phase II project is providing better attractants for the Asian Citrus Psyllid, Diaphorina citri. The attractants can be combined with control products such as Spinosad to generate improved bait-and-kill stations. D. citri vectors greening disease and is the most serious pest faced by the US citrus industry. The psyllid has severely affected Florida citrus production, and has spread to Texas and California, where citrus is a billion-dollar industry with a history dating back to the 19th century. However, current control approaches lack effective attractants for use in bait-and-kill stations and traps. The approach described uses rational design to isolate compounds that bind to critical psyllid chemosensory proteins, and then identifies thosecompounds capable of attracting psyllids. These novel attractants can then be incorporated into bait-and-kill stations containing a control product such as Spinosad. This project incorporates several innovative platform technologies comprising a high throughput assay system for rapidly identifying molecules that interact with insect chemosensory proteins. The project also involves the development of a novel bait-and-kill station intended specifically to attract D. citri to a control agent capable of limiting psyllid populations.The broader impacts of this project are platform technologies applicable to the development of control products for insect pest species of economic, agricultural, or medicinal significance. A bait-and-kill station enhanced with a highly efficient attractant will provide crucial psyllid population control, and no commercial psyllid attractant currently exists. There is thus a serious need by the US citrus industry for such a product.
Isolating new lead compounds:We plan to screen up to 50 additional compounds that are structurally related to INS6937.ChemBridge (San Diego, CA, USA) provides commercial "hit-to-lead" or lead optimization services tailored to the client's requests, and we have established a working relationship with the firm that is now in its sixth year. Modifications that affect the solubility and the volatility of the lead Arometics may be crucial for successful pest control formulations. Current insect repellents such as DEET are hydrophobic, and are dispensed as aerosols or emulsions in non-aqueous solvents as a result; furthermore, timed-release gels or polymers containing control products such as the hydrophobic mosquito attractant, octanol, are already marketed. Thus, appropriate dispensal systems already exist for an Arometics product with similar physical properties to octanol or DEET. We will use the resources and expertise of ChemBridge chemists in order toensure our Attractant formulations are sufficiently volatile in order to be appropriate for use intimed-release polymers or impregnated materialsEnhancing Arometics with foliar volatiles:Behavioral assays will be conducted to determine whether the addition of naturally-occurring foliar volatiles to test ligands might have an additive or synergistic effect on psyllid probing response. Each of the three test ligands (INS6937, INS0338, and INS2511) will be tested with a panel of foliar volatiles that frequently occur in citrus foliage. An initial dosage-response test will be conducted on each of the foliar volatiles using a semi-log concentration gradient (3μL, 9 μL, 30 μL, and 90 μL/ 10 mL SPLAT). This semi-log dosage design has proven effective in our previous studies of psyllid behavioral response to test compounds.38,40 This test will indicate the optimal concentration to use in follow-up evaluations with the test ligands. Binary mixtures of individual ligands and terpenes will then be tested as described above with the exception that the proportions will be based on the optimal concentrations obtained in the dose-response test. If any of the binary or trinary mixtures of test ligands prove to be highly stimulatory, they will then be tested in combination with foliar volatiles as well.Field testing and trap development:Field tests with yellow sticky card traps will be performed to evaluate the effectiveness ofindividual test ligands, ligand mixtures, and mixtures of ligands and naturally-occurring leaf volatile compounds. The results of the laboratory assays described above and the field trapping tests conducted during Phase 1 will be used to inform the design of the experiments and the formulations of the test compounds. In other words, if laboratory assays reveal that certain ligands + ligand combinations or ligand + naturally-occurring leaf volatiles combinations induce high levels of probing activity, then those treatments will be prioritized for field testing. Since field studies conducted in Phase I already indicated that trap captures were enhanced by the addition of test ligand INS6937, additional field studies will be conducted to determine the optimal dosage level of this test ligand for psyllid trapping. Trap design The traps will measure 20 cm long x 2.5 cm wide, be bright yellow in color, coated on both sides with adhesive, and equipped with a twist tie at the top for suspending the trap from a stem. Test ligands and ligand mixtures will be mixed into 10 mL of fluorescent yellow powder (BioQuip, Inc.) with a vortex mixer and then sprinkled onto both sides of the sticky card trap. Approximately 0.5 mL of the powder + ligand mixture will be applied to each trap with a shaker device. In tests that include natural plant volatiles, the test compounds, which are liquids at ambient temperatures, will be dispensed from 0.4 mL narrow bore polyethylene microcentrifugetubes attached to the trap's twist tie. A blank trap, sprinkled only with yellow fluorescentpowder, will be included in the design of all experiments. Depending on the design of theexperiment, 12-25 traps will be placed at each study site. Following placement at the study sites, the traps will be left exposed for seven days and then collected.
Target Audience
We have dicussed our results with several members of the California Citrus Reserach Board and persued additional collaborations to expand the testing of our psyllid attractants.This work was also presented at several meetings and two papers were published describing the research results.
Changes / Problems
Nothing Reported
Training & Professional Development
During the course of this project we have trained 3 technicans and 1 undergraduate. The training has improved the work skills of the individuals involved and the undergraduate student has pre4sented the resutls at local research symposia.
Dissemination Streams
The results from the project has been presented at two international conferences by Dr. Patt and described in a publication. Dr. Woods has presented the results to the California Citrus Research Board and was in discussion with Bayer Crop Science to licence the attractants.
Next Reporting Steps
Nothing Reported
Target Audience
We have dicussed our results with several members of the California Citrus Reserach Board and persued additional collaborations to expandthe testing of our psyllid attractants.
Changes / Problems
Nothing Reported
Training & Professional Development
Nothing Reported
Dissemination Streams
Nothing Reported
Next Reporting Steps
Nothing Reported
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<br>What was accomplished under these goals? Objective 1: Enhancing the attractant by identifying additional Arometics. While INS6937 is our best attractant candidate thus far it has low volatility at lower temperatures. We will continue screening DcitSAP4 with additional small molecules. We have chosen to focus on DcitSAP4 because of the high level of positive compounds (20%) that came from the initial Mini-Target Test (MTT). We have identified 51 additional compounds in the Hit2Lead library that are at least 95% similar to INS6937. Eleven compounds with low logP (to improve solubility) were selected and screened in the Attenu assay for binding to DcitSAP4. Eight compounds demonstrated binding to the protein and are being screened in the MTT assay to test for a behavioral response in ACP. Up to 3 positive compounds will be utilized in small-scale field trials as described below. Objective 2. Enhancing the Arometic Attractant by testing for compound synergy. These experiments will be performed in the second year of testing. Our primary goal this year has been developing a formulation that gives reproducible positive attraction of the psyllids (see below). Objective 3. Enhancing the Arometic Attractant by improving the formulation. While INS6937 exhibits strong attraction the MTT assay, the compound is not naturally volatile and thus requires special formulation for field applications. Therefore we have developed several different methods of formulations to field test for Psyllid attraction of INS6937. The first of these involved dispersing the compound in a mixture of detergent/water. This has previously been shown to increase volatility of chemicals. In the second formulation we have incorporated the compound into SPLAT, a polymer utilized to dispense many pheromones. Tests were conducted at several field sites to evaluate the various formulations of INS6937 to enhance the capture of ACP on yellow sticky card traps. The tests were conducted in orange jasmine hedges at three study sites in Fort Pierce, FL. Orange jasmine is a favored host plant of ACP. The study sites were as follows: 1) USDA Farm--an 1100' long orange jasmine hedge located at the USDA-ARS research farm (USDA Farm); 2) Delaware Ave.--a private residence with a hedge containing 150 orange jasmine plants; and 3) Citrus Ave.-- an office building with an extensive orange jasmine hedge planted around the property perimeter. A second set of traps were placed in a citrus grove in Pauma Valley, California. This was done in collaboration with the California Citrus Research Board. Florida Test Site. The yellow sticky traps were obtained from Alpha-Scents Inc. They were cut to measure 20 cm long x 2.5 cm wide and were placed inside a cylinder constructed of black ¼-inch plastic mesh to exclude lizards and prevent foliage from adhering to the trap surface. The cylinder was capped with a 5 cm-dia. pvc pipe plug to help prevent rain water from wetting the trap surface. The traps were attached to the stems with a twist tie. The test ligand was dispensed from a plastic microscope cover slip placed on the trap surface. Each trap contained two cover slip dispensers, one on each side. The test ligand was dissolved in acetone (40 mg test ligand/10 mL acetone) and a 50-μL aliquot of the solution was applied to each cover slip. Sufficient time was permitted for the acetone to evaporate prior to attaching the cover slip dispenser to the trap. Each test consisted of traps with no additional stimuli (control), the test ligand, and two other test lures, one a generalized mixture of citrus volatiles (general mix) and the other containing three compounds and formulated to mimic the odor emitted by growing orange jasmine foliage (OJ mix). The general mix and OJ were dispensed from 1 mL plastic centrifuge tubes. The tubes were placed inside the screen cylinder and the tube caps perforated to allow the scent mixture to disperse. Tests with two treatments had 16 traps/treatment, tests with three treatments had 10 traps/treatment, and tests with four treatments had 8 traps/treatment. Orange jasmine plants with traps were situated 2 m from each other. Each test lasted for 7 days. Results--There was an approximately 2-fold difference between the mean trap captures on the control trap and the INS6937 treatment. The other scent treatments showed a smaller increase, however overall capture levels were low (Average of 13 ACP/trap). During the course of the study there have been several extended dry periods, which might explain the low numbers of trap captures. Testing was suspended during the winter months because psyllids are generally very inactive at this time of the year. A formulation of the test ligand in SPLAT has been developed and further testing will be conducted using this as a dispenser medium. California Test Site. Double-sided sticky yellow card (18 X14cm) were used in the Pauma Valley test site. The INS6937 was mixed into SPLAT at three concentrations: 0.5, 1 and 5%. The mixture was applied to one side of the sticky card and attached to the stem with a twist tie. A total of nine cards were placed in every fourth tree along a row. The every third trap was a control lacking INS6937. Traps are replaced every week and the placement of the control varied. Results--At the lower concentration (0.5 and 1%) there is up to a 3-fold increase in ACP capture compared to the control. At higher concentrations (5%) the capture rates are similar between control and INS6937. However the overall capture rates are low, and average of 5 ACP per card. <br><br><b>Publications</b><br>