Progress 07/01/18 to 02/28/19

Outputs
Target Audience:With this project, ISCA aims to provide a solution to the threat posed by multiple species of invasive ambrosia beetles, including RAB, BSB, and PSHB. These pests, and the fungal organisms they transmit, pose a serious threat to a wide range of forestry, landscape, and crop trees, and have the potential to cause high levels of ecological and economic damage across the US. Ambro Repel is designed to provide a specific, effective, and environmentally sound method for protecting susceptible trees from attack by these ambrosia beetle pests, whether in forests, urban or suburban landscapes, or crop fields. If Ambro Repel proves effective in protecting ambrosia beetle-susceptible tree stands as well as single trees, the prospective markets are large, involving both large governmental and institutional users as well as consumers that want to protect only a few trees. ISCA Technologies will sell Ambro REPEL to foresters interested in protecting ambrosia beetle host trees and tree stands, in both urban and rural settings, belonging to the private and governmental markets. Ambro Repel can also be marketed as a low cost, efficient alternative to insecticides for commercial growers to protect economically important trees that have been shown to be vulnerable to attack by the target pests, such as apple growers in the eastern US, where BSB is now infesting commercial orchards, and avocado growers in Florida and California seeking to protect their trees from RAB and/or PSHB. Changes/Problems: Nothing Reported What opportunities for training and professional development has the project provided? Nothing Reported How have the results been disseminated to communities of interest?A major component of ISCA's Phase I and Phase II projects for the USDA SBIR program consists of establishing and maintaining informative and productive relationships with the various stakeholder groups affected by a particular insect management problem or opportunity, from agricultural producers and their customers, manufactures and distributors, to government officials and pest control advisors. What do you plan to do during the next reporting period to accomplish the goals? Nothing Reported

Impacts
What was accomplished under these goals? Task 1. Optimize Ambro Repel formulations with a field life of a minimum of 8-12 weeks. Task 1.1. Develop the Proposed Ambro Repel Formulations: Several prototype formulations of Ambro Repel were created, incorporating ISCA's two repellent AIs, verbenone and methyl salicylate into wax matrices of varying characteristics, including wax type and dose of the semiochemical blend. An Ambro Repel prototype containing 10% verbenone and 10% methyl salicylate, applied as 3.0 g dollops was selected for further testing under this task. Task 1.2 Characterize the Field Release Profile of Ambro Repel: Three-gram point sources of Ambro Repel [10% (+)-verbenone and 10% methyl salicylate] were aged in the lab for a period of 3 weeks, compared to equally sized dollops of SPLAT blank (no AI) used as a control. Samples were subjected to GC-FID analysis using a standard analytical protocol, revealing that the concentration remaining of the two repellent AIs decreased with time elapsed since application. On Day 0, analysis revealed the AI concentration to be 9.99% (+)-verbenone and 10.36% methyl salicylate, dropping to 4.15% and 1.55%, respectively, after 7 days of aging in the lab, and to near 0 after 3 weeks. Task 2. Field trials to demonstrate that Ambro Repel suppresses attraction of adult ambrosia beetles. Several field trials assessing the performance of Ambro Repel were conducted, beginning with a study on several prototype formulations to determine their capacity to suppress trap capture rates of adult ambrosia beetles in Tennessee. These trap suppression studies were followed by assessments of the impacts of Ambro Repel applications on ambrosia beetle infestation rates at the single tree and small stand level. Task 2.1. Trap Catch Suppression. A pair of field trials, conducted in May and June 2018 by Drs. Jason Oliver and Karla Addesso (Tennessee State University, Otis Floyd Nursery Research Center, McMinnville, TN), assessed several ISCA repellent formulations for ambrosia beetles, containing (-)-verbenone (the more common isomer of this compound), (+)-verbenone (the rarer and more difficult verbenone isomer to produce), methyl salicylate (MeSa), or a combination of these two AIs. All trap treatments except the blank trap received one ethanol bait. Traps consisted of soda bottles with three slot openings, suspended upside down and attached to a soda bottle blank via a tornado tube (with the center of the tube drilled out) and filled with 15 mL of Splash RV and Marine Antifreeze, mixed with tap water at a 50:50 ratio. Traps were spaced 30 m apart, with 50 m between replicates. Five replicates were established for each treatment in a randomized block design. Differences were observed in the number of Xylosandrus crassiusculus (χ2(6) = 68.39, P < 0.0001), Cnestus mutilatus (χ2(6) = 12.67, P = 0.0485) and Xylosandrus germanus (χ2(6) = 78.32, P < 0.0001) captured by the different treatments. X. crassiusculus is the most damaging species in Tennessee production, therefore the two best repellents for that species, SPLAT Verb MeSA (10%/10%) and SPLAT Verb+ MeSA (10%/10%), which were also effective against two additional ambrosia beetles of concern, were selected for further investigation. These formulations were evaluated in two bolt assays initiated in May and June, 2018, assessing the capacity of the two repellents to reduce ambrosia beetle attraction to ethanol-treated bolts of Zelkova serrata, commonly known as Japanese zelkova. In both trials, the SPLAT Verb/MeSA and SPLAT Verb+/MeSA reduced ambrosia beetle attacks compared to the ethanol checks (Trial 1: χ2(2) = 44.45, P < 0.0001; Trial 2: χ2(2) = 47.26, P < 0.0001). SPLAT Verb/MeSA reduced attacks to a greater degree than Verb+/MeSA in the second trial, but the difference was not significant according to a Tukey-Kramer pair-wise separation test. Task 2.2. Individual tree protection trials. Dr. Agnello, together with his colleague Dr. David Comb (Entomology, Cornell AgriTech), also tested several ISCA repellent formulations for their capacity to suppress infestation and damage by BSB in apple plants. This research team tested trunk applications of several ISCA repellents on 2-year-old potted apple trees waterlogged to stress them to produce ethanol and placed in wooded areas directly adjacent to orchard sites. Separate ethanol lures were also attached to each tree. Different topical formulations and rates of methyl salicylate, alone and combined with verbenone, were applied to the trunk of each potted plant. Two additional products were also tested, to compare the efficacy of these experimental treatments to commercially available alternatives: Actigard (acibenzolar-S-methyl), a Systemic Acquired Resistance (SAR) activator; and Lorsban (chlorpyrifos), the growers' standard insecticide for management of BSB. Trunk and tree damage was assessed among the different treatments on July 10, after the end of the first adult flight, and on August 23, as the second flight was subsiding, to determine what effect these treatments had in preventing attacks by this beetle. On each date, half the trees in each treatment group were uprooted and brought to the lab, where they were dissected to count and characterize the infestation levels, based on number of infestation sites, gallery contents (adults and/or brood), and number of empty or aborted galleries. On August 23, all repellent treatments had fewer infestation sites than the untreated control. There was a similar trend on the earlier evaluation date, but not at statistically significant levels. The fewest number of galleries containing adults was seen in trees treated with Actigard and all repellent treatments, especially on the 23 August evaluation date. No brood was present in galleries on July 10. On August 23, lower brood numbers were seen in all treatments than in the untreated control except the plants treated with Lorsban, with zero brood observed in trees subjected to all treatments containing methyl salicylate. With regard to number of aborted galleries, lower numbers were observed in trees treated with the combination of verbenone and MeSa, particularly when the trees were treated at higher rates. In general, all the repellent treatments had fewer infestation sites than the untreated controls. The combined verbenone+MeSa treatments had the lowest incidences of galleries containing adults or brood; effects were more pronounced according to rate. Task 2.3. Field trials to demonstrate the efficacy of the Ambro Repel in protecting tree stands from Ambrosia Beetle attacks. We conducted a second field study, beginning in summer 2018, to determine the efficacy of Ambro Repel for controlling stands/orchards of trees of each of the three beetle species' preferred target tree. Two treatments were compared: 1) untreated control and 2) Ambro Repel. Treatment plots consisted of 1-ha areas under high ambrosia beetle population pressure. Four replicate plots were assigned to each treatment, and each plot had at least a 200-m untreated buffer zone. Treatment assignment to plots was completely random. Prior to applying treatments, trees within each plot were mapped, the trunk circumference measured, and characterized as healthy (free of infestation), infested, and/or dead. Only those trees that were healthy and susceptible to attack were treated. Ambro Repel treatments were applied once at the start of the trial. Treatments were applied directly to the bark of the tree at a height of 2-3 m. We also determined the rate of tree attack for each of the plots through visual assessments. Task 3. Involve stakeholders in problem identification and implementation of results. See response to later question re: Dissemination of project results to communities of interest.

Publications