Progress 09/01/21 to 08/31/23

Outputs
Target Audience:This Phase II project will provide an effective repellent to control multiple invasive ambrosia beetle pests of forestry and agriculture. Ambrosia beetles are closely related to bark beetles and share many of the same behaviors, digging galleries into the wood of trees in which to lay eggs and rear their young. Unlike bark beetles, ambrosia beetles do not feed directly on the tree's tissues but instead use them as substrates to cultivate ambrosia fungi, which they rely on for sustenance. Redbay ambrosia beetle (Xyleborus glabratus) is a key pest of redbay trees (Persea borbonia) and other lauraceous plants, and transmits the deadly fungal pathogen, laurel wilt, Raffaelea lauricola. This disease has devastated the forest ecosystems of the southeastern US, killing over half a billion trees since RAB was first detected in Georgia in 2002, and is a serious threat to the Florida avocado industry. Black stem borer (Xylosandrus germanus) has become a major pest of ornamental tree nurseries and landscapes across the US, transmitting its associated fungus, Ambrosiella hartigii and the pathogenic Fusarium spp., which can damage timber and cause stem cankers, dieback, and in some cases, tree death. The polyphagous shot hole borer has recently emerged as a serious threat to multiple tree species in California through transmission of Fusarium dieback, which interferes with the conduction of food and water through the tree, placing it understress and eventually leading to dieback. Current control techniques for ambrosia beetles are limited, consisting of keeping trees as healthy as possible to minimize their susceptibility to attacking beetles and fungal infection. Conventional pesticide treatments have generally not been effective against these pests. ISCA proposes to develop a new control product to meet this challenge: Ambro Repel, a potent repellent that will prevent attack on vulnerable forest and crop trees by ambrosia beetles. This product will combine naturally occurring repellent compounds such as the anti-aggregation pheromone and plant-produced volatiles with ISCA's controlled-released matrix, SPLAT® (Specialized Pheromone & Lure Application Technology), to produce a formulation that will provide effective, longlasting protection of treated trees and small stands, and will also be amenable to quick, inexpensive application via a range of manual and mechanized application equipment. Ambro Repel would allow forestry personnel, landscapers, and farmers to efficiently promote area-wide control to eradicate new invasive populations, control established populations, and slow the spread of ambrosia beetles to new areas without resorting to additional inputs of environmentally hazardous, resistanceprone conventional pesticides. Changes/Problems:The disruption of the supply chain has made it difficult to obtain active ingredients, especially verbenone. For example, an order to our CMO in India for one metric ton verbenone AI that we put in September 2022that was to be delivered late in November 2022, they delivered 100kg in February, and are promising another 100kg of AI per every other two weeks. In the past it would take 2 to 3 months for them to deliver such quantities. Same with DJI, from where we order extra batteries for the drone trials, which right now are not available. These disruptions and delays are permeating every aspect of our projects, including this. We are planning to anticipate the production of all the materials necessary for this project, so we avoid problems with disruption of the supply chain. What opportunities for training and professional development has the project provided? Nothing Reported How have the results been disseminated to communities of interest? Nothing Reported 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 formulation. Ambro Repel is designed to be applied in large dollops to the bark of trees using a wide variety of manual or mechanized application technologies (backpack sprayer, caulking gun, vehicle-mounted applicator, etc.) and to cure and become rainfast within a few hours. The active ingredients (AIs) are released into the air around the tree at biologically active rates, sending a chemical signal to any host-seeking ambrosia beetles that the tree is not a suitable host for colonization. Prototype formulations of Ambro Repel containing verbenone, methyl salicylate, or both have been subjected to field trials and have shown efficacy in reducing trap captures and/or attacks on susceptible trees by seven species of ambrosia beetles (black stem borer, redbay ambrosia beetle, Cnestus mutilatus, Xylosandrus crassiusculus, Xyleborus volvulus, and X. bispinatus) in both forestry (Florida redbay groves) and agricultural settings (avocado groves, potted apple trees). ISCA now has evaluated varying formulations of Ambro Repel, containing the following single AIs andcombinations thereof: methyl salicylate; (-)-verbenone, the more common of the beetle anti-aggregation pheromone; and (+)-verbenone, the rarer isomer, which has proven more effective against certain species of wood-boring beetles. Task 2: Optimize Ambro Repel application strategy for orchards. For this task, ISCA has partnered with Agridrones to develop a strategy to apply SPLAT products using specialized applicators affixed to unmanned aerial vehicles (UAVs), also known as drones. There are many advantages to drone-based pesticide applications. Drones are available at much lower costs than fixed-wing aircraft, making them a viable option on farms of virtually any scale. Drones are capable of flying at low altitudes and hovering stationary for long periods, enabling them to deliver the SPLAT formulation, like Ambro Repel/Beetle Guard, at more precise rates and locations, making the application safer, more cost effective, and less wasteful. Our early estimates indicate a drone can apply a given SPLAT payload in one-fifth the time it would take manual applicators to deliver the same payload over the same area at a reduced cost. This leads to two desirable outcomes: reduced cost of application, resulting in a net increase in productivity; and reduced impacts on the surrounding environment and ecosystem. Drones can also deliver SPLAT formulations to areas near trees, cliffsides, and other obstacles that make the other mechanical operations difficult or unsafe. The lack of any need for an onboard pilot to control this type of aircraft also precludes the possibility of injury or death should the vehicle malfunction, while also doing away with the hazards associated with the operation of ground-based application equipment, which can be difficult and dangerous on steep or rocky terrain. The first field trials with the drone application have been done on flowering almonds and the effectiveness of the application (measured as the percent of dollops remaining on the tree instead of dropping on the ground) varied between 88% in young orchards with no foliage, to 98% in orchards with large trees and flowers in bloom. Application of this formulation needs to be adapted to the numerous situations where the product needs to be deployed. We believe that drone application will be a viable form of application of Ambro Repel/Beetle Guard in certain situations, especially in areas of difficult access, like hills, bogs, etc. But we also believe that there are situations where a manual application is called for, as well as mechanical ground application. We are building a mechanical system of applying SPLAT, called "splatigator," that can be mounted on the back of a gator, a tractor, or a pickup for the convenient application of the formulation in large areas. More work will be done to make sure that we provide the user with at least one effective way of applying Ambro Repel/Beetle Guard that is optimal for their situation. Task 3: Optimize Ambro Repelapplication strategy for nurseries. Work conducted on this task to date has been done in collaboration with Karla Addesso and Jason Oliver(Tennessee State University) at Moore Nursery, Irving College, TN. Under their supervision, a small-scale field trial was implemented to assess the capacity of an Ambro Repel prototypeto repel ambrosia beetles from nursery field plots, as measured by ethanol-baited traps placed within the field. Four plots of dogwood trees were used in the trial, each one measuring approximately 35 x 250 ft and containing five tree rows (40 trees per row). Two plots were assigned to receive treatment with Ambro Repel, and the remaining two were left untreated to serve as controls. In treated plots, Ambro Repel was applied in 3-g point sources placed in small cups, which were attached to a low branch of each tree in the plot (~200 trees per plot). To assess the presence of ambrosia beetles in treated and untreated plots, three soda bottle traps were placed for each plot: one 5 ft outside the plot (one tree space), one 5 ft inside the plot, and one in the center of the plot, on a middle tree of the third row. Each trap was baited with an Agbio ethanol lure, and the catch tubes were filled with antifreeze to preserve the beetles for later counting and identification. Traps were left in place from May 11 to June 8, 2021, and were serviced at 2 and 4 weeks. Treatment effects were evaluated on the bases of the number of ambrosia beetles captured in the ethanol baited traps. Target species for this trial included black stem borer, granulate ambrosia beetle (Xylosandrus crassiusculus Motschulsky), camphor shot borer (Cnestus mutilatus Blandford), fruit-tree pinhole borer (Xyleborinus saxesenii Ratzeburg), and any other ambrosia beetles that were readily identifiable in the trap captures. Results: Total number of ambrosia beetles captured across all detected species, total number of captured X. crassiusculus, and total number of captured X. germanus did not differ between untreated plots and plots treated with the Ambro Repel prototype. More beetles were captured in traps placed at the plot center and 5 ft inside the plot than those placed outside its border. These data do not suggest that the application of Ambro Repel exerted a repellent effect on the ambrosia beetles present in and around the nursery at the time of the trial. Possible reasons for this lack of repellent activity include an insufficient application rate of the formulation, ineffective timing of the application (the repellent may prove more effective if applied during bud break, when ambrosia beetles are seeking a host plant after overwintering), and the characteristics of the test site. This trial was conducted on older nursery plots, where a native ambrosia beetle population may already have already become established prior to the installation of the experiment. In addition, ISCA's collaborators for this trial report that repellents that are effective in Ohio and Virginia X. crassiusculus populations consistently prove less effective when tested in Tennessee populations. Finally, the test design itself may not be conducive to assessing the efficacy of Ambro Repel. The beetles may have been caught in the traps because they detected a positive signal (ethanol) emanating from the trap point, absent a negative signal (Ambro Repel), whereas when they approach a tree, the repellent signal would become stronger. We have been able to task 1. Optimize and check the Ambro Repel formulation in the LAB, task 2. Optimize Ambro Repel application strategy for orchards, where we have been able to verify that manual application and drone applications are viable for Ambro Repel, and task 3. we have been able to start the optimization of Ambro Repel application strategy for nurseries.

Publications

Progress 09/01/21 to 08/31/22

Outputs
Target Audience:This Phase II project will provide an effective repellent to control multiple invasive ambrosia beetle pests of forestry and agriculture. Ambrosia beetles are closely related to bark beetles and share many of the same behaviors, digging galleries into the wood of trees in which to lay eggs and rear their young. Unlike bark beetles, ambrosia beetles do not feed directly on the tree's tissues but instead use them as substrates to cultivate ambrosia fungi, which they rely on for sustenance. Redbay ambrosia beetle (Xyleborus glabratus) is a key pest of redbay trees (Persea borbonia) and other lauraceous plants, and transmits the deadly fungal pathogen, laurel wilt, Raffaelea lauricola. This disease has devastated the forest ecosystems of the southeastern US, killing over half a billion trees since RAB was first detected in Georgia in 2002, and is a serious threat to the Florida avocado industry. Black stem borer (Xylosandrus germanus) has become a major pest of ornamental tree nurseries and landscapes across the US, transmitting its associated fungus, Ambrosiella hartigii and the pathogenic Fusarium spp., which can damage timber and cause stem cankers, dieback, and in some cases, tree death. The polyphagous shot hole borer has recently emerged as a serious threat to multiple tree species in California through transmission of Fusarium dieback, which interferes with the conduction of food and water through the tree, placing it under stress and eventually leading to dieback. Current control techniques for ambrosia beetles are limited, consisting of keeping trees as healthy as possible to minimize their susceptibility to attacking beetles and fungal infection. Conventional pesticide treatments have generally not been effective against these pests. ISCA proposes to develop a new control product to meet this challenge: Ambro Repel, a potent repellent that will prevent attack on vulnerable forest and crop trees by ambrosia beetles. This product will combine naturally occurring repellent compounds such as the anti-aggregation pheromone and plant-produced volatiles with ISCA's controlled-released matrix, SPLAT® (Specialized Pheromone & Lure Application Technology), to produce a formulation that will provide effective, long-lasting protection of treated trees and small stands, and will also be amenable to quick, inexpensive application via a range of manual and mechanized application equipment. Ambro Repel would allow forestry personnel, landscapers, and farmers to efficiently promote area-wide control to eradicate new invasive populations, control established populations, and slow the spread of ambrosia beetles to new areas without resorting to additional inputs of environmentally hazardous, resistance-prone conventional pesticides. Changes/Problems:Supply chain and covid restrictions have made it more difficult to deal with the materials needed for further field trials and lab work.For example, active ingredients needed for the Ambro Repel formulationthat used to take two to three months to obtain the raw materials and manufacture, today it is taking over 6 to 9 months just obtain the raw materials. What opportunities for training and professional development has the project provided?Our academic partners are training undergraduate and graduate students using our Ambro Repel system. Results will be published. How have the results been disseminated to communities of interest?We are disseminating the information to communities of interest, including avocado growers in FL and CA, pome and stone fruit growers in NY, macadamia growers in HI, and others. What do you plan to do during the next reporting period to accomplish the goals?The research will continue to optimize Ambro Repel through a five target effort: 1) Further Optimize Ambro Repel formulation. 2) Optimize Ambro Repel application strategy for orchards and forestry. 3) Optimize Ambro Repel application strategy for nurseries. 4) Further diversify commercial opportunities by testing Ambro Repel against additional species of importance in Florida and California avocado groves, and New York pome fruit orchards. 5) continue to involve stakeholders in problem identification and implementation of results.

Impacts
What was accomplished under these goals? Ambro Repel is a safe, effective, and sustainable method of protection for a vast range of tree species, of importance to both US forestry and agriculture, against a group of invasive insect pests that has thus far proven exceptionally resilient to conventional control efforts. The formulation of Ambro Repel is being optimized to fit the needs of different application methods needed to protect this vast range of tree species.

Publications