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INVESTIGATOR: Addesso, K.

PERFORMING INSTITUTION:
TENNESSEE STATE UNIVERSITY
3500 JOHN A. MERRITT BLVD
NASHVILLE, TENNESSEE 37209

BEHAVIORAL-BASED CONTROL METHODS FOR AMBROSIA BEETLE MANAGEMENT IN ORNAMENTAL NURSERIES

NON-TECHNICAL SUMMARY: Nursery and greenhouse crops account for $16 billion of the United States economy. The nursery industry is complex due to the large number of plant species grown, which makes nursery pest management research challenging. Pests that have large economic impacts, attack multiple, high production genera and are quarantine restricted are key targets for research. One major problem impacting the nursery industry today is ambrosia beetles. These beetles feed and reproduce within living or dead trees. The beetles are difficult to control with conventional pesticides, and in the nursery industry, even cosmetic damage from the beetles can result in trees that are unsalable. The overall goal of this project is to investigate new tools for the integrated management of ambrosia beetles in nursery crops. The three areas of inquiry include (1) the identification of new attractants and repellents, (2) applications of the plant defensive hormones methyl salicylate or methyl jasmonate as nursery tree protective treatments, and (3) the recruitment of predators into nursery stands as a biological control option. These objectives will be accomplished through field and laboratory assays conducted at Tennessee State University's Nursery Research Center, located at the center of Tennessee's nursery production industry. The availability of additional management approaches based on any combination of attractants, repellents, plant hormone treatments and natural enemy augmentation will move the nursery industry toward an integrated approach to ambrosia beetle management and away from sole reliance on conventional pesticides.

OBJECTIVES: The overall goal of this research program is to increase understanding of the behavior and chemical ecology of important ambrosia beetle species, their host plants and predators. This goal will be achieved by investigating three objectives. 1. Identification of attractive and repellent compounds for use in ambrosia beetle management. 2. Effect of plant defensive hormone treatments on ambrosia beetle attacks and plant health. 3. Recruitment of ambrosia beetle predators into nursery stands.

APPROACH: Objective 1. Identification of new attractive and repellent compounds for use in ambrosia beetle management 1A. Initial surveys will begin with a simple laboratory bioassay to determine if the test has any attractant or repellent behavioral effects on the beetles. In these assays, tree bolts from host plants will be soaked in water for 4 hours. Test compounds will be applied to the surface of the bolt. Bolts will be placed in containers with vented lids under a fume hood. Five beetles will be placed in each container and number of attacks will be recorded after 24h. Ambrosia beetle attacks will be compared between treatments. Ifbeetles do not function effectively in the aforementioned assays, more labor-intensive field trials of test bolts will be conducted to screen for activity. 1B. Active essential oils that demonstrate potential activity against ambrosia beetles will be exposed to the antennae of X. crassiusculus using a GC-electroantennogram detection system to identify constituents in the test treatments responsible for the activity. Active test constituents will be identified using GC-mass spectrometry. Compound identity will be verified with synthetic standards and tested in behavioral assays to confirm activity. 1C. Compounds found to be the most attractive in screening will be tested in field bolt tests. Bolts injected with 5% ethanol will be placed on metal trap rods around the research center adjacent to forests or at a location known to harbor ambrosia beetle populations during the early spring and summer. A minimum of 8 bolt replications per treatment and untreated control bolts will be tested. Release rates from vials or from bolts will be determined in the laboratory. Bolts will be monitored daily for 7 days. New galleries will be identified for each observation date. All beetle galleries on a sub-sample of at least 4 bolt replicates will be dissected and the beetles identified to give an estimate of species composition. Total ambrosia beetle attacks will be compared between treatments. 1 D. Repellent compounds will be tested on trees injected with 5% ethanol against untreated and pesticide treated controls. A minimum of 8 tree replications per treatment will be run. Trees will be monitored daily for 7 days. New galleries will be identified for each observation date. At the end of the test, all beetle galleries on a sub-sample of at least 4 tree replicates will be dissected and the beetles identified to give an estimate of species composition. Total ambrosia beetle attacks will be compared between treatments. Objective 2. Effect of plant defensive hormone treatments on ambrosia beetle attacks and plant health. 2A. Trees will be treated 10 days before the experiment with methyl jasmonate solution, methyl salicylate or the control solution. The trunk of five dogwood trees will be surface treated. At 10 days, trees will be flood stressed by placing them in water-filled buckets so the pots cannot drain. Treatments will be arranged in a randomized complete block design. The number of attacks on trees will be recorded every 2-3 days for 3 weeks. Total ambrosia beetle attacks and species attacking will be compared between treatments. 2B. Attractiveness of volatiles from treated and untreated tree bark and foliage will be tested in behavioral bioassays. Volatiles will be collected from tree branches with foliage using a portable vacuum pump and specially-designed Teflon bags. Tree volatiles will be presented to X. crassiusculus in behavioral assays following the methods of Objective 1A to determine whether hormone treatments increased or decreased the attractiveness of the host plant volatiles. 3C. Five treated and untreated trees will be sampled 10 and 30 d following application of methyl jasmonate or methyl salicylate in order to measure changes in plant physiological characteristics related to defense and growth. The following parameters will be measured for differences between treatments: Plant Volatile Production. Volatiles from wood and leaf tissues will be collected on Super Q filters using a portable volatile collection pump for 3 hours. Samples will be characterized by GC and GC-MS following the methods described in Objective 1B. Sap Flow. Sap flow will be measured using a Dynamax Flow 32 sap flow meter to assess water usage and transpiration rates. Photosynthesis and Chlorophyll a & b Content. CO2 exchange and chlorophyll fluorescence will be measured using a LiCOR 6400. Chlorophyll a & b content of leaf tissue will be quantified on a UV/Vis microplate reader (Lichtenthaler and Buschmann 2001). Total Phenolics. Total phenolics will be measured on 1-cm diameter cores removed from the trunk at the center of where the plant hormone treatments were applied and 1-foot above the treatment line on the same tree. Total phenolics in trunk cores and 100 g of dried foliage will be measured using the Folin-Ciocalteu Assay (Singleton et al. 1999). Total Protein and Defensive Enzyme Activity. Total protein and defensive enzyme activity will be measured in 5 grams of foliage and trunk cores removed from the center of the treatment area and 1-foot above the treatment line following the methods of Rangasamy et al. (2009) and Pascholati et al. (1986). Total protein will be measured using the Bradford Assay (Bradford 1976). Peroxidase (POX) activity will be measured with guaiacol as a substrate. Increase in absorbance at 470 nm will be monitored for 2 min using a protocol modified from Hildebrand et al. (1986) and Hori et al. (1997). Polyphenol oxidase (PPO) activity will be measured with a protocol modified from Hori et al. (1997). Lipoxygenase (LOX) activity will be determined using a protocol modified from Skorzynska-Polit and Krupa (2002) with linoleic acid substrate prepared by following a protocol described in Koch et al. (1992). Phenylalanine ammonia-lyase (PAL) activity will be measured following the methods of Pascholati et al. (1986). Enzyme values for each sample tree will be analyzed twice and the values averaged. Mean enzyme values for each treatment will be compared to controls using t-tests. If raw or square root (X + 0.5) transformed data do not meet the requirements for parametric analysis, a non-parametric equivalent will be used. Objective 3: Recruitment of ambrosia beetle predators into nursery stands 4A. This study will test if it is possible to increase the number of ambrosia beetle predators, specifically the ubiquitous clerid beetle Thanasimus dubius (Fabricius), in stands of deciduous nursery trees using attractants normally encountered by the predator in coniferous forests. Individual compounds and commercially available lures known to attract T. dubius and related species will be tested. Soda bottle traps with lures will be hung in 5 trap islands at two separate sites the last week of March and monitored weekly for 10 weeks. Antifreeze will be placed in the collection cups to prevent predators from eating trapped beetles. Traps will be placed 20 m apart. The number of T. dubius captured by each lure will be recorded. Other beetles will be identified to family, genus and species if possible. 4B.The best lure from the first experiment will be used in test plots of 1-inch caliper dogwood trees. Four treatments will be tested: (1) untreated trees without a lure, (2) untreated trees with a lure, (3) 5% ethanol injected trees without a lure and (4) ethanol injected trees with a lure. Each treatment replication will consist of a plot with four trees with each plot spaced 20 m apart. In lure treatments, lures will be hung from a pole at the center of the four test trees. Two replications of each treatment will be conducted in two locations. The number of T. dubius present in each test plot based on foraging observations and the number of new ambrosia beetle attacks will be recorded twice a week for four weeks. Tests will be conducted at a different site from 4A to avoid bias from predator removal.

PROGRESS: 2013/09 TO 2016/08
Target Audience:The target audience for this reporting period included student interns and grower stakeholders. Students learned field and laboratory data collection and analysis approaches. Extension and outreach was performed for growers in the form of a talk delivered at an industry field day and a educational display at the TNLA Green Industry Field Day and MTNA industry field days, Master Gardeners of Rutherford County as well as additional presentations local and national meetings. Changes/Problems: Nothing Reported What opportunities for training and professional development has the project provided?This season three graduate students helped with this project while also working on their masters theses. This exposed the students to techniques in chemical ecology and nursery pest management. One masters student has shifted his plan of study to continue with the stress-mitigation work. How have the results been disseminated to communities of interest?The information from this research has been shared with the Tennessee Nursery and Landscape Association, Middle Tennessee Nursery and Landscape Association, Entomological Society of America, Tennessee Entomological Society, Mississippi Entomological Society, Southern Nursery Association. Publications are in preparation for field-relevant journals. What do you plan to do during the next reporting period to accomplish the goals? Nothing Reported

IMPACT: 2013/09 TO 2016/08
What was accomplished under these goals? 1. Several compounds have been tested in field trials and lab assays for incorporation into the current ethanol lure. Only one compound, conopthorin, showed significant increases in catches for our target species. Conopthorin is comercially available, but it is rather costly and may not make a significant enough increase in trap captures to warrent the cost to nursery growers. Some of the compounds did increase captures of non-target species and may be useful in monitoring other groups of ambrosia beetles in forest systems. 2. This objective was altered to include the use of other stress-mitigating and blocking products to reducel ethanol emission from plants with the goal of deacreasing ambrosia beetle attacks. One fungicide product applied prior to flooding of redbud and yellow poplar trees reduced ambrosia beetle attacks by 90%. Despite our prediction that the product would reduce ethanol emission from trees (thereby explaining the reduction in ambrosia beetle attacks), both treated and untreated plants released sufficient amounts of ethanol to attract beetles. The active incredient and other related compounds will be the focus of further investigations as a tool for ambrosia beetle management. Future research will include both pre- and post-flood efficiacy trials. More ethanol monitoring and a closer investigation of ambrosia beetle behavior relating to why the beetles are rejecting treated trees. A second study evaluated the use of a compound that can be mixed with kaolin clay (Surround WP) and applied to the surface of the trees to bind ethanol. This product successfully blocked ethanol in tree bolt and container trials. The next hurdle is to perfect a formulation that will be more rainfast. This will be the focus of future research in this area.

PUBLICATIONS (not previously reported): 2013/09 TO 2016/08
1. Type: Conference Papers and Presentations Status: Published Year Published: 2016 Citation: Evaluation of potentially attractive compounds for wood-boring beetle management in Tennessee and Connecticut. Alicia Bray, Karla Addesso, Jason Oliver and Chris Ranger. International Congress of Entomology/Entomological Society of America Meeting, Orlando, FL, September 24th-30th.
2. Type: Conference Papers and Presentations Status: Published Year Published: 2016 Citation: Stress signal reduction as a tool for managing ambrosia beetles in ornamental nursery. Karla M. Addesso, Fulya Baysal-Gurel, Paul A. O'Neal and Jason B. Oliver. International Congress of Entomology/Entomological Society of America Meeting, Orlando, FL, September 24th-30th.
3. Type: Conference Papers and Presentations Status: Published Year Published: 2016 Citation: A stress-mitigating product to reduce ambrosia beetle attacks on trees. Karla M. Addesso, Fulya Baysal-Gurel, Paul A. O'Neal and Jason B. Oliver. Tennessee Entomological Society, Knoxville, TN, Oct 6th-7th, 2016.
4. Type: Journal Articles Status: Published Year Published: 2016 Citation: Christopher M. Ranger, Michael E. Reding, Peter B. Schultz, Jason B. Oliver, Steve D. Frank, Karla Addesso, Juang Hong Chong, Blair Sampson, Christopher Werle, and Stanton Gill. Biology, Ecology, and Management of Xylosandrus spp. Ambrosia Beetles (Coleoptera: Curculionidae: Scolytinae) in Ornamental Tree Nurseries. Journal of Integrated Pest Management.
5. Type: Journal Articles Status: Other Year Published: 2017 Citation: Addesso, K.M. Oliver, J. B. and Bray, A. Casting the widest net: optimizing lures to survey for bark and ambrosia beetles in a state park. Insects. In preparation.
6. Type: Journal Articles Status: Other Year Published: 2017 Citation: Addesso, K.M., Baysal-Gurel, F. and Oliver, J. B. Pre-treatment of a stress mitigating fungicide to protect trees from ambrosia beetles. Environmental Entomology. In preparation.
7. Type: Journal Articles Status: Other Year Published: 2017 Citation: Addesso, K.M. and Oliver, J. B. Blocking ethanol as a method for managing ambrosia beetles in woody ornamental nurseries. HortScience. In preparation.

PROGRESS: 2014/09/01 TO 2015/08/31
Target Audience:The target audience for this reporting period included student interns and grower stakeholders. Students learned field and laboratory data collection and analysis approaches. Extension and outreach was performed for growers in the form of a talk delivered at an industry field day and a educational display at the TNLA Green Industry Field Day as well as additional presentations local and national meetings. Changes/Problems:2. We will be shifting our focus to two products: one which blocks ethanol emission and another that is a fungicide with growth/stress mitigation potential since the application of plant defense hormones directly to trees was not successful in supressing ambrosia beetle attacks. What opportunities for training and professional development has the project provided?The project supported 2 undergraduate summer interns for 10 weeks. The project also suppored the PI in a trip to Portland, OR to attend the Entomological Society of America Annual Meeting in 2014. How have the results been disseminated to communities of interest?Several presentations have been given at local landscape shortcourse, field days to growers and landscapers in Tennessee. Scientific results have been presented at the national Entomological Society of America Meeting. What do you plan to do during the next reporting period to accomplish the goals?1. We will be testing some additional compounds in the spring of 2016 for lures which have shown activity in the lab. 2. We will be testing a new fungicide product, which is reported to mitigate plant stress and increase growth as well as prevent fungal infections. We will test this in place of the hormone applications for use in mitigating ambrosia beetle attacks. We will also be continuing to test a 'blocker' product trialed in 2015 for blocking ethanol emission from trees as a potential management tool for ambrosia beetles.

IMPACT: 2014/09/01 TO 2015/08/31
What was accomplished under these goals? 1. Identification of attractive and repellent compounds for use in ambrosia beetle management. Threee oils were tested in combination with ethanol standard in the field using white trifold panel traps for a second year in both TN and CT for four weeks, with 8 replicates per treatment. Beetles from TN have been identified for both 2014 and 2015. CT samples are currently being processed and will be identified to family focusing on Scolytidae. Ambrosia beetles will be identificed to species where possible with a focus on the four most common ambrosia beetles nursery pests: granulate ambrosia beetle, black stem borer, fruti-tree pinhole borer and camphor shot borer. In the current year in TN we captured a total of 3,273 beetles. The most populous species were Xylosandrus crassisusculus (1,599), X. germanus (482), Xyloborinus saxeseni (356), Cnestus mutilaus (297), Euwallaceae validus (140), Xyleborus intrusus (94) and Xyleborus xylographus (49). All other species were caught with 20 or fewer specimens. X. crassiusculus showed the strongest preference for conopthorin+EtOH (362 EtOH vs 611 EtOH+conopthorin captures). Xylosandrus germanus, Xyloborus xylographus and Xyloborinus saxeseni also had more trap captured in the conopthorin traps. Cnestus mutilatus showed the opposite trend (132 EtOH vs. 33 EtOH+conopthorin). Only Xyleborus intrusus appeared to prefer the juniperberry oil (10 EtOH vs 65 EtOH+juniperberry). Repellency tests were also re-run this year in TN. 2014 trap captures were extremely low and did not provide much information on preferences. 2015 data is currently being processed. 2. Effect of plant defensive hormone treatments on ambrosia beetle attacks and plant health. This year there were no statistical differences in the number of hits between trees pre-treated with MeJA and controls (25.6 control vs 33 MeJA mean hits), (13.4 vs. 16.4 gallaries established). Nor were there any differences in chlorophyll reading via SPAD on Day 0 or Day 14 post stress treatment. 3. Recruitment of ambrosia beetle predators into nursery stands. The data from the clerid beetle attraction test has been fully processed. A number of different bark beetle species were collected using the 4 lures. Very few bark beetle predators were collected. In ten weeks of collecting we identified 6 specimens of Thanasimus dubius, our clerid of interest and 5 specimens of Enoclerus nigripes.

PUBLICATIONS: 2014/09/01 TO 2015/08/31
1. Type: Conference Papers and Presentations Status: Accepted Year Published: 2014 Citation: Non-native ambrosia beetles as indicators of living, but weakened trees. Christopher Ranger (presenter), Peter B. Schultz, Steven D. Frank, Michael E. Reding, Patrick Tobin, Jason B. Oliver, Karla Addesso. P-IE Section Symposium: Exploring Complex Interactions among Non-Native Bark and Ambrosia Beetles (Coleoptera: Scolytinae), their Associated Fungi, and Na?ve Hosts. Entomological Society of America Annual Meeting, Portland, OR, November 15-19th, 2014.
2. Type: Other Status: Accepted Year Published: 2015 Citation: Woody Ornamental Pest Management: Safer Solutions for Challenging Pests in Nursery and Landscape. Karla M. Addesso and Jason B. Oliver. University of Tennessee Entomology and Plant Pathology Department Seminar Series.
3. Type: Other Status: Accepted Year Published: 2015 Citation: Systemics, Organics and Alternative Methods for Pest Control in the Landscape." Karla Addesso. Middle Tennessee Landscape and Grounds Management Shortcourse.
4. Type: Other Status: Accepted Year Published: 2015 Citation: "Ornamental Nursery and Landscape Pests of Concern in Tennessee," Tennessee Nursery and Landscape Association and Tennessee Nursery and Green Industry Educational Field Day, TSU Nursery Research Center, June 3rd, 2014.
5. Type: Conference Papers and Presentations Status: Accepted Year Published: 2014 Citation: Kaolin clay application as a deterrent for ambrosia beetle (Curculionidae: Scolytinae) attack at ornamental nurseries. Chris Werle (student), Karla Addesso, Blair Sampson and John J. Adamczyk. Entomological Society of America Annual Meeting, Portland, OR, November 15-19th, 2014.
6. Type: Conference Papers and Presentations Status: Accepted Year Published: 2014 Citation: Calling all wood-boring beetles: laboratory and in-field bioassays with potentially attractive compounds for beetle management. Alicia Bray, Karla Addesso, Jason Oliver and Paul A. O'Neal. Entomological Society of America Annual Meeting, Portland, OR, November 15-19th, 2014.
7. Type: Conference Papers and Presentations Status: Accepted Year Published: 2014 Citation: Tree proximity and ethanol dose as factors in trap tree strategy for exotic ambrosia beetles. Jason B. Oliver, Karla Addesso, Nadeer Youssef, Paul A. O'Neal, Christopher Ranger, Michael E. Reding, Peter B. Schultz, Blair Sampson, Joshua P. Basham, Joseph Lampley and Debbie Eskandarnia. Entomological Society of America Annual Meeting, Portland, OR, November 15-19th, 2014.

PROGRESS: 2013/09/01 TO 2014/08/31
Target Audience: The target audience for this reporting periodincluded student interns and grower stakeholders.Students learned field and laboratory data collection and alalysis approaches.Extension and outreach was performed for growers in the form od a talk delivered at an indistry field day and a educational display at the university Small Farms Expo. Changes/Problems: Objective 1B. Our GC-EAD is currently not operational. We are supplementing our screeing procedures with more behavioral assays of individual compounds to compensate for the loss of equipment. Objective 2. We plan to makechanges to objective 2to better focus our efforts on methyl jasmonate, which showed some effect onambrosia beetle behavior this year. We will not repeat the analysis of methyl salicylate. Wewill not conduct a sap flow meter analysis as preliminary tests showed no difference in water usage. We will increase our replications of trees for controls and MeJA treated trees in the attack analysis. What opportunities for training and professional development has the project provided? The project supported three undergraduate summer interns for 10 weeks. The project allowed for one technical and one professional staff member to attend the Bark and Ambrosia Beetle Academy heldat University of Florida inGainesville, FL in May 2014. How have the results been disseminated to communities of interest? A presentation was given to the Tennessee Nursery and Landscape Association members during their Green Industry Field Day on June 4th, 2014. "Ornamental Nursery and Landscape Pests of Concern in Tennessee." What do you plan to do during the next reporting period to accomplish the goals? 1. Identification of attractive and repellent compounds for use in ambrosia beetle management. We will continue to evaluate oils and individual compounds that show promise in small arena assays. 2. Effect of plant defensive hormone treatments on ambrosia beetle attacks and plant health. We will repeat our evaluation of MeJA at previous year's and a higher rate to assess effect on beetle behaviorand tree physiology. 3. Recruitment of ambrosia beetle predators into nursery stands. We will repeat the survey of ambrosia beetle predators and determine which lure, if any, has potential for drawing in predators to nursery stands.

IMPACT: 2013/09/01 TO 2014/08/31
What was accomplished under these goals? 1. Identification of attractive and repellent compounds for use in ambrosia beetle management. A total of 54 essential oils and compounds were tested in small arena assays for attractive or repellent activity against granulate ambrosia beetle. Two oils and two additional compounds displayed repellent effects (> 60% repellency). Nine demonstrated attractive properties (> 60% attraction). Oilsdemonstrating repellent and attractive properties were analyzed by GC-MS and major constituents identified by library search to create a list of potential compounds for further testing. Six targeted oils/compounds were tested in the field using white trifold panel traps in combination with ethanol lures to see if the oils could increase or decrease trap catches of ambrosia beetles and other woodborer families when compared to an ethanol standard. Two tests were run - one containing putative attractants, and the other, repellents -for four weeks, with 8 replicates per treatment.Beetles are currently being processed from traps and will be identified to family focusing on Scolytidae, Buprestidae and Cerembycidae. Ambrosia beetles will be identificed to species where possible with a focus on thefour most common ambrosia beetles nursery pests: granulate ambrosia beetle, black stem borer, fruti-tree pinhole borerand camphor shot borer. 2. Effect of plant defensive hormone treatments on ambrosia beetle attacks and plant health. Trees were surface treated witha0.01% solution of one of two plant defense hormonesor a control. Plants were then drenched with ethanol to inducea stressed state and randomly arrayed along a woodlot with known ambrosia beetle populations. Ambrosia beetle hits were recorded every three days for two weeks. There werefewer hits inthemethyljasomonate treatment compared to the control (47 vs 27 hits;z = 2.3, p = 0.02), but no differencebetween control andmethyl salicylate (47 vs 41;z = 0.64, p = 0.52). The rate of methyl salicilate used, however, did not prevent beetles from attacking treated trees. SPAD meter (chlorophyll) readingsofyoung, full leaves was performedon alltrees in each treatment, but readings were quite variable, even within single leaves. Two of the leaves from each tree sampled by SPAD were collected forchlorophyll analysisprior to hormone treatment, on Day 0 when ethanol stressor was applied and onDay 18 following the ethanol treatment.UV-VIS analysis of chlorophyllshowed no change in estimated chlorophyll a or chlorophyll b in the MeJA and MeSA treated trees between pretreatment and Day 18. There was a slight increase in estimated chlorophyll a content in control trees (z = 2.43,p = 0.015). Additionally, there was an increase in estimated carotenoids in control (z =3.99, p < 0.0001) and MeJA (z = 2.78, p = 0.006)treated trees. Day 0 leaf samples are currently being processed. Coorelationsbeween SPADmeter readings andtotal chlorophyllhad an R-value of 0.56for thepretreatment readings and 0.89 atDay 18. We suspectthe interference of moisture on leaves caused the poor coorrelationin thefirstset of readings. 3. Recruitment of ambrosia beetle predators into nursery stands. We set out lindgren funnel traps in six randomized replicates containing pine oil and lures typically used to monitor coniferous bark beetles in the field in order to assess predator classes and numbers with a particular emphasis on Clerid and Scolytid beetles. Samples from traps are currently being sorted.

PUBLICATIONS: 2013/09/01 TO 2014/08/31
No publications reported this period.