This is the final report for this project. Exceptional cooperation between the University of Florida, ISCA Technologies Inc. and USDA-ARS was made possible through funding provided by CRDF. ARS and UF researchers identified optimal pheromone blends for attraction and disruption of the citrus leafminer, Phyllocnistis citrella, and for control of the incidence and severity of citrus canker disease. Extensive field trials were carried out in commercial citrus groves thanks to support provided by The Packers of Indian River, TRB Groves and Golden River Fruit Company. Experiments employing small (~1 acre) plots consistently showed trap catch disruption using an off-ratio blend consisting of a single pheromone component that was equal to or better than the ‘natural’ 3:1 blend of two pheromone components. Our results showed that the 3:1 blend was necessary and sufficient for attraction of males (for use in traps) but the triene-only single component dispenser was superior and more cost-effective than dispensers of the blend. Male leafminers are not attracted to the off-ratio blend used in the commercial DCEPT CLM product thereby avoiding attraction of males from outside treated areas. Manufacture of DCEPT CLM is simplified because DCEPT CLM requires only one of the aldehyde pheromone compounds. Identification of optimal blends for attraction and disruption was the result of application of advanced stastistical methods including multivariate analysis and response surface modeling. Reduction of citrus canker incidence in disrupted plots was demonstrated in small plots but is expected to be even greater when large contiguous areas of citrus are treated with pheromone. Experiments conducted under this project are documented in 11 peer-reviewed scientific publications and a twelfth is in preparation. This project will now be carried forward through an agreement with the Commercial Product Deliv ery Committee of the CRDF. 1. Lapointe et al. 2014. J. Econ. Entomol. 107: 718-726. 2. Keathley and Lapointe. 2014. Fla. Entomol. 97: 291-294. 3. Keathley et al. 2014. Fla. Entomol. (in press). 4. Keathley et al. 2013. Fla Entomol. 96: 877-886. 5. Kawahara et al. 2013. Fla Entomol. 96: 1213-1215. 6. Lapointe and Stelinski. 2011. Entomol. Exp. et Appl. 141: 145-153. 7. Stelinski et al. 2010. J. Appl. Entomol. 134: 512-520. 8. Lapointe et al. 2011. J. Econ. Entomol. 104: 540-547. 9. Lapointe et al. 2009. J. Chem. Ecol. 35: 896-903. 10. Lapointe and W. S. Leal. 2007. Fla Entomol. 90: 710-714. 11. Lapointe et al. 2006. Fla Entomol. 89: 274-276.
Feeding behavior of ACP is being studied on P. trifoliata and trifoliate hybrids using an electronic feeding monitor, and choice and no-choice assays to study host selection and probing behavior. 1. Colonization by ACP on certain accessions of P. trifoliata was low in free-choice greenhouse experiments compared with Citrus and hybrids. 2. Electrical penetration graph (EPG) recordings are performed using trifoliate and trifoliate hybrid accessions to study xylem and phloem feeding. EPG waveforms provide information on stylet penetration into leaf tissues including saliva excretion and ingestion from xylem or phloem and hypothesized physical barriers based on electron microscope images. Frequency and duration of probing and feeding from xylem and phloem of trifoliates will be studied. Results show that ACP adults have difficulty in achieving ingestion from phloem of trifoliates. 3. Feeding assays showed that the number of ACP adults feeding on trifoliate leaves and subsequent survival were low compared with hybrids or Citrus. 4. Choice and no-choice assays were designed using ground leaf added to a wax matrix (SPLAT’). Choice assays studied olfactory/gustatory ability of ACP to differentiate between different trifoliates or its hybrids. Salivary sheaths excreted in the SPLAT treatments were stained and counted. ACP produced fewer sheaths on several trifoliates preparations compared with preparations containing citrus or hybrids. No-choice assays are underway.
This project is in furtherance of the commercial release of a new product for control of the citrus leafminer and associated spread of citrus canker disease based on a deployment device for the sex pheromone of the leafminer, DCEPT CLM’ (ISCA Technologies, Inc.). Under an agreement with the Commercial Product Delivery Committee of the CRDF, funds are provided to ISCA to subsidize two years of production of DCEPT CLM sufficient to treat 3,000 acres of citrus, mostly grapefruit, at three locations in St. Lucie and Charlotte counties in April/May of this year. The remaining cost of the product is provided by the growers. Funds are also provide to ARS and University of Florida to support monitoring and analysis of the experiments at the three locations. In addition to monitoring efficacy and longevity, the experiments will provide data on the effect of immigration of gravid female leafminers from outside of the treated areas. The application of DCEPT CLM at one location (Emerald Grove, The Packers of Indian River) is contiguous with untreated citrus, a source of immigrant females. Another site (VPI-5, Golden River Citrus Co.) is isolated and surrounded by natural area and pasture and therefore should receive few immigrants. Efficacy and longevity will be compared between these sites to estimate the magnitude of the effect of immigration. Another variable that is known to affect the ability to establish mating disruption in a grove is the structure of the tree canopy. Declining trees, replants or new plantings allow for increased air movement and presumed loss of pheromone compared with complete mature canopies. This variable will also be considered. Data collected so far indicate that trap catch disruption at the VPI-5 site is excellent; trap catch disruption at Emerald is good but may be compromised by low canopy vigor in some locations. Leaf samples will be analyzed to determine actual mining damage at all locations. Finally, the effect of intentional skip rows is being studied at the TRB site. Data to date indicate that trap catch disruption in skip rows is significantly lower than adjacent rows treated with DCEPT CLM. We hypothesize that this is related to the release dynamics from the solid pheromone dispenser compared to previous experience with SPLAT. The dissipation of pheromone from the DCEPT dispenser is being followed by extracting and analyzing DCEPT devices deployed in the field for varying periods of time. A linear decline in pheromone content has been seen over 12 weeks. This will be followed for up to one year to document pheromone evolution.
The objective of this research was to evaluate non-neurotoxic insecticides against Asian citrus psyllid (ACP), in order to identify additional tools for management of ACP in Florida. We investigated several non-neurotoxic insecticides that were known to be effective against insect pests similar to ACP. These additional tools may not only prove effective against ACP, but also could assist in ACP resistance management programs as needed tools for effective rotation with current insecticides. Methoprene is a juvenile hormone (JH) analog that acts as a growth regulator. We evaluated this compound for its ability to inhibit ACP egg hatch of eggs of various ages. We treated 0-48 hrs and 49-96 hrs aged eggs with six different concentrations of methoprene ranging from 0-320 ‘g/ml. For 0-48 hrs aged eggs, we observed egg hatch inhibitions of 14, 24, 31, 41, 65 and 84 percent for concentrations of 0, 10, 20, 40, 80 and 160 ‘g/ml, respectively. Similarly, the concentrations of 0, 10, 20, 40, 80, 160 and 320 ‘g/ml led to 13, 23, 29, 34, 39, 62 and 95 percent egg hatch inhibitions of 49-96 hrs aged eggs. These results show that treatment with methoprene successfully inhibited ACP egg hatch. We investigated the effects of cyantraniliprole against ACP. The contact toxicity of cyantraniliprole was 297 fold higher against ACP than its primary parasitoid, Tamarixia radiata. ACP settled and fed less on cyantraniliprole-treated plants than controls at concentrations as low as 0.025 and 0.125 ‘g AI mL-1, respectively. ACP egg production, first instar emergence and adult emergence were significantly reduced on plants treated with 0.25, 0.02 and 0.25 ‘g AI mL-1 of cyantraniliprole, respectively, when compared with control plants. Sub-lethal effects of cyantraniliprole were observed by comparing ACP settling behavior on treated vs. control plants. During the first 48 h of the experiment, there was no clear trend; however, at 72 h fewer adults settled on plants treated at the 0.025 ‘g AI mL-1 rate than on control plants. We investigated diofenolan to determine inhibition of egg hatch. Percent egg hatch inhibition of 7, 18, 31, 42, 65 and 91 % was observed when eggs were treated with concentrations of 0, 20, 40, 80, 160 and 360 ‘g/ml, respectively. We investigated the effects of diofenolan (Juvenile hormone analog) on survival of various developmental stages of ACP. We treated first instar nymphs with concentrations ranging between 0-360 ‘g/ml of diofenolan. We observed 95, 69, 38, 27, 23, 8 percent survival of ACP into the second instar for 0, 20, 40, 80, 160, 320 ‘g/ml concentrations, respectively. We investigated the effect of diofenolan on the development of third and fifth instars nymphs. We found that this compound significantly reduced development of these nymphal stages. We also investigated the effect of diofenolan on fertility and fecundity of ACP females. We found that this compound significantly reduced both fertility and fecundity. There were no significant differences in the population of adult ACP after treatment with Confirm 2F and Intrepid 2F as compared with the untreated control, whereas the Provado positive control (imidaclopried) significantly reduced the adult population as compared with the control. Intrepid 2 F caused the highest reduction of nymphs followed by Provado, and then Confirm as compared with the control. Therefore, these insect growth regulators do appear to have field activity against ACP immature stages. In general summary, the insect growth regulators tested were effective at suppressing ACP immature stage development. Also, novel chemistries, such as cyantraniliprole, were effective against ACP.
The main objective of this research is to determine both abiotic and biotic factors that regulate Asian citrus psyllid (ACP) acceptance of plants and pathogen transmission. The goal is to use this information to interfere with the vector’s capability of transmitting pathogen between citrus trees. Since April, we examined the effect of wind breaks on psyllid population density. We have investigated five different groves. In these groves, we sampled the edge that was facing the wind break, and the opposite edge that was not facing the wind break. We also sampled middle rows as a type of control. In four out of the five groves, psyllid abundance was significantly lower on the edge row that was facing the wind break compared with rows without a wind break. This difference was up to 87% in some groves. One hypothesis that may explain this difference was that windbreaks may act as a habitat reservoir for natural enemies. To test this hypothesis, we recorded the number of psyllid predators found on edges. We did not find a difference in the number of ladybeetles, Chrysopidae or spiders on the border edge facing a windbreak versus edges without windbreaks. We plan to record the level of parasitism for ACP nymphs on the edges of these different treatments to test if the parasitic biological control agent Tamarixia radiata might be more present in edges facing wind breaks as compared with edge without windbreaks. Other hypotheses that we are considering are that wind breaks provide shade and therefore reduce ACP numbers or prevent ACP from colonizing edge rows as compared with rows without wind breaks. We started the second year of our field experiment investigating the effect of grove architecture (solid reset plantings versus mature groves with resets) on psyllid population densities. Last year, we observed that reset trees in an environment consisting of a mixture of mature and reset trees contained fewer ACP and had lower HLB infection levels than reset trees planted in a grove solely composed of reset trees. To confirm these results, we will conduct this study in three different groves, each divided into two plots consisting of a reset planting and the other of mature trees containing resets sporadically placed in between mature trees. Trees will consist of ‘Hamlin’, ‘Valencia’, and Grapefruit.
This research seeks to determine whether young trees infected with CLas and displaying typical HLB symptoms can be brought to maturity and produce an economically viable yield. This will be achieved by managing a 58 acre grove of 3-year-old ‘Valencia’ / Kuharske Carrizo trees using a combination of three different foliar and three different ground applied nutritional programs. Factorial AxB treatments consist of A) ground-applied: 1) Liquid/dry+Ca (BHG standard), 2) Liquid+Ca, 3) Liquid/dry-Ca B) foliar-applied: 1) BHG standard-Ca, 2) BHG standard+Ca, 3) “Prescription”(+Ca). The prescription treatment was designed to be dynamic, customized for optimization, with feedback based on frequent leaf tissue analyses, visual symptoms, and the growth of the tree canopies and yield. There are six replications of treatments, with two being pure replications. The grove still continues to look better, despite the nearly 100% HLB incidence. Foliation of canopies is dense, and leaf color going into Summer season was a healthy green. Tree canopy sizes were measured in June 2014, and analyzed. Selected data is summarized below: Table 1. Tree canopy volume (cu.ft/tree) FOLIAR FERT STD-Ca STD+Ca Prescrip SOIL FERT Liq/Dry 397 315 429 Liq 350 303 423 Liq/Dry-Ca 384 312 313 The routine leaf tissue analyses for this project are behind schedule because we were prevented from submitting samples to our regular analytical lab due to restrictions on the movement of plant materials and soil. These rules are being enforced by DPI as part of the citrus quarantine for Florida. Once the necessary permitting is in place, we will resume leaf nutrient reporting.
The objective of the current project is the identification of a Bacillus thuringiensis (Bt) crystal toxin with basal toxicity against Asian Citrus Psyllid (ACP) and enhancement of this toxin by addition of an ACP gut binding peptide. A phage display library will be screened to identify peptides that bind to the gut of the ACP. Addition of the gut binding peptide to the Bt toxin will increase toxin binding and associated toxicity against the ACP. During the reporting period, the proteolytic profile of 15 Bt isolates provided by Dr. Michael Blackburn, USDA Maryland was characterized at Iowa State University. Briefly, Bt toxins were solubilized using sodium carbonate pH 10.5, 10mM DTT, lysozyme 200 micrograms per milliliter for 3 to 16 hours at 37’C, according to the solubility of each strain. The samples were dialyzed for 21 hours with three buffer exchanges against 50 mM Tris-Cl pH 8.5. Ten micrograms of Bt toxins were incubated with bovine trypsin at a final concentration of 10% of the toxin concentration at 37 ‘C for 1 or 2 hours for determining the proteolytic profile. Finally a 1 hour activation of a high protein concentration sample was performed to obtain sufficient toxin for toxicity assays. Removal of trypsin was carried out using benzamidine sepharose incubating the toxin sample at room temperature for 30 minutes with removal of the sepharose by centrifugation. The samples were verified by SDS-PAGE and the protein concentration was quantified by Bradford assay. Samples were shipped to USDA ARS Florida for testing by Dr. David Hall . ACP bioassays of five of the Bt isolates have been conducted at USDA ARS Florida. However, the psyllid mortality rates exposed to diet alone confounded conclusions from these assays. A series of experiments were conducted to address the high mortality in the negative control treatment. The psyllid survival rates have now improved, apparently because of seasonal effects on psyllid acceptance of the artificial diet, with low survival in the winter months. Additional challenges associated with the psyllid bioassays were 1) the amounts of Bt isolates provided only allowed for 4 replications of each dose; Additional replications may minimize the impact of variation between replicates. 2) The diet typically includes a yellow-green food coloring dye, but this dye precipitated when the Bt samples were added. The dye is now omitted from the diet and something green placed behind the diet sachet to promote feeding. 3) Bt toxins activated at Iowa State University are supplied in buffer (Tris-HCl pH 8.8). Assays are underway with this buffer to test for toxicity against psyllids. An appropriate amount of buffer will be added to control diet for future assays.
The objective of this study was to determine how enhanced nutrition of citrus plants may affect Asian citrus psyllid (ACP) biology. We conducted this study with complementary field and laboratory experiments. The enhanced grant allowed us to investigate a second field site to test the effect of plant nutrient supplements on Asian citrus psyllid (ACP) populations densities. This experimental field consisted of mature trees and was an addition to our investigations with solid reset plantings. Similarly to our other experiment, we applied a supplemental nutrition program following Keplex’ recommendations. We monitored ACP nymph and adult populations on a weekly basis. Our results indicated that the ACP populations very similar in plots that were assigned to nutritional treatments and the untreated, control plots at the onset of the investigation. However, we found that ACP populations increased significantly in nutrient-treated plots as compared with the controls as the experiment progressed. If this difference remains constant during the summer, this second field experiment will confirm results obtained in the first set of field experiments. We plan to pursue the monitoring of this experiment throughout the summer to confirm the results. However, all available data currently suggest that trees treated with nutritional supplements attract more ACP than untreated trees. This is perhaps not totally surprising, since trees with supplemental fertilization are likely better hosts for ACP as compared with untreated trees. We are also investigating the effect of enhanced phosphorous (P) and potassium (K) on ACP preference for and performance on citrus. We recently analyzed two years of field data confirming that ACP tend to be more abundant on citrus varieties with high levels of P and K in leaves. We are currently finishing a laboratory experiment where plants are treated with red phosphorous in order to artificially increase P and K levels in leaves. Subsequently, these citrus plants with enhanced levels of P and K will be tested for ACP oviposition preference, and nymph development as compared with untreated control trees.
The objective of this study has been to evaluate non-neurotoxic insecticides against Asian citrus psyllid (ACP) and to provide information to growers regarding insecticides of varying modes of action for management of ACP. We investigated non-neurotoxic and other insecticides that have shown promise against insect pests similar to ACP. These insecticides may not only prove effective against ACP, but also may assist in ACP resistance management. This enhancement allowed us to expand our investigation with regard to the number of treatments tested. We investigated diofenolan to determine inhibition of egg hatch. Percent egg hatch inhibition of 7, 18, 31, 42, 65 and 91 % was observed when eggs were treated with concentrations of 0, 20, 40, 80, 160 and 360 ‘g/ml, respectively. To determine the effects of diofenolan (Juvenile hormone analog) on survival of various developmental stages of ACP. We treated first instar nymphs with concentrations ranging between 0-360 ‘g/ml of diofenolan. We observed 95, 69, 38, 27, 23, 8 percent survival of ACP into the second instar for 0, 20, 40, 80, 160, 320 ‘g/ml concentrations, respectively. We investigated the effect of diofenolan on the development of third and fifth instars nymphs. We found that this compound significantly reduced development of these nymphal stages. We also investigated the effect of diofenolan on fertility and fecundity of ACP females. We found that this compound significantly reduced both fertility and fecundity. The results of our research suggest that non-neurotoxic insecticides are effective in disrupting growth of the immature stages of Asian citrus psyllid and are likely useful tools as part of an integrated program for ACP. In addition, as part of this investigation, we were able to test sulfoxaflor. Sulfoxaflor is a systemic insecticide which is considered an insect neurotoxin. It is also the only member a class of chemicals called the sulfoximines which act on the central nervous system of insects with much lower toxicity to mammals (as compared with traditional neurotoxons) and is similar to neonicotinoids. This molecule interferes with neural transmitters in the insect nervous system. Specifically, it blocks the nicotinergic neuronal pathway. While this is not a non-neurotoxic insecticide by definition, its low mammalian toxicity and apparent low toxicity to biological control agents made it an attractive candidate to evaluate for practical management of ACP as part of this study. Also, since field effectiveness of this molecule against ACP in Florida has been proven and since this will likely be a useful tool for ACP management in Florida, we decided to investigate it in more depth. In leaf disc bioassays sulfoxaflor was as toxic as imidacloprid to ACP adults with LC50 values of 8.17 .g A.I. per ml and 5.7 .g A.I. per ml. There was a significant reduction of eggs laid on plants treated with sulfoxaflor compared with controls and significantly fewer adults were produced. Sulfoxaflor significantly reduced adult feeding as measured by honeydew production and mortality of adults on plants sprayed with sulfoxaflor was the same as those sprayed with imidacloprid for up to 28 days after treatment. The toxicity of sulfoxaflor was evaluated in petri dish assays for the parasitoid of ACP, Tamarixia radiata. Sulfoxaflor was toxic to T. radiata with a LC50 of 27.12 .g A.I. per ml. This is about three times higher than the LC50 measured for ACP. Additional field trials with sulfoxaflor at rates of 0.2 and 0.3 L per ha showed significant mortality compared with the untreated controls, but only for about one week after treatment.
The objective of this study was to determine how enhanced nutrition of citrus plants may affect Asian citrus psyllid (ACP) biology. We have conducted this study with complementary field and laboratory experiments. Laboratory experiments indicated a significant preference of ACP for plants supplemented with nutrient regimes as compared with untreated controls. During two choice experiments, ACP settled more on HLB-infected plants supplemented with nutrients than on HLB-infected and non-supplemented control plants. We also performed an experiment where ACP were forced to settle on an initial plant and allowed to disperse toward a newly introduced plant subsequently. We found that ACP dispersed less from HLB-infected plants that were supplemented with nutrients than from a HLB-infected plants that were not. Overall, the laboratory experiments indicated that ACP plants prefer nutritionally supplemented plants. These results were confirmed in the field where adult and nymph densities of ACP were higher on nutrient supplemented plants than on untreated control plants. The differences between the two treatments appeared 6 months after the beginning of the nutrient regime and were consistent over the two years of the study. In 2013, the citrus trees supplemented with nutriments harbored on average 20% more ACP than control trees. We also assessed the HLB infection status of trees with qPCR and after one year of nutritional supplement application, we found no difference in the proportion of HLB-infected trees between control and nutritionally supplemented trees. These results indicate that nutritional supplements do not protect citrus trees against HLB infection, but may not increase the risk of HLB infection either. To confirm these results, we performed an experiment in the laboratory where citrus resets were placed in a Las-infected ACP colony (30% to 55% of the psyllid were Las positive in average during the experiment). Half of these citrus trees were nutritionally supplemented and half of them were sprayed with water (control). Control citrus trees were positive for HLB after 3 months on average, whereas the nutrient-supplemented trees became infected after 6 months. We believe that this difference observed under controlled conditions is likely too small to have a real benefit in the field. We also examined the effect of nutrient sprays on Las acquisition by ACP. We performed the experiment under both field and laboratory conditions. Under field conditions, we did not find significant differences in Las acquisition by ACP when nymphs or adults were exposed to HLB infected trees treated with nutritional supplements versus the water control. In laboratory experiments, we found a significant reduction of Las acquisition by ACP adults after 10 days of exposure on nutrient supplemented trees as compared to control trees. We did not find a difference in bacterial acquisition by ACP between supplemented trees and control trees 28 days after exposure. In summary, our data indicate that nutritional supplements significantly increase ACP population densities in areas that are treated as compared to untreated control trees. However, we did not find a significant correlation between nutritional supplementation and HLB infection levels in the field or bacterial acquisition by ACP in the laboratory.
This project helped initiate development of a novel insect behavior-modifying product to control the ACP. Decoy is a biodegradable emulsion capable of releasing methyl salicylate (MeSA) at rates sufficient to disrupt ACP behavior and transmission of HLB. In laboratory studies were able to confirm that MeSA dispensers reduced the capability of ACP to find infected citrus plant as compared with control. The formulation is being considered by ISCA Technologies for development. This investigation demonstrated that the ACP parasitoid, Tamarixia radiata exploits a phytopathogen-induced volatile (MeSA) to increase parasitization of ACP. T. radiata wasps were attracted by volatiles emitted by Las-infected citrus plants, as compared with uninfected citrus plants. Also, Las infection caused approximately a five-fold increase in ACP nymph parasitization on infected plants by wasps as compared with that observed on uninfected plants. When wasps were simultaneously presented with odors of two psyllid-infested plants (one infected with pathogen and the other uninfected), they did not exhibit a behavioral preference. This suggests that simultaneous pathogen infection and herbivore-induced damage did not increase attractiveness of citrus plants to the wasp parasitoids additively as compared with either factor alone. However, in the absence of herbivores, pathogen infection appears to essentially mimic the effect of herbivore damage with respect to attraction of the vector’s parasitoid. A field investigation will be needed to complement these laboratory results and could further elucidate whether T. radiata parasitize more ACP nymphs on Las-infected than on uninfected citrus plants. Release of MeSA may explain enhanced attractiveness of Las-infected, as compared with uninfected, plants to T. radiata wasps. MeSA release is induced in citrus by infection with Las or by ACP feeding damage, and we found that MeSA was attractive to T. radiata in the currently described behavioral assays. Also, lures that released MeSA caused increased parasitization of ACP by T. radiata on uninfected and MeSA-baited plants at a rate nearly identical to that observed as a result of Las infection. MeSA is a ubiquitous compound found in leaves of many plant species, and its emission is induced by herbivore damage or pathogen infection. MeSA is attractive to some natural enemies of herbivores, including parasitoids, but can also be either neutral or even repellent. In the currently described system, MeSA is also an attractant for the ACP vector and may indicate to psyllids the presence of a suitable host, and/or the presence of conspecifics to favor mating and reproduction. It is possible that MeSA is not the only induced chemical affecting the behavior of T. radiata in this system and further work to address potential blends is needed. As part of this investigation, we have revealed that it is possible to produce an attractant comprised of a number of compounds in defined abundance ratios by utilizing information about VOC alterations to mimic volatile output of pathogen-infected plants. Working with colleagues from the University of California, we combined analytical chemistry, the Attenu assay system for chemosensory proteins, and behavioral testing to identify biomarkers characterizing infected plants that attract the vector. By doing so, we developed a synthetic lure for ACP that was more attractive to ACP than odors emanating from uninfected citrus trees. This strategy could provide a new route to produce chemical lures for vector population control for a variety of plant and/or animal systems. A patent for this lure is in progress (led by UC) and there is interest from the commercial sector to pursue this as a possible ACP attractant.
Our objective for this project has been to evaluate botanical compounds as repellents for Asian citrus psyllid (ACP) with the purpose of developing possible repellent formulations for use in the field. Over the course of this research, we have identified several botanical insecticides that may be useful for management of ACP. Initially, we found that a sesquiterpenoid, citronellol, from lemon grass oil repelled psyllids. However, it was not toxic to ACP with topical applications. Beta-damascone, 9-decen-ol, and geraniol did not influence psyllid behavior. We found that clove oil was the most toxic botanical oil tested and killed nearly 100% of psyllids at a10ng/mL concentration. While both citronella and litsea oils caused 100% mortality at a 100ng/mL dosage, the lethal concentration was lower. Fir and camphor oils exhibited minimal mortality at 100ng/mL suggesting that these oils are not very toxic to psyllids. We selected five botanical oils with known repellency to other insects, to evaluate behavioral activity on ACP. In olfactometer assays, fir oil was repellent to female ACP. However, clove and camphor oils were attractive, while litsea and citronella oils elicited no response from ACP females. In no-choice settling experiments, neither the low (5mg/day) nor high (9.5 mg/day) fir oil dosages tested deterred ACP from settling on citrus. Subsequently, ACP were presented with a choice test between control plants and plants treated with the high dose of fir oil. ACP disproportionately settled on control plants, avoiding fir oil-treated plants completely. We conducted two field trials to determine if these oils affected ACP behavior in citrus groves. We found no attraction of ACP to yellow sticky traps baited with clove or camphor oil deployed from polyethylene vials as compared to unbaited controls. We found no repellency of ACP from sweet orange resets when treated with high release fir oil devices (10 g/day/tree). Our results suggest that ACP behavior may not be easily modified under field conditions by olfactory cues from botanical oil treatments. Volatiles from essential oils of coriander, lavender, rose, thyme, tea tree oil and 2-undecanone, a major constituent of rue oil repelled ACP adults compared with clean air. Also, coriander, lavender, rose and thyme oil inhibited the response of ACP when co-presented with citrus leaves. Volatiles from eugenol, eucalyptol, carvacrol, b-caryophyllene, a-pinene, a-gurjunene and linalool did not repel ACP adults compared with clean air. In an effort to isolate the repellents and toxicants from effective essential oils, the headspace components of coriander and lavender oil were analysed by gas chromatography-mass spectrometry and revealed that a-pinene and linalool were the primary volatiles present in coriander oil while linalool and linalyl acetate were the primary volatiles present in lavender oil. Coriander, lavender and garlic chive oils were also highly toxic to ACP when evaluated as contact action insecticides using a topical application technique. The LC50 values for these three oils ranged between 0.16 and 0.25 lg/ACP adult while LC50 values for rose and thyme oil ranged between 2.45 and 17.26 lg/insect. The repellent essential oil volatiles have potential for development into filed-based tools. In the field experiments conducted throughout the study, results were mixed and often negative. We cooperated with multiple companies in an effort to develop dispensers for field deployment of botanical insecticides. However, in most cases, the devices investigated to date were not comparable to standard toxic insecticides with respect to efficacy and population control of ACP in Florida.
In this enhancement phase of the Asian citrus psyllid vibration trap study, heavy-duty speakers have been operated with amplifier systems that produce airborne signals of 75-97-dB amplitudes with the goal of inducing vibrations in citrus trees that interfere with the mating duets of male and female Asian citrus psyllids. Tests have been conducted with signals produced at multiple harmonics of 200 Hz, i. e., at 200, 400, 600, . . ., up to 4000 Hz, to mimic the harmonics of the signals produced by the psyllids themselves. As expected, we found that additional harmonics are generated by the tree structure itself and the relative amplitudes of the harmonics are affected by the size and shape of the tree. Surprisingly, however the frequencies between 200 and 2000 Hz elicit high rates of calls by males on the tree, but they did not elicit movement. In eight tests completed with male and female psyllids placed on a citrus tree in the laboratory, no mating occurred while the speakers were producing high-amplitude sounds at any of the tested frequencies, but males often produced calls at when the speaker signals were produced between 200 and 2000 Hz. In one case, mating occurred after the speakers were turned off. These studies on the feasibility of interference with psyllid mating are continuing.
In this phase of the vibration trap research project, four different designs of trapping surfaces were tested in the laboratory with a “Bugphone” microcontroller device that detects Asian citrus psyllid male vibrational calls, plays back female replies, and attracts the males to the trap location. Three of the traps employed sticky surfaces to retain the males that were attracted and one used a trap door that shut when the male entered the trap. None of the new designs were considered effective enough to develop further for field usage. Consequently, we have begun consideration of a second option to count the numbers of male calls detected over time and save this information on a secure digital (SD) non-volatile memory card so that it can be retrieved later when the trap is serviced and the microcontroller system battery is replaced. A count of the rate of calls then could be used to estimate the number of males within the detection range of the trap. SD cards have been added to multiple Bugphones and testing will be done to determine their effectiveness in providing estimates of the psyllid population. In addition, electrical engineering colleagues at the University of Florida have begun studies to improve operation of the Bugphone by reducing its energy usage. We have constructed and are beginning tests of a version of the Bugphone that is expected to operate on three AA batteries for a week instead of the previous version that worked for four days on eight AA batteries. Ultimately the goal is to enable operation for a month or more without battery replacement.
The large-scale validation of citrus leafminer (CLM) disruption with the ISCA Dcept CLM technology is underway. Since the project began in the spring (2014) we assisted in applying the Dcept CLM product by hand to approximately 3,000 acres at three locations in southeast and southwest Florida. Our objective has been to determine how scale of application and proximity of pheromone-treated groves to untreated sources of mated CLM females will affect the efficacy of mating disruption with this product. Therefore, the tests have been established at three contrasting locations. For example, Emerald grove (The Packers of Indian River) in NW St. Lucie County is surrounded by extensive citrus plantations. VPI% (Golden River Fruit Co.) in SW St. Lucie County is surrounded by natural areas or pastures and receives relatively few immigrating females representing what we may expect from area-wide application of pheromone. To date, we have been collecting efficacy data by trapping CLM males with pheromone traps as a surrogate measure of mating disruption. Thus far it appears that efficacy is lowest on border areas and areas where there are a significant number of missing trees, as we would predict. However, thus far we have found the treatments to be efficacious overall. In addition to continuing monitoring of CLM populations with trapping in the field, we will conduct flush damage assessments in the next quarter.
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