Issue date for this grant was 13 July 2012. Objectives are: 1. Assess effects of abiotic factors (light quality, photoperiod, air flow, temperature fluctuations) on psyllid movement, 2. Evaluate physiological limits and biotic factors effecting of movement including feeding, egg load, infection status, and population density, 3. Evaluate techniques for tracking psyllid movement in the field for mark recapture studies, 4. Characterize seasonal patterns of ACP distribution and movement at different scales in the field, 5. Develop strategies to protect young trees from colonization by ACP utilizing UV reflection for repellency and insecticide treated trap crops (such as Bergera koenigii) to attract and kill. Objective 1. Bioasssays using a wind tunnel to determine ACP response to different wind induced stimuli were put on hold until native field populations of ACP return to a sufficient level to allow adequate numbers to be collected for testing. Objective 2. Dr. Stellinski’s lab tested the hypothesis that Las-infected psyllids disperse more to uninfested plants than uninfected psyllids. In a laboratory cage experiments, 150 psyllids were allowed to settle on citrus resets for three days. After three days, four new uninfested resets were introduced into the same cages. Over the course of four days, introduced psyllids that dispersed to uninfested plants were counted and removed. They repeated this experiment with psyllids originating from a colony free of Las pathogen, and with psyllids originating from a colony infected with the Las pathogen. They found that psyllids originating from the Las-infected colony dispersed more than psyllids originating from the uninfected colony. This result was corroborated with data obtained using a flight mill apparatus that directly measures duration of psyllid flight. Las-infected psyllids were found to be more prone to flight than uninfected psyllids when placed on the flight mill, irrespectively of their sex. However, distance and duration of flight did not differ between Las-infected and uninfected psyllids. Increased dispersal and flight as a result of Las-infection may be interpreted as a host-phenotype manipulation by the pathogen. By causing the vector to disperse more, the pathogen may increase chances of inoculating a nearby non-infected host. Objective 3. Additional mark release trials were conducted with some success (2% of 2000 ACP released were observed). ACP were observed in the trees at night upwind of the release point but no ACP have been found on sticky cards outside the release point. In addition, the releases have not increased the extremely low levels of native ACP populations in this grove, so additional mark recapture trials can be conducted at this location with minimal residual ACP population interference. Objective 4. Two + years of data from yellow sticky traps set at three canopy heights is being processed. We find more ACP in the upper canopy in the older Charleston grove and a more even distribution at the younger and more uniform Bob Paul grove that still slightly favors the higher sticky card locations. However, we see more captures on the east than the west side of trees at both locations. In general, ACP captures are greatest adjacent to a hammock or wind break. Traps at Bob Paul set to assess ACP movement between insecticide sprayed and unsprayed blocks with low and high populations of ACP respectively showed no significant difference. Objective 5. A paper reporting positive results using UV reflective mulch to repel ACP the first 2 years after planting has been published in the refereed journal Pest Management Science. Subsequent results indicate that the 5 ft strip of metalized mulch is not effective on trees more than 5 ft tall. Trials to determine the impact of reflective mulch on the height of ACP flight were put on hold due to low ACP activity. This trial will be resumed when warmer weather patterns return in the spring which also increase ACP movement. UV reflective plastic ribbon hung in young trees was found to provide minimal protection against ACP colonization of young trees.
Citrus leafminer (CLM) is a major pest of citrus, causing direct damage and increased incidence/intensity of citrus canker. Pheromone traps are valuabe for monitoring CLM but need further evaluation to optimize use for management. The objectives of the proposed project include: (1) assess species composition of trap catches, (2) evaluate spatial and temporal optimization of pheromone traps. Species composition in trap captures is being evaluated using an anchored hybrid enrichment probe set from Illumina HiSeq designed to capture a tiled, 250 bp region of the 658 base pair CO1 ‘barcode region’. Blast-searches against the comprehensive BOLD sequence library will allow a faster, bulk-sequencing approach to the identification of leafminers. Results during the last 3 months using pairwise distances from 117 new sequences suggests greatest similarity with P. citrella (the majority) plus some P. insignis, P. vitigenella and 2 other putative species that differ from the rest by at least 3%. A second trial of the ‘Time Lure Degradation’ study was conducted in summer-fall 2013. We began aging sets of 2 commercial lures” 1) Alpha Scents lure, and 2) ISCA lure on July 15 which were set out in Delta traps (ISCA) on 9 Sep in a 39 ac block of ‘pineapple’ orange at Duda & Sons, Inc., Hendry County, Florida. Three treatments (4-week-old, 6-week-old and 8-week-old lures at the beginning of the experiment) plus a control were tested in a RCB designed with three replicates for each lure brand during a 7 week-period. Pheromone traps were dispensed at central locations in the plots and data collected at 1-week intervals for comparison of trap catch by age and manufacture. Traps with their lure changed on a weekly basis served as a positive control. Lure degradation was estimated for each brand by linear regression as the weekly variation on the proportion of CLM captures with respect to the control treatments. Estimated half-lives, time when capture efficacy is reduced in 50%, were obtained. Results were compared to tthose obtained in spring to discern potential differences in lure degradation depending on brand, season and temperature. In summer-fall, a linear regression model significantly explained the efficacy decay of Isca lure with time (F = 90.97; df = 1, 83; P < 0.0001; r2 = 0.53). The estimated time half-life was of 6.65 weeks (CL95: 5.97-7.33). Alpha lure decay was better explained by a second order equation (F = 149.11; df = 2, 83; P < 0.0001; r2 = 0.79). In this case lure efficacy rapidly decayed so that after 8 weeks almost no captures were observed. Estimated half-life was of 2.88 weeks (CL95: 2.39 ' 3.39) and differences in lure degradation between brands were significant (F = 23.19; df = 1; P < 0.0001). The average mean temperature for the summer-fall experiment, from July 15 to October 31, (T = 26.30 ' 0.15. C) was significantly higher than during the spring experiment, from March 25 to June 21, (T = 23.83 ' 0.33. C) (F = 52.42; df = 1, 196; P < 0.0001). However, no differences in lure degradation were found between spring and summer-fall experiments for ISCA lures (F = 0.03; df = 1; P = 0.8591). In contrast, efficacy of Alpha lures differed significantly between the summer-fall and the spring experiments (F = 122.83; df = 1; P < 0.0001), with more rapid degradation in summer-fall. It appears that either the Alpha lures used in summer-fall were defective or they were more sensitive to high temperatures. In any case, results obtained in this last experiment confirm that for accurate ACP monitoring, lures must be replaced more frequently than the recommended 8-12 weeks.
Issue date for this grant was 13 July 2012. Objectives are: 1. Assess effects of abiotic factors (light quality, photoperiod, air flow, temperature fluctuations) on psyllid movement, 2. Evaluate physiological limits and biotic factors effecting of ACP movement including feeding, egg load, infection status, and population density, 3. Evaluate techniques for tracking ACP movement in the field using mark recapture, 4. Characterize seasonal patterns of ACP distribution and movement at different scales in the field, 5. Develop strategies to protect young trees from colonization by ACP utilizing UV reflection for repellency and insecticide treated trap crops to attract and kill. Objective 1. Movement up wind is typical although movement was suppressed by wind speeds above 2mph in wind tunnel assays. These observations were confirmed in the field. However, ACP movement rate in the wind tunnel was increased with the addition of a host plant upwind. Objective 2. Dr. Stelinski’s laboratory continued to investigate movement capability of ACP using laboratory flight mills. These devices allow direct measurement of ACP flight capability. They found that Candidatus liberibacter asiaticus (Clas) -infected psyllids perform long duration flights more frequently than uninfected counterparts. Infection was quantified with qPCR and estimates of CLas DNA titer per psyllid with emerging techniques that have been developed specifically for this project. Thus far, no correlation was found between amount of CLas DNA carried by an ACP and duration of flight. The hypothesis that flight capability is correlated with dispersal behavior is being tested by allowing psylldis to settle for 3 days on citrus plants, then introducing a new plant into the cage and collecting dispersing psyllids daily. Thereafter, dispersing psyllids are tested on the flight mill. Thus far, they have not found a significant correlation between flight capability on the mill and dispersal behavior. They have also found that green/blue morphs display long distance flights at a significantly greater frequency than brown/gray morph psyllids. Additionally, they are currently working on a protocol to measure the amount of protein, sugar and lipids within psyllids, with the objective of determining how these various energetic reserves contribute to flight/dispersal behavior. Objective 3. ACP marked with fluorescent dye released in a semi-abandoned ‘Murcott’ block were found at night in nearby trees upwind although none were found on sticky cards outside the release point. Releases have not increased the extremely low levels of native ACP populations so additional mark recapture trials can be conducted at this location with minimal residual ACP population interference. ACP have also been observed in what appears to be feeding position on weeds within the grove. Further investigation is underway to evaluate use of non-host plant species by ACP. Objective 4. Yellow sticky traps set at three canopy heights captured a total 1657 ACP; more in the upper canopy in the older Charleston grove but more evenly distributed within the canopy in the younger and more uniform Bob Paul grove. More ACP are captured on the east side compared to the west side of trees at both locations. ACP captures are greatest adjacent to a hammock or wind breaks at these and a third (English Grove) location. Traps set up at Bob Paul to assess ACP movement between insecticide sprayed and unsprayed blocks with low and high populations of ACP respectively indicate no significant directional differences over 136 captures to date. Objective 5. A paper reporting positive results using UV reflective mulch to repel ACP the first 2 years after planting was published in the refereed journal Pest Management Science. Subsequent results indicate that the 5 ft strip of metalized mulch is not effective on trees more than 6 ft tall. Trials to determine the impact of reflective mulch on the height of ACP flight were resumed with warmer spring weather during which ACP movement generally increases.
Citrus leafminer (CLM) is a major pest of citrus, causing direct damage and increased incidence/intensity of citrus canker. Pheromone traps are valuabe for monitoring CLM but need further evaluation to optimize use for management. The objectives of the proposed project include: (1) assess species composition of trap catches, (2) optimize spatial and temporal distribution of pheromone traps. Species composition in trap captures is being evaluated by analysis for Cytochrome Oxidase 1 (CO1) sequence generation incorporating an anchored hybrid enrichment probe set from Illumina HiSeq data with the Lemmon Lab at FSU. The probe set is designed to capture a tiled, 250 bp region of the 658 bp CO1 ‘barcode region’. Blast-searches against the comprehensive BOLD sequence library has allowed a faster, bulk-sequencing approach to the identification of leafminers. Since January 2014, CO1 genes were sequenced from a total of 95 samples of which 65 were recently added to the maximum likelihood phylogenetic analysis. DNA from an additional 70 samples from Silver Strand B9 block, Okaloacoochee Slough State Forest, and Charleston Grove was extracted, analyzed by PCR and is currently being sequenced. Each sample is associated with locality, collection date, type of storage (dry or ethanol), and trap number. Some samples also include morphological data. Two specimens from OK Slough appear in the clade of Phyllocnistis new species 1, and a third appears in the clade of Phyllocnistis new species 2. This is the first time that these two new species have been collected at the Slough. All specimens from the other two localities were citrus leaf miners. Samples collected in 2013-2014 and received in March 2014 are currently being processed, so the current analysis does not include any samples collected during this time frame. Part of the processing will include photography of the specimens prior to extraction. Although many species of Phyllocnistis are cryptic, it is possible that the new species collected in OK Slough can be distinguished from P. citrella by external morphological characters. Photographs of the specimens will help elucidate this in order to provide more accurate identifications. Vince Ficarrotta recently trained David Plotkin in the Kawahara Lab to serve as a part-time assistant through the end of the summer of 2014. David will focus on photographing moths through a microscope and also generate COI sequence data. Almost two years of CLM monitoring for a study to evaluate trap densities and lure efficacy decay under field conditions demonstrated that densities of 1 trap per 2 ha (5 acres) were sufficient to obtain reliable moth counts. Lure efficacy of the two pheromone brands tested declined by 25% after 3-5 weeks and 50% by 6-10 weeks during the spring through fall growing season in Florida. Therefore, large correction factors need to be applied to trap catch readings if traps are replaced at 8-13 weeks. Results obtained will help growers to optimize CLM monitoring programs. A manuscript was submitted to the Journal of Economic Entomology. A formal evaluation of marking techniques was concluded; comparable survivorship of CLM adults powdered or not (P > 0.05) and when exposed to a honey-water solution spiked with blue dye or not (P > 0.05), was demonstrated. Given the shorter period required to affix a mark on CLM with fluorescent powder, this technique will be incorporated. The mark-recapture component is set to begin in April, 2014.
Objectives of this project include: 1) scale up production of Tamarixia radiata to levels that will allow better assessment of the potential impact of augmentation on ACP populations and ultimately the spread of HLB, 2) use genetic techniques to identify parasitoids recovered from the field to demonstrate establishment and effectiveness of released strains, and 3) pass on rearing technology to both private and public sectors to encourage production and an adequate supply of these parasitoids into the future. This quarter a total of 271,828 T. radiata were produced at facilities in DPI Gainesville and SWFREC Immokalee. At DPI 70,775, 32,704, 57,016 and 61,995 T. radiata were produced in colonies from South China, Pakistan, North Vietnam and Florida strain previously established from Taiwan and South Vietnam. The colony at SWFREC produced another 49,338 “Florida Strain” T. radiata. Wasps from these colonies were used for maintaining colonies, research and to release in conventional and organic citrus groves in Lake, Indian River, St Lucie, Polk, Hardee, Collier and Hendry counties. Experiments were conducted by Ph.D. candidate Xulin Chen to study behavior of T. radiata female under six densities of 10, 20, 30, 40, 50, and 60 fourth instar nymphs of Diaphorina citri. Three day old single females were exposed to respective densities in petri dishes and observed for 30 minutes. Initial observations indicated that handling time per host was constant under the six host densities, which corresponded to the type II functional response; searching time was negatively correlated with host density; and probing for host searching was significantly longer than probing for parasitization. At SWFREC, 1,700 and 2,600 Tamarixia were released in Oct and Nov, respectively. In Nov, parasitism averaged 32% (n= 206) in the release block compared to 24 % (0-43%) (n= 22-42) in no release blocks observed mainly from young blocks producing new growth required for nymphal survival. Insecticides were used in the release and no release blocks on need. Tamarixia releases were initiated in a 10 acre block of ‘Hamlin’ orange on ‘Carrizo’ rootstock at A. Duda & Sons in Hendry County planted Jul 2012 to evaluate insecticides only, foliar nutrition only, insecticides + foliar nutrition and untreated control for effects on ACP and HLB. Plots designated for insecticides did not require a treatment during this quarter. In Oct, 3,000 Tamarixia were released followed by 5,800 in Nov although only 1,200 and 1,400 were released prior to sampling in each month. An average of 16% parasitism was observed in the samples collected before the start of releases in Oct. Parasitism in the release and no release plots averaged 63% (n=122) and 35% (n=147), respectively, in Oct and 38% (n=233) and 54% (n=396), respectively, in Nov, indicating spread to no-release areas. Parasitism in the insecticide treated and untreated plots averaged 53% (n=102) and 47% (n=527), respectively, from samples collected in Nov. Chen, X and P. Stansly. 2013. Effect of holding diet on egg formation of Tamarixia radiata (Hymenoptera: Eulophidae), parasitoid of Diaphorina citri (Hemiptera: psyllidae). Florida Entomologist (accepted)
The overall objective of this research is to develop a push-pull system for the Asian citrus psyllid (ACP) that can complement integrated management systems in young citrus plantings. Towards that goal, visual factors that affect psyllid takeoff into flight and landing are being examined to guide development of an optimal pull component. Polarized reflection patterns from host plants are known to guide attraction to host plants for several insect species. The contrast of polarized light reflected from leaves to transmitted through leaves against unpolarized skylight may guide ACP dispersing to new host plants. While ACP are strongly diurnal insects with a strong attraction to light, the role of polarization remains unknown. In nature ACP may be exposed to unpolarized skylight or polarized light transmitted through leaves as well as reflected from leaves, bark, fruit, etc. In particular, responses of dispersing ACP are of greatest interest because of their potential in infesting new trees. In this study, the effect of light polarization in conjunction with color was examined in a flight assay as well as a walking assay. A vertical flight attraction assay was developed to examine activation of ACP from sitting to flight and landing on a target illuminated with filtered light. This assay simulates the response of ACP settled on vegetation to visual stimuli that could be provided by sunlight, or light reflected from or transmitted through leaves. ACP responses were strongest to white light closely followed by a long wavelength UV. Responses to blue/green and green filtered light were moderate and did not differ. The lowest responses were to blue and a mid-range UV light. Polarization of light did not affect responses to white or UV light. Attraction to green, however, was dramatically reduced with polarization and the impact of this finding on behavior and ACP ecology will further examined. Additionally, these assays are being continued to determine if differences between different colored individuals vary in response. A horizontal walking assay was developed to simulate walking responses of ACP on vegetation and this assay measured direction of movement as well as speed of movement. Strong and rapid walking responses towards the light sources were obtained with UV and blue light. Walking responses under green, yellow and orange light were very low and there was no clear indication of movement towards or away from the light. Increasing intensity of light increased movement overall both towards and away from the light source depending on the color of light. The distance and speed of walking towards a white light was decreased when the light was polarized. Walking towards polarized UV light tended to be moderately faster and covered a greater distance than to unpolarized UV. In contrast, polarization increased directional walking distance and speed towards green light. In general for walking assays, males and females responded similarly with little difference between brown- or green- colored individuals. Orange-colored individuals (particularly females) tended to be much less active with lower speed and movement towards any light source. These assays are continuing and will be used to compare responses of ACP reared under conditions that may contribute to production of dispersing individuals (i.e., photoperiod, density, host plant condition). Additionally comparisons will be made between newly emerged unmated female ACP and older gravid females.
The overall goal of this 3-year research project is to efficiently deliver antimicrobial molecules into citrus phloem against HLB bacteria. This quarters (from Sep. to Dec. 2013) research continued to evaluate the penetrants based on our cuticle assay and to prepare the nano-formulations coupled with penetrant for foliar spray or bark-application. The results showed that the cuticle was difficult to isolate from HLB symptomatic (Ct=23.60’1.36) leaves by pectinase and cellulase, compared to asymptomatic leaves (Ct=30.56’0.66). Cuticles are more difficult to isolate from Valencia than those from Lemon, Grapefruit other oranges. Pectinase activities increased by 29.5% and decreased by 3.1% in response to leaf extraction from asymptomatic and symptomatic leaves, respectively. However, cellulase activities increased up to 112.0 % and 188.9 % by asymptomatic and symptomatic leaf extraction, respectively. Difficulty of cuticle isolation from HLB symptomatic leaves may be closely related to Zinc and sugar metabolism. Eight penetrants were tested using the isolated culticles. Compared to the control, seven compounds had more than several fold penetration ability. However, one compound was not effective to promote penetration. Several formulations in W/O and O/W were prepared and applied in HLB-affected citrus by foliar spray and bark-painting. The future work will be focused on the followings: 1) Continuing to optimize the final formulations by combinations of the penetrants and high drug loading capacity formulations; 2) Evaluating of drug loading capacity using the optimized nanoemulsion formulations; 3) Treating the HLB-affected citrus by foliar-spray and bark-application.
Based on results from the previous Contest Project (CRDF#400), eleven compounds have been screened by the contest committee as candidates for further research to determine their efficacy for control of HLB based on their ability to substantially reduce the titers of the bacterium Candidatus Liberibacter in our grafted citrus assay, lack of phytotoxicity to citrus and potential for registration. After first evaluating combinations of these molecules using the graft-based chemotherapy method under a separate agreement, this project extension will evaluate these 11 compounds as control agents for the HLB bacterium individually and in combination using infected, container-grown citrus and HLB-affected scions. The objective of this project is to determine an optimum chemical formulation that may be registered for field control of HLB. In this quarter (Sep to Dec, 2013), 80 treatments from 11 compounds or combinations have been applied to HLB-affected potted-plants by soil-soaking coupled with three heat treatments (Tm=40, 42 and 45). Las bacterial titers in all these treatments will be tested in every 2 months. In this quarter, the second test has been done. The preliminary results showed that heat treatment promoted the growth of new flushes from the seriously HLB-affected citrus. No bacterium was detected in the new leaves but present in the old leaves two months after treatment with some of the compounds coupled with the heat treatments. No great differences in Las bacteria were found among the heat-treatments (Tm=40, 42 and 45). Four months later, several treatments were effective in reducing the Las bacterium both in the new and the old leaves, especially SDX, AMP. The research is ongoing.
The objective of this project is to optimize attractancy of visual targets and to determine what segment of the ACP population is responding to traps to better interpret surveillance data and to better target collection of dispersing psyllids. Knowledge of the factors that enhance psyllid attraction to a target as well as enhance detection by psyllids of the target against the background will result in optimization of the design and placement of a visually targeted trap. A series of experiments will evaluate the role of UV reflectance, translucence, color contrast patterns, background contrast, polarization and color saturation. Laboratory studies have been initiated on attraction of ACP to transmitted yellow or green light of different wavelengths to determine the optimal colors for use in field trials. Data are being collected in the Materials for constructing traps (acrylic sheets, colored films, paints, tangletrap, polarization filters) have been or are being purchased. Paint mixtures with different levels of UV reflectance are being formulated and reflectance characterized with a radiometer. These will be evaluated in laboratory assays and then in the field. Levels of transmitted light through colored films have also been characterized prior to use in laboratory assays.
Progress on development of an acoustic trap to capture male Asian citrus psyllids (ACP) searching for females on branches of citrus trees continued in the fourth quarter of 2013. New versions of the BugPhone trap were constructed for field testing. The Bugphone is an Arduino Uno microcontroller device that detects male vibrational calls and plays back female replies, attracting the males to a sticky cone attached to the tree. The operation of the Arduino was described in a paper published in the December 2013 issue of Florida Entomologist. One of the devices is being tested in the laboratory in preparation for use when ACP populations begin building up in early Spring. Some revisions have been included in the new BugPhone, including a more noise-free signal amplifier, lower power usage, and an option to include a memory chip so that signals obtained during a 1-2-week test period can be retained and examined for comparison with trap catches. In a new phase of this study, we have identified suppliers of robust devices that can produce that can produce signals to confuse duetting male and female ACP in field environments and have begun a search for a postdoctoral associate to assist in development of mating disruption techniques.
The objective of this research is to evaluate non-neurotoxic insecticides against Asian citrus psyllid (ACP), and provide growers with additional tools for management of ACP. We are investigating those non-neurotoxic insecticides that have shown promise 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. In the past quarter, we have been evaluating methoprene and diofenolan in the laboratory. Methoprene and diofenolan are juvenile hormone (JH) analogs that acts as growth regulators. We evaluated developmental inhibition of 1st, 3rd and 5th nymphal instar ACP to the subsequent developmental stage by application of methoprene. We treated 1st, 3rd and 5th nymphal instars with six different concentrations of methoprene ranging from 0 – 256 ‘g/ml. For 1st nymphal instars, we observed 87, 51, 41, 33, 30 and 13 percent development into second instars for concentrations of 0, 16, 32, 64, 128, 256 ‘g/ml, respectively. Similarly, concentrations of 0, 16, 32, 64, 128, 256 ‘g/ml resulted in 84, 71, 65, 53, 46 and 22 percent survival of third instars into fourth instars. When 5th nymphal instars were treated with concentrations of 0, 16, 32, 64, 128, 256 ‘g/ml, percent survivals were 84, 71, 70, 62, 59, 47%, respectively. These results indicate that treatment with methoprene successfully reduced the survival of the nymphal stages of ACP. We also investigated the residual contact toxicity of methoprene on eggs laid on plants after treatment. The plants treated with concentrations of 0, 20, 40, 80, 160 and 320 ‘g/ml resulted in egg hatch inhibition of 5, 14, 42, 63, 86 and 93 percent. Settling behavior of ACP on treated plants was also investigated under laboratory conditions. ACP showed no preference between the treated plants with various concentrations of methoprene ranging from 0-256 ‘g/ml. Currently we are investigating feeding behavior of ACP adults on plants treated with methoprene and the effect of methoprene on ACP reproduction. Diofenolan was evaluated for its ability to inhibit egg hatch of 0-48 hr aged eggs. Percent egg hatch inhibitions of 8, 27, 33, 39, 46, 68, and 96 percent were observed when eggs were treated with concentrations of 0, 10, 20, 40, 80, 160 and 360 ‘g/ml. Currently we are investigating the effect of diofenolan on 48-72 old ACP eggs. We will also investigate the effect of diofenolan on nymphal survival and ACP reproduction. Following completion of evaluations of methoprene and diofenolan (JH analogs), we will begin an evaluation of tebufenozide and methoxyfenozide (ecdysteriod agonists). The results of our research thus far suggest that several non-neurotoxic insecticides are effective in disrupting growth of the immature stages of Asian citrus psyllid.
Management of huanglongbing (HLB) is linked with effective management of the vector, the Asian citrus psyllid (ACP). Recently, tree nutrition supplement programs have been developed that may maintain the health and productivity of HLB-infected citrus trees; however, their impact on ACP biology, pathogen transmission, and insecticide management efficacy are either unknown or there is limited information. The objective of this study is to determine how nutritional supplement programs may affect ACP biology. The outputs are important because nutritional supplement programs are currently implemented by many growers and removal of HLB-infected trees is no longer a popular practice. Outputs for this research may be improved by adding a mature citrus grove experimental field site. Also, we have initiated additional laboratory experiments with other nutrients based on our current progress and available information. Currently, we are focusing most of our field research on one highly replicated experiment at the Mid-Florida Citrus Foundation Grove at Conserv II. This experimental is being conducted within 2-year old resets and allows us to address the question of how nutritional programs affect ACP and pathogen transmission within replanted groves and young tree care. We are in the process of initiating a similar experiment within a planting of mature citrus trees with a mix of HLB-infected and non-infected trees to enhance this investigation. This new field experiment will be composed of mature uninfected and HLB-infected trees. The treatments will be the KeyPlex program versus a non-supplemented control. Before establishing the plots, we are beginning to perform qPCR on approximately 200 trees to determine baseline HLB infection. Also, ACP adult density monitoring is beginning with weekly tap sampling, and nymph density will be monitored with bi-weekly flush examination. We will perform a bi-annual qPCR of the 200 trees, and an annual leaf sample nutrient analysis. The expected output of this additional field experiment should be a better understanding of how nutritional supplement programs affect psyllid population dynamics and pathogen transmission in mature trees. We have begun purchasing materials, including: ‘ qPCR on 200 trees 4 times= total of 1000 qPCR ‘ Nutritional analyses 80 trees, 2 times= total of 160 nutritional analysis ‘ From March to September at least 40 trips to the field ‘ Cost of field maintenance (irrigation, fertilizer) Laboratory experiment materials: ‘ 40 Valencia plants
Over the past few years, we have conducted several investigations focusing on the behavior of Asian citrus psyllid (ACP) with the goal of identifying specific behavior modifying chemicals. The purpose is to formulate these chemicals into controlled release devices for slow release in the field. The approach has focused on three areas: 1) attractants, 2) repellents, and 3) chemicals that prevent ACP from identifying pathogen-infected trees. Throughout these investigations, we have collaborated with industry, working with three companies: ISCA Technologies, Alpha Scents, and Chemtica. With each company, we have either developed new prototype devices or we have evaluated exiting promising technologies. From this work, two of the companies have made significant progress with developing ACP attractants and the next step goal is to bring to market more effective monitoring traps and attract-and-kill devices. Also, two repellent formulations are being pursued by two separate companies; one of which is in the later stages of development. Finally, we have evaluated one tool for preventing psyllids from finding infected trees with promising results. We have initiated 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, while attracting ACP insect predators and parasites to treated plots for three months. The formulation is under development by ISCA Technologies. The repellency of Decoy prototype formulations will be determined in laboratory-based cage studies. We have also begun increasing experiments examining new possible ACP repellents by investigating extracts from noxious plants that have been previously identified as insect repellents in the studies given below: G’k’e, A., L. L. Stelinski, R. Isaacs, and M. E. Whalon. 2006. Behavioral and electrophysiological responses of grape berry moth (Lepidoptera: Tortricidae) to selected plant extracts. Journal of Applied Entomology 130: 509-514. G’k’e, A., L. L. Stelinski, and M. E. Whalon. 2005. Behavioral and electrophysiological responses of leafroller moths to selected plant extracts. Environmental Entomology 34: 1426-1432.
Currently we are evaluating insecticide resistance in the Asian citrus psyllid (ACP) against insecticides with different modes of action across the state of Florida. Starting in 2009, an increase in resistance against the neonicotinoid, imidacloprid, was noted. Recently, we determined that this rise in resistance is primarily mediated by an increase in enzymatic activity of a class of enzymes known as cytochrome P450 monooxygenases. In that same study, it was noted that several populations of ACP had increased resistance against another insecticide, cypermethrin, which is a pyrethroid. Pyrethroids mediate their effects by modulating the kinetics of sodium channels within the nervous system called voltage-gated sodium ion channels. This increase in resistance could be due to increased enzymatic activity as found for neonicotinoids, or could be due to target-site mutations of the channel, rendering it insensitive to the insecticide. The course of action to mitigate the rise in insecticide resistance will depend on which mechanism confers resistance. It is particularly important to determine if the resistance is enzymatically based due to the fact that enzymes typically metabolize insecticides across different classes, e.g., cytochrome P450 monooxygenases can break down organophosphates, neonicotinoids, pyrethroids, along with others. Therefore, continued use of a particular insecticide may drive resistance through a population that will affect the efficacy of not only that particular insecticide, but those from other classes. We have begun to expand current work to include the determination if increased resistance to cypermethrin is due to enzymatic activity, target-site insensitivity, or a combination thereof. A better understanding of the underlying mechanism conferring resistance against this compound and other compounds leads to better management to delay resistance against currently utilized insecticides. We have collected insects ACP from field populations where cypermethrin had been used. All experiments with field populations of ACP are being compared against our susceptible laboratory strain. Two types of assays are being developed to determine the basis for resistance against this compound, topical and enzymatic. Topical assays with different sub-classes of pyrethroids and DDT are being developed to determine if resistance is mediated by target-site insensitivity. Enzymatic assays will be used to determine if there is an increase in enzymatic activity of the three major classes of enzymes known to mediate insecticide resistance, esterases, cytochrome P450 monooxygenases, and S-glutathione transferases. Protocols for these assays have already been established and were those used in our studies to elucidate the mechanism behind imidacloprid resistance. For enzymatic studies, insects will be tested under normal and induced conditions to determine response of enzymes to insecticidal exposure. Response of enzymatic pathways to insecticidal exposure will be evaluated with qPCR to measure changes in mRNA abundance levels and biochemical assays to detect changes in enzyme levels. We have also begun purchasing necessary materials such as: ‘ Substrates and standards for biochemical enzymatic assays ‘ Insecticides ‘ mRNA isolation and cDNA synthesis kits
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. We are working through a list of non-neurotoxic products and determining their effects on psyllid population development including effects on development, fecundity, fertility, and life table development as a result of each chemical. As a result of this research, we have identified some non-neurotoxic chemicals that are effective against ACP. Some of these were previously registered for use on tree fruit, some have gained alternative application use registration, and some are possible compounds that may be useful in the future. Since beginning our work, we have nearly completed research with some of these, such as Novaluron, for example. This compound may be a tool for ACP control. We have conducted both laboratory and greenhouse studies to determine the efficacy of novaluron against various life-stages of ACP. Rimon’ is a commercial blend of novaluron, which is an insect growth regulator used widely in insect management, but is not registered for citrus. The compound was tested on two stages of ACP–fifth instar nymphs and eggs, at three field rates used for similar insect pests, 10, 15 and 20 fl oz/acre. Flush with approximately 15 fifth instar nymphs were dipped into the three preparations of insecticide and mortality was determined 72 hours post-treatment. The three field rates of 10, 15 and 20 fl oz/acre induced 30, 60 and 62% mortality, respectively. For effects on egg hatch, flush with an average of 25 eggs between 0-3 days old were dipped into the insecticide dilutions and the number of live nymphs was determined every 24 hours for eight days. At the 96 hour time point when eggs began to hatch, controls had 1.8-fold more live nymphs over the 10 fl oz/acre treatment and 2.2-fold over 15 and 20 fl oz/acre treatments. Bioassays are also underway to evaluate the viability of eggs from treated females. The initial data suggest that the non-neurotoxic insecticide novaluron is effective against the immature stages of ACP in laboratory assays, showing similar effects to other known insect growth regulators against this pest. We have initiated expanded the above research at a faster pace with other insect growth regulators, including allostatins and ecdysteroid agonists. We have begun purchasing materials including: ‘ Chemicals ‘ Plants for Insect colonies ‘ Plants for experiments ‘ Pots ‘ Soil ‘ Fertilizer
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