Recent efforts to disrupt mating of the leafminer Phyllocnistis citrella have focused on the use of SPLAT’, a flowable wax emulsion intended to serve as a slow-release matrix for pheromones. Early success with this approach was overshadowed by the expense and difficulty of application, and variation in wax component chemistry that contributed to reduced longevity of pheromone emission in the field. Solid elastomer dispensers (DCEPT CLM’, ISCA Technologies, Inc.) loaded with a 3:1 blend of (Z,Z,E)-7,11,13-hexadecatrienal and (Z,Z)-7,11-hexadecadienal, the major components of P. citrella sex pheromone, provided disruption of trap catch in commercial citrus orchards for periods exceeding 30 weeks. The triene component alone worked as well or better than the 3:1 blend. The height of dispensers placed by hand in the tree canopy had a significant effect on trap shutdown. Dispensers placed low (0.6 m) in the canopy resulted in a reduction of trap shutdown in the upper third (>3 m) of the canopy suggesting that the net movement of pheromone molecules was downward during the period of active moth flight. Although moth flight appeared equivalent among the heights tested, placement of dispensers higher in the canopy appears more effective given this downward movement of pheromone plumes. These studies suggest that season-long trap catch disruption can be attained in citrus with a single application of a hand-applied dispenser. Efficacy of trap catch disruption was excellent in the 3 field trials and compares favorably with longevity of disruption previously reported for SPLAT CLM. The ability of the pheromone blend or triene-only formulation to disrupt trap catch of males in traps baited with the ‘natural’ 3:1 blend agrees closely with what we have observed and reported previously for SPLAT-based formulations. The longevity of disruption provided by DCEPT far exceeded results obtained with SPLAT and raises the possibility that a single application of emitters may provide year-long disruption of mating for P. citrella. Dispensers loaded with the triene component performed equally well regardless of the presence of (Z,Z)-7,11-hexadecadienal. The two formulations tested contained the same amount of triene; the addition of the diene component did not increase disruption. We conclude that inclusion of the diene in mating disruption products for this species has no benefit that would justify the additional expense of its synthesis and formulation. Also, the fact that highly effective trap catch disruption was obtained with an off-ratio blend supports the conclusion that disruption in this species falls into the category of non-competitive mechanisms because the triene alone is not attractive to males. The height of traps did not affect the number of males caught suggesting that males were active throughout the canopy. But the location of pheromone dispensers in the tree canopy did have a significant effect on trap catch disruption. Dispensers placed low in the canopy (<0.6 m) resulted in decreased disruption in the center and tops of the canopy compared with disruption achieved low in the canopy. Similarly, dispensers located in the canopy center (2m) were more effective at disruption of traps located low or in the center of the canopy; less disruption was achieved in the canopy top. These results suggest that net displacement of pheromone was downward perhaps due to the fact that the triene (molecular weight 234) is heavier than air and would tend to settle under calmer conditions that prevail at the time of greatest flight activity by males.
Laboratory and field trials failed to demonstrate attraction of males or females to artificial aggregations of males, females or male-female pairs. During the period, we focussed our efforts on collecting headspace volatiles over groups of adult males and females of known age. The adult weevils were collected directly from diet cups and maintained in cages segregated by sex. Headspace volatiles are being collected weekly from cohorts for analysis by GC-MS and for electroantennography. We have been able to confirm the presence of the pheromone methyl (.)-3-(2-hydroxyethyl)-4 methyl-2-pentanoate in headspace (by SPME) and in jar washings from 8-week-old males. Headspace collections from females are being stored (-20’C) for later analysis by GC-coupled electroantennography (GC-EAD) to look for female-produced compounds with antennal activity. However, at this juncture, the most parsimonious explanation of our results to date is that the Diaprepes root weevil does not rely upon a sex pheromone for locating conspecifics in the field.
Canker disease on fruit and foliage is under evaluation in the following 2015 trials: 1) Canker management in grapefruit: Foliar applications of novel bactericidal formulations will be compared with standard film-forming copper and zinc formulations: 1) Nordox 75WG, 2) Nordox 30/30 with 0.5 lb of metallic copper and an equivalent amount of metallic Zn, 3) Nordox 45/15 WG, 4) 2 Zinkicide formulations 5) 6 T-sol formulations; 6) CuSiNG-Quat-P (Cu+Quat), 7) Fixed-Quat DDAC, 8) CS-CuSiNP, 9) SG0025 (treatments 4-9 are experimental formulations from Dr. Swadesh Santra, a Nano-chemist at Univ. Central Florida), 10) Untreated check 2) Canker management in Hamlin: Foliar applications of standard film-forming copper formulations and soluble copper formulations will be compared in 4 yr old trees 3) Greenhouse assay to evaluate local systemic and residual activity of selected UCF formulations and FireWall will be against Xanthomonas citri subsp. citri on potted Pineapple orange trees inoculated by injection infiltration 4) Assessment of Cu bioavailability and residual activity on grapefruit for copper oxide (Nordox 75 WG) and the copper-zinc formulation Nordox 30-30 during two periods of summer season (July and August) to test whether zinc may be enhancing the bioavailability of Cu from the copper oxide in the 30/30 and 45/15 formulations compared to 75 WG copper oxide alone 5) Selected results from 2014 trials were presented at the Florida Citrus Show in January and Citrus Institute in April and published in the April 2015 issue of Citrus Industry magazine
The canker field trial of Zinkicide compared to industry standard management on grapefruit showed better control of canker on fruit than all other treatments. We also observed control of some fungal pathogens. A second year of this field trial has begun to confirm the results and test the consistency of control. Yield and juice quality data from the HLB trial in young Valencia trees has been collected. The timing of first treatment and the trees flush cycle make it difficult to determine efficacy in the first year of treatment. An unexpected problem occurred with a very strong gradient of Las titer in the small tree field trial that was not apparent based on an even distribution of symptoms in the plot layout. This gradient is confounding titer analysis and we are trying to determine the cause. While HLB field trials will continue, they will be performed at a larger scale in a project funded by the SCRI and are no longer a part of this project. HLB symptom ratings were also collected for infected trees in the canker trial because treatment began earlier. The data is promising, however, it was not the main goal of this trial, so initial HLB disease assessment was not done. Additionally, the data may be biased because the reduction in canker could reduce leaf drop and cause the trees to look healthier. Dr. Santra’s group has improved synthesis of Zinkicide to reduce the amount of some ingredients needed for synthesis while maintaining efficacy. This improvement is aimed at reducing cost of production.
Irrigation water acidification (target pH, 7.5, 6.0, 5.0, and 4.0) continues at two citrus groves (one a 20 year-old Hamlin sweet orange trees predominately on Swingle rootstock and the second a three year old Hamlin sweet orange trees on Swingle rootstock). A second sulfur application was made to selected treatment blocks in June 2015. The rainy season started early this year (May 10) has been very wet (419.1 mm of rain at site one and 345.4 mm of rain at site two). Thus, little irrigation has been applied at either site since mid-May. Soil samples taken prior to second sulfur application in June indicate that plots receiving both acid injection and sulfur have similar soil pH to plots receiving only irrigation water acidification. Soil samples taken in December after sulfur application indicated significantly lower soil pH in plots receiving both irrigation water acidification and sulfur application. These results would indicate that the relatively slow release sulfur product (Tiger 90) applied in December reduced soil pH below that achieved by irrigation water acidification only but lasted less than six months. Root density samples taken in June indicate a significantly greater root length density with lower soil pH. These results indicate a positive correlation between root density and reduction in soil pH from greater than 7.0 to less and 6.0. Leaf N, K, and Ca were greater for trees treated with both irrigation water acidification and sulfur application compared with irrigation water acidification only. However, leaf N and Ca were greater in trees receiving only irrigation water acidification to 5.0 and 4.0 compared with trees receiving no acidification (pH 7.0). Tree size and fruit drop measurements to be made in the nest quarter will determine if these increases in root density and leaf nutrient concentrations result in greater size and reduced drop. If this occurs, we should be able to demonstrate positive impact of soil acidification on yield. Average Hamlin and Valencia tree water uptake under greenhouse conditions have not varies greatly from one another for the past year. However, there is some evidence of reduced water uptake for trees receiving water supplemented with calcium bicarbonate. Reduced water uptake by trees receiving calcium carbonate in irrigation water would account for reduced leaf area and trunk diameter. A talk on methods of irrigation water acidification and expected improvements on citrus nutrient status was given on April 7, 2015 to more than 200 attendees of the Citrus Institute. The data was displayed at three grower meetings on citrus nutrition in May and June. Since these presentation, numerous personal contacts have been made with individual citrus growers to continue, alter or initiate soil acidification projects in groves throughout the state.
Two citrus groves, one – 20 year-old Hamlin sweet orange trees predominately on Swingle rootstock and a second consisting of three year old Hamlin sweet orange trees on Swingle rootstock have received acid injection to selected blocks with and without sulfur applications for nine months. Irrigation water was acidified at one of four target water pH (7.5, 6.0, 5.0, and 4.0). A controlled release form of elemental sulfur was applied to half of the trees in each pH treatment (main effect) including the non-acidified control (pH~7.5). Soil pH at both sites have decreased to levels near those of the irrigation water target pH and lower pH in treatments plots receiving elemental sulfur. At the young tree site, no significant difference in root density has been found. However, root density at the mature tree grove has significantly increased with lower soil pH. Root length density increase to 1.7 to 1.8 cm/cm3 for pH treatments of 5.0 adn 4.0 with and without sulfur application compared with 1.3 to 1.6 cm/cm3 for control and 6.0 treatments. To date, little difference in leaf nutrient levels have been found in plots with reduced water and/or soil pH. The increase in root density at the mature site may be indicative of greater root development by mature trees compared with younger trees. It has been demonstrated that citrus trees have a great capacity to store nutrients in their woody tissues thus reducing the effect of differential nutrient studies until these reserve nutrients have been sufficiently reduced to a level that nutrients from the wood tissues can not supplement nutrients provided by fertilizers. The lack of leaf nutrient concentration response for either the young or mature trees may be evidence that citrus trees demonstrating nutrient deficient symptoms consistent with HLB positive trees have lower nutrient concentration in their woody tissues and thus can not provide nutrients to leaves until these reserves are replenished by higher nutrient availability presumed at lower soil pH levels (5.5 to 6.5). If the process of replenishing nutrients to the woody tissue is true we should see greater leaf nutrient concentrations in the next few quarters. Average Hamlin and Valencia tree water uptake under greenhouse conditions have not varies greatly from one another for the past year. However, there is some evidence of reduced water uptake for trees receiving water supplemented with calcium bicarbonate. Reduced water uptake by trees receiving calcium carbonate in irrigation water would account for reduced leaf area and trunk diameter.
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 adult response to different stimuli have yielded temperature threshold estimates for ACP movement at 67-74 F, light above 250 lux, wind speeds below 4.25 mph, and humidity below 70%. Movement was not affected by host plants placed within the wind tunnel. ACP tend to move with the wind when all other factors are equal but can also move into the wind. ACP movement direction is mostly dictated by wind direction with greater wind speeds creating movement patterns more strongly downwind. Increased light intensity above the threshold did not increase ACP movement. Objective 2. Dr. Stelinski’s lab showed that female ACP avoid flush pre-infested with ACP on a density dependent basis. They also demonstrated that females were attracted by herbivore-induced volatiles. The herbivore-induced plant volatile attractant and the female-specific odor repellent appeared to be complementary foraging cues providing psyllids with information for choosing a plant potentially harboring male conspecifics for mating and to reduce intra-conspecific competition by identifying previously exploited resources. They also developed a flight mill specifically adapted to ACP flight capability. This allowed them to establish physiological ACP flight limits of 2.4 km. Flight capability was associated with ACP color morph where green/blue ACP abdomens were more prone to long distance flights than other morphs. Within green/blue morphotypes, 32% of ACP tested exhibited long distance of flight. The average distance of flight for long distance flight was 320 m. Additionally, psyllids infected with CLas dispersed more, initiated flight earlier on the flight mill and performed more long distance flights than non-infected psyllids. Objective 3. Fluorescent dust mark recapture studies were shown to work under laboratory settings without impact on ACP movement patterns nor lifespan. This technique was useful for visualizing ACP within wind tunnel assays reducing error. Field evaluations proved ACP could be visualized using the fluorescent dust but was not an effective method do to the difficultly in observing small ACP with tree canopies. Objective 4. Captures on yellow sticky cards and by suction traps peaked in the spring and and to a lesser extent in fall. Sticky cards set on posts in citrus groves captured more at 1m than 2m except near wind breaks where there was little difference, indicating that ACP flew low to avoid strong winds. Sticky traps indicated preference for the side and upper upper portions of mature trees. Movement of ACP into and out of citrus blocks as judged by sticky card captures is greatest near wind breaks and in one block, on the east and west side although densest populations by tap sample resided on north. Preferences for location and direction of movement did not change over the year. 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. Sampling has reveled the mulch still works to reduce the ACP populations for up to 3 years. In another trial, repellency was demonstrated by the result that fewer ACP were captured on sticky traps set at 1 m in reflective mulch compared to 2 m, where as the opposite was true over white mulch or no mulch (grass) where more ACP were captured more at 1m than 2m.
Issue date for this grant was 13 July 2012. Objectives were: 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 by Ph.D. student Scott Croxton using a wind tunnel to determine ACP adult response to different stimuli. Threshold estimates were: temperature 67-74 F, light above 250 lux, wind speeds below 4.25 mph, and humidity below 70%. Movement was not affected by host plants placed within the wind tunnel. Most movement was with the wind. Light intensity above the threshold did not increase ACP movement. Sticky card captures in the field peaked late morning, and mid-afternoon. Correlations were positive with light and temperature. There were no captures at night or during rain events. Graduate student Thomson Paris found that UV light enhance attraction to green and yellow but not blue. Objective 2. Dr. Stelinski’s lab showed that female ACP avoid flush pre-infested with ACP on a density dependent basis. They also demonstrated that females were attracted by herbivore-induced volatiles. The herbivore-induced plant volatile attractant and a female-specific odor repellent appeared to be complementary foraging cues providing psyllids with information for choosing a plant potentially harboring male conspecifics for mating and to reduce intra-conspecific competition by identifying previously exploited resources. They also developed a flight mill specifically adapted to ACP flight capability with which they established a physiological ACP flight limit of 2.4 km. ACP with green/blue abdomens were more prone to long distance flights than other color morphs, with 32% exhibiting long distance of flights of an average distance of 320 m. Additionally, psyllids infected with CLas dispersed more, initiated flight earlier on the flight mill and performed more long distance flights than non-infected psyllids. Objective 3. Marking with fluorescent dust did not alter ACP movement patterns nor affect lifespan. This technique was useful for visualizing ACP within wind tunnel assays reducing error. Marked ACP released in the field were easily visualized at night and on sticky cards. However, less than 0.5% of the 6000 released ACP were found outside the release area which resulted in too few observations for statistical analysis of the released ACP. Objective 4. Captures on yellow sticky cards and by suction traps peaked in the spring and secondarily in fall. Sticky cards set on posts in citrus groves captured more at 1m than 2m except near wind breaks. More ACP were captured on the east side and upper upper canopy of mature trees. Sticky cards captured more ACP on the east and west side of one block although densest populations by tap sample resided on north. Preferences for location and direction of movement did not change over the year. PUBLICATIONS: Objective 5. Metalized polyethylene mulch repelled ACP for up to 3 years after planting, and reduced incidence of HLB. Croxton, S. D., & Stansly, P. A. 2014. Metalized polyethylene mulch to repel Asian citrus psyllid, slow spread of huanglongbing and improve growth of new citrus plantings. Pest management Sci, 70: 318-323 Paris,TM, Croxton, SD, Stansly, PA, & Allan, SA. 2015. Temporal response and attraction of Diaphorina citri to visual stimuli. Entomologia Experimentalis et Applicata, DOI: 10.1111/eea.12294 Martini, X., M. Hoffmann, M.R. Coy, L.L. Stelinski, & K.S. Pelz-Stelinski. 2015. Infection of an insect vector with a bacterial plant pathogen increases its propensity for dispersal. PloS ONE. 10(6): Dissertations by S. Croxton and T. Paris.
This project, in general, seeks to determine if there are any observable influences of foliar fertilizers on HLB-affected grapefruit in the Indian River marketing district. To-date, this project has established 3 separate research trials in commercial grapefruit groves and university research farms in St. Lucie county, Florida. Trial #1 is ~25 years old of ‘Flame’ grapefruit on Swingle rootstock. Trial #2 utilizes ~6 year old ‘Ruby Red’ on Sour orange trees. The first 2 trials are located in commercial groves. Trial #3 seeks to determine the effect(s) that foliar fertilizers have on young tree growth and their ability to protract HLB disease symptoms in 1 year old ‘Ray Ruby’ grapefruit on Kuharske rootstock. For trial #1, there are a total of 30 replicated experimental units; for trial #2, there are a total of 50 replicated experimental units; and, for the trial #3, there are a total of 24 replicated experimental units. Foliar fertilizer applications have been started since February 2014. Tree growth data, leaf nutrition data, and CLas titer measurements have been made. Fruit drop monitoring will begin in September 2014 in trials #1 and #2. In prosecution of these efforts, 2-part-time OPS employees have been hired to do our foliar sprays and collect data observations. As of January 2015, the first year of data collection on the commercial grove plots (trials #2 and #3) as well as the young tree trial at the IRREC (trial #1). First-year fruit yields were gathered in the commercial trials (trials #2 and #3) in December 2014. In addition, all of the foliar fertilizer applications were made (once per quarter) to all trials. Currently, data are being analyzed from the first growing season (2014). In 2015, foliar fertilizer treatments, tree growth measurements, and leaf nutrient analyses have commenced as of January and March, 2015, and will continue throughout the year in a manner similar to the calendar of activities last year (2014).
The overall objective of this project is to investigate the feeding deterrence provide by reflective colored kaolin clays on citrus plants from Asian Citrus Psyllid. Previously we reported the development of colored kaolin clays with different dyes, covering the visible spectrum. During this reporting period, experiments have been conducted to determine how the coating with colored clays modifies the reflectance of citrus leaves between 300 -700nm. Reflectance experiments performed using ten modified kaolin clays show a significant improvement in reflective properties as compared to standard kaolin. Of the colored kaolin clays applied, kaolin modified with basic red and violet dyes also altered the reflectance of citrus foliage in the ultraviolet range. In addition, citrus foliage coated with colored kaolin clays, have been categorized using a portable MiniScan spectrophotometer (HunterLab). MiniScan measurements are made based on CIE L*a*b* color space approach. Measurements were made on citrus foliage before and after application of colored coatings. Sites of measurements were marked on the leaves to minimize the error due to variability in the leaves. All colored kaolin clays were sprayed and allowed to completely dry on the leave surface. When comparing the color index measurements before and after coating, each colored clay showed to have a significant change in color strength and showed changes differing in magnitude in L*a*b* scale. For example Kaolin clay colored with violet dye showed significant change in color strength, particularly in b* value, indicating a change in the blue color strength. Further experiments and analysis of change in color index with different coatings are underway. Also experiments to increase the rainfastness of the coatings are underway. To promote rainfastness, clay formulations modified with agricultural grade spreaders and stickers have been prepared with minimal effect on the dyes. In addition, formulations are being prepared with adjuvants that can prevent the run off and enhance the adhesion of clays to the citrus foliage. Additional experiments such as effect of coatings on gas exchange and ACP are planned for coming months.
The overall objective of this research project is to develop a potential non-phytotoxic, environmentally friendly film-forming ACP repellent solution for preventing HLB infection. Earlier we developed a series of Organo-Silica Composite Film (OSCF; ACP repellent) materials using various polymers and cross-linkers. These film materials were screened for temperature safety (heat trapping) and phytotoxicity. Out of which, two best performers, OS-SG 6 and OS-SG 10 were delivered for evaluating ACP deterrent efficacy in field conditions. Field trial results did not show any promising efficacy when compared with control. After receiving feedback from field trial results, we have developed two new OSCF materials (OS-SG-11 and 12) using EPA approved polymers and particulate fillers to increase rainfastness and roughness. and environmentally safe inert ingredients to increase rainfastness. Dynamic Light Scattering (DLS) characterization results revealed formation of large multi-micron composites (~ 1 – 5 microns). Scanning Electron Microscopy (SEM) shows the film with rough surface morphology. UV-Vis and NMR spectroscopy results of OS-SG 11 and 12 show strong interaction among film components. The introduction of filler material improved film rainfastness and roughness. Both OSCF 11 and 12 were non-phytotoxic when tested on ornamental plant Vinca sp (highly susceptible to phytotoxicity). Phytotoxicity experiments were conducted using Panasonic Environmental Test Chamber (Model MLR- 352H) that simulated typical FL summer conditions (85% RH; 35 degree C).
Work continues towards estimation of baseline LD50s and LD80s of susceptible CLM some commonly used insecticides. Larvae and adults are exposed for 48 h to different doses (0; 0.01; 0.03; 0.1; 0.3; 1; 3; 10; 30; 50; 100, 300, 600 and 1000 ppm) of: Actara 25WG, Agri-Mek 0.15EC, Cyazypyr, Danitol, Delegate WG, Dimethoate, and Micromite 80WGS. Intrepid (methoxyfenozide) was evaluated on leaves immediately transferred to a water source after excision and evaluated after 96 h due to allow effects of this compound’s mode of action (growth regulator) to become apparent. We found this method to be more efficient than caging plants or artificial diets and feel that results are representative of commercial applications. Updated results for CLM larvae: Actara (thiamethoxam) LD50=4.18 (CL95: 1.86-8.51) ppm, LD80=524.12 (CL95 209.70-1827) (n = 1047; .2 = 9.54; d.f = 10; Heterogeneity = 0.95); Agri-Mek (abamectin): LD50= 0.360 ppm (CL95: 0.14-0.80), LD80=34.6 ppm (CL95: 11.7-189.7) (n = 1350; .2 = 13.10; d.f = 9; Heterogeneity = 1.46); Cyazypyr (cyantraniliprole): LD50=43.36 ppm (CL95: 15.80-131.55), LD80=49,413 (CL95: 0.19E+07) ppm, (n = 1,196; .2 = 7.64; d.f = 8; Heterogeneity = 0.96); Danitol (zeta-cypermethrin): LD50=381.78 (CL95: 86.33-11,771), LD80= 37, 220 (CL95: 31.70-53761.1) ppm (n = 561; .2 = 5.29; d.f = 9; Heterogeneity = 0.59); Delegate (spinetoram): LD50=2.49 (CL95: 1.00-5.19), LD80= 86.58 (CL95: 36.60-308.13) ppm (n = 964; .2 = 10.08; d.f = 8; Heterogeneity = 1.26); Dimethoate LD50=2.49 ppm (CL95: 1.01-5.19) and LD80=86.58 ppm (CL95: 36.60-308.13) (n = 629; .2 = 10.08; d.f = 8; Heterogeneity = 1.26); and Micromite (diflubenzuron): LD50=74.30 (CL95: 13.63-285.15), LD80=5,723 ppm (CL95: 994-0.129E+07), (n = 816; .2 = 19.92; d.f = 9; Heterogeneity = 2.21). For Intrepid, LD50=2.95 (CL95: 0.24-10.30) LD80=64.11 ppm (CL95: 18.36-814.8), (n = 125; .2 = 4.42; d.f = 6; Heterogeneity = 0.736). Updated results for CLM adults: residual contact toxicity of Actara has now been estimated as LD50=16.75 (CL95: 5.00-91.73) LD80=4153 ppm (CL95: 417.4-0.241 E+07), (n = 377; .2 = 0.688; d.f = 3; Heterogeneity = 0.229). Micromite – a further 240 adult CLM were tested at various concentrations up to 1000 ppm since the last report: LD50=476.02 (CL95: 192-1630) LD80=39,483 ppm (CL95: 7988-63,435), (n = 756; .2 = 4.42; d.f = 6; Heterogeneity = 0.736). Delegate (spinetoram) replicates also increased: LD50=24.93 (CL95: 5.37-358), LD80= 742.5 (CL95: 92.76-0.48E+06) ppm (n = 743; .2 = 12.02; d.f = 5; Heterogeneity = 2.4). Agri-Mek LD50=74.51 (CL95: 45.31-165.33), LD80= 364.67 (CL95: 164.59-1974.1) ppm (n = 240; .2 = 4.35; d.f = 3; Heterogeneity = 0.145). Contact toxicity for Intrepid LD50 and LD80 values remain inestimable for CLM adults. Evidence for resistance has not been found in CLM larvae collected from commercial groves.
Citrus leafminer (CLM) is a major pest of citrus, causing direct damage and increased incidence/intensity of citrus canker. Pheromone traps are valuable 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. Laboratory Technician, Vincent Ficarrotta has continued to extract DNA from P. citrella samples. An additional 196 samples have been sequenced for COI barcoding to target the 658 bp region of COI gene. We recently switched extraction methods from QIAGEN DNA Blood Tissue Kit to OmniPrep. The dataset was merged with our previously published genetic dataset (Kawahara et al. 2013) generated from this project. The OK Slough had the highest number of samples sequenced, and within these samples, P. citrella was the most common, followed by P. vitegenella. Other species, such as the two new Phyllocnistis species, are lured to traps at a low frequency (’11 specimens/year) in all years sampled thus far. While the numbers show strong bias towards P. citrella, it is clear that in all years, multiple species of Phyllocnistis readily come to CLM lures, following the initial finding of Kawahara et al. (2013). We also generated a new ML tree with additional samples analyzed on UF’s High Performance Computing Cluster (HPC). This tree shows largely similar results as Kawahara et al. (2013), where the majority of samples obtained grouped with already-known specimens of P. citrella. Bootstrap values for clades were generally low for most groups (<50%), which is comparable to prior results based on a single marker (e.g., DePrins and Kawahara [2012]). However, there were some notable results, such as the strong sister group relationship of P. citrella (North American) to P. citrella (Asia). Further, there is strong support for the monophyly of P. citrella (NA and Asia), P. insignis, and Phyllocnistis new sp. 1. Other taxa that were included in the dataset, but not generated from this particular project, also show strong bootstrap support for their monophyly, which will be relevant to future studies on non - P. citrella related species in the genus (e.g., P. hyperpersea (100% BP), P. longipalpus (100% BP), P. perseafolia (100% BP), P. populiella (93% BP), P. saligna (100% BP)). We also see relatively strong genetic distance between each group (>2-4% in many cases), supporting the hypothesis that these clades should be treated as separate species. Finally, 212 sequences of the 658 bp COI barcode region that were recently sequenced were submitted to GenBank (www.ncbi.org) as proposed in the original project proposal. The GenBank numbers are being acquired and verified by NCBI staff. We are awaiting results of review and a decision on “Placement Density and Longevity of Pheromone Traps for Monitoring of Citrus Leafminer, Phyllocnistis citrella (Lepidoptera: Gracillariidae)” by P. Vanaclocha, M. M. Jones, C. Monz’ and P. A. Stansly. Mark-release-recapture work using fluorescent powders has indicated that male moths are capable of flying 140 m in 24 h. We determined that release immediately after application of powders is essential to capturing marked moths. We have replicated traps at the distances over four release dates and are preparing to deploy traps at 280 m and 420 m from the release site. We are also preparing to sample Rb-spiked citrus to evaluate Rb uptake and detection in CLM for mark-recapture applications.
Previously, we have reported that acquisition and transmission of Las are less efficient at temperatures below 22oC and above 27oC, and this effect also occurs when psyllids are pre-exposed to temperature for 2 or 4 days prior to acquisition experiments. Observations of cuticular melanization in psyllids exposed to extreme temperatures suggest that this process may be associated with corresponding reductions in acquisition and transmission efficiency. Seven target genes from five gene families (rhodopsin, bursicon, Yellow C, DcAWD, and multi-copper oxidase) have been identified to evaluate how temperature-mediated gene expression influence Las transmission (objective 1). Quantitative polymerase chain reaction (qPCR) assays were developed for each of the target genes, and the relative expression of each gene among treatments was compared using the 2-..CT method by normalizing to psyllid wg gene expression, followed by normalization to the treatment based on the lowest gene expression. Subsequently, we have identified changes in the relative expression of four genes from this group in association with melanization. Currently, experiments are underway to evaluate temperature mediate changes in expression of these genes, and their correlation with melanization and Las transmission. Whole plant experiments are ongoing, but initial results suggest that that down regulation of at least one gene is associated with reduced acquisition at 16oC compared with 27oC. We will further evaluate the role of this gene in Las acquisition using dsRNA. Establishment, maintenance, and characterization of psyllid isolines from Florida is ongoing (objective 3). We have identified one isoline in particular that exhibits increased egg laying compared with other populations. Currently, we are developing a restriction enzyme-base assay to rapidly screen for Wolbachia strains in these isolines to determine their association with reproductive fitness. An additional component of this project is germ-line transformation of psyllids. Optimization of egg injections is ongoing. Survival of nymphs following egg injections in Petri dishes was low. Successful egg hatches were obtained using an artificial diet as an alternative approach, but was not significantly greater than the petri dishes procedure.
The goal of this research has been to monitor insecticide susceptibility in Florida populations of Asian citrus psyllid (ACP) and to determine the molecular mechanisms of resistance. We have completed our investigation of the resistance mechanisms to pyrethroids in ACP. The voltage-gated sodium channel (VGSC) is the primary target of pyrethroids, and is prone to mutations which render pyrethroids ineffective through target-site insensitivity. Mutations to the channel have been reported in a wide diversity of arthropod species. We characterized the VGSC of ACP to determine the potential of the channel to develop similar mutations. Pyrethroids are an important component of management rotations for this pest species and their loss would significantly hinder control efforts. The VGSC in ACP shows as much potential for alternative splicing as any other characterized insect, including variants which have been shown to have reduced pyrethroid sensitivity. Furthermore, it shows significant potential to develop target-site insensitivity through kdr mutations, with the greatest potential at kdr position M918. Previous studies demonstrated that ACP has an exquisite enzymatic arsenal to detoxify insecticides resulting in reduced efficacy. The results from this study demonstrate that target-site insensitivity is also a probable basis for insecticide resistance to pyrethroids in this insect species. The VGSC sequence and its molecular characterization should facilitate early elucidation of the underlying cause of an established case of resistance if it should occur. We have evaluated insecticide resistance in field populations of Asian citrus psyllid (ACP) for approximately the past 7 years. This has allowed us to determine the fluctuation of resistance in Florida ACP populations, develop and recommend appropriate rotation schedules, and determine the specific mechanisms mediating resistance in ACP populations. We have already documented reduced susceptibility in regional Florida ACP populations, where prescribed MOAs sometimes are applied up to 12 times/yr to suppress re-infection of trees. By 2011, there was a measurable reduction of insecticide susceptibility among all ACP populations that we examined across the state of Florida. Also, there was a trend for increased resistance from 2009 to 2012. The highest levels of decreased susceptibility found in field and laboratory in Florida ACP varied between 35 to 100 fold. However, at no point was this reduction of susceptibility sufficiently high so as to result in product failure in commercial groves. We estimate that approximately a 100-fold reduction in susceptibility may be associated with product failure based on our research in non-commercial citrus and the laboratory. The coordination of treatments for ACP is meant to enhance insecticide use by preventing rapid re-infestation by psyllids from non-sprayed areas. By the end of 2010, there were 10 CHMAs established. Our 2010 survey was conducted during that inception period of CHMAs. Insecticide resistance was beginning to be a problem at that time. From 2013 to 2014, there has been a progressive drop in resistance observed among ACP populations in Florida, according to our surveys. In fact, based on our sampling capabilities, it appears that since 2013 we have returned to pre-2009 levels of ACP insecticide susceptibility across the state. The number of active CHMAs has risen from the initial 10 to a current 52. We can hope that coordination and effective rotations of insecticides for ACP have contributed to this decline in resistance. However, given that we know that up to 4,000-fold resistance to the very important neonicotinoid insecticides can occur in ACP populations (V zquez-Garc a et al. 2013), we must remain vigilant by rotating MOAs and monitoring resistance.
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