Natural ACP populations were sampled in trees to identify alternative hosts in major Florida citrus growing regions (specific to the dormant winter season). We completed a detailed survey of ACP presence in Lake Kissimmee State Park (Polk Country, FL), where we captured ACP consistently throughout a season. In this forest, occurrence of citrus is particularly rare (only three tangerine trees), and no Rutaceae were found in the trapping locations or at nearby (‘500 m) areas to where ACP were captured. In addition to trap capture data, complementary laboratory no-choice feeding bioassays for ACP performed using Red bay, gallberry, and blueberry. These were selected as possible alternative hosts, given their presence in locations where ACP were captured. The results suggest that ACP may have a wider alternative host feeding range than previously thought, which may allow for significant dispersal even through dense forests in Florida up to 2.3 km from large-area plantings of citrus. We have recently completed temperature bioassays evaluating the effects of temperature on the insecticide susceptibility and gene expression of ACP to address the final objective of the proposed project. RNA collected from insects exposed to heat (simulating spring, summer conditions) and cold (simulating winter conditions) have been collected and analyzed using quantitative reverse-transcriptase- (qRT-) PCR. Over two consecutive winters, we examined the abundance of D. citri in 52 groves throughout Florida, in response to four different management regimes defined as: conventional, intermittent, unmanaged, and organic. During both years, the winter abundance of D. citri in groves under intermittent management was greater than in groves under other management regimes. These findings suggest that only aggressive management was able to control D. citri population during winter periods. In contrast, intermittent management was associated with higher D. citri populations. The abundance of D. citri during winter periods was correlated with high levels of potassium and phosphorous in citrus leaves. Geographic information system analysis indicated that D. citri abundance was higher when more than 20% of the surrounding landscape was urbanized. Citrus variety had a significant effect on D. citri abundance; we found almost no psyllids in grapefruit groves in winter seasons, D. citri had higher abundance in sweet orange varieties . Number of rows and grove area significantly affected D. citri distribution; we found fewer psyllids in larger sampled groves. The interaction between row orientation and elevation had a significant positive effect on numbers of D. citri found in groves . While the number of straight edges (shape score) alone had no significant effect on D. citri abundance, the interaction between management regime and grove shape had a significant effect on insect abundance. Management regime affected the shape of groves; specifically abandoned and intermittently managed groves exhibited fewer straight edges due to patches of open space breaking the edge of groves.
Asian Citrus Psyllid (ACP) is the vector of HLB which is primarily responsible for the disease spread. Controlling ACP invasion could potentially minimize the disease spread and crop loss. This project is aiming at developing an organo-silica based composite film (OSCF) barrier material which will serve as an ACP protectant once spray applied. Preliminary ACP trial results conducted on some of the earlier versions of OSCF materials were promising. The OSCF material was applied on 20 citrus plants on every 2-3 weeks and compared against an industrially available compound, ActigardTM (applied every 30 or 60 days interval). Comparable efficacy was observed with an infection rate as low as 68% for OSCF and 55 ‘ 75% for ActigardTM. Both the OSCF and ActigardTM materials reduced infection in comparison to the untreated control group which expressed an infection rate of 85%. In this reporting period, we have further optimized the OSCF materials which involved two different USEPA approved environmentally-friendly polymers and plant micronutrient based ionic cross-linkers. A total of six different version of OSCF materials have been evaluated for phytotoxicity using Vinca sp (an ornamental model plant which is highly susceptible to phytotoxicity) and Persian Lime. As expected, none of these OSCF materials showed any tissue damage. To address any concerns related to heat trapping potential of OSCF film material once spray applied, we have performed a series of studies on Persian Lime. Surround’, a recommended pest deterrent based on Kaolin Clay was used as a standard control. Four of these OSCF materials showed plant temperature comparable to Surround’ standard after application. Further optimization process will involve adjustment of OSCF spray rate. A couple of the most promising OSCF formulations will be delivered for greenhouse testing against ACPs by the end of this year.
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. Species composition in trap captures continues to be 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. CO1 genes continue to be sequenced from samples to contribute to a maximum likelihood phylogenetic analysis. Sampling from Silver Strand B9 block, Okaloacoochee Slough State Forest, and Charleston Grove continues; several months’ samples are currently being prepared to ship to FSU for analysis. Photographs are taken of specimens prior to extraction, so that new species collected in OK Slough can be distinguished from P. citrella by external morphological characters, also continues. Photographs of the specimens will help elucidate these characters in order to provide more accurate identifications. Vince Ficarrotta presented some of this work at the Lepidopterists’ Society Annual meeting in Park City, UT in June 2014. Vince’s assistant, David Plotkin, now a graduate student, continued work photographing moths through a microscope and generating COI sequence data. David will be presenting some of the leaf-miner work at the upcoming ESA meeting in Portland Oregon. Recently, an undergraduate senior student, Emma Roulette, was added to the project to focus further on dissecting specimens of the two unknown species of Phyllocnistis that are attracted to lures. She plans to dissect and illustrate the genitalia and compare them with type specimens in the National Museum of Natural History to verify their identities. This portion of the project was initially conducted by Q. Jia in 2013, but we are continuing this work and examining additional specimens. A manuscript describing trapping efficiencies was submitted to the Journal of Economic Entomology. We are currently incorporating reviewer comments. Work towards marking techniques continues. Due to inconsistencies with surface-applied powders and diet-incorporated dyes, we will evaluate marking CLM with Rb as a dietary supplement. We are currently evaluating application techniques for this substance including work towards an artificial diet. Once a calibration curve is established, releases will be made and captures compared to background Rb concentrations to establish recapture rates and spatial features of CLM populations.
We have completed our annual survey for the insecticidal response of field populations of the Asian citrus psyllid (ACP) to a panel of insecticides with various modes of action. In years 2009 through 2012, we began to see a drop in susceptibility to all major insecticide classes in a number of ACP populations across central and southern Florida. In the surveys conducted in 2013 and 2014, we found a reversal in the LD50 response of all field populations to levels that were not significantly different from the reference laboratory strain. However, most population dose-response curves were significantly different from the laboratory strain and between one another in both years, suggesting that there is divergence in insecticide response between populations that may have a genetic basis. These results indicate that certain populations of ACP may be primed for resistance development if insecticide use does not continue to be managed strategically and conservatively. We will continue to monitor the insecticidal response in ACP closely. This year we added two additional sites in the survey for select insecticides, and plan to add additional ones next year as well to further our understanding of the natural and induced response of ACP to insecticidal exposure. Overall, it appears that rotations have been effective in decreasing the incidence of insecticide resistance in populations of ACP.
Successful area-wide management of the Asian citrus psyllid (ACP) and Huanglongbing (HLB) requires effective control of the psyllid in abandoned citrus groves. Since ACP adults are highly mobile, they can disperse from abandoned to productive citrus groves and spread HLB. ACP is susceptible to Isaria fumosorosea (Ifr), a native entomopathogenic fungi. ‘Autodissemination’ is insect dispersal of a pathogen to members of its own population. Our project goals are to develop and field test an ‘autodissemination’ system for inoculating ACP with Ifr and use these infected psyllids to instigate epizootics that will rapidly reduce ACP populations in abandoned citrus groves. During the summer (June-July) of 2014, we collaborated with Paramount Citrus to conduct field trials in Hildago County with three of their managed groves and three adjacent abandoned groves. The first site was a managed Rio Red Grapefruit grove next to an abandoned Rio Red Grapefruit grove. The second site was a managed Valencia Orange grove next to an abandoned Rio Red Grapefruit grove. The third site was a managed Rio Red Grapefruit grove next to an abandoned Valencia Orange grove. At each site, we evaluated the impact of Ifr autodisseminators on ACP movement and infestation in plots of abandoned citrus trees and managed citrus trees. For treatment plots, we hung pairs of autodisseminators coated with Ifr spore formulation and equipped with citrus-blend lures in trees on the edge rows of the abandoned groves. The Ifr formulation was provided by Dr. Mark Jackson (USDA-ARS, Peoria, IL) and based on a south Texas isolate (Ifr 3581). For control plots, we hung autodisseminators with only citrus-blend lures. To monitor ACP movement and populations, we released up to 1200 ACP adults marked with fluorescent powder on abandoned trees every week for four consecutive weeks and hung ACP sticky traps on the managed trees directly across from the abandoned trees. We used different fluorescent colors to distinguish psyllids released in treatment plots from psyllids released in control plots. Every seven days during the first to fourth weeks of the trials, we replaced dispensers and inspected ACP traps. During the fifth week, we also ‘tap sampled’ managed trees and abandoned trees. Marked psyllids released within treatment plots were not recovered on either managed trees or abandoned trees. Marked psyllids released within control plots were recovered on abandoned trees that the psyllids were not released on; however, only unmarked psyllids were captured by ACP traps on managed trees. We are repeating our trials in the three Paramount Citrus groves during the fall and winter (October to December) of 2014. To improve recovery of marked psyllids, we will tap-sample trees during each week of these trials and / or increase ACP trap densities. To facilitate future scalability and commercial production, the autodisseminators used for our summer field trials were assembled from durable plastic components fabricated by AlphaScent Inc. To reduce the cost for end users, AlphaScent designed and provided us with cardboard versions of key autodisseminator components. During August and September of 2014, we field-tested the cardboard version and plastic version of our autodisseminator and found that the cardboard version was adversely affected by heavy rain. As a result, we will use only the plastic version for our fall and winter trials. By the second week of October 2014, we expect to receive sufficient autodisseminator components from AlphaScent for our fall and winter trials.
Work continues towards calculation of LD50 and LD80s for some commonly used insecticides for management of citrus pests in Florida for a susceptible CLM colony. These values are being compared to wild populations to assess for possible resistance to the tested insecticides. Larvae and adults from the susceptible colony were 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 the following insecticides: Actara 25WG, Agri-Mek 0.15EC, Cyazypyr, Danitol, Delegate WG, Dimethoate, Intrepid 2F and Micromite 80WGS. The following baselines for active specific ingredients against CLM larvae have been completed: 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= 0.314 ppm (CL95: 0.13-0.69), LD80=16.68 ppm (CL95: 6.14-78.56) (n = 1249; .2 = 13.17; d.f = 8; Heterogeneity = 1.65); 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=2.67 (CL95: 0.87-6.42), LD80= 93.75 (CL95: 33.89-489.58) ppm (n = 938; .2 = 10.69; d.f = 7; Heterogeneity = 1.53); Dimethoate (dimethoate) LD50=1.56 ppm (CL95: 0.15-56.31) and LD80=497.45 ppm (CL95: 22.27-0.37E+09) (n = 546; .2 = 13.07; d.f = 6; Heterogeneity = 2.18); 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). Because methoxyfenozide (Intrepid) is an insect growth regulator with apparently relatively low contact efficacy, its bioassay requires time to allow CLM larvae to develop through at least one instar. We are currently experimenting with modifications of the established bioassay to allow this time. LD50 and LD80 values for CLM adults with tested compounds are also currently being estimated. Preliminary results for Delegate (spinetoram) indicate LD50=2.84 (CL95: 0.77-73.74), LD80= 183.52 (CL95: 16.47-0.44E+07) ppm (n = 430; .2 = 6.30; d.f = 4; Heterogeneity = 1.58). Intrepid and Micromite LD50 and LD80 values are thus far inestimable (n= 703, n=381) as no treatment-associated mortalities have differed significantly from controls. This work continues. Since the last report, no incidents of putative resistance in field populations have been identified. However, we will continue to sample and test populations from commercial groves. A revision of the manuscript “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 is beng prepared for submission to the Journal of Economic Entomology
The overall goal of this three-year research project is to efficiently deliver antimicrobial molecules into citrus phloem against HLB bacteria. This quarter’s (from June to October 2014) research was focused on evaluating the effectiveness of 14 compounds in combinations and alone at various concentration against HLB bacterium. These compounds were formulated in O/W nanoemulsion for bark application. In addition, the enhanced-delivery field trial was also continued on evaluating efficiencies of these nano-formulations for combating HLB bacterium by bark application and gravity bag infusion. The results from greenhouse experiments showed that several treatments can suppress HLB bacterium with no phytotoxicity at 2 months after initial treatments, including Act, Va, Act+Va, penicillin and Zs. Eleven compounds have been treated by bark application in the potted plants, including Carv, Pcy, Pcy+Carv, Al(OH)3, Amp, KLS, SD, Proud, Act+Sdx, qui, and qui+Sdx. The qPCR analysis will be in the next quarter. In field trials, Pen and SDX in nanoemulsions can be effectively delivered into citrus phloem through bark application. After 2 months from initial treatment, HLB bacterial titer was reduced significantly, and no phytotoxicity appeared in these treated citrus trees. However, the bacterial titer increased a little after 6 months from initial treatment. Meanwhile, in gravity bag infusion field trials, the HLB bacterial titer was still lower after 6 months with Pen solution treatment , compared with other three treatments (combination of Pen and insecticide, SDX, and combination of SDX and insecticide). Due to the water insolubility of the insecticide, the compounds coupled with insecticide could not be effectively delivered into citrus phloem by gravity bag infusion. Therefore, the combination of compounds and insecticide treatment was replaced with the compounds alone. Insecticide was foliar- sprayed alone if necessary. In the future, all effective compounds in combination and alone against HLB bacterial will continue to be evaluated using a O/W nano-delivery system in the field. In the field trial, effectiveness of the compounds (Pen and SDX) against HLB bacterium by bark application and gravity bag infusion will be evaluated further, and agronomic performance of HLB-affected citrus treated with Pen and SDX will be investigated in next quarter.
This is the second year of a 3-year project. The overall goal is to evaluate the efficacy of 11 compounds individually and in combination for control of the HLB disease based on reducing titers of HLB bacterium in HLB-affected citrus. In this quarter (from June 2014 to October 2014), effectiveness of 11 compounds individually and in combination against HLB bacterial was continued to be evaluated by bark application using our nano-delivery system. After 4 months and 6 months from initial treatment, the HLB bacterial titer was significantly reduced by Act+VA and Amp treatment and no phytotoxicity appeared in treated trees, which was the same as our previous results in the last quarter. The formulation Act+VA will be applied in a field trial next quarter. Samples will be taken every two months for PCR analysis. Six new compounds have been tested by our graft-based test in the greenhouse. All tests will be analyzed next quarter. The temporal phytotoxicity in trees from the trunk-injection treatments have been restored to normal growth in this quarter. They will be treated using lower concentrations of compounds in the next quarter.
This is the first year of a three-year project. The overall objective was focused on determining the optimum combination of chemotherapy, thermotherapy, and nutrient therapy that can registered for control of HLB in the field. In this quarter (June 2014- October 2014), eighty-one HLB-affected citrus trees with diameters of 10-15 cm were treated once every one month by chemical treatments (Carv and Pcy) formulated with O/W nanoemulsion using both bark application and trunk-injection by gravity bag infusion, using a randomized block complete design. After 2 months and 4 months from initial treatment, the HLB bacterial titer was still high in the treated citrus trees while no phytotoxicity appeared in the trees. In next quarter, more HLB-affected citrus trees will be treated using the other effective compounds and coupled with heat-treatment and site-fertilization. The agronomic performance of treated trees will be investigated.
The large-scale validation of citrus leafminer (CLM) disruption with the ISCA DCEPT CLM technology has continued. Our group has continued in assisting of collecting data to monitor the efficacy of the product during the project. We have been assisting in monitoring certain areas of the ca. 3,000 acres that were treated at three locations in southeast and southwest Florida. We assisted with collection of efficacy data by trapping CLM males through September with pheromone traps as a surrogate measure of mating disruption. Thus far, it appears that the product has been effective.
For the second year, we compared the densities of Asian citrus psyllid (ACP) on reset trees planted in solid set plantings (all trees planted within the last two years) versus the densities of psyllids on resets (trees planted within the last two years to replace mature trees) present within mature groves. This year, the experiment was conducted in three groves of three different citrus varieties (Hamelin, Grapefruit and Valencia). More psyllids were found in the solid set plantings as compared to the resets within mature groves. These data confirm last year’s results. Overall, this study indicates that solid set re-plantings will require more intense vector management than resets within mature groves. Regarding laboratory experiments, we investigated the effect of abiotic and biotic factors on host acceptance of the ACP. We know that ACP are attracted by HLB-infected citrus, and that this attraction is achieved through the emission of Methyl salicylate (MeSA) by the plant. We now investigated how abiotic factors such as drought stress and time since Clas infection may affect the behavior of ACP and the release of volatiles by citrus plants. We found that HLB-infected plants that were more attractive than non-infected plants during olfactometer assays in control conditions, were not more attractive as compared with non-infected plants if they were submitted to drought stress. Leaf volatile collections and GC-MS analyses revealed that HLB-infected plants under drought stress were characterized by a decrease of MeSA emitted, compared to HLB-infected plants under control conditions. In our experiments, drought stress consisted of decreasing soil moisture from 95% to 35% of the water carrying capacity of the pot. We also found that plants infected with the Clas pathogen for over 18 months (‘old infection’) were no longer attractive in olfactometer assays as compared with control plants; whereas, citrus plants infected for 12 months (‘young infection’) were strongly attractive to ACP as compared to control plants. This was confirmed by settling experiments showing that ACP preferred to settle on infected versus non-infected plants, but also preferred settling on young infected plants rather than on old infected plants. Again, these behavioral results were associated with a reduction in the amount of MeSA emitted by plants infected with the pathogen for more than 18 months. We are currently measuring the gene expression of methyle-transferase in citrus plants depending of the time since infection, to investigate if this reduction in MeSA emission is associated with a reduction of the mRNA abundance of the salicylate methyltranferase gene (CsSAMT) that we have identified in the citrus genome.
Because of the natural disease cycle of citrus canker, field trials for citrus canker were initiated before the official start of the proposal to adequately determine efficacy. Trials are well underway in a grapefruit block that is a well established site for canker management trials. As the project was just initiated at the beginning of June, we are in the very early stages of a field trial for HLB. A grove of 3 year old infected Valencia has been identified, scouted for disease rating and sampled for starting Liberibacter titer. The first application has been applied testing multiple application methods compared to untreated controls in a randomized trial.
The canker field trial of Zinkicide compared to industry standard management is ongoing with preliminary disease assessment performed and the data currently being analyzed. Final disease assessment will not be done until all canker-induced fruit drop has occurred. The HLB trial in young Valencia trees continues with regular treatment and disease assessment. The timing of first treatment and the trees flush cycle make it difficult to determine efficacy this soon after initial treatment. Sampling and measurement of the initial titer response to Zinkicide application in these trees is currently underway. The ability to detect any symptom or tree recovery differences are not expected until strong symptom expression in the fall or the spring flush, respectively.
This project terminated 30 August, 2014 and this constitutes the final report for this project. During the course of the project, we developed reliable, replicable and efficient methods for recording from the antennae of live, whole Asian citrus psyllids using a specially-designed microscope stage, an immobilization technique to secure individual psyllids, micromanipulators to place glass capillary electrodes and a Faraday cage to eliminate noise. The use of live whole psyllids is superior to using detached antennae or antennae-head preparations because the intact psyllid remains alive for long periods, sometimes exceeding 24 h and therefore allows for extended use in GC-EAD and EAG studies, and the signal does not degrade over time as commonly occurs in antennae-only preparations as the antenna dies. We then proceeded to characterize the volatile profiles of Citrus and Poncirus genotypes and to test the major and minor organic volatile compounds produced by each. Very few compounds elicited antennal responses. By far, the largest and only consistent antennal responses were obtained from degradation products of E-B-ocimene and citral, namely formic and acetic acids. This serendipitous discovery demonstrated how oxidation of volatile compounds occurs spontaneously in the presence of oxygen resulting in the rapid degradation of ocimene and citral and release of acetic and formic acids. Asian citrus psyllid antennae are highly responsive to these two acids and we were able to demonstrate a behavior response as well. Psyllids more readily probed a wax substrate containing the acids compared with wax substrate alone. Degradation of volatile organic compounds (VOCs) is common in nature and results in the release of acetic and formic acids into the atmosphere. In one study, acetic acid comprised 8.4% of VOCs above a citrus grove. Degradation of VOCs can produce acetic and formic acids at rates 200 to 300% higher than the precursor compounds. In our studies, ACP adults probed a substrate impregnated with formic and acetic acids more frequently compared with blank controls and other citrus VOCs. ACP orientation to host plants may involve acetic and formic acid alone or in blends with other compounds. We will continue to study the behavioral response and orientation of Asian citrus psyllid to these compounds alone and in blends containing other citrus compounds that we have shown to be weakly active by electroantennography.
Feeding behavior of ACP is being studied on P. trifoliata and trifoliate hybrids using an electronic penetration graph (EPG), and choice and no-choice assays to study host selection and probing behavior. We continue to record ACP feeding by EPG and are beginning to describe differences in the number and duration of ingestion waveforms on resistant and susceptible genotypes. We have the capacity to simultaneously record the feeding behavior of 8 ACP using a Giga8 DC monitor and anticipate acquisition of a 4-channel AC-DC monitor that provides improved waveform resolution. A bottleneck to both methods is the time-consuming and onerous analysis of the waveforms. We propose to automate analysis of EPG ingestion waveforms by means of unsupervised learning technique, Hidden Markov Models, to automate temporal pattern recognition of the digital output. In collaboration with PhD candidate Denis Willett in the Stelinski lab at UF, IFAS, Lake Alfred, we are generating EPG digital output files from resistant and susceptible genotypes that have been manually scored for waveform type, a process that typically requires >8h to process one 21-hour run by one psyllid. By comparing automated recognition with manually scored runs, and subsequent confirmation of automated determination by visual inspection of machine-identified waveforms, we will validate the model for multiple psyllids on resistant and susceptible citrus, and for multiple plant species to confirm that the model can function across plant taxa. In addition to contributing to identification of resistant accessions and possible mechanisms of resistant, the addition of rapid, real-time analysis of waveforms will contribute to rapid screening of progenies generated by traditional or novel methods. Of particular interest is the application of EPG to describe the effect of stylet sheath inhibitors on ACP feeding behavior. Greenhouse trials continue to confirm resistance to oviposition. Current tests address the hypothesis that a lack of stimulatory volatiles in resistant P. trifoliata contributes to resistance to oviposition. We will attempt to show that oviposition on resistant P. trifoliata genotypes can be increased in the presence of volatiles from susceptible citrus, e.g., C. macrophylla.
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