The overall goal is to determine the effect of antimicrobials on ACP biology, vector capacity, and behavior. Objective 1: Quantify the effect of citrus antimicrobials on vector fitness. As previously reported, this objective has been completed. Data analysis is underway and a manuscript is being prepared for publication. Objective 2: Determine the effect of antimicrobials on Las transmission. This objective will determine whether ACP feeding on antibiotic treated infected citrus plants will be less likely to transmit Las. Experiment 2.1 Acquisition assays. Several more replicates of these experiments are needed to complete this experiment. These replicates be conducted begining July 2020.Experiment 2.2. Field study. Eight-year-old CLas-infected citrus trees have received six foliar applications (May 2019 May 2020) of streptomycin, oxytetracycline (Treatments), or receive no antimicrobials (Control). Ten CLas-free insects per plant from a laboratory colony were caged on young leaves (flush) of treatment and control trees to analyze ACP survival, CLas-acquisition in ACP P1 and F1 progeny, the total trees sampled consisted of 5 individual trees per treatment. In microcentrifuge tubes containing 1 mL of 80% ethanol, ACP adults were collected individually and then stored at -20°C for subsequent CLas detection using real-time PCR. The survival of ACP and CLas-acquisition were replicated twice from June 2019 to March 2020. However, the UF/IFAS Citrus Research and Education Center (CREC) closed on March 23rd due to COVID-19, limiting our access to the center and equipment. The second sampling (July 2019), the third (September 2019), fourth (October 2019), the fifth (November 2019), the sixth (January 2020), and the seventh (March 2020) replicates were collected and are being processed to analyze the CLas-infection rate.Objective 3: Determine the effect of antimicrobials on plant response and associated ACP behavior. Experiment 3.1 Host choice bioassays. We compared the responses of Asian citrus psyllid (ACP) adults to the odor sources from sweet orange seedings that had been treated with Fireline (oxytetracycline HCL) or Firewall (streptomycin sulfate) versus a water (blank control). All treatments were applied to seedings as foliar sprays at proportionally adjusted label rates. Each treatment was compared relative to an untreated control by placing plants into glass chambers with air throughput delivered into a psyllid two-choice (T-maze) behavioral assay. In this manner, ACP were tested to determine their response to treated versus control plants. Separate experiments were conducted with uninfected and Las-infected plants. Four replicate plants were tested for the uninfected plants and three replicate plants were completed for Las-infected plants plants. The response of 30 ACP adults were evaluated with the T-maze olfactometer per replicate to determine whether antimicrobials affect ACP preferences for antimicrobial-treated versus untreated plants. The majority (> 85 %) of ACP responded to the odors of either uninfected or Las-infected citrus plants compared with a blank control. Also, more ACP were attracted to the odors from Las-infected plants than uninfected plants. These resuls confirmed our previous findings and verified that our experimental set up was working properly. When comparing seedlings that were treated with antimicrobial treatments versus untreated plants, there was no difference in ACP attraction to Fireline-treated versus untreated plants, whether or not they were infected with CLas. Similarly, there was no difference in response of ACP adults to Firewall-treated versus control plants when tests compared all uninfected plants and when tests compared all Las-infected plants. These initial results would suggest that treating plants with antimicrobials should not recruit psyllids from a distance, using odors as cues, to those treated plants and thus should not create greater psyllid infestation on treated plants. Followup investigations are currently under way to determine if psyllid host preference between antimicrobial treated versus untreated plants differs when psyllids are allowed to choose between treated and untreated plants in open air cage experiments. Although the laboratory olfactometer experiments would suggest that antimicrobial treatments did not affect the odor of the plants to indirectly affect psyllid preference, psyllids also use several other senses, in addition to smell, when selecting hosts for feeding and egg-laying. In fact, in recent years, we have come to understand the vision and taste are in many cases more important than smell for psyllids to select an appropiate host. We are evaluating the same treatments as above, comparing psyllid response to antimicrobial-treated and untreated plants, in custom made cage assay currently. Our initial tests in these cages are being conducted with all infected plants, based on the initial olfactometer studies and since the treatments are in practice intended for treated CLas-infected plants.
Objective 1: Quantify the effect of citrus antimicrobials on vector fitness. We expect to observe reduced longevity, reduced fecundity, and longer development times among ACP exposed to antimicrobial treatments as compared with unexposed ACP. We previously reported (March 2019) on the effect of dietary antibiotic treatments on ACP survival (Obj. 1.1). During the past quarter, we evaluated the fecundity and fertility of ACP exposed to antibiotic treatments. The reproductive output of D. citri exposed to oxytetracycline or streptomycin treated plants was evaluated in a greenhouse assay. Five-month-old Citrus plants reared in an insect free greenhouse without exposure to insecticide received foliar applications of streptomycin (FireWall 50WP (Agrosource), or oxytetracycline (Agrosource,) or 1.0 mg ml-1 imidacloprid (Bayer CropScience LP), and water for a control. Females were allowed to oviposit on citrus plants with new growth (flush) over a 25-d period. Total eggs laid (fecundity) were counted under a stereoscope each 5-d period, then transferred to newly treated plants with flush to encourage oviposition. To determine if compounds containing oxytetracycline or streptomycin had an effect on the number of hatched eggs (fertility), plants were maintained as previously described for 6-d after adult removal. The total number of nymphs on plants were counted every 3-d under a stereoscope and recorded. This experiment is currently wrapping up and data analysis is underway. Objective 2. Determine the effect of antimicrobials on Las transmission. We expect that ACP feeding on antibiotic treated infected citrus plants will be less likely to transmit Las. We initiated field experiments to evaluate the hypothesis that ACP will be less capable of transmitting Las after feeding on antimicrobials, because trees treated with antimicrobials are more likely to have lower Las titers for acquisition (Objective 2.2). An experiment was initiated in mature, infected citrus trees located in a research at the CREC to determine whether field applications of foliar antimicrobials are also capable of suppressing acquisition of Las. Trees will be received an initial treatment with streptomycin, oxytetracycline, or receive no antimicrobial treatment. Ten insects from uninfected laboratory cultures were caged on young leaf growth (flush) of antimicrobial-treated or untreated infected trees using mesh bags for oviposition. Treatment were be replicated 10 times on individual trees. Survival of females is be monitored for two weeks. Females will be collected at the end of this quarter (late June) and preserved in 80% ethanol at -20°C for subsequent analysis and CLas detection. Egg clutches will remain on trees enclosed in mesh sleeves. After the nymphs reach the adult stage (approximately 15 days), adult psyllids and three leaves exposed to the psyllids will be collected for analysis.
The objective of this study was to develop an insecticide rotation with different modes of action as a resistance management strategy for Asian citrus psyllid (ACP) in Florida. We selected two large scale experimental sites in two citrus groves in central Florida. One site was in Lake Alfred (Polk County) on CREC property planted with the cultivar Hamlin. The second site was in Wachula (Hardee County) and was planted with Valencia selections. The Lake Alfred site has trees that are bearing fruit. At the Wauchula site, the trees are non-bearing. At each location, three rotation schemes were established ion 4.9 acres each in Lake Alfred and on 4.2 acres each in Wachula. For sampling, we established 6 replicates per treatment in Lake Alfred and 4 replicates in Wachula.
The insecticides were applied in two rotational schemes with five different modes of action. The third treatment was to apply different neonicotinoids with no change in the mode of action. Rotation A consisted of dimethoate followed by cyantraniliprole, fenpropathrin, diflubenzuron and imidacloprid. Rotation B consisted of fenpropathrin followed by dimethoate, imidacloprid and diflubenzuron. No rotation consisted of thiamethoxam, clothianidin, thiamethoxam, imidacloprid and clothianidin. At this point we have completed two applications at both locations.
Prior to the insecticide applications, the plots were monitored for ACP adults using tap sampling. Weekly monitoring was initiated on March 20, 2019 in Wachula and April 1, 2019 in Lake Alfred. When the average number of adults per tap reached 0.4 insects the appropriate insecticide was applied using an airblast sprayer by the cooperator. Also, ACP adults were collected and a baseline insecticide susceptibility was determined and compared with our susceptible laboratory population using a leaf dip assay. Five to six concentrations of each insecticide was tested and replicated 5 times. We found low to moderate resistance for thiamethoxam (RR > 20), imdaclorprid (RR > 10), clothianidin (RR > 10) and dimethoate (RR > 10). We found very low resistance to the other insecticides in the field populations (RR < 5). Finally, before application, morphological measurements on adult ACP were made At least 50 individuals were measured. Body length, abdominal length, wing length, femur length and head width were measured for laboratory susceptible cultures and for both field populations. The results indicated that the abdominal length, wing length and femur length was greater in the laboratory population compared with both field populations. The current investigation is ongoing. It will continue to monitor ACP management and resistance among the treatments and locations described above. Our goal is to find a more refined method of effective insecticide resistance management of ACP that also shows highest efficacy. Our newly developed protocols on suppression of ACP populations by stabilizing or reducing resistance will be communicated with growers when the results are properly verified experimentally.
The current study was conducted as a risk assessment of potential evolution of insecticide resistance phenotypes and genotypes in Asian citrus psyllid (ACP) populations to fenpropathrin as a result of laboratory selection. We also investigated cross resistance of ACP to fenpropathrin with to other insecticide modes of action. The obtained results should contribute to development improved resistance management strategies.
First, assays and selection were performed with adults from a field-collected strain from Wauchula, FL on July 15, 2018 (WF). The bottle bioassay technique was used to determine susceptibility levels of adults of WF strain ACP to fenpropathrin. The chemical residues were achieved by pipetting 200 µL of acetone into a bottle and by rotating the bottle until the acetone evaporated. Subsequently, 5- 10 ACP adults were aspirated and transferred to the treated bottle. The LC50 was determined. Surviving individuals were reared on plants for eight generations. For each generation, adults of the WF strain were exposed to the LC50 concentration. We determined the risk assessment of ACP phenotypical resistance to fenpropathrin. After eight selected generations, the realized heritability of resistance (h2) to fenpropathrin was determined. The estimated h2 to fenpropathrin was 0.10 by the end of selection. The h2 of fenpropathrin resistance was 0.17 and 0.44 during the first and the second rounds of selection, respectively. The h2 values obtained at second round of selection are very high and could indicated a high level of risk in the field population for development of resistance to fenpropathrin. The results also suggest that a brief selection experiment may be sufficient to detect the potential for the development of resistance.
Second, we investigated level of resistance to pyrethoids in laboratory and investigated the possible mechanism involved cross resistance to two relatively commonly used insecticides, dimethoate and imidacloprid. Results indicated that there was no evidence of high cross resistance to imidaclorpid (RR = 1.54) and dimethoate (RR = 4.36) for the WF fenpropathrin-resistant strain. At this point, rotation of fenpropathrin, dimethoate and imidacloprid should not increase insecticide resistance. These results are particular important for verifying the effectiveness of the rotation schedules we are putting into practice in the field.
Third, we investigated pyrethroid resistance levesl and the associated molecular mechanisms in fenpropathrin resistant strain of ACP. The relative gene expression of six cytochrome P450s (CYP6A1, CYP6A2-1, CYP6A13, CYP6A14, CYP6J1 and CYP6K1) and four glutathione S-transferases (GST1, GST2B, GST3 and GST4) were quantified in the selected population, and compared with the laboratory susceptible population. qRT-PCR analysis showed that expression of CYP6A2-1 had significantly increased in the selected population relative to the laboratory susceptible population. Our results indicated that increased target insensitivity and cytochrome P450 metabolic detoxification could be mechanisms responsible for the ACP resistance to the pyrethroid fenpropathrin.
The results further confirmed that the multiple resistance mechanism following artificial selection on the field strain did not confer significant cross resistance to insecticides with other modes of action. Thus, we are able to recommend, based on a large body of evidence, that fenpropathrin, imidacloprid and dimethoate can be effectively rotated in sequence as an effective resistance management protocol for ACP.
The purpose is to evaluate the control effect of bactericides via trunk injection. This proposal addresses the following CRDF CPDC-18 Research Priorities: 1A, 1C, and 1D. To achieve the goal of the research, we are conducting the following objectives:Objective 1. To illustrate whether application of bactericides via trunk injection could efficiently manage citrus HLB and how bactericides via trunk injection affects Las and HLB diseased trees.Three field trials have begun to investigate how the application of bactericides via trunk injection affects citrus growth, production, HLB symptom development, and Las population in different aged trees at different levels of HLB disease severity. We evaluated the inhibitory activity of OTC against Las in greenhouse and field experiments. Citrus trees were trunk-injected with OTC, and leaves were inspected for Las populations and OTC residues using qPCR and HPLC assays respectively, at various times after OTC treatment. We have acquired data about the MBC of OTC in planta. A manuscript is under preparation. Objective 2. To examine the dynamics and residues of bactericide injected into citrus and systemic movement within the vascular system of trees and characterize the degradation metabolites of bactericides in citrus. A field trial has begun to determine the concentrations of bactericides in leaf, stem, root, flower, and fruit using HPLC at the following time points: 2, 7, 14, 28 days, 2, 4, 6, 8, 10, 12 months after injection at different doses. The leaf samples are being collected at different time points for testing. Objective 3. To determine whether trunk injection of bactericides could decrease Las acquisition by Asian citrus psyllids (ACP).In this objective, we will determine whether trunk injection of bactericides at three different doses could decrease Las acquisition by ACP in greenhouse and in the field. We are conducting the experiment right now. Objective 4. To monitor resistance development in Las against bactericides and evaluate potential side effects of trunk injection of bactericides. Las-specific primers were designed to target the putative binding sites of OTC in 16SrRNA gene of Las. Plant genomic DNA was extracted from citrus trees received OTC injection for three years. PCR were performed with the primers and DNA samples, and the products were purified and subjected to DNA sequencing. No mutation was identified yet. We will continue to monitor the resistance development against OTC and Streptomycin. We have collected more samples from multiple citrus groves.
Use of RNAi delivered by Citrus Tristeza Virus Viral Vector to control the Asian Citrus Psyllid 2019 First Quarter Report1. In February, sentinel trees were tested using ELISA to detect the presence of CTV and gel electrophoresis and rtPCR to detect the presence and stability of CTVvv-RNAi.2. A repetition the experiment began in early March and will continue for a period of five weeks. The protocol established is as follows:a. Selected trees were scouted for flush or pruned to induce flushing.b. Areas of flush were bagged and inoculated with 20 parent ACP.c. Parent ACP were removed from bagged flush after two weeks.d. First generation ACP were counted two weeks after removal of parent ACP.e. The presence of native ACP life stages also documented.3. Aphid scouting continues on a biweekly basis. The presence of brown aphid has not been detected.
April 2019 The objectives for this proposal are 1) Conduct ground and aerial applications of fungicides to determine the efficacy and economics of fungicide treatments; 2) Determine if Luna Sensation has enough systemic activity to protect flowers from before they fully develop and open; 3) Determine if the period flowering of trees affected by huanglongbing can be narrowed to eliminate the offseason bloom that contributes to the PFD inoculum increase in groves. In 2019, a field trial was set up and treatments were applied in a Valencia grove in Ft. Meade. There were few predicted infection events this year and only one application was made based on the PFD-FAD or CAS model predictions. Button counts will be collected soon now that flowering has finished. Trees with flowers at different stages were treated in the field with Luna Sensation and branches were collected for inoculation in the lab. The inoculations were done but the petals fell off the open flowers rapidly so it will be difficult to determine how well the open flowers were protected. The third year of bloom synchronization was undertaken in 2019. All the foliar PGR applications were completed. The initial preflowering button counts were done from January to March. In January, the flower counts were started and are still underway. Harvest of the ‘Valencia’ trees was anticipated in March, but, the grower collaborators have delayed harvest until April. Navels were harvested and we are currently in the process of analyzing and interpreting the ‘Navel’ harvest data. Trials were done in 2 sites on ‘Valencia’ trees in Ft. Meade and Dundee, FL to compare the model outputs for predicting infection events. In Ft. Mead, one application per the CAS model was applied, one application per the PFD-FAD model, and three weekly applications. In Dundee, there were no applications with the CAS, one application per the PFD-FAD model, and 2 weekly applications. No infected flowers were found in either grove on the major blooms. Button and fruit counts will be taken later in the year. Finally, a manuscript is in preparation for the Citrus Advisory System (CAS). It is approximately 50% completed and is hoped to be fully completed by the end of May.
April 2019 The objectives for this proposal are 1) Conduct field trials of new products and fungicide programs for PFD management as well as validation trials for the Citrus Advisory System (CAS); 2) Investigate the reasons for the movement of Postbloom fruit drop (PFD) to new areas and recent major outbreaks; 3) Evaluate methods for initial inoculum reduction on leaves so that early fungicide applications could be more effective and identify the constituents of the flower extracts using omics techniques. Objective 1 was covered by project 16-010C and activities are reported there. Contact has been made with the USDA to start on the experiments with the wind tunnel that is located at the ARS facilities in Ft. Pierce. Unfortunately, we have not already started because authorizations were delayed by the federal government shut down. The paperwork to get the permissions has been filed and we are waiting for finalization on that front.In the meantime, work on modelling of leaf wetness to better predict PFD outbreaks in Florida has been started. Five FAWN weather stations were selected for this work and are equiped with leaf wetness sensors. The recorded data was compared to the output of four leaf wetness estimation models, singly and in combination. The most accurate models were considered for modifying the citrus advisory system (CAS). Further analysis to look at the number of recommended sprays and model accuracy are planned. Experiments to assess the effect of available fungicides on the secondary conidiation of C. acutatum on citrus leaves. In the intial experiment, pyraclostrobin was used and no difference was seen in the secondary conidiation regardless if sugar or water were used for stimulation of conidia. In subsequent experiments, ferbam was used instead. To stimulate sporulation, leaves were exposed to sterile water or 2.5% sucrose solutions with or without ferbam. Once the treatments were complete, the leaves were coated with nail polish and the conidia and appressoria stripped from the leaf surface and counted. Ferbam affected the viability of the spores but not the number produced. It also signficantly reduced the number of appressoria. A similar technique will be used to evaluate the effect of flower extracts on conidiation to determine whchi extracts to investigate. Flowers were collected and extracted using water, methanol and ethyl acetate. The yields of all the extracts have been calculated as the future reference. All the extracts have been dried for testing their antifungal effects. Once we receive the antifungal effects of the extracts, we will start the composition analyses.
The goal of this project is to develop integrated, sustainable and economically viable psyllid management programs for conventional and organic citrus producers and improved yields. Conventional growers are facing issues of unsatisfactory control, insecticide resistance, increased costs, and groves without beneficial insects. This is not only impacting management of ACP-HLB complex but also other pests which colonize citrus and their management. In our studies some organic programs have shown potential to provide significant reductions in psyllid populations and produce yields comparable to conventional program. These organic programs used insecticides such as Azera, Aza-direct, Entrust, Grandevo, Microthiol, Pyganic, Surround, Venerate and in combination with HMO FL 435-66. Naturally occurring populations of the predators such as spiders, lacewings and ladybeetles and parasitoid Tamatixia radiata released on regular basis in these programs were additional factors which contributed to psyllid control and yields. This project is focused on developing IPM programs for conventional and organic growers to control ACP. The proposed IPM program for conventional growers will include synthetic and organic insecticides including biopesticide products to complement conventional products. These rotations will help reduce the risk of insecticide resistance which results from continuous exposure to insecticides of similar modes of action, and thus reduce the need for more applications and cost. The IPM program for organic producers will include organic insecticides including biopesticides and oils. It will benefit conventional growers by demonstrating the strengths and limitations of such programs. The conventional only and HMO only programs will serve as comparison against IPM programs with more tools available from both production systems to evaluate costs and benefits. These programs will also provide opportunities for beneficial insects to enhance biological control, which has been diminishing through the constant use of the hard chemistry insecticides. Naturally occurring populations of predators and parasitoids and additional introductions of commercial predators and mass reared Tamarixia will further strengthen these program. Sampling of the experimental block designated for IPM programs in the Gulf region revealed psyllid populations at 0.3 adults per tap sample. Spiders and lacewings averaged at 0.14 and 0.05 per tap sample, respectively. Spray applications in different IPM programs will begin after the block is harvested in April. Positions for the postdoc and temporary assistant were announced and applicants being evaluated.
The future of the Florida citrus industry depends on successful replacement of the present bearing tree inventory with new plantings. This would be a relatively routine process were it not for the ubiquitous presence of huanglongbing (HLB) and its Asian citrus psyllid (ACP) vector. Bringing young trees into production is a challenge in the presence of HLB. The key to protecting young trees from HLB is effective and consistent psyllid control. ACP populations have been steadily increasing statewide for the last few years, increasing pressure on young plantings. Wide spread resistance to the neonicotinoid insecticides, formally pillars of ACP management on young trees, is the other major factor affecting control on young trees. Reduced effectiveness of insecticides to adequately control ACP in young trees is a serious problem requiring new strategies to protect vulnerable young trees from HLB. Reflective mulches have long been used in vegetable production to deter dayflying pests like aphids and thrips, presumably through disruption of horizontal orientation by reflecting sunlight to the bottom of the eye rather than the top. Flight is disrupted when the insect sees sky below and becomes disoriented. Some recent trials have shown significant effectiveness of UV reflective mulches in reducing the incidence of ACP and HLB. The mulch planting system besides insect repellence may provide some additional advantages such as efficient use of water and fertilizer, reduced use of herbicides by virtue of weed suppression, reduced psyllid resistance to insecticides and increased soil and canopy temperature. However, all these variables need to be better quantified and evaluated on different soils. The mulch system will also allow us to test a strategy of more efficient ACP control than previously possible by using irrigation to synchronize flush. It would thus be possible to start psyllid control with a new flush at bud break with no egg or nymph stages and only adult ACP. This is analogous to the situation during tree dormancy and targetting psyllid adults in winter prior to spring growth which we already demonstrated. Evaluation of young tree production on reflective mulches is being conducted in Gulf, Ridge, and River regions of the state. Study locations and plants suitable for all three locations are being prepared. Evaluations include 1) assessment of effects of UV reflective mulch on ACP control, HLB incidence and severity, tree growth and ultimately fruit production, and 2) assessment of ACP control and resistance to insecticides in response to flush synchronization for ACP control using mulch/drip irrigation system on three different soils types. Economic analysis summarizing 3-year and projected costs and benefits of mulch system with and without flush control will be conducted. Positions for the postdoc and temporary assistant were announced and applicants being evaluated.
The long term field trial continues with weekly psyllid counts and quarterly CLas infection testing. Treatments continue to have similar effects on ACP counts, however, the differences between red and white kaolin in CLas infection no longer persist. The two kaolin treatments, however, now have approximately 50% infection, while the control and the foliar insecticide treatment, now have nearly 100% infection. Plants in both of the kaolin treatments continue to show higher growth rates than the other two treatments. The red treatment has the highest growth rate, trunk cross-sectional area, and canopy volume. Kaolin treated trees that are infected grow more than untreated-infected trees, but less than treated uninfected trees. The Master’s student funded by this project completed his thesis and defended it successfully. For this work he completed 3 experiments, and is now assisting in implementation of 2 more repetitions of these to ensure that results are consistent. We now have substantial evidence that both kaolin treatments improve growth, while the red improves water use efficiency. In potted studies whole-plant water loss was dramatically reduced through application of the red particle film. Despite this, we know that growth is increased in the field. We submitted an additional IP disclosure to include the possibility of manipulating whole plant water use through the color of the particle film. We are still exploring options for commercialization. The current experimental repetitions use the earlier designs, but they use adjusted rates so that all the particle films allow the same quantity of light, so that only light quality is different.
A major focus of this research is to screen combinatorial small molecule libraries for activity against the Asian citrus psyllid using feeding bioassays. The first library that will be screened is a peptide library synthesized by Torrey Pines Institute for Molecular Studies (TPIMS). The TPIMS researchers have finished the first round of peptide synthesis and purification. The first peptides are designed to serve as negative controls for experiments involving the Torrey Pines combinatorial libraries. In order to design a negative control, the concept of a random sequence was employed. The best approach to making a negative control when nothing is known about the system is to make randomly selected sequences. 20 different peptides were selected for synthesis. While the sequences chosen were random, 10 of the peptides were biased to contain residues that made the final peptides more likely to be soluble in water. Of the 10 water soluble biased peptides 3 were purified and provided to USDA for testing. The USDA researchers have initiated feeding trials with these peptides and are optimizing concentration ranges. Also, specific proteins within the psyllid saliva secreted during feeding have been identified using the USDA bioassay to collect salivary components. This has provided insight into how the psyllids target the citrus phloem as their food source. This information is being used to develop a screen for inhibitors of this enzyme as a means of blocking the feeding process. Psyllid colony maintenance has been ramped up to provide enough adult psyllids for the bioassay survey and experiments have been initiated to optimize the diet for feeding immature psyllid life stages. Optimizing the diets for the immature stages will allow testing of inhibitors for activity across all stages of the psyllid that feed in citrus.
Our project is examining phloem gene expression changes in response to CLas infection in HLB-susceptible sweet orange and HLB-resistant Poncirus and Carrizo (a sweet orange – Poncirus cross). We are using a recently developed methodology for woody crops that allows gene expression profiling of phloem tissues. The method leverages a translating ribosome affinity purification strategy (called TRAP) to isolate and characterize translating mRNAs from phloem specific tissues. Our approach is unlike other gene expression profiling methods in that it only samples gene transcripts that are actively being transcribed into proteins and is thus a better representation of active cellular processes than total cellular mRNA. Sweet orange, and HLB-resistant Poncirus and Carrizo (sweet orange x Poncirus) will be transformed to express the tagged ribosomal proteins under the control of characterized phloem-specific promoters; tagged ribosomal proteins under control of the nearly ubiquitous CaMV 35S promoter will be used as a control. Transgenic plants will be exposed to CLas+ or CLas- ACP and leaves sampled 1, 2, 4, 8, and 12 weeks later. Ribosome-associated mRNA will be sequenced and analyzed to identify differentially regulated genes at each time point and between each citrus cultivar. Comparisons of susceptible and resistant phloem cell responses to CLas will identify those genes that are differentially regulated during these host responses. Identified genes will represent unique phloem specific targets for CRISPR knockout or overexpression, permitting the generation of HLB-resistant variants of major citrus cultivars. This is the 1st year, 1st quarter progress report; our grant started December 1, 2018. In the last three months, we have processed all the paperwork needed to establish the grant and begin spending funds at ARS. We have identified a qualified and interested post-doctoral researcher, Dr. Tamara D. Collum, who we will be hiring with grant funding. However, all ARS hiring actions, even those using soft funds, are currently on hold at the Department of Agriculture level. Now that the department has a full-year budget, we hope this hold will be lifted shortly so we can bring Tami on board in the next couple weeks. Objective 1 (development of transgenic constructs) is close to completion and work has begun in the Stover lab on objective 2 (production of transgenic citrus lines). For objective 6 (Additional Approach: Phloem limited citrus tristeza virus vectors will be used to express the His-FLAG-tagged ribosomal protein in healthy and CLas infected citrus) inserts have been assembled and sent to Dr. Dawson’s lab for inclusion in CTV vectors and subsequent introduction into citrus.
The objective of this study is to determine how different rotation schedules of commonly used insecticides with different modes of action such as dimethoate (acetylcholinesterase inhibitor), imidacloprid and thiamethoxam (acetylcholine receptor), diflubenzuron (inhibitor of chitin biosynthesis), abamectin 3 % + thiamethoxam 13.9 % (chloride channel allosteric modulator and acetylcholine receptor), fenpropathrin (sodium channel modulator), and cyantraniliprole (ryanodine receptor modulator) may impact the level of insecticide resistance Asian citrus psyllid (ACP) populations. An associated goal is to determine if ACP populations can be managed to reduce resistance in those populations where it already exists to a particular insecticide under rotations. Finally, the rotations must be effective in managing existing ACP populations to acceptable grower standards. We have selected two locations where resistance to neonicotinoid insecticides has been demonstrated and is known to exist. At each location, three rotational schemes of insecticides for ACP management will be established in 5 acre plots in Lake Alfred and 4.2 acre plots in Wauchula. The trees are 1-2 years old Hamlin trees with a variety of rootstocks. We are collecting adult ACP currently from these locations to determine their baseline insecticide resistance levels compared with a susceptible laboratory population of ACP using a leaf dip assay. Field populations have been collected from the Wauchula site and bioassays are underway currently. We will be collecting psyllids from the Lake Alfred site shortly. We will use commercial formulations of dimethoate, fenpropathrin, imidacloprid and cyantraniliprole to determine baseline resistance levels for these populations. Five to six concentrations of each insecticide will be tested and replicated 5 times. We will begin insecticide applications to fully evaluate our rotation treatments in early April 2019. We will collect samples chosen at random from the central rows for both the Lake Alfred and Wauchula sites. The plots will be sampled weekly beginning in late March 2019. The tap sample method will be used to sample adults. Ten samples will be taken per plot. For eggs and nymphs, 10 randomly selected flush samples will be taken per plots and number of eggs and nymph per flush samples will be counted. When counts of adults, eggs or nymphs in any plot reaches a predetermined threshold, a spray will be applied with the next insecticide in the rotation. Also, we will collect adults from the rotation sites to determine the relative expression of ten CYP4 and six GST genes that are implicated in insecticide resistance in ACP compared with the laboratory susceptable population. Finally, our goal is the development of a more refined method of an effective insecticide resistance management strategy. Our newly developed methods will be have positive impact on suppression of ACP populations by stabilizing or reducing resistance and will be economically viable.
A study was designed to investigate the capacity of field collected Asian citrus psyllid (ACP) Diaphorina citri Kuwayama (Hemiptera: Liviidae) to develop resistance to the pyrethroid insecticide, fenpropathrin and determine the biochemical and genetic mechanisms of resistance to this popularly used chemistry for ACP management. We established an insecticide resistant strain of ACP in the greenhouse. The selected adult ACP population was originally collected from commercial citrus groves from Wachula, FL on July 15, 2018. They were treated with the LC50 concentration of fenpropathrin for nine generations of continuous rearing and then for a subsequent seven generations at a higher insecticide concentration. Bioassays were conducted using the bottle bioassay to assess the resistance of adults during each successive generation. Insecticides were dissolved to make 5-7 concentrations in acetone that gave 0 to 100% mortality. Control ACP were treated with acetone. Selection was performed by exposing adults to treated glass vials at the LC50 concentration. The ACP that survived were released in rearing cages to serve as parents of the next generation. The value of LC50 was increased from 0.12 to 3.71 ng/ L after nine generations. The resistance ratio was 30.91 fold. Biochemical assays were performed with detoxifying enzymes, namely esterase (EST), glutathione S-transferase (GST) and cytochrome P450 monooxygenase (P450). These were quantified every two generations and compared with the laboratory susceptible population. The activity ratio of EST enzymes was 1.530 fold higher for the selected population compared with the laboratory population at eight generations and there were significant differences between the two populations (p < 0.001). The activity of GST enzyme was 1.486 fold higher, and for P450 the activity ratio was 1.10 fold higher for the selected population compared to the laboratory population. There were significant differences between the two populations for GST activity (p = 0.038) and for P450 activity (p = 0.045). Future experiments are planned to better understand the genetic basis of resistance by determining the expression levels of genes involved in the detoxification of pyrethorid insecticides. The results of this study provide insight into the development of insecticide resistance and designing appropriate resistance management strategies for ACP.
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