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.
Psyllids feeding on treated plants may ingest antimicrobials, which may have the potential to harm psyllids due to their reliance on bacterial endosymbionts for survival. Antimicrobial treatments may negatively affect a variety of psyllid biological features, including fecundity, transmission capacity, life span, developmental time, and behavior. The current objective was to evaluate the effect of dietary exposure to antibiotics on D. citri survival in a greenhouse bioassay. Feeding solutions containing oxytetracycline (Fireline) or streptomycin (Firewall) were orally administered to D. citri. D. citri mortality was significantly higher when insects fed on artificial diet solutions containing 1mg ml-1 oxytetracycline, 5mg ml-1 oxytetracycline, or imidacloprid. Approximate 40% and 100% mortality occurred among D. citri that fed on 5 mg ml-1 of oxytetracycline after 3-d and 10-d, respectively. Greater mortality occurred in response to the high oxytetracycline concentration than the low concentration on day 3 (25%) and day 10 (63%). D. citri mortality in response to untreated diet was approximately 20% and 30% on days 3 and 10, respectively. Neither 5 mg ml-1 nor 1 mg ml-1 streptomycin exhiwas associated with significant D. citri mortality as compared with untreated diet. After 30d, 40% of D. citri that fed on untreated or streptomycin diets survived. Subsequent assays will evaluate the effect of foliar applied antimicrobial treatments on D. citri survival and CLas transmission.
During this initial 4-month period of the Project, spanning from December 1st 2018 to March, we have followed the chronology developed in our proposal. Our Project has 4 main objectives: Objective 1. Assessing tree growth and absence of psyllids and HLB disease symptoms (including CLas bacteria titer) under protective covering. Objective 2. Assessment of alternative netting approaches involved in targeted , alternated or patterned setup of IPC in groves for more cost-effective protection. Objective 3. Monitoring the transition from vegetative to reproductive stage in the covered trees as compared to the uncovered. Objective 4. Comparing IPC with CUPS-like systems. Objective 1: We assessed the trees (Valencia on Swingle) planted in our pilot study 14 months ago for HLB . Whereas 1/3 of the uncovered trees in the trial are already positive for HLB, all trees covered with IPC have tested negative. In addition we are quantifying leaf drop and comparing leaf drop in both treatments. We are now in the process of planting trees of the varieties Tango , SugarBelle and US Early Pride with our collaborators in Polk County, as described in the proposal. Objective 2: This objective will start later this spring in both SWFREC grove and in our Polk County locations. Objective 3: The trial for this objective starts this month in Polk county and has started already in our grove at SWFREC. We have seen an significant advancement in flushing timing in IPC-covered trees. We have not seen any differences in blooming rate or intensity in IPC trees as compared to uncovered trees. We are assessing and rating the type of flushes each tree individually bears. Objective 4: We are currently planting 100 trees from Tango , SugarBelle and US Early Pride in our CUPS facility, after trellis installation. As expected, a Ms student, Susmita Gaire, has joined the Project and is performing part of this work for her Masters degree. Results from this Project have been already presented at the Citrus Show in Fort Pierce this past January, and an update will be presented in Riverside at the IRCHLB meeting later this month.
March 2019 Objective 1: Evaluate the optimal spray timing for Florida and investigate if tree skirting or alternative products improves fungicidal control of citrus black spot.Objective 3: A MAT-1-1 isolate may enter Florida and allow for the production of ascospores. The industry needs to know if this happens, as it will affect management practices. Additionally, the existing asexual population may be more diverse than currently measured. If multiple clonal linages exist, then there may be different sensitivities to fungicides or other phenotypic traits. We also need to determine whether P. paracitricarpa or P. paracapitalensis are present in Florida for regulatory concerns due to misidentification. We plan to survey for the MAT-1-1 mating type, unique clonal lineages, and two closely related Phyllosticta spp. In this first quarter, we found a field site with sufficient black spot to conduct the skirting and fungicide timing trial. We scouted 100 rows for presence/abscence of black spot and chose the best 96. The plots have been laid out and calculations were done for the fungicides and organized with the grower to apply with an airblast sprayer. Two sites have been scouted for a fungicide spray trial. The details will be determined once the incidence and severity have been collected. We plan to be able to use at least 5 products and possibly more. The remaining activities have been bureaucratic in nature. The postdoctoral researcher in South Africa has been appointed from 1 March 2019, and preparations for genotyping-by-sequencing of a collection of Phyllosticta citricarpa isolates from USA has been initiated. There are also continuing contract negotiation between CRI and UF which we hope will be resolved shortly so funds can be received and the postdoc can continue to work on the project. As we work to collect isolates for objective 3, Jeff Rollins has been working with the UF administration to make sure all of the necessary permits are in place for him to be able to travel to Cuba and collect isolates to determine the mating type and species identifications. We are waiting to recieve MAT 1-1 DNA from Australia and South Africa to use as positive controls for our experiments.
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. We will repeat this experiment. 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. 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.
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