In this project we are profiling the new scions and rootstocks for their tolerance to HLB by studying the metabolite content by GC-MS, and challenging new varieties with psyllids and HLB.Objective(s) pursued: 1. To understand the mechanism behind the tolerance of different varieties toward HLB. The comparison between the varietal responses will allow us to determine the mechanism of tolerance to CLas. 2. To understand the role of rootstocks in citrus tolerance to HLB. The comparison between rootstock metabolites will allow us to determine the best scion/rootstock combination for tolerating CLas. Progress on Objectives: This quarter we focused in three areas: 1) Marathon Mandarin analyses; 2) CUPS varieties; 3) Lucky biology and plant response.1) For the evaluation of the new mandarin hybrid Marathon, finally we have some cuttings that are growing in the greenhouse. In addition, the source plant is growing very well and we were able to sample many leaves for analysis of volatiles and polar metabolites. These samples have been run on the GCs and need integration.2) The samples from the new varieties we collected from the CUPS which were used for cuttings did not root well. It may be because we did this in the winter. They remain in the mist bed, except for UF 411 an UF 711 did root. The leaves collected for volatiles and metabolites are being prepared now for extraction (they must be diced into 0.1g aliquots). We estimate there will be approximately 150 samples for this phase. 3) For Lucky and its parents Sugar Belle and Nava x Osceola, we began the biology experiment (detailed in the previous report) on 11/30/20 and ended it Jan 30th, which was one month longer than planned because the weather during December was so cold, we did not have good colonization. We ended the experiment by collecting leaf samples from all plants to assess the plant response to ACP, sprayed any remaining insects, and returned them back to their outside cage to recover. The plants did not look well, so we trimmed, repotted and fertilized them. The leaf metabolites from the ACP-exposed plants will be compared to non-infested controls.In addition to these efforts, the seeds from the USDA (US-802, 812, 897, 942, 1283, 1284, 1516) for metabolite profiling and HLB screening were received, planted, and the germination rate was good. It will be some months however before we can do anything with them in terms of sample collection.
Objective 2. Determine the effect of antimicrobials on Las transmission. Hypothesis: ACP will be less capable of transmitting CLas after feeding on antimicrobials because trees treated with antimicrobials are more likely to have lower CLas titers for acquisition. Eight-year old CLas-infected citrus trees have received six foliar applications (May-December) 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 tree 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. Survival of ACP and CLas-acquisition were replicated twice from June 2019 to March 2020. During the first replicate, ACP P1 adults were collected on the 26th of June. Approximately two weeks later, five to ten ACP adults corresponding to the F1 progeny were collected. The adults collected on the first replicate in June showed higher CT means (> 38.5) and low copy numbers (< 3); indicating that ACP were unable to acquire the pathogen from treated trees. The second (July), the third (September), fourth (October), the fifth (November), the sixth (January), and the seventh (March) replicates were collected using the same conditions previously described. Concurrently, the titer of CLas had been monitored at the same time-points using three leaves per tree to determine the CLas-infection rate. Currently, psyllids collected from late June through March are being processed to analyze the CLas-infection rate. Objective 3: Determine the effect of antimicrobials on plant response and associated ACP behavior. The objective of this experiment is to determine whether antimicrobial treatments applied to citrus plants affect behavior of Asian citrus psyllid that may change plant susceptibility to ACP infestation or pathogen inoculation. Two antimicrobial treatments are being investigated. These are Fireline (oxytetracycline HCL) and Firewall (streptomycin sulfate). Each is being applied to trees at label recommended rates with recommended adjuvants. To date, all treatments have been applied as foliar sprays; however, experiments are in progress. Treatments were applied to two-year-old Citrus sinensis L. Osbeck cv Valencia grafted onto US-812 rootstock. As described in our previous report, experiments comparing all uninfected (treated with antimicrobials versus untreated) versus all infected plants are ongoing using the T-maze olfactometer to determine whether Fireline affected ACP preferences for antimicrobial-treated versus untreated plants. Experiments are still in progress with Firewall to determine whether application will induce an effect on plants that would cause a consequential change in the behavior of the vector to increase or decrease their preference for treated versus untreated trees.
Objective: Develop threshold-based models for current use in Florida citrus. The objective of this experiment is to optimize management of Asian citrus psyllid (ACP) by implementing an economic threshold for need-based timing of insecticide applications. The most recent experiment we are establishing as part of this project also involves insecticide resistance management. We will compare implementation of three economic thresholds by also comparing various insecticide rotation schemes that we have developed based on previous research on managing resistance for ACP. These two factors will be investigated simultaneously. The rotations we will test are comprised of various insecticide modes of action that are currently registered and used for ACP control; these are: acetylcholinesterase inhibitor, acetylcholine receptor agonist, an inhibitor of chitin biosynthesis, chloride channel allosteric modulator, sodium channel modulator and a ryanodine receptor modulator. By rotating these modes of action, we should be able to prevent development of resistance in ACP entirely while implementing an economic threshold. Since the thresholds will determine timing and frequency of applications, it will be important for us to determine how this affects the order of rotating modes of action. Our purpose is to determine the economic threshold level that controls ACP populations most effectively and economically, as well as, which insecticide rotation works best with implementation of such a threshold. Previously, we have established a large-scale experiment at two sites: a commercial site in Frostproof with an estimated size of 80 hectares with 15-25 years old Valencias and Hamlins. There are 8 replicate blocks at this site. We have continued monitoring ACP and flush production at this location since January. The second, newly established site is located in Lake Alfred. Trees at this location consist of 2-3 year old Hamlins. This site was chosen because it has historically shown some of the highest levels of insecticide resistance documented statewide. For example, resistance to neonicotinoid insecticides and associated product failures of imidacloprid and thiamethoxam have been demonstrated at this site. Three economic threshold levels (0.2, 0.5, and 1 ACP per tap sample) will be evaluated for two different insecticide rotation schedules at this site. Each threshold will be evaluated in replicated plots with four replicates per treatment. Ten trees will be selected in each replicate plot to monitor psyllid densities following insecticide applications. Also, these trees will be used for collecting psyllid samples that will be used to determine changes in ACP susceptibility to insecticides in the laboratory. We have already begun collecting adult ACP from these plots to determine the baseline insecticide resistance levels compared to the susceptible laboratory population of ACP using a leaf dip assay. Field populations have been collected, and bioassays are currently being conducted. We use commercial formulations of dimethoate, fenpropathrin, imidacloprid, and cyantraniliprole for this testing. Five to six concentrations of each insecticide are tested and replicated five times. We will begin insecticide applications when adult tap numbers reach the experimental threshold. We will continue to collect samples chosen at random from the central rows of each plot. The plots will be sampled weekly, beginning in late March 2020. The tap sample method will be used to determine the treatment threshold. Ten samples will be taken per plot to determine an everage ACP count. For eggs and nymphs, 10 randomly selected flush samples will be collected per plot, and the number of eggs and nymphs per flush samples will be counted. Leaf samples will be collected from each plot as well to determine the HLB infection rate. When counts of ACP adults in any plot reach a previously defined threshold level, a spray will be applied with the next insecticide in the rotation. We will determine toxicity and dynamic insecticide resistant development. Also, we will collect adult ACP from the rotation sites to determine the relative expression of ten CYP4 and six GST genes compared to the laboratory population. Genes will be selected based on our previous research which has identified specific genes associated with insecticide resistance in ACP. These will severe as diagnostic tools for helping us identify the specific mechanisms conferring resistance. Finally, our goal is to investigate determine the most effective threshold ACP population level required to trigger a management spray within the context of effective insecticide resistance management. In this manner, we will develop an economical and sustainable management strategy for ACP with insecticide, which is still currently lacking in Florida. Our newly developed methods will be having a positive impact on the management of Asian citrus psyllid populations by stabilizing or reducing resistance and focusing on economical viability of spraying.
The following progress has been made toward Objective 2 of this proposal: -Objective 2: An insecticide resistance management protocol will be developed for young tree protection in Florida citrus that: 1) could be deployed in areas where insecticide resistance is already present and reduce ACP populations and 2) would allow return to normal susceptibility levels for insecticides that have been compromised in effectiveness due to resistance. Thiamethoxam (Platinum 75 SG) has been one of the most overused neonicotinoids for Asian citrus psyllid (ACP) management. Among insecticides currently in use for Asian citrus psyllid, resistance is greatest for neonicotinoids, like thiamethoxam. First, we investigated the possible mechanisms involved in thiamethoxam resistance and the stability of thiamethoxam resistance in the laboratory by establishing populations of field collected resistant ACP. Second, we investgated how to reverse a resistant population back to susceptibility in the field by implementing rotation schedules. Investigations were conducted in areas that were previously treated with consecutive neonicotinoid applications and where the resistance ratio (RR) to thiamethoxam reached between 1266.20 and 1395.00 fold compared with a susceptable population. Finally, we developed a protocol that can be implemented in the field to reverse resistance to thiamethoxam in areas where it is found. First, RNA was extracted using a total RNeasy mini prep kit from adult ACP collected from specific areas where resistance to thiamethoxam was present at two locations: Lake Alfred and Wauchula, FL. A laboratory susceptible population was used as a control for comparison. For each population, ten tubes containing ten ACP adults were used for RNA extractions. The quality of RNA from each sample was assessed on a Nano Drop 2000 Spectrophotometer using A260/A280 ratios, at approximately 2.0. Thereafter, 500 ng of total RNA from the two insecticide resistant populations and the laboratory susceptible population were used for cDNA synthesis. Relative expression of three CYP4, three GST, and one EST gene(s) in each population was compared by qPCR using SYBR Green Power Up PCR master mix in an ABI 7500 Real Time PCR system. The production of the gene specific product and absence of primer dimers was verified by 1% agarose electrophoresis in Tris-acetate EDTA buffer with gel red. qPCR was performed in a 15 µL reaction volume containing 7.50 µL of SYBR green PCR master mix, 1 µl of cDNA, 0.45 µL of each forward and reverse primers and 5.60 µL of nuclease free water. Amplification cycles consisted of an initial denaturing step at 95°C for 10 min, followed by 40 cycles of 95°C for the 30s, 60°C for 30s and 72°C for 30s, and final melting curve step. Three biological replicates were performed for each gene. Actin was used as a reference gene, and to normalize changes in specific gene expression to the ACP laboratory colony. Transcription levels of three CYP, three GST, and one EST genes that are potentially involved in metabolic resistance to insecticides were compared between the laboratory and field selected strains. Second, recovery to susceptibility was determined for two ACP populations where thiamethoxam resistance was present by quantifying the LC50 value of every generation after constant selection for neonicotinoid resistance was ceased. The treatments (rotational schemes) were: dimethoate followed by cyantraniliprole, fenpropathrin, and diflubenzuron (Rotation A) and fenpropathrin followed by dimethoate, cyantraniliprole, and imidacloprid (Rotation B). The third treatment consisted of thiamethoxam followed by clothianidin, thiamethoxam, and imidacloprid (no mode of action rotation). After week 13, the order in rotation A was thiamethoxam, spinetoram, fenpropathrin and abamectin+thiamethoxam. In rotation B, the order was diflubenzuron, dimethoate, abamectin+thiamethoxam, fenpropathrin, and spinetoram. The third area where only neonicotinoids had been applied, received a recovery order consisting of: fenpropathrin, dimethoate, spinetoram, cyantraniliprole, and diflubenzuron. Insecticides were diluted in water at maximal label rates and applied by air-blast sprayer delivering approximately of 100 g of carrier/acre. Adult ACP were collected in the field for the Lake Alfred and Wauchula sites and placed into 60 × 60 × 90 cm insect-proof cages on four citrus plants and maintained in a greenhouse under the rearing conditions. The initial population in each cage contained at least 400 adult ACP from each no rotation treatment and location. In the field, we modified the rotation schedule to cease neonicotinoid applications in plots that indicated high levels of neonicotinoid resistance. After each application, the toxicity of thiamethoxam to ACP was determined to assess how levels of resistance changed over time. The resistance ratios decreased from 1266.20 to 21. 57 and from 1395.00 to 28.71 after four applications of the recovery mode of action rotation order at Lake Alfred and Wauchula, respectively. Under laboratory conditions, the resistance ratios decreased from 1266. 20 to 28.86 and 1395 to 36.71 for Lake Alfred and Wauchula populations, respectively, after five generations of no insecticide exposure. The qPCR analysis showed that expression of CYP4C67 was significantly increased in both resistant populations relative to the laboratory susceptible population. We also showed that resistance in ACP to thiamethoxam declined significantly in the absence of selection pressure under laboratory conditions and rotation application in field. Both laboratory and field investigations indicated susceptibility to thiamethoxam fully recovered after five generations. Three main mechanisms of insecticide resistance typical among insects are: metabolic detoxification, target site modification, and reduced penetration/increased excretion. Previous studies reported five cytochrome P450s (CYP4C67, CYP4DA1, CYP4C68, CYP4DB1, and CYP4G70) analyzed in this investigation that are known to be upregulated after ACP obtain sub-lethal dosages of imidacloprid. In addition, CYP4 genes have been previously implicated in ACP resistance to imidacloprid. There are several cases where up-regulation of one or several detoxification enzyme genes has been attributed to neonicotinoid insecticide resistance or insecticide detoxification among insects. In the present investigation, populations of ACP exhibiting high neonicotinoid resistance were associated with upregulated expression of CYP4C67, implicating the gene product in neonicotinoid resistance in ACP. We compared the expression of CYP, GST and EST genes between known neonicotinoid resistant and susceptible ACP. Similar to the trend in CYP4 expression, GST and EST genes generally exhibited overexpression in insecticide resistant ACP as compared to susceptible counterparts, although these differences were not always statistically significant. Our findings suggest that populations of ACP that exhibited resistance to neonicotinoids in Florida also showed evidence of overexpression of genes implicated in metabolic detoxification. Further research is necessary to determine whether these differences among field populations of ACP have become genetically fixed. Our current focus is on developing an RNA-seq based database to further understand the mechanism(s) underlying thiamethoxam resistance in field selected populations of ACP. Our results revealed that ACP populations develop high levels of resistance to thiamethoxam under continuous selection by label rate applications in cultivated citrus. A high level of resistance occurred following only 3-4 consecutive neonicotinoid sprays and within five egg to adult generations and was associated with subsequent product failure. We also showed that resistance in ACP to thiamethoxam declined significantly in the absence of selection pressure under laboratory conditions and when modes of action rotation was implemented under field conditions. Recovery to a susceptable state under rotation in the field was more rapid than under no selection in the laboratory population. These results suggest that thiamethoxam resistance is likely unstable under the field conditions. Collectively, our results indicate that rotation of thiamethoxam with insecticides from other chemical classes, including cyantraniliprole, fenpropathrin, dimethoate, spinetoram and diflubenzuron effectively mitigates neonicotinoid resistance in areas where ACP are managed with insecticides.
This project evaluates ACP control using various combinations of conventional and organic insecticides and biological control agents. Four Integrated Pest Management (IPM) programs include 1) conventional and organic insecticides plus biological control 2) organic insecticides and Horticultural Mineral Oil (HMO) plus biological control, 3) conventional insecticides plus biological control, and 4) HMO plus biological control. Program 5 is based on biological control only. Populations of Asian citrus psyllid and predators were monitored using tap sampling and suction sampling methods. Tap sampling was conducted twice a month for a total of 6 times and 2,160 tap samples to detect ACP and predators. ACP populations were very low across all programs averaging below 0.1 adults per tap sample. Shoot infestation averaged 2-4% across all programs from a sample of 300-400 shoots available from each program. A total of 24,000 Tamarixia radiata were released across all programs. A cohort study to evaluate contribution of natural mortality factors in suppression of psyllid population in all programs was conducted in November and data being analyzed. Lacewings and spiders were common predators. Ceraeochrysa cubana was dominant species, 76-82% of all lacewings collected from any program. Considering that psyllids averaged below treatment threshold of 0.1 adult per tap sample across all programs, no sprays were conducted except dormant season spray. The dormant sprays which do not require a treatment threshold were applied in Programs 1-4 in December. Imidan was sprayed in programs 1 and 3, Pyganic + 435 oil in program 2 and 435 oil only in program 4.
This project evaluates young tree protection from ACP/HLB using approaches to integrate ground cover, insecticides, and irrigation management under four treatments at three locations. Treatments include 1) soil applied neonicotinoids interspersed with sprays of different mode of action on a calendar basis to trees on UV reflective mulch, 2) rotation of insecticide modes of action sprayed twice on each major flush to trees on UV reflective mulch, 3) soil applied neonicotinoids interspersed with sprays of different mode of action on a calendar basis to trees on bare ground, 4) rotation of insecticide modes of action sprayed twice on each major flush to trees on bare ground. Designated soil and spray applications of insecticide treatments were made to the trees planted on mulch and bare ground at the Gulf and Ridge locations in Immokalee (SWFREC) and Lake Alfred (CREC), respectively. In Immokalee, 1148 trees observations each on mulch and bare ground trees were made overtime during this quarter to look for the plant colonization with adult psyllids and plant infestation with eggs and nymphs. Only 2 trees on mulch were observed to contain adult psyllids compared to 7 on the bare ground, averaging 0.002 and 0.006 adults per plant, respectively. Only one plant on mulch was infested with 2 eggs but no nymphs (0.09% infestation rate) compared with 5 plants on bare ground infested with both eggs and nymphs (0.4% infestation rate). In Lake Alfred, 432 trees observations each on mulch and bare ground trees were made during this quarter to look for the plant colonization with adult psyllids and infestation with eggs and nymphs. Adult psyllids were observed on 3 trees on mulch, averaging 0.02 adults per plant, and on 23 trees on the bare ground, averaging 0.2 adults per plant. Thirteen plants on mulch were observed with eggs and nymphs, infestation rate of 3% compared with 93 plants on bare ground, infestation rate of 22%. Soil moisture sensors were installed in plots with and without the reflective mulch at CREC and SWFREC this quarter. Data will be collected continuously during the duration of the project to document water savings and applicability of the irrigation schedules and rates in a commercial setting. Irrigation installation is underway in Vero Beach, at a grower site and tree planting will be completed in the next quarter. Deficit irrigation treatments to regulate tree flushing will be imposed in Spring 2020, to compare flush sprays against rotation of soil and calendar spray applications. Trees were young and planted late, therefore, allowed to establish through spring 2020 before subjecting to reduced irrigation to synchronize flush and evaluate spray applications.
Objective: Develop threshold-based models for current use in Florida citrus.
This objective is in the process of being established. We began scouting for appropriate field sites in Janaury of 2020. During this time, we secured adequate field sites to establish experiments and mapped specific plots for subsequent trials. In 2020, we initiated monitoring of ACP population levels at these locations in preparation for the investigation. We have also aquired all necessary materials to initiate the research and have finished developing necesarry protocols for monitoring. There are a total of four replicates established in the experiment for each treatment threshold that will be investigated. In addition to monitoring for ACP, we have protocols for monitoring plant health, tree productivity, and pathogen levels.
Objective 1: Compare the effectiveness of biological control in commercial orchards in FL and determine if abiotic factors or management practices influence effectiveness. Use this knowledge to improve the effect of biological control.
The goal of this research is to better understand the contribution of natural enemies on ACP population regulation among citrus groves characterized by differing management inputs in Florida. Two ACP management practices were compared: i) Organic and ii) Low input (2-4 annual sprays) conventional insecticide treatment. Trees were 10-12 year old sweet orange ‘Valencia’. The study was conducted in 2019 form March to June (Spring) and July to September (Summer). We released two pairs of ACP sentinels with and without exclusion cages to measure mortality of ACP cuased by natural enemies. The number of living and dead ACP were counted for three weeks after deployment. We also monitored abundance of ACP and natural enemy abundance in all groves. Natural enemies were recorded during 2-minute visual inspections. Sixteen trees were sampled at each site per sampling date. Tap samples were used to monitor the abundance of ACP. Numbers of ACP adults were monitored weekly by sampling twenty trees per replicate block per week. Mortality of uncaged sentinel ACP was higher than that of psyllids within exclusion cages indicating that natural enemies were killing ACP. Mortality of uncaged ACP was significantly higher than caged ACP during summer in organically managed groves, but not so in conventionally treated groves. Among the natural enemies collected, spiders were most prevalent followed by Dolichopodid flies and coccinellids (lady beetles). Adult ACP populations were significantly higher at sites that were intermittently sprayed with conventional insecticides than in organically managed groves throughout the season. Populations of natural enemies were similar in groves sprayed intermittently with conventional insecticides vs. groves managed organically and without use of toxicants for ACP. However, populations of ACP adults were higher in conventionally treated than organic groves. These results suggest that the level of insecticide input was insufficient to reduce ACP populations, but may have negatively impacted the effect of biological control even though we could not document this directly by survey of natural enemy densities. Our results also suggest that ACP populations can be regulated more effectively by the action of natural enemies than by intermittent spraying under conditions of endemic HLB where curtailing spread of disease is not intended.
Objective 2. Revise insecticide resistance management for psyllid IPM in new plantings
In order to effectively implement resitance management, we have to understand the mechanisms causing it. We therefore initiated a global transcriptome-based analysis of Asian citrus psyllid (ACP) involved in the neonicotinoid resistance using bioinformatics techniques coupled with high throughput RNA-sequencing. First, we conducted insecticide toxicity bioassays on ACP collected from two field populations where neonicotinoid insecticides were used considerably. We used the leaf dipping bioassay technique to determine the level of existing resistance to thiamethoxam at these two sites. We determined that the populations of ACP were 1,394 and 1,266 times less sensitive to thiamethoxam at these two locations as compared with our laboratory susceptable control population. Therefore, we collected adult ACP samples from these to locations for analysis and also from the laboratory susceptible population as a comparison. These sample were immediately frozen and stored at -80°C. Then the total RNA was extracted from these samples using Trizol according to the manufacture’s protocol. RNA samples of acceptable quality were used to construt non-strand-specfic sequening libraries with the TruSeq RNA sample prep kit. These libraries were sequenced using the PE150 mode on an Illumina HiSeq 3000 platform at GENEWIZ. We are in the process of analyzing and interpreting these data. We believe that this transcriptomic profiles will contribute to a comprehensive understanding of the mechnisms of neonicotinoid resistance in ACP. In the future, we will analyze data by mapping to the ACP referrence genome and compare differential gene and transcription factor expression between susceptable and resistant ACP populations. To date, our results indicate that increased cytochrome P450 metabolic detoxification is the mechanism responsible for ACP resistance to neonicotonoids. However, some of our recent findings also suggest that target site insensitivity should be re-investigated; it is possible that over 12 years of selection pressure (continued insecticide application), the mechanism by which ACP are developing resistance could have changed. Understanding this possible shift will allow us to continue to develop the best possible rotation schedules for mitigating resistance.
Objective 2. Determine the effect of antimicrobials on Las transmission.
Hypothesis: ACP will be less capable of transmitting CLas after feeding on antimicrobials because trees treated with antimicrobials are more likely to have lower CLas titers for acquisition.
Eight-year old CLas-infected citrus trees have received six foliar applications (May-December) 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 tree per group. Survival of ACP and CLas-acquisition were replicated twice from June to November. During the first replicate, ACP P1 adults were collected on the 26th of June. Approximately two weeks later, five to ten ACP adults corresponding to the F1 progeny were collected. The second (July), the third (September), fourth (October), and the fifth (November) replicates were collected using the same conditions previously described. 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. Concurrently, the titer of CLas had been monitored at the same time-points using three leaves per tree to determine the CLas-infection rate. Currently, psyllids collected from June through November are being processed to analyze the CLas-infection rate.
Currently, psyllids collected from June through November are being processed to analyze the CLas-infection rate.
Objective 3: Determine the effect of antimicrobials on plant response and associated ACP behavior.
The objective of this experiment is to determine whether antimicrobial treatments applied to citrus plants affect behavior of Asian citrus psyllid that may change plant susceptibility to ACP infestation or pathogen inoculation. Two antimicrobial treatments are being investigated. These are Fireline (oxytetracycline HCL) and Firewall (streptomycin sulfate). Each is being applied to trees at label recommended rates with recommended adjuvants. To date, all treatments have been applied as foliar sprays; however, experiments are in progress and other methods of treatment application will be explored. Treatments are being applied to two year old Citrus sinensis L. Osbeck cv Valencia grafted onto US-812 rootstock. Separate experiments are underway comparing all uninfected (treated with antimicrobials versus untreated) versus all infected plants. In the first experiment, we compared response of ACP to the odors of treated and control plants 4, 6, and 8 weeks after treatment with Fireline. C. sinensis plants were placed in 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 using either all uninfected or all infected plants. ACP response was evaluated with the T-maze olfactometer to determine whether Fireline affected ACP preferences for antimicrobial-treated versus untreated plants. There was no difference in behavioral response of ACP to plants treated with Fireline versus untreated controls 4, 6, and 8 weeks after treatment. These results were consistent when both uninfected and CLas-infected treatment and control plants were compared. Experiments are still in progress with Firewall. Thus far, it does not appear that application of antimicrobial treatments (Fireline) to citrus should induce an effect on plants that would cause a consequential change in the behavior of the vector to increase or decrease their preference for treated versus untreated trees, based on the odors released by trees.
During this 4th quarter of the Project we have continued our work as predicted in the Chronogram.
Objective 1: We continued monitoring tree trunk diameter (rootstock and scion) and canopy areas. So far, no differences were found in trunk diameter, but leaf and canopy areas are bigger in IP-covered trees. All IPC-covered trees are still HLB-negative. After replacing the old 4-ft IPCs with new 8-ft covers, donated by The Tree Defender, Inc, canopy area expanded by branch unfolding. We have documented this by photography and also by leaf are index measurements. Objective 2. We have already planted most of the 700 trees of SugarBelle, Tango and Early Pride mandarins. After performing initial measurements of the tree parameters (trunk diameter, and leaf sampling, for CLas, chlorophyll and sugar analysis), we will continue regularly with these analysis.
Objectives 3 and 4. We are continuing monitoring fruit development inside the IPCs and comparing this with our CUPS planting. We are going to assay this winter deficit irrigation to induce blooming in both IPC and CUPS. We have set up an irrigation system that will allow to perform these studies.
Outreach, Professional Presentations and Extension Activities for this quarter : – A CUPS Day.“CUPS, mini-CUPS and other strategies to manage HLB”. Talk on “Individual Protective Covers” . SWFREC, to be delivered on Dec 17. 45 people registered. -International invited seminar at IVIA, Valencia, Spain. “Living with HLB. The new reality of Florida Citriculture”. -Industry Magazine Article: “Individual Protective Covers for Psyllid Exclusion and HLB Disease Prevention in Young Trees”. Citrus Industry, October 2019.
This main objective of this project is to manage ACP using various combinations of conventional and organic insecticides and biological control agents. Four Integrated Pest Management (IPM) programs were established. These included 1) conventional and organic insecticides plus biological control 2) organic insecticides and Horticultural Mineral Oil (HMO) plus biological control, 3) conventional insecticides plus biological control, and 4) HMO plus biological control. Program 5 relied only on biological control. Between July-September, there were six sampling events in which 2,160 tap samples were conducted to detect ACP and predators and 2,267 shoots examined for infestation with ACP immatures. ACP populations were very low across all programs averaging below treatment threshold of 0.1 adults per tap sample. Shoot infestation rate averaged 7%. No spray applications were made in any program. Psyllid adults averaged 0.05 per tap sample in the program 5 compared to 0.01 adults per tap sample across programs 1-4, showing a significant reduction of 80, which persisted from previous applications as no new sprays were conducted due to low populations. Shoot infestation averaged 18% in program 5, and 2-10% across programs 1-4. Lacewings, spiders, and ants averaged 0.05, 0.006, and 0.32, respectively, in program 5, and 0.05, 0.002, and 0.19, respectively, across programs 1-4. A total of 12,000 Tamarixia radiata were released across all programs. Nymphs were not available to evaluate parasitism.
This project is focused on young tree protection from HLB through reduction in populations of vector ACP and irrigation management for improved tree health. Metalized polyethylene mulch and irrigation management can alleviate the problem by repelling ACP and controlling flush cycles to improve ACP spray timing and efficiency. During this quarter we were able to plant at the Gulf region location. The orange trees planted at the Immokalee location were Valencia on Swingle rootstock. The four treatments to be applied to these trees include 1) soil applied neonicotinoids interspersed with sprays of different mode of action on a calendar basis to trees on mulch, 2) rotation of insecticide modes of action sprayed twice on each major flush to trees on mulch, 3) soil applied neonicotinoids interspersed with sprays of different mode of action on a calendar basis to trees on bare ground, 4) rotation of insecticide modes of action sprayed twice on each major flush to trees on bare ground. Trees were planted late due to logistics and therefore it was not possible to establish a drought period to manage flush for the treatments 2 and 4, which includes spraying on flush. Therefore, all plots will be allowed to establish a first spring flush and then the drought treatment will be applied to synchronize flush and evaluate spray applications. Soil application of Admire Pro were made in treatments 1 and 3. Sprays of Delegate were made to trees in treatments 2 and 4 in September and Transform to all trees in October. So far, 3 ACP detected throughout the experiment one each in treatments 2, 3 and 4. We covered twenty-four trees with tree defenders, and these will be compared with unprotected trees for ACP and HLB incidence and tree health. Experiment at the Ridge location was planted during the previous quarter. In September, 2 adult ACP and 2 infested flush were observed on a plant in treatment 4.
Objective 2. Determine the effect of antimicrobials on Las transmission.
Objective 2.2 Hypothesis: ACP will be less capable of transmitting CLas after feeding on antimicrobials because trees treated with antimicrobials are more likely to have lower CLas titers for acquisition.
Eight-year old CLas-infected citrus trees have received three foliar applications (May-July) of streptomycin, oxytetracycline (Treatments), or 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 and CLas-acquisition in ACP P1 and F1 progeny. Acquisition from five individual individual trees per replicate was evaluated. ACP survival and CLas-acquisition were repeated in June and July. During the first trial, ACP P1 adults were collected on the 26th of June. Approximately two weeks later, five to ten ACP adults corresponding to the F1 progeny were collected. The second trial began on July using the same conditions previously described, ACP P1 adults exposed to CLas-infected trees were collected on the 15th of July and its F1 progeny was collected on 22nd of July; ACP adults were collected individually in microcentrifuge tubes containing 1 mL of 80% ethanol and then stored at -20°C for subsequent CLas detection using real-time PCR. Concurrently, the titer of CLas had been monitored at the same time-points using three leaves per tree to determine the CLas-infection rate. The third trial is scheduled to begin in September. ACP from the first two trials are currently being processed to quantify acquisition.
August 31, 2019 – In this quarter, we have continued to work on objectives outlined in our chronogram.
Objective 1. We have completed assessment of trees planted in our pilot study (planted 22 months ago) for CLas infection and HLB symptoms. All the non-covered trees are PCR-positive for CLas whereas all trees covered with IPC have tested negative. We are continuing with quantification of leaf drop and comparing leaf drop in both treatments; 6-month cumulative data show no significant differences in leaf drop in IPC-covered trees compared with non-covered trees. Interestingly, when counted seasonally, in spring leaf drop was significantly higher in non-covered trees as compared to IPC trees, whereas in summer, it was slightly higher inside IPCs. This fact points out a seasonal component that we will investigate as the project progresses.
In August, we have replaced the old 4-ft IPCs with new 8-ft covers, donated by The Tree Defender, Inc, because the trees had filled the volume of the cover completely. This also has opened the possibility of studying the dynamics of branch unfolding, which we are doing visually (photography documentation) and by measuring canopy growth and leaf area index. We have also assessed other pest and disease incidences inside the IPCs. We have found less incidences of canker inside IPCs and approximately equal incidences of greasy spot. However, greasy spot severity is higher inside the IPCs. We have found more incidence of other pests such as mites, armyworms, and leafrollers inside the IPCs, and a total absence of predators (beneficials). This suggests that relying only on IPC for insect control is not sufficient, and insect management must still be conducted. No psyillid have been found inside the IPCs.
Objective 2. To study the edge effect in different IPC layouts, we are now preparing to plant 700 trees of SugarBelle, Tango and Early Pride mandarins and using 3 different arrangements (targeted, alternated and patterned, as described in the proposal) of IPC. We have performed initial measurements of the tree parameters (trunk diameter, and leaf sampling, for CLas, cholorophyll and sugar analysis).
Objectives 3 and 4. We are continuing to measure fruit set and development inside the IPCs and comparing this with our CUPS planting. We are taking fruitlet and fruit samples regularly for biochemical analysis.
Outreach, Professional Presentations and Extension Activities for this quarter :
-Grower Presentation: “Growing Young Citrus Trees Under Individual Protective Covers (IPCs): What We Know After 18 Months” Citrus Expo 2019, August 15, Fort Myers, Fl.
-Industry Magazine Article: “Individual Protective Covers for Psyllid Exclusion and HLB Disease Prevention in Young Trees”. Article submitted to Citrus Industry Magazine in July to be published in October issue.
-Our Project was also noted in the September’s issue of Citrus Industry Mag’s UF/IFAS. The Citrus State Opinion Column by Jack Payne highlighted this work as an example of collaboration between growers, extension agents, and scientists in Florida. The column was entitled “Collaboration breeds solutions”.
Our objective during this past quarter was to investigate the evolution of insecticide resistant phenotypes (physical characteristics) in Asian citrus psyllid (ACP) and their underlying genotype (genes responsible for those characteristics). Specifically, we investigated resistance to the neonicotinoid insecticide, thiamethoxam, in a field investigation and also addressed cross-resistance to other insecticide modes of action by investigating field populations of ACP where we identified and documented neonicotinoid resistance. The obtained results are contributing to development of rotation strategies for improved resistance management of ACP.
Two types of rotation models have been developed by us over time based on our understanding of how resistance develops in ACP. These two rotations of insecticide modes of action are meant to prevent development of resistance in the field. While any rotation of modes of action is better than not rotating at all, our data to date with ACP from the laboratory indicate that certain rotation schemes should be superior to others in preventing resistance. This is because there can be varying levels of multiple-resistance between modes of action. Also, previous exposure to a certain mode of action in a sequence can effect the response of an ACP population to a second modes of action. These two rotation schedules compared here are referred to as “A” and “B”. First, insecticides were applied in these two rotational schemes (treatments), each consisting of four different insecticide modes of action (rotations A and B). The third treatment was a type of control in which neonicotinoid insecticides were applied in sequence four times with no rotation of mode of action (this is referred to as no rotation or NR). Rotation A consisted of dimethoate followed by cyantraniliprole, fenpropathrin, and diflubenzuron. Rotation B consisted of fenpropathrin followed by dimethoate, cyantraniliprole, and imidacloprid. NR consisted of thiamethoxam followed by clothianidin, thiamethoxam, and imidacloprid. Although different chemicals were rotated in the NR treatment, these are all different types of neonicotinoids. The field experiment consisted of five randomized replicate blocks and was conducted in two different groves: Grove 1 and Grove 2.
Insecticide toxicity bioassay were performed on ACP collected from the replicated treatment blocks from each of the three treatments compared: 1) NR, 2) rotation treatment A, and 3) rotation treatment B. A leaf dip bioassay technique was used to determine susceptibility levels of field-collected adult ACP after each insecticide application for two full rotations of all 4 insecticides comprising each treatment for a total of 9 evaluations, which included a pre-treatment evaluation. During each of the evaluations, the susceptibility of ACP adults from each replicated treatment plot was compared with the susceptibility of a known susceptible laboratory population maintained at the Citrus Research and Education Center. In Grove 1, we found that resistance of the ACP population in the NR control treatment, where neonicotinoids were applied in sequence rose 1,394 fold after 4 consecutive applications of neonicotonoids. In Grove 2, resistance of the ACP to thiamethoxam in the NR (no rotation) treatment rose by 1,266 fold after only three consecutive applications of neonicotinoids. However, the susceptibility of ACP to thiamethoxam in plots that were treated with rotations A and B only changed by 1.71 and 4.57 fold in Grove 1, respectively, and by 3.71 and 5.28 fold in Grove 2, respectively. Our results indicate that we have developed two rotation schedules that can robustly prevent development of resistance to neonicotinoids in locations where resistance to these insecticides has been previously documented for ACP in Florida. Furthermore, the results indicate that rotation A is slightly more effective than rotation B.
In addition, we have investigated the possible underlying mechanisms that have caused resistance in the populations of ACP in the NR (no rotation) treatment plots. We have also investigated the mechanism(s) involved in possible cross-resistance to different insecticide modes of action after an ACP populations develops resistance to neonicotinoids. We investigated susceptibility of ACP to the insecticides: dimethoate, cyantraniliprole, fenpropathrin, clothianidin, and imidacloprid from each of the three treatments evaluated in both Grove 1 and Grove 2. Resistance ratio (RR) were calculated by comparing the susceptibility of ACP to insecticides in the field with that our our laboratory susceptible strain reared at CREC. The results indicated that we did not find an increase in resistance that would cause product failure to fenpropathrin (RR=8.19); cyantraniliprole (RR=1.57); and dimethoate (RR=9.72) in Grove 1, and fenpropathrin (RR=4.24); cyantraniliprole (RR=1.58); and dimethoate (RR=7.22) in Grove 2. However, we documented significantly higher resistance for imidacloprid (RR=1059.65 in Grove 1 and RR = 1595.43 in Grove 2) and clothianidin (RR = 1798.77 in Grove 1 and RR = 1270.57 in Grove 2). These results indicate that there was significant cross-resistance developed between the neonicotinoid insecticides in plots where neonicotinoids were not rotated; however, the results demonstrate that multiple resistance did not occur, where a decrease in susceptibility to neonicotinoids did not impact the susceptibility of neonicotinoid-resistant ACP to other insecticide modes of action.
Currently and into the future, we are continuing to monitor stability of insecticide resistance to thiamethoxam in ACP populations using combined laboratory and field experiments. We have plans to evaluate a new rotation schedule treated by a different sequence of modes of action insecticide. Second, we will be analyzing the expression of seven P450, four GST and one EST gene and comparing their expression between the laboratory susceptible populations and identified resistant strains from the field. Finally, we will systematically examine differential gene expression using RNA sequencing (RNA-seq) to identify genes involved in general insecticide resistance in ACP. Again, here we will compare the laboratory susceptible strain with populations that are resistant to neonicotinoids. Furthermore, we are developing a new method to compare genes involved ACP fitness and reproduction, that are affected by development of resistance to thiamethoxam. The assembly, transcriptome annotation, the sequencing provides valuable genomic resources for further understanding the molecular basis of resistance and will allow us to precisely define the mechanisms conferring insecticide resistance in ACP. Figuring out the underlying genetic mechanisms confirming resistance allows us to tailor effective rotation schedules for management of resistance.
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