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.
Asian citrus psyllid (ACP), Diaphorina citri Kuwayama (Hemiptera: Liviidae), adults were collected from eight citrus groves across central Florida and the level of insecticide resistance to ten insecticides was measured by using a bottle bioassay. The gene expression of five cytochrome P450 CYP4 (CYP4C67, CYP4DA1, CYP4DB1, CYP4G70 and CYP4C68) and three glutathione S-transferase (GSTD1, GSTE2 and GSTE1) genes was characterized in seven field populations of ACP and compared with the laboratory population. Finally, we reared four neonicotinoid insecticide resistant field populations in the laboratory and observed susceptibility changes to insecticides without exposure to insecticides over multiple generations. The eight field populations of ACP adults showed no and very low levels of resistance (RR = 1 and 2-10) to dimethoate, chlorprifos, carbaryl, fenpropathrin, bifenthrin, flupyradifurone, and spinetoram. Very low to low resistance was found to imidacloprid and cyantraniliprole (RR = 2-10 and RR = 10-20). Moderate to high resistance was found for thiamethoxam (RR = 20-50 and RR = 50-100). The CYP4G70 and CYP4C68 genes were expressed at a higher level in field populations as compared with the laboratory population. Also, three separate field populations exhibited higher expression of all target genes compared to the laboratory population. For imidacloprid and thiamethoxam, there was a decline in susceptibility by 6.62-and 6.42-fold, respectively, compared to the initial results. These results indicate that the insecticide resistance may reverse in the field if insecticide selection pressure is removed from the spray schedule or with use of a rotational scheme with insecticides of different modes of action. Also the results support use of a survey program combined with effective rotation for integrated insecticide resistance management of ACP where huanglongbing (HLB) management includes vector suppression with insecticides. Importantly, this investigation verified that insecticide resistance in the Asian citrus psyllid carries a significant fitness cost for resistant populations. Thus, in the absence of selection for 5-6 months, psyllid populations return to normal levels of susceptibility. Our investigation demonstrated the value of implementing MOA rotation to manage resistance for ACP, and shows that resistance can be managed effectively by rotating 5 modes of action. These results may have been even more marked if conducted on a larger scale to prevent the local population from inbreeding between treatment applications. Rotation of insecticide modes of action does prevent or delay onset of resistance in ACP populations, and the effects of even relatively short duration selection pressure can be observed even under extreme circumstances where inbreeding with non-selected psyllids is possible. Our results indicate that even brief failures to rotate modes of action during ACP management with insecticides in Florida citrus production may establish localized populations of insecticide resistant ACP.
Use of RNAi delivered by Citrus Tristeza Virus Viral Vector to control the Asian Citrus Psyllid – 2019 Second Quarter Report1. In April all trial trees were sampled and tested using ELISA to detect the presence of CTV and gel electrophoresis and rtPCR to detect the presence and stability of CTVvv-RNAi.2. All trial trees were also sampled and tested using qPCR for the presence of HLB in April. 3. Aphid scouting continues on a biweekly basis. The presence of brown aphid has not been detected.
This project is focused on developing Asian citrus psyllid (ACP) management programs for conventional and organic growers. Programs include sprays of organic or conventional insecticides alone or combined and with use of biological control. Studies were initiated in a 15-acre block of mature Valencia orange in the Gulf region. After the harvest in spring 2019, the experimental block was treated with insecticide Portal at 32 oz per acre rate to bring the psyllid populations to the same level throughout the block before dividing into different management programs. Four Integrated Pest Management (IPM) programs focused on controlling the ACP were initiated. 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, 4) HMO plus biological control and 5) Only biological control program. Programs 1 and 3 received Apta 17 oz per acre rate and program 2 and 4 received HMO at 3% of the total application volume which was 100 gallons per acre. A total of 360 tap samples were conducted to detect ACP adults and predators and 694 shoots examined for infestation with ACP immatures. Psyllid adults averaged 0.3 per tap sample in the program 5 where no insecticide or HMO sprays were conducted compared to a range of 0-0.06 adults per tap sample across programs 1-4, showing a significant reduction of 80-100%. Shoot infestation with ACP immatures averaged less than 10% between programs 1-4 significantly less than 40% in program 5. Spiders averaged 0.03-0.08 per tap sample and ants averaged 0.06-0.81 per tap sample across all programs. Postdoctoral associate and temporary assistant were hired.
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. Replicated experiments planned for the Ridge, Gulf and River regions using a split plot factorial design to compare costs and benefits of bare ground to mulch (main plots), and two ACP management programs (subplots) based on soil applied insecticides compared to spraying on flush were initiated. The mulch experiment at Ridge location (Lake Alfred) is already planted. The orange trees planted were Valencia on Carrizo rootstock with 19 ft spacing between rows and 6 ft spacing between trees. Mulch is also installed at the Gulf location (Immokalee) and plants ready to be planted in August. Mulch and plants are also ready for the River (Vero Beach) location which will also be planted in August. We are using 96 inch wide metalized reflective mulch (Shine N’ Ripe XL, the original MRM) instead of 72 inch wide mulch used in the previous experiments and therefore expect more reduction in ACP populations over longer period. Data collection will include periodic pest scouting and counts, HLB incidence, soil moisture/temperature and tree growth variables. Specific objectives 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.
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.
(863) 956-8817
(863) 956-5894
700 Experiment Station Road
Lake Alfred, FL 33850
Email Us
