ACP Vector

Sustainable Management of Asian citrus psyllid (ACP) and Citrus Production

This project evaluates four Integrated Pest Management (IPM) programs for ACP, including 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 biological control only. Between July-September, biweekly tap sampling was conducted in all programs. However, the number of Asian citrus psyllid adults averaged less than our treatment threshold of 0.1 per tap sample except program 4 employing HMO plus biological control which needed a spray application in September. In July, 10,000 Tamarixia radiata wasps were released across all programs, followed by 6,000 in August and 4,000 in September. However, due to the low psyllid populations during this quarter nymphal samples were not available to assess the parasitism rates. We also released 20,000 predatory mites Amblyseius swirskii across all IPM programs in the month of September and will be evaluating their establishment. We continued suction sampling during this quarter which showed spiders as the most abundant predators at 69% of all collections followed by the lacewings at 31%. We made collections of ACP from our IPM programs to start testing for insecticide resistance. Conisdering the abundance of lacewings in these programs, we made some field collections and established their colonies and initiated some studies to look at their tolerance to commonly used insecticides. We will be able to report progress with these experiments at a later time.       

Optimizing Benefits of UV Reflective Mulch in Solid Block Citrus Plantings

This project evaluates young tree protection from ACP/HLB using approaches to integrate ground cover, insecticides, and irrigation management at three locations 1) Southwest Florida Research and Education Center (SWFREC), Immokalee, FL, 2) Citrus Research and Education Center (CREC), Lake Alfred, Fl, and 3) Florida Research Center for Agricultural Sustainability, Vero Beach Florida. Treatments include 1) soil-applied neonicotinoids interspersed with sprays of a 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 a 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. Treatments of insecticides in soil and sprays were conducted to control ACP in trees planted on mulch and bare ground in all three experiments at the SWFREC, CREC, and Vero Beach locations. However, the irrigation deficit treatments to synchronize flush were not implemented due to avoid confounding effects of rains. At SWFREC, ACP adults were significantly less in the plants planted on the mulch than those on the bare ground determined using the tap sampling method. For treatments 2 and 4, we made spray applications when most flush was observed. ACP suppression was more in the treatments timed to target flush than the treatment using soil applications of neonicotinoids intercepted with foliar sprays. Another set of leaf samples was collected and submitted for HLB analysis. At CREC, the sampling date influenced the presence of all life stages of ACP, which is logical, given the biology of the pest. Only the ground cover influenced the number of ACP eggs and nymphs on the plants. Between the two locations, the influence of ground cover and spray timing on the psyllid populations adults or progeny suggests that mulch and timing spray applications to key flush periods provide good psyllid control, and the latter will save growers money in terms of insecticide applications and psyllid suppression. We were able to continue data monitoring for soil moisture, tree size and leaf nutrient status in the experimental blocks. Virtual results show vigorous tree growth in the reflective mulch treatments over bare ground. We will follow up with quantitative statistics in the near future to show the benefits of reflective mulch. In the next quarter, we will collect leaf and soil samples to document soil and tissue nutrient status and changes according to treatments. The trial at Vero Beach suffered significant damage from rains resulting in 36% dead plants on the mulch and 12% on bare ground.       

Disrupting transmission of Candidatus Liberbacter asiaticus with antimicrobial therapy

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. Laboratory acquisition assays were completed in July 2020. Analysis of CLas acquisioton will be completed following DNA extraction of insect  tissues and PCR to determine CLas infection.Field acquisition assays continued during this quarter. Eight-year-old CLas-infected citrus trees have received foliar applications (May 2019 – September 2020) of streptomycin, oxytetracycline, or receive no antimicrobials (Control). One day after the application, 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. CLas-acquisition experiments were replicated from June 2019 to September 2020. All sample replicates have been collected according to schedule, and are currently being processed to analyze the CLas-infection rate. The final results will be available in the next report. Objective 3: Determine the effect of antimicrobials on plant response and associated ACP behavior.  Bioassays were carried out using 4 year old Citrus sinensis L. Osbeck cv Valencia grafted onto US-812 rootstocks, maintained at 23 ± 3 °C, 60RH, and a 16:8 h (Light: Dark) photoperiod. Trees were watered twice per week, and fertilized once per month with an alternating schedule of a 24-8-16 (Nitrogen–Phosphorus–Potassium) Miracle-Gro All Purpose Plant Food (Scotts Miracle-Gro Products, Marysville, OH) and a 10-10-10 (N–P–K) granular fertilizer (Growers Fertilizer Corp., Lake Alfred, FL) Colonies of CLas-free of Asian citrus psyllid (ACP) were maintained on C. sinensis L. Osbeck cv Valencia at 26 ± 2°C, 60-65% RH, and a 16:8 h (Light: Dark) photoperiod in a greenhouse. In order to determine the presence/absence of the CLas pathogen in our lab reared ACP, a sub-sample of 40 adult insects were collected for DNA extraction and later TaqMan qPCR assay were performed according with laboratory protocols. Experiments were conducted  to determine whether the antibiotic applications to sweet orange, C. sinensis, affect subsequent insect host preference and acceptance behaviors. Six trees (biological replicates) were evaluated per treatment. Individual trees were sprayed with FireWall (Streptomycin sulfate), FireLine (Oxytetracycline), or control (adjuvant); and then relocated into a growth chamber maintained at 23 ± 3 °C, 60RH, and a 16:8 h (Light: Dark) photoperiod until further bioassays. To test the insect choice response 20 days post-treatment, a pair of antibiotic- and control- plants were transferred to a behavioral chamber (dimensions?) with 70 ACP adults. Insects were allowed 24 hours to search out and settle on plants. Afterward, all insects found feeding  or control plants were counted.   Our results showed that antibiotic treatments rendered treated plants significantly less acceptable to infestation by ACP adults than comparable controls. In direct comparisons with control plants, Streptomycin Streptomycin (FireWall), Oxytetracycline (FireLine), and the combination of Streptomycin/Oxytetracycline (FireWall/FireLine) each reduced plant acceptability to released psyllids by 57-63%. Currently, we are investigating whether longer-terms treatment with antibiotics further reduces plant acceptability to the vector. We are also investigating the possible mechanisms explaining why plants treated with antibiotics are less acceptable to the vector. Overall, the results indicate that antibiotic treatments may have beneficial impact by reducing vector feeding on plants (which should reduce pathogen onoculation) in addition to their direct effects on the pathogen.   

Preventing young trees from psyllids and infection with CLas through use of protective netting

1. Please state project objectives and what work was done this quarter to address them: As in the last quarter, work in this quarter has been affected by the UF work restrictions due to the COVID-19. However, as activities have been gradually resumed, we have been able to accomplish most of the work that was scheduled. These are: Objective 1. Assessing tree growth and absence of psyllids and HLB disease symptoms (including CLas bacteria titer) under protective covering (i.e., IPC). We have continued monitoring trunk diameter and canopy area. Trunk diameters in IPC covered trees continue to show bigger diameters with statistical significance. All IPC trees are still HLB-negative and show less incidence in canker. We have continued monitoring the incidence of pests and psyllids. In August 2020, we removed the IPC from the first experiment and we are now monitoring for potential HLB infection of the uncovered trees in real time (i.e., monthly). Objective 2. Assessment of alternative netting approaches involved in ‘targeted’, ‘alternated’ or ‘patterned’ setup of IPC in groves for more cost-effective protection. After planting the new 700 trees last quarter in an alternated pattern we are monitoring for CLas in trees adjacent to the IPC-covered trees. In addition we started collaborating with some commercial growers who are also evaluating different netting layouts. We plan to collect data on psyllid populations and HLB incidence. Objective 3. Monitoring the transition from vegetative to reproductive stage in the covered trees as compared to the uncovered. We are starting to collect data on parallel experiments at SWFREC, Hendry Co, and Central Florida using Bingo, Early Pride, and Tango trees. These experiments will allow us to determine the ability of these varieties to set fruit in the absence of pollinators. Objective 4. Comparing IPC with CUPS-like systems. We performed last season an experiment on the effect of deficit irrigation in all the varieties and we already saw that we can control blooming: we saw more bloom inside CUPS and IPCs after applying deficit irrigation. After June drop, we can now say that the same is true for fruit set. Outreach activities performed in this quarter:-Alferez, F. Online Presentation at Citrus Expo: “IPCs. New data on tree performance and lessons learned”. Fort Myers, August 2020. Impact: 146 views so far. -Gaire, S, Alferez, F and Albrecht, U. Use of protective covering and its effect on citrus tree physiology and HLB develpoment. Oral presentation at ASHS annual meeting. August 2020. Publications:-Individual Protective Covers for Psyllid Exclusion and HLB Disease Prevention in Young Trees. Fernando Alferez, Susmita Gaire, Ute Albrecht, Ozgur Batuman, Jawwad Qureshi and Mongi Zekri. Submitted to Citrus Industry Magazine. -Individual Protective Covers’ by Alferez, F, Gaire, S., Albrecht, U., Batuman, O., Qureshi, J., Zekri, M., IN PREPARATION,to be submitted to EDIS. -Gaire, S., Albrecht, U., Batuman, O., Qureshi, J., Zekri, M., Alferez, F. 2020. Horticultural performance of citrus trees grown under Individual Protective Covers (IPCs). IN PREPARATION, too be submitted to Plants, MDPI  2. Please state what work is anticipated for next quarter: Objective 1.We will continue monitoring parameters described in the first section. Also we will monitor HLB progression after IPC removal in the first experiment.Objective 2. We will start collecting data on psyllid populations and HLB incidence in the different netting layoutsObjectives 3 and 4.We will start to collect data on fruit quality and yield. Outreach: -F.Alferez, Citrus greening. Where are we now? Invited talk at the Southeast Regional Master Gardener Volunteer Virtual  Conference 2020. September 2020. -Protected citrus growing systems: from healthy trees to high quality fruit. F. Alferez, SWFREC Zoom seminar series.  -Canopy growth and physiological assesment of Valencia orange trees with and without protective covers. Gaire,S, Alferez, F and Albrecht, U. To be presented at FSHS annual meeting, October 2020.   3. Please state budget status (underspend or overspend, and why): We have spent about 40% of the budget. As in the last quarter, this means some underspending. The reasons are the same as in the last quarter:1) Budgeted meetings (national and international) and publications have not occurred yet, and 2) some expensive reactives for analysis have not been purchased as sampling has not been completed yet. Some delays in spending due to COVID ocurred, but as we have been resuming activities we are increasing our expenditure to normal levels.Budgeted amounts for salaries and student stipend and tuition are being spent as predicted. 

Preventing young trees from psyllids and infection with CLas through use of protective netting

1. Please state project objectives and what work was done this quarter to address them: As activities have been gradually resumed after COVID-19 pause, we have been able to accomplish the work that was scheduled. These are: Objective 1. Assessing tree growth and absence of psyllids and HLB disease symptoms (including CLas bacteria titer) under protective covering (i.e., IPC). As in the last quarter, we have continued monitoring trunk diameter and canopy area. Trunk diameters in IPC covered trees continue to show greater diameters with statistical significance. All IPC trees are still HLB-negative and show less incidence of canker. We have continued monitoring the incidences of other pests and psyllids. After IPC removal in August 2020, we have been monitoring CLas infection of the uncovered trees in real time (monthly). We have not found significant results yet. Objective 2. Assessment of alternative netting approaches involved in ‘targeted’, ‘alternated’ or ‘patterned’ setup of IPC in groves for more cost-effective protection. As stated in our last report, after planting the new 700 trees last quarter in an alternated pattern we are monitoring for CLas in trees adjacent to the IPC-covered trees. Also, we are collecting data on psyllid populations and HLB incidence from  a few  commercial collaborators who are also evaluating different netting layouts under the CRAFT program. These results will yield more significant and quantifiable data in spring and early summer, when psyllid populations are yypically more abundant. Objective 3. Monitoring the transition from vegetative to reproductive stage in the covered trees as compared to the uncovered. We are collecting data on parallel experiments at SWFREC, Hendry Co, and Central Florida using Bingo, Early Pride, and Tango trees. These experiments will allow us to determine the ability of these varieties to set fruit in the absence of pollinators. We are seeing differences in fruit set. For instance, Early Pride and Tango are able to set fruit in a pollen free environment, but GA application increases this capacity. This effect is more evident in SugarBelle mandarins. Objective 4. Comparing IPC with CUPS-like systems. We followed up on the experiment we performed last season on the effect of deficit irrigation in all the varieties; in our last quarterly report  we communicated what we saw, that is we can control blooming: we saw more bloom  and fruit st inside CUPS and IPCs after applying deficit irrigation. We have seen a significant increase in yield and also, unexpectedly in fruit internal quality and peel color. We plan to continue these treatments as they may help develop better color in varieties resistant to degreening. Outreach activities performed in this quarter: -Alferez, F. Individual Protective Covers (IPCs): Ventajas y Problemas (In Spanish). Invited talk at the Citrus IPM Forum,  November 25, 2020, University of Puerto Rico.-Protected citrus growing systems: from healthy trees to high quality fruit. F. Alferez, SWFREC Zoom seminar series, October 2020.-F.Alferez, Citrus greening. Where are we now? Invited talk at the Southeast Regional Master Gardener Volunteer Virtual  Conference 2020. September 2020.-Canopy growth and physiological assesment of Valencia orange trees with and without protective covers. Gaire,S, Alferez, F and Albrecht, U. Presented at FSHS annual meeting, October 2020. This communication won the third position award in the Best Student Oral Presentation Competition.-Batuman, O. Individual and direct contact with CRAFT applicants to establish and evaluate IPC trials for psyllid and HLB control.   Publications:-Individual Protective Covers for Psyllid Exclusion and HLB Disease Prevention in Young Trees. Fernando Alferez, Susmita Gaire, Ute Albrecht, Ozgur Batuman, Jawwad Qureshi and Mongi Zekri.  Citrus Industry Magazine, November. -Gaire, S., Albrecht, U., Batuman, O., Qureshi, J., Zekri, M., Alferez, F. 2020. Horticultural performance of citrus trees grown under Individual Protective Covers (IPCs). Manuscript ready to be submitted to HORTSCIENCE    2. Please state what work is anticipated for next quarter: Objective 1.We will continue monitoring parameters described in the first section. Also we will monitor HLB progression after IPC removal in the first experiment.Objective 2. We will continue collecting data on psyllid populations and HLB incidence in the different netting layoutsObjectives 3 and 4.We will finish collecting data on fruit quality and yield for this sesaon and apply new irrigation deficit treatments. Outreach: -Individual Protective Covers’ by Alferez, F, Gaire, S., Albrecht, U., Batuman, O., Qureshi, J., Zekri, M., IN PREPARATION,to be submitted to EDIS.      3. Please state budget status (underspend or overspend, and why): We have spent about 60% of the budget. This is putting us on track of spending after COVID pause.Budgeted amounts for salaries and student stipend and tuition are being spent as predicted. 

Functional IPM for Asian citrus psyllid under circumstances of chronic HLB.

 Objective 2. Revise insecticide resistance management for psyllid IPM in new plantings An insecticide resistance management protocol is being 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.   ACP has developed resistance to neonictinoids in some Florida citrus groves. Therefore, we assessed the mechanism(s) conferring resistance in these ACP populations, compared to the highly susceptible laboratory colony of ACP. Knowing the mechanisms allows development of methods to reverse the problem where it exists and prevent it where it has not yet developed. Previously, we determined that resistance to the chemical fenpropathrin (pyrethoroid insecticide) occurs among populations of Asian citrus psyllid (ACP) after continuous exposure to this insecticide for 10 egg to adult generations of ACP. We also conducted a risk assesment for resistance  development to this insecticide in Florida, and compared relative expression of the gene CYP6A2-1 between insecticide resistant (RR) populations of ACP and a laboratory susceptible population (SS). Our results indicated that continuous exposure of ACP populations to fenpropathrin can generate  high levels of resistance (100 fold increase) and that this is, in part, caused by increased expression of cytochrome P450 (CYP) genes. Cytochrome P450 genes aid in metabolic detoxification and are one of the mechanisms contributing to fenpropathrin resistance in ACP. In our most recent experiments, we used the same populations of ACP that are known to be resistant to fenpropathrin to assess the stability of fenpropathrin resistance in ACP. We compared ACP populations with differing initial frequencies of resistance to understand the reversal process back to susceptability. In addition, we performed molecular analysis of fenpropathrin resistance by targeting the specific receptor that confers fenpropathrin insensitivity and also conducted biochemical analysis to determine how metabolic detoxification of fenpropathrin differs between resistant and susceptable populations of ACP.  First,  we collected ACP  from commercial citrus groves that exhibit resistance to fenpropathrin (RR)  and crossed (mated) these psyllids with ACP from the laboratory susceptible population (SS). The ratios of crossed populations that were established were 100RR+0SS, 75RR+25SS, 50RR+50SS, 25RR+75SS and 0RR+100SS. The five populations were set up on March 23, 2020 in a greenhouse environment without exposure to insecticides. Thereafter, we performed bi-monthly leaf dip bioassays assesing insect mortality in response to insecticide exposure to assess the stability of fenpropathrin resisitance over time. The leaf-dip bioassays were performed on June 12 and July 29, 2020 for the five crossed populations that were established. The bioassays determined the level of resistance to fenpropathrin in each population. These laboratory bioassays included 5 to 10 concentrations of each insecticide tested with 3-4 replications per concentration. Mortality counts of the insects were taken 48 h after being transferred into a room under the same environmental conditions used for insect rearing.   Our results showed no consistent changes in susceptibility of the established strains that consisted of 100% initially resistant individuals (100RR and 0SS cross) or 100% susceptible individuals (0RR and 100SS strain) after four months of rearing without insecticide exposure. In the resistant population (100RR and 0SS cross), resistance to fenpropathrin remained very high (RR > 100), while sensitivity of the susceptible population (0RR and 100SS strain) did not change over 4 months. However, resistance was not stable in populations of ACP that resulted from cross breeding of 25% resistant (RR) and 75% susceptible (SS) and 50% RR and 50% SS. In the case of the populations that were established with different initial frequencies of resistant ACP, fenpropathrin resistance declined over the course of four months in the absence of selection pressure. These results indicate that if a population of ACP develops resitance to pyrethroids in the field, this resistance can be bred out of the populaton through an influx of susceptible genes, if susceptible psyllids breed with the resistant individuals. Second, we worked to identify the specific knockdown (kdr) gene that confers resistance to fenpropathrin by cloning and sequencing genes from the laboratory population and then comparing them to the field population. For pyrethoids like fenpropathirn, a common mechanism confering resistance is a mutation in the sodium channel caused by differences in the kdr gene. Identification of specific mutations in the sodium channel gene required extracting genomic DNA from ACP samples from the SS population and then conducting PCR on three regions of the sodium channel gene designated as regions highly responsive to amino acid substitutions (V410, V930 and F1530). Primers for reach region were designed 100 base pairs (bp) up and downstream of the predicted substitution site (site of the mutation). PCR products were purified using a universal DNA purified kit, and the purified product was ligated into the pGEMT-easy vector. The recombinant plasmid was cloned into JM109 competent cells. The competent cells were spread on Luria-Bertani (LB) solid medium, selected based on color, and cultured to turbidity in LB broth media. We used gel electroporesis to analize the gene sequence. We found gene regions containing the V410, V930, and F1530 positions, and the specific amino acids containing substitutions within the kdr gene were amplified in the susceptible (SS) population. The results suggest that fenpropathrin resistance is likely unstable under field conditions, primarily due to the presence of localized susceptible ACP populations throughout Florida that periodically interbreed with those populations of ACP that develop resistance to fenpropathrin when sprays are not appropriately rotated. Also, the gene regions of the sodium channel that contain the V419, V930, and F1530 positions may have a role in resistance to pyrethroids in ACP.  In  future studies, we will continue to monitor insecticide resistance in populations of ACP in Florida citrus groves, as well as, the expression of the CYP6A2-1 gene using laboratory and field selected populations. Also, we will extract genomic DNA from 20 individuals from all five cross-resistance and susceptible populations after six or eight-months of monitoring insecticide resistance levels. While analyzing the three regions (V410, V930, and F1530) of the sodium channel gene that undergo mutation, we will determine how the mutations lead to fenpropathrin resistance. The ultimate goal is to understand how the mechanisms of resistance [metabolic totoxification vs. target site (sodium channel)] insensitivity interplay to confer stability of fenpropathrin resistance in ACP. If we can figure out how each mechanism or the combination of the two influences stability of resistance to this very important class of insecticides, we could then more effectively destabilize it, so that it can be quickly reversed in cases where resistance to pyrethroids shows up in the field.       

Why spray if you don't need to? Putting the IPM back into cItrus IPM by ground truthing spray thresholds

Objective: Develop threshold-based models for current use in Florida citrus. Background: In areas where huanglongbing is widespread, the occurrence of disease can reach a 90-100% within citrus groves. The use of insecticides addresses none of the symptoms of disease but instead reduces ACP populations. The objective of this experiment has been to optimize management of ACP using an economic threshold to time insecticide applications that also involves insecticide resistance management. We are investigating various insecticide rotation programs that we have developed based on previous research on managing resistance for ACP. These rotations are comprised of insecticides of various 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. Our purpose is to determine the economic threshold level that controls ACP populations effectively and whether this population level can be effectively maintained with an insecticide rotation that will prevent resistance development. Ongoing work and preliminary data In our first report, we described two areas where experiments are being carried out (Frostproof and Lake Alfred). In this report, we focus on the partial results obtained from the Frostproof location.  This is a commercial site with an estimated size of 80 hectares. Eight 30-tree blocks were established for monitoring. These consisted of either ‘Hamlin’ or ‘Valencia’ trees. There are 8 blocks in total with 4 blocks per variety. Each block is being monitored weekly by counting psyllid densities and feather flush intensity per block. Within each tree variety, two insecticide rotations are being compared and a treatment threshold of 0.5 ACP per tap sample is in place for triggering sprays in each rotation. Insecticides are applied when ACP populations reach approximately 0.5 ACP per tap. We are investigating the possible differences in ACP densities and feather flush intensity between these citrus varieties (‘Valencias’ and ‘Hamlins’) when using this threshold and two insecticide rotations. There were no differences in ACP densities between the four blocks of Valencias during the month of January 2020 prior to when treatments were made with ACP populations in all blocks averageing approximately 0.1 ACP per tap. The first insecticide application was made during the first week of March 2020 after ACP reached the 0.5/tap threshold population in the four blocks of Valencias. Two of them were sprayed with Movento (spirotetramat, inhibitor of acetyl CoA carboxylase) as part of ‘rotation A’ and other two blocks were sprayed using Exirel (anthranilic diamide, ryanodine receptor modulator) as part of ‘rotation B’. After insecticide applications, ACP populations were not reduced in blocks sprayed with Movento and weekly counts reached 0.4 ACP per tap. A second insecticide (Minecto Pro) application was triggered in these blocks on June 2 2020. In contrast, blocks sprayed with Exirel nearly completely eliminated the ACP population until May 21th, 2020.  The results observed in Hamlin have been different. The first insecticide (Exirel) application in these blocks was triggered by the 0.5 ACP/tap threshold in March 2020. Interestingly, in these blocks, the ACP population has remained below the thredhold until August, 2020.  Thus far, we have observed differences in ACP populations due to choice of insecticide that were not entirely unexpected. Exirel is a much more effective ACP adulticide than Movento, and the results have confirmed that Exirel reduces populations more effectively than Movento after application. Even though Movento can be a useful early season choice for application during bloom, the results suggest that ACP populations must be effectively eliminated after budbreak and prior to bloom instead of relying on the bee-safe Movento during bloom, if a threshold is going to be used for subsequent timing of sprays. The difference in ACP population densities between Valencia and Hamlin blocks treated with the same insecticide (Exirel) is more unexpected. These results were not related to differences in flush intensity between the two varieties. Flush intensity was similar in Valencia and Hamlin blocks during this period. Numerically, flush intensity was actually higher in the Hamlins than Valencias during some of the sampling dates, depite higher psyllid numbers in the Valencias than Hamlins. We will continue to monitor this pattern as the experiment progresses. To date, the data appear to indicate that the 0.5 ACP/ tap threshold is equivalently effective in Hamlins and Valencia, if a potent adulticide, such as Exirel is applied during the dormant period to knock down psyllids before trees begin flushing heavily. In constrast, if Movento is used an an early season treatment, as a means to comply with bloom period restrictions for bee safelty, psyllid populations are not effectively reduced and remain high throughout the spring and summer, triggering more sprays and sooner. These results appear to confirm that a broad spectrum and potent insecticide needs to be scheduled and fit in soon after flushing and before bloom and harvest in order to maintain ACP low during bloom and the subsequent early spring period. Without effectively suppressing ACP populations in the spring, a subsequent threshold-based psyllid management program is unlikely to be successful.        

Use of RNAi delivered by Citrus Tristeza Virus Vector to control the Asian Citrus Psyllid

The purpose of this project was to evaluate the use of Citrus Tristeza Virus viral vectors (CTVvv) containing promising RNAi constructs against the Asian citrus psyllid (ACP) in a small scale field trial in an attempt to duplicate the results obtained in growth chamber experiments. The goals of the project were to:

1. Determine if selected target sequences are effective in controlling ACP
2. Determine the effectiveness of the CTVvv as a delivery method of RNAi
3. Determine the effect of CTVvv+RNAi on CLas
4. Determine the effect of CTVvv+RNAi on the spread of HLB.

The field trial was established 8/23/2017 in a randomized complete block design with 4 different CTVvv-RNAi constructs targeting different ACP genes . At periodic intervals in 2018, 2019 and 2020, adult ACP (from a CLas infected colony) were caged on selected branches of the test trees. ACP survival, ACP reproduction, and citrus greening infection were assessed at 14 and 28 at each date and compared to trees with a CTVvv construct containing an antimicrobial peptide and to untreated control trees. In addition to the introduced caged ACP, tree branches that were naturally infested with wild ACP were also caged and subjected to the same assessments for ACP mortality and reproduction. In addition to the ACP and CLas assays, the status of the CTVvv+RNAi contructs was assayed at each assay date with respect to the presence of CTV in the trees and to determine if the CTVvv retained the RNAi insert or if it had reverted to a wild type CTV isolate (i.e. was the insert stable withing the vector).
With the exception of the initial sample date in April-2018, there were no differences in ACP mortality and reproduction rates for any of the RNAi constructs compared to the untreated controls. Similarly, there were no differences in CLas infection rates for any of the RNAi constructs compared to the untreated and controls. After 3 years in the field, essentially 100% of the trees were infected with citrus greening (under extreme challenge pressure). Vector stability appeared to be an issue for most of the RNAi constructs. By the end of the experiment, most of the CTVVvv+RNAi constructs had thrown out the gene target insert. The one contruct that did show some activity early on, was also apparently the most stable construct. The lack of contruct stability did not appear to be seasonal.

Sustainable Management of Asian citrus psyllid (ACP) and Citrus Production

This project evaluates four Integrated Pest Management (IPM) programs for ACP, including 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 biological control only. During this quarter, project activities were significantly impacted due to COVID-19. We had to stop all sampling and applications between March and May. When we sampled in the middle of May, ACP numbers were well above our treatment threshold of 0.1 adults per tap sample in all programs. Therefore, sprays were conducted immediately in programs 1-4. Program 1 plots were sprayed with Microthiol, program 3 with Voliam Flexi, while, program 2 and 4 plots were sprayed with 2% 435 oil. All these treatments provided some level of suppression during the next 2-3 weeks, but only Voliam Flexi treatment was able to bring the ACP populations down to 0.1 adults per tap sample for about two weeks. The next treatments warranted in all programs were delayed due to the continuing situation with COVID-19, and sprays were not applied until the last week of June. At that time, Program 1 and 3 plots were sprayed with Delegate, Program 2 with Entrust, and Program 4 with 2% 435 oil. We were able to measure the direct contact effect of all these treatments on ACP by caging the adults on the trees in the respective treatments. At 24 h after treatment application, 98-100% caged adults were dead in programs 1-3 and 85% in program 4. We started rereleasing the parasitoid Tamarixia in the second quarter of May at the biweekly rate of 800 adults per program. In June, we conducted exclusion experiments to observe the effect of natural mortality factors on ACP populations in all programs. The developing colonies of ACP nymphs were protected with the sleeve cages or left exposed to the natural mortality factors. The natural mortality factors impacted the nymphal populations of ACP, which was reflected in the less adult emergence in the uncaged colonies compared with caged colonies in all programs. Adult emergence from nymphs in the programs 1, 2, 3, 4, and 5, averaged 19%, 33%, 15%, 38% and 41%, respectively, in the uncaged colonies, and 92%, 80%, 62%, 80% and 76%, respectively, in the caged colonies. We were expecting less impact of natural mortality factors such as predators in programs 1 and 3 due to the negative impact of conventional insecticides on their populations; however, that was not the case. It seems that disruption in sprays between March and May diluted the previous effects of program-specific sprays on the populations of beneficial organisms, and they were able to spread across all programs. Spiders and the lacewing, particularly Ceraeochrysa cubana, were the most abundant predators and common in all programs.      

Optimizing Benefits of UV Reflective Mulch in Solid Block Citrus Plantings

This project evaluates young tree protection from ACP/HLB using approaches to integrate ground cover, insecticides, and irrigation management at three locations 1) Southwest Florida Research and Education Center (SWFREC), Immokalee, FL, 2) Citrus Research and Education Center (CREC), Lake Alfred, Fl, and 3) Florida Research Center for Agricultural Sustainability, Vero Beach Florida. Treatments include 1) soil-applied neonicotinoids interspersed with sprays of a 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 a 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. At the SWFREC and CREC locations, trees planted on mulch and bare ground received deficit irrigation and full irrigation treatments during this quarter. However, due to the COVID-19 restrictions, we were not able to follow up with the treatments for ACP control using sprays on the plots treated with deficit irrigation intended to produce synchronize flush and soil-applied neonicotinoids interspersed with sprays of a different mode of action insecticide on the plots receiving full irrigation until the middle of the May. All data collection activities were also suspended during that time. Six weekly samplings were conducted during remainder of May and June at SWFREC. There was an average of 4.5 flushes per plant and not different between those on mulch and bare ground or between the deficit and full irrigation treatments. Trunk diameter measurements were taken in June. Trunk diameter of both the rootstock and scion of trees on mulch was significantly larger than those on the bare ground, and those receiving full irrigation had a larger diameter than the ones receiving deficit irrigation. We are also testing tree defenders on the plants in bare ground. Trunk diameter of both the rootstock and scion of trees covered with tree defenders was not different from those planted on mulch but was larger than those planted on the bare ground but not covered with tree defenders. Soil moisture monitoring continued in the past and this quarter at SWFREC and CREC sites. Soil moisture data are being recorded continuously every 30 minutes at 6 inch and 18-inch depth.  There was 5-10% relatively greater soil moisture under mulch than the bare ground at the CREC location. Plants at the Vero Beach location were planted in March and are currently maintained at full irrigation until they are well established and ready for deficit irrigation treatment.At SWFREC, ACP adults were significantly less in the plants planted on the mulch than those planted on the bare ground averaging 0.3 and 0.1 per tap sample, respectively, but did not differ between the treatments of foliar sprays and soil applications of neonicotinoids intercepted with foliar sprays. Significantly less flush was infested with eggs of ACP on mulch than bare ground, averaging 9% and 16%, respectively. A similar effect was observed for flush infestation with nymphs, averaging 13% and 17%, respectively. However, there was no difference in flush infestation with eggs and nymphs between plants receiving treatments of foliar sprays and soil applications of neonicotinoids intercepted with foliar sprays. Sampling on ACP infestation was also conducted at CREC.      

Preventing young trees from psyllids and infection with CLas through use of protective netting

1. Please state project objectives and what work was done this quarter to address them:  Work in this quarter has been affected by the restrictions due to the COVID-19 situation. However, we have accomplished most of the work that was scheduled. Objective 1. Assessing tree growth and absence of psyllids and HLB disease symptoms (including CLas bacteria titer) under protective covering. We have continued monitoring trunk diameter and canopy area. Trunk diameters in IPC covered trees are starting to show bigger diameters with statistical significance. All IPC trees are still HLB-negative and show less incidence in canker. We have continued monitoring the incidence of pests and psyllids. We have shown that leaf drop is seasonally affected but under IPC is significantly less. Objective 2. Assessment of alternative netting approaches involved in ‘targeted’, ‘alternated’ or ‘patterned’ setup of IPC in groves for more cost-effective protection. After planting the new 700 trees last quarter in an alternated pattern we are monitoring CLas in trees adjacent to the IPC-covered trees. New plantings are expected to study the effect of patterned IPC disposition in the grove. Objective 3. Monitoring the transition from vegetative to reproductive stage in the covered trees as compared to the uncovered . Parallel experiments at SWFREC, Hendry Co, and Central Florida have been performed using Bingo , Early Pride, and Tango trees to determine the ability of these varieties to set fruit in the absence of pollinators. We will be collecting data on these trials starting next quarter. Objective 4. Comparing IPC with CUPS-like systems. We are monitoring the effect of deficit irrigation in all the varieties and we have seen that we can control blooming: we see more bloom inside CUPS and IPCs after applying deficit irrigation. Outreach activities  performed this quarter:-Tango Grower meeting by zoom, May 26. 40 growers attended.-Bingo Grower meeting by zoom, May 19, 26 growers attended.-Bingo Grower Working Group. Kickstart meeting at CREC, February 27.-In ServiceTraining for Extension Agents online: “Horticultural practices: IPCs”. 2. Please state what work is anticipated for next quarter: Objective 1. We will continue monitoring parameters described in the first section. Also we will continue chlorophyll and starch analysis. We will remove the covers in the trees from the first experiment to monitor HLB start and incidence rate.Objective 2. New plantings will be adopting the patterned IPC configuration.Objective 3. We will assess fruit set success after June drop and compare outside-grown trees with IPC- and CUPS-grown trees.Objective 4. We will continue to monitor growth and physiological parameters for comparison in CUPS, IPC, and outside-grown trees after deficit irrigation experiments. -Presentation at Citrus Expo: “IPCs. New data on tree performance and lessons learned”. Fort Myers, August 2020. Publications:-Individual Protective Covers’ by Alferez, F, Gaire, S., Albrecht, U., Batuman, O., Qureshi, J., Zekri, M., to be submitted to EDIS.-Gaire, S., Albrecht, U., Batuman, O., Qureshi, J., Zekri, M., Alferez, F. 2020. Horticultural performance of citrus trees grown under Individual Protective Covers (IPCs). To be submitted to Plants, MDPI      3. Please state budget status (underspend or overspend, and why): So far, we have spent about 33% of the budget. We are in the middle of the project duration, so we have underspend so far. This is due to two main things: 1) Budgeted meetings (national and international) and publications have not occurred yet, and 2) some expensive reactives for analysis have not been purchased as sampling has not been completed yet. Some delays in spending due to COVID have occurred as well.Budgeted amounts for salaries and student stipend and tuition are being spent as predicted.   

Use of RNAi delivered by Citrus Tristeza Virus Viral Vector to control the Asian Citrus Psyllid

1. Please state project objectives and what work was done this quarter to address them: 1.         A repetition of the experiment was conducted in late January through February over a period of five weeks. The protocol established is as follows:a.         Selected trees were scouted for flush or pruned to induce flushing.b.         Areas of flush were bagged and inoculated with 20 parent ACP.c.         Parent ACP were removed from bagged flush after two weeks.d.         First generation ACP were counted two weeks after removal of parent ACP.e.         The presence of native ACP life stages also documented.2.         In March all sentinel trees were sampled and tested using gel electrophoresis and rtPCR to confirm the absence of CTVvv-RNAi per permit requirements. Sentinels were also tested via qPCR for the presence of HLB.3.         Trial trees were selected for the next repetition of the experiment. These trees were also tested using gel electrophoresis and rtPCR to confirm the presence of CTVvv-RNAi, and qPCR for the presence of HLB.4.         Aphid scouting continues on a biweekly basis. The presence of brown aphid has not been detected.  2. Please state what work is anticipated for next quarter: The project will terminate and a final report will be issued   3. Please state budget status (underspend or overspend, and why):  On budget  

Functional IPM for Asian citrus psyllid under circumstances of chronic HLB.

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. We have continued to investigate the contribution of natural enemies on population regulation of Asian citrus psyllid (ACP) by measuring psyllid mortality of psyllids in response to natural enemies, where sentinel psyllids were deployed monthly on tree branches with or without screen exclusion cages. During the previous quarter, we have been able to process and analyze abundant data collected during previous quarters. We found that the mortality of uncaged ACP, where natural enemies had access to flush with eggs and nymphs, was higher than of caged ACP at organic sites during all seasons analyzed i.e. Summer, Spring and Fall. These results demonstrated that natural enemies reduced ACP population in organic management groves with no chemical input, which is consistent with previous investigations employing exclusion cages to measure the impact of biological control. As anticipated, psyllid mortality was not different between caged and uncaged sentinel psyllids in conventionally managed groves that received sprays of neurotoxins during the majority of our monitoring period. These results suggest that natural enemies may have contributed to regulation of ACP populations in organic groves more so than under intermittent use of conventional insecticides. We measured populations of ACP under both management regimes and recorded more ACP in areas treated intermittently with conventional insecticides than in organic blocks, suggesting that the level of insecticide input was insufficient to adequately reduce ACP populations. Furthermore, populations of natural enemies densities may have been impacted by the intermittent insecticide applications made in the conventional groves. Natural enemies’ population were similar in groves sprayed intermittently with conventional insecticides vs. groves managed organically and without use of chemical inputs for ACP. In both management groves, we found six different natural enemies including predators such as ladybeetles (Coccinellidae), syrphid flies (Syrphidae), green lace wings (Chrysopidae), long-legged flies (Dolichopidae), and spiders (Aranae) and parasitic wasp T. radiata (Eulophidae). Among these natural enemies, spiders were the dominant group of predators observed in both management groves. The mortality of ACP in an open cages in the exclusion experiment was higher even though other natural enemies were relatively fewer which may have been caused by abundant spiders. Another dominant natural enemy observed were Dolichopodids, which are predaceous; these especially feed on adult psyllids. The predatory Dolichopodid flies were found in higher numbers after spiders in both management sites. Ladybeetles are an important predator that contributes to mortality of ACP and were the third most frequently observed natural enemy in this study. During our study period, the abundance of ladybeetles was also not affected by the management regime. Tamarixa radiata is one of the key natural enemies of ACP, but we found very few wasps in both organic and conventional groves and occurrence of parasitism was rare. In addition to parasitism, female T. radiata also kill a proportion of psyllid nymphs by host feeding. The low numbers of Tamarixia could be due to indirect effects of predators such as Coccinellidae and Dolichopodidae. In previous work conducted in Central Florida, T. radiata was found to suffer mortality (64-95%) due to predation of parasitized nymphs by ladybeetles. Dolichopodids also compete for resources with Tamarixia and eat them when encountered during their searching behavior.  Objective 2. Revise insecticide resistance management for psyllid IPM in new plantings 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.   ACP has developed resistance to neonictinoids in some groves in Florida citrus groves. Therefore, we assessed the mechanism(s) conferring resistance in these ACP populations, compared to the highly susceptible laboratory colony of ACP. Knowing the mechanisms allows development of methods to reverse the problem where it exists and prevent it where it has not yet developed. In the current study, we took three genotypes (groups where individuals within groups are genetically similar, but where the groups differ from one another) of ACP with varying levels of resistance and susceptibility to neonictinoids, and comparatively investigated them. Specifically, we performed a detailed  transcriptional analysis comparing all of the ACP genes between resistant and susceptible populations. By figuring out which of these differences are important to development of resistance, we can better understand how the resistant populations changed because of resistance development and therefore what we need to do to reverse it. First, we collected psyllids from these areas, extracted their DNA, and conducted a data analysis called RNA-seq. We identified what are called differentially expressed genes (DEGs) between the resistant populations and our susceptable baseline (‘wild type’) population. These DEGs indicate differences between the resistant and susceptable populations. We found a total of 388 DEGs that distinguish the resistant Lake Alfred population from a known susceptable ACP population and 368 DEGs that distinguish the Wachula resistant psyllids from the susceptible population.  After identifying the differences between resistant and susceptable populations (the DEGs), we determinted the molecular function of the DEGs using a Web Gene Ontology Annotation Plot. The Gene Ontology (GO) classifications were used to predict and identify the specific functions for the identified DEGs. In other words, we determined what type(s) of protein differences are produced by the genetic differences between susceptable and resistant ACP. This tells us what the ‘genes do’ for the insect. Three hundred genes were annotated in this manner for the Lake Alfred population and 290 DEGs were assigned a function for the Wauchula insecticide resistant population. The cellular process, including cell and catalytic activity terms contained 120, 94, and 61 DEGs in Lake Alfred and were dominant in each of the three main categories such as biological process, cellular component, and molecular function. Likewise, 104, 84, and 59 DEGs were assigned to cellular proces, cell, and catalytic activity in the Wauchula population. The functions of some genes involved in metabolic processing, immune system processing, enzyme regulator activity, receptor activity, and catalytic activity were specifically related to expression of insecticide resistance development in ACP. Third, we conducted a procedure called functional enrichment analysis to identify which DEGs were significantly enriched, statistically, in the gene ontology GO using the Kyoto Encyclopedia of Gene and Genomes (KEGG). We did this to visualize the functional involvement of DEGs (differences between resistant and susceptable ACP) in various biological pathways in psyllids. Pairwise comparisons (Lake Alfred vs Wauchula) of the KEGG annotated proteins revealed two proteins absent in Lake Alfred, namely purine-nucleoside phosphorylase, and nicotinamide mononucleotide adenylyltransferase which are critical for the insect’s ability to metabolize (break down) nicotinates and nicotinamides (like neonicotinoid insecticides). In addition, three proteins that are in the cytochrome P450 family, including glutathione S-transferase and carbonyl reductase 1, were found present in high levels the Lake Alfred populations. In insects, these genes are specifically responsible for metabolism of xenobiotics, including insecticides. For the Wachula population, we identified one glutathione S-transferase that distinguishes this population from normal susceptable ACP populations.  Glutathione S-transferases are known specific proteins in insects that break down insecticides, including pyrethroids. To summarize, we identified very specific differences between two populations of ACP in Florida that show significant resistance to several insecticides as compared with ‘wild type’ susceptable ACP. We know that some of these genes are specific to neonicotinoids, which is congruent with our findings that neonicotinoids are most often the mode of action exhibiting significant resistance, but also found that more general detoxifying enzyme groups are more active in resistant psyllids. These results suggest that our current recommendations for rotation of five modes of action should continue working in Florida as a method for reversing and preventing resistance and that the situation does not appear to have worsened. However, the finding that the majority of the transcripts that we identified in this investigation are novel, and have not been previously implicated in insecticide resistance mechanisms in ACP and other insects, are surprising. Our new results  suggests that resistance development to neonicotinoids, such as thiamethoxam, in ACP is more complex than previously described. Our new results implicate the participation of a broad array of novel resistance genes and possible resistance mechanisms working in concert. We continue conducting more detailed  quantification of expression of the selected genes we have identified for the first time. These results will be incorporated into our insecticide rotations protocols, and if they improve resistance management, they will be incorporated into our spray recommendations for FL citrus.    

Why spray if you don't need to? Putting the IPM back into cItrus IPM by ground truthing spray thresholds

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.  We investigated the relationship between insecticide rotation using various modes of action and implementation of specific pest threshold levels for timing of these insecticide applications. Our goal is to determine how these factors influence ACP and insecticide resistance management, as well as their economic viability. First, we monitored adult psyllid populations before and after application for three threshold levels by tap sampling (0.2, 0.5, and 1 adult per tap sample). There are two rotation schemes in replicated plots in an experimental grove in Lake Alfred. The effectiveness was evaluated by weekly counts of ACP adults. Tap samples were taken from 20 trees per replicate and there were 4 replicates for each treatment. Second, we are using an insecticide bioassay to monitor changes in susceptibility to insecticides before and at the end of each rotation modules. The difference in susceptibility before and after each application is being used to determine if susceptibility to a given insecticide is shifting in populations exposed to a given module. For each bioassay, 5 insecticide representing different chemistries and mode of action are being evaluated. For each insecticide, five to seven concentrations were tested that have mortality levels between 1 and 99% based on preliminary studies. Test the effect of concentration for each insecticide, we are using commercially formulated products diluted in distilled water on the day of testing. Before application, we found low to moderate resistance for thiamethoxam (RR = 5.25), imidacloprid (RR = 5.97), cyantraniliprole RR = 1.79) (RR=resistance ratio).  Third, we have been collecting samples beginning in March 23, 2020. Leaf samples have also been collected from each plot to determine the presence of Candidatus liberibacter asiaticus in the trees and determine the infection rate. When counts of ACP adults in any plot reach a previously defined threshold level, a spray has been applied within the insecticide rotation developed for that particular treatment. On April 21, 2020, the 0.2 ACP per tap treatment threshold was exceeded, and therefore on May 5, 2020, we applied dimethoate for one rotation scheme and fenpropathrin for the other. After application, the ACP populations in all areas that were treated decreased to low levels.  Finally, our goal is to investigate the correlation between the threshold ACP population level required to trigger a management spay within the context of effective insecticide resistance management. For each insecticide application, the insecticide mode of action, we are testing differences in population wide levels of acetylcholinesterase, nicotinic acetylcholine receptors, inhibitors of chitin biosynthesis, glutamate gated chloride channels, allosteric modulators, and voltage gate sodium ion channels. In this manner, we will develop an economical and sustainable management strategy for ACP with insecticides, which is still currently lacking in Florida. Our newly developed methods should have a positive impact on the management of Asian citrus psyllid populations by stabilizing or reducing resistance and increasing their economic viability.           

Optimizing Benefits of UV Reflective Mulch in Solid Block Citrus Plantings

This project evaluates young tree protection from ACP/HLB using approaches to integrate ground cover, insecticides, and irrigation management. Treatments include 1) soil-applied neonicotinoids interspersed with sprays of a 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 a 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. This experiment is planted at three locations 1) Southwest Florida Research and Education Center (SWFREC), Immokalee, FL, 2) Citrus Research and Education Center (CREC), Lake Alfred, Fl, and 3) Florida Research Center for Agricultural Sustainability, Vero Beach Florida. During this quarter, irrigation set up for the experiment was completed, metalized reflective mulch installed, and 600 Ruby Red grapefruit trees on US 897 rootstock planted at the location in Vero Beach. These trees will receive irrigation at the same rate for a couple of months so that they are well established before they are subjected to deficit irrigation treatment to synchronize flush and start comparing the treatment of soil-applied insecticides rotation with foliar sprays against sprays on synchronized flush. The locations at SWFREC and CREC were already planted and were subjected to treatments of soil and spray applications for ACP control. However, the treatments to control irrigation to synchronize flush were initiated in February this year. In Immokalee, observations were made overtime during this quarter to look for the plant colonization with adult psyllids and plant infestation with eggs and nymphs. For plants on mulch, there was an average of 0.02 adults per plant (n = 984) much less than those on the bare ground averaging 0.08 adults per plant (n = 864). On mulch and bare ground plantings, the percentage of plants observed with adults was 0.6% and 4.7%, respectively. The plants treated with soil-applied neonicotinoids rotation with spray applications averaged 0.04 adults per plant (n = 930) and those treated with spray applications only averaged 0.06 adults per plant (918). There was no difference in the flushes between the mulch and bare ground treatments. There was not much impact of the deficit irrigation treatment to regulate tree flushing probably because the time to measure the impact was not enough for the reported period, or the irrigation schedules need to be adjusted. The percentage of flush infested with psyllid immatures averaged 1% on the mulch, and 10% on the bare ground planted trees. Plant tissue samples (n = 80) were collected and processed from the SWFREC experiment for PCR analysis, and none were positive for HLB. Data on plant growth and incidence of ACP was also recorded at the CREC location. We have also been continuously collecting data on soil moisture, and soil temperature changes by treatments at both locations and soil moisture sensors were installed at the Vero Beach location this quarter.