ACP Vector

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:  Objective 1. Assessing tree growth and absence of psyllids and HLB disease symptoms (including CLas bacteria titer) under protective covering (i.e., IPC). The most striking finding in this objective during the last quarter has been the finding that fruit retention was significantly improved in trees that were covered by IPCs, resulting in larger yields and improved internal quality. As a reminder, we removed covers in August 2020. These trees bloomed and set fruit for the first time in February 2020; therefore, this represented the first crop. Fruit drop was non-existent in trees that had been protected by the IPCs, whereas non-covered trees showed 60% fruit drop. Fruit quality was also dramatically improved by use of IPCs: Brix was 10.9 in IPC trees whereas in non covered trees Brix was 7.5.   Objective 2. Assessment of alternative netting approaches involved in ‘targeted’, ‘alternated’ or ‘patterned’ setup of IPC in groves for more cost-effective protection. We have started analyzing samples to determine HLB incidence in the different layouts and if layout dictates different psyllid colonization.  Objective 3. Monitoring the transition from vegetative to reproductive stage in the covered trees as compared to the non-covered trees.In the new plots, after having a good bloom in all three varieties (SugarBelle, Early Pride and Tango) covered by IPCs, we assessed fruit set and did not find significant differences compared to non-covered trees. This is promising, since it shows that these varieties do not need to be exposed to pollinators to set fruit, and IPC protection could potentially be prolonged to get the trees well into the productive age, producing high quality fruit, as we have shown in Objective 1. By applying brassinosteroids we expect to  prolong further tree health and produce a commercial-size crop of good quality fruit.    Objective 4. Comparing IPC with CUPS-like systems. We have collected data on bloom and fruit set on deficit irrigation plots for the second year, and we were able to confirm more bloom and fruit set in protected trees. We installed an automated irrigation system that is helping us to perform these treatments consistently. We have also installed several moisture probes to fine tune the treatments.  Outreach for this quarter:-Alferez, F. Citrus Institute 2021. Virtual. April 6.Individual Protective Covers (IPCs) influence on tree performance, fruit production, pests, and diseases. -Alferez, F. Invited presentation at CRDF BOD meeting. April 26, Arcadia. -Alferez, F, Batuman, O, Gaire, S, Albrecht, U, Qureshi, J. Assesing spatial patterns of IPCs deployment in young citrus. Submitted to Citrus Industry -Batuman, Alferez, Qureshi: Assesing spatial patterns of IPCs deployment in young citrus. CRAFT TWG meetings and one-on-one grower meetings. 2. Please state what work is anticipated for next quarter:Objective 1. We will perform regular work on horticultural/pathology parameters in all plots.Objective 2. We will continue collecting data on psyllid populations and HLB incidence in the different netting layouts. Our idea here is to find any seasonal differences that can be affected by the different patterns.Objectives 3 and 4.We will continue collecting data on fruit set and fruit growth and maturation for this second season of deficit irrigation treatments. We will also monitor early fruit drop, if it occurs.  Outreach:-Gaire, S, Alferez, F, Albrecht, U. Horticultural attributes of SugarBelle, Tango and Early Pride mandarin trees grafted on two different rootstocks grown with and without individual protective covers (IPCs). ASHS Annual meeting. August 5-9, 2021, Denver CO   3. Please state budget status (underspend or overspend, and why):We are on track with activities  and spending. Budgeted amounts for salaries and student stipend and tuition are being spent as predicted. We expect to spent more this quarter, as we will be paying publication fees for our first paper accepted and registration for the ASHS annual meeting in Denver, CO.   

Disrupting transmission of Candidatus Liberbacter asiaticus with antimicrobial therapy

1. Please state project objectives and what work was done this quarter to address them:Objectives1. Quantify the effect of citrus antimicrobials on vector fitness.  2. Determine the effect of antimicrobials on Las transmission.  3. Determine the effect of antimicrobials on plant response and associated ACP behavior.   The goal of this proposal was to determine whether antimicrobial treatments interfere with Asian citrus psyllid (ACP) populations and their capacity to transmit Candidatus Liberibacter asiaticus (Las), the causal agent of citrus greening (HLB). The premise of this technique is to kill Las with application of registered antimicrobials, streptomycin (Firewall) and oxytetracyline (Fireline) to the tree beyond the pathogen’s capacity to survive. These data suggest that antimicrobials used for CLas management, particularly oxytetracycline, elicit lethal and sublethal effects in D. citri. We determined that antibiotic use has a negative impact on ACP, therefore there is utility of these applications for ACP management in addition to any effects on Las in trees. ACP feeding on bactericide treated trees could be less capable of transmitting CLas to healthy trees. This can be due to 1) trees treated with bactericides are more likely to have lower CLas titers for acquisition and 2) CLas in infected will be reduced or eliminated when ACP feeds on bactericides, and 3) trees treated with bactericides prior to ACP will be protected from CLas inoculation. Given the limited efficacy of antimicrobial treatments to eliminate CLas completely in infected trees, it appears that their greatest benefit may due to their negative effects on the ACP vector and CLas transmission.  2. Please state what work is anticipated for next quarter: The project is complete. The final work will consist of completing data analysis and submitting manuscripts for publication. 3. Please state budget status (underspend or overspend, and why): Our budget was slightly underspent due to orders for materials to extract DNa not received by the project end date.    

Developing near and long-term management strategies for Lebbeck mealybug (Nipaecoccus viridis) in Florida citrus

Work done this quarter (continued on Word document)(1a) Develop monitoring methods to time management actionsWe have developed methods for following the seasonal populations of lebbeck mealybugs and are working with 7 growers throughout central Florida to monitor populations. Based on data collection through the end of May 2021, there is some consistency in the onset of population development, with mealybug populations increasing in March and April concurrent with early fruit development. Lebbeck mealybugs appear to be highly attracted to developing fruit and establish populations around the calyx at fruit set. In our study and in a fruit drop study by a colleague, we are seeing high levels of fruit drop related to the mealybugs not related to physiological fruit drop. While we are not quantifying it in our phenology study, Dr. Vashisth has added this quantification to her fruit drop trials.Along with population monitoring, we are evaluating the potential use of virgin females as a lure for males with the intention of using these data to develop a targeted lure in the future to help reduce field populations and detect cryptic populations before they increase to damaging populations. No data yet, this method is in the trouble shooting stage.(1b) Adjuvant screeningPreliminary trials have been conducted with adjuvants alone to determine their lethality to lebbeck mealybug. A total of 10 adjuvants, including and 4 from Helena Agri Enterprises were mixed with DI water at label rates and sprayed until dripping on Volk lemon leaves with mealybugs attached. 9 of the adjuvants resulted in significantly greater mealybug mortality over a 7-day period compared to a DI water control. (1c) Evaluate promising materials in open grove setting: We began a field trial at a 10-acre commercial citrus planting that was heavily infested in 2020. First insecticide applications occurred as a pre-bloom prophylactic spray of a systemic material, with a second spray planned in the end of April.(1d) Ant Management We are testing a variety of methods to remove red imported fire ants (Solenopsis invicta) from citrus groves. Four experimental treatments were tested and compared to an untreated control: Chlorpyrifos drench, Clinch ant bait (Abamectin), Extinguish ant bait (S-methoprene), and spot treating ant colonies with hot water. Fire ant abundance was assessed by counting the number of surviving colonies, and by determining presence/absence of foragers with pecan sandie baits. Natural enemy abundance was assessed by dissecting mealybug clusters for predator larvae. Sampling for each of these methods took place every 2 weeks.Currently, all treatments have been applied and we are conducting follow-up surveys to determine their efficacy. Preliminary data shows that both insecticidal baits and hot water treatments significantly reduced fire ant colony abundance, while the chlorpyrifos drench had no effect on colony abundance.Impacts on mealybug predator population development is currently being quantified now that populations are starting to establish.(1e) Evaluate management options for IPCsEntomopathenognic Fungi (EPF) data shared last quarter. More work to come.(2a) Predator assessmentsPredators who directly consume prey:The commercially available predators Cryptolaemus montrouzieri, Orius insidiosus, Adalia bipuctata, Hippodamia convergens, and Chrysoperla carnea have been screened using no-choice assays to determine if they will feed on larval lebbeck mealybugs, and on mealybug ovisacs. Additionally, several wild-caught and lab reared predators have also been screened, including Harmonia axyridis, larva of the genus Ceraeochrysa (colloquially called trash bugs), Diomus austrinus, and Coccinella septempunctata.  Both adult and larval C. montrouzieri readily feed on lebbeck mealybug larvae and ovisacs, as do larval Ceraeochrysa. Larval Chrysoperla carnea and adult Diomus austrinus consistently consume mealybug larvae, but do not consume ovisacs. Adult Orius insidiosus, early instar Adalia bipunctata, and adult and larval Harmonia axyridis do not feed on lebbeck mealybug larvae or ovisacs. Adult Hippodamia convergens, Coccinella septempunctata, and Adalia bipunctata do feed on some lebbeck mealybug larvae, but appear to attack and kill the larvae more than fully consuming them. Neither feed on ovisacs. From our results, only Cryptolaemus montrouzieri shows promise as a commercially available biological control agent for lebbeck mealybug, although Ceraeochrysa larvae may act as natural biological control in the grove. Chrysoperla carnea may be useful for controlling mealybug larvae, but not reproductive adult females.Parasitoids:Limited numbers of Anagyrus pseudococci, a commonly used parasitoid for mealybugs in greenhouses, were tested to determine if they would parasitize lebbeck mealybug adults and ovisacs. A. pseudococci adults were placed in arenas with adult lebbeck mealybugs, or ovisacs and left for 40-48 hrs. Surviving parasitoids were then transferred to adult Citrus mealybugs and given an opportunity to parasitize them, to act as a positive control. Mealybugs were checked for parasitism after 16 days. The majority of parasitoids died in arenas with lebbeck mealybugs, and none parasitized adults or ovisacs. However, half of those that survived went on to parasitize Citrus mealybug adults. These preliminary results suggest that A. pseudococci will not readily parasitize lebbeck mealybug, however further trials with more individuals and life stages of mealybugs are needed.Gut content assays of field-collected potential predatorsSpecies-specific primers for lebbeck mealybugs and a DNA extraction protocol have been established and used in feeding bioassays with the predator mealybug destroyers (Cryptolaemus montrouzieri). Using DNA extracted from the predators after they fed on the mealybug in PCR, we were able to model the length of time that the mealybug DNA fragment associated with our primers is detectable in mealybug destroyers. We found that the lebbeck mealybugs amplicon was detectable in 100% of mealybug destroyers for 4 hours after feeding, in 40% of mealybug destroyers up to 56 hours after feeding and was no longer detectable in mealybug destroyers 60 hours after feeding. Field collections were executed in mealybug infested groves August 2019 through December 2020. Species identified as potential predators of lebbeck mealybugs were selected for DNA extraction and PCR using the lebbeck mealybugs primers. So far we have found that green lacewings have the lowest abundance but the highest rate of positive detection of lebbeck mealybugs DNA. Field-collected mealybug destroyers has the next lowest abundance and the next highest rate of positive detection of lebbeck mealybugs DNA. The most abundant predators are spiders, but which have a far lower rate of positive detection of N. lebbeck mealybugs DNA. The spider which tests positive most frequently is the jumping spider, Hentzia palmarum. Predatory flies in the family Dolichopodidae will also be screened for lebbeck mealybugs DNA using our primers.  (2b) Determine how to implement mealybug management concurrent with other pest management programsStarting in July 2021 for field efficacy then planning to test programs in Spring 2022(2c) Determine what insecticide chemistries inhibit feedingWe have completed EPG documentation of lebbeck mealybug to determine wavelength correlations on one host plant and will be starting on a second host plant once plants are voided of insecticides (3-4 month holding process in the greenhouse to). Baseline feeding interaction data are necessary for a minimum of 3 host plants prior to including insecticides to look at feeding inhibition and the ability to kill the adult female in her ovisac. (2d) Develop tools to minimize spreadKilling crawlers with isopropyl alcoholWe tested different concentrations of isopropyl alcohol to determine how lethal they are to 1st instar lebbeck mealybugs. 50%, 70%, and 90% solutions of isopropanol were sprayed onto mealybug crawlers placed on cloth swatches, and compared to a DI water control. Both 1 spray at each concentration, and 2 sprays at each concentration were tested. Mortality was assessed after 5, 10, and 15 minutes. The test was repeated, this time assessing mortality at 30 min, 1hr, and 2hrs. For all concentrations of isopropanol, 1 spray resulted in significantly greater mealybug mortality compared to the control. However, several mealybug crawlers remained alive and active after a single spray for all concentrations. 2 sprays of each concentration resulted in almost 100% mortality or incapacitation at all time points. Using steam to kill adult mealybugs and ovisacsAdult mealybugs and ovisacs may be accidentally transferred from infested groves on tools and equipment. Steam treatments may be an effective method of sanitizing equipment and killing both adults and ovisacs. Using a steam cabinet on-station, we steam treated adults and ovisacs at 100, 120, and 130 degrees Fahrenheit for 5, 10, and 15 minutes to determine mortality. Previous preliminary experiments showed steam treatments at 130 degrees Fahrenheit for 15 and 30 minutes resulted in 100% mortality. Mortality was assessed at 0, 3, and 5 days after treatment, and ovisacs were held for an additional 2 weeks to see if crawlers emerged. At 100 degrees F for all times points, adult mealybug and ovisac mortality was not significantly different than the control. At 120 degrees F for 5 minutes, mortality was also functionally 0%. However, mortality rose to 100% at 120 degrees F for 10 and 15 minutes. At 130 degrees F, mortality was 100% for all time points. This week we have begin testing ovisacs embedded in IPCs, which are insulating the ovisacs, requiring higher temperatures and longer time points. 2. Please state what work is anticipated for next quarter:(1a) Seasonal population monitoring will occur at 7 groves for one calendar year. Virgin female lure traps will be deployed once seasonal rains end.(1b) Adjuvant screening will continue to determine optimal adjuvants to work in synergism with insecticide sprays. We plan to take the most effective adjuvants and add them to an insecticide with good, but not great, efficacy on its own.(1c) We will continue treating and scouting the grove we have started a management comparison trial at.(1d) The ant management project will continue throughout the upcoming quarter with the addition of monitoring for predatory insect establishment.(1e) IPC management trials will continue, incorporating conventional materials for management and spray penetration by tractor mounted sprayers (this was delayed from our work plan last quarter by unexpected challenges in establishing infestation).(2a) Working with FDACS, we have permission to deploy sentinel infested materials to screen more broadly for predators that may be present in the system which we missed with haphazard rearing from infested grove sites. We plan to do this starting in late summer 2021, once higher populations have established, which would attract a higher abundance and diversity of predatory insects.(2c) Feeding mechanisms will continue to be worked out using EPG with the future plan to determine if we can interfere with their feeding via chemical intervention leading to reduced offspring production.(2d) We will continue evaluating sanitation options and are planning to look at vehicle & equipment sanitation in the next quarter. 3. Please state budget status (underspend or overspend, and why): on target  

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

The objective of this experiment is to determine the effect of incorporating action thresholds for ACP on populations of natural enemies as well as secondary pests of citrus. ACP vector control is a basic component of HLB management even in situations where disease incidence is high. The use of action thresholds is a possible strategy to reduce unnecessary insecticide input to improve economic return, and reduce potential for insecticide resistance. The idea is to spray only when one reaches a population level of ACP that cause economic damage rather than spraying for ACP all the time. We suspect that an ancillary benefit of using action thresholds will be to reduce the negative impact of insecticides on populations of natural enemies and/or reduce secondary pest infestations because we spray less for ACP control. This may provide further benefit by improving activity of biological control. While the main goal of this investigation is to establish economic thresholds for ACP control to optimize returns on investment, we investigated this ancillary question of whether reducing insecticide input for ACP control may affect populations of beneficial natural enemies in citrus groves.  A year-long study was initiated on March 5, 2021 in an experimental sweet orange grove under standard cultural practices located in Lake Alfred and with an estimated initial HLB infection level of 100 %. Prior to the start of the experiment, the entire site was sprayed with pyrethroid. Four replicates were established for each threshold treatment. Insecticide sprays for ACP were based on nominal thresholds of 0.2, 0.5, or 1.0 adult per stem tap and ACP treatment sprays were made of different insecticide modes of action in rotations. There were seven applications for the 0.2 adults per tap threshold treatment, five applications for the 0.5 adults per tap threshold treatment, and two applications for the 1.0 adult per tap threshold treatment. Also, a calendar based positive control treatment was established where ACP sprays were applied once per month, irrespective of ACP population density. ACP were monitored by calculating the mean number of adults per tap from ten randomly selected trees across all four plots for each treatment. Tap samples were made by holding a 22 × 28 cm plastic laminated white paper sheet horizontally underneath a randomly chosen branch, which was then struck sharply three times with a 40 cm length of PVC pipe. ACP adults falling on the sheet were quickly counted to obtain the number of ACP adults per stem-tap and tree. If the means reached or exceeded the target economic threshold, all replicate plots assigned to that treatment threshold were sprayed. ACP sampling occurred every 7 to 15 d.  Populations of ACP and natural enemies were assessed every week from March 19 to April 28, 2021 using the stem tap sample described above. Spiders (Araneae), arboreal ants (Hymnoptera: Formicidae), lady beetles (Coleoptera: Coccinellidae) and lacewings (Neuroptera) previously identified as key natural enemies of ACP or other important citrus pests were counted. Counts were made by visually inspecting randomly selected tree branches in earch plot for 2 minutes per sampling period and counting all arthropods found during that period. Inspections were conducted weekly. Arthropods were identified to species level where possible. Ants were collected with pointed round paint brushes and preserved in 70% ethanol and identifications were made subsequently.  Cumulative numbers for Asian citrus psyllid were 156, 129, 143 and 135 for the 0.2, 0.5, and 1.0 psyllid per tap, and calendar treatments, respectively. There were 8, 11, 8 and 4 spiders found for the 0.2, 0.5, and 1.0 psyllid per tap, and calendar treatments, respectively. We counted 25, 50, 41, and 51 arboreal ants for the 0.2, 0.5, and 1.0 psyllid per tap, and calendar treatments, respectively. There were 3, 1, 0. and 0 lacewings for the 0.2, 0.5, and 1.0 psyllid per tap, and calendar treatments, respectively. There were 10, 16, 16, 22 ladybird beetles for the 0.2, 0.5, and 1.0 psyllid per tap, and calendar treatments, respectively. Finally, there were 1, 4, 2 and 1 citrus root weevils for the 0.2, 0.5, and 1.0 psyllid per tap, and calendar treatments, respectively. This experiment will run throughout the remainder of the summer. At this early point in the esperiment, we have not yet observed an effect of reducing sprays by employing treatment thresholds on populations of natural enemies as compared with plots treated with calendar sprays. However, we predict that differences may emerge as the investigation progresses, based on our previous experience. The possible improvement in the effect of biological control as a consequence of using treatment thresholds for timing sprays against ACP may prove to be a means of integrating insecticides with biological control for psyllid management.                                 

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

In this quarter, monitoring and management activities continued in all of the Integrated Pest Management (IPM) programs for ACP which include: 1.  conventional and organic insecticides plus biological control, 2.  organic insecticides, and Horticultural Mineral Oil (HMO) plus biological control, 3.  conventional insecticides plus biological control 4.  HMO plus biological control. 5.  biological control only. The dormant spray applications in February included Danitol in programs 1 and 3, Pyganic + 435 oil (2%) in program 2, and 435 oil (2%) in program 4. The March sprays included Microthiol in program 1, 435 oil (2%) in program 2 and 4, and Movento in program 3. Nine samplings were conducted to monitor for ACP and beneficial arthropods. February sprays conducted in the middle of the month kept psyllids below treatment threshold of 0.1 adults per tap sample across all programs until the second week of March. After March sprays a decreasing trend in the populations of ACP was seen in all programs and numbers dropping from 0.2-0.3 adults per tap sample to 0.1 adults per tap sample by the end of the month. Within the predator guild, spiders and lacewings continue to be the dominant groups and among lacewings Ceraeochrysa cubana most dominant. In the laboratory, C. cubana larvae were more tolerant to imidacloprid than C. claveri or D. citri. Ceraeochrysa cubana seems to have a significant potential to be used in biological control against D. citri.    A preliminary laboratory study testing a predator prey ratio of 1:1 (mite: ACP egg) showed that the predatory mite Amblyseius swirskii provided 50% reduction in ACP. Follow up experiments were initiated in the greenhouse using different densities of this predator against ACP. Residual toxicity of March sprays was evaluated against A. swirskii. Three days after sprays leaves were collected from the treated trees and exposed to the predatory mite in the experimental arenas in the laboratory. After, 24 h of exposure 12% mortality was observed in the Movento treatment, and 8% in the Microthiol and 435 oil treatments. Amblyseius swirskii were also released in the field within a week of March sprays. Approximately 5,000 mites per plot and total of 20,000 per program were released. No predatory mites were observed in a sample conducted before the release. After release, 20% of the sampled trees in program 3 sprayed with Movento were found to contain predatory mites, 30-32.5% in the program 2 and 4% sprayed with 435 oil, 35% in program 1 sprayed with Microthiol and 40% in the program 5 which is untreated control.      Colonies of field collected ACP from all programs were established in the laboratory and their subsequent generations tested for tolerance to dimethoate, fenpropathrin, and imidacloprid in comparison with a laboratory colony. The concentration required to kill 50% of the field population averaged 2, 4 and 8 times more for dimethoate, fenpropathrin, and imidacloprid, respectively, than the concentration required to kill same percentage of ACP from the laboratory colony. Leaf samples were collected from all programs and submitted for HLB analysis in March. Analysis of the leaves collected in Fall 2020 showed that 80-95% samples collected across all programs were HLB positive. Although, no Tamarixia radiata was released in the programs during this quarter due to the problems with the colonies at the FDACS-DPI, Dundee, facility, the previous work on the parasitism activity of T. radiata tested at different times of the day and temperatures was written and a presentation on the same work given at the Southeastern Branch Meeting of the Entomological Society of America and Florida Citrus Grower Institute.    

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, FL. Treatments of 1) soil-applied neonicotinoids interspersed with sprays of a different mode of action insecticides on a calendar basis, and 2) rotation of insecticide modes of action sprayed twice on each major flush are being implemented to the trees on UV reflective and bare ground. The irrigation deficit treatments are also functional at the SWFREC and CREC locations to trees on UV reflective and bare ground to synchronize flush to target spray applications on major flushes. Sampling was conducted to monitor flush abundance and psyllid populations at three experimental sites. At the Vero Beach location, significantly more flush was observed in the trees on mulch compared to the trees on bare ground averaging 4.09 and 3.47 per plant, respectively. The diameter of the trunk of the rootstock and scion of the trees on the mulch was significantly more than the trees on the bare ground. An average rootstock diameter of 33.79 mm on the mulch and 28.20 mm on the bare ground was observed. Scion diameter averaged 24.04 mm on the mulch and 21.12 mm on the bare ground. At the CREC location, on average, there was also significantly more flush observed on mulched trees than on bare ground trees. Analysis of data from September 2020 to March 2021 showed significantly fewer adults on trees with mulch than with no mulch. Trees with insecticides applied based on a calendar schedule also had fewer adult ACP than those that experienced insecticide applications based on flush. ACP adult abundance was weakly but positively dependent on flush abundance. Peaks of adult counts occurred between March to June as well as September to October in 2020 and this is where treatment differences were starkest. Significantly fewer eggs and nymphs were observed on the trees with mulch than those on the bare ground. At SWFREC, trends on flush abundance across treatments and ACP response to the treatments were similar to other two locations. During this quarter, the mulched and non-mulched treatments’ soil moisture contents varied between 0.11±0.02 m3 m-3 and 0.10±0.01 m3 m-3 showing not much difference in water storage with the root zone. Overall, canopy density appears to be greater on mulched than non-mulched trees. In the next quarter, we will do leaf and soil nutrient analysis and canopy size determinations to see changes if any in the treatment effects.  At SWFREC, several experiments were conducted using the remote-controlled and manual ground-penetrating radar (GPR) operation to evaluate the effort needed to perform the GPR scanning, measure the required time to complete the scanning process, evaluate precision and accuracy of the scanning circles, and determine if the remote-controlled system can perform better for non-invasive root detection than the manual process. The scan line data collected from both the remote-controlled and manual process were compared after processing the data using an auto root detection software (Tree Radar Inc., Maryland, USA.) The results of root detection for both processes were almost the same, and there were no significant differences between them. The depth of the detected roots in the remote-controlled process was same as of the manual process. These experiments show that the remote-controlled process can reduce the required application time by 3 times when compared to a manual process. It can also reduce the human effort required and increase the precision of the data collection process. A Qualtrics survey to obtain growers input on the reflective mulch technology was developed and distributed through several channels.         

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

In this quarter, monitoring and management activities continued in all of the Integrated Pest Management (IPM) programs for ACP which include: 1.  conventional and organic insecticides plus biological control, 2.  organic insecticides, and Horticultural Mineral Oil (HMO) plus biological control, 3.  conventional insecticides plus biological control 4.  HMO plus biological control. 5.  biological control only. The dormant spray applications in February included Danitol in programs 1 and 3, Pyganic + 435 oil (2%) in program 2, and 435 oil (2%) in program 4. The March sprays included Microthiol in program 1, 435 oil (2%) in program 2 and 4, and Movento in program 3. Nine samplings were conducted to monitor for ACP and beneficial arthropods. February sprays conducted in the middle of the month kept psyllids below treatment threshold of 0.1 adults per tap sample across all programs until the second week of March. After March sprays a decreasing trend in the populations of ACP was seen in all programs and numbers dropping from 0.2-0.3 adults per tap sample to 0.1 adults per tap sample by the end of the month. Within the predator guild, spiders and lacewings continue to be the dominant groups and among lacewings Ceraeochrysa cubana most dominant. In the laboratory, C. cubana larvae were more tolerant to imidacloprid than C. claveri or D. citri. Ceraeochrysa cubana seems to have a significant potential to be used in biological control against D. citri.    A preliminary laboratory study testing a predator prey ratio of 1:1 (mite: ACP egg) showed that the predatory mite Amblyseius swirskii provided 50% reduction in ACP. Follow up experiments were initiated in the greenhouse using different densities of this predator against ACP. Residual toxicity of March sprays was evaluated against A. swirskii. Three days after sprays leaves were collected from the treated trees and exposed to the predatory mite in the experimental arenas in the laboratory. After, 24 h of exposure 12% mortality was observed in the Movento treatment, and 8% in the Microthiol and 435 oil treatments. Amblyseius swirskii were also released in the field within a week of March sprays. Approximately 5,000 mites per plot and total of 20,000 per program were released. No predatory mites were observed in a sample conducted before the release. After release, 20% of the sampled trees in program 3 sprayed with Movento were found to contain predatory mites, 30-32.5% in the program 2 and 4% sprayed with 435 oil, 35% in program 1 sprayed with Microthiol and 40% in the program 5 which is untreated control.      Colonies of field collected ACP from all programs were established in the laboratory and their subsequent generations tested for tolerance to dimethoate, fenpropathrin, and imidacloprid in comparison with a laboratory colony. The concentration required to kill 50% of the field population averaged 2, 4 and 8 times more for dimethoate, fenpropathrin, and imidacloprid, respectively, than the concentration required to kill same percentage of ACP from the laboratory colony. Leaf samples were collected from all programs and submitted for HLB analysis in March. Analysis of the leaves collected in Fall 2020 showed that 80-95% samples collected across all programs were HLB positive. Although, no Tamarixia radiata was released in the programs during this quarter due to the problems with the colonies at the FDACS-DPI, Dundee, facility, the previous work on the parasitism activity of T. radiata tested at different times of the day and temperatures was written and a presentation on the same work given at the Southeastern Branch Meeting of the Entomological Society of America and Florida Citrus Grower Institute.    

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, FL. Treatments of 1) soil-applied neonicotinoids interspersed with sprays of a different mode of action insecticides on a calendar basis, and 2) rotation of insecticide modes of action sprayed twice on each major flush are being implemented to the trees on UV reflective and bare ground. The irrigation deficit treatments are also functional at the SWFREC and CREC locations to trees on UV reflective and bare ground to synchronize flush to target spray applications on major flushes. Sampling was conducted to monitor flush abundance and psyllid populations at three experimental sites. At the Vero Beach location, significantly more flush was observed in the trees on mulch compared to the trees on bare ground averaging 4.09 and 3.47 per plant, respectively. The diameter of the trunk of the rootstock and scion of the trees on the mulch was significantly more than the trees on the bare ground. An average rootstock diameter of 33.79 mm on the mulch and 28.20 mm on the bare ground was observed. Scion diameter averaged 24.04 mm on the mulch and 21.12 mm on the bare ground. At the CREC location, on average, there was also significantly more flush observed on mulched trees than on bare ground trees. Analysis of data from September 2020 to March 2021 showed significantly fewer adults on trees with mulch than with no mulch. Trees with insecticides applied based on a calendar schedule also had fewer adult ACP than those that experienced insecticide applications based on flush. ACP adult abundance was weakly but positively dependent on flush abundance. Peaks of adult counts occurred between March to June as well as September to October in 2020 and this is where treatment differences were starkest. Significantly fewer eggs and nymphs were observed on the trees with mulch than those on the bare ground. At SWFREC, trends on flush abundance across treatments and ACP response to the treatments were similar to other two locations. During this quarter, the mulched and non-mulched treatments’ soil moisture contents varied between 0.11±0.02 m3 m-3 and 0.10±0.01 m3 m-3 showing not much difference in water storage with the root zone. Overall, canopy density appears to be greater on mulched than non-mulched trees. In the next quarter, we will do leaf and soil nutrient analysis and canopy size determinations to see changes if any in the treatment effects.  At SWFREC, several experiments were conducted using the remote-controlled and manual ground-penetrating radar (GPR) operation to evaluate the effort needed to perform the GPR scanning, measure the required time to complete the scanning process, evaluate precision and accuracy of the scanning circles, and determine if the remote-controlled system can perform better for non-invasive root detection than the manual process. The scan line data collected from both the remote-controlled and manual process were compared after processing the data using an auto root detection software (Tree Radar Inc., Maryland, USA.) The results of root detection for both processes were almost the same, and there were no significant differences between them. The depth of the detected roots in the remote-controlled process was same as of the manual process. These experiments show that the remote-controlled process can reduce the required application time by 3 times when compared to a manual process. It can also reduce the human effort required and increase the precision of the data collection process. A Qualtrics survey to obtain growers input on the reflective mulch technology was developed and distributed through several channels.         

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:   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 as well as flushing and blooming dynamics in the new plots (700 trees planted last year). After IPC removal in the original plot in August 2020, we have been monitoring CLas infection of the uncovered trees in real time. We are now processing these samples for real time PCR CLas detection so we can document the rate of infection once IPCs are removed from trees.  Objective 2. Assessment of alternative netting approaches involved in ‘targeted’, ‘alternated’ or ‘patterned’ setup of IPC in groves for more cost-effective protection. We have continued monitoring the new 700 trees mentioned in the Objective 1 planted last year that are arranged in an alternated pattern we are monitoring for CLas in trees adjacent to the IPC-covered trees. Also, we have continued working with  several commercial collaborators who are also evaluating different netting layouts under the CRAFT program.  Objective 3. Monitoring the transition from vegetative to reproductive stage in the covered trees as compared to the uncovered. We are continuing data collection on Bingo, Early Pride, and Tango trees. This is the second year in documenting blooming on these varieties. We have also documented different blooming rate in the trees from our first experiment that were uncovered last August. These trees are blooming more profusely than the always-uncovered trees. We are counting flowers and we will assess fruit set in the coming weeks. Objective 4. Comparing IPC with CUPS-like systems. We have performed for the second year deficit irrigation. As in last year, we have induced more bloom. We are also  finishing regular quality analysis from fruit that matured this year. Outreach for this quarter: -Batuman, O. Individual and direct contact with CRAFT applicants to establish and evaluate IPC trials for psyllid and HLB control.  -Gaire, S., Albrecht, U., Batuman, O., Qureshi, J., Zekri, M., Alferez, F. 2020. Horticultural performance of citrus trees grown under Individual Protective Covers (IPCs). Crop Protection, under review.  -Individual Protective Covers’ by Alferez, F, Gaire, S., Albrecht, U., Batuman, O., Qureshi, J., Zekri, M., 2021-2022 Citrus Production Guide, EDIS. Under Review. -Gaire, S. Evaluation of individual protective covers for preventing vector transmission of Candidatus Liberibacter asiaticus and effects on growth and physiology of young citrus trees. Master’s Thesis Defense, March 11 2021, UF, Horticultural Sciences Dept. -Gaire, S. Evaluation of individual protective covers for preventing vector transmission of Candidatus Liberibacter asiaticus and effects on growth and physiology of young citrus trees. Oral Presentation at the Southern Fruit Workers 3 Minute Thesis Competition, ASHS, 2nd Prize winner.  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 monitoring HLB progression after IPC removal in the first experiment.and fruit yield and quality to compare fruit from IPC and non-IPC trees.Objective 2. We will continue collecting data on psyllid populations and HLB incidence in the different netting layouts.Objectives 3 and 4.We will start collecting data on on bloom and fruit set for this second season of deficit irrigation treatments. Outreach:-Alferez, F. Invited speaker at the Citrus Institute 2021. Virtual. April 6.Individual Protective Covers (IPCs) influence on tree performance, fruit production, pests, and diseases.    3. Please state budget status (underspend or overspend, and why):  As mentioned in the last report, we are on track with activities  and spending after the COVID pause. Budgeted amounts for salaries and student stipend and tuition are being spent as predicted.   

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

We investigated the potential of a usable economic threshold to increase sustainability of Asian citrus psyllid (ACP) management in citrus under conditions of high huanglongbing (HLB) incidence. A year-long study was initiated in the spring of 2020 in a young planting of “Hamlin” sweet orange grove under standard agricultural practices for citrus, including mowing and fertilization, located in Lake Alfred, Florida. ACP management strategies were tested in a randomized complete block design with four replicates. Insecticidal sprays for ACP were based on either of three nominal thresholds of 0.2, 0.5, and 1 adult per stem tap and ACP treatment sprays were comprised of two rotations of different MoAs designated as rotations A and B. There were seven applications for the 0.2 adults per tap threshold treatment; five applications for the 0.5 adults per tap threshold treatment; and two applications for the 1.0 adult per tap threshold treatment (Table 1). ACP were monitored by calculating the mean number of adults per tap across all four plots for each treatment. If the means reached or exceeded the target economic threshold, all replicate plots assigned to that treatment were sprayed. ACP sampling occurred every 7 to 15 d. If the mean ACP density for a threshold treatment was within ± 0.05 per tap below the target threshold, a decision was often made to spray that treatment rather than waiting until the following week. Sprays were made after plots were sampled, and insecticide susceptibility to thiamethoxam was determined. Thiamethoxam was chosen as bellwether to test for resistance development given that resistance in ACP is predominantly metabolic and this MoA typically predicts subsequent multiple resistance within populations of this pest. An insecticide program was designed to maintain ACP numbers as close to zero as possible in the treatments considering potential impacts on resistance risks. Broad-spectrum insecticides were restricted to the ‘dormant’ season, and when activity of natural enemies was expected to be reduced, whereas more selective insecticides were used during the primary growing season. Table 1. Description of insecticide rotation programs according to different economic thresholds to manage Asian citrus psyllid                                                              Rotation A                                                         Rotation B  App Date                         0.2*                         0.5                     1.0                             0.2                       0.5                     1.0 5-May-20                              dimethoate                   ————                  ————                      fenpropathrin             ————                 ———— Jun 9 10, 2020                     cyantraniliprole            dimethoate                    ————                      dimethoate              fenpropathrin             ———— Jul 7-10, 2020                      fenpropathrin              cyantraniliprole             dimethoate                       cyantraniliprole         dimethoate                 fenpropathrin      12-Aug-20                            thiamethoxam             fenpropathrin                 ————                       diflubenzuron          cyantraniliprole           ———— 24-Sep-20                            spinetoram                  thiamethoxam              cyantraniliprole                  thiamethoxam          diflubenzuron              dimethoate    28-Oct-20                             diflubenzuron               ————-                    ————                       spinetoram                ————                  ———— 18-Dec-20                             abamectin                   spinetopram                   ————                       abamectin                thiamethoxam              ———— *: 0.2, 0.5 and 1.0 was the designated action (economic) threshold that triggered treatment with insecticide  The average number ACP eggs, nymphs, and adults counted was higher in plots where the 1 adult/tap threshold was implemented than in plots where the 0.2 or 0.5 D. citri/tap economic thresholds were implemented for both rotation A and rotation B. There were no differences in treatment efficacy between the two rotations. Overall, there were no statistically significant changes in susceptibility of D. citri following the completion of the either rotation schedule triggered by either of the three treatment thresholds tested. Furthermore, GSTE1, GST1, EST6, CYP4D1, and CYP4C67 gene expression levels were not significantly different in ACP collected from different threshold treatment populations as compared with the susceptible control. These results allow us to conclude with confidence that resistance was effectively kept in check throughout the trial. The incidence of HLB was determined by the level of CLas pathogen in each of the economic threshold treatment plots. There was 100 % infection of trees before the first application in the early spring. Therefore, the scoring and decline index of HLB was observed after harvesting and nutritional applications. All 480 trees were scored for each economic threshold treatment after harvest on a 0-4 scale, where category 0 = no HLB symptoms, normal growth flushes; category 1 = some HLB symptoms, mostly normal growth of flush; category 2 = some HLB symptoms, some normal growth; category 3 = obvious symptoms with no flush growth. category 4 = obvious HLB symptoms, including small leaves and dead wood and no new growth. The decline index was calculated for each treatment.  Furthermore, for rotation A, greater input of insecticides (lower action threshold) was correlated with lower incidence of HLB. We determined insecticide application costs and fruit drop rate for each economic threshold treatment. Insecticide costs were compiled from the University of Florida extension reports and the 2020-2021 Florida Citrus Production Guide. All prices were based on the products used in our tests. For fruit drop counts, each treatment threshold, 20 trees were counted per replicate plot and 80 trees total were counted per economic threshold treatment to determine mature fruit drop. Dropped fruit numbers were counted weekly for four weeks before harvesting. To estimate the level of CLas pathogen infection among treatment plots, three trees were randomly chosen for sampling per plot for a total of 24 trees for each rotation x treatment threshold treatment combination. Every tree sampled from each treatment plot was CLas positive. Similarly, all trees exhibited  HLB symptoms, The results indicated that trees had similar symptoms of HLB in all treatment plots and ranged between categories 1-4. The input costs of spraying at the 0.2 adults per tap economic threshold were estimated at $451.50 and $ 451.93/hectare for rotations A and B, respectively. The costs associated with the 0.5 adults per tap economic threshold were estimated at $ 288.88 and $ 284.38 per hectare for rotations A and B, respectively. Finally, at the 1.0 adult per tap economic threshold treatment, costs were estimated at $ 101.12 and $ 35.62 per hectare for rotations A and B, respectively (Table 6 and Supplementary data Table 7 and 8). There were no significant differences in fruit drop between each economic threshold treatment compared. However, we have no yet finished analyzing the yield data that were collected. In summary, our results indicate that an economic threshold could be implemented as a decision tool for timing insecticide applications in Florida and is compatible with a range of possible rotations of available insecticides for ACP management. Such rotational programs can maintain psyllids below population levels that negatively impact yield but can reduce the number of insecticide sprays needed per season. In the current investigation, although psyllid populations were reduced more effectively with the lower threshold that necessitated the most insecticide sprays per year (7), there was no difference observed in fruit drop or tree health between plots treated 2 times / year using the 1.0 psyllid / tap threshold and those sprayed 7 times/ year using the 0.2 psyllids/ tap threshold. As we continue this research, we will continue to validate use of economic thresholds in mature trees and determine how use of economic thresholds impacts secondary pest and beneficial arthropod populations. Also, we plan on compating profits between the management programs evaluated by including the yield component into our calculations.                          

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

In this quarter, monitoring of ACP and beneficial insects continued in all the Integrated Pest Management (IPM) programs established for ACP control under this project, 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 4.         HMO plus biological control. 5.            biological control only. Six biweekly samplings were conducted between October-December. As expected for Fall, trees were not flushing; therefore, ACP populations low. ACP adults averaged 0.1 or less per tap sample across all programs; therefore, spray treatments were not required until December, when the first dormant spray was conducted. The dormant applications included Imidan in programs 1 and 3, Pyganic + 435 oil (2%) in program 2, and 435 oil (2%) in program 4. No ACP adults were detected in the tap sampling conducted two weeks after the dormant spray application in programs 1-4; however, an average of 0.5 adults per tap sample was detected in program 5, which does not include insecticide use. We screened five populations from the five programs established in the field and started testing those for resistance against commonly used insecticides, including a population from a colony established at the SWFREC for several years.  Spiders and lacewings continue to be the two dominant groups of predators present across all programs. This quarter spiders averaged at 60% of the collected specimens and lacewings at 30%. We used the field-collected population to establish the colonies of three lacewings species that we found in the field. Testing the lacewings on ACP nymphs’ diet revealed that 75% Ceraeochrysa cubana, and 65% Ceraeochrysa claveri, developed to adulthood. We also initiated experiments to test the lacewings and the parasitoid Tamarixia radiata for their tolerance to different insecticides used in the citrus groves. Tamarixia radiata was also released in all programs; however, evaluations on parasitism rates were not possible due to the non-availability of nymphs. We also initiated collecting data on the fruit drop, which will continue into the next quarter. Findings from these programs were presented at the Annual meeting of the Entomological Society of America and grower meetings.  In the next quarter, we will be monitoring the population of ACP and making spray applications as needed. Studies on biological control will include monitoring the predatory insects’ natural populations, releasing, and evaluating the commercially available predators and the parasitoid Tamarixia radiata, tolerance/resistance of ACP, lacewings, and parasitoid to commonly used insecticides. We will also collect the leaf samples to do qPCR analysis to determine the incidence of HLB. We also hope to get the harvest done in the next quarter and obtain yield data.        

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, FL. Treatments of 1) soil-applied neonicotinoids interspersed with sprays of a different mode of action insecticides on a calendar basis, and 2) rotation of insecticide modes of action sprayed twice on each major flush were applied to trees on UV reflective and bare ground at the three locations. The irrigation deficit treatments were implemented at SWFREC and CREC locations to trees on UV reflective and bare ground to synchronize flush to make insecticide spray applications twice on each major flush.We monitored flush abundance and psyllid populations at CREC and SWFREC. Analysis of data at CREC revealed that ACP adult abundance responded to all the factors tested, including ground cover (bare vs. mulch) and insecticide application timing (calendar vs. flush applied). There were fewer ACP adults on trees with mulch than with bare ground. Trees with insecticides applied based on a calendar schedule also had fewer adult ACP than those that experienced insecticide applications based on flush. Lastly, ACP adult abundance was weakly but positively dependent on flush abundance, which was impacted by the ground cover treatment and the date of sampling. On average, there was fewer flush observed on bare ground trees than on mulched trees. We have reported similar findings from the data collected at SWFREC on these variables in the previous reports. However, effects during this quarter were not as pronounced as observed at the CREC location. We also measured trunk growth and analyzed HLB incidence in the trees at SWFREC. The diameter of the trunk of the rootstock and scion of the trees on the mulch was significantly more than the trees on the bare ground. An average rootstock diameter of 45.6 mm on the mulch and 39.7 mm on the bare ground was observed. Scion diameter averaged 29.9 mm on the mulch and 26.5 mm on the bare ground. As part of this project, we are also evaluating tree defenders on trees planted on bare ground. Interestingly, the trees’ rootstock or scion trunk diameter on mulch did not differ significantly from the trees covered with tree defenders on bare ground. HLB incidence was significantly higher in the trees planted on the bare ground compared to the trees on mulch, an average of 55% and 22% HLB positive trees, respectively; however, disease incidence did not differ between the trees treated with insecticides applied based on a calendar schedule (foliar sprays interspersed with soil applications) and those that experienced insecticide spray applications based on flush. Data on soil moisture and irrigation treatments have been continuously monitored every 30 minutes at the CREC and SWFREC sites. The next report will provide some updates on the status of available water in the treatment with regular or deficit irrigation. A significant number of trees at the Vero Beach locations were damaged by rains and died. We have already replaced 100 trees and are in the process of replacing another 50-60 trees. One challenge observed to-date is the difficulty in imposing irrigation regime (regular over deficit irrigation) treatments at this location due to the presence of a perched water table that results in water upflux, confounding the effect of irrigation in the reflective mulch treatments. At this point, we have discussed with the collaborator and are maintaining the same level of irrigation to trees on mulch and bare ground to be able to see any differences between the main treatments and wait to evaluate deficit irrigation treatment to synchronize flush at a later time based on findings.An economics student was hired that will work on this grant, though but funded through other sources. The student had a delayed start in the fall semester but has been working on this project and has now completed a draft of the survey instrument and begun data collection for partial budgeting analysis. The instrument has IRB approval and will go through grower and team reviews in 2021. Because of COVID-related travel restrictions, the team has to change the survey approach. Project spending was delayed but will begin with funding the new survey approach and hiring a postdoc to conduct analysis and help to create educational material. We will continue our activities reported here into the next quarter.          

Developing near and long-term management strategies for Lebbeck mealybug (Nipaecoccus viridis) in Florida citrus

1. Please state project objectives and what work was done this quarter to address them:   (1a) Field monitoring: Redesigned traps evaluated and will work for population monitoring. Pheromone attraction protocol has been designed and works in a laboratory setting. (1b) Laboratory screening of conventional insecticides and entomopathenogenic fungi has been completed and accepted for publication. Adjuvant screening started in December 2020. (1d) Ant management: Preliminary data shows a greater abundance of known and potential predators associated with the mealybugs when ants were prevented from accessing the bugs, the most common ant collected in both groves was the red imported fire ant. A field trial for removal of fire ants was prepared and ant density was evaluated, trial had to be delayed due to key collaborator getting COVID. (1e) Entomopathenogenic Fungi (EPF) field tests for use in IPCs was completed with the final samples being evaluated presently. Preliminary data suggest that EPFs may provide 3-4 weeks of control when deployed in IPCs. (2a) Assessment of predators for mealybugs: two years of field data have been collected to determine what predators are present in two locations, samples are still being sorted and identified. MS student K. Gaines created a DNA primer to enable evaluation of gut contents for lebbeck mealybug. This DNA primer has been fully vetted and works well in our control trials. Predator screening assays for potential predators that could be added to the system from what is commercially available for release has been started). (2c) EPG library for mealybug feeding has been started and we are making progress. This information will be necessary to evaluate chemistries that may inhibit feeding. (2d) Minimize spread: We have resumed working on developing recommendations to reduce spread, with a focus on reducing likelihood of accidental movement on individuals. 2. Please state what work is anticipated for next quarter: (1a) Mealybug population monitoring to begin January 2021. Pheromone trapping with live females and new lures from a collaborator will be tested in groves. (1b) Soil drench materials will be evaluated under laboratory conditions for prevention of infestation starting in late January/early February 2021. (1c) Evaluate promising materials in open grove setting: will deployed in March/April. (1d) Ant management: trial is scheduled to be deployed in February 2021. (1e) Management options for IPCs: field plots will be infested in late January 2021 for clean-up testing once populations have established. (2a) Molecular marker for lebbeck mealybug presence in guts of field collected predators will be started. Predator assays for potential mealybug predators to be released will continue. We plan to perform a field survey for other predators using methods used for Pink Hibiscus Mealybug and intend to begin this in the spring or early summer. (2c) We will continue working on the EPG library to enable the feeding inhibition trials to occur. (2d) Minimize spread: We will complete the testing of materials to reduce spread on people and resume steam sanitation work.     3. Please state budget status (underspend or overspend, and why): We are back on track with budget spending as we were able to resume focus on this project once COVID protocols were in place    

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

Objective 1: Survival of sentinel Asian citrus psyllids (ACP) was substantially higher, as compared to sentinels that could be accessed by natural enemies, in organic groves. ACP nymphs are suitable prey for a wide range of generalist predators. Although natural enemies may have occurred at lower densities in organic than conventional groves during our survey, our exclusion cage experiments indicated an overall effect of natural enemies on mortality of ACP in organic than conventional groves. It is also possible that cryptic or nocturnal predators could have contributed to the difference in predation observed between treatments in our exclusion cage experiments. For example, adult syrphid flies are nocturnal and larvae are cryptic as well as active mostly during dusk and dawn (Hagen et al., 1999), which may have been overlooked during our visual observations. In future studies, we could improve the sampling of natural enemies using additional methods such as sticky traps or vacuums, in addition to visual observations in order to better estimate natural enemy populations. Further investigations elucidating the impact of ants and their interaction with generalist predators on biological control of ACP could further improve management practices for this insect vector. The current study suggest that intermittent applications of insecticides sprays for management of ACP could sufficiently disrupt activity of  natural enemy populations to reduce normal population regulation of ACP, even if populaitons of natural enemies are not eliminated entirely populations. In constrast, undesurbed populations of natural enemies in organically managed groves in Florida can maintain populations of ACP at levels lower than observed in nearby conventional groves. Objective 2: Continuous selection imposed on a field-collected population of ACP with fenpropathrin for ten generations caused development of moderate to high levels of resistance (96.67-fold). Our investigation revealed that ACP has the capacity to develop a high level of fenpropathrin resistance as a result of continuous and persistent selection. Given the apparent lack of cross resistance between pyrethroids and other commonly used modes of action against ACP such an organophosphates and neonicotinoids, mode of action rotation should remain an effective model for managing resistance in D. citri as demonstrated in field studies. Expression variability analysis of detoxification related genes indicates that elevated levels of CYP enzymes are associated with fenpropathrin resistance. Furthermore, our results specifically implicate the CYP6A2-1 gene with fenpropathrin resistance. Objective 3: ACP populations develop high levels of resistance to thiamethoxam under continuous selection by label rate applications in cultivated citrus. A high level of resistance occurred following only 3-4 consecutive neonicotinoid sprays and within five egg to adult generations and was associated with subsequent product failure. We also showed that resistance in ACP to thiamethoxam declined significantly in the absence of selection pressure under laboratory conditions and when modes of action rotation was implemented after initially selecting for resistance under field conditions. Recovery to a susceptable state under rotation in the field was more rapid than under no selection in the laboratory population. These results suggest that thiamethoxam resistance is likely unstable under the field conditions. Collectively, our results indicate that rotation of thiamethoxam with insecticides from other chemical classes, including cyantraniliprole, fenpropathrin, dimethoate, spinetoram and diflubenzuron should mitigate neonicotinoid resistance in areas where ACP are managed with insecticides.   

Evaluation of the tolerance of newly developed citrus cultivars, on different rootstocks, to Huanglongbing

Ww are evaluating the new scions and rootstocks for their tolerance to HLB by studying the metabolite content by GC-MS, and challenging new varieties with psyllids and HLB.

Objective(s) pursued:

1. To understand the mechanism behind the tolerance of different varieties toward HLB. The comparison between the varietal responses will allow us to determine the mechanism of tolerance to CLas.

2. To understand the role of rootstocks in citrus tolerance to HLB. The comparison between rootstock metabolites will allow us to determine the best scion/rootstock combination for tolerating CLas.

Progress on Objectives:

Scion evaluations

For “Lucky” and its parents Sugar Belle and Nava x Osceola, we started a biology experiment to determine the response to psyllid infestation (5 plants per scion x 50 insects). After ACPs have colonized the plants for one month, the psyllids will be removed and samples taken to evaluate the citrus response to infestation. One month later, we will graft with HLB-infected budwood to establish CLas bacteria in these scions.

More scions

In cooperation with Dr. Schumann’s lab we received permission to take samples of some varieties in CUPS. This will comprise a separate set of experiments under CUPS/Semifield conditions.

Group 1 – Scions under CUPS conditions (includes Sugar Belle, Bingo, Early Pride) along with Ray Ruby GF, Ruby Red GF, Persian lime Minneola, Dancy, and Murcott. Set 1a – leaves only for VOC and metabolite evaluations; Set 1b – we will graft these onto Swingle or Carrizo RS. Sugar Belle will be considered the HLB-tolerant control while Minneola will be considered the HLB-susceptible control.

Group 2 – Duncan GF on 4 rootstocks (US-897, Cleopatra mandarin, Volk, BS/BO) for RS comparison

Group 3 – Ray Ruby GF on 3 RS (X639, Sour orange/US897) for RS comparisons

We hope to sample these in early December.

More Rootstock evaluations

In addition to those already in progress, we will receive some more rootstocks from USDA in Ft. Pierce. These include US-802, 812, 897, 942, 1283, 1284, 1516 for metabolite profiling and HLB screening.

* we have published a paper about the evalution of new hybrids that show attraction to ACP and could be used as awindbreak trees.

18- Killiny N, Jones SE, Hijaz F, Kishk A, Santos-Ortega Y, NehelaY, Omar AA, Yu Q, Gmitter FG, Grosser JW, Dutt M. 2020. Metabolic Profiling of Hybrids Generated from Pummelo and Citrus latipes in Relation to Their Attraction to Diaphorina citri, the Vector of Huanglongbing. Metabolites 10: 477.