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.
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.
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.
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.
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
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.
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. Schumanns 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.
The objective of this study is to provide a model for determining the population level (spray threshold) of Asian citrus psyllid (ACP) that would require insecticide treatment. Such a threshold could optimize the cost of ACP management in groves under conditions of high huanglongbing (HLB) incidence. We are trying to further optimize the economic threshold based on different insecticide mode of action rotation strategies such that use of this threshold can be integrated with resistance management. Ultimately, we will determine how various levels of pest control input using different insecticide rotation strategies affect both ACP densities and citrus yield in replicated field plots. First, the action thresholds of 0.2, 0.5 and 1.0 ACP adults per tap sample were assigned to field plots of Hamilin citrus. The plots were approximately 4 acres in size. Furthermore, the plots were split into two groups and each group was assigned to one of two insecticide rotation schemes. The rotations were: 1) dimethoate, cyantraniliprole, fenpropathrin, thiamethoxam, spinetoram, dimethoate, fenpropathrin, abamectin, and imidacloprid for rotation A; and 2) fenpropathrin, dimethoate, cyantraniliprole, diflubenzuron, thiamethoxam, abamectin, dimethoate, spinetoram and imidacloprid for rotation B. We collected samples chosen at random from the central rows of each plot sampling ACP adults. When the tapping number reached the 0.2, 0.5 or 1 ACP adult per tap threshold, an insecticide spray occurred. For the 0.2 adult threshold treatment, the following applications were made: The first treatment was dimethoate followed by cyantraniliprole, fenpropathrin, thiamethoxam, spinetoram and diflubenzuron for rotation A and fenpropathrin, dimethoate, cyantraniliprole, diflubenuron, thiamethoxam and spinetoram for rotation B. For the 0.5 adult per tap threshold treatment, the first treatment was dimethoate followed by cyantraniliprole, fenpropathrin and thiamethoxam for rotation A. For the second rotational scheme, we applied fenpropathrin, dimethoate, cyantraniliprole and diflubenuron for rotation B when the 0.5 ACP/tap thressholds were triggered. For the 1 adult threshold treatment, the first spray was dimethoate followed by cyantraniliprole for rotational A and fenpropathrin and dimethoate for rotational B. There were 6 applications for the 0.2 adult per tap threshold treatment, 4 applications for the 0.5 adult per tap threshold treatment, and 2 application for the 1 adult per tap threshold treatment. Second, we monitored ACP adults, eggs and nymphs to determine insecticide efficacy. Experimental treatments were evaluated by weekly counts before and after insecticide applications. For eggs and nymphs, 10 randomly selected flush samples were collected from each plot weekly and eggs and nymphs were quantified by a ranking method. The rankings for eggs were 0 = 0; 1= 1-20; 2 = 21-40; 3 > 41 and nymphs were 0 = 0; 1= 1-5; 2 = 6-10; 3 > 11. Adults were monitored by the tapping method by tapping 20 trees per plot. Densities of ACP adults were monitored every week from March 23, 2020 and eggs and nymphs were monitored weekly starting May 22, 2020. The results indicated that there were significant differences in numbers of ACP adults counted between rotation A and rotation B (df = 1; F = 6.35; p = 0.01). Furthermore, there were significantly differences in numbers of adults between each threshold treatment for 0.2, 0.5 and 1 adult per tap (df = 5; F = 27.51; p < 0.001). The average ACP count was higher in plots where the 1 adult/tap threshold was implemented than in plots where the 0.2 or 0.5 ACP/tap thresholds were implemented for both rotation A (0.42 ± 0.85) and rotation B (0.47 ± 0.95). There were no significant differences in numbers of ACP eggs between rotation A and rotation B (df = 1; F = 0.05; p = 0.81); However, there were significant differences between each threshold treatment for the numbers of eggs counted per flush (df = 4. F= 9.03; p < 0.001). More ACP eggs occurred in plots where the 1 adult/tap was used for rotation A (0.48 ± 0.86) and rotation B (0.50 ± 0.81) than in plots where the 0.2 and 0.5 ACP adults/tap thresholds were implemented. There were no significant differences in ACP nymph numbers between rotation A and rotation B (df = 1; F = 1.11; p = 0.29). However, there were significant differences between each threshold treatment (0.2, 0.5 and 1 ACP adults/tap) in the numbers of nymphs counted per flush (df = 4; F = 20.99; p < 0.001). Significantly more nymphs were counted in plots where the 1 ACP adult/tap threshold was implemented in both plots treated with rotation A (0.92 ± 1.17) and rotation B (0.80 ± 1.01) than in plots where the 0.2 or 0.5 ACP adults/tap thresholds were implemented3. Third, we use an insecticide bioassay to monitor changes in toxicity to thiamethoxam to field ACP after each application. This allowed us to determine if our field insecticide schedules are having an effect on resistance development of field ACP. Also, the incidence of the Candidatus Liberibacter asiaticus pathogen was determined before insecticide applications. For each bioassay, a total 5-8 concentrations were selected for testing. In plots that were managed with the 0.2 ACP adults/tap threshold, the resistance ratios ranged between 2.75-5.25 for rotation A and 1.63-5.12 for rotation B. For the 0.5 ACP adults/tap threshold, the resistance ratios varied from 1.6-6.75 for rotation A and 1.75-5.25 for rotation B. For the 1 ACP adult/tap threshold, the resistance ratios ranged between 3.13-4 for rotation A and 2.25-3.25 for rotation B. Overall, these results indicated that there were no great differences in susceptibility of ACP between the two rotations and various treatment thresholds and that all of our management schemes were effectively keeping resistance levels in check. To determine the level of CLas pathogen in treatment plots, three trees were sampled per plot for a total of 24 trees for each rotation. The presence of Candidatus Liberibacter asiaticus was determined by qPCR. We found that 100 % of the trees on this study were HLB positive. In the future, we will continue monitoring ACP populations and the HLB infection rate. An economic analysis will be used to calculate cost per acre and total cost per field for each insecticide application, as well as total insecticide costs for the growing season. The yield will be determined from each plot. Juice quality will be also be measured. Economic viability of each threshold treatment will be analyzed using yield data from all harvests.
In this project we are profiling the new scions and rootstocks for their tolerance to citrus greening pathogen by studying the metabolite content using gas-chromatography mass spectrometry (GC-MS), along with biological assays such as challenging new varieties with psyllids and HLB.Since our last report, we began sampling the new rootstocks and scions that we have propagated thus far. The analyses for these samples include stored volatiles and leaf polar metabolites.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. Methods: This quarter we sampled the mature leaves from the new scions and rootstocks, and did two different extractions – 1) hexane for leaf volatiles such as limonene, linalool and caryophyllene; and 2) methanol- chloroform-water for non-volatile metabolites such as sugars, amino acids and organic acids. Progress on Objectives: Scion evaluations `Marathon’ mandarin – we were only able to obtain one tree from a nursery, so we made cuttings for the mist bed. Although some of the cuttings did grow roots, when transplanted, the cuttings have not grown well. Our trial showed clearly that compared with many other citrus varieties with which we have experience with vegetative propagation, `Marathon’ is much less easily propagated. The source tree is growing fairly well and for this reason, we anticipate completing the metabolite work using only one biological sample, but 5 or more technical replicates.For Lucky, Sugar Belle, Nava x Osceola, and Grapefruit 914:1. Stored leaf volatiles (hexane extracts): 20 samples were run on the GC-MS (4 varieties x 5 biological samples), resulting in good chromatograms with approximately 50 volatile organic compounds identified. The peaks are being integrated and quantified currently.2. Polar leaf metabolites (TMS) 20 samples were run on the GC-MS (4 varieties x 5 biological samples), the chromatograms look good, with approximately 60 peaks. Integration and data analysis is in progress. Rootstock evaluationsFor the eight rootstocks we propagated and that are growing well: UFR-1, -2, -4, -5, -15 and -17; 46 x 20-04-6; 46 x 20-04-29:1. Stored leaf volatiles (hexane extracts): 40 samples were run on the GC-MS (8 varieties x 5 biological samples), resulting in good chromatograms. The integration and data analysis has not been started yet.2. Polar leaf metabolites (TMS) – 40 samples were run on the GC-MS (8 varieties x 5 biological samples), and the data analysis is waiting to be analyzed.Nematode susceptibility – Because the evaluation of new rootstocks should include tolerance to underground pests and pathogens such as Phytophthera and nematodes – we planted 6 of each of the 7 UFR rootstock seedlings in sandy soil for evaluation to burning nematodes. The soil was inoculated with burning nematodes and we are beginning our evaluations for resistance to nematodes. Our initial findings are that all of the UFR rootstocks have stunted growth, and damage in the root cortex with subsequent yellowing foliage compared to control plants.UFR-6 is not growing well, when it looks good we will analyze this one. Next quarter work1. We will complete the GC-MS data analysis for the first two sets of scions and rootstocks (8 rootstocks, four scions x two methods).2. `Marathon’ mandarin may be ready for GC-MS analysis.3. We should obtain more scions for evaluation. We will receive more rootstock seeds from the USDA.4. We will begin the biological evaluation of three scions (Lucky, and its parents, Nava x Osceola, and Sugar Belle) by challenging with ACPs to inoculate with HLB, as well as complete host preference studies.5. We will sample the foliage of the 6 UFR rootstocks under nematode pressure and their healthy controls for volatile and non-volatile metabolite analyses.
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.
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.
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 (NitrogenPhosphorusPotassium) Miracle-Gro All Purpose Plant Food (Scotts Miracle-Gro Products, Marysville, OH) and a 10-10-10 (NPK) 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.
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.
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.
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.
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