1. Please state project objectives and what work was done this quarter to address them: Objective 1: Using callose inhibitors to improve systemic uptake and reduce HLB symptoms.Objective 2: Maintaining water-saturated injection site: Sealed trunk injection ports that stay functional by avoiding woundresponses.Objective 3: Targeted root delivery.The field experiment initiated on sweet orange ~8 years old (Citrus x sinensis) trees. We selected and labeled treesaccording to 8 different treatments: (1) injected control (water), (2) Oxytetracycline (2 g per tree), (3) DDG (0.1 mM), (4)DDG (0.1mM) + Oxytetracycline (2 g per tree), (5) DDG (1mM), (6) 3AB (0.1mM), (7) 3AB (0.1mM) + Oxytetracycline (2 gper tree), (8) 3AB (1mM). Then we designed the appropriate map for the experiment. Before applying treatments,baseline stomatal conductance data and data related to trunk diameter, canopy volume and canopy density wascollected. Furthermore, six mature leaf samples per tree were collected to measure the CLas titer and quantify calloselevel of each tree. Then we employed the trunk injection (as described in (Vincent et al. 2022)). One week after applyingtreatments, we collected stomata conductance data to assess the treatment impact on leaf health. Leaf samples werecollected to measure the CLas titre and quantify the callose level of trees 1 and 2 weeks after treatments.We will evaluate the trees for callose level and Clas titre monthly. We will also measure the canopy volume and density,fruit drops via monthly data collection to determine the effect of injections on tree health. We will begin experiments for onobjectives 2 and 3. 2. Please state what work is anticipated for next quarter:We will evaluate the trees for callose level and Clas titre monthly. We will also measure the canopy volume and density,fruit drops via monthly data collection to determine the effect of injections on tree health. We will begin experiments foron objectives 2 and 3. 3. Please state budget status (underspend or overspend, and why):Underspend- since it took some time to initiate the work 4. Please show all potential commercialization products resulting from this research, and the status of each:We are still evaluating the effect of the callose inhibitor in the field trials
1. Please state project objectives and what work was done this quarter to address them: Objective 1: Screen FANA antisense oligonucleotide targeting CLas for efficacy in a field trial.During this quarter, we conducted field trials to assess the efficacy of FANA antisense oligonucleotides (ASOs) targeting CLas in infected citrus trees. We designed and synthesized FANA ASOs complementary to two essential CLas genes: the CLas NAD-dependent DNA Ligase gene (LigA) and the CLas DNA B-Helicase gene. Additionally, a scramble sequence FANA ASO was used as a negative control. Treatments were applied to 10-year-old, CLas-infected ‘Hamlin’ trees in 1-acre plots, with each treatment replicated three times in a randomized complete block design. The FANAs were delivered via microinjection, following the dosages determined in previous greenhouse assays.We collected data from the third replicate of the experiment, which encompassed the fall of 2022 and the spring of 2023. In the fall of 2022, CLas infection declined significantly after seven days in trees treated with LigA-FANA and antibiotics. At the 45-days after treament, CLas infection was observed in FANA ASO and antibiotic treatments but not in the Control-FANA treated trees. After 90 days, the lowest CLas infections were observed in LigA-FANA treated trees and antibiotic-treated trees. Preliminary data from spring of 2023 indicate that CLas infection significantly declined in antibiotic-treated trees from 0 to 60 days following application. Objective 2: Evaluate FANA antisense oligonucleotide targeting CLas to reduce vector transmission.We performed acquisition and inoculation assays to evaluate the impact of FANA ASOs on CLas transmission by the Asian citrus psyllid (ACP), the vector of citrus greening disease. In the acquisition assay, ACP nymphs were allowed to feed on FANA-treated infected citrus trees. In the inoculation assay, ACP collected from treated trees were transferred to uninfected citrus seedlings for inoculation feeding, and the development of CLas infection in the plants was monitored.In the fall of 2022, a reduction in CLas acquisition by ACP adults and their offspring feeding on FANA ASO-treated trees was observed. The lowest CLas infection rates were found in ACP feeding on antibiotic-treated trees, followed by LigA-FANA treated trees. Similar trends were observed in the spring of 2023, with reduced CLas acquisition by ACP adults feeding on Helicase-B-FANA treated trees and antibiotic-treated trees. This experiment was replicated in spring 2023 and data will be reported in a future report. 2. Please state what work is anticipated for next quarter: Next quarter, we plan to continue field trials to further assess the efficacy of FANA ASOs targeting CLas. A fourth and final applications of treatments will be applied to trees during this quarter. We will focus on analyzing the long-term effects of the treatments and evaluating the sustainability of the observed reductions in CLas infection rates. We will collect additional data on CLas titer, ACP transmission, and plant infection rates, extending the analysis timeline. Moreover, we aim to perform statistical comparisons between treatments to determine significant differences and evaluate treatment efficacy. 3. Please state budget status (underspend or overspend, and why): Spending is currently on track. We requested to use salary savings on the project to purchase additional treatments and conduct two full years of treatments.
April 20231. Please state project objectives and what work was done this quarter to address them:The objectives are to 2) determine if the flush cycle and infection period for Z. citri-griseum have changed due to the influence of HLB on citrus physiology or changing environmental factors; 4) evaluate the potential promotion of greasy-green symptoms related to nutrition programs or to peel reactions like a chemical burn from different pesticide and combinations of pesticide tank mixes; and 5) evaluate if postharvest degreening treatments might be modified to adequately remove the green coloration while mitigating poor shelf life from anticipated longer degreening times. Objective 2: To determine whether the flush cycle and infection period for Zasmidium citri-griseum have changed due to the influence of HLB on citrus physiology and other factors such as the changing environment, a site located in Fort Pierce was selected based on feedback from growers. Last year, two blocks with different grapefruit varieties were selected at the site for monitoring. Within each block, two groups of twenty mature grapefruit trees with similar canopy health status were selected and ten flush per tree was tagged. The same blocks were used for our second year of the field trial, which has been layed out. The data from last year’s experiment, including the flush cycle and fruit size, has been processed. Preliminary results of flush stage and fruit diameter per site (east and west) were assessed every two weeks using a shoot maturity index and caliper, respectively. In the summer flush cycle, there was a significant difference in the flush stage over time. Fruit size significantly increased over time starting in May (fruit size started above 3cm) as was expected. In the white grapefruit variety, fruit were slightly larger on the west side compared to the east side. The flush stage and fruit size data will be combined with the fungal data in the once it is fully processed. Evaluation of epiphytic growth on fruit and leaves is still ongoing. For the epiphytic growth on fruit, 210 slides were assessed, and the results suggest that epiphytic growth started in September on both white and red grapefruit which was unexpected. A total of 1200 leaf discs obtained from leaves collected in both blocks from June to July were evaluated and suggested that the epiphytic growth on leaves started in June on both varieties as reported in previous studies. When the trees were sampled, there were very few symptomatic leaves present in the grove, however greasy green symptoms were present on the fruit. Objective 4: We are also trying to meet with growers to compare programs from greasy-green affected and non- or less-affected blocks. The response is that there is not much difference between blocks. Some have indicated a willing to meet, but the actual dates for those meetings are still pending. Objective 5: We evaluated Red and white grapefruit from greasy-green affected blocks in January after initial degreening treatment in December. Degreening (with or without the cold treatment) significantly improved peel color after 5 days, however, even the best performing treatment was not very good (still negative a/b ratio). 4 treatments of each grapefruit type. Treatment 1: Straight into degreening room – degreen for 2 days (83F, 4 ppm ethylene) Treatment 2: Cold treat at 38F for 24 hrs. + degreen for 1 day (83F, 4 ppm ethylene)Treatment 3: Cold treat at 38F for 24 hrs. + transfer to ambient conditions without ethylene) – no degreeningTreatment 4: control (hold at ambient conditions without ethylene. (high humidity) For both types of grapefruit, treatments 1 and 2 had the greatest color change (P < 0.0001) although the a/b ratio was still negative, indicating a green color to the rind 5 days post-treatment. In February, we also evaluated color development of greasy-green Red and white fruit from the IMG blocks with different lengths of degreening. While degreening improved color, even after 20 days ambient storage, peel color was still not great . The data are still under analysis 2. Please state what work is anticipated for next quarter: We will continue to collect the second season of field data in Fort Pierce. Data analysis is underway on the the previous seasons samples. The degreening experiments will be further analyzed. We hope to be able to get some data from growers about their practices this season rather than empty promises. It will be difficult to generate testable hypotheses about interactions of products in the grove without this information. 3. Please state budget status (underspend or overspend, and why): No over or underspend on budget currently 4. Please show all potential commercialization products resulting from this research, and the status of each: None at this date.
1. Please state project objectives and what work was done this quarter to address them: CLas inhibition with antisense oligonucleotides for management of citrus greening disease Objectives: 1. Screen FANA antisense oligonucleotide targeting CLas for efficacy in a field trial. Our working hypothesis is that CLas-specific FANAs can be delivered using microinjection developed for RNAi-based technologies to reduce CLas in infected citrus trees. 2. Evaluate FANA antisense oligonucleotide targeting CLas in order to reduce vector transmission. Our working hypothesis is that CLas will be specifically inhibited in psyllids by using CLas-specific FANAs, resulting in a reduction in Las acquisition and transmission by ACP in a field setting. 1. Please state project objectives and what work was done this quarter to address them: Objective 1. Screen FANA antisense oligonucleotide targeting CLas for efficacy in a field trial. Field trials were conducted in research groves at the UF Citrus Research and Education Center. Treatments were applied to 10-year-old, CLas-infected ‘Hamlin trees of a standard size and CLas titer in September 2022. The following treatments were applied as trunk injetions: FANA ASOs (LigA) and (Hel), nontarget control FANA ASO, and oxytetracycline (OTC; FireLine). Injected treatments were compared with an insecticide-only control treatments. Each treatment was applied applied to 15 trees in 1-acre plots, replicated three times in a randomized complete block design. Treatments were applied to both sides of the tree canopy using microinjection of dosages determined in our previous greenhouse assays. All FANAs dosages were 625 ppm per tree. To monitor the effect of treatments on trees CLas titers as assessed 0, 2, 7, 30, 60 and 90 days post-treatment by qRT-PCR analysis. Currently, we are processing and analyzing the data from treatments applied inSeptember 2022. A third treatment application was applied in March 2023 Fruit were harvested to assess fruit quality and yield in November 2022. After Hurricane Ian on October 11, 2022, fruit drop was quantified by installing 2×3 ft2 PVC pipe squares around each tree and counting and removing all the fruit inside the square. On November 2, 2023, fruit drop was quantified again to assess pre-harvest fruit drop. On November 10, 2022, we harvested the treatment plots based on the maturity of Hamlin fruit. Each tree was harvested entirely, and fruit was placed into individual bushel bags. Fruit were processing at the CREC Pilot Plant in Lake Alfred to obtain fruit and juice quality analyses. Fruit count, weight, and size the fruit were assessed per tree. Juice Brix/acid ratio and color were also quantified for each treatment. Results: Fruit drop. The greatest fruit drop occurred during October 2022 due to Hurricane Ian. In November, however, fruit drop was minimal. Among the trees treated with LigA-FANA, control-FANA, and Helicase-B-FANA, a total of 287, 263, and 254 fruit were collected, respectively. These treatments were the most severely affected by the hurricane and had the highest fruit drop in 2022. Trees treated with OTC had the lowest total fruit drop, followed by trees treated with insecticides only. Furthermore, the OTC-treated trees had the lowest mean fruit drop per tree, which was only 11.26 fruit/tree. In comparison, average fruit drop per tree was 17-19 fruit/tree in FANA-treated trees and 14 fruit/tree in insecticide-treated trees. The percent fruit drop was lowest in OTC-treatments (22%), followed by control-FANA (66%), LigA-FANA (69%), Helicase-B-FANA (71%), and insecticide (72%) treamtments. Fruit yield. In 2022, the highest citrus fruit yield was obtained from OTC-treated treatments (697), followed by LigA-FANA (169), control-FANA (153), insecticide (147), and Helicase-B-FANA (146) treatments. The highest mean fruit yield per tree (46 fruit/tree), occurred in the OTC treamtent, followed by Helicase-B-FANA (12.17 fruit/tree), LigA-FANA (12.07 fruit/tree), insecticide (11.31 fruit/tree), and control-FANA (10.2 fruit/tree) treatments. Citrus fruit weight (in pounds) was greatest in OTC-treated trees, with a total of 156.07 pounds, followed by LigA-FANA (34.00), insecticide (31.30), Helicase-B-FANA (28.60), and control-FANA (28.30) treated trees. The mean fruit weight per tree was the highest in the OTC treatment, with 10.40 pounds per tree, followed by LigA-FANA (2.43), insecticide (2.40), Helicase-B-FANA (2.38), and control-FANA (1.88) treatments. In addition, OTC-treated trees also produced the highest juice weight (in pounds), with a total of 78.47 pounds produced in 2022, followed by LigA-FANA (16.27), insecticide (15.19), Helicase-B-FANA (13.65), and control-FANA (13.58) treated trees. The mean juice weight per tree was the highest in OTC-treated trees with 5.23 pounds of juice per tree, followed by LigA-FANA (1.16), insecticide (1.16), Helicase-B-FANA (1.14), and control-FANA (0.90) treatments. External and internal fruit quality. The mean fruit diameter was largest from trees treated with OTC injections, (19 cm/fruit), followed by LigA-FANA (17.53 cm/fruit), Helicase-B-FANA (17.35 cm/fruit), control-FANA (16.99 cm/fruit), and insecticide-only (16.29 cm/fruit) treatments. The mean Brixº/acid ratio (15.79) was also highest in juice from OTC-treated trees, followed by Helicase-B-FANA (13.08 Brix/Acid), control -FANA (13.05 Brix/Acid), insecticide (13.02 Brix/Acid), and LigA-FANA (11.81 Brix/Acid) treated trees. No significant differences were found in the juice color among treatments. The juice from OTC-treated trees had the highest color score (30.73), followed by Helicase-B-FANA (30.35 score), control -FANA (30.34 score), LigA-FANA (30.20 score), and insecticide (30.13 score) treated trees. Overall, preliminary data from 2022 suggest that trunk injection of OTC was associated with lower fruit drop, increased yield, and improved external and internal fruit quality parameters as compared with FANA and insecticide treatments. The highest fruit production and lowest percent fruit drop were observed in response to OTC injection. Notably, fruit drop, yield, and diameter were also higher in response to FANA treatments as compared with insecticide-treated trees. No difference in Brix/Acid ratio and juice quality was observed between FANA and insecticide-treated trees. Overall, trunk injections of antibiotics appear promising for reducing fruit drop and increasing citrus yield while improving fruit quality characteristics, with FANA treatments contributing to improved fruit quality.Objective 2. Evaluate FANA antisense oligonucleotide targeting CLas in order to reduce vector transmission. Acquisition and inoculation assays were replicated in fall 2022. Samples are currently being processed. 2. Please state what work is anticipated for next quarter: Objective 1:Samples from the second and third round of treatment applications will be processed and reported during the next quarter. Objective 2: Preliminary data from the second replicate of these experiments will be reported next quarter will be reported next quarter. 3. Please state budget status (underspend or overspend, and why): Our budget is on track for the project. FANA treatments will be purchased ffor the final treatment application will be purchased in the upcoming quarter. Remaining budget will be spent on field use charges and for payroll to complete sample analysis, PCR of field samples, ACP assays and colony maintenance, and fruit processing.
We aimed to investigate 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. Specifically, we measured efficacy of three nominal thresholds by relating ACP densities to cost of application and yield. Moreover, two spray programs of insecticide MoAs were compared in a region of Florida where insecticide resistance in ACP was previously identified in an effort to evaluate the need for an effective dormant season spray and to combine integrated pest management (IPM) with integrated resistance management (IRM) practices. The highest yield was observed with the 0.2 ACP / tap threshold that required 7 annual sprays, while reducing the number of sprays to 5 and below with higher thresholds caused a significant decline in yield. The estimated profit obtained with using the 0.2 ACP / tap threshold was higher than with the two higher thresholds tested in this study, indicating that we were unable to reduce the number of sprays below 7 per year under this set of circumstances without compromising yield. Fewest adults were observed in plots with the lowest threshold evaluated, which required seven annual sprays. ACP populations were lowest overall in plots where treatments were triggered by the lowest threshold evauated (0.2 ACP/tap). The input cost of spraying at the 0.2 adults per tap threshold were estimated at $451.93/hectare. The costs associated with the 0.5 adults per tap economic threshold were estimated at $284.38 per hectare. Finally, at the 1.0 adult per tap economic threshold treatment, costs were $35.62 per hectare. There were no significant differences in fruit drop between the three threshold treatments compared. Susceptibility of ACP to thiamethoxam in treated field plots after each insecticide application was monitored with an insecticide bioassay. In treatments that were managed with the 0.2 ACP/ adults / tap threshold, resistance ratios ranged between 1.63-5.25. For the 0.5 ACP adults / tap threshold, the resistance ratios varied from 1.75-5.25. For the 1 ACP adult / tap threshold, the resistance ratios ranged between 2.89-3.25. Overall, there were no statistically significant changes in susceptibility of ACP following the three treatment thresholds tested. Significant differences were observed between two insecticide rotation programs depending on whether or not an effective dormant season insecticide spray was applied near budbreak of the first seasonal flush. High counts of ACP adults were associated with the presence of feather flush structures on Valencia and Hamlin citrus trees. Also, differences in vegetative growth intensity were observed between Valencia and Hamlin varieties which could have an impact on ACP vector control programs if ACP adults are able to migrate to new groves where feather flush structures are present. Our results indicate that ACP management is most critical during the period between January to March, when citrus is characterized by flowering, fruit maturation (final stage), and the need for safe harvesting. Our current results also indicate that the 0.2 psyllid per tap threshold shown to effectively predict need for ACP treatment application in mature citrus may also be useful in young trees, but going above this threshold may not be possible without reducing yield. This outcome may have been affected by the frequent flushing that occurred in young trees combined with the small size of our treatment plots, which likely promoted re-colonizaiton of treated areas due to psyllid movement. Our results also suggest that combining an action threshold with an appropriate rotation program can effectively prevent development of insecticide resisitance among psyllid populatons. Furthermore, our results indicate that a highly effective dormant season insecticide spray targeting both adult and immature psyllids near budbreak of the first seasonal flush will be required in order to implement a low (0.2 psyllds/tap) treatment threshold during the remainder of the season. Implemention of thresholds to predict need for ACP sprays could allow for transitioning away from calander-based spray programs and development of more sustainable citrus management programs. Further research on the consequences of using economic thresholds on populations of secondary pests and beneficial arthropod species in Florida is warranted.
1. Please state project objectives and what work was done this quarter to address them: Objective 1: Determine the level of foliar phosphate fertilization required to reduce phloem citrate levels to less than 0.5 mM, a level that does not support L.crescens growth. A phosphate spray of 6mM reduced citrate levels in the phloem significantly more than other treatments, to a level of ~28mM. Objective 3: Use the phosphate level from Objective 1 in the field trials to demonstrate the reduction in phloem citrate levels and reduction in phloem CLas titers as well as effects on fruit quality and yield. CLas titers were not significantly reduced by phosphate spray treatments of 0.667, 2.0, 6.0, 18.0mM when compared to a 0.0mM control in 2019, 2020, and 2021. Effects on fruit quality and yield were measured using Leaf Area Index (LAI), fruit number/m^2, and fruit weight/10 fruit (kg) in two separate field trials (Immokalee and Hamilton field trials). Hamilton results for LAI show an increase in leaf area index from 2019 to 2020 for all treatments, and a slight decrease from 2020 levels in 2021. Yield results are lower from 2020 to 2021. For the Immokalee trial, LAI results improve and are more uniform in 2021 compared to 2019 and 2020. Yield results improve for all treatments from 2020 to 2021. 2. Please state what work is anticipated for next quarter:CLas titers will continue to be monitored in our field trials. LAI measurements and yield data will be collected seeing that there is an improvemet over the years and in 2021. Furthermore, compared to 2020, in the year 2021 all tree managment was resumed to pre-COVID 19 pandemic regimes therefore we expect to have definitive data in 2022. A no-cost extension would allow us to complete the objectives. In particularly, we want to see if CLas titer in psyllids has declined – which is an objective of the proposal. In addition, we need to measure citrate levels in leaf petioles in our groves.We also need to complete three manuscripts. 3. Please state budget status (underspend or overspend, and why): The project took time to get off the ground. It took longer than expected to identify the grove we could use in two locations. The budget remaining is $55,341.70 in direct costs and $6,641.09 in indirect costs.
1. Please state project objectives and what work was done this quarter to address them: 1. Quantify the effect of citrus antimicrobials on vector fitness. This objective is to determine if ACP exposed to antibiotic treatments will have reduced lifespans, fecundity, fertility or development time as compared with untreated ACP.Survival and fertility assays were completed previously. Replicate development time and fecundity assays were initiated at during this quarter. 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. We expect that ACP will be less capable of transmitting Las after feeding on antimicrobials, because 1) trees treated with antimicrobials are more likely to have lower Las titers for acquisition and 2) Las in infected psyllids will be reduced or eliminated when psyllids feed on antimicrobials.This experiment was performed in mature, infected citrus trees located in a research grove located at Lake Alfred, FL to determine whether field applications of foliar antimicrobials were capable of suppressing the acquisition of CLas. Eight-year-old CLas-infected citrus trees received foliar applications (Sept October 2020) of streptomycin, oxytetracycline, or no treatment (Control). All trees were treated with monthly insecticide sprays. One day after the application, ten CLas-free insects (5 females and 5 males) per plant from a CLas-negative 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. Survival of ACP adults was monitored the day after inoculation. After one-week, parental adults were collected 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. Egg clutches remained on trees enclosed in mesh sleeves after parental removal. After the nymphs reached the adult stage (approximately 2 weeks after ACP inoculation), adult psyllids were collected for analysis. Objective 3: Determine the effect of antimicrobials on plant response and associated ACP behavior. Insect choice bioassays were completed. Antibiotic treatments were applied to sweet orange, C. sinensis, trees to determine if they affect ACP host plant selection and acceptance behavior. Six trees (biological replicates) were individually sprayed per treatment. The chemicals evaluated were: FireWall (Streptomycin sulfate), FireLine (Oxytetracycline), and control (adjuvant only). The following treatments were compared with adjuvant only control in paired choice tests: 1) two FirelLne applications, 2) two FireWall applications, 3) one FireWall followed by one FireLine application, 4) one FireLine followed by one FireWall application. In certain treatments, plants that received an initial treatment with FireWall were subsequently sprayed with FireLine and vice versa. In other treatments, plants received two successive applications of FireWall or FireLine (Figures 1 and 2). After treatment applications, all plants were relocated into a growth chamber maintained at 23 ± 3 °C, 60RH, and a 16:8 h (Light: Dark) photoperiod until further bioassays. Psyllid response to treatment versus control (application of adjuvant only) plants was assayed twice. The first assay took place 25 days after the initial treatment applications were made and the second behavioral assay took place another 25 days after the second application of treatments was made (50 days after the experiment was initiated). During bioassays, paired treatment and control plants were placed into sealed bioassay chambers into which ACP adults were released. During assays, 70 D. citri adults were released into a behavioral chamber containing plant pairs for a period of 24 hours. The number of psyllid adults landing and initiating feeding on treated versus control plants was recorded 24 hours after insect release.2. Please state what work is anticipated for next quarter:Obj. 1 Conduct final replicate of development time and fecundity assays and complete data analysis.Obj. 2. Replicate field application of foliar antimicrobials to evaluate supression of CLas acquisition by ACP caged on treated trees. ACP will be collected following acquisition assays. All insects from this replicate and previous replicates, and tree samples, will be processed to determine CLas titers in response to treatments (DNA extraction and PCR) during the final two quarters of the project.Obj. 3. Complete data analysis. 3. Please state budget status (underspend or overspend, and why): We were underspent this quarter due to previouly being unable to spend funds on emplowyees working in the lab due. The funding needed for materials and personnel to perform experiments and process samples is still needed to complete the objectives, but was effected by work delays. Many purchases were in process when the project ended because samples were still being processed on the final project ended date in November. We have received a no cost extension to complete the experiments, sample processing, and data analysis.
This project is focused on conducting research in four Integrated Pest Management (IPM) programs and biological control only program for ACP. The programs are listed below: 1. conventional and organic insecticides plus biological control,2. organic insecticides, and Horticultural Mineral Oil (HMO) plus biological control,3. conventional insecticides plus biological control4. HMO plus biological control.5. biological control only.This report describes the activities from July to September 2021, which include monitoring the populations of ACP and beneficial insects in all programs as well as releases and evaluation of the parasitoid Tamarixia radiata and two commercially available predators (Ladybeetle Rhyzobius lophanthae and Brown lacewing Sympherobius barberi) against ACP. ACP populations remained well below our spray treatment threshold of 0.1 adults per tap sample and therefore no spray applications were required this quarter. Tamarixia radiata were released in all programs biweekly at the rate of 200 adults per plot for a total of 800 per program. At least three shoots infested with ACP nymphs (4th and 5th instar) were checked per plot for a total of 12 samples per treatment to recover parasitoids. However, the nymphal populations were very low, and it was only in August when limited samples were available and the parasitism rates observed were between 2-3%. Rhyzobius lophanthae were released at the rate of 200 individuals per replicate (800/treatment) in July and 500 individuals per replicate (2,000/treatment) in September. Sympherobius barberi releases included 200 individuals per replicate (800/treatment) in July, 500 individuals per replicate (2,000/treatment) in August, and 300 individuals per replicate (1,200/treatment) in September. The potential of each predator to control ACP was also evaluated. In the laboratory conditions, ACP nymphs at six densities (1, 5, 10, 15, 20, and 40 nymphs) were offered to each predator (R. lophanthae or S. barberi) and the prey consumption was evaluated after 24 hours. The experimental unit was a citrus shoot infested with ACP nymphs with one predator inside a sleeve cage. Prey consumption by each predator increased with the increasing density of the prey, however, there was no significant difference in the consumption rate between the two highest densities (20 and 40 nymphs per shoots) for either species. The consumption rate averaged 12.70 ± 0.63 nymphs per R. lophanthae and 11.80 ± 0.95 nymphs per S. barberi. We also tested three release rates of both predators. In the field, citrus shoots infested with ACP nymphs were enclosed with 1, 3, or 5 individuals of R. lophanthae or S. barberi in the sleeve cages. The consumption rate increased with the increasing density of the predator averaging 28-64% for the R. lophanthae and 56-84% for the S. barberi. S. barberi were also captured in the yellow sticky traps that we installed to monitor their dispersal in the experimental area following releases. Findings on R. lophanthae or S. barberi suggest that both species are good predators of ACP nymphs. Among the naturally occurring predators in the field, we continue to see the predominance of lacewing particularly Ceraeochrysa cubana and spiders. It seems that the naturally occurring predators as well as releases of the two commercial predators and T. radiata were negatively impacting ACP populations which remained below the treatment threshold of 0.1 adults per tap sample during this quarter and therefore no spray applications were made in any programs.
This project evaluates young tree protection from ACP/HLB using approaches such as 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. In this quarter 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 implemented to the trees on UV reflective and bare ground. The irrigation deficit treatments to manage flush were also functional at the Immokalee and Lake Alfred locations to trees on UV reflective mulch and bare ground to synchronize flush to target spray applications on major flushes. All the trees were drip-irrigated with two emitters. A separate irrigation treatment using a microsprinkler was also evaluated at the Lake Alfred location.Sampling was conducted to monitor psyllid populations and flush abundance at all three experimental sites. Significant effects of ground cover on psyllid populations were observed at all three locations. At Immokalee, a reduction of 61% in the adult ACP numbers in the plants on mulch compared to bare ground was observed with an average of 0.20 and 0.51 adults per tap sample, respectively. The same level of reduction (61%) was observed at Vero Beach, with an average of 0.07 and 0.18 adults per tap sample on mulch and bare ground, respectively. An overall reduction of 69% using all data on adults was observed at Lake Alfred. At Immokalee, flush infestation with psyllid immatures averaged 22% on mulch and 36% on bare ground, a reduction of 39% on mulch. An average of 5% shoots on mulch and 9% on bare ground were infested at Vero Beach, a reduction of 44% on mulch.Data from April 2021 showed higher soil moisture averages from mulch treatment at all layers (8, 15 and, 45 cm) compared with bare ground. However, these differences were masked by rains in summer 2021. Soil analysis from Immokalee location showed that except for Mg, K, and B, all the other nutrient concentrations were higher in the mulch plots suggesting better nutrient distribution within the root zone and minimal leaching threat. The flush count was impacted by the ground cover treatment (mulch vs. bare) and the date of sampling. On average, more flush was observed on mulched trees than on unmulched trees. The trees on irrigation deficit treatments produced less flush compared to those on the full or conventional irrigation treatment and that trend persisted on the mulch or bare ground.Trees on the UV reflective mulch showed a significant difference in growth. At Vero Beach, compared with last measurements in March, an increase in the growth of rootstock averaged 20.9 mm and 16.8 mm on mulch and bare ground, respectively, whereas scion growth averaged 13.5 and 11.6 mm, respectively. Similar effects on tree health were observed at other locations. Overall, tree canopy density appears to be greater on mulched than bare ground trees at all locations and went up by 40% at the Lake Alfred and 30% at Immokalee. A detailed article Implementing UV reflective mulch and flush timed sprays for managing Asian citrus psyllid was prepared and submitted for publication in Citrus Industry magazine. We are continuing measurements on multiple variables relating to psyllid, HLB and tree health from all locations and hope to better understand the impact of mulch and flush treatments on tree growth and yield in the coming years. The start of this project was delayed significantly due to the logistics involved in setting up the trials in three regions. Therefore, we will need one more year after the end date of December 2021 for the successful completion of this project.
Importance of the dormant spray with regard to establishing threshold-based annual spray program. Previous research has demonstrated the importance of reducing ACP populations during the dormant winter period as an effective method to initiate the citrus growing season with low psyllid populations. Our initial results indicated that it may be difficult to implement the use of treatment thresholds without effectively managing psyllids during the dormant period using appropriately timed insecticides that are effective against both psyllid adults and nymphs. Because of the differences in dormant season spray applications between commercial growers, we were able to investigate this question by monitoring psyllid populations and associated citrus flushing in groves that were managed differently, yet located in close proximity to one another. In doing so, we were able to compare psyllid populations in groves: 1) lacking an effective dormant season application (Grove 1 below) versus those 2) where an effective dormant season treatment had been applied (Grove 2 below). These applications were made because of various goals and constraints (harvest period, available budget) facing each particular grower involved. Six and five insecticide sprays were performed at Grove 1 and Grove 2 groves, respectively, during the course of monitoring. Two insecticide rotations were compared; the first rotation (HB) consisted of Movento, Timectin, Minecto Pro, Timectin, and Micromite. The second rotation consisted of Exirel, Movento, Agri-Flex, Minecto Pro, and Apta. Grove 1 did not receive an effective dormant season application, while Grove 2 did. In Grove 1, Movento was sprayed during the dormant season and this spray occurred more than 10 d after budbreak, because of the difficulty in timing the spray in between harvest and bloom. Grove 2 did receive an Exirel spray during the dormant period, which is highly effective against all stages of ACP. This proved to be a highly effective dormant spray even though it had not been timed perfectly and occurred a week after budbreak of the first flush. Grove 2 was then sprayed by keeping ACP near the 0.2 psyllids/tap threshold the remainder of the season. In Grove 1, it was not possible to maintain ACP near the 0.2 ACP/tap threshold despite application of several sprays. In Grove 1, there was no opportunity for an untreated area given that monitoring of treatment effects was conducted within commercial citrus and the main purpose was to compare Groves 1 and 2 with the main treatment difference being an effective dormant season application. Therefore, all of Grove 1 blocks were treated the same and had relatively similar ACP populations. The most intense periods of vegetative growth (feather flush structures) occurred from January to March during both years, 2020-2021. The presence of feather flush structures at the beginning of 2020 was associated to high counts of ACP adults. However; during the same period on 2021, this association between vegetative growth and the number of psyllids was disrupted, and a significant reduction in adults was observed. To analyze the interaction between ACP adults with vegetative growth, autocorrelation analyses were performed. We found that between 0 to 3 weeks after vegetative growth was detected on trees, there was an increase in ACP adults with a statistical association value of 0.5 between occurrence of flush and psyllids. This result means that at least 50% of the adult ACP population present on these Valencia trees emerged from the eggs oviposited during the previous vegetative growth. Also, the effectiveness of each pesticide used in Grove 1 rotation was analyzed, indicating that the Movento spray was the least effective insecticide in the rotation program. Following application of Movento, the number of ACP adults on trees did not change. However, after Timectin and Minecto Pro sprays, there was a significant reduction in ACP adults observed in citrus trees. Six blocks consisting of Valencia (2 blocks) and Hamlin (4 blocks) citrus trees were examined. Similar intense periods of vegetative growth (feather flush structures) were observed on Valencia and Hamlin trees from January to March during both years, 2020-2021. Similar to the results obtained on Valencia trees in Grove 1, high counts of ACP adults were observed at the beginning of 2020 associated with high intensity of feather flush structures. However, during the same period in 2021, this association was disrupted. Autocorrelation analyses showed that 1 to 2 weeks after vegetative growth, a high number of ACP adults were detected on flushing Valencia and Hamlin citrus trees. The data revealed a higher association value of 0.8 between occurrence of flush and psyllids, which indicates that 80% of the ACP population present in these trees was explained by the occurrence of flush. The effectiveness of each pesticide used in the Grove 2 rotation was evaluated. We found that the Exirel spray to Valencia and Hamlin trees was the most effective insecticide used in the rotation program. The results indicated that Exirel eliminated ACP adults from treated trees for more than 30 days and ACP populations remained low (less than 0.1 ACP/tap) during 2020, except in one Valencia block. Significant differences were observed between the two insecticide rotation programs. High counts of ACP adults were associated with the presence of feather flush structures on Valencia and Hamlin citrus trees. Also, differences in vegetative growth intensity were observed between Valencia and Hamlin varieties, which could have an impact on ACP vector control programs if ACP adults are able to migrate to new groves where feather flush structures are present. Our results indicate that ACP management is most critical during the period between January to March, when citrus is characterized by flowering, fruit maturation (final stage), and the need for safe harvesting. When comparing the two rotation programs, significantly more ACP adults were observed in plots in Grove 1 that did not have an effective dormant season spray than plots in Groves 2, where an effective dormant season spray was applied. Our results highlight the importance of applying a highly effective insecticide, in this case Exirel, during the dormant winter period and soon after initial budbreak of the first seasonal flush.
1. Please state project objectives and what work was done this quarter to address them: The objectives for this proposal are 1) Conduct field trials of new products and fungicide programs for PFD management as well as validation trials for the Citrus Advisory System (CAS); 2) Investigate the reasons for the movement of Postbloom fruit drop (PFD) to new areas and recent major outbreaks; 3) Evaluate methods for initial inoculum reduction on leaves so that early fungicide applications could be more effective and identify the constituents of the flower extracts using omics techniques. We conducted 7 field trials to evaluate how well the Citrus Advisory System (CAS) predicted PFD outbreaks from 2017 to 2021. During this period there were very few PFD outbreaks. In all four years, only one fungicide application was recommended by CAS. This was in 2019 in Fort Meade. In most seasons, there was no significant difference among the treatments, applications recommended by CAS, the older model PFD-FAD, Weekly applications and an untreated control. This indicates that the recommendation of no fungicide application was correct. We also looked at the economic savings of using CAS over the other fungicide application recommendation methods and found there were considerable savings. We also conducted five fungicide trials from 2018 to 2021. Unfortunately, we were not able to make solid conclusions about fungicide efficacy from these trials because the disease was not at high enough levels to statistically separate the treatments reliably. We investigated how far the conidia of Colletotrichum acutatum (syn. C. abscissum) can travel under different conditions: 5, 10, and 20 m/s with and without rain in a laminar flow windtunnel. We also used a turbulent wind tunnel at 5 and 7 m/s with and without rain. We found that the secondary conidia formed on leaves could travel at least 15 m at the 15 and 20 m/s winds with and without rain. This has not been observed before. We were unable to test primary conidia from flowers in the laminar flow tunnel because of COVID-19 and a mechanical breakdown. In the turbulent wind tunnel, the conidia from the leaves did not travel as far but could still move further than if from simple splash with and without rain. We determined that the high polarity fraction of the sugars from citrus (flowers and leaves) can stimulate the germination of C. acutatum conidia nearly as well as the raw floral and leaf extracts. We did not expect leaf extracts to stimulate or stimulate as much as floral extracts, but surprisingly they did. We have tried most of the consituents of the high polarity sugar blend but no single consituent seems to work as well as the original extract. We tested the stimulation of conidia production on leaves with and without fungicide. Ferbam completely inhibits the production of conidia and Headline partially inhibited it. We are trying to figure out why the production of conidia are not stimulated by leaves in the field. Ultimately, we would like to test whether we could suppress inoculum by stimulating it and suppressing it with fungicide before it could infect flowers. 2. Please state what work is anticipated for next quarter: None. This is the final report 3. Please state budget status (underspend or overspend, and why): The budget is closed and no further spending will occur
In this project we are profiling the new scions and rootstocks for their tolerance to HLB by studying the metabolite content by GC-MS, and challenging new varieties with psyllids and HLB.Progress on Objectives: Objective 1.1. 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. This quarter we focused in three areas: 1) Marathon Mandarin analyses; 2) CUPS Mandarins, 3) new Valencia varieties.a. For the evaluation of the new mandarin hybrid Marathon, the leaf samples for analysis of volatiles and polar metabolites have been run on the GC-MS and integrated. The analysis found eucalyptol in Marathon, which we have not detected before in other citrus varieties. However, overall profile strongly suggests that Marathon is unfortunately susceptible and does not behave like Sugar belle. Challenging with CLas by grafting confirmed that. b. For the leaf samples from inside the CUPS taken in March 2021, we collected all of the varieties of Mandarins or mandarin hybrids that were available. The VOC analysis is completed for these. Findings: From the VOC analysis we found two distinct chemotaxonomic groups – those that produce thymol and those that don’t. Previously we found that leaves of Sugar Belle, a mandarin hybrid considered tolerant to HLB, contained thymol and its methyl ester, and leaves of most sweet orange (considered HLB susceptible) do not. The group that contained thymol and/or its methyl ester included Sugar Belle, Dancy tangerine (a parent of Sugar Belle), Minneola (also a parent of Sugar Belle), BB4-8-20, and UF711. We associated thymol with tolerance to HLB based on its well-known antimicrobial properties.The thymol non-producers were UF950 clementine, Murcott, Bingo, Early Pride, UF411, and Kinnow. These thymol non-producers also did not produce gamma-terpinene, and had high levels of sabinene. We will look at the ratios of thymol and other blends of VOCs to detect any trends that might indicate HLB tolerance. We expect that robust data analysis will help separate the varietal differences and identify key volatiles involved in ACP deterrence or action against CLas bacteria. We hope to be able to predict or screen for HLB tolerance based on the chemometric profiles of the new varieties if we can develop an accurate model.c. For Lucky and its parents Sugar Belle and Nava × Osceola, we repeated this experiment in June using CLas-infected ACPs. We will measure the response to ACP infestation and the acquisition of CLas over time. New work:d. We obtained Valencia and two new varieties from Southern Citrus (5 each of Valencia, Valquarius, and Vernia) for a small study. So far the VOC profile of the three varieties are nearly identical. We will look at non-volatile metabolites next and perform some ACP challenges. Objective 2.2. 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 combinations for tolerating CLas. a. The rootstock seeds from the USDA (US-802, 812, 897, 942, 1283, 1284, 1516) for metabolite profiling and HLB/nematode screening are about three months old and ready to be moved outside to encourage growth.b. We continue evaluating the previous rootstocks for growth habits and HLB tolerance.We greatly appreciate the Schumann Lab for granting access to the CUPS at CREC for sampling for this part of the study. They are presently one of the few sources of healthy mature citrus trees.
In this project we are profiling the new scions and rootstocks for their tolerance to HLB by studying the metabolite content by GC-MS, and challenging new varieties with psyllids and HLB.Progress on Objectives: Objective 1.1. 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. This quarter we focused in three areas: 1) Marathon Mandarin analyses; 2) CUPS Mandarins, 3) new Valencia varieties.a. For the evaluation of the new mandarin hybrid Marathon, the leaf samples for analysis of volatiles and polar metabolites have been run on the GC-MS and integrated. The analysis found eucalyptol in Marathon, which we have not detected before in other citrus varieties. However, overall profile strongly suggests that Marathon is unfortunately susceptible and does not behave like Sugar belle. Challenging with CLas by grafting confirmed that. b. For the leaf samples from inside the CUPS taken in March 2021, we collected all of the varieties of Mandarins or mandarin hybrids that were available. The VOC analysis is completed for these. Findings: From the VOC analysis we found two distinct chemotaxonomic groups – those that produce thymol and those that don’t. Previously we found that leaves of Sugar Belle, a mandarin hybrid considered tolerant to HLB, contained thymol and its methyl ester, and leaves of most sweet orange (considered HLB susceptible) do not. The group that contained thymol and/or its methyl ester included Sugar Belle, Dancy tangerine (a parent of Sugar Belle), Minneola (also a parent of Sugar Belle), BB4-8-20, and UF711. We associated thymol with tolerance to HLB based on its well-known antimicrobial properties.The thymol non-producers were UF950 clementine, Murcott, Bingo, Early Pride, UF411, and Kinnow. These thymol non-producers also did not produce gamma-terpinene, and had high levels of sabinene. We will look at the ratios of thymol and other blends of VOCs to detect any trends that might indicate HLB tolerance. We expect that robust data analysis will help separate the varietal differences and identify key volatiles involved in ACP deterrence or action against CLas bacteria. We hope to be able to predict or screen for HLB tolerance based on the chemometric profiles of the new varieties if we can develop an accurate model.c. For Lucky and its parents Sugar Belle and Nava × Osceola, we repeated this experiment in June using CLas-infected ACPs. We will measure the response to ACP infestation and the acquisition of CLas over time. New work:d. We obtained Valencia and two new varieties from Southern Citrus (5 each of Valencia, Valquarius, and Vernia) for a small study. So far the VOC profile of the three varieties are nearly identical. We will look at non-volatile metabolites next and perform some ACP challenges. Objective 2.2. 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 combinations for tolerating CLas. a. The rootstock seeds from the USDA (US-802, 812, 897, 942, 1283, 1284, 1516) for metabolite profiling and HLB/nematode screening are about three months old and ready to be moved outside to encourage growth.b. We continue evaluating the previous rootstocks for growth habits and HLB tolerance.We greatly appreciate the Schumann Lab for granting access to the CUPS at CREC for sampling for this part of the study. They are presently one of the few sources of healthy mature citrus trees.
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.
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 pathogens 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.
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