1. Please state project objectives and what work was done this quarter to address them: Objective: Determine whether Vismax treatment promotes resistance to other major citrus diseases, specifically citrus canker and phytophthora root rot in greenhouse assays with Dr. Evan Johnson Progress (Year1 Q1; 3/4 – 6/11/2021)a. Canker trial #1: Status = Complete. Trial included foliar and soil application methods for Vismax, 7 days prior to inoculation of sweet orange with X. citri. Rate testing of foliar applications (1X, 2X, and 4X concentration) was performed. Trial was successful with controls performing as expected. Raw data and preliminary report received.b. Phytophthora trial #1 (traditional method; potted trees): Status = in progress, propagule counts. Trial includes foliar and soil applications of Vismax, and Ridomil Gold control applications.c. Phytophthora trial #2 (traditional method; Rhizotron): Status = in progress, imaged weekly for root growth. Trial includes foliar and soil applications of Vismax, and Ridomil Gold control applications.d. Trials were visited by EE personnel in May 2021 (no cost to grant)2. Please state what work is anticipated for next quarter:a. Completion of Phytophthora trials #1 and #2, including data anlaysisb. Second canker trials to be repeated to confirm the results of Canker trial #1c. Disease-free seedlings on order for repeat of Phytophthora trials. 3. Please state budget status (underspend or overspend, and why):Status = on budget ($14,462 of $30,600 budgeted in Year 1 – March 1, 2021- Feb 28, 2022) $14,462 paid to University of Florida on 04/05/2021 per University of Florida – Elemental Enzymes Ag & Turf LLC agreement to initiate trials
April 2021The 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. The two validation trials for CAS have been laid out in the Fort Mead area and a small amount of disease has been observed on early bloom in at least one site. We hope for at least one positive site. There were 3 weekly applications made and 2 applications according to the PFD-FAD. The fungicide trial had one application made on May 8th after a rain period. There were no triggers for disease this year at the site and this was the most likely infection period. Fruit data should be recorded in June or early July. A manuscript is in preparation for all the years of CAS validation. The majority of analysis for the leaf wetness models is now completed and manuscript preparation is continuing. We compared the output of leaf wetness sensors to combinations of predictive models for accuracy and sensitivity. As for all models, reliability was heavily influenced by the quality of the weather station data. Reasonable accuracy was found for one, three, or four models in combination but with just two models, the predictive capacity was poor. We are in discussions for how this information could be used to improve CAS predictions. We are working on evaluating PFD risks via an analysis for prediction accuracy. So far, we have tested floral extracts of pinhead, popcorn, and open citrus flowers. All floral extracts stimulated Colletotrichum abscissum conidial germination, and stimuli were compared among these treatments. We analyzed the sugar composition of the floral extracts. Then, we prepared sugar solutions with the same sugar concentration and composition to that of the floral extracts. The germination stimulus of the sugar solutions was higher than the water control, but less than the floral extracts. We tested the stimulus posed by leaf extracts prepared with new and old leaves on the pathogen. Leaf extracts were as good as floral extracts in stimulating the pathogen. As leaves are easier to obtain and available year-round, we decided to investigate the leaf extracts further, to pinpoint what portion of it was responsible for the stimulus considering polarity. We found that the highly polar portion of the leaf extract was responsible for the stimulus. An untargeted metabolomic analysis revealed that there are thirteen main components of the high-polarity portion of the leaf extract. These components will be tested soon.We have been in conversation with the USDA and found out that they have only just repaired their wind tunnel and had slightly more access to the facilities. In the mean time, as we were unsure how soon we could use the equipment, we decided to build a similar tunnel at CREC. We have done a complete set of experiments with the new tunnel with inoculated leaves and flowers with and without rain. We are analyzing the data and adding it to what we observed with the USDA wind tunnel. The manuscript is in preparation and we may do a few follow up experiments as questions arise during the analysis
1. Please state project objectives and what work was done this quarter to address them: This report is for the continued support of the Southern Gardens Citrus Diagnostic Laboratory that provides testing for citrus greening for researchers, growers, and homeowners. The current report is for the 3rd quarter of year 2 of a 2-year project. For the 3rd quarter of year 2, a total of 10,230 samples were processed and assayed. This brings the total for the project-to-date to 48,550 samples assayed, or 3,550 over the budgeted amount for the whole project (8% over). The projection for the full 2-year period is for the lab to have processed ~23% more than the budgeted amount. There is a growing trend for sample submitters to request copy number determination instead of just a Ct value (52 % of the samples were provided with copy number for this quarter, 59% the previous quarter). This is likely due to the fact that the majority of the samples being submitted are coming from researcher trials where quantitative data are desired. All of the samples for the current period were plant samples. No ACP samples were submitted. 2. Please state what work is anticipated for next quarter: It is anticipated that a higher than budgeted amount of samples will continue to be submitted. This is based on an increase in the number of trials being established that require extensive sampling and to several researchers bringing in backlogged samples. 3. Please state budget status (underspend or overspend, and why): Given that we are running 8% above the budgeted amounts for the whole project (123% over if prorated for 7 quarters), it is likely that we will be over the budgeted number of samples for the project. As has been done in the past, we will wait until the end of year 2 of the project and adjust the final invoice either up or down depending on the total number of samples run during the project. If the number of samples exceeds the budgeted amount, the final invoice will be Increased upwards to cover the cost of the consumables. If the number of samples is below the budgeted amount, the final invoice will be reduced to reflect the reduced amount of consumables used.
Work done this quarter:(1b) Adjuvant screeningPreliminary trials have been conducted with adjuvants alone to determine their lethality to lebbeck mealybug. A total of 9 adjuvants, including and 4 from Helena Agri Enterprises were mixed with DI water at label rates and sprayed until dripping on Volk lemon leaves with mealybugs attached. 8 of the adjuvants resulted in significantly greater mealybug mortality over a 7-day period compared to a DI water control. (1c) Evaluate promising materials in open grove setting: We began a field trial at a 10-acre commercial citrus planting that was heavily infested in 2020. First insecticide applications occurred as a pre-bloom prophylactic spray of a systemic material, with a second spray planned in the end of April.(1d) Ant Management · We are testing a variety of methods to remove red imported fire ants (Solenopsis invicta) from citrus groves. Removing the ants can allow natural enemies to prey on and parasitize lebbeck mealybug without interference, and provide more effective biological control. Our research goals are:1. Reduce abundance of fire ants associated with lebbeck mealybug (Nipaecoccus viridis) in central FL citrus groves. · 2. Determine long-term efficacy of different treatment types at reducing fire ant abundance · 3. Assess time and cost of different treatment types · 4. Determine effects of ant treatment on predators abundance within trees and within N. viridis clusters. We are collaborating with the King Lab from the University of Central Florida, and are conducting the experiment in the grove of a local citrus grower. Four experimental treatments will be tested and compared to an untreated control: Chlorpyrifos drench, Clinch ant bait (Abamectin), Extinguish ant bait (S-methoprene), and spot treating ant colonies with hot water. Fire ant abundance will be assessed by counting the number of surviving colonies, and by determining presence/absence of foragers with pecan sandie baits. Natural enemy abundance will be assessed by dissecting mealybug clusters for predator larvae. Sampling for each of these methods will take place every 2 weeks for a total of 3 months post-treatments. Currently, all treatments have been applied and we are conducting follow-up surveys to determine their efficacy. (1e) Evaluate management options for IPCsWe recently completed the study to evaluate several commercially available entomopathogenic fungi (EPF) products as a potential tool to control lebbeck mealybug infestation on young citrus trees within individual protective covers (IPCs). Findings of the study indicate that EPF can cause death of mealybugs and EPF conidia are viable on citrus trees within IPCs up to 42 days after treatment. Thus, EPF can offer control of mealybug for up to 1.5 months after treatment. In February, we began infesting trees in our research planting to run a similar comparison of topical insecticides for clean-up, with pretreatment drenches planned prior to the next set of trees to be infested. Additionally, a controlled greenhouse comparison of drench materials including aldicarb is planned once trees develop sufficient leaf mass to enable testing of residual post application of all chemistries used in addition to impact on mealybugs. We ran a methods trial and found that malathion lasted 2-3 times longer under IPCs compared to open field settings, suggesting that materials sprayed in IPCs that are known to have rapid UV breakdown may persist much longer and remain effective longer under IPCs. (2a) Predator assessmentsPredatory insects have been reared from locally infested plant materials the past two years, with the majority of predators emerging being parasitic flies, which consume a variety of piercing-sucking pest insects, mealybug destroyers, and one species of lacewing. A primer has been developed and evaluated to enable the detection of mealybugs in the guts of predators. Post-feeding retention time in a known predator was assessed and we feel confident that this tool will now enable screening of field caught arthropods that may play a role in the suppression of lebbeck mealybug. The commercially available predators Cryptolaemus montrouzieri, Orius insidiosus, Adalia bipuctata, and Hippodamia convergens have been screened using no-choice assays to determine if they will feed on larval lebbeck mealybugs, and on mealybug ovisacs. Additionally, several wild-caught predators have also been screened, including Harmonia axyridis and larva of the genus Ceraeochrysa (colloquially called trash bugs). Both adult and larval C. montrouzieri readily feed on lebbeck mealybug larvae and ovisacs, as do larval Ceraeochrysa. Adult Orius insidiosus, early instar Adalia bipunctata, and adult and larval Harmonia axyridis do not feed on lebbeck mealybug larvae or ovisacs. Adult Hippodamia convergens and Adalia bipunctata do feed on some lebbeck mealybug larvae, but appear to attack and kill the larvae more than fully consuming them. Neither feed on ovisacs. From our results, only Cryptolaemus montrouzieri shows promise as a commercially available biological control agent for lebbeck mealybug, although Ceraeochrysa larvae may act as natural biological control in the grove. (2d) Develop tools to minimize spread· Killing lebbeck mealybug with isopropyl alcoholWe tested different concentrations of isopropyl alcohol to determine how lethal they are to 1st instar lebbeck mealybugs. 50%, 70%, and 90% solutions of isopropanol were sprayed onto mealybug crawlers placed on cloth swatches, and compared to a DI water control. Both 1 spray at each concentration, and 2 sprays at each concentration were tested. Mortality was assessed after 5, 10, and 15 minutes. The test was repeated, this time assessing mortality at 30 min, 1hr, and 2hrs. For all concentrations of isopropanol, 1 spray resulted in significantly greater mealybug mortality compared to the control. However, several mealybug crawlers remained alive and active after a single spray for all concentrations. 2 sprays of each concentration resulted in almost 100% mortality or incapacitation at all time points. · Using steam to kill adult mealybugs and ovisacsAdult mealybugs and ovisacs may be accidentally transferred from infested groves on tools and equipment. Steam treatments may be an effective method of sanitizing equipment and killing both adults and ovisacs. Using a steam cabinet on-station, we steam treated adults and ovisacs at 100 and 120 degrees Fahrenheit for 5, 10, and 15 minutes to determine mortality. Previous preliminary experiments showed steam treatments at 130 degrees Fahrenheit for 15 and 30 minutes resulted in 100% mortality. Mortality was assessed at 0, 3, and 5 days after treatment, and ovisacs were held for an additional 2 weeks to see if crawlers emerged. At 100 degrees F for all times points, adult mealybug and ovisac mortality was not significantly different than the control. At 120 degrees F for 5 minutes, mortality also functionally 0%. However, mortality rose to 100% at 120 degrees F for 10 and 15 minutes. Currently, we are running trials on adults and ovisacs treated at 130 degrees F for 5 and 10 minutes, and preliminary results show 100% mortality. Work planned for next quarter(1b) Adjuvant screening will continue to determine optimal adjuvants to work in synergism with insecticide sprays.(1c) We will continue treating and scouting the grove we have started a management comparison trial at.(1d) The ant management project will continue throughout the upcoming quarter with the addition of monitoring for predatory insect establishment.(1e) IPC management management trials will continue, looking towards conventional materials for management and spray penetration by tractor mounted sprayers.(2a) Working with FDACS, we have permission to deploy sentinel infested materials to screen more broadly for predators that may be present in the system which we missed with haphazard rearing from infested grove sites.(2c) Feeding mechanisms will continue to be worked out using EPG with the future plan to determine if we can interfere with their feeding via chemical intervention leading to reduced offspring production.(2d) We will continue evaluating sanitation options and are planning to look at vehicle sanitation in the next quarter.
Work done this quarter:(1b) Adjuvant screeningPreliminary trials have been conducted with adjuvants alone to determine their lethality to lebbeck mealybug. A total of 9 adjuvants, including and 4 from Helena Agri Enterprises were mixed with DI water at label rates and sprayed until dripping on Volk lemon leaves with mealybugs attached. 8 of the adjuvants resulted in significantly greater mealybug mortality over a 7-day period compared to a DI water control. (1c) Evaluate promising materials in open grove setting: We began a field trial at a 10-acre commercial citrus planting that was heavily infested in 2020. First insecticide applications occurred as a pre-bloom prophylactic spray of a systemic material, with a second spray planned in the end of April.(1d) Ant Management · We are testing a variety of methods to remove red imported fire ants (Solenopsis invicta) from citrus groves. Removing the ants can allow natural enemies to prey on and parasitize lebbeck mealybug without interference, and provide more effective biological control. Our research goals are:1. Reduce abundance of fire ants associated with lebbeck mealybug (Nipaecoccus viridis) in central FL citrus groves. · 2. Determine long-term efficacy of different treatment types at reducing fire ant abundance · 3. Assess time and cost of different treatment types · 4. Determine effects of ant treatment on predators abundance within trees and within N. viridis clusters. We are collaborating with the King Lab from the University of Central Florida, and are conducting the experiment in the grove of a local citrus grower. Four experimental treatments will be tested and compared to an untreated control: Chlorpyrifos drench, Clinch ant bait (Abamectin), Extinguish ant bait (S-methoprene), and spot treating ant colonies with hot water. Fire ant abundance will be assessed by counting the number of surviving colonies, and by determining presence/absence of foragers with pecan sandie baits. Natural enemy abundance will be assessed by dissecting mealybug clusters for predator larvae. Sampling for each of these methods will take place every 2 weeks for a total of 3 months post-treatments. Currently, all treatments have been applied and we are conducting follow-up surveys to determine their efficacy. (1e) Evaluate management options for IPCsWe recently completed the study to evaluate several commercially available entomopathogenic fungi (EPF) products as a potential tool to control lebbeck mealybug infestation on young citrus trees within individual protective covers (IPCs). Findings of the study indicate that EPF can cause death of mealybugs and EPF conidia are viable on citrus trees within IPCs up to 42 days after treatment. Thus, EPF can offer control of mealybug for up to 1.5 months after treatment. In February, we began infesting trees in our research planting to run a similar comparison of topical insecticides for clean-up, with pretreatment drenches planned prior to the next set of trees to be infested. Additionally, a controlled greenhouse comparison of drench materials including aldicarb is planned once trees develop sufficient leaf mass to enable testing of residual post application of all chemistries used in addition to impact on mealybugs. We ran a methods trial and found that malathion lasted 2-3 times longer under IPCs compared to open field settings, suggesting that materials sprayed in IPCs that are known to have rapid UV breakdown may persist much longer and remain effective longer under IPCs. (2a) Predator assessmentsPredatory insects have been reared from locally infested plant materials the past two years, with the majority of predators emerging being parasitic flies, which consume a variety of piercing-sucking pest insects, mealybug destroyers, and one species of lacewing. A primer has been developed and evaluated to enable the detection of mealybugs in the guts of predators. Post-feeding retention time in a known predator was assessed and we feel confident that this tool will now enable screening of field caught arthropods that may play a role in the suppression of lebbeck mealybug. The commercially available predators Cryptolaemus montrouzieri, Orius insidiosus, Adalia bipuctata, and Hippodamia convergens have been screened using no-choice assays to determine if they will feed on larval lebbeck mealybugs, and on mealybug ovisacs. Additionally, several wild-caught predators have also been screened, including Harmonia axyridis and larva of the genus Ceraeochrysa (colloquially called trash bugs). Both adult and larval C. montrouzieri readily feed on lebbeck mealybug larvae and ovisacs, as do larval Ceraeochrysa. Adult Orius insidiosus, early instar Adalia bipunctata, and adult and larval Harmonia axyridis do not feed on lebbeck mealybug larvae or ovisacs. Adult Hippodamia convergens and Adalia bipunctata do feed on some lebbeck mealybug larvae, but appear to attack and kill the larvae more than fully consuming them. Neither feed on ovisacs. From our results, only Cryptolaemus montrouzieri shows promise as a commercially available biological control agent for lebbeck mealybug, although Ceraeochrysa larvae may act as natural biological control in the grove. (2d) Develop tools to minimize spread· Killing lebbeck mealybug with isopropyl alcoholWe tested different concentrations of isopropyl alcohol to determine how lethal they are to 1st instar lebbeck mealybugs. 50%, 70%, and 90% solutions of isopropanol were sprayed onto mealybug crawlers placed on cloth swatches, and compared to a DI water control. Both 1 spray at each concentration, and 2 sprays at each concentration were tested. Mortality was assessed after 5, 10, and 15 minutes. The test was repeated, this time assessing mortality at 30 min, 1hr, and 2hrs. For all concentrations of isopropanol, 1 spray resulted in significantly greater mealybug mortality compared to the control. However, several mealybug crawlers remained alive and active after a single spray for all concentrations. 2 sprays of each concentration resulted in almost 100% mortality or incapacitation at all time points. · Using steam to kill adult mealybugs and ovisacsAdult mealybugs and ovisacs may be accidentally transferred from infested groves on tools and equipment. Steam treatments may be an effective method of sanitizing equipment and killing both adults and ovisacs. Using a steam cabinet on-station, we steam treated adults and ovisacs at 100 and 120 degrees Fahrenheit for 5, 10, and 15 minutes to determine mortality. Previous preliminary experiments showed steam treatments at 130 degrees Fahrenheit for 15 and 30 minutes resulted in 100% mortality. Mortality was assessed at 0, 3, and 5 days after treatment, and ovisacs were held for an additional 2 weeks to see if crawlers emerged. At 100 degrees F for all times points, adult mealybug and ovisac mortality was not significantly different than the control. At 120 degrees F for 5 minutes, mortality also functionally 0%. However, mortality rose to 100% at 120 degrees F for 10 and 15 minutes. Currently, we are running trials on adults and ovisacs treated at 130 degrees F for 5 and 10 minutes, and preliminary results show 100% mortality. Work planned for next quarter(1b) Adjuvant screening will continue to determine optimal adjuvants to work in synergism with insecticide sprays.(1c) We will continue treating and scouting the grove we have started a management comparison trial at.(1d) The ant management project will continue throughout the upcoming quarter with the addition of monitoring for predatory insect establishment.(1e) IPC management management trials will continue, looking towards conventional materials for management and spray penetration by tractor mounted sprayers.(2a) Working with FDACS, we have permission to deploy sentinel infested materials to screen more broadly for predators that may be present in the system which we missed with haphazard rearing from infested grove sites.(2c) Feeding mechanisms will continue to be worked out using EPG with the future plan to determine if we can interfere with their feeding via chemical intervention leading to reduced offspring production.(2d) We will continue evaluating sanitation options and are planning to look at vehicle sanitation in the next quarter.
March 2021Objective 1: Evaluate the optimal spray timing for Florida and investigate if tree skirting or alternative products improves fungicidal control of citrus black spot.Objective 3: A MAT-1-1 isolate may enter Florida and allow for the production of ascospores. The industry needs to know if this happens, as it will affect management practices. Additionally, the existing asexual population may be more diverse than currently measured. If multiple clonal linages exist, then there may be different sensitivities to fungicides or other phenotypic traits. We also need to determine whether P. paracitricarpa or P. paracapitalensis are present in Florida for regulatory concerns due to misidentification. We plan to survey for the MAT-1-1 mating type, unique clonal lineages, and two closely related Phyllosticta spp. We collected data from the large spray timing and skirting trial in March. We evaluated 50 fruit each for disease severity on approximately 125 trees in 32 rows. We made significant progress on the incidence analysis of the data and are close to finalizing the analysis. We found that fungicide program significantly reduced the black spot incidence compared to the control. The greatest reduction was from the Florida standard timing with applications from May to September. Skirting had no significant effect on the disease incidence but the interaction between fungicide timing and skirting did have a significant effect. The Florida standard timing with skirting was the best performing treatment. For the late fungicide timing (May to October), there was a slight decrease in incidence with the addition of skirting. It should be noted that the 2019-2020 season was light for black spot incidence and severity. From initial scouting, it appears that black spot severity is greater this year and there may be more differences among the treatments that will hopefully support the conclusions from the first year. The minor plots were re-randomized within the main plots and we were able to get the trial re-flagged just in time for the early spray (delayed by 2 weeks but no rain occurred from the first of April until after the early application). All applications were made on time in the spray trial. We will be collecting the data at the end of March, 2021. We were unable to set up the second planned fungicide trial this year because of the COVID-19 shut down. We will be collecting pre-treatment data at the end of March and plan to move forward with the trial. The second season of trials in which different fungicide products are tested for their efficacy to protect citrus fruit from CBS infection, is currently underway. Products being evaluated include Enable (Indar) and Luna experience sprayed on their own as well as Copper hydroxide sprayed in alternation with either Amistar Top or Headline (Cabrio). Fungicides are applied every 4 weeks from October 2020 until March 2021. The fungicides are being tested in a ‘Valencia’ orange orchard with a history of CBS. The trials will be evaluated at the end of August 2021. An additional 8 South African and 8 global (from Argentina and Swaziland) isolates have been sequenced. As the next-generation sequencing data becomes available, the analysis and results are continuously updated to include the new data. DNA from 16 isolates (Argentina, Australia, Brazil and China) passed QC and is in the process of being sequenced. More isolates from Brazil and China are being cultured for DNA extraction and sequencing. We aim to sequence 6 isolates from each of the 5 South African provinces where CBS is found (North West, Limpopo, Mpumalanga, Kwa-Zulu Natal and Eastern Cape) and 6 each from the other countries (Argentina, Australia, Brazil, China, Swaziland and USA) by end of March. Objective 3 (Survey for the MAT-1-1 mating type and two closely related Phyllosticta spp.). AStudies on the diversity of Phyllosticta spp. associated with citrus in Florida has progressed. Our collection of P. citricarpa isolates were obtained from citrus fruit in different areas under quarantine from 2010 to 2020. These isolates were previously screened by morphology to remove nonpathogenic P. capitalensis isolates. We are now screening the remaining 202 isolates using molecular techniques to determine if we have cryptic species that may have been misidentified as P. citricarpa. To date we have screened 125 isolates by amplifying and DNA sequencing the tef-1a (Translation elongation factor-alpha) locus. This screen has revealed that two isolates (Gc-6 and Gc-7) match to a Phyllosticta species not previously reported in association with citrus. The best sequence match based on ITS and tef-1a is to a species reported previously as a pathogen on a member of the Amaryllidaceae family (Hymenocallis littoralis) in Australia. Numerous inoculations of citrus have determined that these isolates do not cause citrus black spot. Multilocus analysis and leaf inoculations on Hymenocallis species are ongoing. These studies will allow us to conclusively determine the identity of these two isolates. Vegetative and sexual compatibility assays (sandwich mating) were performed to determine the mating type of the two isolates. The results showed that Gc6 and Gc-7 isolates are heterothallic and belong to the same mating type, as neither isolate was a capable of producing pseudothecia in solo cultures or in paired cultures with one another. The screening and further characterization of these new species is continuing to obtain robust information on the diversity of Phyllosticta species and determine the presence of cryptic species in Florida.We screened an additional 26 isolates of our P. citricarpa isolates for mating-type (MAT1-1 and MAT1-2). During this period we have screened an additional 26 new isolates and, as shown in Figures 1, only the MAT1-2-1 mating type is detected in new isolates collected in 2020. We conclude that the MAT1-1 mating type is still absent in the Floridian population.To determine the phenology of fruit susceptibility inoculation studies of citrus fruit (Meyer lemon) were performed in a quarantine greenhouse at the Florida Department of Agriculture and Consumer Services (DPI) in Gainesville. A total of 97 fruit were used in this experiment. Of these 97 fruits, 25 served as controls and 72 were inoculated with the Gc-12 isolate of P. citricarpa. Disease assessments were performed weekly for a full calendar year. A total of 50 fruit produced symptoms in this period. All 50 were from fruit inoculated with the Gc-12 isolate, and no symptoms were observed in the control treatment. Therefore, from all inoculated citrus fruit, 69.4% produced symptoms, and 30.5% remained asymptomatic. The indications from this one experiment suggest that citrus fruit are susceptible at all stages of their development regardless of their maturation time. A second trial will be conducted in 2021 to determine the period of fruit susceptibility to P. citricarpa.
1. Please state project objectives and what work was done this quarter to address them:
This report is for the continued funding of the Southern Gardens Diagnostic Laboratory that provides testing for citrus greening for researchers, growers, and homeowners. The curent report is for the 2nd quarter of year 2 of a 2-year project. For the second quarter of year 2, a total of 5,536 samples were processed and assayed. This brings the total for the project to date to 38,320 or approximately 13.5% over the amount budgeted for the first 6 quarters of the project )or 85.2% of the total number of samples budgeted for the whole project). Of the grower samples run for the period, 59% of the samples were reported with copy number as well as CT values. All of the samples for the current period were plant samples. No ACP samples were submitted.
2. Please state what work is anticipated for next quarter:
Based on communications with repeat customers, it is anticipated that the sample load will be at or above the budgeted amount. Depending on the timing, it could be substantially above the budgeted amount.
3. Please state budget status (underspend or overspend, and why):
Given that we are running 13.56% above the budgeted amounts, it is likely that we will be over the budgeted number of samples for the project. As has been done in the past, we will wait until the end of year 2 of the project and adjust the final invoice either up or down depending on the total number of samples run during the project. If the number of samples exceeds the budgeted amount, the final invoice will be Increased upwards to cover the cost of the consumables. If the number of samples is below the budgeted amount, the final invoice will be reduced to reflect the reduced amount of consumables used.
Five multi-metal bactericides containing Cu as minor component, Cu-Mg (MM25C75M), Cu-Zn (MM25C75Z), Cu-ZnO (MM20C80Z), Cu-ZnS (MM17C83Z), Cu-Mg-Zn (MM17C17Mg66Zn) and four respective controls without Cu (i.e. coated Mg, Zn, ZnO, ZnS) were included 2020 field trial. 8 year-old ‘Ray Ruby’ grapefruit block with windbreaks consisting of approx. 25 ft. tall Corymbia torelliana was was selected in Vero Beach area. Foliar application rate included one gallon per tree (equivalent to 145 trees per acre, a total of eight foliar applications) starting June 1 and ending October 26, 2020. Early spring applications were missed because of COVID-19 work restrictions preventing delivery of materials. Disease evaluation assessed the incidence of fruit with canker lesions for 100 fruit per treatment from the middle 3 trees in each plot. Melanose and scab were also assessed at time of canker rating. Any phytotoxicity (peel burn) was noted and recorded at harvest. Yield was measured as lbs fruit per tree for the middle 3 trees in each plot. A subset of fruit from each plot was sized and tested for juice quality at the CREC packing house/pilot plant facility. No monthly rainfall was detected in March 2020 when initial infection of the spring leaf flush would be expected. Monthly rainfall was about average from April to June and then dropped well below average from July through September. The lack of rainfall during the spring flush probably reduced inoculum buildup in the leaves and early fruit infection. Despite the low rainfall early in the season, the incidence of fruit canker lesions increased slightly to 12.4% from 8.2% the previous year in the UTC. All experimental treatments performed well and comparable to the standard Cu, Kocide 3000. Although not significant, there was an apparent dose effect of ZnO with the lower rate having the highest incidence of canker other than the UTC. Most products performed numerically better than Kocide 3000, but this could be due to rate differences as the only product tested at multiple rates showed a rate response trend. COVID-19 prevented early season treatments, so the full effect of the products on yield cannot be determined; however there was a significant retention of fruit compared to the UTC resulting in higher yields. The largest effect on yield this season was canker management. The lack of a significant yield difference between bactericidal products prevents direct inference about HLB efficacy; however, Kocide 3000 had one of the lowest yields of the bactericides, suggesting that some of the others may have some efficacy for HLB and improvements may require a couple years to become significant. No significant differences were found in internal fruit quality based on brix-acid ratio or total soluble solids. Insufficient scab and melanose were observed to collect useful data this year. Fruit size and juice quality data are being analyzed and will be reported in future report.
The purpose if this project is to reveal the mechanisms of bactericide uptake and transport in citrus plant and establish a theoretical basis for developing technologies to improve the efficacy of bactericides, which is helpful to provide potential solution to the development of effective chemotherapeutic tools for HLB management. Three application methods, foliar spraying, tree trunk injection and root administration, for bactericides (oxytetracycline and streptomycin) currently suggested to be used in citrus HLB management have been employed in this study with greenhouse and field tests. The data obtained in the test will be used to: 1) compare the delivery efficacy of the bactericides applied by the three application methods into the citrus plants; 2) to compare the distributions and concentrations of the bactericides in different citrus tissues and; 3) to compare the effect of citrus variety and age on the delivery of bactericides applied by the three methods and up taken concentrations of bactericides by the plants. This project officially started on December 1 2018. This is the 9th quarterly progress report covering December 1, 2020 to February 28, 2021. During this period, we have conducted and/or completed the following work/research tasks:1) Field study has been completed based on greenhouse study results. The study included two citrus varieties, Grapefruit and Valencia orange, in two farms located in the Fort Pierce area. Oxytetracycline and streptomycin applied into/onto citrus plants by foliage spraying, tree trunk-injection and root administration methods. Samples of citrus leaves, stems, roots and fruits from all the tested plants collected on the day 0, 7, 15 and 30 after treatments and brought back to the Lab for processing. 2) All the samples collected from the field study have been processed for extraction of oxytetracycline and streptomycin analysis purpose. The procedure used for the extraction of oxytetracycline and streptomycin included washing, air drying, cutting, liquid nitrogen grinding, weighing out, and solvent extracting, SPE column cleaning up nitrogen-air dying and bring up to targeting volume with special solvent for HLPC-MASS analysis. 3) The extracted samples for oxytetracycline and streptomycin analysis have been sent to cooperated institute, the Citrus Research and Education Center (CREC), UF for analysis purpose. However, the analysis has not been finished yet. The work planned for the next quarter (March 1 to May 31, 2021): The major research tasks for the next three months include: 1) Completing the analyses of the extracted samples 2) Processing of the data from the field trials.3) Write the final report for this project.4) Preparation of manuscripts from the data obtained from the greenhouse and field experiments..
January 2021The 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. The two validation trials for CAS have been laid out in the Fort Mead area and a small amount of disease has been observed on early bloom in at least one site. We hope for at least one positive site. The fungicide trial is in the planning stages and will be laid out in the next few weeks. The majority of analysis for the leaf wetness models is now completed and manuscript preparation is underway. We compared the output of leaf wetness sensors to combinations of predictive models for accuracy and sensitivity. As will all models, reliability was heavily influenced by the quality of the weather station data. Reasonable accuracy was found for one, three, or four models in combination but with just two models, the predictive capacity was poor. We are in discussions for how this information could be used to improve CAS predictions. We are working on evaluating PFD risks via an analysis for prediction accuracy. When we extracted leaf compounds from citrus to evaluate them for stimulation of appresoria and conidia production, we were greatly surprised to find they stimulated germination and conidia production as much as the floral extracts. The mature citrus leaves from Valencia tree were extracted by sonication with water at room temperature for three hours. The extract was filtered and concentrated under reduced pressure to yield a dried sample. The concentrated extract was then chromatographed over silica gel (3.5 × 30 cm; 40-63 µm particle size) and eluted with gradient mixtures of n-hexane/acetone/MeOH (10:1:0 . 0:0:1, each 2 L) to afford seven sub-fractions (F17). Seven fractions were analyzed by UHPLC to analyze their polarity. The UHPLC system was following: 0 -5 min, 8% B; 5-16 min, 8-90% B; 16-17 min, 90-100% B; 17-24 min, 100% B. The mobile phase included 0.1 % formic acid aqueous solution (A) and 0.1 % formic acid containing acetonitrile (B). According to their polarity, seven subfractions were combined into two fractions. One fraction contains highly polar compounds and the other one possesses medium polar compounds. The high-polarity extract greatly induceds conidia germination in 12 hours, even more than floral extracts. This is counter our hypothesis that sugars were responsible for the stimulation as the extracts that contain most sugars did not stimulate germination to the same extent. We are in conversation with the USDA to recommence work in their wind tunnel to conduct the experiment with conidia on flowers for the final comparison. However, it is not clear when the USDA will reopen their facilities. We hope it will be soon.
1. Please state project objectives and what work was done this quarter to address them: This report is for the continued funding of the Southern Gardens Diagnostic Laboratory that provides testing for citrus greening for researchers, growers and homeowners. The current report is for the 1st quarter of year 2 of a 2 year project. For the first quarter of year 2, a total of 6255 samples were processed and sent out. This brings the total for the project to date to 32,625, or approximately 16% over the amount budgeted for the first 5 quarters of the project. Of the samples run for the period, results for 33% of the samples were reported with copy number as well as Ct values. All of the samples for the current period were plant samples. No psyllid samples were submitted. 2. Please state what work is anticipated for next quarter: The submission of samples is completely at the discretion of the submitters and as such is somewhat variable. The trend for the last few quarters has been to receive large groups of samples from research trials, sometimes upwards of 1000 samples per submission. If this trend continues, we would expect to be above the budgeted amount of samples. 3. Please state budget status (underspend or overspend, and why): Given that we are running 16% above the budgeted amounts, it is likely that we will be over the budgeted number of samples for the project. As has been done in the past, we will wait until the end of year 2 of the project and adjust the final invoice either up or down depending on the total number of samples run during the project. If the number of samples exceeds the budgeted amount, the final invoice will be increased upwards to cover the cost of the consumables. If the number of samples is below the budgeted amount, the final invoice will be reduced to reflect the reduced amount of consumables used.
December 2020Objective 1: Evaluate the optimal spray timing for Florida and investigate if tree skirting or alternative products improves fungicidal control of citrus black spot.Objective 3: A MAT-1-1 isolate may enter Florida and allow for the production of ascospores. The industry needs to know if this happens, as it will affect management practices. Additionally, the existing asexual population may be more diverse than currently measured. If multiple clonal linages exist, then there may be different sensitivities to fungicides or other phenotypic traits. We also need to determine whether P. paracitricarpa or P. paracapitalensis are present in Florida for regulatory concerns due to misidentification. We plan to survey for the MAT-1-1 mating type, unique clonal lineages, and two closely related Phyllosticta spp. We collected data from the large spray timing and skirting trial in March. We evaluated 50 fruit each for disease severity on approximately 125 trees in 32 rows. The data entered for analysis but only very preliminary analysis has begun. The treatments were in a randomized split-plot design with skirting as the main plot and fungicide timings (early, standard, late) along with an untreated control were the minor plots. The data were verified and formatted for analysis. We are in the process of analysing the incidence data of this large data set. The minor plots were re-randomized within the main plots and we were able to get the trial re-flagged just in time for the early spray (delayed by 2 weeks but no rain occurred from the first of April until after the early application). All applications were made on time in the spray trial. We were unable to set up the second planned fungicide trial this year because of the COVID-19 shut down. It occurred just as the pre-treatment data should have been collected but we plan to conduct the trial next year if a no cost extension is granted. In South Africa, the trial in which different fungicides were tested for their efficacy to protect Valencia orange fruit from CBS infection was evaluated in August 2020. All the fungicides tested were effective in protecting fruit as they achieved more than 97% CBS free fruit, except for Luna Sensation (applied 6x on its own), which achieved 67.5% clean fruit. More than 99% CBS free fruit were yielded by applying Enable (Indar) 6 times on its own and also applying Amistar Top in alternation with copper hydroxide. The highest percentage (100%) of CBS free fruit was achieved with standard program consisting of copper oxychloride followed by the application of two sprays of azoxystrobin + copper oxychloride + mineral oil and lastly copper oxychloride. Trees that were sprayed with copper hydroxide in alternation with Cabrio (Headline) yielded 97.3% CBS fruit. The trial site was, however, characterised by a low incidence of citrus black spot during the 2019-20 season with the untreated trees yielding 60.2% fruit without CBS lesions. With the exception of programs alternating copper hydroxide with either Cabrio (92% fruit showing no phytotoxicity) or Amistar Top (97.8% fruit showing no phytotoxicity), the experimental fungicides did not produce any phytotoxicity on fruit. Due to the unavailability in South Africa, Miravis, Miravis Top, PhD, Priaxor and Luna Experience were not tested in this trial. Twenty-three isolates were sequenced (6 from South Africa and 17 from the USA) using the Ion Torrent System. The genomes of all the isolates were successfully assembled and analysed using a customised bioinformatics pipeline. Previous genotypes obtained with SSR primers were confirmed and new SSR primers were developed in silico. To date, mapping and SNP variant statistics as well as in silico genotyping data revealed significantly less variation between the USA isolates than between the isolates from South Africa. To investigate the fine-scale genetic differences within the USA P. citricarpa population, the assembled genomes were annotated by mapping the reference genes to the assembled contig sets, using GMAP. The variant calling results together with the annotations were further analysed using SNPeff, to detect putative variable genes. In silico detection of mating types were also performed and confirmed that only one mating type is present in the USA. A manuscript was accepted and published by Physiological and Molecular Plant Pathology describing the analysis and results from the USA isolates (https://doi.org/10.1016/j.pmpp.2020.101591).Eight of the 12 South African isolates sent for 200bp sequencing passed the quality control checks and were sequenced. Quality and completeness of the genome assemblies will be assessed, as well as number of SSRs that can be detected, to determine whether 200bp sequencing is a viable and more cost-effective sequencing approach. Our research facilities were closed from mid-March to end of May due to the COVID-19 pandemic, causing the delay in evaluating the 200bp sequencing. The isolates that failed quality control, as well as additional South African isolates, are in the process of being cultured and DNA will by re-extracted and sequenced. NGS data from the eight samples were received and are in the process of being analyzed. Eight more samples were submitted for NGS. In total, 11 South African isolates have been sequenced and analysed in the same manner as the USA isolates, to investigate the population structure of P. citricarpa in South Africa. Twelve more South African isolates have been cultured, DNA extracted, and are in the process of being sequenced. A more cost-effective sequencing approach (200bp rather than 600bp sequencing) are currently investigated.Objective 3 (Survey for the MAT-1-1 mating type and two closely related Phyllosticta spp.). A study on the diversity of Phyllosticta species is ongoing to determine which species (pathogenic and endophyte) are associated with citrus in Florida. Currently, 59 isolates were extracted and high-quality DNA purified. Based on tef1, ITS and actA sequences, two isolates (Gc-6 and Gc-7) demonstrated polymorphism with P. capitalensis and P. citricarpa, and the highest sequence identity was found with P. hymenocallidicola. This finding strongly suggests that the two isolates are identical to P. hymenocallidicola. Additional multi-locus phylogenetic analysis of GPDH sequence of these two isolates is underway to support our preliminary result and reject any possibility of misidentification. Sexual and vegetative compatibility test of isolate Gc-6 and Gc-7 determined that both isolates are likely the same clone. Moreover, isolates Gc-6 and Gc-7 failed to produce symptoms in citrus when tested on lemons in quarantine. Leaf inoculation of Amaryllis species is underway for a pathogenicity test of both isolates. Hymenocallis littoralis will be tested as well since P. hymenocallidicola was originally described from this host. It was difficult to get the H. littoralis plants but they are now growing in our screenhouse in preparation for inoculation. To obtain robust information on the diversity of species of Phyllosticta in Florida. Further Phyllosticta isolates are being prepared for screening using tef1 primers. To date, no P. paracitricarpa or P. paracapitalensis have been identified but using the tef1 primers will identify any isolates that maybe part of our remaining collection. Out of 202 samples, 125 isolates are from our Phyllosticta collection, 66 isolates were isolated from fruit lesions collected from different locations under quarantine in Florida (samples provided by Dr. Hector Urbina from the Division of Plant Industry), and 11 isolates isolated from fruit lesions collected in groves, in the La Belle area on the leading northern edge of the spring 2020 quarantine zones. To better understand the diversity of P. citricarpa in the region our partnership with Cuban researchers has been strengthened. Permits for the acquisition of genomic DNA from Cuban P. citricarpa isolates has been approved. We are, amidst current travel restriction, devising plans to have the DNA shipped from Cuba. DNA representing these isolates will be screened for mating type and used within a larger analysis of the global P. citricarpa population structure. Additional efforts have been focused on determining the role of fruit developmental etiology on susceptibility to CBS. Our established quarantine greenhouse experiment with fruit-bearing Myer lemon trees recently finished. Ninety-seven fruit of varying developmental stages, as well as controls, were inoculated in December 2019. There were 25 water controls and 73 were inoculated with isolate Gc-12. At the end of the experiment, 50 fruit were symptomatic. The data are being analaysed. A poster of the phylogenetic results was prepared and presented at the virtual annual meeting of APS in August.
The purpose of this project is to reveal the mechanisms of bactericide uptake and transport in citrus plant and establish a theoretical basis for developing technologies to improve the efficacy of bactericides, which is helpful to provide potential solution to the development of effective chemotherapeutic tools for HLB management. Achieving this outcome will require progress in the following three tasks: (1) to compare the delivery efficacy of bactericides with three application methods (foliar spraying, truck injection, and root administration) based on the uptake and dynamic movement/distribution of the bactericide within the tree; (2) to clarify the systemic movement and transportation mechanisms of bactericides within the phloem of tree; and (3) to investigate the effects of citrus variety and age on the delivery efficacy of bactericides. This project requires a combination of greenhouse studies and field trials. Prior to conducting these experiments, a sensitive and accurate quantifying method of bactericides (oxytetracycline and streptomycin) in citrus tissues is needed. This project officially started on December 1, 2018. This is the 8th quarterly progress report covering September 01 to November 30, 2020. During this period, we have conducted and/or completed the following work/research tasks:1) The samples from the trunk injection and root administration experiments in greenhouse were analyzed. The results show that after trunk injection, oxytetracycline (OTC) was detected in leaves on day 7, 15 and 30; the concentration of OTC in leaves was highest on day 7 and decreased with time. The OTC in stems and roots was detected only on day 7, whilst streptomycin (STR) was not detected in leaves, stems or roots. The results of root administration using a root-split technique showed that both OTC and STR could be horizontally transported from the treated roots to untreated roots. The absorption efficiency of STR was higher than that of OTC. Both antibiotics could be detected in leaves and roots on day 30.2) Among the three tested application methods, root administration by the split technique (RS) has the highest delivery efficiency for OTC, followed by trunk injection (TI), and least with foliar spraying: RS > TI >FS. The results for STR are inconsistent. The results from greenhouse experiments remain to be confirmed by the field trials.3) The field trials with three application methods, i.e. foliar spraying, trunk injection and root administration, were conducted in two different citrus groves, one grapefruit and one orange. So far we have completed the treatments and collection of leaf, stem and root samples on the 7th, 15th and 30th days after the application of OTC or STR.The work planned for the next quarter (December 1, to February 28, 2021):The major research tasks for the next three months include: 1) Continuing the field experiments.2) Preparation of the samples from the field trials prior to analysis of OTC and STR by LC-MS, including grinding, extraction, purification and concentration.3) Analysis of the pretreated samples for concentration of OTC and STR using the LC-MS.4) Processing of the data from the field trials.5) Preparation of reports and/or manuscripts from the data obtained from the greenhouse experiments.
Field trial samples (Mg-Sol, Mg-Sol_Cu, NAC-ZnO, NAC-ZnO_Cu, NAC-ZnS, NAC-ZnS_Cu) and commercial controls (Zinc nitrate, MagnaBond and Kocide) were tested for rainfastness following a published protocol [1] with minor modifications.
Briefly, citrus leaves were collected from citrus seedlings, washed with DI water and air dried. Then, leaf surface area was measured using the LeafByte App. Leaves were individually sprayed with treatment at the same rate they are applied in the field. Same rate was used for Zn and Cu controls. After air drying, leaves were individually dipped in a conical tube containing 30 ml of DI water for 30 seconds under agitation in order to simulate rain (Wash 1 – W1). Leaves were dried and the washing procedure was repeated twice in order to have W2 and W3. A fourth wash (W4) was done using 30ml of ethanol-acid solution for 60 seconds under agitation aiming to remove all the remaining metal on the surface of the leaves. Then, the metal concentration in each wash was measured by Atomic Absorption Spectroscopy. The total metal content per leaf was calculated by summing W1, W2, W3 and W4 content and dividing by the leaf surface area. The percentage of metal released in each wash was calculated by dividing the wash metal content by the total metal content. Triplicates were done and the average was used to report the results on Table 1.
In terms of Mg release, around 60% of the total magnesium content was released in the first wash (W1) for Mg-Sol and Mg-Sol Cu. The presence of Cu in Mg-Sol_Cu seems to have increased the amount of Mg release in W2 and W3 in comparison to Mg-Sol.
In terms of Cu release, Mg-Sol_Cu also released around 60% of the Cu in the first wash (W1) while NAC-ZnS_Cu released more than 80% of the Cu in W1. NAC-ZnO_Cu showed similar behavior of Kocide, releasing only 12% of the Cu in the first wash. The remaining Cu found in W4 for both Kocide and NAC-ZnO_Cu was around 87%, indicating Cu can be sustained released from those samples.
In terms of Zn release, NAC-ZnS and NAC-ZnS_Cu released 90% of the Zn in the first wash, while NAC-ZnO, NAC-ZnO_Cu released around 70% of the Zn, similarly to the control Zn nitrate.
[1] Impact of (nano) formulations on the distribution and wash-off of copper pesticides and fertilisers applied on citrus leaves. M Kah, D Navarro, RS Kookana, JK Kirby, S Santra, A Ozcan, S Kabiri. Environmental Chemistry 16 (6), 401-410 (2019).
This project started on Nov. 1st, 2019. So, this is the fourth quarterly report (8/1/20-11/15/20). There are two objectives in this project. The progress of each objective is listed here:
Objective 1. Evaluation of blended juice using released HLB-tolerant sweet orange/mandarin cultivars via analyses of sensory and consumer acceptance.
Sensory for Sugar Belle blended with Hamlin were completed in the second quarter. At the end of April, we have harvested Valencia and have processed Valencia. Sensory and consumer study of Valencia mixing with Sugar Belle has been completed in this quarter. The completed sensory and consumer study included 1) a commercial product 2) 90% Valencia blended with 10% Sugar Belle 3) 50% Valencia blended with 50% Sugar Belle 4) 100% Valencia 5) 100% Sugar Belle. The best overall liking and flavor liking is 50% Valencia blended with 50% Sugar Belle then flowed by 100% Valencia. The sensory attributes included: sweetness, sourness, bitterness, overall flavor, orange flavor, overall liking and flavor liking etc. The consumer attitude toward mandarin blended orange juice was included in the surveyIn addition, they provided the price they would like to pay for each sample, which could provide more information on consumer liking regard each sample.
Objective 2. Identify more tolerant cultivars resembling the quality of Valencia for the juice market, and identify a chemistry definition of consumer accepted orange flavor
We collected about 20 mandarin hybrids, pure mandarins and sweet orange in the first quarter and these cultivars have been identified with the great field performance. The trained sensory panel study started in the first quarter but stopped for the second quarter due to COVID-19. In the third quarter, we continued trained panel study for differentiating citrus flavor attributes such as sweet, citrusy, fruity, and fl-ora etc. In the fourth quarter, we have completed all the sensory studies for 20 cultivars collected. In the fourth quarter, we have also completed all the Gas-Chromatography analysis for all the aromas of 20 samples. We are still in the process of completing all non-volatile (e.g. taste compounds) analysis and hope can be done in the next quarter.
We expect to correlate trained sensory data with analytical data (aroma and taste) at the end. In this way, a chemical definition of mandarin and orange flavor can be provided.
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