October 2020The 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. Nothing further was conducted on the fungicide or CAS validation field trials. We were unable to apply our fungicides during bloom because of the COVID-19 shutdown. We plan to start the planning and set up in the next quarter for these trials in 2021 if we are granted our requested no cost extension.Further work on leaf wetness has been done to investigate how well individual sensors work for leaf wetness duration estimation. This will be presented as a oral presentation at the virtual FSHS meeting in 2020 along with a written document. It was found that the simpler-to-use Deccagon sensors would be less sensitive for leaf wetness duration estimation compared to Campbell sensors in FAWN stations but that they are adequate for risk determination. The risk mapping has been continuing and we are gathering Postbloom fruit drop risks for major citrus producing locations in Florida and intend to analyze data shortly.We are currently setting up several trials assessing the effect of floral extracts on important metabolic processes of the life cycle of C. acutatum. Dr. Wang’s lab has prepared floral extracts using flowers at different developmental stages – pinhead (small white floral buds), popcorn (expanded floral buds), and open flowers. All three extracts from the different floral stages greatly stimulated pathogen development. Based on previous studies, we anticipated that the sugar content on the extracts was responsible for stimulating the pathogen to become active and cause disease. Therefore, the sugar composition of the floral extracts was analyzed and model solutions containing the same proportion of different sugars were prepared. In vitro assays tested the effect of the sugar solutions and compared them to the floral extracts. Although the sugar solutions stimulated the pathogen, the stimulus was not nearly as large as that posed by the floral extracts. Our team is waiting forthe flowering period to collect more flower samples and perform a bioactivity-guided fractionation and identification of the flower extracts. Such fractionation aims to precisely identify the compound or compounds of flowers responsible for pathogen stimulation. Citrus leaves (young and mature leaves from Valencia trees) were also collected and provided to Dr. Wang’s lab so an extract could be obtained and tested in vitro to check for pathogen stimulation. Any common compounds will not be included in our flower extract evaluation if no stimulation is observed, as we expect based on what is observed in the field. 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. We were unable to acheive our field or wind tunnel portions of the project as planned due to the shut down and travel ban. These were extenuating circumstances and we have requested a no cost extension so we can undertake the objectives next season. Accordingly, I have removed my technical staff from the project as they are unable to work on the project as had been anticipated.
June 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 as of March 13. We evaluated 50 fruit each for disease severity on approximately 125 trees in 32 rows. The data entered for analysis but only very preliminarly 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. However, all the treatments appear to have had significantly better disease management than the untreated control. 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). Applications have been 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 occured just as the pre-treatment data collection should have done but we plan to conduct the trial next year if a no cost extension is granted. 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 have been 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 submitted describing the analysis and results from the USA isolates and we are still waiting for a decision by the journal of Molecular Plant Pathology.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. 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, fifty-nine isolates have been 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 is underway to determine if both isolates are the same clone or not. 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. 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 88 samples, 63 isolates are from our Phyllosticta collection, 14 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 is ongoing. Eighty-six fruit of varying developmental stages, as well as controls, continue to be monitored following inoculation in December. The final data are expected to be collected in October. Data are being collected on temperature, relative humidity and light intensity in addition to monitoring for symptom development to determine developmental and environmental parameters of symptom development. 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 (oxytetracycline and streptomycin), 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 bactericides 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 in citrus tissues is needed. This project officially started on December 1, 2018. This is the 7th quarterly progress report covering June 01 to August 31, 2020. During this period, we conducted and/or completed the following work/research tasks: 1) The samples from the foliar spraying experiments in greenhouse were analyzed. The preliminary results show that after foliar application, the concentration of oxytetracycline (OTC) and streptomycin (STR) decreased in the order of leaf > stem > root. Leaf concentration of the bactericides decreased with time, whereas their concentration in stem increased and reached peak at day 15. The concentration of antibiotics in the root was very low, only the STR was detected at day 7. 2) The samples from the treatments of trunk injection and root administration in the greenhouse experiments were all pretreated and are currently under analysis using the LC-MS, and hopefully we can obtain additional data by the end of September. 3) Preparations were made for field trials, including chemicals and tools for field applications of bactericides. The work planned for the next quarter (September 1, to November 30, 2020):The major research tasks for the next three months are to: 1) Analyze the data to be obtained and evaluate the delivery efficacy of three different application methods for bactericides in greenhouse experiments. 2) Implement field experiments, based on the greenhouse results, 3) Conduct treatments in fields, including foliar spraying, trunk injection and root administration.
1. Please state project objectives and what work was done this quarter to address them:
Obj1 1: Near term management
(1a) Field Monitoring with mobile stages: All methods field tested to date have failed for monitoring tools, our team has redesigned the methodology using lab colonies and will be testing in mid to late September.
Field Monitoring with pheromone attraction: Protocol is designed and will be deployed when field populations appear to become more active; (1b) Screening of effective materials and adjuvants: Entomopathenogenic Fungi (EPF) screening completed for late nymphal instars, look like an ideal candidate for use in IPCs. Screening of commercially available inseciticides labelled for use in FL citrus completed, has been presented and in preparation for publication; (1c) Evaluate promising materials in open grove setting: grower validation of some materials that worked well in lab assays (not formal trials); (1d) Evaluate ant management as part of graove management plants: documentation of ant species that are present and impacts on predator establishment (ongoing work, unpublished); (1e) Evaluate management options for IPCs: 3 field sites located for testing EPFs, sites prepared and will be treated once weather improves (need a drier day to complete applications and sampling). (2a) Assessment of predators: Collection of potential predators continued through summer, primers have been deisgned and validation is almost complete; (2b) Determine how to implement mealybug management concurrent with other pest management (anticipated start summer 2021); (2c) Determine what chemistries inhibit feeding: Feeding behaviors in the process of quantification, will continue into fall; (2d) Develop tools to minimize spread: ongoing evaluation of sterilization methods, trying to find what works and is reasonable for growers to implement
2. Please state what work is anticipated for next quarter:
(1a) We anticipate taking monitoring tools for mobile stages to the field in mid to late September and field validating mating pheromone monioring method if we see male cocoons in field populations this fall, if not that will occur in Spring 2021. (1b) Expand life stages tested in conventional insecticide tests, develop assays for determining wax
penetration/degradation by adjuvants; (1c) Tests planned in research planting in Lake Placid this fall; (1d) no work planned for fall, will work on in Spring 2021; (1e) Field sites located and prepared, waiting for a few dry days to treat; (2a) Field collection of potential predators will continue through fall 2020, Validation of primers on known predators (lab fed) will be completed by early October. We anticipate running mealybug primers on subsets of likely predators from field collections starting late October; (2c) We anticipate completing the description of lebbeck mealybug feeding and begin studying influence of insecticides on these behaviors in Fall 2020; (2d) We plan to optimize suggested methods for sterilization of large and small field equipment to minimize spread.
3. Please state budget status (underspend or overspend, and why):
Our budget is slightly underspent, mostly on labor as the postdoc to be hired does not start until late September. Additionally, COVID-19 limited some of the work we wanted to complete this summer, so some field tests could not be performed. We expect to be able to complete much of this work over next 6 months as it appears the mealybug is active, at least in the lab colonies, throughout the winter.
July 2020The 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. Nothing further was conducted on the fungicide or CAS validation field trials. We were unable to apply our fungicides during bloom because of the COVID-19 shutdown. We plan to start the planning and set up in the fall for these trials in 2021.We found that in the polar extracts of the pin head, popcorn, and open flowers greatly stimulated the production of conidia compared to sterile distilled water and 2.5% sucrose solution. The length of the germ tubes and their diameter were greatly increase, an unexpected result. There were also a greater propotion that germinated when compared to the water and sucrose solutions. We plan to test to see if this makes the spores more or less infectious on flowers during bloom. Tests with the non-polar extracts are planned. Further tests with specific constituents of the extracts will be tested based on Dr. Wang’s analysis.Flowers of different floral stages were collected and delivered to Dr. Yu Wang’s program so a new set of floral extracts are obtained and the experiments repeated. Since the partial re-opening, Dr. Wang’s program has been able to conduct polar and non-polar extractions of the 3 flower stages (pin head, popcorn, and open bloom). They are analysing the constituents for further experimentation. 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 were unable to acheive our field portions of the project as planned due to the shut down and travel ban. These were extenuating circumstances and we hope to be able to receive a no cost extension so we can undertake the objectives next season. Accordingly, I have removed my technical staff from the project as the are unable to work on the project as had been anticipated.
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 4th quarter of year 1 of a 2 year project. For the 4th quarter of year 1, a total of 8,343 samples were processed and sent out. This brings the total for year 1 to 26,370 or approximately 15% over the budgeted amount (22,500) for the period. 2. Please state what work is anticipated for next quarter: The submission of samples is completely at the discretion of the submitter and as such is somewhat variable. During the initial days of the Covid-19 pandemic, the number of samples was somewhat reduced. However, since University and others have been allowed to return back to work, the number of samples has increased substantially. It is unclear if these are “catch up” samples or if this will reflect the trend for the coming year. I project that we will probably be at or above the budgeted amount of samples (22,500) for year 2. 3. Please state budget status (underspend or overspend, and why): For year 1 of the project, we processed approximately 15% more samples than were budgeted. 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.
This project started on Nov. 1st, 2019. So, this is the third quarterly report (5/1/20-7/31/20). 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 study will be conducted in the coming Fall semester due to COVID-19, but in the third quarter, we have submitted IRB paperwork for the approval of running sensory with special measures during the pandemic. All the paperwork has been approved, so we are clear for the following processes.
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 second quarter, we started analytical chemistry. Volatile compounds (i.e. aromas) have been analyzing using gas chromatographymass spectrometry (GC-MS). Non-volatile compounds (i.e. sweet, sour, bitter compounds) have been analyzing using Liquid chromatographymass spectrometry (LC-MS). In the third quarter, we continued trained panel study for differentiating citrus flavor attributes such as sweet, citrusy, fruity, and flora etc. Different from sweet oranges, mandarins were mainly characterized with bitter, sour and terpene like attributes, which is consistent with the fact that terpenes play essential roles in the characterization of mandarin flavor. However, some mandarin hybrids were more sweet-orange like. Their sensory profile was much similar to the sweet orange profile. We have been continuing working on volatile and non-volatile analysis in the third quarter.
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 synthesized and delivered to UF collaborator Dr. Johnson to support 2020 field trial. A relatively new Ray Ruby’ grapefruit block with windbreaks was selected in Vero Beach area. In vitro antimicrobial studies were performed using Xanthomonas alfafae as citrus canker surrogate as part of QC protocol. The minimal inhibitory concentration (MIC) was in the range of 128ppm Mg / 32ppm Cu for MM25C75M C, 32ppm Zn / 8ppm Cu for MM25C75Z, 32ppm Zn / 16ppm Cu / 16ppm Mg for MM17C17M66Z, 16ppm Zn / 4ppm Cu for MM20C80Z, and 512ppm Zn / 103ppm Cu for MM17C83Z. Additional studies on Minimal Bactericidal Concentration (MBC) and Colony Forming Units (CFU) were also performed. MM20C80Z and MM25C75Z demonstrated bactericidal activity at 32ppm Zn / 8ppm Cu, while MM25C75M and MM17C1766Z showed MBC at 512ppm Mg / 128ppm Cu and 64ppm Zn / 16ppm Cu / 16ppm Mg, respectively. MM17C83Z did not present bactericidal activity. Quality of all the treatments remain the same as the last year. All treatments are being evaluated this season (metallic Cu at 0.5 lb/acre) for yield, fruit size and disease incidence. Trial results will be reported by the end of the trial season. Additional material characterization results on systemic activity will be reported in coming months.
June 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 as of March 13. We evaluated 50 fruit each for disease severity on approximately 125 trees in 32 rows. The data entered for analysis but analysis has not yet 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. However, all the treatments appear to have had significantly better disease management than the untreated control. 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). The delay was caused by the travel and work restrictions cause by COVID-19. Several subsequent sprays have been applied. During the 2019 -2020 growing season, 6 applications of fungicide were made. There was no major delay or issue with any of our treatments. In our trials, we had nine fungicides along with a water control total of 10 treatments. On March 9-13, 2020, we completed our first trial’s evaluations. We inspected 50 fruit per tree in all treated trees using a 0 (no disease) to 5 (high severity) rating scale. The data are entered and a very preliminary analysis shows all products or product rotations had significantly greater odds of no black spot compared to the untreated control. We were unable to set up the second planned fungicide trial this year because of the pre-treatment data collection needed but we plan to conduct the trial next year if a no cost extension is granted. New fungicide products are being tested for the management of CBS in South Africa. Products being evaluated include Kocide (copper hydroxide) sprayed in alternation with Amistar Top, Luna Sensation or Headline. The final monthly application of fungicide has been completed. The fungicides are being tested in a ‘Valencia’ orange orchard with a history of CBS. The trial evaluation is planned for the end of August. Despite this, 23 isolates were sequenced (6 from South Africa and 17 from the USA) using the Ion Torrent System. The genomes of all the isolates have been 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.Eight of the 12 South African isolates send 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. A manuscript was submitted describing the analysis and results from the USA isolates.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.). Studies on the diversity of Phyllosticta species in Florida is ongoing to determine which species (pathogenic and endophyte) are associated with citrus. High quality genomic DNA has been extracted and purified for fifty-nine of the 125 single-spored Phyllosticta isolates in our collection. Based on tef1 sequences, two isolates (Gc-6 and Gc-7) demonstrated polymorphism distinct from P. capitalensis and P. citricarpa. Greatest sequence identity was found with P. hymenocallidicola. Additional multi-locus phylogenetic analysis of ITS, LSU, and actA sequence of these two isolates was conducted. Analysese of these loci against a larger diversity of Phyllosticta species confirmed that the isolates Gc-6 and Gc-7 uniquely clade with P. hymenocallidicola. This finding strongly suggests that these two isolates from citrus are identical to one another and are P. hymenocallidicola. Collaboration with Dr. Hector Urbina and colleagues at the Division of Plant Industry is ongoing to assess the mating type of isolates collected at the expanding margins of the quarantine zone. Fifteen isolates from 2019 -2020 have been single spored and are being prepped for DNA extraction to confirm species identification and to determine mating type. Isolates from additional sites in Southwest Florida were collected and under going single spore isolates. 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 is ongoing. Eighty-six fruit of varying developmental stages, as well as controls, continue to be monitored following inoculation in December. Data is being collected on temperature, relative humidity and light intensity in addition to monitoring for symptom development to determine developmental and environmental parameters of symptom development. Little progress has been made other than isolate maintenance and monitoring for fruit symptoms since the last report due to COVID-19 restrictions. We hope to be able to accomplish more very soon. A poster of the phylogenetic results is being prepared for the virtual annual meeting of APS this 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 6th quarterly progress report covering March/01 to May/31, 2020. During this period, all the faculty/staffs/graduate students at the University of Florida were required to work at home without accessing the laboratory due to COVID-19 pandemic. Therefore, we have conducted and/or completed only a part of the planned work/research tasks:1. Preparation for field trials including: 1) purchase of equipment and chemicals, 2) creation of questionaires for field survey to the citrus growers, and 3) selection of field sites.2. Performance of literrature review related to this study. The work planned for the next quarter: The major goals of research for the next three months are to analyze the data and evaluate the delivery efficacy of three different application methods for bactericides in greenhouse experiments. Based on the above results, field experiments will be started and the following research tasks will be performed during the next quarter (June 1, 2020 to August 31, 2020):1) collection and analysis of the obtained data from greenhouse experiments; 2) implementation of field experiments; 3) conducting foliar spraying experiments in the field sites; 4) preparation of trunk injection treatments for the field experiments; and 5) preparation of the next quarterly progress report.
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 6th quarterly progress report covering March/01 to May/31, 2020. During this period, all the faculty/staffs/graduate students at the University of Florida were required to work at home without accessing the laboratory due to COVID-19 pandemic. Therefore, we have conducted and/or completed only a part of the planned work/research tasks:1. Preparation for field trials including: 1) purchase of equipment and chemicals, 2) creation of questionaires for field survey to the citrus growers, and 3) selection of field sites.2. Performance of literrature review related to this study. The work planned for the next quarter: The major goals of research for the next three months are to analyze the data and evaluate the delivery efficacy of three different application methods for bactericides in greenhouse experiments. Based on the above results, field experiments will be started and the following research tasks will be performed during the next quarter (June 1, 2020 to August 31, 2020):1) collection and analysis of the obtained data from greenhouse experiments; 2) implementation of field experiments; 3) conducting foliar spraying experiments in the field sites; 4) preparation of trunk injection treatments for the field experiments; and 5) preparation of the next quarterly progress report.
April 2020The 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 were not able to conduct the trials we had started to plan in January because of the work stopage due to COVID-19. The plots were flagged and we were waiting for the major bloom to open. The shutdown occurred at a particularly sensitive time for the trial just as bloom was starting to open. As a consequence, I have moved my technical employees from this grant at this time because we were not able to acheive our objective. I plan to apply for a no cost extension to be able to do the trials we had planned for the spring of 2020. We did monitor the weather during the shut down and despite several PFD infections periods prior to bloom, no infection periods were predicted during bloom in 2020. Despite obtaining interesting data in the wind tunnel trials, we failed to obtain enough flowers to study the dispersal of C. acutatum from infected flowers. It would be very interesting to better understand the dispersal of conidia produced during this life stage of the pathogen, since it is the most important for inoculum production and PFD epidemics. We were in contact with our USDA collaborators responsible for the wind tunnel in the USDA laboratory in Fort Pierce. We were coordinating with their team to start the trials assessing the potential of dispersal of C. acutatum at the beginning of March to initiate the flower inoculations. However, also due to the COVID-19 pandemic, we were unable to do the trials. Their station has closed before ours and we lost contact. We plan to carry the trials out by the end of this year, if sufficient early bloom occurs. During the 2020 season, we set up two field trials in commercial fields in Florida for the Citrus Advisory System validation trials. They were both located in Ft. Meade, in two different blocks within the same commercial operation. One weekly treatment application was applied, but we were unable to continue the trials due to restrictions enforced by the University of Florida regarding travel within the State of Florida during the COVID-19 pandemic. The monitoring of the weather conditions and the model outputs was not interrupted, nonetheless ,but we could not verify events that were conducive for PFD for the area in which the trials were set up. Due to our inability to visit the fields during the pandemic, we could not verify the occurrence of symptoms in the treated areas. The trials assessing the effect of fungicide applications on the secondary conidiation were repeated. We have added an additional inoculum concentration (a higher one) and verified that the fungicide does not hold back conidia production when inoculum levels are high. This is an interesting result that we intend to repeat shortly. The effect of floral extracts of different floral stages has also been assessed in preliminary trials. We have verified that extracts should be filter sterilized prior to the experiments and that a concentration of 1:100 is the ideal one to conduct our trials and avoid saprophyte proliferation. We will be continuting these experiments as soon as permission is granted from the University of Florida. Flowers of different floral stages were collected and delivered to Dr. Yu Wang’s program so a new set of floral extracts are obtained and the experiments repeated. We will analyze if the pattern of the composition of the extracts holds the same proportion of sugars we verified last year. We will also repeat the experiments with the conidia to check if the pattern we are currently observing holds the same. The different stage flowers were freeze dried and will be extracted as soon as permission is granted by the University of Florida and the new flower extracts analyzed for their constituents We were unable to acheive our field portions of the project as planned due to the shut down and travel ban. These were extenuating circumstances and we hope to be able to receive a no cost extension so we can undertake the objectives next season. Accordingly, I have removed my technical staff from the project as the are unable to work on the project as had been anticipated.
This project started on Nov. 1st, 2019. So, this is the second quarterly report (2/1/20-4/30/20). 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. Sugar Belle and Hamlin were processed in the first quarter. In February, we did the sensory and consumer study at both locations of Gainesville and Lake Alfred and in total we recruited about 150 panels. All the tests were conducted based on what we proposed including understanding the demographic difference of consumers, sensory attributes of blended juice, consumer insight of marketing certain blends. There were five groups of samples were tested: 1)100% Hamlin juice 2) 10% Sugar Belle Juice blended with 90% Hamlin juice 3) 50% Sugar Belle juice blended with 50% Hamlin 4) 100% Sugar Belle Juice 5) A commercial juice purchased from grocery store. As a result, 50% Sugar Belle blended with 50% Hamlin was the most favorite juice with the most favorable flavor and taste attributes such as well-balanced brix/acid and no bitter taste. However, the commercial juice was the least favorite one. In addition, the price that panel were willing to pay for 50% Sugar Belle and 50% Hamlin was 1 dollar more than that of the commercial product. Shelf life study was also set up in March, which would take 6 months to get results. At the end of April, we have harvested Valencia and have processed Valencia. Sensory study will be conducted using human subject when COVID issue is eased. 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 in the first quarter and these cultivars have been identified a great field performance. The trained sensory panel study was conducted in fist quarter. In the second quarter, we have been focusing on analytical chemistry. Volatile compounds (i.e. aromas) have been analyzing using gas chromatographymass spectrometry (GC-MS). Non-volatile compounds (i.e. sweet, sour, bitter compounds) have been analyzing using Liquid chromatographymass spectrometry (LC-MS). It will take another one or two quarters to complete the chemistry work and data analysis.
This project started on Nov. 1st, 2019. So, this is the sixth quarterly report (1/31/21-4/30/21). 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.
Brief summary of the achievements from the previous quarters: sensory for Sugar Belle blended with Hamlin and Valencia were completed in the first year. For this fruit season 2020- 2021, the second year of our project, we have been repeating the process and sensory study to determine the quality consistent. Early season cultivar Hamlin was processed and blended with Sugar Belle for the sensory study.
In this quarter, late season Valencia was harvested and processed. Sensory study has been scheduled on May 26th. The completed sensory and consumer study will include 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 sensory attributes will include: sweetness, sourness, bitterness, overall flavor, orange flavor, overall liking and flavor liking etc. The consumer attitude toward mandarin blended orange juice will be included in the survey. In addition, the price consumer would like to pay for each sample, which could provide more information on consumer liking regard each sample will be included.
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
Brief summary of the achievements from the previous quarters: we collected about 20 mandarin hybrids, pure mandarins and sweet orange, which have been identified with the great field performance. In the previous year, we have completed all the sensory studies all the Gas-Chromatography analysis for all the aromas.
In this quarter, we have completed LC-MS/MS data and correlated all flavor data with analytical data to understand the chemistry definition of sweet orange and mandarin. From the results, we could clearly conclude the major difference between sweet orange flavor and mandarin flavor. For example, more aldehydes were correlated with sweet orange flavor but more terpenoids were correlated with mandarin flavor. A manuscript about this study is in the preparation. Hope could be published by the next quarter.
Project summary: The equipment obtained through this grant is anticipated to have the capacity to separate harvested fruit into HLB-symptomatic and non-symptomatic categories, along with data on fruit color, size and weigh distribution, fruit density, internal Brix, and other quality data specifically related to healthy fruit. Historically, most trials report total yield from research plots and lack the ability to segregate healthy fruit from HLB symptomatic fruit. Knowing how treatments impacts fruit quality, especially marketable fruit, is important as growers must improve fruit quality to enhance revenue. CRDF funded acquisition of the optical sorting/grading equipment. The ultimate goal is to support CRDF researchers by mounting the equipment to a mobile platform (with support team) for in-field determination of “healthy” vs HLB-affected fruit. Accomplishments: The Compac sorting equipment for the optical sizing/grading unit was ordered and had the latest software, computers, and electronic communication equipment installed by the manufacturer before shipment. The equipment was delivered to the Indian River Research and Education Center the first week of March. A three-phase, 480-volt generator was purchased by the research center that is dedicated to power the main motors of the unit. A Compaq technician set the machine up on March 19, 20, & 24th and it is now operational. Compaq will provide approximately 5 days of advanced training on the machine and software operation/adjustments when travel is possible after COVID-19 restrictions are lifted. The software adjustments are vital to determine and establish the parameters that will be used to separate health vs. HLB-affected fruit. CRDF funding was for the purchase of the grading equipment and the PI has prepared a separate grant proposal for additional funding for a trailer, modifications and mounting of the equipment to the trailer, to lease a truck, and to hire a team to transport and operate the equipment at field locations and to work with individual researchers in evaluating their specific treatments and fruit quality factors. Currently, CRDF has forward this supplemental proposal to another agency for funding consideration.
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