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.
This project started on Nov. 1st, 2019. So, this is the first quarterly report (11/1/19-1/31/20). 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. In November 2019, all PIs hired three students which has been budgeted for this project and discussed the details of protocols. At the end of December 2019, Sugar Belle was harvested and in early January, Hamlin was harvested. At the end of January, Sugar Belle and Hamlin were processed and pasteurized at CREC packing house. 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. we have been doing the data analyses in the second quarter and the results will be provided in the second quarterly report. In addition, shelf life study was also set up, which would take 6 months to get results. All the data such as brix, brix/acid and oil content was recorded. 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 November and December 2019 and these cultivars have been identified a great field performance. The trained sensory panel study was conducted in January, February and March 2020. In addition, we started the analytical study in February 2020. Some results could be provided in the second quarterly report.
This project started on Nov. 1st, 2019. So, this is the fifth quarterly report (11/15/20-2/15/21). 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.
Sensory for Sugar Belle blended with Hamlin and Valencia were completed in the first year. For this fruit season, the second year of our project, we have been repeating the processing and sensory evaluation in order to see the quality consistence among different years. Bt the end of December 2020, we harvested Sugar Belle and Hamlin (as the early cultivar). In mid January, we processed both Sugar Belle and Hamlin juice. The sensory evaluation has been scheduled next week in February. The completed sensory and consumer study will include 1) a commercial product; 2) 90% Hamlin blended with 10% Sugar Belle; 3) 50% Hamlin blended with 50% Sugar Belle; 4) 100% Hamlin; 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, they provided the price they would like to pay for each sample, which could provide more information on consumer liking regard each sample. The stability of Sugar Belle juice will be conducted in March.
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 flora 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. In the current quarter (5th quarter), we have completed all non-volatile (e.g. taste compounds) analysis using LC-MS/MS. We are now correlating trained sensory data with analytical data (aroma and taste). Hope we can complete the correlation work by the end of next quarter. In addition, in this current quarter, we start to implement machine learning into defining mandarin and orange flavor profile using all published data. We are currently collecting data and trying the machine learning models. This part is expected to complete by the end of this project.
This project started on Nov. 1st, 2019. So, this is the fifth quarterly report (11/15/20-2/15/21). 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.
Sensory for Sugar Belle blended with Hamlin and Valencia were completed in the first year. For this fruit season, the second year of our project, we have been repeating the processing and sensory evaluation in order to see the quality consistence among different years. Bt the end of December 2020, we harvested Sugar Belle and Hamlin (as the early cultivar). In mid January, we processed both Sugar Belle and Hamlin juice. The sensory evaluation has been scheduled next week in February. The completed sensory and consumer study will include 1) a commercial product; 2) 90% Hamlin blended with 10% Sugar Belle; 3) 50% Hamlin blended with 50% Sugar Belle; 4) 100% Hamlin; 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, they provided the price they would like to pay for each sample, which could provide more information on consumer liking regard each sample. The stability of Sugar Belle juice will be conducted in March.
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 flora 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. In the current quarter (5th quarter), we have completed all non-volatile (e.g. taste compounds) analysis using LC-MS/MS. We are now correlating trained sensory data with analytical data (aroma and taste). Hope we can complete the correlation work by the end of next quarter. In addition, in this current quarter, we start to implement machine learning into defining mandarin and orange flavor profile using all published data. We are currently collecting data and trying the machine learning models. This part is expected to complete by the end of this project.
Crystallographic and morphological characterization results
In this report, we focused on the crystallographic and morphological characterization of as-prepared multi-metal materials, using X-Ray Diffractometry (XRD) and Scanning Electron Microscopy (SEM).
(1) X-Ray crystal structure of purified MM25C75M powder was determined using X-ray diffraction (PANalytical Empyrean; Cu Kα radiation with wavelength equal to 1.5406 Å and two-dimensional area detector). The XRD results showed the diffraction pattern of MM25C75M with peaks at 2Ɵ = 178.993°, 33.182°, 37.963°, 50.414°, 58.766°, 61.944°, and 72.019° well matched with brucite Mg(OH)2 (JCPDS 44-1482). No additional phase peak was observed in the XRD patterns. The peak position of MM25C75M (5000 µg/ml metallic Cu, 15000 µg/ml metallic Mg) along (001) direction was shifted to the lower value (2Ɵ = 17.993°) in comparison to reagent-grade Mg(OH)2 (2Ɵ = 18.665°), possibly due to lattice expansion. This lattice expansion could be attributed to the higher ionic radius of Cu compared to the ionic radius of Mg. It indicates that Cu ions substituted Mg ions in the MM25C75M without changing the brucite Mg(OH)2 structure.
(2) The morphology of MM25C75M and MM10C45M45Z (5000 µg/ml metallic Cu, 22500 µg/ml metallic Mg, 22500 µg/ml metallic Zn) was observed under SEM. MM25C75M showed a pyramid-like structure. The particle size was around 253 nm. MM10C45M45Z were small lamellar shaped flakes and the size was around 100 nm. The addition of a Zn caused changes in the morphology, suggesting interactions among the metals and structural reorganization. SEM data can be correlated to the average particle size obtained by Dynamic Light Scattering (DLS) technique previously reported.
January 2020
The objectives for this proposal are 1) Conduct field trials of new products and fungicide programs for PFD management as well as validation trials for the Citrus Advisory System (CAS); 2) Investigate the reasons for the movement of Postbloom fruit drop (PFD) to new areas and recent major outbreaks; 3) Evaluate methods for initial inoculum reduction on leaves so that early fungicide applications could be more effective and identify the constituents of the flower extracts using “omics” techniques.
Planning commenced for the upcoming spring’s field trial. Some infected flowers and persistent calyxes have been seen in groves in the state. We are looking for a site for the timing trial that is a hot spot for PFD in priority but hopefully another site for a fungicide trial. Treatments are being decided upon.
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 are currently attempting to obtain flowers from greenhouse plants. We intend to test the same treatments as assessed before in the wind tunnels with PFD-symptomatic flowers.
We have yet to finish developing the risk map for PFD in partnership with UF’s agricultural and biological engineering department.
We gathered data on the number of alerts within a season for several locations and seasons. We intend to develop a database to show growers the importance of weather to PFD epidemics. The database will be the base for the development a risk map for PFD in the future. So far, we have noticed that recent seasons were not very conducive to PFD development, especially when compared to 2015/16. We have shared this info with some growers recently. We calculated daily PFD risks using the model adapted from Lima et al. (2011) and currently used in the Citrus Advisory System. Our data corroborates with our hypothesis that the disease was more important to FL growers when conditions were more conducive to PFD according to our model.
The experiments with the flower extracts did not work because of unexpected fungal contamination of the extracts twice. New trials are to be started shortly after filter sterilizing the extracts to remove what are apparent contaminents. All other components were sterile and so we think the extracts were the source of the fungal contaminents.
Dr. Yu Wang’s lab was starting a more comprehensive analysis of the consitituents of the water extracted flowers from the different developmental stages.
2019 Citrus canker trial results
In this report, we focused on the 2019 citrus canker trial results. The citrus canker trial was performed in a 7-year old `Ray Ruby’ grapefruit block with windbreaks (Vero Beach, FL). The multi-metal materials were applied at 0.5 lb. metallic Cu per acre on April 2, April 23, May 13, June 3, June 24, July 15, August 5, August 26, September 16, October 7, and October 28. Disease ratings were performed on July 15, August 26, October 7, and November 11 based on incidence of fruit lesions for 100 fruit per treatment from the middle 3 trees in each plot (0.17 acres/plot). Harvest was done on November 11 and the yield was measured as lb. fruit per tree in each plot. A subset of fruit from each tree was sized and tested for juice quality at CREC packing house/pilot plant facility (Lake Alfred, FL). MM25C75M, MM25C75Z, and MM10C45M45Z treated grapefruits showed 0% of fruit with canker lesion present throughout this trial, which was significantly better than the untreated control (0.8%, 1.2%, 4.6%, and 8.2% at successive disease ratings). Monthly rainfall in 2019 was below average, so early season leaf inoculum and subsequently fruit lesions were low throughout the trial making it difficult to differentiate between moderate and strong efficacy. The yield of MM25C75M, MM25C75Z, and MM10C45M45Z treated grapefruits was 50.6, 60.5, and 49 lb/tree respectively. The yield of MM25C75Z treated grapefruits showed a statistic difference compare with untreated grapefruits (43.8 lb/tree). The mean fruit diameter for MM25C75M, MM25C75Z, and MM10C45M45Z treated grapefruits of 86.9, 83.6, and 85 mm, respectively, was not significantly different from the untreated controls (85.8 mm). No significant differences were found in internal fruit quality based on brix acid ratio or total soluble solids. Greenhouse results of the most recent formulations are not available because the inoculation occurred too late in the fall to get clear results even with the Blockade positive control. A new trial will be initiated around spring flush when day length has increased to provide robust greenhouse flush.
The contract with Compac Sorting Equipment LTD and University of Florida was developed and signed by both parties for the optical sizing/grading unit. The equipment has been serviced by Compaq, including updating the operating system to the latest software, computers, and electronic communication. They replaced the electronics because the chips used in the dedicated PLC’s are no longer available. Their engineers designed a new system to replace the legacy systems. This is a key to limiting the possibility of expensive repairs down the road. The payment from UF should be sent soon and the equipment delivered shortly after that. We anticipated receiving the equipment by the end of February at the very latest.
This single-lane machine has extensive capabilities with both visible spectrum and near infrared (NIR) cameras to measure fruit color, dimensions, weight, and internal constituents. Computing algorithms can be utilized the calculate fruit volume, density, internal Brix concentrations, defects, and the manufacturer believes it can accurately detect and separate HLB-affected fruit. The unit has three drops for separating different classifications of fruit.
Sept 2015
Objective 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 are still waiting for symptom experssion in the fungicide and skirting trials. As explained in the previous quarterly report, we expect symptom expression in early 2020 as Valencia, a late harvested cultivar, start to ripen. We have marked off products that should not be harvested and have occassionally visited the trials to make sure that no symptoms have started to express unexpected. None have been observed so far.
The subcontract with CRI is finally complete. 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.
Five more South African isolates is in the process of being sequenced. For some of the isolates selected for the next round of sequencing, the available DNA are not of high enough quality, and new cultures of these isolates are grown and DNA will be extracted.
New fungicide products are being tested for the management of CBS in South Africa. Products being evaluated include Enable (copper hydroxide) sprayed in alternation with Amistar Top, Luna Sensation or Headline, which is also tested in alternation with copper hydroxide. Fungicides are applied every 4 weeks from October 2019 until March 2020. Additional fungicides including Miravis, Miravis Top, Ph-D, Priaxor and Luna experience could not be tested due to their unavailability in South Africa. The fungicides are being tested in a ‘Valencia’ orange orchard with a history of CBS.
Within Objective 3 (Survey for the MAT-1-1 mating type and two closely related Phyllosticta spp.), the culture collection of P. citricarpa isolates from Florida which contains more than 125 single-speed isolates has been plated and individual isolates cultured for DNA extraction. Thirty-six isolates have been extracted and high quality DNA purified. PCR conditions for amplifying the tef1 sequence from Phylosticta spp. are currently under evaluation and optimization. Amplicons will be sequenced and compared to determine if the cryptic P. paracitricarpa and P. paracapitalensis species exist within our collections. In regards to screening for matting type, all isolates from our current collection have been screened and determined to be of the MAT1-2 mating type. Screens are ongoing at the Division of Plant Industry to evaluate mating types of new isolates coming from outside of the current quarantine zone and we are coordinating with Dr. Hector Urbina and colleagues to monitor the findings. Beyond Florida, permits have been approved from both the US and Cuba to receive extracted DNA samples from the Cuban collection of P. citricarpa isolates. DNA representing these isolates will be screened for mating type and used later for analysis of global P. citricarpa population structure. Additional efforts have been focused on determining the role of fruit developmental etiology on susceptibility to CBS. A quarantine greenhouse experiment has been established with fruit-bearing Myer lemon trees. Eighty-six fruit of varying developmental stages as well as controls have been inoculated. We are currently monitoring for symptom development and collecting data on environmental parameters and fruit development.
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