The purpose if this project is to reveal the mechanisms of bactericide uptake and transport in citrus plant and establish a theoretical basis for developing technologies to improve the efficacy of bactericides, which is helpful to provide potential solution to the development of effective chemotherapeutic tools for HLB management. Three application methods, foliar spraying, tree trunk injection and root administration, for bactericides (oxytetracycline and streptomycin) currently suggested to be used in citrus HLB management have been employed in this study with greenhouse and field tests. The data obtained in the test will be used to: 1) compare the delivery efficacy of the bactericides applied by the three application methods into the citrus plants; 2) to compare the distributions and concentrations of the bactericides in different citrus tissues and; 3) to compare the effect of citrus variety and age on the delivery of bactericides applied by the three methods and up taken concentrations of bactericides by the plants. This project officially started on December 1 2018. This is the 9th quarterly progress report covering December 1, 2020 to February 28, 2021. During this period, we have conducted and/or completed the following work/research tasks:1) Field study has been completed based on greenhouse study results. The study included two citrus varieties, Grapefruit and Valencia orange, in two farms located in the Fort Pierce area. Oxytetracycline and streptomycin applied into/onto citrus plants by foliage spraying, tree trunk-injection and root administration methods. Samples of citrus leaves, stems, roots and fruits from all the tested plants collected on the day 0, 7, 15 and 30 after treatments and brought back to the Lab for processing. 2) All the samples collected from the field study have been processed for extraction of oxytetracycline and streptomycin analysis purpose. The procedure used for the extraction of oxytetracycline and streptomycin included washing, air drying, cutting, liquid nitrogen grinding, weighing out, and solvent extracting, SPE column cleaning up nitrogen-air dying and bring up to targeting volume with special solvent for HLPC-MASS analysis. 3) The extracted samples for oxytetracycline and streptomycin analysis have been sent to cooperated institute, the Citrus Research and Education Center (CREC), UF for analysis purpose. However, the analysis has not been finished yet. The work planned for the next quarter (March 1 to May 31, 2021): The major research tasks for the next three months include: 1) Completing the analyses of the extracted samples 2) Processing of the data from the field trials.3) Write the final report for this project.4) Preparation of manuscripts from the data obtained from the greenhouse and field experiments..
January 2021The objectives for this proposal are 1) Conduct field trials of new products and fungicide programs for PFD management as well as validation trials for the Citrus Advisory System (CAS); 2) Investigate the reasons for the movement of Postbloom fruit drop (PFD) to new areas and recent major outbreaks; 3) Evaluate methods for initial inoculum reduction on leaves so that early fungicide applications could be more effective and identify the constituents of the flower extracts using omics techniques. The two validation trials for CAS have been laid out in the Fort Mead area and a small amount of disease has been observed on early bloom in at least one site. We hope for at least one positive site. The fungicide trial is in the planning stages and will be laid out in the next few weeks. The majority of analysis for the leaf wetness models is now completed and manuscript preparation is underway. We compared the output of leaf wetness sensors to combinations of predictive models for accuracy and sensitivity. As will all models, reliability was heavily influenced by the quality of the weather station data. Reasonable accuracy was found for one, three, or four models in combination but with just two models, the predictive capacity was poor. We are in discussions for how this information could be used to improve CAS predictions. We are working on evaluating PFD risks via an analysis for prediction accuracy. When we extracted leaf compounds from citrus to evaluate them for stimulation of appresoria and conidia production, we were greatly surprised to find they stimulated germination and conidia production as much as the floral extracts. The mature citrus leaves from Valencia tree were extracted by sonication with water at room temperature for three hours. The extract was filtered and concentrated under reduced pressure to yield a dried sample. The concentrated extract was then chromatographed over silica gel (3.5 × 30 cm; 40-63 µm particle size) and eluted with gradient mixtures of n-hexane/acetone/MeOH (10:1:0 . 0:0:1, each 2 L) to afford seven sub-fractions (F17). Seven fractions were analyzed by UHPLC to analyze their polarity. The UHPLC system was following: 0 -5 min, 8% B; 5-16 min, 8-90% B; 16-17 min, 90-100% B; 17-24 min, 100% B. The mobile phase included 0.1 % formic acid aqueous solution (A) and 0.1 % formic acid containing acetonitrile (B). According to their polarity, seven subfractions were combined into two fractions. One fraction contains highly polar compounds and the other one possesses medium polar compounds. The high-polarity extract greatly induceds conidia germination in 12 hours, even more than floral extracts. This is counter our hypothesis that sugars were responsible for the stimulation as the extracts that contain most sugars did not stimulate germination to the same extent. We are in conversation with the USDA to recommence work in their wind tunnel to conduct the experiment with conidia on flowers for the final comparison. However, it is not clear when the USDA will reopen their facilities. We hope it will be soon.
1. Please state project objectives and what work was done this quarter to address them: This report is for the continued funding of the Southern Gardens Diagnostic Laboratory that provides testing for citrus greening for researchers, growers and homeowners. The current report is for the 1st quarter of year 2 of a 2 year project. For the first quarter of year 2, a total of 6255 samples were processed and sent out. This brings the total for the project to date to 32,625, or approximately 16% over the amount budgeted for the first 5 quarters of the project. Of the samples run for the period, results for 33% of the samples were reported with copy number as well as Ct values. All of the samples for the current period were plant samples. No psyllid samples were submitted. 2. Please state what work is anticipated for next quarter: The submission of samples is completely at the discretion of the submitters and as such is somewhat variable. The trend for the last few quarters has been to receive large groups of samples from research trials, sometimes upwards of 1000 samples per submission. If this trend continues, we would expect to be above the budgeted amount of samples. 3. Please state budget status (underspend or overspend, and why): Given that we are running 16% above the budgeted amounts, it is likely that we will be over the budgeted number of samples for the project. As has been done in the past, we will wait until the end of year 2 of the project and adjust the final invoice either up or down depending on the total number of samples run during the project. If the number of samples exceeds the budgeted amount, the final invoice will be increased upwards to cover the cost of the consumables. If the number of samples is below the budgeted amount, the final invoice will be reduced to reflect the reduced amount of consumables used.
December 2020Objective 1: Evaluate the optimal spray timing for Florida and investigate if tree skirting or alternative products improves fungicidal control of citrus black spot.Objective 3: A MAT-1-1 isolate may enter Florida and allow for the production of ascospores. The industry needs to know if this happens, as it will affect management practices. Additionally, the existing asexual population may be more diverse than currently measured. If multiple clonal linages exist, then there may be different sensitivities to fungicides or other phenotypic traits. We also need to determine whether P. paracitricarpa or P. paracapitalensis are present in Florida for regulatory concerns due to misidentification. We plan to survey for the MAT-1-1 mating type, unique clonal lineages, and two closely related Phyllosticta spp. We collected data from the large spray timing and skirting trial in March. We evaluated 50 fruit each for disease severity on approximately 125 trees in 32 rows. The data entered for analysis but only very preliminary analysis has begun. The treatments were in a randomized split-plot design with skirting as the main plot and fungicide timings (early, standard, late) along with an untreated control were the minor plots. The data were verified and formatted for analysis. We are in the process of analysing the incidence data of this large data set. The minor plots were re-randomized within the main plots and we were able to get the trial re-flagged just in time for the early spray (delayed by 2 weeks but no rain occurred from the first of April until after the early application). All applications were made on time in the spray trial. We were unable to set up the second planned fungicide trial this year because of the COVID-19 shut down. It occurred just as the pre-treatment data should have been collected but we plan to conduct the trial next year if a no cost extension is granted. In South Africa, the trial in which different fungicides were tested for their efficacy to protect Valencia orange fruit from CBS infection was evaluated in August 2020. All the fungicides tested were effective in protecting fruit as they achieved more than 97% CBS free fruit, except for Luna Sensation (applied 6x on its own), which achieved 67.5% clean fruit. More than 99% CBS free fruit were yielded by applying Enable (Indar) 6 times on its own and also applying Amistar Top in alternation with copper hydroxide. The highest percentage (100%) of CBS free fruit was achieved with standard program consisting of copper oxychloride followed by the application of two sprays of azoxystrobin + copper oxychloride + mineral oil and lastly copper oxychloride. Trees that were sprayed with copper hydroxide in alternation with Cabrio (Headline) yielded 97.3% CBS fruit. The trial site was, however, characterised by a low incidence of citrus black spot during the 2019-20 season with the untreated trees yielding 60.2% fruit without CBS lesions. With the exception of programs alternating copper hydroxide with either Cabrio (92% fruit showing no phytotoxicity) or Amistar Top (97.8% fruit showing no phytotoxicity), the experimental fungicides did not produce any phytotoxicity on fruit. Due to the unavailability in South Africa, Miravis, Miravis Top, PhD, Priaxor and Luna Experience were not tested in this trial. Twenty-three isolates were sequenced (6 from South Africa and 17 from the USA) using the Ion Torrent System. The genomes of all the isolates were successfully assembled and analysed using a customised bioinformatics pipeline. Previous genotypes obtained with SSR primers were confirmed and new SSR primers were developed in silico. To date, mapping and SNP variant statistics as well as in silico genotyping data revealed significantly less variation between the USA isolates than between the isolates from South Africa. To investigate the fine-scale genetic differences within the USA P. citricarpa population, the assembled genomes were annotated by mapping the reference genes to the assembled contig sets, using GMAP. The variant calling results together with the annotations were further analysed using SNPeff, to detect putative variable genes. In silico detection of mating types were also performed and confirmed that only one mating type is present in the USA. A manuscript was accepted and published by Physiological and Molecular Plant Pathology describing the analysis and results from the USA isolates (https://doi.org/10.1016/j.pmpp.2020.101591).Eight of the 12 South African isolates sent for 200bp sequencing passed the quality control checks and were sequenced. Quality and completeness of the genome assemblies will be assessed, as well as number of SSRs that can be detected, to determine whether 200bp sequencing is a viable and more cost-effective sequencing approach. Our research facilities were closed from mid-March to end of May due to the COVID-19 pandemic, causing the delay in evaluating the 200bp sequencing. The isolates that failed quality control, as well as additional South African isolates, are in the process of being cultured and DNA will by re-extracted and sequenced. NGS data from the eight samples were received and are in the process of being analyzed. Eight more samples were submitted for NGS. In total, 11 South African isolates have been sequenced and analysed in the same manner as the USA isolates, to investigate the population structure of P. citricarpa in South Africa. Twelve more South African isolates have been cultured, DNA extracted, and are in the process of being sequenced. A more cost-effective sequencing approach (200bp rather than 600bp sequencing) are currently investigated.Objective 3 (Survey for the MAT-1-1 mating type and two closely related Phyllosticta spp.). A study on the diversity of Phyllosticta species is ongoing to determine which species (pathogenic and endophyte) are associated with citrus in Florida. Currently, 59 isolates were extracted and high-quality DNA purified. Based on tef1, ITS and actA sequences, two isolates (Gc-6 and Gc-7) demonstrated polymorphism with P. capitalensis and P. citricarpa, and the highest sequence identity was found with P. hymenocallidicola. This finding strongly suggests that the two isolates are identical to P. hymenocallidicola. Additional multi-locus phylogenetic analysis of GPDH sequence of these two isolates is underway to support our preliminary result and reject any possibility of misidentification. Sexual and vegetative compatibility test of isolate Gc-6 and Gc-7 determined that both isolates are likely the same clone. Moreover, isolates Gc-6 and Gc-7 failed to produce symptoms in citrus when tested on lemons in quarantine. Leaf inoculation of Amaryllis species is underway for a pathogenicity test of both isolates. Hymenocallis littoralis will be tested as well since P. hymenocallidicola was originally described from this host. It was difficult to get the H. littoralis plants but they are now growing in our screenhouse in preparation for inoculation. To obtain robust information on the diversity of species of Phyllosticta in Florida. Further Phyllosticta isolates are being prepared for screening using tef1 primers. To date, no P. paracitricarpa or P. paracapitalensis have been identified but using the tef1 primers will identify any isolates that maybe part of our remaining collection. Out of 202 samples, 125 isolates are from our Phyllosticta collection, 66 isolates were isolated from fruit lesions collected from different locations under quarantine in Florida (samples provided by Dr. Hector Urbina from the Division of Plant Industry), and 11 isolates isolated from fruit lesions collected in groves, in the La Belle area on the leading northern edge of the spring 2020 quarantine zones. To better understand the diversity of P. citricarpa in the region our partnership with Cuban researchers has been strengthened. Permits for the acquisition of genomic DNA from Cuban P. citricarpa isolates has been approved. We are, amidst current travel restriction, devising plans to have the DNA shipped from Cuba. DNA representing these isolates will be screened for mating type and used within a larger analysis of the global P. citricarpa population structure. Additional efforts have been focused on determining the role of fruit developmental etiology on susceptibility to CBS. Our established quarantine greenhouse experiment with fruit-bearing Myer lemon trees recently finished. Ninety-seven fruit of varying developmental stages, as well as controls, were inoculated in December 2019. There were 25 water controls and 73 were inoculated with isolate Gc-12. At the end of the experiment, 50 fruit were symptomatic. The data are being analaysed. A poster of the phylogenetic results was prepared and presented at the virtual annual meeting of APS in August.
The purpose of this project is to reveal the mechanisms of bactericide uptake and transport in citrus plant and establish a theoretical basis for developing technologies to improve the efficacy of bactericides, which is helpful to provide potential solution to the development of effective chemotherapeutic tools for HLB management. Achieving this outcome will require progress in the following three tasks: (1) to compare the delivery efficacy of bactericides with three application methods (foliar spraying, truck injection, and root administration) based on the uptake and dynamic movement/distribution of the bactericide within the tree; (2) to clarify the systemic movement and transportation mechanisms of bactericides within the phloem of tree; and (3) to investigate the effects of citrus variety and age on the delivery efficacy of bactericides. This project requires a combination of greenhouse studies and field trials. Prior to conducting these experiments, a sensitive and accurate quantifying method of bactericides (oxytetracycline and streptomycin) in citrus tissues is needed. This project officially started on December 1, 2018. This is the 8th quarterly progress report covering September 01 to November 30, 2020. During this period, we have conducted and/or completed the following work/research tasks:1) The samples from the trunk injection and root administration experiments in greenhouse were analyzed. The results show that after trunk injection, oxytetracycline (OTC) was detected in leaves on day 7, 15 and 30; the concentration of OTC in leaves was highest on day 7 and decreased with time. The OTC in stems and roots was detected only on day 7, whilst streptomycin (STR) was not detected in leaves, stems or roots. The results of root administration using a root-split technique showed that both OTC and STR could be horizontally transported from the treated roots to untreated roots. The absorption efficiency of STR was higher than that of OTC. Both antibiotics could be detected in leaves and roots on day 30.2) Among the three tested application methods, root administration by the split technique (RS) has the highest delivery efficiency for OTC, followed by trunk injection (TI), and least with foliar spraying: RS > TI >FS. The results for STR are inconsistent. The results from greenhouse experiments remain to be confirmed by the field trials.3) The field trials with three application methods, i.e. foliar spraying, trunk injection and root administration, were conducted in two different citrus groves, one grapefruit and one orange. So far we have completed the treatments and collection of leaf, stem and root samples on the 7th, 15th and 30th days after the application of OTC or STR.The work planned for the next quarter (December 1, to February 28, 2021):The major research tasks for the next three months include: 1) Continuing the field experiments.2) Preparation of the samples from the field trials prior to analysis of OTC and STR by LC-MS, including grinding, extraction, purification and concentration.3) Analysis of the pretreated samples for concentration of OTC and STR using the LC-MS.4) Processing of the data from the field trials.5) Preparation of reports and/or manuscripts from the data obtained from the greenhouse experiments.
Field trial samples (Mg-Sol, Mg-Sol_Cu, NAC-ZnO, NAC-ZnO_Cu, NAC-ZnS, NAC-ZnS_Cu) and commercial controls (Zinc nitrate, MagnaBond and Kocide) were tested for rainfastness following a published protocol [1] with minor modifications.
Briefly, citrus leaves were collected from citrus seedlings, washed with DI water and air dried. Then, leaf surface area was measured using the LeafByte App. Leaves were individually sprayed with treatment at the same rate they are applied in the field. Same rate was used for Zn and Cu controls. After air drying, leaves were individually dipped in a conical tube containing 30 ml of DI water for 30 seconds under agitation in order to simulate rain (Wash 1 – W1). Leaves were dried and the washing procedure was repeated twice in order to have W2 and W3. A fourth wash (W4) was done using 30ml of ethanol-acid solution for 60 seconds under agitation aiming to remove all the remaining metal on the surface of the leaves. Then, the metal concentration in each wash was measured by Atomic Absorption Spectroscopy. The total metal content per leaf was calculated by summing W1, W2, W3 and W4 content and dividing by the leaf surface area. The percentage of metal released in each wash was calculated by dividing the wash metal content by the total metal content. Triplicates were done and the average was used to report the results on Table 1.
In terms of Mg release, around 60% of the total magnesium content was released in the first wash (W1) for Mg-Sol and Mg-Sol Cu. The presence of Cu in Mg-Sol_Cu seems to have increased the amount of Mg release in W2 and W3 in comparison to Mg-Sol.
In terms of Cu release, Mg-Sol_Cu also released around 60% of the Cu in the first wash (W1) while NAC-ZnS_Cu released more than 80% of the Cu in W1. NAC-ZnO_Cu showed similar behavior of Kocide, releasing only 12% of the Cu in the first wash. The remaining Cu found in W4 for both Kocide and NAC-ZnO_Cu was around 87%, indicating Cu can be sustained released from those samples.
In terms of Zn release, NAC-ZnS and NAC-ZnS_Cu released 90% of the Zn in the first wash, while NAC-ZnO, NAC-ZnO_Cu released around 70% of the Zn, similarly to the control Zn nitrate.
[1] Impact of (nano) formulations on the distribution and wash-off of copper pesticides and fertilisers applied on citrus leaves. M Kah, D Navarro, RS Kookana, JK Kirby, S Santra, A Ozcan, S Kabiri. Environmental Chemistry 16 (6), 401-410 (2019).
This project started on Nov. 1st, 2019. So, this is the fourth quarterly report (8/1/20-11/15/20). There are two objectives in this project. The progress of each objective is listed here:
Objective 1. Evaluation of blended juice using released HLB-tolerant sweet orange/mandarin cultivars via analyses of sensory and consumer acceptance.
Sensory for Sugar Belle blended with Hamlin were completed in the second quarter. At the end of April, we have harvested Valencia and have processed Valencia. Sensory and consumer study of Valencia mixing with Sugar Belle has been completed in this quarter. The completed sensory and consumer study included 1) a commercial product 2) 90% Valencia blended with 10% Sugar Belle 3) 50% Valencia blended with 50% Sugar Belle 4) 100% Valencia 5) 100% Sugar Belle. The best overall liking and flavor liking is 50% Valencia blended with 50% Sugar Belle then flowed by 100% Valencia. The sensory attributes included: sweetness, sourness, bitterness, overall flavor, orange flavor, overall liking and flavor liking etc. The consumer attitude toward mandarin blended orange juice was included in the surveyIn addition, they provided the price they would like to pay for each sample, which could provide more information on consumer liking regard each sample.
Objective 2. Identify more tolerant cultivars resembling the quality of Valencia for the juice market, and identify a chemistry definition of consumer accepted orange flavor
We collected about 20 mandarin hybrids, pure mandarins and sweet orange in the first quarter and these cultivars have been identified with the great field performance. The trained sensory panel study started in the first quarter but stopped for the second quarter due to COVID-19. In the third quarter, we continued trained panel study for differentiating citrus flavor attributes such as sweet, citrusy, fruity, and fl-ora etc. In the fourth quarter, we have completed all the sensory studies for 20 cultivars collected. In the fourth quarter, we have also completed all the Gas-Chromatography analysis for all the aromas of 20 samples. We are still in the process of completing all non-volatile (e.g. taste compounds) analysis and hope can be done in the next quarter.
We expect to correlate trained sensory data with analytical data (aroma and taste) at the end. In this way, a chemical definition of mandarin and orange flavor can be provided.
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.
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