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.
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 4th quarterly progress report covering 9/01 to 11/31, 2019. During this period, we have conducted and/or completed the following work/research tasks:
1) The procedures of sample preparation method are further improved. Based on the test results of leaves, stems and root samples from foliar spraying, the recovery of both bactericides (streptomycin, oxytetracycline) is approximately 70%, which is within the acceptable range.
2) The greenhouse experiments of citrus tree trunk injection were completed. All the samples were collected and are being prepared with the above-mentioned newly modified method for the analysis of bactericides using a LC-MS. The results are expected to be available at the end of December 2019.
3) The root administration experiments were initiated in greenhouse, and the treatments included two bactericides (streptomycin, oxytetracycline) and three different concentrations for each bactericide on young citrus trees. So far, two batches of samples (leaves, stems and root) were collected and are being prepared for the analysis of bactericides using the LC-MS, and the last batch of samples will be collected on December 20.
The work planned for the next quarter:
The major goals of research for the next three months are to analyze the obtained data and to evaluate the delivery efficacy of three different application methods for bactericides in field. To accomplish the goals, field experiments will be carried out and the following research tasks will be performed during the next quarter (December 1, 2019 to February 29, 2020): 1) Collection and analysis of the obtained data; 2) Selection of desired sites and citrus trees for field experiments; 3) Conducting foliar spraying treatments of both bactericides for the field experiments.
This report is for the first quarter of year 1 of project 19-001C. For the first quarter, 5,866 samples were run which is 241 samples over the budgeted amount for the period. Of these, 97% were plant samples and 3% were psyllid samples. This total also includes approximatley 1000 samples that were run using alternate assays as requested by the customer. Beginning with the 19-001C funding period, the Southern Gardens Diagnostic Laboratory (SGDL) said it would run alternate assays as requested by the customer provided that the customer provides the primer sequences and testing details.
October 2019
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.
The spray trial and CAS validation trial data were collected and analyzed. The data were reported as part of the comprehensive final report for project 16-010C in September. Planning for the next season will be underway in the next quarter.
The wind tunnel experiments continued throughout this quarter. The majority of the data for spores from leaves has been completed. Leaves were inoculated in planta with Colletotrichum acutatum and allowed to form appressoria. The approssoria were stimulated to germinate and for secondary conidia. The trees were then exposed to three windspeeds: 5, 10, and 20 m/s, with and without rain within the wind tunnel at the USDA Tropical Research Unit in Fort Pierce. Petri dishes with selective media were placed a different distances from the inoculated plants prior to the treatments. The number of CFU were counted to assess how well the inoculum moved. The data are in the process of being modelled to select the best fitting models. Even though the data are still being analyzed, it is evident that some inoculum can move up to 15m with just wind and even more inoculum moves when there is wind and rain. This has not been reported for C. acutatum before which is considered to be an exclusively splash dispersed organism. The secondary conidiation inoculum has not been studies in detail previously but is the primary PFD inoculum and responsible for initial outbreaks of disease. We plan to study the movement of the primary inoculum produced on flowers when they become available in the spring.
Little progress has been made with the modeling to better predict PFD outcomes because of the focus on the inoculum movement. The movement was a priority because of the limited access to the USDA facilities.
There was also little work on the germination stimulation with the flower extracts because of the reasons stated above. The experiments have been restarted and progress with the different extracts are expected in time to inform our efforts at flowering this spring.
Dr. Yu Wang’s lab has been busy analysing the constituents of the water extracts of the flowers at pin head, button bloom, and open flowers. All the standards and chemical techniques were obtained and methods optimized. The sugars and sugar alcohols in the flower stages were identified. In agreement with previous studies that sucrose stimulated the germination of appressoria, glucose and fructose concentrations increase with flower maturity. Some sugars and sugar alcohols did not change signficantly among flower maturity stages while some increased at button stage but decreased at open bloom.
Sept 2019
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.
In the fungicide and skirting control trial, seven applications of fungicides have been completed. They were applied approximately every 28 days. Skirting was done on June 4, 2019. We plan to have one more application for one treatment. Activity on this part of the project will be minimal in the next few months as we will need to wait for enough symptom development to rate which is expected no earlier than February based on previous years.
Applications were continued at an approximate 28 day interval for the fungicide screening trial throughout the summer for a total of 6 applications. Again, this part of the project will have minimal activity because we will need to wait for symptom development. Since again the cultivar is Valencia, symptom expression is expected in early 2020.
The subcontracting process with CRI has been slow although some progress has been made and should be complete soon. 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.
Jeff Rollins is still working on travel to Cuba because of changes to Federal rules and regualtions. Trees and fruit have been selected for on tree experiments and inoculum production is underway.
We continued our tree evaluations at the three trial locations (1) SWFREC, (2) Hendry County – Duda & Sons, and (3) Polk County – Peace River Packing Co. during the third quarter of 2019. Tree evaluations included monthly (SWFREC) or bimonthly (Hendry County and Polk County) root growth measurements using rhizotrons and leaf flush ratings. In addition to rhizotron measurements, root cores were collected at each location to determine root densities and other fibrous root properties. Leaves were collected from all trials for nutrient analysis. Statistical data analysis is still in progress.
Two-year tree growth data are currently being collected for trees located at SWFREC, and preparations at SWFREC are in progress for whole tree excavation and detailed root architectural analysis, plant biomass determination, hydraulic conductivity measurements, and measurement of other tree parameters.
Updates on project progress were presented during the Citrus Expo seminar presentation in August (“Bigger is not always better – large-scale rootstock evaluations on a Ridge and flatwoods site”) and poster display “Rootstock propagation methods” with printed educational material. Additional information was presented during a seminar at SWFREC in September titled “Which rootstock to plant in an HLB-endemic environment”. A new EDIS publication “Citrus nursery production guide, chapter 6: Citrus rootstock propagation: traditional techniques and recent advances” was completed and is available at https://edis.ifas.ufl.edu/hs1329.
We are awaiting to plant the fourth trial at a site in Indian River County (FLARES Inc.) once the grower has finalized his decision on the grove location. Trees are field-ready and in the holding stage at the USDA Horticultural Research Laboratory DPI-certified greenhouses in Ft. Pierce.
Progress on objectives 1, 2, 6, and 8 were made as follows:
Objective 1 – The ratio effect of Fe2+ and citric acid concentration and proportion on hydroxyl radical production and stability will be quantified and validated by response surface methods with accompanying mathematical models and ANOVA.
An assay to quantify hydroxyl radical production via ferrous iron induced Fenton reaction was setup and calibrated (i.e., standard curves). The hydroxyl radicals produced by the Fenton reaction degrade the dye methylene blue, and is measured by spectrophotometry. This assay is required to determine the Fe2+/citric acid ratio effects.
Objective 2 – Phytotoxic effects of Fe2+ and citric acid concentration and proportion will be quantified and validated by response surface methods with accompanying mathematical models and ANOVA.
Citron plants for this experiment were established from bud cuttings.
Objective 6 and 8 (Note: there is no objective 7) – Effects of Fe2+ and citric acid treatment on HLB status and horticultural measures of young newly planted trees AND mature HLB-infected field trees are in progress. Prior to treatment applications, the caliper and height for each of the newly trees was measured. Tree condition for the mature trees was visually determined by disease indexing (DI) according to the methods of T. R. Gottwald, B. Aubert and Z. Xue-Yuan. Trees with a similar DI were selected, flagged, randomized and mapped for treatment.
Aerial images were acquired (drone) for the young and mature field planting. Images will provide canopy size, density, and shape data. This data, in combination with ground data, will form a baseline measure for treatment comparisons over time.
The summer cover crop mix was planted in late May/early June at both locations. This mix included buckwheat, brown top millet, and dove millet. Sunnhemp was included in the mixes for the nitrogen-fixation treatments (1/2 of the rows).
Dataloggers and soil moisture probes continued to record soil moisture every hour. Root growth measurements using the mini-rhizotron tubes installed in both groves were performed in March and July 2019. Data on these measurements are currently being analyzed and will continue in Fall 2019.
Weed density and identification measurements were made in March and Aug 2019. Initial analysis of data collected from March 2019 revealed a general (72-84%) reduction in density of weeds in citrus row middles that received cover crop treatments when compared to controls with no cover crop in the row-middles. The next data collection is scheduled towards the end of this year.
Collection of samples for the annual assessment of leaf and soil nutrient concentrations, trunk diameter, canopy size, and soil microbial community composition have begun and will be completed for all treatments at both locations by the end of August. DNA will be extracted from soil samples and sent for sequencing analysis within the next quarter.
The postdoctoral research associate, Dr. Antonio Castellano-Hinojosa, joined the project on July 1. The graduate student under the supervision of Drs. Kadyampakeni and Kanissery, Miurel Brewer, began working in May 2019.
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