The purpose of this project is to optimize the CRISPR technology for citrus genome editing. This study is related to the CRDF RMC-18 Research Priorities 4AB. We are optimizing the CRISPR-Cas9 technology in citrus genome editing by conducting the following three objectives: Objective 1. Expanding the toolbox of citrus genome editing. In this study, we will adapt StCas9, NmCas9, AsCpf1, FnCpf1 and LbCpf1 on genome modification of citrus. Lately, we have shown CRISPR-Cpf1 can be readily used as a powerful tool for citrus genome editing. One manuscript entitled CRISPR-LbCas12a-mediated modification of citrus has been published on Plant Biotechnol J.Objective 2. Optimization of the CRISPR-Cas mediated genome editing of citrus. In this study, we will first test different promoters in driving expression of Cas9 and Cpf1. We have identified one optimized promoter which showed higher efficacy in driving gene expression in citrus than 35S promoter and Arabidopsis U6 promoter. We are further characterizing the promoter and test its efficacy in driving sgRNA and in genome editing of citrus. We have also developed a method to increase the transient expression efficiency. Objective 3. Optimization of the CRISPR technology to generate foreign DNA free genome editing in citrus. We have conducted transient expression of Cas9/sgRNA plasmid and Cas9 protein/sgRNA ribonucleoprotein complex in citrus protoplast. The plasmid-transformed protoplast has 1.7% editing efficiency, and the RNP-transformed samples have approximately 3.4% efficiency. The genome modified protoplast cells are undergoing regeneration. We aim to increase the efficacy to over 20% and eventually generate non-transgenic genome modified citrus. One manuscript is under preparation. One patent has been filed on the CRISPR-Cas mediated genome editing of citrus.
During the second quarter of 2019, we continued our horticultural field trial evaluations as outlined in the proposal. Trees in all three trials are composed of Valencia scion on eight different rootstocks (US-802, US-812, US-897, US-942, US-1516, X-639, Swingle, Cleopatra). The SWFREC trial (Collier County) was established in October 2017. The two commercial trials were established in April 2018 on a typical Ridge site in Polk County and on a flatwoods type-site in southwest FL (Hendry County). Rhizotron root growth measurements at the SWFREC location were continued in monthly intervals. Results showed some differences in root growth among propagation methods within a rootstock cultivar during the first year of growth in the field. But, when averaged across all rootstock cultivars, no differences associated with propagation method were observed. Statistical analyses are still ongoing. After one year of growth, significant differences in tree height were found among trees on different rootstocks independent of the propagation method. The largest tree size was measured for US-1516 and the lowest for US-897. No statistically significant differences in tree size were attributed to the method by which rootstocks were propagated. Other horticultural parameters such as trunk diameter and the ratio of rootstock to scion trunk diameter were significantly influenced by rootstock and by propagation method. Nutrient analysis of leaves showed no significant differences associated with the propagation method, but differences were found among trees on different rootstocks for all nutrients except nitrogen and potassium. The SWFREC trial was demonstrated during a field day in April 2019.During the first year of growth in the commercial locations, no dieback of trees was observed for trees at the Polk County location. A small percentage of trees died within several weeks after transplant at the Hendry County location, but dieback was not related to the propagation method or rootstock cultivar. After one year of growth, no significant differences in tree height, canopy spread, and trunk diameter among trees were observed based on the method by which rootstocks were propagated in both trials. In contrast, significant differences were observed for trees on different rootstocks independent of the propagation method. Data analysis is still ongoing. Rhizotrons for root observations were installed during the previous quarter, and the first measurements were conducted.Tree propagations for the additional field trial to be conducted in Indian River County were completed and trees are maintained at the US Horticultural Research Lab in Ft. Pierce until ready for planting. Trees have Valencia scion and are grafted on four different rootstocks (US-812, US-897, US-942, and US-1516) propagated by seed, cuttings, and tissue culture.
Systemic characterization of three Mg-Zn gel based multi-metal formulations containing Cu A.I. [Cu:Mg:Zn (10%:45%:45%, MM10C45M45Z), Cu:Mg:Zn (15%:42.5%: 42.5%, MM15C42.5M42.5Z) and Cu:Mg:Zn (20%:40%: 40%, MM20C40M40Z)] is in progress. The hydrodynamic size of as-prepared materials was measured through dynamic light scattering (DLS). MM10C45M45Z, MM15C42.5M42.5Z, and MM20C40M40Z showed increased hydrodynamic size with higher Cu content (290.2 nm, 334.7 nm, and 354.2 nm respectively), suggesting Cu could induce the aggregation of primary particles. The primary particle size of multi-metal formulations (MM25C75M, MM25C75Z, MM10C45M45Z, MM15C42.5M42.5Z, and MM20C40M40Z) will be evaluated through electron microscopy. The electron microscopy results for the as-prepared multi-metal formations will be included in the next report.The antimicrobial efficacy of as-prepared materials was tested in vitro against Pseudomonas syringae. To determine the minimum inhibitory concentration (MIC) of the as-prepared materials and selected controls, a macrodilution assay was performed (Nature Protocols, 2008, 3, 163-175). The MIC value was determined by measuring the optical density at 600 nm after 48 h incubation at 27 °C under shaking (150 rpm). The MIC value of MM25C75M, MM15C42.5M42.5Z, and MM20C40M40Z was 80 ppm of metallic Cu, which was similar to copper controls (copper nitrate and Kocide 3000). The MIC value of MM10C45M45Z was 40 ppm of metallic Cu. The enhance antimicrobial efficacy may due to the smaller particle size. The MIC value of MM25C75Z was 5 ppm of metallic Cu. This result suggests that MM25C75Z formulation might contain ultra-small size particles. To determine the minimum bactericidal concentration (MBC) of as-prepared materials and selected controls, a colony-forming unit (CFU) assay was performed (Nature Protocols, 2008, 3, 163-175). The MBC value of MM25C75M, MM25C75Z and MM10C45M45Z was 80, 10 and 40 ppm metallic Cu respectively. All multi-metal formulations showed enhanced bactericidal efficacy when compare with commercial copper bactericide control (Kocide 3000, MBC: 320 ppm metallic Cu).To evaluate the systemic mobility of the Cu A.I., a plant uptake study was performed using Citrus reshni (Cleopatra mandarin) seeding in a growth chamber (Panasonic Environmental Test Chamber, Model MLR-325H, Kadoma, Japan). Temperature and relative humidity were maintained at 35 ºC and 85%. The citrus seeding was foliar-sprayed using a hand-pump sprayer with 300 ml of MM25C75M, MM25C75Z, MM10C45M45Z (500 and 1000 µg/ml metallic Zn concentration) and deionized water as a control. After 48 h incubation in the plant growth chamber, citrus seedlings were removed from the soil and gently washed with 1% cleaning detergent (Alconnox ®, Alconnox Inc.) and 0.1% HCl. Leaves, roots and stem sections were separated after washing and left in an oven for 48 h (45 ºC). Then, the dried leaves, roots, and stems were acid digested (EPA method 3050 B Acid Digestion of sediments, Sludge, and Soil). Cu, Zn and Mg contentions in leaves, stems, and roots will be quantified with Atomic Absorption Spectroscopy (AAS). The AAS results will be included in the next report.
The purpose of this project is to optimize the CRISPR technology for citrus genome editing. This study is related to the CRDF RMC-18 Research Priorities 4AB. We are optimizing the CRISPR-Cas9 technology in citrus genome editing by conducting the following three objectives: Objective 1. Expanding the toolbox of citrus genome editing. In this study, we will adapt StCas9, NmCas9, AsCpf1, FnCpf1 and LbCpf1 on genome modification of citrus. As a proof of concept, CsPDS and/or CsLOB1 are chosen for targeting through transient expression of Cas9-sgRNA or Cpf1-crRNA via Xcc-facilitated agroinfiltration. Recently, we employed CRISPR-LbCpf1, derived from Lachnospiraceae bacterium ND2006, to edit the citrus genome. First, LbCpf1 was successfully used to modify Duncan CsPDS via Xcc-facilitated agroinfiltration. Next, GFP-p1380N-35S-LbCpf1-crRNA-lobp and GFP-p1380N-Yao-LbCpf1-crRNA-lobp were constructed to edit the PthA4 effector binding elements in the CsLOB1 promoter (EBEPthA4-CsLOBP) in transgenic Duncan grapefruit. Totally, seven GFP-p1380N-35S-LbCpf1-crRNA-lobp-transformed Duncan plants were generated, designated as #D35s1 to #D35s7, and ten GFP-p1380N-Yao-LbCpf1-crRNA-lobp-transformed Duncan plants were created, designated as #DYao1 to #DYao10. LbCpf1-directed EBEPthA4-CsLOBP modification was observed in three 35S-LbCpf1-transformed Duncan (#D35s1, #D35s4 and #D35s7). However, no LbCpf1-mediated indels were observed in the Yao-LbCpf1-transformed plants. Importantly, transgenic line #D35s4, containing the highest mutation rate, alleviates Xcc.pthA4:dCsLOB1.4 infection. Therefore, CRISPR-LbCpf1 can be readily used as a powerful tool for citrus genome editing. We have also made progress regarding use other Cas proteins. Objective 2. Optimization of the CRISPR-Cas mediated genome editing of citrus. In this study, we will first test different promoters in driving expression of Cas9 and Cpf1. We have identified one optimized promotors which showed higher efficacy in driving gene expression in citrus than 35S promoter and Arabidopsis U6 promoter. We are further characterizing the promoter and test its efficacy in driving sgRNA and in genome editing of citrus. We have also developed a method to increase the transient expression efficiency. Objective 3. Optimization of the CRISPR technology to generate foreign DNA free genome editing in citrus. We have conducted transient expression of Cas9/sgRNA plasmid and Cas9 protein/sgRNA ribonucleoprotein complex in citrus protoplast. The plasmid-transformed protoplast has 1.7% editing efficiency, and the RNP-transformed samples have approximately 3.4% efficiency. The genome modified protoplast cells are undergoing regeneration. We aim to increase the efficacy to over 20% and eventually generate non-transgenic genome modified citrus. One manuscript is under preparation. We are also filing patent for this technology.
The spring cover crop mix was planted in late February and early March in both locations. This mix included Daikon radish, white clover, crimson clover, buckwheat, oats, and sunflower. Daikon radish germination was particularly good in both locations.
Dataloggers and soil moisture probes continued to record soil moisture every hour. A rain gauge will be installed at each site to record rainfall data. Preparations are underway for the next assessment of root growth via the mini-rhizotron tubes installed in both groves. Weed emergence in row-middles and tree-rows were collected in mid-March from North grove location. Weed emergence data in South grove location will be collected in mid-summer.
Yield and juice quality data was collected for the North grove location March 28-April 4 and for the South grove April 11-18. As treatments had only just begun, this yield and juice data will serve as baseline data for comparison with subsequent years of the project.
The summer cover crop mix is being planted at both locations in the next two weeks. This mix will include dove millet, buckwheat, brown top millet, and sunnhemp.
A postdoctoral research associate will be joining the project in July and a graduate student for Drs. Kadyampakeni and Kanissery will begin this summer. Dr. Wades graduate student will begin this fall.
The next collection of samples for soil and leaf measurements will occur this summer.
The spring cover crop mix was planted in late February and early March in both locations. This mix included Daikon radish, white clover, crimson clover, buckwheat, oats, and sunflower. Daikon radish germination was particularly good in both locations.
Dataloggers and soil moisture probes continued to record soil moisture every hour. A rain gauge will be installed at each site to record rainfall data. Preparations are underway for the next assessment of root growth via the mini-rhizotron tubes installed in both groves. Weed emergence in row-middles and tree-rows were collected in mid-March from North grove location. Weed emergence data in South grove location will be collected in mid-summer.
Yield and juice quality data was collected for the North grove location March 28-April 4 and for the South grove April 11-18. As treatments had only just begun, this yield and juice data will serve as baseline data for comparison with subsequent years of the project.
The summer cover crop mix is being planted at both locations in the next two weeks. This mix will include dove millet, buckwheat, brown top millet, and sunnhemp.
A postdoctoral research associate will be joining the project in July and a graduate student for Drs. Kadyampakeni and Kanissery will begin this summer. Dr. Wades graduate student will begin this fall.
The next collection of samples for soil and leaf measurements will occur this summer.
The spring cover crop mix was planted in late February and early March in both locations. This mix included Daikon radish, white clover, crimson clover, buckwheat, oats, and sunflower. Daikon radish germination was particularly good in both locations.
Dataloggers and soil moisture probes continued to record soil moisture every hour. A rain gauge will be installed at each site to record rainfall data. Preparations are underway for the next assessment of root growth via the mini-rhizotron tubes installed in both groves. Weed emergence in row-middles and tree-rows were collected in mid-March from North grove location. Weed emergence data in South grove location will be collected in mid-summer.
Yield and juice quality data was collected for the North grove location March 28-April 4 and for the South grove April 11-18. As treatments had only just begun, this yield and juice data will serve as baseline data for comparison with subsequent years of the project.
The summer cover crop mix is being planted at both locations in the next two weeks. This mix will include dove millet, buckwheat, brown top millet, and sunnhemp.
A postdoctoral research associate will be joining the project in July and a graduate student for Drs. Kadyampakeni and Kanissery will begin this summer. Dr. Wades graduate student will begin this fall.
The next collection of samples for soil and leaf measurements will occur this summer.
An experiment was designed and setup for Objective 3, Effect of Fe2+ and citric acid treatment on HLB titer of model HLB system determined. The experiment tests Treatment Solution B reported in Patent US 8,945,631, Liquid for treatment of citrus greening disease and treatment method using same. Greenhouse-grown citron plants propagated from HLB-infected citron were tested by RT-PCR. Thirty-six plants that tested positive and showed HLB symptoms were planted into Ruck’s pots. The experiment included 3 treatments applied to three groups of twelve plants. The treatments included Treatment Solution B at two concentrations – 30 ppm Fe and 150 ppm Fe. 30 ppm Fe was the concentration used in the field experiment of the patent. 150 ppm Fe was the upper limit of Treatment Solution B the patent’s authors thought was possible without significant phytotoxicity. Treatment Solution B is applied to foliar and soil at 150 ppm Fe weekly. For soil, 40 mls is added to each pot. The testing of F11 as described in the February, 2019 report continues. A third experiment to determine the phytotoxicity threshold of F11 and Treatment Solution B was initiated. Citron cuttings were taken, planted, and plants selected. Eight iron treatments, including an untreated control, and 12 replicates arranged in a randomized block design were finalized. The 8 iron treatments include 1) F11 applied to foliar-only to runoff; 2) F11 @2X to foliar + soil; 3) F11 @10X to foliar + soil; 4) Fe chelate mixture applied to foliar + soil; 5) Sequestrene 138 (a standard Fe treatment) to foliar + soil; 6) Treatment Solution B applied to foliar + soil; 7) a proprietary iron mixture with high Fe2+ to foliar + soil; and 8) an untreated control receiving standard recommended citrus nutrition. With the exception of the Fe chelate mixture (#4) and Sequestrene 138 (#5), all Fe treatments are high Fe2+ and meet the patent’s specifications for the use of Fe2+ to manage HLB. To determine the number of replicates a power analysis was conducted using tree quality and fruit yield data from the subcontractor’s field site (Florida Research Center for Agricultural Sustainability, Vero Beach, FL) where the field experiments will be conducted. The analysis calculated the number of replicates required to detect a difference between treatments that would be of sufficient size to be useful for managing HLB. For tree condition, measured on a 0-4 scale, an 18% difference can be reliably detected. For boxes/tree, a 124% difference between treatments can be reliably detected. Both measures used 12 replicates and the standard 5% significance and 80% power thresholds. Based on this analysis 96 uniform Red Grapefruit trees on USDA 897 rootstock (P.D. 12/19/18) have been randomly assigned to one of the eight treatments in 12 replicated plots using a Randomized Complete Block (RCB) design. Trees were allowed time to establish root systems and exposure to native population of ACP before treatment applications. This supports Objective 7, Determine the effect of Fe2+ + organic acid solutions on newly planted (<2 years old) field trees.
An experiment was designed and setup for Objective 3, Effect of Fe2+ and citric acid treatment on HLB titer of model HLB system determined. The experiment tests Treatment Solution B reported in Patent US 8,945,631, Liquid for treatment of citrus greening disease and treatment method using same. Greenhouse-grown citron plants propagated from HLB-infected citron were tested by RT-PCR. Thirty-six plants that tested positive and showed HLB symptoms were planted into Ruck’s pots. The experiment included 3 treatments applied to three groups of twelve plants. The treatments included Treatment Solution B at two concentrations – 30 ppm Fe and 150 ppm Fe. 30 ppm Fe was the concentration used in the field experiment of the patent. 150 ppm Fe was the upper limit of Treatment Solution B the patent’s authors thought was possible without significant phytotoxicity. Treatment Solution B is applied to foliar and soil at 150 ppm Fe weekly. For soil, 40 mls is added to each pot. The testing of F11 as described in the February, 2019 report continues. A third experiment to determine the phytotoxicity threshold of F11 and Treatment Solution B was initiated. Citron cuttings were taken, planted, and plants selected. Eight iron treatments, including an untreated control, and 12 replicates arranged in a randomized block design were finalized. The 8 iron treatments include 1) F11 applied to foliar-only to runoff; 2) F11 @2X to foliar + soil; 3) F11 @10X to foliar + soil; 4) Fe chelate mixture applied to foliar + soil; 5) Sequestrene 138 (a standard Fe treatment) to foliar + soil; 6) Treatment Solution B applied to foliar + soil; 7) a proprietary iron mixture with high Fe2+ to foliar + soil; and 8) an untreated control receiving standard recommended citrus nutrition. With the exception of the Fe chelate mixture (#4) and Sequestrene 138 (#5), all Fe treatments are high Fe2+ and meet the patent’s specifications for the use of Fe2+ to manage HLB. To determine the number of replicates a power analysis was conducted using tree quality and fruit yield data from the subcontractor’s field site (Florida Research Center for Agricultural Sustainability, Vero Beach, FL) where the field experiments will be conducted. The analysis calculated the number of replicates required to detect a difference between treatments that would be of sufficient size to be useful for managing HLB. For tree condition, measured on a 0-4 scale, an 18% difference can be reliably detected. For boxes/tree, a 124% difference between treatments can be reliably detected. Both measures used 12 replicates and the standard 5% significance and 80% power thresholds. Based on this analysis 96 uniform Red Grapefruit trees on USDA 897 rootstock (P.D. 12/19/18) have been randomly assigned to one of the eight treatments in 12 replicated plots using a Randomized Complete Block (RCB) design. Trees were allowed time to establish root systems and exposure to native population of ACP before treatment applications. This supports Objective 7, Determine the effect of Fe2+ + organic acid solutions on newly planted (<2 years old) field trees.
An experiment was designed and setup for Objective 3, Effect of Fe2+ and citric acid treatment on HLB titer of model HLB system determined. The experiment tests Treatment Solution B reported in Patent US 8,945,631, Liquid for treatment of citrus greening disease and treatment method using same. Greenhouse-grown citron plants propagated from HLB-infected citron were tested by RT-PCR. Thirty-six plants that tested positive and showed HLB symptoms were planted into Ruck’s pots. The experiment included 3 treatments applied to three groups of twelve plants. The treatments included Treatment Solution B at two concentrations – 30 ppm Fe and 150 ppm Fe. 30 ppm Fe was the concentration used in the field experiment of the patent. 150 ppm Fe was the upper limit of Treatment Solution B the patent’s authors thought was possible without significant phytotoxicity. Treatment Solution B is applied to foliar and soil at 150 ppm Fe weekly. For soil, 40 mls is added to each pot. The testing of F11 as described in the February, 2019 report continues. A third experiment to determine the phytotoxicity threshold of F11 and Treatment Solution B was initiated. Citron cuttings were taken, planted, and plants selected. Eight iron treatments, including an untreated control, and 12 replicates arranged in a randomized block design were finalized. The 8 iron treatments include 1) F11 applied to foliar-only to runoff; 2) F11 @2X to foliar + soil; 3) F11 @10X to foliar + soil; 4) Fe chelate mixture applied to foliar + soil; 5) Sequestrene 138 (a standard Fe treatment) to foliar + soil; 6) Treatment Solution B applied to foliar + soil; 7) a proprietary iron mixture with high Fe2+ to foliar + soil; and 8) an untreated control receiving standard recommended citrus nutrition. With the exception of the Fe chelate mixture (#4) and Sequestrene 138 (#5), all Fe treatments are high Fe2+ and meet the patent’s specifications for the use of Fe2+ to manage HLB. To determine the number of replicates a power analysis was conducted using tree quality and fruit yield data from the subcontractor’s field site (Florida Research Center for Agricultural Sustainability, Vero Beach, FL) where the field experiments will be conducted. The analysis calculated the number of replicates required to detect a difference between treatments that would be of sufficient size to be useful for managing HLB. For tree condition, measured on a 0-4 scale, an 18% difference can be reliably detected. For boxes/tree, a 124% difference between treatments can be reliably detected. Both measures used 12 replicates and the standard 5% significance and 80% power thresholds. Based on this analysis 96 uniform Red Grapefruit trees on USDA 897 rootstock (P.D. 12/19/18) have been randomly assigned to one of the eight treatments in 12 replicated plots using a Randomized Complete Block (RCB) design. Trees were allowed time to establish root systems and exposure to native population of ACP before treatment applications. This supports Objective 7, Determine the effect of Fe2+ + organic acid solutions on newly planted (<2 years old) field trees.
The purpose of the project is to develop new guidelines for restoring root health and improving overall tree nutrition in Florida oranges and grapefruit. The objectives of the project are to:1. Determine optimal nutrient concentrations in roots and leaves for multiple grapefruit and orange varieties.2. Compare and contrast fertigation, soil, and foliar fertilization to identify best application method for uptake of nutrients into both underground and aboveground components.3. Investigate the relationship between root and leaf nutrient contents to tree health, yield, and fruit quality as well as bacteria titer.4. Generate updated and new guidelines for optimal nutrient contents for roots and leaves for HLB-affected trees. Progress to date:The project is being implemented at three sites at Citrus Research and Education Center (CREC), Southern Gardens Citrus near Clewiston, FL and Indian River Research and Education Center (IRREC). First preliminary data collection (on yield, fruit size, juice quality, canopy size, HLB and other disease ratings, soil characteristics and root growth and nutrition) has been completed at the 3 sites in the first and second quarters. First nutritional treatments have been imposed in spring 2019 and will be continued as scheduled. Data collection will continue and analyses will be done as needed. Updates and data will be presented in future extension meetings after about a year or two of data collection and validation of results to get feedback from growers and the citrus industry. Plans for Next QuarterThe team will continue with data collection and reporting on the progress of the project.
Good progress was made to accomplish the objectives for this project during the first quarter in year 1 of this project. One field trial was established at the UF/IFAS SWFREC farm in November 2017. Two commercial trials were established in Collier County and in Polk County in April of last year. Prior to planting, a subset of trees was destructively sampled to assess root system architectures and other plant growth parameters in detail. Data from these measurements are being prepared for publication. Since trial establishment, trees have been closely monitored and horticultural measurements were conducted as outlined in the project proposal. Horticultural measurements include tree height, canopy size, scion and rootstock trunk circumferences, and canopy health. Tree wraps were regularly inspected for ant problems or other issues that may negatively impact tree growth and health. Bases of rootstocks were assessed for potential abnormalities arising from propagation. Root growth has been monitored monthly in the SWFREC trial since trees were planted. Root measurements are non-destructive, making use of rhizotrons (long plastic tubes) and an associated camera and software system. Root measurements were continued during the first quarter of the funding period. Additional rhizotrons were purchased and prepared for installation to the root zone in a subset of trees from the two commercial locations. Rhizotron preparation included post-purchase modifications such as applying paint and sealing openings for blocking out light and moisture. The first set of rhizotrons was installed during the third week of March at the Polk County location (Peace River Packing Co). The second set was installed during the last week of March at the Collier County location (Duda & Sons). In each trial, 102 rhizotrons were installed. Root growth measurements will commence in April once the soil has settled and roots have recovered from any stress that may have been induced during installation. One person (OPS) was hired and began work in the last week of March. Propagation of new plant material in the DPI-approved USHRL greenhouses commenced. Four rootstock cultivars were used. Rootstock liners were generated from seed, from tissue culture, and from cuttings, and were grafted with `Valencia’ scion. Trees are anticipated to be ready for field planting in September of this year.
For this reporting period, different formulations of Mg-hydroxide or Zn-hydroxide loaded with Cu-chelates were successfully synthesized using sol-gel chemistry. The total metal content is 40,000 ppm (wt/V) including up to 50% of Cu-chelates (equivalent to 20000 ppm of metallic Cu; the A.I.): (1) Cu: Mg (10%: 90%, MM10C90M), (2) Cu: Mg (25%: 75%, MM25C75M), (3) Cu: Mg (50%:50%, MM50C50M), (4) Cu: Zn (5%:95%, MM5C95Z), (5) Cu: Zn (10%:80%, MM10C90Z), (6) Cu: Zn (25%:75%, MM25C75Z). The stability of each formula was observed for the duration of 48 h post-synthesis. Most formulations (except for MM50C50M and MM25C75Z) formed a stable colloid as no phase separation was observed. Hydrodynamic diameter of the colloidal solution was estimated using Dynamic Light Scattering (DLS) technique. The hydrodynamic size for MM50C50M, MM25C75Z and MM25C75M was estimated to be around 880 nm, 580 nm and 450 nm, respectively, suggesting aggregation of primary particles. MM25C75M particle size was comparable to MM10C90C size. DLS particle size of MM5C95Z and MM10C90Z could not be reliably estimated due to low scattering intensity (less than 10kcps, below the detection limit). This suggests that MM5C95Z and MM10C90Z formulations might contain highly-dispersible ultra-small size particles. Further study using electron microscopy is needed to confirm primary particle size. Phytotoxicity testing was conducted using a plant growth chamber (Panasonic model # MLR 352H, temperature cycle was set for summer conditions, T > 80oF, RH 60-80%). Ornamental vinca plant was used as a model system due to their high susceptibility for metals. Three different foliar application rates (300, 500 and 900 ppm wt/V of total metal content, similar to field application rate) were tested. Phytotoxicity symptoms were evaluated after 3 days of incubation based on visual observation as described in our previous paper (Plant Disease 2016, 100(12), 2442-2447). All multi-metal treatments showed reduced plant leaf damage at all tested concentrations when compared to copper alone. Future reports will include phytotoxicity and antimicrobial efficacy results.
An experiment was designed and setup for Objective 3, Effect of Fe2+ and citric acid treatment on HLB titer of model HLB system determined. The experiment tests F11, a high Fe2+ product sold only in Japan and which was developed from Patent US 8,945,631, Liquid for treatment of citrus greening disease and treatment method using same. Greenhouse-grown citron plants propagated from HLB-infected citron were tested by RT-PCR. Twenty-two plants that tested positive and showed HLB symptoms were divided into two groups with eleven plants in each group. The plants are growing in Ruck’s pots. One group is being treated with F11 + Siltrate, and one group is being treated with Siltrate only and serves as the control group. F11 is foliar and soil applied at 150 ppm Fe weekly. For soil application 40 mls is added to each pot. Siltrate Advanced (Meherrin Inc., Severn, NC), is a nonionic siloxane surfactant, and is used at 1.3 ml/L. The experiment was interupted by the Federal Government shutdown and plants were not treated. The plants were cut back to 25 cm, cuttings weighed, and treatments resumed. Plants will be treated for 6 months. A similar experiment will be setup using Treatment solution B as described in Patent US 8,945,631. Treatment solution B is a high Fe2+ formulation that includes FeSO4.7H2O and citric acid. The subcontractor is running the field experiments and received his funding last week (week of February 25th) from USDA, ARS. Trees were ordered and have been planted (12/19/18) for Objective 7, Determine the effect of Fe2+ + organic acid solutions on newly planted (<2 years old) field trees.
The purpose of the project is to develop new guidelines for restoring root health and improving overall tree nutrition in Florida oranges and grapefruit. The objectives of the project are to:1. Determine optimal nutrient concentrations in roots and leaves for multiple grapefruit and orange varieties.2. Compare and contrast fertigation, soil, and foliar fertilization to identify best application method for uptake of nutrients into both underground and aboveground components.3. Investigate the relationship between root and leaf nutrient contents to tree health, yield, and fruit quality as well as bacteria titer.4. Generate updated and new guidelines for optimal nutrient contents for roots and leaves for HLB-affected trees. Progress to date:The project is being implemented at three sites at Citrus Research and Education Center (CREC), Southern Gardens Citrus near Clewiston, FL and Indian River Research and Education Center (IRREC). Preliminary data collection (on yield, canopy size, HLB and other disease ratings, soil characteristics and tree and root health and nutrition) is underway and will be reported in the next quarter. Nutritional treatments will be applied starting in spring 2019. One graduate student (CREC), 5 agricultural assistants (IRREC and CREC) have been recruited to work on the project. A search for another graduate student (IRREC) has been completed and the person will start in Fall 2019. Co-PIs (Drs. Kadyampakeni, Rossi, Ferrarezi and Johnson) on the project presented some of their on-going work at the Citrus Show in Fort Pierce, FL and indicated that results from this project will be used to refine current citrus nutrition guidelines. Updates and data will be presented in future extension meetings after about a year or two of data collection and validation of results to get feedback from growers and the citrus industry. Plans for Next QuarterThe team will continue with data collection and reporting on the progress of the project.
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