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.
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. 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. 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.
June 2018 The objectives for this proposal are1) Conduct ground and aerial applications of fungicides to determine the efficacy and economics of fungicide treatments; 2) Determine if Luna Sensation has enough systemic activity to protect flowers from before they fully develop and open; 3) Determine if the period flowering of trees affected by huanglongbing can be narrowed to eliminate the offseason bloom that contributes to the PFD inoculum increase in groves. Trials were conducted to evaluate further fungicides and fungicide programs. We applied 30 treatments in 3 trials at two locations. One site had Navel oranges and the other was Valencia sweet orange. Only one application was made in each site because there was so little rain during bloom. There was rain during bloom but the temperatures were so low during the rain events that limited disease developed and there was one predicted infection event the week of March 7 when using the PFD-FAD forecasting system. Button data from these trials was collected in early June but has not been analysed. Fruit data are expected to be collected in July. Experiments to validate the Brazilian model, now as part of the citrus advisory system (CAS), were carried out in two citrus production areas, one with Valencia and the other one with Navel trees. Treatments were equal to the ones tested in 2017. Due to drier and cooler weather the Brazilian model did not recommend any sprays in both orchards. PFD-FAD-based treatments were sprayed twice, whereas three and two weekly applications were performed in the Valencia and Navel groves, respectively. The difference in number of weekly applications was due to the shorter flowering period of the Navel trees. Button data was collected from these locations in early June but has not been analysed to date. Fruit data will be collected as soon as the fruit are large enough to easily see within the canopy. During this time period, the Valencia harvest and flower count data from spring was analyzed. There was no significant difference in yield were observed as result of treatments. Interestingly, a significant increase in fruit size was observed with GA (20 g a.i.) applied 5 times in season as compared to control and NAA treatment. With the use of GA (20 g a.i.; 5 times), we were able to compress the flowering window and decrease the number of flowering. NAA had no effect on flowering window or number. No changes were made to the web interface of the PFD model, citrus advisory system (CAS) during this period. The manuscript continues to be prepared about the CAS decision support system and will be submitted to the Computers & Electronics Journal when completed. This publication will describe the database infrastructure, system functionalities and applications. The presentation at the annual meetings of the Society of Agriculture Engineers – Florida section in June was done.
Sept 2018 The objectives for this proposal are1) Conduct ground and aerial applications of fungicides to determine the efficacy and economics of fungicide treatments; 2) Determine if Luna Sensation has enough systemic activity to protect flowers from before they fully develop and open; 3) Determine if the period flowering of trees affected by huanglongbing can be narrowed to eliminate the offseason bloom that contributes to the PFD inoculum increase in groves. Trials were conducted to evaluate further fungicides and fungicide programs. We applied 30 treatments in 3 trials at two locations. One site had Navel oranges and the other was Valencia sweet orange. Only one application was made in each site because there was so little rain during bloom. There was rain during bloom but the temperatures were so low during the rain events that limited disease developed. Fruit counts were collected in July but analysis has not been conducted yet this year. Experiments to validate the Brazilian model were carried out in two citrus producing areas, one with Valencia and the other one with Navel trees. Treatments were equal to the ones tested in 2017. Due to drier and cooler weather the Brazilian model did not recommend any sprays in both orchards. PFD-FAD-based treatments were sprayed twice, whereas three and two weekly applications were performed in the Valencia and Navel groves, respectively. The difference in number of weekly applications was due to the shorter flowering period of the Navel trees. Fruit data were collected in June and there were no statistical differences among the treatments. This again supports the earlier conclusion that the Brazilian model is more conservative and accurate to not indicate sprays when conditions are unsuitable for PFD development. It is hoped that there will be at least one season of where disease occurs to ensure that the model predicts disease as well as it predicts no disease. A presentation was given on the citrus advisory system (CAS) at Citrus Expo in August. For the plant growth regulator trials during this time period, the trees were re-flagged, data were collected on canopy volume (2018) and the treatments were started with applications for Fall 2018. We met to discuss the function of the CAS and if there are any further improvements that are immediately needed. Much of the discussion centered around leaf wetness measurements used in the model and how to make them more robust and reliable, a chronic problem for this type of measurements. To address this question, a small project was initiated to evaluate historical data on how well leaf wetness probes perform compared to models and if models would be sufficient in all situations. Data is being collected.
Sept 2016 The objectives for this proposal are1) Conduct ground and aerial applications of fungicides to determine the efficacy and economics of fungicide treatments; 2) Determine if Luna Sensation has enough systemic activity to protect flowers from before they fully develop and open; 3) Determine if the period flowering of trees affected by huanglongbing can be narrowed to eliminate the offseason bloom that contributes to the PFD inoculum increase in groves. The project officially started March 1st, 2016 but site selection and plot layout was initiated prior to the start of the project because PFD was beginning to affect the blocks we were planning to use. Four trials were initiated. They were in the Ona, Polk City, and Fort Meade areas. Four weekly applications were made in March by air and ground in Ona. Two applications were made in Polk City as that only was predicted to be needed by the PFD-FAD prediction system prior to the flowering period finishing. Three applications were made in Fort Meade where the bloom was more attenuated than Polk City. The button data was collected in April and May and the data from the Fort Meade, Polk City, and Luna trials has been analyzed. The fruit data was collected in June from these trials but not from aerial trial in Ona. The fruit data from Ona was collected in July. All of the data were analyzed from the Fort Meade, Polk City, and Luna trials. In both fungicide trials, all treatments performed better than the untreated control and the best treatments involved strobilurin fungides combined with Ferbam. In the Luna trial, the best treatment was that applied at the pin head stage (P < 0.0001). The sites for the plant growth regulators were selected and laid out in September.
Tissue culture. The last experiment for hardening off tissue cultured plantlets was completed and summarized in the October report. Stem cuttings. On August 17, rootstock shoots of US812, US1281, US1282, US802, US1284, UFL2, UFL4 and X639 were offered by Alico for making cuttings. They were also made into single node cuttings and stuck using the 4000 ppm Dip&Gro. A total of 6,107 cuttings were made and stuck from these shoots. Some such as US812, had functional roots within 3 weeks. By late October, 5,229 cutting were determined to be rooted, a success rate of 85% overall. The poorest success rate was with USDA1284 at 29%. The success of most other varieties was around 90%.
Tissue culture. The last set of hardening off of tissue culture plants started on 5-25-2016 and were subjected to the same 4 durations of relative humidity (RH; 2,3,4 and 5 days) as previously. Plants were harvested on 13 July for root and shoot growth. Citrus cultivars consisted of Kuharske, Swingle, C-35, sour orange and US 812. For the most part, survival rates have high on most cultivars, with perhaps more growth under the shorter hardening-off periods. During the last 2 hardening off periods, tissue culture plantlets were placed along side of traditional stem cutting. For both trials, rooting success of tissue culture plants under common mist systems after 6 weeks was ~100%, with growth of both shoots and roots matching or exceeding that of traditional reductions in relative humidity. Stem cuttings. Cuttings of Kurharske, C-54, C-35, US 812 and sour orange were treated with 4000 ppm Dip&Gro on 28 April and 20 May. These last two sets of cutting were harvested 30 June and 21 July 2016. This completed 1 year of initiating the rooting of cutting every 3 weeks. Root development and growth was similar to that in 2015- high. Plants were dried at 70C. Measurement of root growth and dry mass is continuing for both tissue culture and stem cuttings. On June 28 & 29, available cuttings of the UFL HLB resistant trees of 7, 9, 10, 11 and 12 were dipped in the 4000 solution of Dip&Gro and stuck in the same peat:perlite mixture. Most shoots were rooted by 6 weeks and removed and watered by hand. On August 17, rootstock shoots of US812, US1281, US1282, US802 and US1284 were offered by Alico. They were also made into cuttings and stuck using the 4000 ppm Dip&Gro. Some such as US812, had functional roots within 3 weeks. Over 4500 cuttings of these desirable root stocks were propagated by mid-October for distribution to nurseries.
For this reporting period, rainfastness experiment was conducted on heirloom tomato plants (heat tolerant variety Florida 91) in triplicates. Plants were sprayed with agri-grade TSOL-UP (field trial samples) at foliar spray rate of 800 ppm of metallic zinc. Plants were then washed following an industry recommended protocol with simulated rainfall for 3 consecutive days. Whole plants were then harvested, dried, digested with aqua regia solution (a mixture of nitric acid and hydrochloric acid in a molar ratio of 1:3) and analyzed for zinc content by atomic absorption spectroscopy (AAS) technique. Our results suggested that maximum zinc content was observed after 1st wash (2.4 mg/gram dry wt. of plant sample) while the zinc content was stabilized for 2nd and 3rd wash (1.2 and 1.3 mg/gram dry wt. of treated plant sample respectively over the water-sprayed control). Colony forming unit (CFU) or viability assay was performed with both TSOL-U and TSOL-UP (field trial agri-grade samples). For both the versions of TSOL, the MIC appeared to be at 80 ppm (metallic zinc) for Escherichia coli bacteria. In case of Xanthomonas alfalfae, the MIC of TSOL-U was found to be 80 ppm and for TSOL-UP the MIC was 40 ppm. The concentration of samples picked for CFU study was one concentration above and one below MIC values. For example, in case of E. coli, the three concentrations chosen for both TSOL-US and TSOL-UP are 40, 80 and 160 ppm of metallic zinc (because the MIC was 80 ppm of metallic zinc). Based on the CFU studies, it appears that at MIC concentration (80 ppm in E. coli), the bacterial load was reduced by 4 logs (>2 log reduction is considered significant) compared to the untreated control. At 160 ppm, there was complete killing of bacteria whereas at 20 ppm (below MIC), there was no significant killing observed as expected. For the upcoming reporting period we plan on extracting plant sap and analyzing it further to detect the presence of zinc content along with other components.
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