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.
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.
The citrus pathogen Xanthomonas axonopodis pv. citri (Xac) can cause extensive damage to twigs, leaves, and fruit of susceptible varieties. The overall objective of this research program has been to develop a bio-control system for citrus canker that utilizes virulent (lytic) phages and/or phage components. Our approach has been to develop a bank of virulent phages and/or antibacterial particles called tailocins , which are derived from phages. We have isolated a pool of both type IV and non-type IV dependent phages and tailocins active against Xac. In greenhouse studies, both phage and tailocin cocktails showed efficacy in reducing canker symptoms. We have continued further characterization of phages to confirm their virulent lifestyle by conducting lysogeny testing and sequencing of the genomic termini to determine packaging. Phages CCP504, 505, 511 and 513 have been determined to be virulent. More recently, we have focused on characterization of myophages CCP509 and CCP519 that are both type IV pili dependent. Although both phages are type IV pili dependent, the two phages exhibit differential plating activity, which indicates that their secondary receptor sites are different. Phage CCP509 exhibits activity against 9/13 Xac strains including Xac306, whereas CCP519 has activity against 7/13 Xac strains with some overlap, which makes them good candidates for a phage cocktail. Tailocins XT-1 and XT-4 exhibit broad host range activity, killing 13/13 Xac isolates in vitro and have shown efficacy in greenhouse studies. A tailocin cocktail composed of XT-1 and XT-4 reduced lesion formation by an average of 51%, as compared to non-tailocin challenged plants inoculated only with Xac. In order to minimize the protocol necessary for production of tailocins, we evaluated Carbadox (0-8.5 mM) and hydrogen peroxide (0 -100 mM) two known inducing agents. Carbadox did not induce tailocin production, but 25 mM hydrogen peroxide added to midlog cultures resulted in production of XT-1 and XT-4 equivalent to that observed for UV induction. Our results indicate that hydrogen peroxide can be used as alternative method to UV induction of tailocins, which reduces the steps involved in the production of antibacterial cocktails.
Tissue culture. Tissue culture plants started on 3-09-2016 were subjected to the same 4 durations of relative humidity (RH; 2,3,4 and 5 days) as previously. Plants were harvested on 28 April for root and shoot growth. Citrus cultivars consisted of C-35, C-54 and sour orange. Dry mass measurements are continuing. The last set of tissue culture plants for hardening were started on 25 May 2016. These will be harvested on 13 July. This set includes Kuharske, Swingle, C-35, sour orange and US 812. For the most part, survival rates have remained high on most cultivars, with perhaps more growth under the shorter hardening-off periods. Stem cuttings. Cuttings of Kurharske, C-54, C-35, US 812 and sour orange were treated with 4000 ppm Dip&Gro on 8 April, 28 April, 20 May and 10 June. This completed 1 year of initiating the rooting of cutting every 3 weeks. Concurrently stem cutting stuck previously were harvested for set #13 on April 6, with the remaining harvested on May 3, May 20, June 10 and the last replication harvested on June 30. With the harvest of the April 8 cuttings, root development and growth was similar to that early summer 2015, and was continued until the end of the experimental year. All plants were dried at 70C. Measurement of root growth and dry mass is continuing. On 25 March, all available cuttings of the HLB resistant trees in Citra were taken and brought to the citrus propagation bay at MREC Apopka. These were treated with the 4000 ppm auxin and stuck in seedling trays. On 25 April cuttings were selectively harvested from MREC block and stuck. Total HLB resistant cuttings stuck this spring were UFR7 216, UFR9 473, UFR10 153, UFR11 374 and UFR12 143. Additional cuttings will be propagated as required by Dr. Grosser.
July 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. This data has not been analyzed to date. Economic analysis will begin one the fruit data has been statistically analyzed. The field trials for the plant growth regulators are in the planning and site selection stage.
This research project aims at developing an alternative to Cu biocides in the form of Quaternary ammonium compounds (Quat) as an antimicrobial agent. While Quat compounds are powerful antimicrobial agents they are not known to be used directly on plants because of potential toxicity to plant tissue. Fixed-Quat is a non-phytotoxic Cu-alternative antimicrobial formulation in which Quat is combined with other inactive ingredients (such as silica gel and silica particles). In the previous reporting period, a new nanoformulation, Fixed-Quat E nanogel was synthesized with a quat concentration of 13,500 ppm ( For Food Use Quat used). In this reporting period, Fixed-Quat A II and Fixed-Quat E were further optimized to a quat concentration of 20,000 ppm ( For Food Use Quat used). FTIR was used to confirm silica and Quat interaction of the concentrated formulation and peak shifts similar to previous batches were observed. The Fixed-Quat A-II and E nanogel s safety was tested by phytotoxicity studies carried out in a Panasonic Environmental Test Chamber (Model MLR- 352H) which allowed for controlled day/night cycling temperatures, light intensity and humidity to simulate summer weather conditions (biocide application season). Studies conducted on Sour orange, a common citrus variety and Roma Tomato sp, an ornamental plant revealed no sign of plant injury when tested with Quat concentration as high as 1000 ppm. It is noted that EPA maximum concentration for Quat industrial use (as surfactant/flocculating agent) is 200 ppm, indicating a large therapeutic window. Antimicrobial studies of optimized Fixed-Quat E nanogel was conducted against several model bacterial species, Xanthomonas alfalfae subsp. citrumelonis (Citrus Canker Surrogate), Pseudomonas syringae pv syringae, a gram negative causative agent of bacterial speck in citrus and tomato sp and Clavibacter michiganensis subsp michiganensis, a gram positive causative agent of canker and systemic infections in tomato. Studies were conducted to determine the Minimum Inhibitory Concentration (MIC) and compared against Kocide 3000 and copper sulfate. MICs of Fixed-Quat A-II and E were found to be = 1.0 g/mL for X. alfalfae, = 1.0 g/mL for P. syringae and = 1.0 g/mL for C. michiganensis. MIC results indicate no loss in efficacy when combined with different silica sources, thus displaying strong potential for commercial usage. Optimized versions of Fixed-Quat A-II and Fixed-Quat E are currently undergoing Citrus Canker trials on Ruby Ray grapefruit in Vero Beach, Florida.
In this reporting period, nine different T-SOL variants made with agriculture-grade chemicals were included for plant uptake studies. Citrus seedlings (Cleopatra sp.) were used as model plants. The T-SOL variants involved three different Zn chelating agents and three different concentrations of a plant surface permeability enhancer (1, 0.5 and 1M with respect to metallic Zn). Citrus seedlings were treated for 24hours with 800ppm dose of different T-SOL variants. After 24hours of treatment whole seedlings were washed thoroughly with 5L of deionized water to clean the additional materials sticking to plant surface. Whole plant samples were then oven dried for 5days at 60 C. The treated plant samples were then ground to fine powder in a dry blender (Cuisinart, Model SG-10). 0.5gms of the dried samples were then digested in 20mLs of aqua regia solution to find out the zinc uptake by plants. Zinc uptake varied from 0-0.8mg/gram dry weight for different treatments compared to untreated control samples (0.09mg Zn/ gram dry weight). Maximum uptake of up to 0.8mg Zn/gram dry weight of plant sample was observed. It was observed that plant micronutrient based Zn chelating agent exhibited maximum uptake. In the coming reporting period, plant uptake studies will be conducted to determine the uptake and translocation of T-SOL inside different plant tissue which can help further to select the most effective T-SOL variant prepared with agri-grade chemicals that can be later used in green house and field trial studies.
The program research objectives are to develop an effective and sustainable bacteriophage (phage)-based biocontrol system for Xanthomonas axonopodis pv. citri (Xac), the causal agent of citrus canker. Our approach has been to develop a bank of virulent (lytic) phages and/or antibacterial particles called tailocins , which are derived from phages. We have identified seven tailocins with activity against Xac and developed a large bank of virulent phages representative of the Caudovirales (tailed phages). The tailocins are protein assemblages that function like phage tails and kill the target bacterial cell by adsorbing and puncturing the cell envelope. In two independent experiments, a cocktail composed of tailocins XT-1 and XT-4 showed efficacy in reducing canker symptoms, when applied as a foliar spray, post application of Xac. Current efforts are directed towards isolating additional tailocin producing strains that are active against Xac. As reported previously, we are also focused on completing the characterization of Xac phages. We have determined that the burst size for CCP504, a virulent KMV-like phage, is ~70 PFU/cell and that the burst size for CCP513, a siphophage, is ~80 PFU/ cell. Abortive lysogeny tests are ongoing to reconfirm virulent status of cocktail phages. Further characterization of the two non-type IV pilus dependent phages, previously reported, determined that both phages had limited host ranges and would not be candidates for development of phage cocktails.
April 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. It is too early to start taking disease data yet and so no economic analysis has begun. The field trials for the plant growth regulators are in the planning stage.
The objective of this research will 1) characterize Pr-D (FP3) and its role and disease suppression; 2) investigate the dynamics of the prophages/phages in Las bacteria by revealing the variations in gene expression and recombination; and 3) identify critical elements, such as heat and chemical stress that facilitates lytic activities of the prophages. In addition, we will demonstrate whether or not if the ‘cross protection’ using mild strains of Las bacteria will work for the HLB pathosystem along with quantitative detection protocols for prophage-based strain differentiation. We harvested various Las-infected citrus and periwinkle samples showing symptoms ranging from mild to severe, and used for isolation and enrichment of prophage/phage apart from plant and bacterial host materials by differential centrifugation, PEG precipitation, and CsCl density fractionation. Absolute and relative amounts of prophage/phage and Las bacteria existed in total DNA and in various fractions have been evaluated by using specific LJ900 primers targeting the repeat sequence in the LasAI gene located within a prophage region of the Las genome and 16S rDNA primers for detecting Las genomic DNA. We developed a protocol that could isolate and enrich lytic phages from fresh plant tissues. The Las phages were enriched in certain fractions of the purification process, particularly in PEG precipitated pellet and certain ClCs fractions, consistent with the biochemical and biophysical property of free phages. For further confirmation, these fractions were directly examined by electron microscopy, and we were able to find some particles consisting of head-tail fiber structure typical of small bacteriophages, with diameter of the heads varies from 50-70 nm and the length and the width of the tails varies from 140-190 nm and 10-20 nm respectively. We have establish a digital PCR (dPCR) system for accurate quantification of HLB and Las prophage/phage. After optimization, we are able to detect as low as 1-2 copy numbers of targeted DNA molecule/.L sample. To further improve the quantification accuracy of absolute and relative amounts of phages and the bacterium, we designed new sets of primers and probes targeting only single copy genes. Based on Las genome analysis, we targeted gene 05560 in the prophage region, .-operon gene in the Las genome, and a CitLGT gene (limonoid UDP-glucosyltransferase-like) in the citrus genome as an internal measurement reference. The specificity and sensitivity of the new primers is currently being tested and we are optimizing an dPCR-based assay for accurate measurement of lytic phage activities in Las-infected materials. Duan Lab to detect bacterial transcripts in mixed eukaryotic/ prokaryotic samples at set time points throughout a typical course of thermotherapy treatment. Overall, the analysis revealed that, depending upon the time at which the samples was taken, between 4% and 9% of the total predicted genes for Las appear to be differentially regulated during the thermotherapy process compared to a sample taken at time zero. These genes provide initial evidence of how the bacteria itself is modifying its transcriptional activity in response to the increase in temperature. Although a majority of the regulated genes found are defined as hypothetical, several do have a predicted function and their contributions to the effects of heat therapy are now under investigation. Their regulation has now been confirmed and transcripts are being quantified via real-time PCR. Additional plants have also been stressed with heat for verification purposes and to ensure the accuracy of the RNA-seq results. Identification of these genes is leading the way towards deciphering the molecular mechanisms behind thermotherapy in an effort to find alternative methods of achieving the same reduction in Las titer as seen with thermotherapy that will work in the roots as well as the foliage. Construction of a transcriptional reporter system is also currently in progress for the final verification of the genes identified as being involved in stress response to heat in plants subjected to thermotherapy. This system will also allow future experimentation to rapidly identify other catalysts that can produce the same reduction in bacterial numbers as thermotherapy. Purification of the ~10Kb FP3 region has been achieved from both periwinkle and citrus, though the amount purified from citrus appears to be less than that from periwinkle (as would be expected from the lower bacterial titer found in citrus). This should allow the region to be sequenced in its entirety and comparisons made to help characterize the role of FP3 found in citrus vs periwinkle.
Tissue cultivation Tissue culture plants started on 12-14-2015 were subjected to the same 4 durations of relative humidity (RH; 2,3,4,5) as previously. This group also included 3 reps of each cultivar hardened-off on heated benches (80 F). On 1 Feb. these plants were harvested for root and shoot growth. Citrus cultivars consisted of C-35, C-54 and sour orange. The next set of tissue culture plants for hardening were started on 9 March 2016. These will be harvested in late April. This set also includes 3 replications on 80 F heated benches. This set includes Kuharske, Swingle, C-35, sour orange and US 812. For the most part, survival rates have remained high on most cultivars, with perhaps more growth under the shorter hardening-off periods. Stem cuttings Kurharske cuttings stuck in the propagation benches under mist produced using the same protocols as in the past produced no roots or shoots when stuck on 1-4-16, nor when stuck on 1-12-16. This was independent whether there was bottom heat or not. In contrast, cutting similarly stuck on 2-5-16 once again began to produce some roots over the 6 week rooting period that ended on 3-28-16. Continuing in the sequence, cuttings of Kurharske stuck on 2-23-16 produced roots when harvested on 3-29-16. Cuttings of both X639 and C-54 were also stuck with this group of Kurharske. They also produced roots. These plants were harvested, but dry weights have not been measured. The last set of cuttings were stuck on 3-18-16. These consisted of Kurharske, X639, C-35 and US812. In late March, all available cuttings of the HLB resistant trees in Citra were taken and brought to the citrus propagation bay at MREC Apopka. These were treated with an auxin blend of 4000 ppm auxin and stuck in seedling trays.
This research project aims at developing Fixed-Quat as an alternative to Cu biocides. While Quat compounds are powerful antimicrobial agents they are not known to be used directly on plants because of potential toxicity to plant tissue. However, combining Quat with other inactive ingredients such as our silica gel delivery makes it safer (non-phytotoxic) thus producing Fixed-Quat. In the previous reporting period, a new nanoformulation, Fixed-Quat E nanogel was synthesized with a quat concentration of 13,500 ppm ( For Food Use Quat used). In this reporting period, Fixed-Quat A II and Fixed-Quat E were further optimized to a quat concentration of 20,000 ppm ( For Food Use Quat used). FTIR was used to confirm silica and Quat interaction of the concentrated formulation and peak shifts similar to previous batches were observed. The Fixed-Quat A-II and E nanogel s safety was tested by phytotoxicity studies carried out in a Panasonic Environmental Test Chamber (Model MLR- 352H) which allowed for controlled day/night cycling temperatures, light intensity and humidity to simulate summer weather conditions (biocide application season). Studies conducted on Sour orange, a common citrus variety and Roma Tomato sp, an ornamental plant revealed no sign of plant injury when tested with Quat concentration as high as 1000 ppm. It is noted that EPA maximum concentration for Quat industrial use (as surfactant/flocculating agent) is 200 ppm, indicating a large therapeutic window. Antimicrobial studies of optimized Fixed-Quat E nanogel was conducted against several model bacterial species, Xanthomonas alfalfae subsp. citrumelonis (Citrus Canker Surrogate), Pseudomonas syringae pv syringae, a gram negative causative agent of bacterial speck in citrus and tomato sp and Clavibacter michiganensis subsp michiganensis, a gram positive causative agent of canker and systemic infections in tomato. Studies were conducted to determine the Minimum Inhibitory Concentration (MIC) and compared against Kocide 3000 and copper sulfate. MICs of Fixed-Quat A-II and E were found to be = 1.0 g/mL for X. alfalfae, = 1.0 g/mL for P. syringae and = 1.0 g/mL for C. michiganensis. MIC results indicate no loss in efficacy when combined with different silica sources, thus displaying strong potential for commercial usage. Optimized versions of Fixed-Quat A-II and Fixed-Quat E are currently undergoing Citrus Canker trials on Ruby Ray grapefruit in Vero Beach, Florida.
In this reporting period, nine different variants were included for the optimization of T-SOL with agriculture-grade chemicals. These variants involved three different Zn chelating agents and three different concentrations of a plant surface permeability enhancer (with respect to metallic Zn). Interaction of metal chelating agents with Zn was characterized by UV-Vis and FT-IR spectroscopy suggested binding of metal ions with the chelate functional groups (such as carboxyls, hydroxyls and amines) which resembles the data collected with lab-grade chemicals. Microplate Alamar blue assay was used to determine the minimal inhibitory concentration (MIC) of the different variants of the T-SOL. Antimicrobial properties of the newly made T-SOL samples (from agri-grade chemicals) were assessed by running a Minimal Inhibitory concentration (MIC) assay as delineated by Clinical Laboratory and Standards Institute. The MIC values of the three variants of T-SOL containing chelating agent- (1, 0.5 and 0.1) were 75 ppm against Escherichia coli, whereas the MIC values for the T-SOL variants containing chelating agents 2 and 3 was 150 ppm for E. coli. But, the MIC values for all the nine variants were 75 ppm when treated against Xanthomonas alfalfae, values similar to the reagent grade chemicals. Phytotoxicity study was conducted with citrus plants (Sp. Cleo) at a field spray (800ppm) rate with different T-SOL variants. No toxicity was observed for all the treated variants. TSOL materials were tested for their efficacy in protecting a citrus variety in the Vero Beach, Indian River County area of Florida. Six different materials were tested using 3 different capping agents and with /without a surface modifying agent. Materials were sprayed on 8 yr.-old ‘Ray Ruby’ grapefruit trees at an application rate of 0.5 lbs/ acre (750-800 ppm) every 21 days from April to September. Materials were compared against standard Cu and/or (Cu+Zn) commercial products. The untreated control displayed a total infection incidence of 60 % while T-SOL materials reduced the infection incidence to 17-24 %. T-SOL materials performed better to commercial products which displayed comparable protection. The two best performing T-SOL variants will be delivered this month for canker and HLB field trial. In the coming reporting period, plant uptake studies will be conducted to determine the uptake and translocation of TSOL inside different plant tissue which can help further to select the most effective T-SOL variant prepared with agri-grade chemicals that can be later used in green house and field trial studies.
This research project aims to develop an alternative to Cu biocides in the form of Quaternary ammonium compounds (Quat) as an antimicrobial agent. While Quat compounds are powerful antimicrobial agents they are not known to be used directly on plants because of potential toxicity to plant tissue. However, combining Quat with other inactive ingredients such as our silica gel delivery makes it safer by producing Fixed-Quat. In the previous reporting period, a new nanoformulation, Fixed-Quat E nanogel was synthesized with a quat concentration of 9,000 ppm (“For Food Use” Quat used). The formulation composition and interactions between the components (silica and Quat) was confirmed using Fourier Transform Infrared Spectroscopy (FTIR), with Si-O stretching and SiO-H stretching confirming the presence of silica. The Si-O stretching frequency changed from 1039 cm (-1) to 1031 cm (-1) whereas the SiO-H frequency changed from 3391 cm (-1) to 3388 cm (-1), suggesting interaction of positively charged Quat with the silica gel. The Quat N-H bending frequency changed from 1468 cm (-1) to 1431 cm (-1). This change also supports the interaction of Quat with silica gel. In this reporting period, Fixed-Quat E was optimized to a quat concentration of 13,500 ppm (“For Food Use” Quat used). FTIR was used to confirm silica and Quat interaction of the concentrated formulation and similar peak shifts were observed. The Fixed-Quat E nanogel’s safety was tested by phytotoxicity studies carried out in a Panasonic Environmental Test Chamber (Model MLR- 352H) which allowed for controlled day/night cycling temperatures, light intensity and humidity to simulate summer weather conditions (biocide application season). Studies conducted on Cleopatra orange, a common citrus variety and Tomato sp, an ornamental plant revealed no sign of plant injury when tested with Quat concentration as high as 1000 ppm. It is noted that EPA maximum concentration for Quat industrial use (as surfactant/flocculating agent) is 200 ppm, indicating a large therapeutic window. Antimicrobial studies of optimized Fixed-Quat E nanogel was conducted against several model bacterial species, Xanthomonas alfalfae subsp. citrumelonis (Citrus Canker Surrogate), Pseudomonas syringae pv syringae, a gram negative causative agent of bacterial speck in citrus and tomato sp and Clavibacter michiganensis subsp michiganensis, a gram positive causative agent of canker and systemic infections in tomato. Studies were conducted to determine the Minimum Inhibitory Concentration (MIC) and compared against Kocide 3000 and copper sulfate. MICs of Fixed-Quat E were found to be = 1.0 ug/mL for X. alfalfae, = 1.0 ug/mL for P. syringae and = 1.0 ug/mL for C. michiganensis. Optimized versions of Fixed-Quat A-II and Fixed-Quat E have been prepared for 2016 Citrus Canker trials on Ruby Ray grapefruit.
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