The goal of the proposed study is to characterize the effect of application of beneficial bacteria (MICROBE Program) on management of HLB. Currently, we are setting up the experiments to test different Microbe Products in management of HLB. Assay for compatibility between isolates using antagonistic survival tests showed that all the selected beneficial bacteria are compatible with each other. Plant growth promoting activity of six selected isolates was evaluated using the model plant Arabidopsis grown in vitro. The results suggested that three isolates could promote plant growth. The plant growth promoting activity of these six isolates was tested using citrus (grapefruit) seedlings in greenhouse. Greenhouse assays suggested that a consortium of three Bacillus and relative isolates (AY16, PT6 and PT26A) may delay the development of both HLB symptoms and pathogen population on citrus leaves after root inoculation. The potential of the consortium to recover the tree decline from HLB infection is being evaluated in greenhouse. The growth conditions of the three strains were optimized using a small fermenter. Three antifoam agents, A204, PPG200 or M-Oil did not affect the growth of the three bacterial strains. The initial neutral to alkaline pH values (7.0 ~ 8.0) favor growth of the three bacteria in LB, while acidic pH (5.0 ~ 6.0) suppress bacterial growth. The optimal cultural temperature was determined to be around 30C with average bacterial population of 109-1010 cfu/ml after 20-hour incubation, although the bacteria may grow slowly under room temperature (~ 23C). The shelf life of three different formulations of the bacterial culture is being evaluated under room temperature. In a six-month time course, the bacterial populations in LB broth, OPB broth and tape water are comparatively stable with initial and final both at ~ 108cfu/ml. Four field trials are being conducted including more beneficial bacteria. For one of the field trial, six applications have been performed. We are evaluating the survival of the beneficial bacteria in the soil. The application method has been changed during application to improve the survival of microbes in the soil. For two trials, four applications were conducted. For another field trial, the application started in February, 2014. We have surveyed the HLB disease incidence, and Las population. We are investigating the survival of the applied microbes in the soil.
Management of phloem-limited bacterial diseases is very challenging. These bacteria employ unusual and sometimes unique strategies by which to optimize their niche occupation and obtain their nourishment from the host plant. Their location within the living (sieve tubes) plant cells, rather than in the intercellular spaces, offers different challenges and opportunities for them to avoid the host plant’s defense system. Phloem is also difficult for any bactericides to reach to control the pathogen population. Among the phloem-limited bacterial diseases, citrus Huanglongbing (HLB, greening) is one of the most devastating diseases. The current management strategy of HLB is to chemically control psyllids and scout for and remove infected trees. However, the current management practices have not been able to control HLB and stop spreading of Candidatus Liberibacter asiaticus (Las). The goal of the proposed study is to develop HLB management strategies which boost plant defense to protect citrus from HLB by exploiting the interaction between Las and citrus and understanding how Las manipulates plant defense. Recently, we compared the gene expression of PR1, PR2 and PR5 in healthy trees and Las infected citrus plants. The expression of PR1, PR2 and PR5 was significantly reduced in HLB diseased grapefruit as compared to healthy grapefruit after inoculation with Xac AW. We also tested whether infection by Las can make citrus more susceptible to infection by Xanthomonas citri subsp. citri. We also sprayed four times with different chemicals in 17 different combinations on citrus to test their effect in controlling HLB in one grove. Multiple compounds showed control effect. To further test those compounds, we have selected two more groves to expand the field test. The disease index of the two groves have been investigated and treatments already started. The follow up investigations are ongoing, including monitoring the HLB symptoms, disease incidence and Las titer in leaves. We compared the SA levels in HLB infected and healthy grapefruit after the inoculation with Xac AW. We also compared the SA levels in HLB infected and healthy Valencia citrus. We are continuing to evaluate the effect of different compounds on management of HLB both in greenhouse and in citrus grove. We have applied different compounds at three separate field trials. Four compounds were shown to have positive effect on controlling HLB based on two year field test results. We are also testing the mechanism of those compounds showing positive effect on HLB control. For example, the analysis of gene expression of pathogenicity related genes and callose synthase CalS1 in treated plants and nontreated plants is underway. We have investigated the effect of those compounds on disease severity, yield, juice content and quality. We will repeat those treatments for one more year. Currently, the treatments are being conducted.
The goal of the research is to control citrus HLB using small molecules which target essential proteins of Candidatus Liberibacter asiaticus (Las). In our previous study, structure-based virtual screening has been used successfully to identify five lead antimicrobial compounds against Las by targeting SecA. SecA is one essential component of the Sec machinery. Those compounds showed promising antimicrobial activity. However, further work is needed to apply the compounds. We will evaluate the important characteristics of our antimicrobial compounds including solvents and adjuvants, phytotoxicity, antimicrobial activities against multiple Rhizobia, antimicrobial activity against Las, application approaches, and control of HLB. Those information are critical to for the practical application of those antimicrobial compounds in controlling HLB. We also propose to further optimize the five lead compounds. In addition, we propose to develop antimicrobial compounds against lipid A of Las. The lipid A substructure of the lipopolysaccharides (LPS) of Sinorhizobium meliloti, which is closely related to Las, suppresses the plant defense response. Las contains the complete genetic pathway for synthesis of lipid A. We hypothesized that Las uses lipid A to suppress plant defense. Thus, targeting lipid A could activate plant defense response. Lipid A is also an ideal target and has been targeted for screening antimicrobial compounds for multiple pathogenic bacteria. We are optimizing the compounds in collaboration with IBM. Two compounds with slightly higher binding affinity than C16 were identified. Currently, we are evaluating the best range of composition ratio among each component (%weight) of AIs, solvents and surfactants. The following characteristics are being evaluated: 1) emulsion stability and ease of emulsion; 2) stability of diluted concentrate; 3) freeze-thaw stability; and 4) phytotoxicity to citrus species. We have successfully identified one formulation suitable for all five compounds without phytotoxicity. Using the formulation, we have tested all five compounds against eight different bacterial species including Liberibacter crescens. We are repeating the results for one more time. Field test is being conducted. We have sprayed once. Greenhouse trial is being conducted.
At the request of the CRB, Grafton-Cardwell and Morse merged their core entomology research efforts under a single project, 5500-501 (Morse’s portion of the project is 5500-501b). We have always coordinated our research efforts but this arrangement formalizes the situation. This report summarizes the Morse lab’s recent research under this coordinated project (all arthropod research except ACP which is a separate project, i.e. 5500-189). Our major effort this last year has focused on helping the industry to deal with Fuller rose beetle (FRB) in relation to citrus exports to Korea. For the last several years, Korea has put increasing pressure on the CA industry to reduce egg mass levels on export fruit and there is a concern that in 2014-15, loads found to be infested with viable egg masses may be denied entry into that country. Morse and Dr. Andy Cline of CDFA conducted 6 training sessions for county agricultural inspectors in different areas of California during Nov. 2013 — at the prompting of APHIS, county inspectors have agreed that all loads of citrus shipped to Korea would be sampled to determine the percent of fruit infested with unhatched FRB egg masses. Field FRB control trials were run at Lindcove by Grafton-Cardwell (15 treatments evaluated) and in Pauma Valley by Morse (10 treatments evaluated). None of the treatments results in 100% control but it appears that 2 treatments are relatively effective — either 2 ground sprays of bifenthrin, 2 trunk sprays of bifenthrin, or 2 foliar sprays of combinations of carbaryl, cryolite, and/or thiamethoxam. A method of analyzing bifenthrin residues on trunks was developed in collaboration with Dr. Jay Gan (UCR Dept. of Environmental Sciences). Studies were conducted on 3 dates to determine bifenthrin levels on trunks necessary to cause beetle paralysis after they walk over the trunk spray (“LC”50 of 0.04874 ug/cm2). The persistence of 5 formulations of bifenthrin trunk sprays were evaluated in Riverside by taking residue samples 6, 12, and 18 weeks after trunk application. Citrus thrips resistance to Delegate has been confirmed in the San Joaquin Valley. Two products nearing registration on citrus that will be useful in control of citrus thrips (as well as other pests) are Bexar and Closer. To better study Delegate resistance, we started a greenhouse colony from an area reporting poor control and have confirmed that the population is resistant to Delegate (very flat dose-response line). So that we can study the mechanism and genetics of resistance, we will select for higher levels of resistance. We are examining contaminants of export citrus using 10 randomly selected cartons per load from a variety of citrus packing houses. To date, we have processed 29 such loads with a diversity of commercial varieties examined. Genetic identification of insects appears fairly routine but we will have to continue to work on methods for mite identification — initial extractions have been problematic.
At the request of the CRB, Grafton-Cardwell and Morse merged their core entomology research efforts under a single project, 5500-501 (Morse’s portion of the project is 5500-501b). We have always coordinated our research efforts but this arrangement formalizes the situation. This report summarizes the Morse lab’s recent research under this coordinated project (all arthropod research except ACP which is a separate project, i.e. 5500-189). Our major effort this last year has focused on helping the industry to deal with Fuller rose beetle (FRB) in relation to citrus exports to Korea. For the last several years, Korea has put increasing pressure on the CA industry to reduce egg mass levels on export fruit and there is a concern that in 2014-15, loads found to be infested with viable egg masses may be denied entry into that country. Morse and Dr. Andy Cline of CDFA conducted 6 training sessions for county agricultural inspectors in different areas of California during Nov. 2013 — at the prompting of APHIS, county inspectors have agreed that all loads of citrus shipped to Korea would be sampled to determine the percent of fruit infested with unhatched FRB egg masses. Field FRB control trials were run at Lindcove by Grafton-Cardwell (15 treatments evaluated) and in Pauma Valley by Morse (10 treatments evaluated). None of the treatments results in 100% control but it appears that 2 treatments are relatively effective — either 2 ground sprays of bifenthrin, 2 trunk sprays of bifenthrin, or 2 foliar sprays of combinations of carbaryl, cryolite, and/or thiamethoxam. A method of analyzing bifenthrin residues on trunks was developed in collaboration with Dr. Jay Gan (UCR Dept. of Environmental Sciences). Studies were conducted on 3 dates to determine bifenthrin levels on trunks necessary to cause beetle paralysis after they walk over the trunk spray (“LC”50 of 0.04874 ug/cm2). The persistence of 5 formulations of bifenthrin trunk sprays were evaluated in Riverside by taking residue samples 6, 12, and 18 weeks after trunk application. Citrus thrips resistance to Delegate has been confirmed in the San Joaquin Valley. Two products nearing registration on citrus that will be useful in control of citrus thrips (as well as other pests) are Bexar and Closer. To better study Delegate resistance, we started a greenhouse colony from an area reporting poor control and have confirmed that the population is resistant to Delegate (very flat dose-response line). So that we can study the mechanism and genetics of resistance, we will select for higher levels of resistance. We are examining contaminants of export citrus using 10 randomly selected cartons per load from a variety of citrus packing houses. To date, we have processed 29 such loads with a diversity of commercial varieties examined. Genetic identification of insects appears fairly routine but we will have to continue to work on methods for mite identification — initial extractions have been problematic.
Mid Florida Citrus Foundation (MFCF) a 501c5 not for profit organization which has supported (past 25 years) and currently supports citrus research efforts of scientists from the University of Florida, USDA and private industry. The MFCF supports citrus research through the employment of a full time grove manager whom works closely with researchers to ensure that their projects are handled properly and that the grove is an excellent condition. The management of this grove requires extra financial commitment as grove care costs tend to be higher than commercial groves due to the nature of many of the research projects. Current projects being conducted at the MFCF are Asian citrus psyllid (ACP) pesticide evaluation control trials, low volume applicator trials, windbreak evaluation, HLB nutritional programs, new and existing herbicide trials, variety and rootstock evaluation trials. During the recently completed quarter (October 1 to December 31, 2013), the following highlights occurred at the Mid Florida Citrus Foundation ‘ A.H. Krezdorn Research Grove: ‘ Plant Improvement Team o Scion for HLB tolerance under evaluation o Evaluation of Valencia clones on new rootstocks established o Sugarbelle harvested ‘ Dr. Singh continued to evaluate herbicide tolerance of selected USDA rootstocks to various residual herbicides continues ‘ UAS of America evaluation on supplemental materials applied to the soil and/or foliage to increase tolerance to the affects of HLB and citrus canker continues ‘ Dr. Futch evaluations: o Continued evaluations of trifoliate rootstocks for HLB tolerance ‘ Applications of the ‘Boyd Program’, Keyplex and Ben Hill Griffin programs continued in the ‘commercial scale’ nutritional trial. ‘ Conducting late summer/early fall fertilizer and pest management programs for the groves o Herbicide program on schedule o Dormant psyllid management continued o Initiated mechanical weed/middle management for evaluation ‘ Applications of seven nutritional treatments continue in MFCF replicated nutritional programs evaluation and plant growth data taken in July ‘ MFCF evaluating topping treatments evaluated ‘ Commercial Trials: o Eurofins evaluations on disease and insect management continue o Evaluations of Agri Quest Citrus Root Health Improvement Project continue o Keyplex nutritional trial evaluations continue o DuPont demonstration for row middles management with Matrix Herbicide established o Syntech trial for GLP evaluation ‘ Drs. Stelinski and Rogers have continued evaluations of Asian citrus psyllid and citrus leafminer management in their areas. ‘ Drs. Albrigo and Wong have continued to evaluate antibiotics to manage HLB
We have continued to focus on the treatments and analysis of naturally infected trees in the field to evaluate the efficacy of our treatments. Twenty four trees were chosen and first sprayed with our 8 spray treatments that included 6 therapeutic treatments and 2 control treatments. The plot was first sprayed on June 25, 2013 and then again in October 30 2013 with 3 trees per treatment. The therapeutic treatments included: 2, L- Arginine, 2, gibberellin in combination with 6-benzyl adenine (BA), and 2, atrazine in combination with sucrose. The remaining two treatments were control treatments that accounted for the surfactant Silwet, K-phite and fertilizer LDKP3XTRA that were added to all treatments. After the spray treatments the trees were sampled at day 0, 3 and 6 collecting 6 leaves from around each of the trees. The leaves from each tree was pooled and extracted for DNA, RNA and protein. The first spray yielded 72 samples that have been processed. The analysis of the DNA revealed that 40% of the plants were infected and that infected trees were unfortunately not always randomly distributed among each of the three replicates for each treatment. To overcome this in October 30, 2013 we sprayed an additional 6 infected trees per treatment. We have analyzed 10 biomarkers at both 3 and 6 days after treatment: 5 related to immune responses pathways (EDS1, PR1, WRKY70, WRKY48, WRKY54), 2 involved in gibberellin pathways and 3 related to sucrose and starch metabolism, a pathway highly linked with HLB syndrome. As we expected, Gibberellin-2-oxydase was clearly induced at Time 6 days in response to GA + BA treatment. Same trend was observed for one arginine and one atrazine + sucrose treatment. Relating to genes involved in sucrose and starch metabolism, we interestingly observed that at least at one concentration, the six treatments were able to reverse the trend of expression of invertase and sucrose synthase induced by HLB infections. GPT2 is strongly induced by HLB in leaf tissues and it is believed to be strongly associated with the starch accumulation in infected leaves. GPT2 is a transporter present in the chloroplast and responsible of the glucose-6-phosphate transport leading to the HLB-induced starch biosynthesis. Interestingly, we observed a strong down regulation of this gene in all six treatments only at three days after treatments. Data showed that some treatments may allow boosting Citrus immune responses. Indeed, WRKY70, a well-known transcription factor involved in biotic stress response was clearly enhanced by BA + GA spray and one Arginine treatment at both Time 3 and 6 days. Arginine and atrazine treatments induced expression of PR1, a SAR-related protein at Time 6 days. Conversely, WRKY48 was not significantly regulated by any six spray conditions. Another key player in biotic stress responses, WRKY54, was higher in abundance in both arginine and atrazine treatments. One concentration of GA + BA showed similar effect. The transcript abundance of EDS1 was unchanged in comparison to both controls. Taken together, these data look promising and indicated that, at least at one concentration, the three therapeutic strategies were able to significantly affect expression of HLB-regulated key biomarkers. However this effect is temporary and will need to be validated by the next gene expression data performed at different season time. Based on this finding we have planned another experiment integrating the eight conditions in two treatments (Arginine + GA + BA or Atrazine + GA + BA) and two control (K-phite and LDKP3XTRA fertilizer that were included also in the other two treatments). These 4 treatments were sprayed on March 11, 2014. We have performed a preliminary proteomic analysis for few treatment samples that allowed identifying around 250 proteins, mostly belonging to Citrus host response. Few pathogen proteins were identified and might be considered possible target to improve early detection. Different protein extraction protocols were tested and we are focusing on one that was the most effective allowing ~1300 proteins to be detected. These data will be functionally analyzed with data mining tools, integrated with previous transcriptomic findings to depict an overall view of the HLB-related syndrome in infected leaves.
Our earlier report showed (Dec-2014) the induction of new flush and flowering in HLB-infected trees following the first spray (Oct-2004) application of 10 ‘M strigolactone (SL). The second spray in January-2015 further induced the vegetative and reproductive growth in greenhouse grown HLB-infected ‘Valencia’ trees. We observed SL induced spring flush in fall and summer flush in spring. The number of vegetative branches were more numerous in SL treated HLB-infected trees in comparison to all other treatments (Healthy, Healthy + SL, HLB alone). SL can be used to regulate canopy architecture in citrus. Early induction of vegetative growth during dormant period of psyllid activity would prevent transmission of Clas through young foliage. Due to early induction of reproductive growth the fruit diameter is larger in SL treated HLB-infected trees in comparison to other treatments. In addition number of fruit and number of flower were also higher in SL treated HLB-infected trees in comparison to untreated HLB trees. Following 2 spray applications of SL over a period of 7 months the number of fruits remained higher in SL treated HLB-infected trees. Field evaluation of SL activity on fruit drop was also conducted on ‘Valencia’ tress at CREC and Weirsdale, Marion County. The fruit drop was 50% in HLB-infected tress. However, only 30% fruit drop was observed in SL treated HLB trees. At anatomical level, SL induced the formation of novel phloem in stem of ‘Valencia’ tress in greenhouse conditions as well as in field grown sweet orange ‘Pineapple’ trees. New phloem tissue in SL treated HLB trees is approximately five times larger in diameter than non-treated HLB trees. Phloem tissue remained functional without any plugging under SL treatment. Both cambium and phloem tissues were poorly visible in HLB-infected trees. SL also induced the starch mobilization at ultrastructural level. Lower accumulation of starch was observed in stems of SL treated sweet orange ‘Pineapple’ trees in comparison to untreated HLB trees. Starch mobilization provides indirect evidences for the availability of inorganic phosphate in cells. Third spray application of SL was done in Apr-2015 on greenhouse grown ‘Valencia’ trees and data will be collected in July-2015 for fibrous root growth, development, and anatomy.
HLB is characterized by impaired phloem performance, hormonal imbalance, poor root function and architecture, limited nutrient supply resulting in immature fruit drop and ill tree health/death. Our goal is to restore the proper functioning of the phloem tissue and consequently restore tree health by using the newest plant hormone ‘Strigolactones’ (SL). As opposed to other growth regulators, SL are carotenoids derived hormones demonstrated to induce cambial activity, fruit growth and development and symbiotic arbuscular mycorrhizal (AM) fungi association in many crops. SL applications have resulted in substantial increases in vascular tissue including phloem. Besides SL regulates root and shoot architecture by increased formation of primary roots, lateral roots, and elongation of root hairs. Application of SL either foliar and/or drench to citrus trees in nano- to micro- mole quantities, should to induce new phloem, roots in and regulate shoot architecture of HLB trees resulting in restore tree health. month intervals. Strigolactone (SL) application in green house trial resulted in early induction of new flush and flowering in HLB-infected trees in comparison to control tress. Similar results were observed in HLB-infected trees in field experiments. Second, SL spray application was done on green house plants after an interval of three months to observe the effect of SL on fruit retention and development. In addition, SL was also applied on individual branches of Sweet orange ‘Pineapple’ to further confirm the vegetative growth promontory role of SL. Control trees were also sprayed with SL in Wiersdale, Marion County. We are also developing a model system which included one leaf-one stem-one root system of citrus to observe the effect of SL under greenhouse conditions. For all field and greenhouse experiments, measurements of fruit size and petiole diameter have been recorded. In addition, tissue samples were collected for further anatomical studies.
HLB is characterized by impaired phloem performance, hormonal imbalance, poor root function and architecture, limited nutrient supply resulting in immature fruit drop and ill tree health/death. Our goal is to restore the proper functioning of the phloem tissue and consequently restore tree health by using the newest plant hormone ‘Strigolactones’ (SL). As opposed to other growth regulators, SL are carotenoids derived hormones demonstrated to induce cambial activity, fruit growth and development and symbiotic arbuscular mycorrhizal (AM) fungi association in many crops. SL applications have resulted in substantial increases in vascular tissue including phloem. Besides SL regulates root and shoot architecture by increased formation of primary roots, lateral roots, and elongation of root hairs. Application of SL either foliar and/or drench to citrus trees in nano- to micro- mole quantities, should to induce new phloem, roots in and regulate shoot architecture of HLB trees resulting in restore tree health. month intervals. To carry out this project, a Postdoctoral associate was hired. The individual is already established in the lab and commenced with the preparations for all types of experiments. Experimentally, both parts of the project have been started. Greenhouse experiments: For the greenhouse trial,s 50 Valencia on Carrizo trees were purchased. Trees were planted on 3.5 gal pots and placed in a greenhouse. An automatic irrigation system was established and trees will be allowed to acclimate for 30 days prior to the start of treatments. Field experiments: Field grown Hamlin HLB-affected trees have been selected in a block at CREC. Trees have been flagged, area under the tree has been cleaned, fruit per tree counted and experimental fruit flagged. We have purchased the growth regulator strigolactone and are poised to begin the first sprays during the first week in September.
The goal of the project is to identify a Bacillus thuringiensis (Bt) toxin with toxicity against Asian Citrus Psyllid (ACP) and to improve its toxicity by genetic modification with a psyllid gut binding peptide. The peptide will be identified by screening a phage display library for peptides that bind to the gut of the ACP. Addition of the selected peptide to the identified Bt toxin will result in significant enhancement of toxicity of the modified toxin relative to the wild type toxin toward the ACP. Durign the current reporting period, the focus was on trypsin activation of the partially purified Bt toxins from selected Bt strains provided by USDA ARS, MD. Trypsin activation of Bt toxins was carried out at Iowa State University. Briefly, Bt toxins were solubilized using sodium carbonate pH 10.5 + 10mM DTT for three hours at 37’C and dialyzed against 50 mM Tris-Cl pH 8.5. Bt toxins were then incubated with bovine trypsin at a final concentration of 10% of the toxin concentration at 37 ‘C for 1 h. Removal of trypsin was carried out using benzamidine sepharose. The samples were boiled in denaturing SDS sample buffer for 5 min, separated on 10% (wt/vol) SDS/PAGE and stained with Coomassie blue. Different SDS-PAGE profiles of trypsin-treated Bt toxins were apparent with multiple molecular weight protein bands ranging from ~18 kDa to ~150 kDa. Based on the similarity of the molecular mass of the toxin profiles, eleven Bt strains are classified into the following six groups. These groups are distinct from the five groups reported in the previous update. Group one: Five Bt strains, each with three activated toxin bands of ~60, ~65 and ~70 kDa. Group two: Two Bt strains, each with two activated toxin bands of ~60 kDa. Group three: One Bt strain with one activated toxin band of ~60 and ~65 kDa. Group four: One Bt strain with one activated toxin band of ~70 and~73 kDa. Group five: One Bt strain with activated toxin bands of ~70, ~90, and ~150 kDa. Group six: One Bt strain with activated toxin bands of ~18, ~20, ~22, ~28,~30, ~35, ~49, ~60, and ~70 kDa. Two milligrams of each of four trypsin-activated Bt toxin samples were sent to Dr. David G. Hall, USDA-ARS for ACP membrane feeding assays for toxicity analysis.
ACPS -grapefruit research at IRREC: The overarching goal of this component is to develop ACPS and high-density plantings for commercial grapefruit on the east-coast. To date, the progress of this project is as follows: Irrigation installation was completed in October 2013. We are currently testing and evaluating the irrigation systems to fine-tune their performance and eliminate inefficiencies and any errors made during installation. The first experimental block of citrus was planted in November 2013. ‘Ray Ruby’ grapefruit trees on ‘Sour orange’ and ‘US-897’ rootstocks were planted on 8 acres on the citrus research grove at the UF-IFAS-IRREC station in Ft. Pierce. ‘Ray Ruby’ / ‘Sour orange’ trees were planted at a density of 152 trees/acre with microsprinklers and will be fertilized with granular, dry fertilizer: these plantings will serve as the ‘grower standard’ control treatment. ‘Ray Ruby’ trees on both ‘Sour orange’ and ‘US-897’ rootstocks were planted in staggered-set, tramline configurations at a high-density of 421 trees/acre: half of these plots will be fertigated with microsprinklers and half will be fertigated with in-line drip tubing. Funds from this project were also used to partially support the construction of a 5-acre high-density block of ‘Ray Ruby’ / ‘Kuharske’ grapefruit at the IRREC. In this block, all trees are being irrigated with microsprinklers but are planted at 3 different densities: 126, 189, and 421 trees/acre. Funds from other sources were used to cover the costs to complete this research block. These trees were also planted in November 2013. Data collection on tree growth, psyllid abundance, and CLas presence will begin in late December 2013. We plan to hold a field day for stakeholders in late winter/early spring of 2014. This field day will exhibit the two trials listed above. A Ph.D. graduate student was recruited and hired through the UF-Horticultural Sciences Department to assist in executing this objective. Currently, this student is in Gainesville completing her required coursework. The student will be available to monitor the progress of the research blocks full-time starting in the summer of 2014. A full-time OPS worker was hired and began work in August 2013. The balance of this worker’s wages came from other funding sources. This employee has been extremely instrumental in completing the irrigation installation and maintenance of the research block. This employee will continue to help maintain these research blocks and assist the graduate student (described above) with data collection.
Experiments to determine the efficacy of different nano-particle systems to deliver nutrients and antimicrobial molecules to citrus leaves: The objective of this study is to deliver water-soluble antimicrobial compounds into citrus plants affected by greening disease. The delivery of the antimicrobial compounds will be accomplished using nanoparticulate carrier systems (e.g. liposomes and polymer nanoparticles). In the first quarter of this project, dye-doped lipid and polymer nanoparticles that have encapsulated water-soluble dyes as surrogate for antibiotics have been prepared. Liposomes have been prepared using e.g. 1,2-distearoyl-sn-glycero-3-phosphocholine, 1,2-dioleoyl-3-trimethylammonium-propane, and cholesterol through the thin lipid film hydration method. Initial studies have been made to encapsulate water-soluble dyes such as sulforhodamine B and fluorescein. In order to minimize the leaching of the dyes, cationic lipids are incorporated in the lipid composition. Primarily, the particle size is being restricted to less than 100nm by the use of appropriate filtration membrane and the choice of hydrating solvent. Characterization of the developed dye-doped lipid structures as well as optimization to minimize leaching is under progress. In addition, dye-doped polymeric nanoparticles comprised of environmentally safe materials such as poly (lactic-co-glycolic acid) have also been prepared. Initial studies have been performed to encapsulate hydrophilic dyes in the polymer matrix. Using the double emulsion method, polymer nanoparticles that have successfully encapsulated fluorescent dyes have been synthesized. The size of the dye-doped particles lies between 200-300 nm. The engineered particles are being characterized for their particles size, distribution, absorbance, and fluorescence properties. Future studies involve the encapsulation of select dyes that have low interference from the auto-fluorescence of citrus plants and to study their uptake.
Experiments to determine the efficacy of different nano-particle systems to deliver nutrients and antimicrobial molecules to citrus leaves: The objective of this study is to deliver water-soluble antimicrobial compounds into citrus plants affected by greening disease. The delivery of the antimicrobial compounds will be accomplished using nanoparticulate carrier systems (e.g. liposomes and polymer nanoparticles). In the first quarter of this project, dye-doped lipid and polymer nanoparticles that have encapsulated water-soluble dyes as surrogate for antibiotics have been prepared. Liposomes have been prepared using e.g. 1,2-distearoyl-sn-glycero-3-phosphocholine, 1,2-dioleoyl-3-trimethylammonium-propane, and cholesterol through the thin lipid film hydration method. Initial studies have been made to encapsulate water-soluble dyes such as sulforhodamine B and fluorescein. In order to minimize the leaching of the dyes, cationic lipids are incorporated in the lipid composition. Primarily, the particle size is being restricted to less than 100nm by the use of appropriate filtration membrane and the choice of hydrating solvent. Characterization of the developed dye-doped lipid structures as well as optimization to minimize leaching is under progress. In addition, dye-doped polymeric nanoparticles comprised of environmentally safe materials such as poly (lactic-co-glycolic acid) have also been prepared. Initial studies have been performed to encapsulate hydrophilic dyes in the polymer matrix. Using the double emulsion method, polymer nanoparticles that have successfully encapsulated fluorescent dyes have been synthesized. The size of the dye-doped particles lies between 200-300 nm. The engineered particles are being characterized for their particles size, distribution, absorbance, and fluorescence properties. Future studies involve the encapsulation of select dyes that have low interference from the auto-fluorescence of citrus plants and to study their uptake.
A new high-capacity indirect hot air portable heater was purchased for further study on the effect of heat treatment of HLB inoculum as well as tree health. The heater works by convection, heating the air as it passes over a flame while a fan moves the heated air to the heat treatment tent as opposed to the electric heaters used in the previous treatments which use radiant heat transfer. The goal of using the dry hot air heater was to provide supplementary heat to increase the temperature more rapidly and test the ability of heat treatment late in fall and winter as well as to resolve the issues that were created by electric heaters used in the first part of this study. In November, five trees were tested at temperatures ranging from 50-55’C (122-131’F). A new portable tent was built to accommodate the new hot air heater and the larger trees subjected to the test. In all five tests, it was observed that the temperature within the tent rose much more rapidly than when the electric heaters were used. However, at this point, it is too early to assess the differences in leaf damage due to heat stress. It seems that hot air can increase the temperature more uniformly and rapidly than electric heaters, but it also increases the amount of leaf drop in the canopy after treatment. The original 36 trees that were heat treated in the summer are still undergoing physiological tests to evaluate the overall health of the trees. Six month post-treatment tests are currently underway. The goal of these tests is to quantify the change in health of the tree due to the heat treatment. This data will be used to support evidence of fruit yield and fruit quality improvement. A mathematical model was developed using the finite difference method to simulate heat transfer in the citrus canopy. This model will help to predict the amount of heat needed for different sizes of trees. Two types of heat transfer, conduction and convection, were applied within the model. The conduction heat transfer model will simulate heat transfer through the trunk and branches, while the convection heat transfer will simulate heat transfer around the canopy under the enclosure. The input parameters included the trunk diameter, thermal conductivity of the trunk, heat transfer coefficient of air, and air velocity from the fan. The output parameters were temperature and heating duration, in which a mapping of temperature with heating duration was produced. Regressions and model fitting was done based on the plots generated by the data. Model calibration and validation were done by comparing the data from the model with experimental data obtained from the preliminary results.
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