OVERVIEW: The program is designed to provide partial technical salary support to all faculty at the Citrus Center to ensure continuity of the research. PROGRAM OPERATIONS Horticulture/ Breeding: The funds were used to partially support Sonia del Rio. During the reporting period, she worked on: (1) Testing Texas Red grapefruit selections for fruit quality through the season (2) Screening rootstock seedlings for Phytophthora resistance (some survive under intense infection pressure) (3) Embryo recovery of plants from aborted seed of greening-infected fruit for potential resistance Plant Pathology: The funds were used to support Perla Duberney. During this time she worked on the evaluation of the effect of soil conditioners in improving the effectiveness of Mefenoxam treatment for Phytophthora control. The results showed that (1) Phytophthora propagule counts were suppressed in treated plots after 4, 8 and 12 weeks post application; (2) the use of the soil conditioner had a detrimental effect on Mefenoxam effectiveness; (3) fruit size was increased in treated trees compared to untreated trees; (4) application of soil conditioners promote better absorption of micronutrients. Entomology and Plant Physiology will utilize their portions of the grant in the latter portion of the FY.
The central mission of this project is to combine existing fertilization, irrigation, scion and rootstock options in modern, high-density citrus groves that will be more productive sooner, and survive the onslaught of HLB more successfully than conventional Florida citrus groves. The resulting synergistic production system was called the Advanced Citrus Production System, and relied heavily on open hydroponics with liquid fertigation, and computer automation, as well as precocious, dwarfing rootstocks and high planting densities. The overall goals of this three-year project renewal are to continue the existing ACPS experiments at Auburndale (2.5 year old replant) and at the CREC (mature 20 year old ACPS retrofit) in order to obtain long-term data which is crucial for the successful recommendation and adoption of this technology. A demonstration ACPS experiment testing four different rootstocks was established in March 2011 on 5 acres of CREC’s Lake Placid grove, and will also be continued with this project. Finally, two new ACPS experiments will be established to fill gaps and to evolve the new ideas developing from our current research experiments. Specific objectives are: i) To install a ‘Valencia’ juice orange ACPS replant experiment at the CREC, testing two rootstocks (US897, Swingle), and two novel ultra-high planting densities with narrow grove equipment. ii) To install a grapefruit fresh fruit ACPS replant experiment in the Indian River region in order to adapt the technology for regional priorities, conditions and soil types. iii) To continue the existing ACPS experiments at Auburndale, CREC (Lake Alfred), and Lake Placid. The collective results from various ACPS experiments of this project were valuable in demonstrating to citrus growers the need for using higher planting densities and more intensive irrigation and balanced fertilization technologies in order to remain competitive in an HLB-endemic environment. Economic production in the young Hamlin experiment was achieved in the third year (220 boxes/acre), and a year later, reached full production at 622 boxes/acre), with the best combination of rootstock (C35), high density (363 trees/acre), and drip fertigation. Conventionally grown groves would typically reach full production only after 6-10 years. Unfortunately the HLB incidence in this grove grew rapidly, reaching 75% in the fifth year, and causing drastic yield decline of affected trees. The best yield obtained in the fifth year was only 436 boxes/acre, and continued to decline thereafter due to stunted tree canopies and root systems, low fruit set, small fruit size and excessive preharvest fruit drop. The recommended practice of HLB management by destroying HLB-affected trees was futile in this grove because it was surrounded by symptomatic dooryard citrus trees in neighboring properties, over which we had no control. Unfortunately the other trials in this study suffered similar early high HLB incidence rates, leading to unsustainable production scenarios. The new knowledge, and horticultural gains of tree growth and fruit yield made with the ACPS research in this project were remarkable, but were compromised by less successful psyllid control and HLB prevention practices. The outcomes and technologies of this research have been adopted by many Florida growers for replanting new groves, especially with higher densities, precocious rootstocks like C35, and using effective hydroponic fertigation through drip or microsprinkler irrigation systems. Ultimately when HLB-resistant rootstocks or varieties are developed and released, the new resistant groves can be established in the most efficient and rapid method possible by adopting ACPS techniques developed and tested in this project.
Progress made in the second quarter of this study has focused on grove selection and the initial sampling of leaf tissue and soil. Work has also continued regarding development of soil conditions for root hair proliferation Objective 1: Leaf nutrient thresholds All three study areas have selected groves and trees for this phase of the research. The Ridge focus area has selected 11 study grove blocks (round orange) with 5 trees per block. The Indian River area has selected 10 study grove blocks (both round orange and grapefruit). The South Florida area has also selected grove blocks for sampling. The initial measurements that have been taken include LAI, tree volume, PCR, leaf nutrition, leaf starch content, soil nutrition, organic matter content, and physical properties. Leaves for nutrition were measured for SPAD, leaf area, dry mass, and scanned using a flatbed scanner for color as well as leaf circumference and leaf area. Objective 2: Determine soil conditions that favor root hair and VAM proliferation i. As mentioned in Objective 1 above, initial soil sampling at the 0-6 inch and 6-12 inch depth has been completed and analysis is beginning. Percent OM, using Loss On Ignition has been completed for the ridge portion of the survey area and in the next quarter the initial samples from the remainder of the survey areas will be analyzed. Analysis includes, soil nutrient content, pH, CEC, and soil color. ii. The cuttings that were placed in aeroponics tanks are continuing to be monitored and maintained with replacement solutions bi-weekly. The solution chemistries are still being fine-tuned and analyzed. Growth of the cuttings has been slow but is progressing. Analysis of the root development will be the next step of this portion of the study.
Seed decontamination: 1. Develop guidelines to prevent seed contamination by canker. Part 1. Surface disinfection of seeds. Objective is to develop economical and practical cleaning methods for removing Xcc without loss of seed viability. Progress: Thirty freshly squeezed seeds of Kuharske were plated on LB plates. Concurrently rinsate of seeds rinsed in sterile water had serial dilutions plated on media plates viz. V8, PDA, LB and Dox. LB plates showed numerous distinct colonies, whereas the rest showed bacterial smears. Bacterial colonies on LB plates resembled X. citri and other unknown bacteria. Similar results occurred on LB plates plated with freshly squeezed intact seeds. Identity of these bacteria is unknown, but appear to be common environmental bacteria. Identification requires DNA sequencing, planned in the future. Seeds treated with different concentrations of chlorox and PPM appeared to display higher seed germination rates, to be investigated in the future. Part 2. Evaluating the 2006 CHRP decontamination procedure. Progress: Experiments are in progress along with experiments using sodium hypochlorite and PPM for decontamination. Part 3. Development of diagnostic methods for on-site canker detection. Progress: Experiments are ongoing. Cutting and tissue cultivation In October propagation beds with bottom heat were constructed. Sides and separators were installed on 3 benches, producing 12 independent beds. Beds were partially filled with perlite and heating cables installed with independent temperature controls. On 14 October, 4 schedules of acclimation of tissue cultured rootstocks were applied to C-35, C-54 sour orange, US-812, SW-13 and KC-13 root stocks. Any roots were removed and stems transplanted into peat:perlite in seedling trays. Plants were harvested on 11/16. Success was >92% for all rootstocks except sour orange (82%). The next set of tissue culture plants were planted on 12/14, with additional plants placed in 4 of the heated sections; with the same levels of light and heat exclusion. Changes in relative humidity and light levels were the same reported previously. The gap in the sequence was due to insufficient numbers of plantlets the first delivered, with delays in obtaining the remainder. Due to technical issues, humidity levels were insufficient the first week and many plantlets died. Plants in heated benches faired better due to higher humidity. These plants will be harvested in mid-February. Cutting propagation continued with single node cuttings of Kuharski initiated on 10/14, 11/3 and 12/1. Harvest of cuttings have continued to be every 6 weeks, with harvest occurring on 10/14, 11/3, 12/1. Auxin concentrations of 4000 and 7500 ppm have been consistent. Most woody plants require higher auxin concentrations during the winter months for good rooting. Rooted cuttings of the HLB resistant lines were transplanted in 5 tree pots the week before Christmas. Any cutting that had good root systems were transplant, independent of shoot growth. Less than 10 cutting failed to produce roots. Even plants with no additional shoot growth had abundant root growth at transplanting. These plants were placed in the production side of the greenhouse due to insufficient space in the propagation bay.
The objectives of this project are to characterize the molecular interactions between the effectors and the host mitochondrial proteins; to screen for molecules that inhibit the effector functions; and to control HLB using the inhibitor(s) and/or other related molecules. Transgenic Arabidopsis plants expressing lasAI or lasAII showed a different degree of impaired growth. In particular, the LasAI contains domains responsible for abnormal growth of the root and/or meristem. Trangenic citrus plants expressing Las AI also display growth retardation. Meanwhile, to further study the function of LasAI in citrus, transgenic citrus were generated to express LasAI, LasAI N-terminal, LasAI C-terminal, LasAI repeat region, LasAII and GFP control, respectively. We have obtained transgenic citrus plants transformed with different domains of LasAI, Interestingly, transgenic plants show different degree of growth retardation, in particular the full length LasA1 and LasA1 C-terminal shows slower growth compared to the other constructs. Using RNAseq and RT-qPCR, we were able to identify the up- and down- regulations of some important genes involved in host-pathogen interactions and biosynthesis of secondary metabolites in these transgenic plants. Transient expression of LasAI and three different LasAI domains, LasAI-N-terminal, LasAI-repeat, LasAI-C-terminal allowed us to visualize the sub-cellular localizations of different domains. Because of high level expression of these effector proteins, we developed a novel in vitro screening system that evaluates small molecules against these Las effectors. The library consists of more than 30 million compounds obtained from the small molecule libraries of the TPIMS (Torrey Pines Institute for Molecular Studies). Interestingly, a few groups of compounds showed interference activity against the mitochondrial localization of LasAI. Meanwhile, to concert this screening, we developed another in vitro screening system in conjunction with the culture screening using Liberibacter cresence (Lcr). From these screening of 65 scaffold chemicals, we identified a number of chemical groups that disrupted the interaction between LasA1 and mitochondria and inhibit both Las and Lcr growth. We are narrowing down to individual compounds that inhibit the function of the Las AI effector or kill Las bacteria via other pathways, and measured the dosage effect of these potential candidate. The selected chemicals (individual or small groups) are in the evaluation process with graft-based assay. In addition, another hypothetical protein has been expressed in planta via transient and stable transformation, and founded to affect host resistance to a bacterial pathogen. The antibody against this protein was able to detect this antigen both in the transgenic plants and in the Las-infected plants. Meanwhile, the Western blot results revealed unique formation of this protein in E. coli and plants. Citrus plants with high level expression of this transgene displayed HLB-like symptoms, yellow shoot and impaired growth. Further characterization of this effector revealed its unique sub-cellular localizations. We also analyzed the expression of LasA1 in Las-infected citrus plants. RT-PCR results indicate that LasA1 expression is correlated with severity of HLB symptoms. In particular, LasA1 was expressed more in the yellow leaves or the yellow spots than green spots of the symptomatic leaves with blotchy mottle. Since transgenic plants expressing Las A1 showed up-regulation of defense-related genes, these transgenic plants were propagated and graft-inoculated with Las bacteria. We detected LasA1 in Las infected citrus leaves using antibody-based tissue printing. The results indicate that LasA1 was abundant and diffusible in Las-infected vascular tissues. To further characterize LasA1, we identified several citrus proteins that interacted with LasA1 using Yeast Two-Hybrid screening system. It is worth noting that a couple of these proteins were also identified as up-regulated genes in our RNA-seq data as mentioned above.
The overall objective of our research project is to develop an effective and sustainable phage/phage component-based biocontrol system for Xanthomonas axonopodis pv. citri (Xac), the causal agent of citrus canker. We have previously reported on first and second round evaluations of phage cocktails and first round testing of tailocin cocktails in cooperation with Dr. Nian Wang (University of Florida-Lake Alfred). In two independent experiments, when the tailocin cocktail containing tailocins XT-1 and XT-4 was applied to foliage at a multiplicity of killing units (MOKU) of 16 at 6 h post- or pre-inoculation with 5 X 10^7 CFU/ml of Xac, there was a an average reduction of 49% and 53%, respectively, in lesion formation as compared to the plants inoculated only with Xac. It was of interest to determine a dose response curve to the tailocin treatment. Using the same condition as previously described (Jan. 2015 report), Hamlin sweet orange trees were inoculated with 1 10^8 CFU/ml of Xac. After 6 h, the tailocin was applied at a MOKU of 15, 10 or 5. Lesion numbers were assessed 21 days after inoculation with the pathogen for treated and untreated trees. In two independent experiments an average of 39%, 31% and 11% reduction in lesions were observed in leaves of trees treated with tailocin cocktails at a MOKU of 15, 10 and 5, respectively, as compared to non-treated trees. Using a delta-PilA mutant of Xac, we have isolated and purified two non-type IV pilus dependent phages that appear to be virulent. Base on their plaque morphology the phages appear to exhibit depolymerase activity. Ongoing studies will determine the morphology and lifestyle of the two phages. Bioinformatic analysis of Xac phages genomes and tailocin gene clusters is ongoing.
The focus of our project is to develop a detection system for bacteriophage (phage) and/or phage components (tailocins) using Liberibacter crescens strain BT-1. We have accomplished that goal and have moved forward to identify naturally occurring phages and to construct modified tailocins that are active against strain BT-1. Once Candidatus Liberibacter asiaticus (Las) is successfully cultured, the protocols developed for L. crescens can be translated to Las. We have demonstrated that the tail fibers of tailocins can be deleted and complemented. Since the R-type pyocin systems have been well studied, we have initiated studies using the R2 platform and designed fusions between N-terminal tail fiber region of the R2 and C-terminal portions of tail spike from BT-1 prophages. We have identified the BT-1 tail spike protein based on topology. We are continuing the search for naturally occurring phages active against L. crescens. Members of the Rhizobium, Agrobacterium and Liberibacter are in the family Rhizobiaceae. Because of their phylogenetic relationship, it is possible that members of the Rhizobiaceae could share receptors. Rhizobium spp., Agrobacterium spp. or L. crescens BT-1 were used as hosts for enrichments from environmental samples. Several new Agrobacterium and Rhizobium phages have been isolated and purified. Electron microscopy studies will confirm their morphology. It is our experience that growth conditions can affect the bacterial surface, and therefore in vitro testing for phage and tailocin sensitivity. The phages are currently being tested against BT-1 using several modifications of medium BM7.
The objectives of this research are 1) investigate the effect of heat stress on Las; 2) monitor healthy and HLB-affected citrus genome response to heat; and 3) optimize field thermotherapy. To reach our goals in objective 1, we have exposed both HLB-affected and healthy periwinkle (40 C) and citrus (42 C) to heat stress. DNA (to study phage gene copy number) and RNA (to study gene expression) has been extracted. Changes in gene copy number for genes located in the phage region have been studied. Additionally, expression levels for Las genes were studied with RNA-seq and several genes of interested were identified. Reverse-transcription PCR has confirmed the results of the RNA-seq analysis and has suggested a possible mode of action behind the successful elimination of Las seen in thermotherapy. For objective 2, we have conducted a comparison study between field heat-treated and non-heat-treated citrus plants. There were 31 consistent up-regulated genes and 47 down-regulated genes in the citrus trees treated with heating. Additionally, potted Las-positive and negative citrus were exposed to 4 hours for 4 days of 40 C, 85 % relative humidity (similar to heat exposure in field setting) in a controlled greenhouse. RNA-Seq data (from new flush present before heat treatment and the new flush that developed after heat) were analyzed. There were 3,722 differently expressed genes (DE) between Las-negative and positive trees not exposed to heat. Flush that appeared after heat treatment on the positive plants had 294 DE as compared to flush on unheated positive plants and 1308 DE as compare to flush on healthy trees. Some heat shock and oxidative proteins were identified in the DE lists. Additionally, potted Las-positive and negative citrus were exposed to 30 hours of 40 C, 85 % relative humidity and sampled during the exposure. RNA has been extracted from these samples. Time 0 before treatment and time 30 hours (of continuous heat) are being monitored for changes in gene expression for heat shock genes and other identified stress genes using RT-qPCR. As for the objective 3, over 3 years of prior data (tree Las Ct values, treatment procedures, and temperature logs from one location) have been summarized. Extensive analysis of temperature and humidity data using KS nonparametric test, ANOVA, Tukey’s HSD, and other measures have shown that each HLB-affected trees respond uniquely to heat treatment. The greatest decrease in Las titer and overall duration of this decrease varies for each tree (6-18 months) and is not solely dependent on heat but most likely affected by the biology of the tree. When comparing 7 versus 9 days of treatment, the longer treatment did not increase titer reduction. Also, six days was not more effective than a 4 day treatment. Regarding temperature, the greatest effect was present at 40, 41, and 42 C for 5 to 7 hours for 3 out of 4 test plots. A detailed correlation chart indicates other combinations of temperature and durations can also be effective at reducing Las. All statistical analyses shows that the response to heat stress is unique for each Las-infected tree. Although fruit drop did decrease for many of the treated trees, due to the large variation in data, the decrease was not statistically significant. Fruit was harvested and juice made. Volatile production analysis of juice from commercial grove 1 has been finished. A total of 63 aromatic volatile compounds were detected by HS-SPME-GC-MS. Discriminant analysis separated the “no heat” juice from the “heat” juice. Juice made from the product of the heat-treated trees had “fruity” and “pineapple fruit” top-notes. Juice is still being analyzed for sugar/acid, secondary metabolites, and Las titer. Juice quality taste panels are complete and panelists could distinguished between heat treated and no treatment juice sampled juice from the two commercial HLB positive Valencia groves (p<0.05). USDA Picos farm juice was not significantly different (p=0.052). Almost all panelists who differentiated between juice correctly said that heat-treated HLB juice was sweeter, less acidic, less bitter, and had more flavor and body than the juice produced from unheated trees.
Seed decontamination 1. Develop guidelines for seed propagation that prevent contamination of seed by citrus canker. Surface disinfection of seeds. Objective is to develop economical and easy-to-implement seed cleaning methods for removing Xcc bacteria without loss of seed viability. Progress: Citrus seeds obtained from a local grower were free of any X. citri infection. Therefore citrus seeds were artificially inoculated with X. citri . Seeds were soaked in a freshly prepared X.citri culture (OD 0.002) in a beaker for 1 hour. Contaminate seeds were then placed on sterile filter papers and dried at room temperature for 24 hrs. These were then treated with PPM and Chlorox according to the plan of the project. Seeds were rinsed in sterile water and serial dilutions of the rinsed water were plated for bacterial count. Similarly intact seeds were placed on LB for determining the efficiency of decontamination protocols. Some data has been collected, with the rest being collected from the pictures of LB plates. After completion of data collection, data will be analyzed. The same experiment will be repeated once more. Evaluating the 2006 CHRP decontamination procedure. Progress: These experiments are in progress along with experiments using sodium hypochlorite and PPM for decontamination. Development of rapid and sensitive molecular diagnostic methods for on-site detection of Citrus canker. Progress: Initial standardization experiments are going on. The PI met with our collaborators at UF to obtain additional isolates. Cutting and tissue cultivation On 7 July, 4 schedules of acclimation of tissue cultured citrus rootstocks were applied to C-35, C-54 sour orange, US-812, SW-13 and KC-13 root stocks. Any roots were removed and stems transplanted into a peat:perlite blend in seedling trays. Relative humidity (RH) started at 90% under layers of 30% and 50% shade cloth, overlain with reflective R-3 insulation board (from inside out). RH was decreased step-wise every 2, 3, 4 or 5 days until the end of days at RH = 50%. Covers were similarly removed. Success was very high for most varieties at the fastest acclimatization rate, at over 90%; except sour orange. A second set was started in early October. Cutting propagation began on May 6th and have generally been initiated monthly since. Initially cuttings with 1, 2 or 3 nodes were used. No differences in rooting were noted but multiple nodes required pruning to a single node. Thereafter single node cutting have been used. Different auxin concentrations and carriers were also evaluated. Best results were 4000 and 8000 ppm. Both concentrations have been carried forward. Success has been >90% to date using common rootstocks of Kuharski and C-35.
The last quarter of 2015 was dedicated to microscopy with additional time allotted to starting the second year experiments and to write manuscripts. There was a great deal of time dedicated to data analysis and field plus greenhouse preparation. In November, we began with the planned treatments both in the greenhouse and field. We followed the proposed plan of work with the following experiments. I: Effect of drenching application of SL on HLB-infected trees. Soil in potted Valencia trees was drenched with SL at the predetermined concentration. Tree characteristics were noted and changes recorded on a bi-weekly basis. II: Effect of spray application of SL on HLB-infected trees (Repeat experiment). This is a repeat of the experiments involving spraying SL on Valencia potted trees in the greenhouse. Tree characteristics were noted and changes recorded on a bi-weekly basis. III: Effect of SL + Fungicides on Phytophthora growth in HLB-infected trees. This treatment has been postponed. IV: Effect of spray application of SL on other varieties of citrus in groves. ‘Midsweet’ was selected as a second variety to be tested for SL effect on tree health. Tree physical characteristics and fruit drop are being monitored. V: Effect of spray application of SL + other promising compounds on citrus in groves. Additional natural organic compounds, successfully used in other crops, are being tested for the potential effectiveness in citrus trees. So far, diluted sucrose solutions have been effective in enhancing new flush in trees with advanced stages of HLB. For all trees, data is being collected on the appearance of new growth, flowering, fruit drop and vegetative growth in general.
In the initial phase of this research project, development of field work protocol and fertilizer solution chemistries for the greenhouse portion have been the primary focus. Grove test site selection is still underway and once all of the grove sites are identified, the bulk of the field data collection work will begin. Of the four primary objectives, one and two have been the primary focus. Objective 1: Leaf nutrient thresholds We are working to identify and select groves that will be suitable for this phase of the research. One of the groves identified on the CREC campus was used to test the protocol of the work to be done on a regular basis. LAI, tree canopy volume, PCR, soil samples, and leaf tissue nutrition were all measured. Leaves for nutrition were measured for chlorophyll index (SPAD), leaf area, dry mass, and scanned using a flatbed scanner for color as well as leaf circumference and leaf area. Objective 2: Determine soil conditions that favor root health, including root hair and VAM proliferation i. While groves are still being selected for this research, the preliminary work, as mentioned in Objective 1 above, has been done and includes the soil sampling at the 0-6 inch and 6-12 inch depth. ii. Cuttings were taken from both healthy non-infected (HLB-) trees as well as HLB+ infected trees. These cuttings were rooted using plastic seed germinating domes to limit transpiration. After the cuttings rooted, they were switched to aeroponics chambers for further root development using standard fertilizer solutions. In the meantime, solution chemistries were developed to contain the correct mix of nutrients for the target phosphate concentration using soluble phosphorus, tricalcium phosphate (TCP) and rock phosphate (RP). Initial groups of HLB suspect cuttings were placed into three aeroponics chambers with specific nutrient chemistries. Those chambers are, Chamber 1, complete fertilizer minus soluble phosphate and using TCP; Chamber 2, complete fertilizer minus soluble phosphorus and using RP; and Chamber 3, complete fertilizer solution with soluble phosphorus. The purpose behind the use of solid TCP and RP is to establish stable solid-liquid equilibria that regulate concentrations of P and Ca in solution for optimizing root growth and health. We succeeded in establishing stable chemical equilibria with both PR and TCP in the slightly alkaline pH range, and the trees are now being grown in aeroponics to monitor and measure root proliferation.
The objectives of this project are to characterize the molecular interactions between the effectors and the host mitochondrial proteins; to screen for molecules that inhibit the effector functions; and to control HLB using the inhibitor(s) and/or other related molecules. Transgenic Arabidopsis plants expressing lasAI or lasAII showed a different degree of impaired growth. In particular, the LasAI contains domains responsible for abnormal growth of the root and/or meristem. Trangenic citrus plants expressing Las AI also display growth retardation. Meanwhile, to further study the function of LasAI in citrus, transgenic citrus were generated to express LasAI, LasAI N-terminal, LasAI C-terminal, LasAI repeat region, LasAII and GFP control, respectively. We have obtained transgenic citrus plants transformed with different domains of LasAI, Interestingly, transgenic plants show different degree of growth retardation, in particular the full length LasA1 and LasA1 C-terminal shows slower growth compared to the other constructs. Using RNAseq and RT-qPCR, we were able to identify the up- and down- regulations of some important genes involved in host-pathogen interactions and biosynthesis of secondary metabolites in these transgenic plants. Transient expression of LasAI and three different LasAI domains, LasAI-N-terminal, LasAI-repeat, LasAI-C-terminal allowed us to visualize the sub-cellular localizations of different domains. Because of high level expression of these effector proteins, we developed a novel in vitro screening system that can evaluate small molecules against these Las effectors. The library consists of more than 30 million compounds obtained from the small molecule libraries of the TPIMS (Torrey Pines Institute for Molecular Studies). Interestingly, a few groups of compounds showed interference activity against the mitochondrial localization of LasAI. Meanwhile, to concert this screening, we developed another in vitro screening system in conjunction with the culture screening using Liberibacter cresence (Lcr). From these screening of 65 scaffold chemicals, we identified a number of chemical groups that disrupted the interaction between LasA1 and mitochondria and inhibit both Las and Lcr growth. We are narrowing down to individual compounds that inhibit the function of the Las AI effector or kill Las bacteria via other pathways, and measured the dosage effect of these potential candidate. In addition, another hypothetical protein has been expressed in planta via transient and stable transformation, and founded to affect host resistance to a bacterial pathogen. The antibody against this protein was able to detect this antigen both in the transgenic plants and in the Las-infected plants. Meanwhile, the Western blot results revealed unique formation of this protein in E. coli and plants. Citrus plants with high level expression of this transgene displayed HLB-like symptoms, yellow shoot and impaired growth. Further characterization of this effector revealed its unique sub-cellular localizations. Understanding the molecular mechanism of how the effector induces HLB-like symptom is underway. We also analyzed the expression of LasA1 in Las-infected citrus plants. RT-PCR results indicate that LasA1 expression is correlated with severity of HLB symptoms. In particular, LasA1 was expressed more in the yellow leaves or the yellow spots than green spots of the symptomatic leaves with blotchy mottle. We successfully optimized the production of LasA1 polyclonal antibody. The new antibody increased the detection sensitivity and specificity. The new LasA1 antibody can detect LasA1 protein in Las infected periwinkle plants using western blot. Moreover, we detected LasA1 in Las infected citrus leaves in histological localization experiments. From the tissue print results, we identified the presence of a strong diffuse signal in the vascular bundle, indicating that LasA1 is abundant in las infected tissue and that diffuses in the phloem, which indicates that LasA1 was secreted outside of Las bacteria and move in the phloem system.
The objectives of this research are 1) to develop cost effective thermotherapy protocols for field application by optimizing temperature and relative humidity conditions in the tent; 2) to develop a mathematical model derived from our data and grower s data which will be used to determine the best treatment duration in future applications; and 3) to study gene expression of HLB-affected citrus plants that received heat treatment, and identify critical citrus genes that may be induced by heat stress for the benefit of suppressing HLB. To reach our goals in objective 1, we have exposed both HLB-affected and healthy periwinkle (40 C) and citrus (42 C) to heat stress. DNA has been extracted and amplified for Las 16S rRNA and certain phage genes. A standard curve for a normalization gene has been established and data is being analyzed using the delta Ct method. Additionally, changes in expression levels for these genes are being monitored. Reverse-transcription PCR is currently being used to confirm the results of gene expression data. As that Las cannot be cultured, a kill curve of Liberibacter crescens was determined. A dramatic decrease in viability was shown after L. crescens was exposed to 10 minutes of 46 C. For objective 2, we have conducted a comparison study between field heat-treated and non-heat-treated citrus plants. There were 31 consistent up-regulated genes and 47 down-regulated genes in the citrus trees treated with heating. Additionally, potted Las-positive and negative citrus was exposed to 4 hours for 4 days of 40 C, 85 % relative humidity (similar to heat exposure in field setting) in a controlled greenhouse. RNA-Seq data was analyzed using DESeq2 with a FRD of 0.5 and fold change above 2. Using new tender flush as a sample, there were 3,722 differently expressed genes (DE) between Las-negative and positive trees not exposed to heat. Flush that appeared after heat treatment on the positive plants had 294 DE as compared to flush on unheated positive plants and 1308 DE as compare to flush on healthy trees. Some heat shock and oxidative proteins were identified in the DE lists. Analysis and confirmation are ongoing. As for the third objective, over 3 years of prior data (tree Las Ct values, treatment procedures, and temperature logs from one location) have been summarized and are currently being used to determine an algorithm that relates environmental conditions with decreases in Las titer. Extensive analysis of temperature and humidity data using KS nonparametric test, paired T Test, and other measures have shown that each HLB-affected tree responds uniquely to heat treatment. The greatest decrease in Las titer and overall duration of this decrease varies for each tree (6-18 months) and is not solely dependent on heat but most likely affected by the biology of the tree. When comparing 7 versus 9 days of treatment, the longer treatment did not increase titer reduction. Also, six days was not more effective than a 4 day treatment. Regarding temperature, the greatest effect was present at 40, 41, and 42 C for 5 to 7 hours for 3 out of 4 test plots. A detailed correlation chart indicates other combinations of temperature and durations can also be effective at reducing Las. All statistical analysis shows that the response to heat stress is unique for each Las-infected tree. Although fruit drop did decrease for many of the treated trees, due to the large variation in data, the decrease was not statistically significant. Juice quality taste panels have just been completed. Panelists could tell the difference between juice from commercial Valencia trees that were heated and not heated with a 95-99% confidence interval. Further analysis of taste panel data is ongoing. Volatile production analysis for one group of data (out of three) has been finished. A total of 63 aromatic volatile compounds were detected by HS-SPME-GC-MS. Discriminant analysis separated the “no heat” juice from the “heat” juice. Juice made from the product of the heat-treated trees had “fruity” and “pineapple fruit” top-notes.
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). Tailocins XT-1 and XT-4 exhibit broad host 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 51% , as compared to non-tailocin challenge plants inoculated only with Xac. We have previously reported on the identification of the cassette encoding for tailocin XT-1.We now report that the tailocin cassette is 16,880 bps and encodes for 22 ORFs. We have identified genes encoding for the tail tube, tail sheath, tail fiber, baseplate assembly and tail tape measure proteins. Further analysis of the XT-1 cassette will identify all genes and allow for an initial bioinformatic comparison of tailocins XT-1, XT-4 and XT-7. The newly identified XT-7 tailocin shows activity against 10/13 Xac isolates tested including the Xac 306 strain. The type IV pilus dependency of Xac phages CCP504 (podophage), CCP513 (siphophage) and CCP519 (myophage) was confirmed using a pilA gene deletion Xac mutant. The deletion mutant exhibited no twitching motility and was resistant to all three phages, whereas the in trans complement exhibited twitching motility and was sensitive to the three phages.
The focus of the project is to develop a bacteriophage (phage) and/or phage components (tailocins) system with activity against Liberibacter crescens strain BT-1 that will be a model for Candidatus Liberibacter asiaticus . Strain BT-1 harbors two prophages (LC1 and LC2) that we have determined are not inducible when the organism is exposed to UV or oxidative stress, which may indicate that the prophages are defective. Both of the phages are predicted to be podophages (i.e., no separate tail structure). Multiple constructs are being produced to determine the correct fusion that will produce a mature and active tailocin with activity against L. crescens. The assay system we have developed for L. crescens will be used for testing activity. Our goal is to construct tailocins with specific targeting to L. crescens by changing the C-terminal portion of the tail fiber. As a method to determine validity of the approach, we have constructed an unmarked and in-frame deletion mutant of the tail fibers of tailocin Bcep0425 and complemented in-trans with the homologous tail fiber and chaperone genes. We then constructed a series N-terminal tail fiber region derivatives (i.e. 153aa, 183aa and 243aa) of tailocin Bcep0425. When the N-terminal regions were joined with the heterologous C-terminal tail fiber regions of a known prophage we observed no activity against the new target. This is not surprising since the tail fiber structure is an elongated homo-trimers with a fibrous morphology and .-sheet topologies with unusual repetitive folds such as a triple .-helix, so it is necessary to identify the correct fusion. We will continue to make new constructs to identify the correct fusion. We are also continuing to search for naturally occurring phages of L. crescens. Rhizobium spp., Agrobacterium spp. and L. crescens BT-1 were used as hosts for enrichment with weed extracts, water and soil samples. No phages were detected for BT-1, however Rhizobium phages were isolated but showed no activity against L. crescens.
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