The goal of the proposed study is to characterize the effect of using endophytic microbes in controlling HLB. Our hypothesis is the outcome of the interaction among Las, psyllid and citrus is affected by the citrus phytobiome. In order to achieve the goal of this study, the following objectives will be conducted: Objective 1. To characterize the phytobiomes and endophytic microbes from HLB survivor trees and HLB diseased trees. In this objective, we will investigate the phytobiomes and endophytes of survivor trees and HLB diseased trees. Metagenomic approaches will be used to investigate the microbiomes of survivor trees and HLB diseased trees. Metatranscriptomic approach will be employed to characterize the expression profile of the microbiomes of survivor trees and HLB diseased trees. We will culture the most representative or beneficial microbes based on metagenomics information. Objective 2. To illustrate whether the endophytic microbes from survivor trees could efficiently manage citrus HLB. In this objective, we will test whether the endophytic microbes affect citrus attractiveness to psyllids, psyllid feeding, and Las establishment in planta. The endophyte application will be through grafting roots or branches of survivor trees or using cultured representative or beneficial microbes from survivor trees. Grafting allows endophytes in survivor trees to be transmitted to testing trees directly without the culturing step and without risking the loss of microbes that are difficult to cultivate. We have extracted RNA and DNA from the rhizosphere, rhizoplane, and endosphere from healthy and HLB diseased trees. The samples have been sent out for sequencing analysis. We will have a better picture about the microbiome of citrus after analysis of the data.
Our hypothesis is that application of antibacterial-producing bacteria directly to citrus root could suppress Las population in the roots and control Las. Application of antibacterials in this manner will avoid the strict restrictions of application of antibiotics on crops and ease public concerns since those bacteria are naturally present in the soil and are associated with plant roots. In order to achieve the goal, the following objectives will be conducted: Test antibacterial-producing bacteria against Liberibacter crescens and other Rhizobiaceae bacteria which are closely related to Las. We will mainly test the antagonistic effect of Bacillus, Paenibacillus, Streptomyces and Pseudomonas strains Agrobacterium tumefaciens, Sinorhizobium meliloti, and L. cresens; Control HLB using antibacterial-producing bacteria. For the field test, we will investigate how antibacterial-producing bacteria affect HLB disease severity, Las titres, and citrus yield, survival of the antibacterial-producing bacteria in the rhizosphere and expression of the antibacterial biosynthesis genes in vivo. We have isolated Streptomyces spp. Bacillus spp. Paenibacillus spp., and Pseudomonas spp. from Florida groves. Multiple isolates showed antimicrobial production activity. We tested 27 antibacterial compound producing bacteria. These strains had been recovered, purified and confirmed by 16S rDNA sequencing. The antagonistic activity against Agrobacterium, Sionrhizobium meliloti and Xanthomonas citri pv. citri was determined. 5 strains, belonging to Paenibacillus, Burkholderia, Paenibacillus, Streptomyces and Streptomyces showed good antagonistic activity. Three bacteria showing high antimicrobial activities have been sequenced to help us understand the mechanism. Currently, the genome sequencing was finished and we are analyzing the results. Four bacterial strains: two Burkholderia, one Pseudomonas geniculata, one Rhodococcus strains have been tested for their activity in and all showed induced plant defenses and against infection by Xanthomonas citri. To further study the antimicrobial producing bacteria, tow Burkholderia strains have been labeled with GFP tag. Seven other strains are being labeled with GFP or RFP tag. We also investigated the antibiotic genes in nine antimicrobial producing bacteria that we isolated previously. These strains were inoculated to citrus roots and the colonization was determined by inoculation and recover method in lab condition using small citrus seedlings. Around 10E8 cfu were inoculated to each seedling. Approximately 10E4 cfu were recovered from roots 20 days after inoculation (dpi). In a separate experiment, two Burkholderia strains were tested and up to 10E5 cfu/g soil was recovered at five days post inoculation. For the field trial, we have selected the grove and conduct survey on HLB disease severity. We are comparing the different delivery methods to improve the efficacy of beneficial bacteria. A root drench delivery method has been established. This delivery method will apply bacteria close to the roots and reduce the loss during surface application. We continue to isolate and test the antimicrobial producing bacteria from Florida citrus groves. To understand the beneficial traits, we have sequenced multiple bacteria. The data is under analysis.
The goal of this project is to find non-copper treatment options to control citrus canker, caused by Xanthomonas citri ssp. citri (Xcc). The hypothesis of the proposed research is that we can control citrus canker by manipulating the effector binding element (EBE) of citrus susceptibility gene CsLOB1, which is indispensable for citrus canker development upon Xcc infection. We have previously identified that CsLOB1 is the citrus susceptibility gene to Xcc. The dominant pathogenicity gene pthA4 of Xcc encodes a transcription activator-like (TAL) effector which recognizes the EBE in the promoter of CsLOB1 gene, induces gene expression of CsLOB1 and causes citrus canker symptoms. To test whether we can successfully modify the EBE in the promoter region of CsLOB1 gene, we first used Xcc-facilitated agroinfiltration to modify the PthA4-binding site in CsLOB1 promoter via Cas9/sgRNA system. Positive results have been obtained from the Cas9/sgRNA construct, which was introduced into Duncan grapefruit. We analyzed the Cas9/sgRNA-transformed Duncan grapefruit. The PthA4-binding site in CsLOB1 promoter was modified as expected. Currently we are using both Cas9/sgRNA and TALEN methods to modify EBE in sweet orange using transgenic approach. TALEN targeting the promoter of CsLOB1 is also being done using citrus protoplast. Transgenic Duncan and Valencia transformed by Cas9/sgRNA has been established. Totally four transgenic Duncan grapefruit lines have been acquired and confirmed. Mutation rate for the type I CsLOB1 promoter is up to 82%. GUS reporter assay indicated mutation of the EBE of type I CsLOB1 promoter reduces its induction by Xac. The transgenic lines are being grafted to be used for test against citrus canker. In the presence of wild type Xcc, transgenic Duncan grapefruit developed canker symptoms 5 days post inoculation similarly as wild type. An artificially designed dTALE dCsLOB1.3, which specifically recognizes Type I CsLOBP, but not mutated Type I CsLOBP and Type II CsLOBP, was developed to evaluate whether canker symptoms, elicited by Xcc.pthA4:dCsLOB1.3, could be alleviated on Duncan transformants. Both #D18 and #D22 could resist against Xcc.pthA4:dCsLOB1.3, but not wild type Xcc. Our data suggest that activation of a single allele of susceptibility gene CsLOB1 by Xcc-derived PthA4 is enough to induce citrus canker disease and mutation of both alleles of CsLOB1, given that they could not be recognized by PthA4, is required to generate citrus canker resistant plants. The data has been accepted for publication by Plant Biotechnology Journal Transgenic Valencia transformed by Cas9/sgRNA has been established in our lab. Three transformants have been verified by PCR. The PthA4-binding site in CsLOB1 promoter was modified as expected, only one transgenic line seems to be bi-allelic mutant. The EBE modifed transgenic line is being evaluated for resistance against Xac. Currently, we are constructing different sgRNA sequences to target CsLOB1. We are continuing to generate more transgenic lines to get biallelic mutations in the EBE region of the CsLOB1 gene. We have sequenced the EBE regions of several commercial varieties to design appropriate sgRNA for gene editing. We are improving the gene editing efficacy by improving the Cas9/sgRNA constructs. In addition, we are studying the function of the susceptibility gene CsLOB1 and its downstream genes.
The goal of this project is to develop management strategies which boost natural defense mechanisms to control Huanglongbing (HLB) disease by counteracting salicylic acid (SA) hydroxylase of Ca. Liberibacter asiaticus (Las). Our previous study indicate that Las contains a functional SA hydroxylase that degrades SA and its derivatives. SA and its derivatives play important roles in plant defenses. Las employs SA hydroxylase to suppress plant defenses. Our central hypothesis is that we can improve HLB management by counteracting SA hydroxylase. We will focus on counteracting SA hydroxylase using inhibitors based on structure based design. The SA hydroxylase protein is being expressed in E.coli and purified. Several inhibitors identified using structure based design will be tested for their inhibitory effect against SA hydroxyalse.
The goal of the proposed study is to characterize the effect of using endophytic microbes in controlling HLB. Our hypothesis is the outcome of the interaction among Las, psyllid and citrus is affected by the citrus phytobiome. In order to achieve the goal of this study, the following objectives will be conducted: Objective 1. To characterize the phytobiomes and endophytic microbes from HLB survivor trees and HLB diseased trees. In this objective, we will investigate the phytobiomes and endophytes of survivor trees and HLB diseased trees. Metagenomic approaches will be used to investigate the microbiomes of survivor trees and HLB diseased trees. Metatranscriptomic approach will be employed to characterize the expression profile of the microbiomes of survivor trees and HLB diseased trees. We will culture the most representative or beneficial microbes based on metagenomics information. Objective 2. To illustrate whether the endophytic microbes from survivor trees could efficiently manage citrus HLB. In this objective, we will test whether the endophytic microbes affect citrus attractiveness to psyllids, psyllid feeding, and Las establishment in planta. The endophyte application will be through grafting roots or branches of survivor trees or using cultured representative or beneficial microbes from survivor trees. Grafting allows endophytes in survivor trees to be transmitted to testing trees directly without the culturing step and without risking the loss of microbes that are difficult to cultivate. We have extracted RNA and DNA from the rhizosphere, rhizoplane, and endosphere from healthy and HLB diseased trees. The samples have been sent out for sequencing analysis. We will have a better picture about the microbiome of citrus after analysis of the data.
Our hypothesis is that application of antibacterial-producing bacteria directly to citrus root could suppress Las population in the roots and control Las. Application of antibacterials in this manner will avoid the strict restrictions of application of antibiotics on crops and ease public concerns since those bacteria are naturally present in the soil and are associated with plant roots. In order to achieve the goal, the following objectives will be conducted: Test antibacterial-producing bacteria against Liberibacter crescens and other Rhizobiaceae bacteria which are closely related to Las. We will mainly test the antagonistic effect of Bacillus, Paenibacillus, Streptomyces and Pseudomonas strains Agrobacterium tumefaciens, Sinorhizobium meliloti, and L. cresens; Control HLB using antibacterial-producing bacteria. For the field test, we will investigate how antibacterial-producing bacteria affect HLB disease severity, Las titres, and citrus yield, survival of the antibacterial-producing bacteria in the rhizosphere and expression of the antibacterial biosynthesis genes in vivo. We have isolated Streptomyces spp. Bacillus spp. Paenibacillus spp., and Pseudomonas spp. from Florida groves. Multiple isolates showed antimicrobial production activity. We tested 27 antibacterial compound producing bacteria. These strains had been recovered, purified and confirmed by 16S rDNA sequencing. The antagonistic activity against Agrobacterium, Sionrhizobium meliloti and Xanthomonas citri pv. citri was determined. 5 strains, belonging to Paenibacillus, Burkholderia, Paenibacillus, Streptomyces and Streptomyces showed good antagonistic activity. Three bacteria showing high antimicrobial activities have been sequenced to help us understand the mechanism. Currently, the genome sequencing was finished and we are analyzing the results. Four bacterial strains: two Burkholderia, one Pseudomonas geniculata, one Rhodococcus strains have been tested for their activity in and all showed induced plant defenses and against infection by Xanthomonas citri. To further study the antimicrobial producing bacteria, tow Burkholderia strains have been labeled with GFP tag. Seven other strains are being labeled with GFP or RFP tag. We also investigated the antibiotic genes in nine antimicrobial producing bacteria that we isolated previously. These strains were inoculated to citrus roots and the colonization was determined by inoculation and recover method in lab condition using small citrus seedlings. Around 10E8 cfu were inoculated to each seedling. Approximately 10E4 cfu were recovered from roots 20 days after inoculation (dpi). In a separate experiment, two Burkholderia strains were tested and up to 10E5 cfu/g soil was recovered at five days post inoculation. For the field trial, we have selected the grove and conduct survey on HLB disease severity. We are comparing the different delivery methods to improve the efficacy of beneficial bacteria. A root drench delivery method has been established. This delivery method will apply bacteria close to the roots and reduce the loss during surface application. We continue to isolate and test the antimicrobial producing bacteria from Florida citrus groves. To understand the beneficial traits, we have sequenced multiple bacteria. The data is under analysis.
Fruit and vegetative tissue collected and prepared during the first year of experimentation continued to be analyzed under SEM. A notable and consistent observation has been that xylem tissue of HLB affected trees appears severely occluded, with the walls of vessels and fibers displaying abnormal thickening. In fact, in some samples, the xylem tissue appeared to form a semi solid wall. Following this observation, we started a detailed study to investigate this anomaly that is likely affecting water transport in HLB-affected trees. Measurements of fruit size and peduncle diameter were taken as part of the first greenhouse experiments with SL. Trees treated with SL remain decisively healthier and retained fruit compared to untreated HLB trees. Most of the last quarter was dedicated to microscopy with additional time allotted to prepare for the second year experiments and to write manuscripts. Furthermore, results were presented in a scientific meeting. We are preparing plants in the greenhouse for the new cycle of experiments and to repeat our previous experiments. Following is the list of experiments to be started in October-2015. I: Effect of drenching application of SL on HLB-infected trees. II: Effect of spray application of SL on HLB-infected trees (Repeat experiment). III: Effect of SL + Fungicides on Phytophthora growth in HLB-infected trees. IV: Effect of spray application of SL on other varieties of citrus in groves. V: Effect of spray application of SL + other promising compounds on citrus in groves. Prior to start the start the second set of experiments, irrigation lines have been established and most trees repotted, fertilized and prepared for treatment.
Our earlier report (Mar-2015) demonstrated the induction of new flush, flowering, reduction in fruit drop, and mobilization of starch in stem tissue of HLB-infected trees following the spray application of 10 M strigolactone (SL) in greenhouse trees. Further investigation on the ultrastructural anatomy of citrus fibrous roots revealed that roots are infected very early as evidenced by the deterioration of phloem tissue in young tertiary roots. We classified the fibrous roots as primary, secondary, and tertiary roots. Symptoms in HLB-infected primary and secondary fibrous roots are uncertain as in the case of foliage tissue e.g., vascular bundles of the same tissue showed normal functional phloem while others showed blocked and disintegrated phloem. Similarly, accumulation of starch in root cortical tissue is uncertain. Within the same tree, some roots samples showed accumulation of starch while other are completely devoid of starch accumulation. SL application improved the storage of starch in root cortical tissue and generated more functional phloem in HLB-infected trees. SL treated plants showed more roots growth. Roots appear healthy and enriched with new young roots. HLB-infected roots appeared small and dark brown in color. SL application significantly delayed fruit abscission in green house grown trees. At this time, fruits are larger in diameter and remained intact on trees.
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 have identified multiple small molecular ‘or’ peptide inhibitors against LipidA using pharmacophore based methods and are finalizing the list of the compounds for the activity studies. Six lipid A inhibitors have been ordered and are being tested against Las and its relatives. Our preliminary data showed that the lipid A inhibitors have excellent antimicrobial activities against L. crescens and other Las relatives. One manuscript on lipid A inhibitor is being prepared. For SecA inhibitors, we are optimizing the compounds in collaboration with IBM. Two compounds with slightly higher binding affinity than C16 were identified. We also identified multiple SecA inhibitors. The antimicrobial activities of the newly identified SecA inhibitors have been tested. 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 SecA inhibitors without phytotoxicity. Using the formulation, we have tested all five compounds against eight different bacterial species including Liberibacter crescens, E.coli, Agrobacterium, and Sinorhizobium. The formulation has significantly improved the antimicrobial effects of SecA inhibitors, comparable to streptomycin. The data has been summarized in one manuscript being submitted. Overall, this project generated multiple antimicrobials with potential against Las, which were summarized in two manusscripts. One patent has been filed for SecA inhibitors.
The objective of this project is first to identify a Bacillus thuringiensis (Bt) crystal toxin with basal toxicity against Asian citrus psyllid (ACP). The toxicity of the selected toxin will then be enhanced by addition of a peptide that binds to the gut of ACP. This peptide addition to the toxin is expected to enhance both binding and toxicity against ACP. To identify a Bacillus thuringiensis strain with basal toxicity against ACP, bioassays were conducted with solubilized and activated toxins derived from 46 different Bt isolates. Six isolates showed promise with statistically significant ACP mortality at 500ug/ml relative to control treatments. One strain was selected for further identification of individual toxins by MS/MS analysis and the three primary toxins expressed by this strain identified. Additional bioassays are underway to select the toxin with the greatest toxicity against ACP for modification with ACP gut binding peptides. Four ACP gut binding peptides were isolated and the peptide sequences cloned for production of mCherry fusion proteins as described in previous reports. Binding of these fusion peptides to gut proteins was confirmed by pull down analysis with ACP brush border membrane vesicles (BBMV). Two negative control proteins, mCherry alone and a random peptide-mCherry fusion were included as negative controls for binding to ACP BBMV. Replication of 2D gel electrophoresis of BBMV proteins coupled with ligand blot analysis (1 to 3 replicates per peptide-fusion protein) showed that the four peptide-mCherry fusion proteins bind 50kDa, 37kDa and 25kDa proteins all at pI ~9. As negative control, mCherry showed non specific binding to an abundant 50kDa protein, pI ~5. In vivo binding of the fusion peptides and mCherry negative control using fluorescence analysis indicated that fusion proteins with peptides 15 and 18 bind the ACP gut. These two peptides represent promising candidates for use in Bt toxin modification.
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. Although we previously reported that LasAI and LasAII target host mitochondria, we had to co-inoculate the gene silencing suppressor P19 to have a detectable expression of LasAI and LasAII (PLoS ONE8:e68921, 2013). After optimization for a variety of parameters that are critical for efficient gene expression in plants, high expression level of LasAI/LasAII were detected without co-inoculation of P19. 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. These transgenic plants are under evaluation of transgene expression and the host response to the transgene. Using RNAseq, 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. The interactions between LasA1 and the putative host receptors identified are also characterized using the yeast two hybrid systems.
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. 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 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 data is still being determined.
Installation of all pluming, i.e. valves, wires, misters and foggers were completed. A 24 station mist clock was installed and connected to valves for mist control for rooting cutting in mid-May. Tissue culture hardening off A datalogger control system was fabricated and installed. Temperature/humidity sensors were connected to the control system and tested for the 12 tented structures without bottom heat. Layers of shade cloth were insufficient in preventing excessive heat in the tented structures. This was remedied by addition of R5 insulation roofs over the shade cloth. The second set of tissue culture plantlets were placed in tented structures on 7 July. Four humidity reduction procedure are being applied. Once humidity levels are declined to 50% and held there for 4 days, tents will be open, then the insulation roof removed and later the shade cloth layers will be removed Rooting cuttings All cutting research was conducted using Kuharske rootstock. Several different experimental combination were initiated in late May. These consisted of 1, 2 and 3 node cutting, stem position, different auxin concentration and different auxin carriers. Greatest new shoot growth occurred using 8000 ppm Dip & Grow on single node cutting taken 12 to 24 inches below the shoot terminal. Greatest root dry mass consistently occurred with the 12 to 24 inch section of a stem. Interestingly all cutting produced viable root system by 6 weeks, most had self-sustaining roots by 3 weeks. Other data is still being analyzed. Dr. Gul Ali s report on canker elimination on seeds. 1. Develop guidelines for seed propagation that prevent contamination of seed by citrus canker. Part 1. Surface disinfection of seeds. The objective is to develop economical and easy-to-implement seed cleaning methods for removing Xcc bacteria from seeds without loss of seed viability. Progress: Seeds obtained from a local nursery grower were contaminated with Penecillium, which will interfere with our assays using bacterial canker bacteria. We have also isolated seeds from a citrus trees growing in the wild. Fruit and seeds from these trees are also contaminated non-pathogenic fungi. To get around this issue we are reevaluating our experimental strategy and we will most likely remove seed coats from seeds to avoid contamination with non-specific fungi. Part 2. Evaluating the 2006 CHRP decontamination procedure. Progress: We are waiting on getting enough seeds from a local grower. These experiments will be done concurrently with experiments using sodium hypochlorite and PPM for decontamination. Part 3. Development of rapid and sensitive molecular diagnostic methods for on-site detection of Citrus canker. Progress: In order to find several genes that are unique to each of the A, A* and AW strains, genomes of 24 strains representing each of these strains were downloaded from the National Center for Biotechnology Information (NCBI) website. Local BLAST databases were generated for each of the three strains using the CLC genomic software. Initial bioinformatics analyses indicated that at least twenty different genes are unique to each strain groups. These genes are currently being assessed for their suitability for LAMP assays. DNA of type A strain was easy to obtain from locally available Xcc strains. However, DNA of A* and AW requires collaboration with a USDA laboratory for testing our LAMP assays. We are in the process of obtaining DNA of several representative strains with our collaborators at UF.
The research program objectives are to develop an effective and sustainable phage-based biocontrol system for Xanthomonas axonopodis pv. citri (Xac), the causal agent of citrus canker. We have developed both virulent phages and antibacterial particles called tailocins , which are derived from phages that can kill Xac. Phage particles carry their own DNA, so that each phage infection not only kills the target bacterial cell but generates hundreds of progeny particles. Tailocins do not carry DNA and thus each tailocin infection kills one bacterial cell functioning as injectisomes that kill bacteria – one hit one kill. Our greenhouse trials indicate that both are viable candidates for application to reduce canker symptoms. Using Liquid Chromatography- tandem Mass Spectrometry (LC-MS/MS) analysis of the tail sheath and tail tube proteins, and bioinformatics we have identified the cassette encoding for tailocin XT-1 and are in the process of annotating the sequence to identify all components. Information obtained from analysis of XT-1 will be used as a scaffold to characterize other identified tailocins. We have identified a new tailocin designated XT-7 that is currently being characterized for activity and host range. To confirm the type IV pilus dependency of Xac phages in our pool, we have constructed a pilA gene deletion mutant in Xac. Twitch motility and phage sensitivity of the mutant and complement are being conducted.
Liberibacter crescens has been cultured under laboratory conditions and is considered a model system that can be used to develop a biocontrol for Candidatus Liberibacter asiaticus (Las). The focus of our project is to develop a detection system for bacteriophage (phage) and/or phage components (tailocins) using L. crescens strain BT-1. Tailocins, unlike phages, do not carry DNA and do not replicate. Each tailocin adsorbs to and kills one bacterial cell, functioning as injectisomes that kill bacteria – one hit one kill. We are continuing to assay soil, water and plant samples for naturally occurring phages. In addition, as previously reported, we have initiated a recombinant approach to produce tailocins with activity against L. crescens. We have constructed an unmarked and in-frame deletion mutant of tail fibers of tailocin Bcep0425 and complemented in-trans with homologous tail fiber and chaperone genes. Our approach is to understand the junction of the N-terminal region of Bcep0425 that joins with the C-terminal fragment of tail fibers obtained from prophage(s) to form a functional tailocin. Information gained, will allow us to construct tailocins with adsorption specificity to target bacteria. We are currently constructing a series of hybrid complementing plasmids to identify the protein junctions that can assemble to form functional hybrid tailocins, using tail fibers from a known phage that has different host specificity to that of Bcep0425. The ultimate goal is to obtain functional retargeted tailocins with activity against L. crescens.
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