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. During the recently completed quarter (April 1 to June 30, 2015), the following highlights occurred at the Mid Florida Citrus Foundation A.H. Krezdorn Research Grove: Plant Improvement Team o Continue planting new NVDMC trees o Maintaining pomegranates in the North Block for Dr. Castle Deciduous Block o Production practices in progress on Evans Properties Mechanical Harvest study Dr. Futch evaluations: o Continued evaluations of trifoliate rootstocks for HLB tolerance o Continue evaluating two new Dow herbicide trials Conducted spring fertilizer and pest management programs for the groves o Herbicide program on schedule o Psyllid management continued . Participated in coordinated area wide spray (CHMA) in early May Continue to maintain Dr. Bowman s new USDA rootstock plantings Commercial Trials: o Eurofins evaluations on disease and insect management continue . Preparations for new plantings o Florida Ag Solutions continue to make applications to herbicide and insecticide evaluations (5) Drs. Stelinski has established some new studies in the New Block Drs. Albrigo and Wong have continued to evaluate antibiotics to manage HLB
Sequence analysis and comparison of pathogen genomes can provide valuable insights into epidemiological spread, sources of phenotypic variation, and the genetic bases for pathogen survival and interaction with host plant and insect vector. A JBrowse based genome viewer (http://hlbws.sgn.cornell.edu/jbrowse/JBrowse-1.11.4/) was installed on the CG-HLB Genome Resources Website (http://www.citrusgreening.org/) to provide end users with a smoother interface for viewing seven Liberibacter genome sequences and to enhance user ability to visualize different categories of single nucleotide polymorphisms (SNPs). SNP locations can provide a basis for various analyses including strain and isolate tracking, and identification of sources of phenotypic difference. This is particularly valuable information for experimentally challenging organisms such as Liberibacter. Four sequences of CLas are now available including the reference strain CLas psy62, CLas gxpsy, CLas HHCA and CLas A4. Owing to the cessation of funding from CRDF, hosting of the the JBrowse genome viewer, and associated analyses of seven Liberibacter genome sequences has been transferred to servers at the Boyce Thompson Institute in Ithaca NY. The domain name citrusgreening.org has also been transferred. The research group of Lukas Mueller has received funding to host genome resources for citrus, psyllid vectors, and Liberibacter asiaticus strains. Resources and analyses generated by the Lindeberg program with CRDF funding will provide a nucleus for this effort. While the new site at http://www.citrusgreening.org/ is under development, the archived site can still be accessed at http://hlbws.sgn.cornell.edu/archive/.
The goal of this project is to find non-copper treatment options to control citrus canker, caused by Xanthomonas citri ssp. citri (Xcc). Currently, sprays with copper bactericides are the primary mean in controlling citrus canker, which causes adverse effects on the environment by contaminating ground water or accumulating in the soil and affecting tree health. 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 via its 17.5 tandem repeats, 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 submitted to Plant Biotechnology for publication consideration. 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.
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. We have identified genes involved in antimicrobial biosynthesis, e.g., phlD, prnC, phz. 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.
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. 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. We are testing their inhibitory effect against purified SecA right now. 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, 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. Using the optimized formulation, two compounds are being tested in the field trial to determine the effective dose and application frequency.
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. Under room temperature, after a 20 month storage, the population reduced about 10E5 to 10E6 folds. The field trials for estimation for bacterial activity improving the health of HLB diseased trees continued as scheduled. We completed 10 applications through soil drench in the two groves at LAKE WALES: 07/2013, 09/2013, 11/2013, 01/2014, 03/2014, 05/2014, 07/2014, 09/2014 11/2014 and 02/2015 (every 2-3 months); conducted 5 surveys of HLB disease severity and collected leaf samples for Las population analysis using qPCR (03/2013, 09/2013, 03/2014, 09/2014 and 03/2015 (every 6 months)). The data collected suggested that after 10 applications (19 months after 1st application), the three bacteria treatment showed a reduced HLB disease severity with lower values of standard area under the disease progress stairs (sAUDPS) and Las bacterial populations in leaf samples as compared with the non-treated control in one trial that contains relative younger trees with mild HLB symptoms when the experiment was initiated. We will collect data for four trials. A manuscript is in preparation to summarize all the data.
The goal of this study is to understand the role of biofilm formation and quorum sensing (QS) in X. citri ssp. citri infection of citrus fruit and to prevent its infection by interfering with biofilm formation and QS. Three compounds exhibited a significant reduction in biofilm formation both on polystyrene surface and in glass tubes compared to the untreated control, where the level of biofilm formation were reduced to 50% and 60% of control, respectively. Plant test in greenhouse showed that treatment with the three compounds prior to infection could reduce biofilm formation of Xac on leaf surface, reduce the formation of canker lesions on spray-inoculated grapefruit leaves with the wild-type strain. Effects of the three compounds on Xac on detached immature citrus fruit were also tested using spray inoculation. Preliminary results showed that these small molecules affected Xac 306 infection of unwounded and wounded citrus fruits at sub-inhibitory concentrations. We have completed testing the effect of those compounds in different combinations with copper based bactericides in controlling Xac infection of grapefruit plants in the greenhouse. The sensitivity of biofilm and planktonic cells of Xac 306 to copper (copper sulfate) were evaluated by measuring the MICs. Biofilms are less susceptible to copper than planktonic cells. Effect of the selected compounds on sensitivity of Xac planktonic cells and biofilm cells to copper sulfate was also investigated. In the NB medium, planktonic cells exhibited a MIC of 0.50 mM CuSO4 without biofilm inhibitor. In the presence biofilm inhibitors at sub-MIC concentrations , the MICs of CuSO4 against Xac 306 planktonic cells were decreased to 0.25 mM. In a in vitro biofilm system test, the combined use of copper sulfate and the compounds individual or both resulted in significantly increased killing compared to killing by copper sulfate alone. The results have been published by Phytopathology in a manuscript entitled: Foliar application of biofilm formation-inhibiting compounds enhances control of citrus canker caused by Xanthomonas citri subsp. citri. One patent is filed based on the results. We also identified multiple new biofilm inhibitors. The effect of those biofilm inhibitors to control citrus canker is being investigated. We tested the survival of both biofilm deficient and QS mutants on fruit surface. Effects of biofilm formation inhibitors on Xac infection on detached immature citrus fruit were tested using spray inoculation. The inhibitors affected the infection of Xac on both unwounded and wounded citrus fruits. We continue characterizing how quorum sensing and biofilm formation contribute to Xac infection of citrus fruit. Multiple virulence genes involved in quorum sensing and biofilm formation are being investigated. The involvement of ColR, RpfF, and three more genes involved in biofilm formation or quorum sensing is studied in details. We compared the gene expression of Xac RfpF mutant and wild type in planta. Two manuscripts are under preparation. We also identified multiple quorum sensing inhibitors which showed positive effect in controlling citrus canker. The field trial (two different sites) is ongoing to test the effect of the identified biofilm inhibitors to control citrus canker. In the MidFlorida trial, the data collected from the 1st year experiment showed that the application of biofilm inhibitors and quorum sensing inhibitors, individually or in combination, reduced canker disease incidence on leaves and fruit, with lower values of the standard area under the disease progress stairs (sAUDPS) and lower percent of diseased fruit and fruit drop. For the 2nd year experiment, the canker incidence in the MidFlorida grove before treatment application was surveyed and recorded on March 19, 2015. It is expected the experiment will end at the end of 2015.
The goal 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. A phage disulfide-constrained heptapeptide library (Ph.D.-C7C; New England Biolabs) was screened and ACP gut binding peptides isolated as described in the previous report. Four peptide sequences have been cloned fused to mCherry and expressed using the expression vector pBAD. These four fusion peptides have been purified using nickel affinity columns. The fusion peptides were used in pull down assays with ACP brush border membrane vesicles (BBMV) confirming in vitro binding of all four peptides to ACP gut proteins. A negative control peptide SG2, did not bind to ACP BBMV proteins in these assays. Ligand blot analysis showed that the fusion peptides primarily bind to 37kDa and 25kDa BBMV proteins. The negative control, mCherry showed low non specific binding to a single abundant 48kDa protein. In vivo analysis of fusion peptide binding to the psyllid gut is underway for selection of the peptide to be used for modification of a selected Bt-derived toxin against ACP.
The propagation bay was handed over to MREC on 8 April 2015. This was comprised of 12 – 21×4 ft fixed benches, with environmental controls and electrical outlets in place. In anticipation of receiving the bay, construction of 16 tented structures measuring 5 x 4 ft were fabricated from half inch pvc pipe and fittings in the lab. Temperature/ humidity sensors were ordered in early April. Irrigation stub-ups and electrical wires for controlling solenoid valves were installed in mid-March. Irrigation valves and manifolds were fabricated the second week of April. To date, 3 benches have been plumbed. Samples of Elle pots and similar tubes have been ordered, with the Elle pot samples received. First cuttings will likely be started around the first of June, using the Kuharske cultivar to fine tune the system. For the research into sterilizing seed, Dr. Gul Ali has this to report. 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 were obtained from a local nursery grower, and plated on synthetic media plates to check for any bacterial contamination including Xanthomonas spp. No bacterial colonies appeared on the plates after at least 2 months of culture. Upon storage at either room temperature or refrigerators, however seeds got heavily infested by a non-pathogenic fungus, most likely a Penicillium spp. To find out the optimal concentration of sodium hypochlorite and Plant Preservative Mixture (PPM) that results in the least seed lethality (no germination), seeds were treated with five increasing concentrations of sodium hypochlorite (0, 10, 20, 30, 40 and 50%) or PPM (0, 1,2, 3, 6 and 10 %), and plated on seed germination medium. Each treatment consisted of three replication each consisting of 20 seeds. Seeds were observed for any contamination every day for the first two weeks and then every week for the next three months. All non-treated controls displayed fungal growth after 3 days of incubation on plates, but no bacterial colonies. None of the seeds germinated for up to 1 month. Percent seed germinations were 0, 33, 7, 40, 2 and 35% for 0,10, 20, 30, 40 and 50% sodium hypochlorite, and 0, 15, 18, 12 13, and 3% for 0, 1,2, 3, 6 and 10 % PPM, respectively. Part 2. Evaluating the 2006 CHRP decontamination procedure. Progress: These experiments will initiated in the next phase. Part 3. Development of rapid and sensitive molecular diagnostic methods for on-site detection of Citrus canker. Progress: Primers have been designed for several genes for PCR and LAMP. Citrus leaves displaying symptoms of citrus canker were collected from an experimental grove at the Southwest Research and Education Center at Immokalee. Leaf segments showing symtoms were were surface sterilized and plated on media in petri dishes. Characteristic colonies of Xanthomonas developed on these plates. Individual bacterial colonies were recovered and stored at -80 oC. DNA from several independent colonies has been isolated, and we are in the process of accurately identifying the strains. In addition we are in the process of obtaining additional isolates of various Xanthomonas spp.
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, and they are under evaluation of transgene expression and the host response to the transgene. 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. 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 interaction between LasA1 and the putative host receptor identified is 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 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 is 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. This initial data indicates that longer durations of temperatures greater than 41-42’C is more effective with reduction of Las titer. Although, leaving the tent on the tree longer than a week to achieve this duration does not appear to improve thermotherapy results. Eighteen months samples are being analyzed. Extensive analysis of temperature and humidity data is being analyzed to identify why results at plot 2 do not fit the trend usually observed. Despite increasing titers, trees continue to grow well. By April 17th, Valencia will be harvested from these three plots and juiced. Fruit drop (based off of two marked branches) will be determined. Brix, chemical analyses, and a taste panel are in the process.
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 evaluation of phage cocktails in cooperation with Dr. Nian Wang (University of Florida-Lake Alfred). In two independent experiments, when phage cocktails were applied pre- or post-inoculation with Xac, reduced canker lesion numbers were observed, as compared to the Xac only inoculated plants. The most effective treatment was observed using a multiplicity of infection of 20. Similar results were obtained with a tailocin cocktail treatment, composed of tailocins XT-1 and XT-4 using a 17:1 (tailocin killing units:Xac) application. Our results indicate that both phage and tailocin cocktails are viable candidates for treatments that can reduce canker development in citrus plants. We have initiated studies to identify genes encoding for tailocin XT-1. SDS-PAGE analysis of tailocin XT-1 revealed major protein bands of approximately of 42 kDa and 18 kDa that were putatively identified as the tail sheath and tail tube proteins, respectively, based on molecular mass. Additionally, minor protein bands of 100 kDa and 30 kDa were putatively identified as the tail fiber and tail fiber assembly proteins, respectively. The two major protein bands were applied to Liquid Chromatography-Mass Spectrometry (LC-MS/MS) for analysis. Scaffold was used to validate peptide and protein identifications. Analysis of the 42 kDa protein revealed 58% homology, at the amino acid level, to a tail sheath protein found in GenBank. The 18 kDa protein showed 74% homology to a phage major tube protein found in GenBank. We have assembled a draft genome of the XT-1 producing strain that has 61 contigs. We are currently in the process of identifying the genes encoding for XT-1 tail sheath and major tube proteins. To confirm the type IV dependency of Xac phages in our pool, experiments are ongoing to obtain a pilA gene deletion mutant in Xac. Since PilA is the major subunit of type IV pili and is required for pilus formation, the mutant should be devoid of the pili and lack twitching motility.
‘Candidatus Liberibacter asiaticus’, the causal agent of citrus greening, has not been successfully cultured. Our studies are focused on the development of a potential bacteriophage and/or bacteriophage components (tailocins) based control for ‘Candidatus Liberibacter asiaticus’ using the Liberibacter crescens model system. The overlay system developed in our laboratory has been used in a continued effort to identify, from environmental and microbial samples, bacteriophages, tailocins or compounds with activity against L. crescens. We have initiated a recombinant approach to produce tailocins with activity against L. crescens. Using a broad host range tailocin (Bcep0425), identified and characterized in our laboratory, we have completed proof of concept studies. To accomplish the experiments, it was first necessary to have a complete annotated genetic map of the tailocin that identified all functional proteins involved in the structure and assembly (chaperones) of the active tailocin. An allelic exchange system was used to construct an unmarked and in-frame deletion mutant in which the tail fibers and associated chaperones were excised. Confirmed deletion mutants were then complemented in-trans with homologous tail fiber and chaperone genes to obtain active tailocin. Induced cultures of complemented deletion mutants produced lysates of the tailocin with similar titer activity as the wild type, indicating that assembly of an active tailocin was accomplished by in-trans complementation of the tail fiber and related chaperone genes. Retargeting of the tailocin for activity against L. crescens will require that the N-terminal fragment of hybrid tailocin tail fiber be assembled to the Bcep0425 tail baseplate and that the C-terminal fragment of tail fiber from the BT-1 prophage(s) determine the adsorption specificity to L. crescens. Identification of protein junctions that result in the assembly of a functional hybrid tail fiber with adsorption specificity to L. crescens will require an array of constructs.
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. During the recently completed quarter (January 1 to March 31, 2015), the following highlights occurred at the Mid Florida Citrus Foundation ‘ A.H. Krezdorn Research Grove: ‘ Plant Improvement Team o Continue planting new NVDMC trees o Maintaining pomegranates in the North Block for Dr. Castle ‘ Evans Properties is interested in evaluation of harvest equipment in the Pecan Block ‘ Dr. Futch evaluations: o Continued evaluations of trifoliate rootstocks for HLB tolerance o Continue evaluating two new Dow herbicide trials ‘ Conducted spring fertilizer and pest management programs for the groves o Herbicide program on schedule o Psyllid management continued . Participated in coordinated area wide spray (CHMA) in early February ‘ Continue to maintain Dr. Bowman’s new USDA rootstock plantings o Harvest completed ‘ Commercial Trials: o Eurofins evaluations on disease and insect management continue . Preparations for new plantings o Florida Ag Solutions continue to make applications to herbicide and insecticide evaluations (5) ‘ 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 ‘ International scientists visited on various occasions
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. The application for the field trails have been completed as scheduled. Up to 12 applications have been done. Four compounds were shown to have positive effect on controlling HLB based on two year field test results. the Las titer in leaf samples were determined at multiple time points using qPCR assays. Lower Las titers were observed for some treatments. Yield data and fruit quality were also collected. The effect of different compounds on the area under the disease progress curve is investigated. Trunk injection was conducted for SA analogs and other compounds. Compared to spray, the two initial field trials indicate that trunk injection of SA analogs and several other compounds have significantly improved the control efficacy. This result indicate that we can improve the control effect by optimizing the application method. We are currently conducting field trial to test this hypothesis. We are also testing the mechanism of those compounds showing positive effect on HLB control. Our results indicated that those compounds upregulated defense related genes, e.g., PR2, but not PP2 an callose synthase genes, in both field and greenhouse. Las titers were also lower in treated plants than non-treated controls with the four compounds. SA hydroxylase is being expressed and purified. Multiple SA hydroxylase inhibitors have been identified using molecular modeling. The inhibitory effect of the SA hydroxylase inhibitors have been tested using the purified SA hydroxylase. Three compounds showed inhibitory effect against SA hydroxylase. The three SA hydroxylase inhibitors will be used to control HLB in greenhouse or field trials. For the putative sec dependent effectors, we have identified some putative targets using yeast two hybrid assays and confirm the results using immunoprecipitation method. We are testing the possibility of generating HLB resistant plants by modifying the citrus aa residues involved in interaction with Las using Cas9/sgRNA.
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