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. Next, we will use both Cas9/sgRNA and TALEN methods to modify EBE in sweet orange using transgenic approach.
The goal is to develop short term approaches to control citrus Huanglongbing (HLB) using antibacterial-producing bacteria. Recent studies indicate that HLB severely damaged citrus roots. The destructive effect of HLB on roots partly results from Candidatus Liberibacter asiaticus (Las) infection of the roots. In this study, we will conduct the following objectives: Screen bacteria that can produce antimicrobial compounds against Liberibacter crescens and related Rhizobiaceae bacteria; purify and characterize antimicrobial compounds produced by the screened bacteria; illustrate the regulation of antimicrobial production by producing bacteria at different environmental conditions and with different inducers; investigate the growth of the producing bacteria in different conditions; and control HLB by delivering antimicrobial compounds using the producing bacteria in the soil. We have isolated 84 strains of Streptomyces spp., Bacillus spp., Paenibacillus spp., and Pseudomonas spp. producing antibacterials from Florida citrus groves. We have also acquired 28 different species of Streptomyces spp., Bacillus spp., Paenibacillus spp., and Pseudomonas spp. strain producing different antibacterials. The antagonistic activity against Agrobacterium and Sionrhizobium meliloti was determined. The primary screen was done, and 5 strains showed good activity. The second round of antagonism (quantitative) is being performed to quantify the activity. These strains are being inoculated to citrus roots and the colonization is being determined by inoculation and recover method. These strains will also be labeled with GFP and the tissues (roots, branches and leaves) will be observed under florescent microscope to determine the colonization and movement of these strains. The effects of inoculation to the HLB infected plants are being determined.
The goal of the proposed study is to characterize the effect of application of beneficial bacteria (MICROBE Program) on management of HLB. Currently, we are setting up the experiments to test different Microbe Products in management of HLB. Assay for compatibility between isolates using antagonistic survival tests showed that all the selected beneficial bacteria are compatible with each other. Plant growth promoting activity of six selected isolates was evaluated using the model plant Arabidopsis grown in vitro. The results suggested that three isolates could promote plant growth. The plant growth promoting activity of these six isolates was tested using citrus (grapefruit) seedlings in greenhouse. Greenhouse assays suggested that a consortium of three Bacillus and relative isolates (AY16, PT6 and PT26A) may delay the development of both HLB symptoms and pathogen population on citrus leaves after root inoculation. The potential of the consortium to recover the tree decline from HLB infection is being evaluated in greenhouse. The growth conditions of the three strains were optimized using a small fermenter. Three antifoam agents, A204, PPG200 or M-Oil did not affect the growth of the three bacterial strains. The initial neutral to alkaline pH values (7.0 ~ 8.0) favor growth of the three bacteria in LB, while acidic pH (5.0 ~ 6.0) suppress bacterial growth. The optimal cultural temperature was determined to be around 30C with average bacterial population of 109-1010 cfu/ml after 20-hour incubation, although the bacteria may grow slowly under room temperature (~ 23C). The shelf life of three different formulations of the bacterial culture is being evaluated under room temperature. In a six-month time course, the bacterial populations in LB broth, OPB broth and tape water are comparatively stable with initial and final both at ~ 108cfu/ml. Four field trials are being conducted including more beneficial bacteria. For one of the field trial, nine applications have been performed. We are evaluating the survival of the beneficial bacteria in the soil. The application method has been changed during application to improve the survival of microbes in the soil. We are developing new methods to further increase the survival of microbes in the soil and allow the microbes have easy access to the roots. We are also testing the colonization and survival of the tested microbes on citrus roots and in rhizosphere soil in greenhouse using conventional culture-based method and qPCR-based method. We are evaluating plant immunity response by studying the expression of specific genes related to plant defense.
Management of phloem-limited bacterial diseases is very challenging. These bacteria employ unusual and sometimes unique strategies by which to optimize their niche occupation and obtain their nourishment from the host plant. Their location within the living (sieve tubes) plant cells, rather than in the intercellular spaces, offers different challenges and opportunities for them to avoid the host plant’s defense system. Phloem is also difficult for any bactericides to reach to control the pathogen population. Among the phloem-limited bacterial diseases, citrus Huanglongbing (HLB, greening) is one of the most devastating diseases. The current management strategy of HLB is to chemically control psyllids and scout for and remove infected trees. However, the current management practices have not been able to control HLB and stop spreading of Candidatus Liberibacter asiaticus (Las). The goal of the proposed study is to develop HLB management strategies which boost plant defense to protect citrus from HLB by exploiting the interaction between Las and citrus and understanding how Las manipulates plant defense. Recently, we compared the gene expression of PR1, PR2 and PR5 in healthy trees and Las infected citrus plants. The expression of PR1, PR2 and PR5 was significantly reduced in HLB diseased grapefruit as compared to healthy grapefruit after inoculation with Xac AW. We also tested whether infection by Las can make citrus more susceptible to infection by Xanthomonas citri subsp. citri. We also sprayed four times with different chemicals in 17 different combinations on citrus to test their effect in controlling HLB in one grove. Multiple compounds showed control effect. To further test those compounds, we have selected two more groves to expand the field test. The disease index of the two groves have been investigated and treatments already started. The follow up investigations are ongoing, including monitoring the HLB symptoms, disease incidence and Las titer in leaves. We compared the SA levels in HLB infected and healthy grapefruit after the inoculation with Xac AW. We also compared the SA levels in HLB infected and healthy Valencia citrus. We are continuing to evaluate the effect of different compounds on management of HLB both in greenhouse and in citrus grove. We have applied different compounds at three separate field trials. Four compounds were shown to have positive effect on controlling HLB based on two year field test results. We are also testing the mechanism of those compounds showing positive effect on HLB control. 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. We have investigated the effect of those compounds on disease severity, yield, juice content and quality. We will repeat those treatments for one more year. Currently, the treatments are being conducted. SA hydroxylase is being expressed and purified. New SA hydroxylase inhibitors are being identified using molecular modeling.
The goal of the research is to control citrus HLB using small molecules which target essential proteins of Candidatus Liberibacter asiaticus (Las). In our previous study, structure-based virtual screening has been used successfully to identify five lead antimicrobial compounds against Las by targeting SecA. SecA is one essential component of the Sec machinery. Those compounds showed promising antimicrobial activity. However, further work is needed to apply the compounds. We will evaluate the important characteristics of our antimicrobial compounds including solvents and adjuvants, phytotoxicity, antimicrobial activities against multiple Rhizobia, antimicrobial activity against Las, application approaches, and control of HLB. Those information are critical to for the practical application of those antimicrobial compounds in controlling HLB. We also propose to further optimize the five lead compounds. In addition, we propose to develop antimicrobial compounds against lipid A of Las. The lipid A substructure of the lipopolysaccharides (LPS) of Sinorhizobium meliloti, which is closely related to Las, suppresses the plant defense response. Las contains the complete genetic pathway for synthesis of lipid A. We hypothesized that Las uses lipid A to suppress plant defense. Thus, targeting lipid A could activate plant defense response. Lipid A is also an ideal target and has been targeted for screening antimicrobial compounds for multiple pathogenic bacteria. We are identifying the small molecular ‘or’ peptide inhibitors against LipidA using pharmacophore based methods and finalizing the list of the compounds for the activity studies. For SecA inhibitors, we are optimizing the compounds in collaboration with IBM. Two compounds with slightly higher binding affinity than C16 were identified. Currently, we are evaluating the best range of composition ratio among each component (%weight) of AIs, solvents and surfactants. The following characteristics are being evaluated: 1) emulsion stability and ease of emulsion; 2) stability of diluted concentrate; 3) freeze-thaw stability; and 4) phytotoxicity to citrus species. We have successfully identified one formulation suitable for all five compounds without phytotoxicity. Using the formulation, we have tested all five compounds against eight different bacterial species including Liberibacter crescens. Field test is being conducted. We have compared spray and trunk injection. Trunk injection seems to have better efficacy. We are synthesizing two compounds at large amount for field trial.
The routine leaf tissue analyses for this project are behind schedule because we were prevented from submitting samples to our regular analytical lab due to restrictions on the movement of plant materials and soil. These rules are being enforced by DPI as part of the citrus quarantine for Florida. Once the necessary permitting is in place, we will resume leaf nutrient analysis and reporting for this project.
Progress with the new ‘Ray Ruby’ grapefruit ACPS experiment at IRREC, Ft. Pierce: The overarching goal of this component is to develop ACPS and high-density plantings for commercial grapefruit on the east-coast. To date, the progress of this project is as follows: Irrigation installation was completed in October 2013. We are currently testing and evaluating the irrigation systems to fine-tune their performance and eliminate inefficiencies and any errors made during installation. The first experimental block of citrus was planted in November 2013. ‘Ray Ruby’ grapefruit trees on ‘Sour orange’ and ‘US-897’ rootstocks were planted on 8 acres on the citrus research grove at the UF-IFAS-IRREC station in Ft. Pierce. ‘Ray Ruby’ / ‘Sour orange’ trees were planted at a density of 152 trees/acre with microsprinklers and will be fertilized with granular, dry fertilizer: these plantings will serve as the ‘grower standard’ control treatment. ‘Ray Ruby’ trees on both ‘Sour orange’ and ‘US-897’ rootstocks were planted in staggered-set, tramline configurations at a high-density of 421 trees/acre: half of these plots will be fertigated with microsprinklers and half will be fertigated with in-line drip tubing. Funds from this project were also used to partially support the construction of a 5-acre high-density block of ‘Ray Ruby’ / ‘Kuharske’ grapefruit at the IRREC. In this block, all trees are being irrigated with microsprinklers but are planted at 3 different densities: 126, 189, and 421 trees/acre. Funds from other sources were used to cover the costs to complete this research block. These trees were also planted in November 2013. Data collection on tree growth, psyllid abundance, CLas presence began in December 2013. Leaf and soil nutrition monitoring for these blocks began in late May/early June 2014. We plan to hold a field day for stakeholders in late 2014. This field day will exhibit the two trials listed above. In addition, a tour of these plots is planned as part of the annual American Society for Horticultural Science national meeting in July 2014. A Ph.D. graduate student was recruited and hired through the UF-Horticultural Sciences Department to assist in executing this objective. Currently, this student is in Gainesville completing her required coursework. The student will be available to monitor the progress of the research blocks full-time starting in the summer of 2014.
Citrus canker is a devastating disease, caused by Xanthomonas axonopodis pv. citri (Xac). It is well established that the NPR1 gene plays a pivotal role in systemic acquired resistance (SAR) in Arabidopsis. We isolated and characterized an NPR1 homolog from citrus, namely Citrus NPR1 homolog 1 (CtNH1). Sequence alignment and phylogenetic analysis indicate that CtNH1 is closely-related to the Arabidopsis NPR1 gene and its orthologs from rice, grapevine, and cacao. CtNH1 was cloned into the binary vector pTLAB31 under the control of the strong figwort mosaic virus (FMV) promoter. The resultant construct was transformed into ‘Duncan’ grapefruit. We focused on the three transgenic lines, CtNH1-1, CtNH1-3, and CtNH1-5, that showed normal growth phenotypes, high levels of CtNH1 transcripts, and constitutive expression of the pathogenesis-related (PR) gene chitinase 1 (Chi1). The three transgenic lines were inoculated with Xac306 using both leaf infiltration and leaf spray methods. Disease symptoms were scored 14 days after inoculation. In the leaf infiltration experiments, the over-expression lines of CtNH1 developed significantly less severe canker symptoms as compared with the ‘Duncan’ grapefruit plants. Similar results were obtained when the plants were spray inoculated with Xac306. Consistent with the lesion development data, Xac population in CtNH1-1 plants is 104 fold lower than that in ‘Duncan’ grapefruit. These results indicate that overexpression of CtNH1 results in a high level of resistance to citrus canker. Microarray experiments were conducted using the transgenic line CtNH1-1 and ‘Duncan’ grapefruit inoculated with Xac306. A total of 451, 725, and 2144 genes were differentially expressed at 6, 48, and 120 hours post inoculation, respectively. These genes likely regulate transcription, protein degradation, post-translational modification, and the photosynthetic pathway. Much effort was devoted to determine whether CtNH1 confers resistance to citrus Huanglongbing (HLB). The well-characterized CtNH1 lines were graft-inoculated with budwood from HLB pathogen infected citrus trees at both Drs. Yongping Duan (USDA-Fort Pierce) and Nian Wang (UF-CREC)’s labs. Although five of the eight CtNH1 positive plants showed no disease symptoms (four plants) or mild symptoms (one plant), the other three CtNH1 positive plants were diseased. We carried out quantitative-PCR (qPCR) assays to detect the populations of Ca. Liberibacter in the infected plants and the results are largely consistent with the observations from disease assessments. Since there is no correlation between CtNH1 overexpression and reduced or absent HLB symptoms, our results indicate that CtNH1 confer no resistance to the disease. Publication: Chen et al. (2013). Overexpression of the citrus CtNH1 gene confers resistance to bacterial canker disease. Physiological and Molecular Plant Pathology 84: 115-122.
Mid Florida Citrus Foundation (MFCF) a 501c5 not for profit organization which has supported (past 25 years) and currently supports citrus research efforts of scientists from the University of Florida, USDA and private industry. The MFCF supports citrus research through the employment of a full time grove manager whom works closely with researchers to ensure that their projects are handled properly and that the grove is an excellent condition. The management of this grove requires extra financial commitment as grove care costs tend to be higher than commercial groves due to the nature of many of the research projects. Current projects being conducted at the MFCF are Asian citrus psyllid (ACP) pesticide evaluation control trials, low volume applicator trials, windbreak evaluation, HLB nutritional programs, new and existing herbicide trials, variety and rootstock evaluation trials. During the recently completed quarter (April 1 to June 30, 2014), the following highlights occurred at the Mid Florida Citrus Foundation ‘ A.H. Krezdorn Research Grove: ‘ Plant Improvement Team o Site prep for new planting of rootstock and scion cultivars being evaluated for HLB tolerance/resistance o Harvest completed o Grove maintenance in Site 3 trial ‘ Dr. Futch evaluations: o Continued evaluations of trifoliate rootstocks for HLB tolerance o Initiating new Dow herbicide trial ‘ Applications of the ‘Boyd Program’, Keyplex and Ben Hill Griffin programs continued in the ‘commercial scale’ nutritional demonstration. ‘ Conducting spring fertilizer and pest management programs for the groves o Herbicide program on schedule o Psyllid management continued . Participated in coordinated area wide spray in early May o ‘summer oil’ sprays applied o Mechanical weed/middle management evaluation continues o Hedging and topping completed . Also skirted in the east block ‘ Continue to establish Dr. Bowman’s new USDA rootstock plantings. ‘ Commercial Trials: o Eurofins evaluations on disease and insect management continue o Keyplex nutritional trial evaluations continue o DuPont demonstration for row middles management with Matrix Herbicide continues o Syntech evaluations on insect management o Florida Ag Solutions initiated herbicide and insecticide evaluations ‘ 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
The objective of this proposal was to translate the analysis of the transcriptome of HLB infected citrus trees to identify key processes connected to the HLB disease phenotype observed in the field in Florida. Based on this analysis two target processes were identified 1) the deficient citrus innate immune response and 2) the sucrose starch metabolism altered by source-sink disruption both caused by HLB. Literature and metadata analysis of our data as well as existing expression data available in NCBI-GEO formed the basis of choosing 6 chemicals that were formulated as treatments. We tested 8 spray treatments that included 6 therapeutic treatments and 2 control treatments. The therapeutic treatments included: 2, L- Arginine, 2, gibberellin in combination with 6-benzyl adenine (BA), and 2, atrazine in combination with sucrose. The remaining two treatments were control treatments that accounted for the surfactant Silwet, K-phite and fertilizer LDKP3XTRA that were added to all treatments as a nutritional suppliment. These treatments were first successfully tested with greenhouse citrus graft inoculated with HLB. Then these treatments were applied to 24 trees in the field on June 25, 2013 with 3 trees per treatment. In October 2013 these 24 and an additional 48 trees were sprayed with the 8 treatments providing 9 replicate per treatment. The 8 treatments were combined to make 4 , with 2 treatments each containing 4 chemicals and with 2 controls were sprayed on the 72 trees in March 2014. After the spray treatments the trees were sampled at day 0, 3 and 6 collecting 6 leaves from around each of the trees. The leaves from each tree was pooled and extracted for DNA, RNA and protein. The analysis of the DNA was used to determine the bacterial titer. The RNA was used to analyzed 10 biomarkers using qRT-PCR: 5 related to immune responses pathways (EDS1, PR1, WRKY70, WRKY48, WRKY54), 2 involved in gibberellin pathways and 3 related to sucrose and starch metabolism, a pathway highly linked with HLB disease syndrome. As we expected, Gibberellin-2-oxydase was clearly induced with GA + BA treatments. Same trend was observed for one arginine and one atrazine + sucrose treatment. Relating to genes involved in sucrose and starch metabolism, we interestingly observed that at least at one concentration, the six treatments were able to reverse the trend of expression of invertase and sucrose synthase induced by HLB infections. GPT2 is strongly induced by HLB in leaf tissues and it is believed to be strongly associated with the starch accumulation in infected leaves. GPT2 is a transporter present in the chloroplast and responsible of the glucose-6-phosphate transport leading to the HLB-induced starch biosynthesis. Interestingly, we observed a strong down regulation of this gene in all six treatments only at three days after treatments. Data showed that some treatments may allow boosting Citrus immune responses. Indeed, WRKY70, a well-known transcription factor involved in biotic stress response was clearly enhanced by BA + GA spray and one Arginine treatment. Arginine and atrazine treatments induced expression of PR1, a SAR-related protein. Conversely, WRKY48 was not significantly regulated by any six spray conditions. Another key player in biotic stress responses, WRKY54, was in higher abundance in both arginine and atrazine treatments. One concentration of GA + BA showed similar effect. The transcript abundance of EDS1 was unchanged in comparison to both controls. Taken together, these data look promising and indicated that, at least at one concentration, the three therapeutic strategies were able to significantly affect expression of HLB-regulated key biomarkers. Further experiments are needed to be performed with these treatments to evaluate their effect on other important production features like fruit drop, yield and fruit/juice quality.
Fruit harvesting of the ‘Hamlin’ orange ACPS experiment near Auburndale was completed in December 2013. At that time the trees were exactly 5 years old in the ground. The experiment compares two rootstocks (Sw = Swingle; C35 = C35), two fertigation delivery methods (Drip = 2x 0.5 gph drippers/tree; MS = 1x 10.5 gph microsprinkler/tree) and three planting densities (218, 303 and 363 trees/acre). Cumulative 5-year yields of the best ACPS treatment exceeded 1,300 boxes/acre, while that of the grower control almost reached 800 boxes/acre. Average annual fruit yield from the best treatment was 260 boxes/acre, suggesting that it was rapidly approaching the economic break-even point. This important economic milestone in the life of a new grove strongly depends on the costs of production, the fruit yield, and the price of the fruit sold. The factors under evaluation in the ACPS experiment favored a high, early fruit yield and ensured reasonable limits on the production costs. The price of fruit and fruit revenue were not controlled experimental factors. A moderate production cost with the ACPS was possible despite a higher investment in trees at higher planting densities because the grove care involving sprays was more efficient (more canopy area covered per acre at high densities for broadcast pesticide, fungicide and fertilizer sprays). Water and fertilizer use efficiencies were also up to five times higher for the ACPS treatments than the grower control treatment in the first three years, thus saving fertilizer, fuel, and water. Summary fruit yield data (boxes/acre) for the four years 2-5 are shown in the table: Year 2 Year 3 Year 4 Year 5 STD-Sw (218*) 8.57 91.1 321 370.7 MS-OH-Sw (218) 18.23 93.1 395 339.7 Drip-OH-Sw (218) 17.17 115 388 356.9 Drip-OH-Sw (303) 24.71 159.7 471 489.9 Drip-OH-C35 (303) 34.04 200.9 557 478.1 Drip-OH-C35 (363) 38.52 222.2 622 436.5 * trees/acre Unfortunately the yields in this ACPS experiment have already peaked due to rapidly increasing HLB incidence, with many previously productive trees displaying blotchy mottle leaf symptoms, tree dieback and excessive preharvest fruit drop. At the end of five years, the HLB incidence in the experiment was about 70%.
Experiments to determine the efficacy of different nano-particle systems to deliver nutrients and antimicrobial molecules to citrus leaves: Greenhouse experiments to test the efficacy of nano-particles to deliver Mg to citrus trees are underway. Mg deficient citrus trees were treated with three types of nano-particles containing Mg. The nano-particles being tested are: 1.liposomes, 2. dendrimers, 3. kaolin. As control, we used an aqueous solution of Mg at a concentration commonly used in foliar nutrition programs. Trees are being sampled on a weekly basis for their photosynthetic rates as an indication of the effect of Mg in the reconstitution of the photosynthetic apparatus. Anatomical studies should follow completion of the treatments. Concurrently, polymer based nano-particles and liposomes containing a soluble fluorescent marker have been applied to citrus leaves to study the delivery of antimicrobial substances. Application has been performed on both abaxial and adaxial leaf surfaces. Penetration and movement of the fluorescent probe is being analyzed by fluorescent microscopy and laser scanning confocal microscopy.
Experiments to determine the efficacy of different nano-particle systems to deliver nutrients and antimicrobial molecules to citrus leaves: Greenhouse experiments to test the efficacy of nano-particles to deliver Mg to citrus trees are underway. Mg deficient citrus trees were treated with three types of nano-particles containing Mg. The nano-particles being tested are: 1.liposomes, 2. dendrimers, 3. kaolin. As control, we used an aqueous solution of Mg at a concentration commonly used in foliar nutrition programs. Trees are being sampled on a weekly basis for their photosynthetic rates as an indication of the effect of Mg in the reconstitution of the photosynthetic apparatus. Anatomical studies should follow completion of the treatments. Concurrently, polymer based nano-particles and liposomes containing a soluble fluorescent marker have been applied to citrus leaves to study the delivery of antimicrobial substances. Application has been performed on both abaxial and adaxial leaf surfaces. Penetration and movement of the fluorescent probe is being analyzed by fluorescent microscopy and laser scanning confocal microscopy.
Physiological tests and data collection on the original 36 heat treated trees are being continued. Leaf anatomy samples are being processed and embedded in paraffin for cross-sectioning. Looking at the cross-section of the leaf petiole will show the area of the phloem. An effect of HLB is flattening of the phloem cells which limits nutrient transport and leads to the overall decline of the tree. Measuring the phloem area of the leaf petioles will give insight as to whether the heat treatment process has reduced or rid the tree of the bacteria, allowing the phloem to recover and resume their natural shape. Chlorophyll fluorescence measurements will also be done to further quantify the overall health of the tree. A portable steam generator was used in the design of a new heat treatment system in which a retractable plastic tent has been retrofitted to a fruit hauling truck (goat truck). The steam generator is also part of the design of a heat treatment system for tree roots. Injecting steam into the soil will raise the temperature of the roots in order to kill the bacteria in the root system. The heat treatment system for the roots consists of a array of perforated steel pipes which will be pushed into the soil using the goat’s fruit picking hydraulic arm. Field experiments are planned to evaluate the performance of this system. Preliminary tests will determine the amount of steam that can be applied before permanant damage to the roots occurs.
This report pertains to the no-cost extension granted by CRDF for project #564 ending 4/30/2014. Work has continued during the no cost extension period on genome-enabled investigation of Diaphorina citri biology and characterization of strain-to-strain variation among Liberibacter asiaticus species. With regard to D. citri biology we are principally interested in the insect response to Liberibacter infection and to this end have mined the scientific literature for insect gene sets linked to bacterial symbiosis and transmission in other species, identified relevant homologs in the D. citri draft genome, and forwarded sequences to collaborators in the Cilia lab (ARS-Ithaca) to use in directed searches of the D. citri proteome in the presence and absence of Liberibacter infection. During the course of these analyses, proteome sequencing has revealed the presence of proteins that appear to lack corresponding genes in the current D. citri draft genome assembly. The absence of experimentally confirmed genes from the current sequence assembly may result from use of a 1 kb cutoff during the assembly process leading to the discarding of ~4000 sequence fragments. These sequences have been retrieved and are currently being used to improve the genome assembly such that we and others who are relying on this draft sequence will have access to the most complete data available. Availability of closely related Liberibacter asiaticus sequences permits analysis of strain-to-strain variation within these species, applicable to both diagnostics and insight into genome evolution. Alignment of Las psy62 with Chinese strains Las gxpsy and the recently released (but not analyzed) Las A4 reveals only limited differences in gene repertoire, with Las gxpsy having eight predicted genes absent from Las psy62 and Las A4, and twelve genes present in both Las psy62 and Las gxpsy that are absent from Las A4. All genes showing variable distribution are either hypothetical or phage-related, reducing the likelihood of significant differences in biological functioning among the strains. Single nucleotide polymorphisms (SNPs) have also been mapped for genes conserved among strains. Las gxpsy was shown to have 1766 SNPs relative to Las strain psy62 and Las A4 to have 1410 SNPs relative to Las psy62. 15-20% of the genes in the Chinese strains account for all of the SNPs relative to Las psy62. As more strain sequence become available, these strategies can be readily applied to determine how patterns of variation observed for the three strains compare with other isolates.
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