We converted our experience gained from the computational analysis of Candidatus Liberibacter asiaticus proteome into a fully automated web-server for researchers to use. Computational sequence analysis, that is, prediction of local sequence properties, homologs, spatial structure and function from the sequence of a protein, offers an efficient way to obtain needed information about proteins under study. Since reliable prediction is usually based on the consensus of many computer programs, meta-severs have been developed to fit such needs. Most meta-servers focus on one aspect of sequence analysis, while others incorporate more information, such as PredictProtein for local sequence feature predictions, SMART for domain architecture and sequence motif annotation, and GeneSilico for secondary and spatial structure prediction. However, as predictions of local sequence properties, three-dimensional structure and function are usually intertwined, it is beneficial to address them together. We released a MEta-Server for protein Sequence Analysis (MESSA) to facilitate comprehensive protein sequence analysis and gather structural and functional predictions for a protein of interest. For an input sequence, the server exploits a number of select tools to predict local sequence properties, such as secondary structure, structurally disordered regions, coiled coils, signal peptides and transmembrane helices; detect homologous proteins and assign the query to a protein family; identify three-dimensional structure templates and generate structure models; and provide predictive statements about the protein’s function, including functional annotations, Gene Ontology terms, enzyme classification and possible functionally associated proteins. We tested MESSA on the proteome of Candidatus Liberibacter asiaticus. Manual curation shows that three-dimensional structure models generated by MESSA covered around 75% of all the residues in this proteome and the function of 80% of all proteins could be predicted. MESSA is free for non-commercial use at http://prodata.swmed.edu/MESSA/
Website Creation and Development: As previously described, the CG-HLB Genome Resources website hosts a genome viewer for the purpose of providing a convenient interface for accessing Ca. Liberibacter genome features and analysis of predicted proteins (http://www.citrusgreening.org/HLB-GBrowse.html). The genome viewer contains nine tracks specifying various predicted features of the Ca. L. asiaticus psy62 genome, as well as Ca. L. solanacearum CLso-CZ1 and the phage associated region from Ca. L. asiaticus UF506. Addition of new tracks and links to the genome viewer is ongoing as more bioinformatic analyses are performed. Implementation of the GMOD Primer3 program for use in designing primers capable of distinguishing different isolate groups is also in progress. Outreach: Contacts were made with other researchers at the 2nd IRCHLB research conference in Orlando Florida and we are currently assisting Dr. Manjunath Keremane (ARS, Riverside) with assembly and analysis of Ca. Liberibacter psyllaurus, a strain related to Ca. L. asiaticus that promises to be a useful experimental model for understanding the Liberibacter interaction with both psyllid vector and host plant. We are also assisting Dr. Wayne Hunter (ARS, Fort Pierce) with analysis of the Asian citrus psyllid metagenome with a particular focus on Wolbachia, the other alpha-proteobacteria associated with this insect. Characterization of the psyllid metagenome and its accompanying endosymbionts is expected to reveal new insights into how Liberibacter manages to survives while in the vector. Website personnel have gained expertise in a variety of analytic strategies and programs during the course of these analyses. A listing of some of these can be found at the Collaboration Opportunities link accessible from the CG-HLB Genome Resources home page, and researchers in need of assistance with data analysis are urged to contact us. Bioinformatic Analyses: Manjunath Keremane has built a BAC library for Ca. L. psyllaurous composed of 100 BAC clones of sizes ranging from 51 to 90kb. Ongoing analyses of the Ca. L. psyllaurus genome on our part involves assembly of 454 sequences of the BAC clones using MIRA3 and evaluation of different tools for joining the contigs generated from each BAC clones. In collaboration with Dr. Wayne Hunter, Wolbachia reads have been pulled from the psyllid metagenome sequence and a variety of assembly strategies applied. Annotation for the optimized assembly has been generated using RAST with select manual curation. Resulting genome features are being compared with other Wolbachia strains to evaluate potential differences. Regions of high similarity between Liberibacter and Wolbachia will be documented in the genome viewer, providing further guidance for intelligent primer design.
The purpose of this project is to preserve citrus germplasm in Florida that is threatened by loss due to huanglongbing (HLB) and citrus canker. Using input from stakeholders and scientists, a priority list of germplasm for recovery has been made of citrus germplasm in Florida. At Ft. Pierce 64 advance hybrid selections were identified and established in the greenhouse and now test HLB negative following treatment with penicillin-streptomycin. These trees will be tested as sources of budwood for rescuing further material in combination with antibiotic treatment of budwood and thermotherapy of young plants. Selected cultivars will be subjected to STG in Gainesville and forwarded to Riverside for further indexing. Additional accessions from Florida have been received by the USDA ARS Repository, Riverside, CA and are undergoing therapy/indexing. The USDA ARS Repository now has in operation two additional growth chambers to expedite thermotherapy work. Trials are being initiated in Riverside on alternative methods of thermotherapy, using stubborn and the tomato psyllid yellows model system, and on the use of young seedlings to speed up biological indexing for citrus pathogens. In cooperation with USDA ARS, Ft. Collins, CO, the use of cryotherapy to eliminate HLB is being explored.
Work conducted during the first half of 2011 was focused on two main areas. The first part concerned the impact of temperature and storage on mandarin flavor quality. The second portion dealt with evaluation of the use of hand-held devices using either visible-near infrared (NIR) spectroscopy or electrical conductivity to estimate soluble solids concentration (SSC) or titratable acidity (TA) in navel oranges and mandarins. The work on mandarin flavor quality was a continuation of our research of the prior season where we found that storage at a warm temperature of 68’F was extremely detrimental to mandarin flavor. In this testing we attempted to further refine the length of time needed for storage at 68’F to have a negative effect on flavor by evaluating fruit after both 3 and 7 days of 68’F storage. Fruit were obtained for the study in both early February and late March for the testing and stored for 0, 1 or 3 weeks at 41’F prior to placing fruit at 68’F. Fruit that had been stored at 68’F were compared by sensory panelists to fruit that had been stored continuously at 41’F for the same length of time. Panelists were asked if they detect a difference between these paired samples as well as being asked to rate the fruit on 1 to 9 hedonic scale to describe how much they liked eating the fruit. Flavor volatiles content for the various treatments was also determined to help provide an explanation for any flavor differences. The results of this testing are currently being summarized and analyzed. Visible-near infrared spectroscopy was investigated to determine the internal quality of citrus fruits, using a handheld spectrometer (Model NIRVANA-Analytical Spectrometer, Integrated Electronics, Sydney, Australia). Five different varieties of mandarins: China S-9, Fairchild, Nova, Okitsu Wase, and Frost Owari and the Powell navel orange were collected from the orchards over a five month of period, starting from November to March in 2010-11. Spectral measurements from wavelengths 390 to 1041 nm were carried out on each fruit at two randomly selected equatorial positions (180′ apart) and the fruit cut to determine SSC and TA. Regression models on the data indicated that both SSC and BrimA could be well predicted but that the relationship with TA was not as clear. Electrical conductivity measurements were conducted on juice using a device manufactured by G-won, Ltd (Korea) on the same fruit samples as were used for the NIR testing. The device measured conductivity and displayed the results as TA by the use of a preset calibration. We found that the G-won acidity meter generally read close to the actual acidity values (usually within 0.1%). The accuracy was less, however, at low acidities (less than 0.7% acid) for both navel oranges and mandarins. The G-won device was found to be very easy to use and may be of use in both the field and packinghouse. This work has been summarized and submitted to Citrograph for publication.
During the third period, we continued manual analyses of protein sequences, structures and functions with the emphasis on the data integration over Candidatus Liberibacter asiaticus (C. L. asiaticus) proteome. Based on these analyses, we started to construct a list of potential virulence factor and initiated building a model of the metabolic network of C. L. asiaticus (in collaboration with Dr. Andrei Osterman). For each protein in C. L. asiaticus proteome, we selected the closest homologous structure as the template for structure modeling, analyzed the domain architecture of the protein and collected evidence to support function prediction by the consensus of all information, including homology, structure models, the context of the sequence in the genome, and the integrity of metabolic and signaling pathways. For about 80% of all proteins, we generated confident structure prediction based on homology modeling. For around 60% of all proteins, we obtained specific function predictions and for 20% only general function predictions could be achieved. Remaining 20% are currently unassigned. Based on the function prediction and manual analyses, we classified proteins into different categories as described in details at: http://prodata.swmed.edu/congqian/category.html. One important category is potential virulence factors. Based on the local sequence properties (virulence factors are likely to be secreted or exposed on the surface of the bacterium), evolutionary history and predicted functions, we assembled a list of potential effectors, which may help us to understand the mechanism of C. L. asiaticus infection. Another important category is the proteins in metabolic pathways, for which we built the metabolic network model of this bacterium in collaboration with Dr. Andrei Osterman. This model not only offered insights into metabolic properties of the bacterium, but also provided further validation of the functions of these proteins. Presently, we are still optimizing the metabolic model and predicting proteins essential for the survival of C. L. asiaticus. Among them we will determine potential drug target if certain essential proteins lack orthologs in the plant or their sequence and structure properties are different enough to allow specific interaction between a drug and a bacterial protein. In the next period, we will incorporate manually verified structure and function predictions into a public website. We will work on further identification of potential virulence factors or proteins that are relevant to important processes during infection by two approaches: (1) comparative analysis between C. L. asiaticus and the closely related proteome, Candidatus Liberibacter solanacearum, (2) homology relationship between C. L. asiaticus proteins with known virulence factors. We hope that our hypotheses about the virulence factors can lead to advances in understanding and controlling of citrus greening.
Mid Florida Citrus Foundation (MFCF) a 501c3 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 cost tend to be higher than a commercial grove 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. In 2010 MFCF installed fertilizer tanks, pumps, timers, micro sprinkler irrigation, windbreaks and planted trees on an additional 17 acres of grove to allow more research work to be conducted at the facility. This was made possible due to the fact that the MFCF received grant support from the FCRPAC and an EQIP grant from the USDA NRCS. These federal dollar are cost share dollars which require the implementation of windbreaks around existing or planned citrus groves and for the conversion of water guns to micro-sprinklers before being reimbursed for fifty percent of the cost. In 2011 MFCF expansion projects include an economic study of high density citrus rooted cuttings for early production of citrus in the presence of greening, remedial and preventive tests for HLB infection, new herbicide trials,variety evaluation with HLB tolerance in Florida and ACP studies. A total of 31 acres has been prepared for planting with 25 acres planted this May and the remaining acreage to be planted this year. This is in addition to the 17 acres that were planted last year. This large planting of young trees also requires added young tree care expenses and an increase in grove care costs without any fruit to support continuing operations. Future plans for 2011 with FCPRAC support include the following purchases; a new laptop for farm manager, a slightly used utility vehicle, the improvement of interior roads with clay. Additionally the MFCF is has two more annual payments of tractor and sprayer that was purchased in 2009. In this past year over twenty citrus research trials have been conducted at the MFCF. Additionally MFCF supports extension efforts. These efforts included three citrus grower field days to relay research results, visits by international scientists and extension personnel, community outreach efforts such as 4-H and Master Gardener trainings. The 2011 MFCF field day had an estimated 250+ people attend. Additionally private companies with a vested interest in the citrus industry have made considerable donations of materials and irrigation equipment for these new projects. This is a leverage of research dollars that does not get recognized. Between private donations and EQIP grant monies, the CRDF is getting approximately 2.00+ dollars towards research projects for every 1.00 dollar in grant monies awarded.
During this quarter, the young ‘Hamlin’ orange trees in the Ridge experiment encountered very demanding dry spring conditions after flowering and fruit set. The computerized drip irrigation using two drippers per tree kept up with the high transpiration demand with the help of a highly developed proliferating root mass growing in the wetted zone. Photos of the roots can be viewed on the Photos page of the web site http://128.227.177.113/ACPS/index.html When we originally decided to try the open hydroponics (OH) technique in Florida, there was some doubt about the ability to grow such a specialized, concentrated root ‘ball” or “pad” in subtropical Florida conditions due to the frequent, abundant rain and excessively sandy soil. Our results now conclusively show that the root system of citrus trees grown on the Florida Ridge can be manipulated to grow around the drippers in exactly the same way that they would in dry or mediterranean climates. Such dedicated root systems have important implications for the success of the OH system because they allow the most efficient uptake of water and nutrients by the tree from the fertigation stream and also offer opportunities for manipulating leaf flushes and tree canopy size when needed later in the production cycle.
The purpose of this project is to preserve citrus germplasm in Florida that is threatened by loss due to huanglongbing (HLB) and citrus canker. Using input from stakeholders and scientists, a priority list of germplasm for recovery has been made of citrus germplasm in Florida. At USDA ARS USSHL Ft. Pierce the penicillin-streptomycin treatment shown to be effective by Zhang et al. has been widely used in the rescue of budwood in Florida. Vacuum infiltration into buds ready for propagation has been shown to have negligible phytotoxicity compared to non-treated control buds and has resulted in nearly 100% bud take. Sixty four unique hybrid advance selection field trees from the USDA citrus breeding program have been rescued by propagation into the greenhouse. Experiments led by the Yongping Duan lab, USSHL Ft. Pierce, have shown a 50X reduction in Clas from HLB-infected unique hybrid field trees. These trees will be tested as sources of budwood for rescuing further material in combination with antibiotic treatment of budwood and thermotherapy of young plants. Selected cultivars will be subjected to STG in Gainesville and forwarded to Riverside for further indexing. Several accessions have been received by the USDA ARS Repository, Riverside, CA and five are in final stages of therapy/indexing for release from quarantine. The USDA ARS Repository, from other sources of funds, has been able to purchase two growth chambers for thermotherapy use, a better microscope for shoot tip grafting, and to upgrade the greenhouse cooling/heating system to better extend the time period in which cool temperature biological indexing may be performed. This will improve the ability to perform the required biological indexes for release from quarantine following preliminary therapy of the targeted accession in Florida. Research is continuing on the use of antibiotics and/or cool temperature treatments to eliminate HLB from budwood, with citrus stubborn and tomato psyllid yellows being used as model systems in California. In cooperation with USDA ARS, Ft. Collins, CO, the use of cryotherapy to eliminate HLB is being explored.
Website Creation and Development As previously described, the CG-HLB Genome Resources website hosts a genome viewer for the purpose of providing a convenient interface for accessing Ca. Liberibacter genome features and analysis of predicted proteins (http://www.citrusgreening.org/HLB-GBrowse.html). The genome viewer is composed of 10+ “tracks” specifying various predicted features of the genome sequence. Public release of the genome sequence of Ca. Liberibacter solanacearum CLso-CZ1 and a sizable phage-associated region for Ca. Liberibacter asiaticus UF506 has led to incorporation of these additional sequences into the genome viewer along with predictions for the genome features found therein. Addition of new tracks and links to the genome viewer is ongoing as more bioinformatic analyses are performed. In addition, a continuously updated list of research publications on Liberibacter has been added as one of the additional resources provided by the CG-HLB website. Bioinformatic Analyses In conjunction with incorporation of the Lso CLso-CZ1 and Las UF506 phage sequences into the Liberibacter genome browser, more attention is being given to how sequence variation among strains and species might best be represented and used in design of diagnostic primers. Currently, regions of the Las genome determined to be unique relative to the Lso genome (E value cutoff of 1e-5) are shown in a track on the genome viewer. However, we are working on different approaches for displaying variation on a finer scale. This viewer is extendable to displaying variation among multiple strains as they become available. Outreach Information on the CG-HLB Genome Resources web site and associated genome viewer was presented at the 2nd IRCHLB research conference in Orlando Florida and contacts made with researchers sequencing additional Ca. Liberibacter strains. We are also assisting Dr. Wayne Hunter (ARS, Fort Pierce) with metagenome analysis of the Asian citrus psyllid with a particular focus on Wolbachia, the other alpha-proteobacteria associated with this insect. Regions of high similarity between Liberibacter and Wolbachia will be documented in the genome viewer and predicted metabolic capabilities compared between the two bacteria to evaluate the likelihood of complementary metabolism.
Objective 1. Localization of Liberibacter asiaticus (Las) in the Asian citrus psyllid (ACP). [A] Our Q-PCR study on dissected ACP organs will be published soon in the Annals of Entomological Society of America (2011, Vol. 104, in press). Our results provided the first PCR confirmation of Las in the alimentary canal and salivary glands of ACP and strongly suggested that the salivary glands constitute an important transmission barrier to Las in the psyllid vector. These results also suggested that Las may replicate or accumulate in both the alimentary canal and salivary glands of ACP. [B] We finished testing several fluorescent in situ hybridization (FISH) protocols with three Las-specific probes for the localization of Las in hemolymph smears and dissected organs of ACP that fed on HLB-infected citrus trees in the field or laboratory, and in sections from HLB-infected citrus leaves. Las was detected by FISH in the filter chamber, midgut, Malpighian tubules, hemolymph, salivary glands, ovaries and in muscle and fat tissues of HLB-infected ACP, as well as in the phloem of infected citrus leaves, but not in healthy control leaves or psyllids. FISH results indicated that the proportion of Las-infected salivary glands were significantly lower than that of infected alimentary canals or other organs. Our results showed the near systemic infection of ACP by Las and confirmed our previous PCR results suggesting that the salivary glands constitute a significant barrier to Las infection and/or transmission by ACP. Objective 2. Elucidation of various acquisition and transmission parameters between ACP and Las. [A] We finished two experiments to study the effects of various acquisition access periods on HLB-infected citrus plants, between 1 day and 2 weeks, on both acquisition and transmission of Las by ACP nymphs and adults, as well as on Las replication in both life stages. But we are still analyzing hundreds of individual ACP’s from these experiments with Q-PCR. Results will be discussed in coming reports. [B] We continued to develop and test a new ‘detached-leaf assay’ method that can potentially speed up Las-inoculativity tests on psyllids considerably by shortening this period from 3-12 months to 2-3 weeks. Young adults of ACP, reared as nymphs on HLB-infected citrus plants, were tested for inoculativity by caging them singly or in small groups (5 or 10 per leaf) in 50-ml polypropylene tubes on detached healthy leaves of sweet orange as described by Ammar and Hall (2011, Florida Entomol. 94:340-342). In two trials in which 10, 5 or 1 infected adult ACP per leaf were fed on detached leaves for 7 days, percentages of PCR-positive leaves were 40, 18.8 and 4.4% respectively, using Li primers, and 60, 40.6 and 11.1% respectively, using the more sensitive LJ900 primers. These results on Las-inoculativity of ACP on detached leaves using the more conventional and less sensitive Li primers are largely comparable to those recently reported for Las transmission when whole citrus seedlings are used for inoculation (Pelz-Stelinski et al. 2010, J. Econ. Entomol. 103:1531-1541). Our results suggest that detached citrus leaves can be used to test the inoculativity of single or small groups of ACP and that using more sensitive Las primers can increase the usefulness of this method. [C] Among ACP adults collected from HLB-infected citrus trees in Picos Farm (Fort Pierce, FL) since Feb. 2009, there have been no strong trends observed over time with respect to percentages of Las-infected ACP or titers of the pathogen. However, during late summer 2010, there were eight consecutive weeks in which percentages of ACP testing PCR-positive for the Las bacterium averaged below 10%. Statistical analyses indicated that increased rainfall and excessively high air temperatures during these weeks were significantly correlated with the lower percentages of Las-infected ACP.
Panelist could detect bitterness in HLB juice even though the levels of the bitter compounds were below their reported thresholds in most cases. Use of electronic nose and electronic tongue on processed orange juice samples was conducted on both Hamlin and Valencia juices from the last year. The juice had been frozen and was thawed for testing. The effect of nutritional spray systems (Keyplex, Diamond R and the Maury Boyd cocktail) were also investigated as it seems many growers are going to this system rather than scouting and removing trees. These nutritional programs are reported to reverse HLB symptoms on the tree but it is not known if they reverse symptoms on the fruits. So samples will be collected from healthy and HLB-affected trees from conventionally sprayed groves and those using the above nutritional sprays and analyzed for chemical and sensory differences. The data will be reported in the final report. There are differences in the volatile profile and in sensory perception of odors between healthy and HLB fruit juice, however the differences are much more subtle than the differences in taste. Differences in taste do correlate with limonin and nomilin values and lower sugars in both asymptomatic and also higher acids in symptomatic fruit, but more so overall in symptomatic fruit. The levels of limonin and nomilin, however, are generally below reported thresholds, even in Hamlin juice which has much higher limonin and nomiln levels than does Valencia, and even early in the season when these values are highest for both varieties. Threshold tests were done in several model juices and in Valencia juice for limonin and nomilin separately and together as well as in the presence of added sugar or acid. The values of limonin at which panelists could detect the compound were 2.32 ppm and nomilin 1.73 ppm in orange juice which was half the level of detection in model juices. When the compounds were tested together, their odor threshold was 1.89 ppm, which explains why bitterness can be detected in HLB juice. We were also interested in how much really bad symptomatic HLB juice could be blended with healthy juice before panelists could distinguish a difference. This was tried in both Hamlin and Valencia juices and determined to be around 25% of symptomatic juice in healthy juice for panelists to detect a difference in both varieties. Since the differences in aroma profiles between HLB and healthy juice are significant by not dramatic and the corresponding olfactory experience resulting in only subtle differences,it was not surprising that the enose was not as good as the etongue at separating HLB uice from healthy juice. It did, however, separate the juices, but with some overlap of some samples. Conversely, the etongue did clearly separate all HLB juices from healthy juice, even for HLB and healthy juices from the nutritional programs. The etongue also separated the different nutritional treatments from each other and from the conventional spray treatments. This instrument could be a useful tool for discrimination of juice samples based on quality.
Mid Florida Citrus Foundation (MFCF) currently supports the research efforts of scientists from the University of Florida, USDA and private industry. The majority of research projects are conducted by UF scientists at the approximately 112 acre citrus grove. 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 cost tend to be higher than a commercial grove 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. Currently MFCF is in the processes of laying irrigation for an additional 35 acres of citrus groves, which will support seven new research projects. This expansion was made possible due to this grant in conjunction with an EQIP grant from the NRCS. The NRCS grant provides for the leveraging of CRDF funds; that is for every dollar spent on expansion projects the NRCS cost shares approximately 50% of the cost. The completion of the establishment of new grove is scheduled for the beginning of May. Continued support is needed for the care and maintenance of these newly established groves into 2011
Management of citrus Huanglongbing (HLB), which is associated with Candidatus Liberibacter asiaticus, could be achieved by application of antimicrobials and stopping the spread of HLB pathogen. Curing Ca. L. asiaticus infected citrus trees is one attractive goal due to the high value of citrus trees and the high cost of citrus tree removal and replanting. Wide spread of HLB throughout Florida renders curing Ca. L. asiaticus infected citrus trees necessary. Treatment of Ca. L. asiaticus infected citrus could be pursued by applying antimicrobials to infected trees. The most common targets for antimicrobial agents include receptors, proteins and enzymes, DNA, RNA and ribosomal targets. Among them, proteins have become the major target due to their druggable characteristics. In this study, we presented our research on screening small molecule inhibitors against SecA. SecA is one essential component of the Sec machinery which provides a major pathway of protein translocation from the cytosol across or into the cytoplasmic membrane. The Sec pathway was also shown to be required for virulence of Ca. L. asiaticus in our study. SecA is the protein translocase ATPase subunit, which involves in pre-protein translocation across and integration into the cellular membrane in bacteria. First we filtered the structures based on their physico-chemical properties, e.g. Molecular Weight, H-Bond Donor, H-Bond Acceptor & Rotatable bonds and structurally similar to adenine moiety. Approximately 5000 structures were retrieved from ~5 million structures of commercially available databases. The identified data set was used for virtual screening by molecular docking method. Based on the dock scores we eliminated about 4500 low scored structures and selected ~500 (10%) for further molecular docking & minimization to evaluate the scoring functions (Dock glide scores, Hydrophilic, Hydrophobic e.t.c). Based on scoring functions, structural diversity, and our chemical intuition we have chosen forty structures for biological activity studies against purified SecA protein. For that purpose, SecA of Ca. L. asiaticus was expressed in E.coli using the pE-SUMO vector and purified. The inhibition assays of the 40 compounds against SecA was done as described previously by Denis et al. 1978. The IC50 ranged from 0.3’M to 100’M. At 20 ‘m seventeen compounds showed >50% inhibition and four compounds had more than 65% inhibition. One potent compound 18 was found with IC50 value of 2.5 ‘m. The differences between the high & low activity structures could be partially explained by the ‘CN groups which are only present in low activity compounds and 1, 3 Di-Methoxy groups which are present in the high activity compounds. The compound 18 is being further tested on Sinorhizobium and in planta. We are further screening the small molecules for activities in nm level against SecA.
Citrus canker is a serious disease of most commercial citrus cultivars in Florida.The goal of the proposed research is to identify and characterize novel and critical genes involved in pathogenicity and copper resistance present in X. axonopodis pv. citri (Xac) and related strains. Identification of critical virulence factors is a crucial step toward a comprehensive understanding of bacterial pathogenesis, host-species specificity, and invasion of different tissues thus to design new management strategies for long term control. Treatment of citrus with copper-based bactericides is one of the most common practices used for control. However, there is potential for horizontal gene transfer of copper resistance genes from other closely and distantly related bacterial strains, which will drastically reduce the efficacy of copper bactericides. Currently, copper resistant strains of other xanthomonads, including X. axonopodis. pv. citrumelo, the citrus bacterial spot pathogen, have been isolated from fields in Florida.Understanding the potential mechanisms of copper resistance in Xac and potential horizontal gene transfer of this resistance to Xac is also important for the long-term management of citrus canker.Currently, five Xac related strains are being sequenced with FL-1195 completed, XacAw close in completion (chromosome completed, plasmids in progress), while three more genomes are in the gap closing stage. Genome sequence of Xac strain Aw was completed. It is currently being checked for any problematic regions. The GenePrimp analysis of the annotation showed 1728 anomalies. Manual curation is currently in progress. Multiple gaps still exists for the two plasmids. Primer walking,cloning and sequencing are being used to close those gaps.The genome sequence has been compared to the reference strain XAC A 306 using MAUVE and global rearrangements were observed. FL-1195 was sequenced using 454-pyrosequening, Illumina (Solexa) sequencing and Opgen optical mapping. The finished sequence of XACM (4,967,469bp) was annotated and curated. Our analysis revealed that XACM lacked plasmids, although they are commonly associated with other strains of xanthomonas. Phylogenetic analysis based on housekeeping genes revealed a close relatedness of XACM to Xanthomonas axonopodis pv. citri str. 306 (XAC) causing citrus canker and Xanthomonas campestris pv. vesicatoria str. 85-10 (XCV) causing bacterial spot in tomato and and pepper. Whole genome comparison revealed a gene order similar to both XAC and XCV. Several genome rearrangements and insertion/deletion regions indicating genome plasticity were found. An all against all BLASTP of the complete proteomes revealed a total of 410 coding sequences unique to XACM. Several type III secretion system effector genes of XACM uniquely shared with the citrus or the tomato pathogen were found. Comparative genomics also showed various other changes in genes encoding cell wall-degrading enzymes, lipopolysaccharides, etc.
In Florida, the use of low volume sprayers has been adopted as a cost effective way to reduce Asian citrus psyllid populations. Low volume sprayers are calibrated to deliver 2 to 5 gallons per acre with a volume mean diameter droplet size of 100 microns. This relative measure of droplet size describes the range of droplet sizes, where 50% are smaller than 100 microns and 50% are larger. Smaller droplets are more susceptible to environmental conditions and drift at the time of application. A review of the literature on drift and droplet size identified a number of environmental conditions that could affect the deposition of these small droplets. This study monitored these environmental conditions in a Florida citrus grove from June to December 2010 and provides some insight into the prevalence of the favorable weather conditions for low volume spray applications. A number of environment conditions were monitored during this study. These measured conditions were then used to develop an estimate for the relative favorability for applying a low volume spray application on a given night. The study evaluated wind speed inside and outside a grove, an air dryness factor developed from the difference between dew point and air temperature measurements and the presence of inversion conditions. The trigger values used in the study were when average wind speeds were 0.0 mph or greater than 10 mph, a difference between the air temperature and calculated wet bulb temperature of greater than 140F and wind speeds less than 2 mph with air temperatures higher at the 25 foot level compared to the five foot level (used to determine the potential for a temperature inversion). From the collected weather measurements, the greatest potential threat to low volume spray applications in Florida could be conditions conducive to the development of temperature inversions. During the study period, the potential for temperature inversion existed on 16 to 32% of the nights. It appears in Florida that there is very little potential for evaporation to volatilize low volume spray droplets based on the established criteria. Wind speed conditions were taken from inside the grove at two locations and compared to observations outside the grove. Typically wind speeds fell into the 0.0 to 10 mph range from 82 to 99% of the time. The average wind speed recorded above the tree canopy at a 20 foot level was 8.75 to 20.5 times greater than the wind speed recorded in the tree row within the grove at a level of five-feet. The wind speed above the tree canopy most closely resembled the average wind speed measurements recorded from outside the grove at five and 25 foot levels. Mature hedgerow citrus groves in Florida appear to act as effective windbreaks, thereby significantly reducing wind speed within the block or grove as compared to outside grove observations. Additional activities were also conducted to offer low volume calibration events to Florida citrus growers with 3 events being conducted during the grant period. These events were conducted in conjunction with USDA ARS. These calibrations events assisted growers in adjusting sprayers to provide droplet sizes to be in the 100 micron range. The ability to effectively calibrate low volume spray equipment is not a task that can easily be accomplished without sophisticated and expensive equipment.
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