As we approach the end of our third year of evaluating the Boyd cocktail of macro-nutrients, micro-nutrients, phosphite, SARs, and hydrogen peroxide we are collecting data that is showing the importance of macro and micro-nutrients, and the phosphite. The ‘Hamlin’ trial was harvested in January and yield results will be available for the next report. The two ‘Valencia’ trials will be harvested in April. The rehabilitation trial begun this year to evaluate the value of buckhorning existing HLB declining trees to rebalance the shoot/root ration and applying foliar nutrients to the regrowth has gotten a lot of interest from growers. The idea of of salvaging existing trees over removal, replanting, and bringing a new planted tree into production has a lot of advantages. One advantage is the cost and another is having the tree out of production only one or two years. Another advantage is having a tree with vigorous regrowth and fully productive canopy. Growth measurements of shoot length and leaf size of the regrowth have shown shoot length to be twice the length of unpruned trees receiving the same nutrients on every flush during the first year. The pruned trees have reached more than half their original height and tree volume in one growing season. Leaf nutrient analysis data has been collected to monitor the status of the vigorous growing trees. The trees in both the Boyd cocktail trials and the rehabilitation trial all look good considering the leaf drop experienced this past October statewide in groves. In addition, we have experienced similar leaf and fruit drop from the freezes in December that other growers have had. We have documented greater leaf and fruit drop from HLB infected trees where the added stress makes the trees more susceptible. HLB makes the citrus tree less tolerant to any stress factor like drought, poor nutrition, freezes, disease, insects, etc., and can result in leaf and fruit drop. We are in the third year of a replicated experiment in a 12-acre experiment commercial block of 8-year-old ‘Valencia’ oranges on ‘Swingle’ to test effects of micro-nutrients + systemic acquired resistance inducers, and Asian citrus psyllid (ACP) chemical control on ACP populations on Can. Libericacter asiaticus (CLas) titer, and plant yield. Since our last report we have conducted two dormant sprays on the treated plots. We are conducting preparation for the third harvest in these plots possibly towards the end of March beginning or April 2011. This third harvest is considered to be key to confirm the observed tendencies from years past. In our last harvest, trees in plots where ACP was managed using insecticides produced significantly more Lb-solid per tree (5.76’0.36 Lb-solid/tree) than trees in untreated plots (4.17’0.65 Lb-solid/tree), continuing a trend seen last year. Also, trees treated with Micro+SAR produced more (5.26’0.55 Lb-solid/tree) compared with untreated trees (4.55’0.64 Lb-solid/tree), following trends seen from the first harvest in the 2008-2009 season. Neither the insecticide treatment nor the nutrients have slowd down the spread of HLB (now virtually 100%), Treatments where ACP is being managed have had consistently lower titer over the two year study, except in our May 2010 where the CT values were not different Treated (24.0’0.3), Untreated (24.1’0.2). In January we are conducting the plant samples for HLB detection. Simultaneously, we are analyzing the data from the last plant sample collection.
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. Currently, we are testing the antimicrobial activities of the top 10 compounds against Sinorhizobium meliloti, Agrobacterium tumefaciens, and E.coli. In addition, those compounds will serve as leads to develop new compounds.
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. Analysis of T3SS effectors revealed presence of unique XopF1 and avrGf1 effector genes which are absent from Xac A 306 strain. Mutant of xopF1 and double mutant of xopF1/avrGf1 are being constructed to determine their roles in host specificity. Complete genome sequence of X. axonopodis pv. citrumelo FL-1195 was completed. At least 48 regions, which were found incorrect during annotation analysis, were rechecked and corrected. Correction was done using the 454 and Illumina read data as well as PCR sequencing of the questionable regions. The CDS prediction was curated using the GenePrimp pipeline. 1485 anomalies were checked and 570 CDS were corrected after being identified as long, short, broken or new genes. The genome sequence has been compared to the reference strain Xac A 306 using MAUVE and global rearrangements were observed. In detail analysis of T3SS, other secretion systems, effectors, LPS, EPS, motility clusters and unique regions different from Xac A 306 strain is currently in progress.
Website Creation and Development A genome viewer feature has been added to the CG-HLB Genome Resources website 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 initial version of the viewer, brought online in the previous quarter has now been moved to a faster and more flexible server hosted by the Cornell Center for Advanced Computing. New features include explanatory pop-up windows to aid novice users and a greater variety of imbedded hyperlinks. Users can search the genome by gene name, locus tag, feature type, or coordinates. Previously implemented tracks show predicted functions and cellular locations of the encoded proteins as well as genome structural features such as predicted operons and repeat sequences. More recent additions to the genome viewer include direct hyperlinks to precomputed lists of similar proteins at NCBI. Links to the metabolic pathways at the BioCyc Database are currently being installed to provide ready access to predicted metabolic roles of Liberibacter proteins. Addition of new tracks and links is ongoing as more bioinformatic analyses are performed. Bioinformatic Analyses Bioinformatic analyses are focused on genome sequence comparison with other bacteria. Of highest priority is comparison of the Las psy62 sequence to the recently released genome sequence of L. solanacearum CLso-CZ1. The two sequences are highly similar and we are exploring different approaches for representing both SNPs (single nucleotide polymorphisms) and insertions and deletions that occur between the two sequences. Our goal is to develop an effective means of visually representing sequence variation that can be scaled up as more Liberibacter sequences become available, providing a tool for rapid design of diagnostic primers that distinguish among strains. Genome sequence comparisons are also being conducted with related free-living bacteria as well as bacteria occupying analogous biological niches. By distinguishing between conserved core regions and those regions or genes that either exhibit faster rates of evolution or are more similar to genes from bacteria in analogous niches, genes critical to adaptation to the insect/phloem niche may be identified. Outreach An announcement of the genome viewer has been sent to all 76 registered users of the CG-HLB Genome Resources web site together with a survey soliciting comments on additional features that researchers might find of use. Information on the CG-HLB Genome Resources web site and associated genome viewer is being presented and additional feedback sought at the upcoming research conference on HLB in Orlando Florida.
The Citrus Greening Bibliographical Database [ http://swfrec.ifas.ufl.edu/hlb/database/ ] managed by the Entomology group at the University of Florida – IFAS in Immokalee with cooperation from the Florida Center for Library Automation in Gainesville has become a widely used source for information on Huanglongbing (HLB) for researchers, growers, and students throughout the world. Entries represent worldwide research on the various aspects of HLB: the associated bacteria (Candidatus Liberibacter spp.), the vectors [Diaphorina citri Kuwayama and Trioza erytrea (Del Guercio)], the effects of the disease on plants and vectors, and the most current management tactics. The database was designed to centralize relevant information in an accessible, user-friendly interface. This past quarter, October to December 2010, we continued to add and update information to the database and cross-reference all information for accuracy. We now have 1968 citations, 81% of which are linked to their original sources. Eighty-six percent of the entries are in English, the remaining 14% are in Spanish, Portuguese, Afrikaans, Japanese, Chinese, French, German, Vietnamese, Dutch, Farsi, Arabic, Czech,Thai, and Hebrew. We have also created an interactive Official Facebook Page [ http://www.facebook.com/pages/HLB-Greening-Database/164498020244442 ] with which you can become a ‘friend’ from the database webpage. This will serve as a forum in which researchers and growers can exchange HLB related information and discuss topics of interest and/or concern, post news and events related to HLB. The development and use of this project has been presented to researchers, students and growers in several national and international meetings in the U.S and in Mexico and has continued to have increased exposure within the research community through citrus research and extension web pages that have published links to our database (see partial list below). Our goal for the upcoming quarter is to continue to improve this service by updating existing documents to ensure accuracy, searching for the most current information from researchers around the world, continuing to link existing entries to their original documents, and promoting participation in the interactive Facebook forum (1) FCPRAC request for proposals 2009 [www.fcprac.com/proposals-2009.html] (2) Florida Entomological Society. [www.flaentsoc.org/]. (3) The Grower’s Citrus Greening Resource Center. [www.growermagazine.com/CitrusGreeningResearchCenter] (4) University of Florida Entomology and Nematology Pest Alert. Gainesville FL. [www.entnemdept.ufl.edu/pestalert/] (5) University of Florida- IFAS- Extension CREC. [www.crec.ifas.ufl.edu/extension/greening/links.htm] (6) USDA- APHIS. Plant Health – Citrus greening [http://www.aphis.usda.gov/plant_health/plant_pest_info/citrus_greening/links.shtml ] Arevalo, H.A., A.B. Fraulo, and P.A. Stansly. 2010. The HLB Bibliographical database: an information tool for growers and researcher. Citrus industry. 91:6. 22-23
Objective 1. Localization of Liberibacter asiaticus (Las) in the Asian citrus psyllid (ACP): [A] We continue our studies to improve the sensitivity and specificity of several FISH protocols to localize Las in hemolymph smears and dissected organs of ACP, and in leaf sections from HLB-infected plants as positive controls. So far, Las has been detected by FISH in the hemolymph, filter chamber and midgut of HLB-infected ACP from our laboratory colonies or from the field. [B] Using Q-PCR on dissected insect organs, Las was detected in the salivary glands, alimentary canals and other body parts of HLB-infected ACP adults. Our results suggest that the salivary glands constitute a major transmission barrier to Las in ACP, and that Las may replicate or accumulate in both the alimentary canal and salivary glands of its vector. Objective 2. Elucidation of various acquisition and transmission parameters between ACP and Las. [A] We have started two large experiments to compare between young (2nd-3rd-instar) nymphs and adults having various acquisition access periods (AAPs) on HLB-infected citrus plants. Previous reports have indicated that ACP nymphs are more efficient in Las acquisition than adults, and that Las probably multiplies in ACP nymphs (not adults), but the effects of various AAPs on nymphal and adult stages in this regard have not been carefully investigated. Our experiments are designed to study the effects of various AAPs on both acquisition and transmission of Las by ACP, as well as on Las replication in both nymphs and adults. We are now analyzing with Q-PCR hundreds of individual ACP from these two experiments. Results will be discussed in coming reports. [B] Since using whole citrus plants for maintaining psyllids when studying pathogen-vector relations takes considerable space, time and other resources, we have developed a new and simpler method for short-term rearing of ACP in conical 50-ml plastic tubes using detached citrus leaves for adults and detached young terminal shoots for nymphs. Survival of young adults was 89, 80 and 75% after 2, 3 and 4 weeks, respectively, on detached leaves changed weekly. Survival and development of 2nd-3rd-instar nymphs reared on detached terminal shoots in these tubes were largely comparable to those previously reported for ACP nymphs reared on whole citrus seedlings. ACP excretion droplets fell down from the leaves and accumulated in the conical bottom of the rearing tubes. Thus, in addition to its potential use for collecting psyllid excretions, this new method allows closer observation and photography of psyllid nymphs and adults, and can save time, space and other resources in various studies on the biology, behavior, management and pathogen-vector relations of ACP and probably other citrus psyllids. [C] We used the above method in devising a new “Detached Leaf Assay” to test the inoculativity of ACP with Las. Our results indicate that after feeding 10, 5 or 1 infected adult ACP/leaf on detached leaves of sweet orange for 7 days, the percentages of Q-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, even using the less sensitive but more commonly used Li primers, are largely similar to those reported earlier on inoculativity/transmission tests of Las-infected ACP using whole citrus seedlings. Using more sensitive primers, however, can increase the usefulness of this method. We suggest that this new ‘Detached-leaf assay’ method can potentially speed up Las-inoculativity tests on ACP from 3-12 months to only 2-3 weeks, which can greatly enhance pathogen-vector relation studies on Las and ACP.
The intent of this study is to examine the effect of windbreaks, copper sprays to reduce infection, and leafminer treatments to determine there individual and combined effects on control of citrus canker in Brazilian commercial citrus and the applicability of this strategy to the US commercial citrus industry. Via a USDA/ARS specific cooperative agreement with the University of Sao Paulo, and the Brazilian cooperator, new replicated plots have now been established at the IAPAR farm, in Xambr’, Parana state, located 350 km west from Londrina and 250 km west from Maring’. The plots consist of the cultivar P’ra on Rangpur lime, two years of age at the beginning of the experiment. Windbreaks have been completed and plants were be established in Mid April 2010. Plots are progressing and the following treatments are being applied: 1) no sprays (control), 2) Cu++ sprays to reduce citrus canker incidence, and 3) insecticide sprays to inhibit infestations of Asian leafminer (secondary effects). Main effects are windbreak versus no windbreaks. Citrus canker incidence is being estimated on multiple branches on each tree treated as the number of leaves per branch infected. Data collection is currently underway. We anticipate running these plots for 2-3 more years. The development of the Programmable leaf wetness controller (PLWC) software was written, debugged, is complete, and the control program is working well. New leaf wetness sensors were designed and constructed. Calibration has been tricky as this is a new type of sensor we are developing and has never been used before by any other research group. The new design of the leaf wetness controller has much greater sensitivity and provides better environmental control, but with these benefits comes added complexity in electronics. A circuit to control the leaf wetness sensor and another to control fans that facilitate wind generation in ambient environments have been completed, tested and calibrated. Initial trials have demonstrated some glitches that we are currently addressing. Once completed, we will continue with studies to examine the survival characteristics of bacterial pathogens under field conditions.
During this quarter, preliminary studies on root development were conducted in the Gapway Groves ACPS experiment. Soil cores were removed around trees in the different treatments and the root densities were quantified. 1) Root length density was greater in the 0-15 cm than 15-30 cm depth for three ACPS treatments. 2) Root length density was greater at the deeper depth (15-30 cm) than the surface in the conventional (grower) treatment. 3) Greatest root length density was nearest the tree in the direction of tree rows. 4) The microsprinkler fertigation treatment had the highest root density uniformity with distance from the tree, indicating more uniform wetting and root growth in areas adjacent to the tree. 5) The conventional grower treatment had greater root density at 15 cm from the tree in the tree row and 15 cm perpendicular to the location 15 cm in the tree row. Lower but similar root length density was found 30 cm from the tree in the row and 15 cm perpendicular to the row at the 30 cm distance. This would indicate greater irrigation near the tree but less at 30 cm from the tree. Daily Water Requirement Calculator A simple on-line web calculator was developed to determine the water requirements for trees. This is a “beta test” version calibrated for the Lake Alfred area. * It is important to know how much soil water to replace on a daily basis according to the amount consumed by the tree. That is the philosophy of the ACPS system, and at least monthly adjustments to the daily irrigation schedule should be made as the trees grow and the evapotranspiration changes during the season. * If you schedule irrigation with soil water sensors to maintain soil water content at near field capacity during daytime hours, you should find agreement between the water delivered per tree per day, and the numbers calculated, assuming that the trees receive no rain during that period. * The calculator was calibrated in an OH system with a high (near 100%) irrigation efficiency because each tree’s root system was dedicated to only two drip emitters; the drip feed line had no other emitters wasting water and nutrients between tree locations (see schematic). If your drip system is different, especially if there are drip emitters located along the feed line and not in the tree’s root zone, you will have to adjust your irrigation schedule to account for those losses in water and lower irrigation efficiency. For all the latest up-to-date information on the ACPS research, especially for photos and calculator which cannot be displayed here, browse to http://128.227.177.113/ACPS/index.html The water calculator is on the Data/Reports page.
The purpose of this project is to preserve citrus germplasm in Florida that is threatened by loss due to huanglongbing (HLB) and citrus canker. A priority list of germplasm has been established and most of the selections have been established in a secure greenhouse at USDA ARS Ft. Pierce. At the USDA ARS Repository, Riverside, two additional growth chambers for use in thermotherapy and a better microscope for use in shoot tip grafting have been purchased from other funds, and will soon be available to help increase the capacity for therapy of selections. In cooperation with USDA ARS Ft. Collins, the use of cryotherapy for elimination of HLB and other graft transmissible pathogens of citrus is being explored at the USDA ARS Ft. Pierce location. In Riverside using the Candidatus Liberibacter psyllaurous in tomato as a model system, buds stored under cool conditions for two weeks are no longer capable of transmitting the bacterium when grafted onto healthy tomato plants. Similar trials are underway using stubborn, a phloem limited prokaryote, as a model system, but it is too early to get a final reading of results. Efforts are continuing to get adoption of the ‘Citrus Passport’ program whereby the three agencies (Florida Citrus Germplasm Introduction Program, Gainesville, FL; California Citrus Clonal Protection Program, Riverside, CA; and USDA ARS National Clonal Germplasm Repository for Citrus and Dates, Riverside, CA) which may import citrus varieties into quarantine for therapy, indexing and release to the citrus industry have harmonized their protocols and recognize the therapy of selections which comes from other programs, with a protocol for short term indexing of common graft transmissible pathogens which may be completed within 6-8 months, and then be able to release the selection to the respective citrus industry. Implementation of this ‘Citrus Passport’ program will facilitate returning clean selections to Florida from this project to recover and preserve Florida citrus germplasm.
No experiments have been conducted yet for the second season as this work is on fresh citrus and those experiments will begin with the fresh season this month. To summarize results from the first season: Between Oct. 5th and 30th, five experiments were conducted that included Fallglo (1 time), sunburst (2 times), red grapefruit (4 times), and navel oranges (1 time). Fruit were treated on a commercial packingline (3 experiments) or on the Indian River Research and Education Center research line (2 experiments). Treatments included 1) full wash (brush bed + high-pressure wash) + waxing (carnauba), 2) full wash, 3) brush bed only, 4) brush bed with brushes rotating half normal speed, 5) high-pressure wash (HPW) only, 6) HPW for 10 seconds, 7) HPW for 5 seconds, 8) running fruit only over PVC rollers, and 9) a control (not washed or waxed). On the commercial line, fruit remained on the brush bed for ~ 1 min. 10 seconds, and on the full HPW for ~35 seconds. Normal brush rotation speed was ~100 rpm. Fruit were also evaluated for how surface dirt obscured the ability to grade the fruit for canker and other grade defects. Fruit from all treatments were degreened under simulated commercial conditions (5 ppm ethylene, 85F, 95% RH) and color development and weight loss measured almost daily. Fruit were subsequently stored and evaluated for the development of decay and disorders during storage under ambient conditions (~70-75F). In general, all very early season Fallglo fruit were relatively clean and did not need washing for adequate grading. This changed somewhat by the end of October when grapefruit that received more extensive washing (i.e., full washing) was significantly easier to grade compared to unwashed fruit. HPW produced fruit with intermediate gradeability. However, even minimally washed fruit were sufficient for adequate grading. These experiments will be repeated this season to determine variability in initial fruit cleanliness from year to year. Washing and waxing the fruit gave the greatest inhibition of degreening, almost stopping color development completely. Compared to preliminary results in 2008, results again showed that full washing of fruit on both the brush washer and HPW, or washing on the brush bed along inhibited degreening significantly more than did washing fruit only as they passed over the HPW. Fruit that were not brushed at all, but only passed over rollers experience a slight, but significant delay in color development compared to the control, but the delay was relatively minor compared to the other washing treatments. As the season progresses, fruit exterior surfaces become more soiled with dirt and sooty mold that makes grading more difficult without washing. Experiments were conducted in July 2010 using late-season red grapefruit, harvested from a block with citrus canker to test how well the different washing methods allowed graders to detect canker and other peel blemishes. Unwashed fruit were run past commercial graders and the number of fruit with any canker or surface defects that would justify elimination based on export grade standards were counted. These fruit were then taken to a commercial packinghouse and either left un-washed (control), or washed over 1) the entire line (brush + HPW), 2) the brush line only, or 3) HPW only. Fruit were then evaluated by commercial graders again. It appears that commercial graders were able to detect canker lesions and other surface defects from even unwashed fruit this season.
Note that this report corresponds to the second of the two additional quarterly updates mandated in amendment #1 of the no-cost extension for the first year of grant #123 Website creation and development To provide a more convenient interface for users to view different aspects of genome characterization for Ca L. asiaticus, A Gbrowse based genome viewer for L. asiaticus has been put in place and can now be publicly accessed from the CG-HLB Genome Resources Website GBrowse page (http://www.citrusgreening.org/HLB-GBrowse.html). The genome viewer is searchable by gene name, locus tag, feature type, or coordinates. Tracks include a coding sequence (CDS) track hyperlinked to corresponding records at NCBI, COG track hyperlinked to the Clusters of Orthologous Groups database at NCBI, proteins with predicted signal peptide or lipopeptide cleavage sites, subcellular locations as predicted using PSORTb, repeat sequences generated using a variety of repeat finders, operons predicted using DOOR, and a gene track hyperlinked to locus tag searches in Google (connecting to KEGG and other databases). An additional track has been added that links to locus pages generated by the Grishin lab, complementing the genome structural features predicted here with in-depth protein motif analyses performed by that group. The Las GBrowse viewer is currently hosted by the WebGbrowse server, after installation on the Cornell Information Technology server proved unworkable. Use of other Cornell servers is currently being explored with the goal of establishing a local installation that can facilitate a wider array of features and higher level of security. Bioinformatic Analyses Comparative analyses with related free-living bacteria are being performed to distinguish between conserved core regions and those exhibiting faster rates of evolution, for the purpose of characterizing regions more closely linked to adaptation to the insect/phloem niche. Outreach The Gbrowse viewer was introduced at the APS meetings this past August and a survey has been added to the Gbrowse page of the CG-HLB Genome Resources web site asking users to evaluate the GBrowse features and suggest additional features of interest. All registered users of the site will be contacted by email to inform them of this new feature.
Exported proteins are expected to be important for virulence and their comprehensive analysis should aid our understanding of the disease. We combined the results of computer programs and manual curation to identify potential transmembrane and extracytoplasmic proteins. We applied 6 TMH predictors (TMHMM, HMMTOP, TOPPRED, MEMSAT, MEMSAT_SVM and Phobius) and two of them (MEMSAT_SVM and Phobius) detect SPs that are likely to be processed by the Sec complex. In addition, we used the well established SignalP3.0, which contains both Hidden Markov Model (SignalP_HMM) and Artificial Neural Network (SignalP_NN) modes for SP prediction. These automatic methods are generally based on the local properties of protein sequences or sequence profiles, resulting in a considerable rate of false predictions. Consequently, we manually inspected all the proteins that are predicted to have TMHs or SPs by any automatic predictors we applied. This broad inclusion can help lower the false negative rate. At the same time, to control the false positive rate, we integrated several lines of evidence, including consensus between predictors, predicted 3D structure (to rule out buried hydrophobic segments in known cytoplasmic proteins) and function (to identify proteins and protein domains known to function outside the cytoplasm or in the membrane), features of a protein’s orthologs (to validate if the SPs and TMHs can be constantly predicted in a orthologous group) and specific information about secretion machineries of Ca. L. asiaticus. After careful analysis of 218 proteins that were predicted to have SPs by any automatic method to identify extracytoplasmic proteins, we hypothesize that 86 proteins with predicted SPs are likely secreted from cytoplasm to periplasm via the Sec machinery. Many proteins from the initial list of 218 candidates were excluded due to the following reasons: (1) the SP cannot be consistently predicted (predicted by only 1 out of 4 methods); (2) the protein is predicted to have multiple TMHs, such as the sensory box/GGDEF family protein (locus: CLIBASIA_01765; gi: 254780468); (3) the confidently predicted function of the protein suggests its localization in the inner membrane or cytoplasm, for example, the ribosomal protein L35, which is predicted to have a SP by 3 out of 4 predictors applied; (4) close homologs likely lack SPs. The resulting list available here: http://prodata.swmed.edu/congqian/paper/supplement_table_S1.pdf will be used to mine for potential virulence factors. We also hypothesize that 21 proteins without SPs are likely targeted to the extracytoplasmic space through Sec-independent mechanisms. In addition, 184 proteins likely locate in the inner membrane of this Gram-negative bacterium. The lists of these proteins are here: http://prodata.swmed.edu/congqian/paper/supplement_table_S2.pdf
During the first period, we finished automatic computational analysis of all Candidatus Liberibacter asiaticus proteins. In order to predict structures of and to gain functional insights into these proteins, we set up a computational pipeline to assemble all relevant information for each protein. The pipeline consists of the following steps: 1) Reliable information about each protein was gathered from various biological databases, such as KEGG and those provided by NCBI. 2) Several essential sequence features were predicted, such as secondary structure, coiled coils, low complexity and disordered regions. Multiple predictors were applied to derive more confident conclusions. Transmembrane helices and signal peptides were specifically detected since they are likely to be present in potential targets for pathogen control. Multiple sequence alignments of closely related proteins were generated to highlight conserved and thus important residues. 3) Close homologs for all analyzed proteins were detected. Close homologs usually preserve the function inherited from the common ancestor and thus offer the most reliable functional inference. Interestingly, we noticed several internal duplication events within this genome, with some proteins over 90% identical to each other. The duplications are present despite the small size of the genome and may indicate important and pathogenic proteins. 4) Since proteins usually consist of one or several conserved sequence domains, such domains were found by RPS-BLAST and HHsearch programs. Identifying conserved domains will help us classify a target into a certain protein family and deduce protein properties from better studied proteins in the same family. 5) Homologs with experimentally determined spatial structures were detected. As of today, homology modeling is the most reliable way of structure prediction. Several software packages were used to infer homologous structures. The combination of these procedures varying in accuracy and sensitivity, will enhance the chance to get a reliable template for homology based structure prediction. 6) Structural domains, the protein folding units, were mapped onto each sequence. The presence of a structural domain usually implies certain function, and domain identification will not only reveal the domain architecture of the target protein, but provide hints about protein function. All this information and results were presented as a web-site where users can freely post comments and suggestions about each protein: http://prodata.swmed.edu/congqian/Candidatus_Liberibacter_genome_home.html Each protein is represented by a web-page, where the results are conveniently formatted for subsequent analysis. With the help of the website, on the next stage we will manually analyze each protein, i.e. check the consensus between different predictions, select the best homologous structure and deduce function. We will attempt to obtain single 3D structure model for each protein, but for difficult cases where no reliable homologous structures were detected, ab initio methods to fold proteins in silico will be applied. Afterwards, important and most challenging proteins, metabolic and regulatory pathways will be studied in details to address interesting questions, e.g. which pathway this bacterium utilizes to secret certain virulence factors. Finally, a list of candidates for HLB control will be provided, and the website will be expanded to include structure models and functional predictions.
Under Objective 1 ( biofilm formation by Xanthomonas citri subsp. citri (Xcc) in comparison with other bacteria that are known to form biofilms). Different assays are being performed to establish how different bacteria form biofilms on abiotic surfaces (glass slides) and whether the aggregates on glass surfaces contain metabolically active bacteria. Development of aggregates was visualized on glass surfaces with confocal microscopy and the use of green fluorescent protein (Gfp) expressing strains. Viability of the bacteria in aggregates is confirmed using strains transformed with a plasmid expressing a labile Gfp protein that breaks down when the bacterium dies. Similar studies on leaf and fruit surfaces indicated that all the bacterial cells in aggregates attached to the surface did not show the same metabolic activity as revealed by comparing stable and labile Gfp variants. Only bacteria that formed biofilms were viable while scattered individual cells died in the initial stages. Differences in bacterial aggregation and biofilm formation were observed between Xcc strains with different host range, A and A*, as well as X. campestris and X. alfalfae susp. citrumelonis. Preliminary results with scanning electron microscopy showed differences in biofilm structure between A strain (wide host range) and A* strain (narrow host range) on fruit and leaf surfaces that confirmed initial results in vitro with violet crystal staining of biofilm formation on plates. These results indicate reduced ability of the narrow host range strain A* to form biofilm. In addition, there were differences in bacterial motility between A and A* strains. A* strain had higher swimming and lower swarming capacity than A strain that may be related to their differences in ability to form biofilm. Under Objective 2 (studies in vitro and in vivo using transformed strains of Xcc subjected to different environmental conditions). Two different types of studies are in progress. One is the effect of different bactericides on the initial stages of biofilm formation and the other is the effect of these compounds as biofilm agents once the biofilm is formed. Aggregates were stable and Xcc in them remained viable after water washes which explains why fruits harbor residual Xcc after disinfection treatments. Fruit wash treatments with toxicants and disinfectants (CuSO4, NaCl, NaOCl and SOPP) were tested for their effect on biofilm formation. Sublethal concentrations of NaCl and SOPP in the initial stage of surface colonization increased aggregation of viable Xcc. This effect was less clear with NaOCl or CuSO4 at the sublethal concentrations tested thus far. Greater aggregation is explained as a bacterial resistance response to chemical stresses. Experiments are in progress to confirm these results. Assays to measure bacterial aggregation and biofilm formation in a quantitative manner using violet crystal staining after exposure to bactericides are in progress in order to repeat experiments performed last year. In addition total RNA is being extracted from treated biofilm forming and planctonic bacteria to evaluate differential expression of genes involved in biofilm formation and aggregation and possibly those related to motility and quorum sensing.
Objective 1: A comparative study of two susceptible hosts, Duncan grapefruit (DG, C. paradisi), and Rough lemon (RL, C. jambhiri) and two resistant cultivars of kumquat (Fortunella spp.), ‘Meiwa’ and ‘Nagami’, evaluated the mechanisms involved in the resistance of kumquat to the citrus canker. MK and Nagami NK developed a hypersensitive response (HR), with necrotic lesions with population of Xanthomonas citri subsp. citri (Xcc) < 5 log units after 168 h in detached leaf and attached leaf assays. Early expression of genes related to programmed cell death associated with HR were identified in MK and NK. The resistance in kumquats has several characteristics associated with HR: 1) Rapid necrosis of leaf tissue in 48-72 h post inoculation in vitro or 72-96h in planta; 2) Disruption of epidermal and mesophyll cells by 72 h; 3) Xcc bacterial ingress limited to few cell layers below the epidermis; 4) Xcc population growth arrested at 72 h coincident with the cellular disruption; 4) Light microscopy and TEM, show death of the cells adjacent to the inoculation site with very few bacteria proliferating; 6) 5) HR-related genes and other putative resistance-related genes expressed early in resistant KN but not in susceptible DG. Behavior of susceptible DG and RL was: 1) No symptoms are detect in susceptible until 72 hr after inoculation and water soaked developes at 168 hr; 2) pustular callus-like lesions erupted through the cuticle by 10-16 days post inoculation (dpi); 4) Xcc populations reach 6 log cfu of Xcc per inoculation site at 168 hr; 5) Xcc population increases up to 15 dpi. Objective 2: Validate the inheritance or resistance for cybrids with susceptible Red grapefruit (RG) and RL with Valencia orange (VO). The putative RL cybrid has been recently been fully analyzed and determined using Single stranded repeat (SSR) analysis to be a cybrid from a mislabeled callus line of Valencia orange and not Meiwa kumquat. Hence the inheritance of resistance is not the HR type but is one of moderate susceptibility compared to high susceptibility in RG. Evidently, there is a definite expression of resistance in the cybrid inherited as a result of presence of the heterologous mitochondrial or chloroplast genome from the VO callus line. Evidence for this is as follows: Intermediate lesion symptoms are observed for RL+VO cybrid in vitro and in-planta. In contrast to development of callus, the inoculated area develops necrosis by 10 dpi. Xcc population plateaus by 10 dpi below the bacteria populations susceptible RL or Red GF. Two types of lesion were observed: necrotic and also callus, suggesting that cell death occurs and arrests the proliferation of Xcc. Expression of HR-related genes is intermediate between MK and RL, further substantiating that some yet to be determined elements of resistance have been inherited in the cybrids. Finally, the current set of Ruby red grapefruit cybrids with VO planted in canker-affected locations on the east coast continue to be more resistant than Red grapefruit trees around them. These RG+VO cybrids will continue to be observed for vigor and eventually fruiting characteristics, as well as resistance to canker.
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