Besides the physical defects due to the destructive Huanglongbing (HLB) citrus greening disease on oranges, the infected fruit and resulting juice have been perceived as being more sour, bitter and off-flavored. In the symptomatic juice, the off-flavor was correlated with lower sugars, and sometimes with higher acids. Nevertheless, differences in flavor between symptomatic and healthy fruit were also correlated with higher limonin levels, although they were below taste thresholds. Thresholds (concentration of a compound at which it can be detected) of limonin and nomilin have been previously reported in water but never in combination and or in a complex mixture, such as a sugar-acid matrix or orange juice. Therefore, as a preliminary step to understand and characterize what the metabolites are that are responsible for the bitter off-favor of HLB orange fruit, thresholds of limonin, nomilin, and their combination in a sugar and acid matrix, as well as in Valencia orange juice were determined by taste panels. Between 16 and 23 panelists, previously trained to differentiate bitter and sour tastes, were asked to participate in the taste panels. Food grade limonin and nomilin were added alone or in combination to a simple matrix solution composed of 6% sucrose and 0.15% citric acid in drinking water; or to a complex matrix solution containing 5.2% sucrose, 2.1% glucose, 2.5 % fructose, 0.75% citric acid and 0.25% malic acid, or added directly into Valencia and commercial orange juice. All stimuli were prepared by dissolving the compounds in solution and once the highest concentrations were prepared, the rest of the samples were diluted in a series by a factor of 2. Each series of stimuli were presented 3 times on different days. A detection threshold was determined using a forced-choice ascending concentration series method of limits described in ASTM E679-91. At each selected concentration, a triangle sample set consisting of one test and two blank samples was presented to subjects; subjects were asked to determine which the test sample was. The subjects were also asked to rate the difference from 0 to 10. The best-estimate threshold for each panelist was considered to be the geometric mean of the concentration at which the last miss occurred and the next higher concentration. In the simple matrix, the threshold of limonin was lower than nomilin, indicating that it would be detected at a lower concentration. Panelists perceived the nomilin bitterness as being more astringent, metallic and lingering than the limonin bitterness, as has been described for HLB juice. The synergetic effect of limonin and nomilin was significant in decreasing their individual thresholds in a sugar and acid matrix. Interestingly, the thresholds of limonin and nomilin were lower in Valencia and commercial orange juice compared to the thresholds measured in the complex matrix. Our current results show that the threshold concentrations of limonin and nomilin in healthy ‘Valencia’ orange juice are higher than the concentrations of both limonin and nomilin found in HLB ‘Valencia’ fruit, which was perceived bitter by a taste panel. Perhaps the lower sugar and higher acid content of HLB fruit decrease the threshold of those bitter compounds which will be explored in further studies. We mixed normal juice with symptomatic HLB juice and found that panelists could detect the symptomatic HLB juice at different levels depending on variety and more information will be forthcoming on this in the next report.
Quarterly report 4-1-11 to 6-30-11 The major program conducted during the quarter was the Florida Citrus Growers’ Institute in Avon Park on April 6, 2011. This program is organized by the citrus extension agents to inform growers about selected citrus topics. Attendance exceeded 300 growers from throughout the state as well as a few international growers. Twelve presentations highlighted psyllid and HLB management, foliar applications of nutritionals and insecticides as well as topics on genomics and cultural management. All presentations (video and viewable) are posted to the citrus agents’ website at: http://citrusagents.ifas.ufl.edu/events/GrowersInstitute2011/GrowersInstitute2011.htm . Additionally, presentations from the 2008, 2009 and 2010 Institute programs are also available to provide reference information from earlier programs. Over the 4-yr period, psyllid and management of greening have been a major focus of the Institute program. Program support for meals is provided by sponsors with the rental fees and video recordings being supported from funding from the CRDF. The Mid Florida Citrus Foundation field day in May near Avalon, FL was the site of an Extension outreach effort highlighting various citrus management issues (HLB, psyllid, weeds and environmental factors). This field day had more than 150 growers participating in the educational program. Citrus extension agents collectively conducted more than 110 field visits during the 3-month period to discuss citrus production management issues with growers. Additionally, meetings and newsletters in each of the agent’s local area continue to provide supporting information and educational opportunities to strengthen citrus management practices and to address local issues and problems. Citrus HLB (greening) photo series continues to post monthly photos of the same trees at selected locations around the state. This web-based series allows growers to view the condition of specific HLB infected trees over time. Each agent collects photos from 4-6 sites within their respective area. All trees have been infected for at least 2-3 years and have tested HLB positive. Efforts are also devoted to ensuring growers have ample opportunity to obtain continuing education units (CEUs) by attending area meetings and by reading and completing tests in Citrus Industry magazine and on the web (Citrus Agents’ web site) to allow renewal of restricted use pesticide licenses. The licenses are essential to maintaining properly trained and licensed employees who must supervise or use insecticides in the battle to suppress psyllid populations. Agents are also actively involved in related areas to ensure urban groups also are aware of HLB and its impact on the Florida citrus industry.
Nutritional spray programs applied directly on the foliage of orange trees for management of the Huanglongbing (HLB) disease have been investigated in the last few years to maintain tree health and yield. The impact of these programs on HLB infected fruit flavor and aroma is unknown. This study looked at the effects of 3 different foliage nutritional spray programs (Maury Boyd, Keyplex and Wettable Powder) applied to healthy and HLB infected trees on the flavor and composition of their fruit, and compared them with the fruit treated with a conventional spray program. Three types of fruit samples were selected: healthy orange fruit from non-infected trees (healthy), asymptomatic fruit from HLB infected trees (HLB), and symptomatic fruit from HLB infected trees (HLBs).Four harvest times and two cultivars (‘Hamlin’ harvested in December 2009, December 2010 and January 2011, and ‘Valencia’ harvested in April 2011) were investigated. For all three harvests of ‘Hamlin’ fruit, HLB and HLBs juice samples for all treatments were significantly different by smell and by taste from the conventionally treated healthy juice, with the exceptions of conventionally treated HLB (harvested in December 2009) and the HLB Maury Boyd treated juice (Harvested in January 2011) which were not significantly different by smell from the healthy control. Generally, the panelists described the differences as being bitter, grapefruit like, more sour, astringent, and metallic. The limonin content was usually higher in the HLB and HLBs juice, regardless of the nutritional programs, while the nomilin content was found higher or at similar levels compared to the conventionally treated healthy juice. For the December 2009 harvest, the sugar content in HLB and HLBs juice samples of all treatments were the same as the levels as those found in conventionally treated healthy juice, while lower sugar content in all HLB and HLBs juice samples (regardless of the treatment) compared with healthy fruit were measured in the December 2010 and January 2011 harvests. The Valencia harvest in April, 2011, exhibited differences between healthy, HLB and HLBs for the conventional spray group, but less and inconsistent differences were observed between healthy, HLB and HLBs for Valencia juice from the nutritional spray groups. In the ‘Valencia’harvest of April 2011, conventionally treated HLBs juice was judged significantly different by taste compared with the healthy control. The HLBs juice was described as being sweeter, more sour, more tart, more bitter, metallic, off-flavored, and having more of a fruity-non-citrus flavor than the control. No significant difference by smell or by taste was observed between the Keyplex treated juice samples (healthy, HLB and HLBs) and the conventionally treated healthy control. Healthy Maury Boyd treated juice did not differ either by taste or smell from the conventionally treated healthy control, but the Maury Boyd HLB and HLBs juice sample were both significantly different by smell and taste. No clear conclusions can be drawn at this point whether the nutritional programs really have a beneficial impact and alleviate the negative effects of HLB disease on fruit flavor and composition. In most cases, the panelists could distinguish between a conventional treated healthy control and a sample that received a nutritional spray. Although, no significant difference in taste or smell were observed in the ‘Valencia’ harvest for the Keyplex nutritional treatment, there was either a difference between the HLB and HLBs samples from the conventional treatment compared with the healthy control. More harvests would be necessary to draw a conclusion. Also the effect of volatiles on juice aroma should also be investigated.
Objective 1. Localization of Liberibacter asiaticus (Las) in the Asian citrus psyllid (ACP). Our qPCR study on dissected organs and tissues of ACP (Ammar et al., 2011, Ann. Entomol. Soc. Amer. 104:526-533) strongly suggested that the salivary glands constitute an important infection and/or transmission barrier to Las in the psyllid vector. This may partly explain why the proportions of Las-positive psyllids (by PCR) are normally much higher than those that can actually inoculate Las into citrus plants. A second study on the distribution of Las in various organs and tissues of ACP using fluorescent in situ hybridization (FISH) was completed and accepted for publication (Ammar et al., 2011, J. of Phytopathology, in press). Specific DNA probes with FISH and confocal laser scanning microscopy detected Las in the filter chamber, midgut, Malpighian tubules, hemolymph, salivary glands, ovaries and in muscle and fat tissues of Las-infected ACP. Our results showed the near-systemic infection of ACP by Las, indicating that Las may replicate and/or translocate into all these organs and tissues, and confirmed our PCR results suggesting that the salivary glands constitute a barrier to Las infection and/or transmission by ACP. Objective 2. Elucidation of various acquisition and transmission parameters between ACP and Las. [A] We developed a new ‘detached-leaf assay’ method that potentially speeds up Las-inoculativity assays on ACP from the current 6-12 months (when using whole plants) to only 2-3 weeks (when using detached leaves). Las-infected ACP adults were tested for inoculativity by caging them singly or in small groups (5 or 10 per leaf) on detached healthy citrus leaves for 1-2 weeks (as described by Ammar & Hall, 2011, Florida Entomologist 94:340-342). These leaves were then assayed by qPCR 1-2 weeks later using two Las primers (Li and LJ900). The results of four trials on detached leaves using the more conventional Li primers are largely comparable to those recently reported for Las transmission when whole citrus seedlings were used for inoculation. Our results, a summary of which appears in “Proceedings of the 2nd. Inter. Res. Confer. on HLB held in Orlando, FL, Jan. 10-14, 2011”, indicate that detached citrus leaves can be used to test the inoculativity of ACP and that this new method can speed up inoculativity tests on citrus psyllids considerably, which should enhance various studies on HLB epidemiology and vector interactions. [B) We conducted two large experiments to study the effects of various acquisition access periods on Las-infected citrus plants (between 1 day and 2 wks) on both acquisition and transmission of Las by ACP nymphs and adults, as well as on Las replication in both life stages. Preliminary results indicate significant differences between nymphs and adults as well as between various AAP’s in the acquisition and transmission of Las by ACP. But we are still analyzing the results on the replication of Las in both nymphs and adults in these experiments that will be repeated for verification. [C] A study on the ultrastructure and morphometrics of ACP stylets and the food and salivary canals (inside the maxillary stylets) is underway using both transmission and scanning electron microscopy. This study is expected to reveal the role of these canals in Las transmission (or lack of) by ACP nymphs and adults. [D] In a field study on ACP adults collected from HLB-diseased citrus trees in 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.
Objective 1. Localization of Liberibacter asiaticus (Las) in the Asian citrus psyllid (ACP). [A] Our qPCR study on dissected ACP organs and tissues 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 infection and/or transmission barrier to Las in the psyllid vector. This partly explains why the proportions of PCR-positive psyllids are normally much higher than those that can actually inoculate Las into citrus plants. Our results (Ammar et al., 2011, Ann. Entomol. Soc. Amer. 104: 526-533) also suggested that Las may replicate or accumulate in both the alimentary canal and salivary glands of ACP. [B] We were finally able to study the distribution of Las in various organs and tissues of ACP by fluorescent in situ hybridization (FISH). Only one of the six protocols tested succeeded in the localization of Las in hemolymph smears and dissected organs of ACP adults that fed on Las-infected plants and in sections from infected leaves. Using specific DNA probes with FISH and confocal laser scanning microscopy, Las was detected in the filter chamber, midgut, Malpighian tubules, hemolymph, salivary glands, ovaries and in muscle and fat tissues of Las-infected ACP, as well as in the phloem of Las-infected leaves. Our results showed the near-systemic infection of ACP, which suggests that Las may replicate in various tissues of ACP, and confirmed our PCR results suggesting that the salivary glands constitute a barrier to Las infection and/or transmission. This study was recently accepted for publication (Ammar et al., 2011, J. of Phytopathology, in press). Objective 2. Elucidation of various acquisition and transmission parameters between ACP and Las. [A] We have developed a new ‘detached-leaf assay’ method that potentially speeds up Las-inoculativity assays on ACP from the current 6-12 months, when using whole plants, to only 2-3 weeks when using detached leaves. Las-infected ACP adults 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 citrus leaves for one week (as described by Ammar & Hall, 2011, Florida Entomologist 94:340-342). These leaves were then assayed by qPCR, 1-2 weeks later, using two Las primers. So far, the results of three trials on Las-inoculativity tests on detached leaves using the more conventional Li primers are largely comparable to those recently reported for Las transmission when whole citrus seedlings were used for inoculation. Our results indicate that detached citrus leaves can be used to test the inoculativity of single or small groups of ACP and that this new method can speed up inoculativity tests on citrus psyllids considerably, which should enhance various studies on HLB epidemiology and vector interactions. An extended abstract on this new method is published in “Proceedings of the 2nd. Inter. Res. Confer. on HLB held in Orlando, FL, Jan. 10-14, 2011”. [B) We conducted two large experiments to study the effects of various acquisition access periods on Las-infected citrus plants (between 1 day and 2 wks) on both acquisition and transmission of Las by ACP nymphs and adults, as well as on Las replication in both life stages. We are still analyzing hundreds of individual psyllids from these two experiments with qPCR using primers to Las and to one of the psyllid genes for comparison. [C] In a field study on ACP adults collected from HLB-diseased citrus trees in 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.
The overall goal of this project is to identify a Bacillus thuringiensis (Bt) crystal toxin with toxicity to Asian Citrus Psyllid (ACP) and to further enhance toxicity by genetic modification. The long term goal of the proposed research is an effective management strategy for the psyllid and associated HLB disease that is more sustainable, less costly and more environmentally benign than the repeated application of broad spectrum insecticides. Our focus for the current reporting period has been on trypsin activation of the partially purified Bt toxins from the selected Bt strains. Trypsin activation of Bt toxins was carried out Iowa State University. Briefly, Partially purified Bt toxins were dialyzed against 50 mM Tris-Cl pH 8.5. Small aliquots of the dialyzed Bt toxins were incubated with bovine trypsin at a final concentration of 10% of the toxin concentration at 37 ‘C for 1 h. Proteolysis was stopped by adding 1 mM phenylmethylsulfonyl fluoride. The samples were boiled in denaturing SDS sample buffer for 5 min, separated on 10% (wt/vol) SDS/PAGE and stained with Coomassie blue. The SDS-PAGE profile of trypsin-treated Bt toxins exhibited multiple high intensity protein bands ranging from ~22 kDa to ~75 kDa . Based on the similarity of activated toxins bands, the toxin profiles of the nine Bt strains are categorized into the following five groups. Group one: Four Bt strains, each with five activated toxin bands of ~22, ~30, ~32, ~50 and ~70 kDa. Group two: Two Bt strains, each with three activated toxin bands of ~65, ~77, and ~85 kDa. Group three: One Bt strain with one activated toxin band of ~65 kDa. Group four: One Bt strain with one activated toxin band of ~85 kDa. Group five: One Bt strain with activated toxin bands of ~22, ~30, ~32, and ~70 kDa. Trypsin activated Bt toxins will be sent to Dr. David G. Hall, USDA-ARS for ACP membrane feeding assays for toxicity analysis.
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. Those findings were published in article entitled: “Discovery of novel SecA inhibitors of Candidatus Liberibacter asiaticus by structure based design” on Bioorganic & Medicinal Chemistry Letters Volume 21, Issue 14, 15 July 2011, Pages 4183-4188. Currently, the second screening was evaluated with twenty compounds identified and five compounds were determined to have nano molar activity against SecA by In-vitro studies. The most active five compounds will be used to apply on HLB infected trees for preliminary study in greenhouse and to identify more active compounds to develop an agrochemical/pesticide for HLB bacterial infections.
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) and annotation is on going, while three more genomes are in the gap closing stage. For XacAw, manual curation is currently in progress. 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. Illumina, 454 sequencing and optical mapping were used to obtain a complete genome sequence of Xacm strain FL1195, 4.9Mb in size. The strain lacks plasmids as compared to other citrus pathogens. Phylogenetic analysis revealed that this pathogen is very close to the tomato bacterial spot pathogen Xcv 85-10 with a completely different host range. We also compared Xacm to the genome of citrus canker pathogen Xac 306. Comparative genomic analysis showed differences in several gene clusters like Type 3 effectors, Type 4 secretion system, lipopolysaccharide synthesis and others. In addition to pthA, effectors such as xopE3, xopAI and hrpW were absent in Xacm while present in Xac. These effectors might be responsible for survival and reduced virulence of this pathogen on citrus compared to Xac. We also identified unique effectors in Xacm that may be related to the different host range as compared to Xac. Xacm also lacks various genes such as syrE1, syrE2 and RTX toxin family genes, which were present in Xac. These may be associated with distinct virulence of Xacm and Xac. Comparison of the complete genome sequence of Xacm to Xac and Xcv provides valuable insights into evolution of xanthomonads and emergence of new strains with different host range and distinct virulence.
Citrus canker is an economically important bacterial disease of most commercial citrus cultivars resulting in significant losses worldwide. Spread of citrus canker has been a severe problem to the citrus industry of Florida. Understanding the genetic mechanism of release of Xanthomonas axonopodis pv. citri (Xac) from citrus canker lesions will help develop effective control and containment strategies to stop citrus canker pathogen from spreading. The goal of the proposed research is to understand the genetic mechanism of release of Xac from citrus canker lesions. The specific objectives are to: 1. characterize critical genes involved in release of X. axonopodis pv. citri from citrus canker lesions; 2. understand the release mechanism by studying the host response of citrus upon infection by Xac wild-type strain and mutant strain(s) affected in release from citrus canker lesions. We have identified 12 EZ-Tn5 transposon mutants of Xac with reduced capacities of release from citrus canker lesions. The citrus canker lesions will not rupture, thus do not release bacteria as normal lesions caused by the wild-type strain Xac. Our preliminary analyses indicate the 12 mutants released onto the citrus leaf surface from water-soaked spots were significantly less compared to the wild-type strain Xac A. Identification of the mutated genes in those mutants is underway.
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.
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