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.
Mid Florida Citrus Foundation (MFCF) a 501c3 not for profit organization which has supported (past 22 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. The MFCF recently installed fertilizer tanks, pumps, timers, microsprinkler 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. The FCPRAC funds have been used to implement these practices which have resulted in a match of grant dollars from the EQIP grant. In essence every dollar of FCPRAC monies used to create additional research grove is matched with additional federal grant funds. 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 and new variety evaluation in Florida. 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. Additional projects have included the upgrading of an irrigation system on a 20 acre block which is subdivided into plots giving the ability to perform state of the art replicated irrigation trials. Also the farm managers office which was in disrepair was repaired and painted. Future expansion plans for 2011 with FCPRAC support include up to fifteen acres of new grove for ACP control trials. ACP control research requires young trees which flush more vigorously. Existing blocks used for ACP research are becoming more mature and less ideal of ACP research work. The newly planned planting would ensure the continued availability for researchers. In this past year over twenty citrus research trials have been conducted at the MFCF. Additionally MFCF supports extension efforts and vice versa. 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.
From July 1, 2009 through June 30, 2010, the Southern Gardens Diagnostic laboratory processed, ran, and reported on 46,272 grower samples. Samples were received from 874 different groves and from 53 different submitters. For the same period of time during the previous year (July 1, 2008 to June 30, 2009), 44985 grower samples were run. Since it’s inception, the Southern Gardens Diagnostic laboratory has run over 141,338 grower samples submitted by growers, homeowners, extension agents and researchers throughout the state. In addition to the grower samples, the lab has processed 16,898 samples for Southern Gardens, 5,703 individual psyllid samples, and approximately 5,000 research samples for an approximate total of 168,939 samples run. The lab continues to make progress to improve efficiency and is in the process of automating the DNA preparation procedures using a liquid handling robot and magnetic bead separation technology. Also a second HLB panel test is in the works, whereby replicated tissue samples (in ETOH) will be sent to laboratories across the US to compare and contrast the various testing methodologies being used by the various laboratories in the US. The lab has provided hands-on training for researchers from multiple labs within the United States and abroad.
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, funds were transferred for collaboration, plot maintainence and experimental conduct. Permission was obtained to use an IAPAR farm, in Xambr’, Parana state, located 350 km west from Londrina and 250 km west from Maring’ where replicated field plots were established. The cultivar used is P’ra on Rangpur lime, two years of age at the beginning of the experiment. Windbreaks were completed and plants were be established in Mid April 2010. The experiment has now been initiated 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 and are in the final stages of calibration. In fact, a whole new design of leaf wetness controller was developed for the project which has much greater sensitivity and provides better environmental control. 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 been designed to examine the survival characteristics of bacterial pathogens under field conditions and have been initiated.
To determine the effect of prewashing on Xcc survival on asymptomatic lemons, apparently healthy fruit were harvested from infected trees in an affected orchard in Tucum’n, Argentina, and grapefruit from Florida. Replications of 5 fruit were harvested for each of the following treatments: 1) non-treated check, 2) chlorine for 2 min, 3) chlorine for 2 min followed by detergent (Neutro Deter N Sinner) for 20 seconds, 4) prewash with water followed by chlorine for 2 min, 5) prewash with water followed by chlorine for 2 min followed by detergent for 20 seconds. To determine the population of canker bacteria the wash solution was assayed by injection-infiltration of the suspension into two immature leaves on greenhouse grown grapefruit seedlings via needle-less syringe into 8 sites on the surface of each leaf. At 14 days after inoculation, the total number lesions per leaf were counted from all injection sites. There were no significant differences among the treatments, but the lesion counts were low (maximum of 3) and the frequency of zero lesions relatively high across all treatments. Nonetheless, there was a trend suggesting that chlorine treatment slightly reduced the number of lesions recovered, and chlorine treatment after prewashing the fruit, with or without detergent was beneficial in reducing the number of Xcc recovered from the fruit. The effectiveness of prewashing fruit with detergent at the same time or immediately after chlorine appears to be beneficial. Results from the two prewash trials, showed that chlorine as a disinfestant alone did not greatly reduce surface bacterial populations. However when chlorine, detergent, or detergent plus chlorine was added as a prewash, followed by a wash usually with SOPP (a disinfestant with detergent activity), there was a reduction in surface bacterial populations. The effect of a prewash was most apparent when SOPP/detergent was included. Prewashing of the fruit, especially with detergent, effectively wets the surface by lowering surface tension, which in turn allows the chlorine greater access to surface Xcc, as well as removing debris such as dirt, sooty mold, and scale insects from the fruit surface that could tie up the chlorine and/or SOPP which potentially reduce the effectiveness of the disinfestation treatment. By removing surface contaminants, the fruit surface was more exposed and, therefore, more effectively disinfested by the chlorine and/or SOPP that followed in the subsequent fruit wash. Currently, in citrus packing lines, the normal procedure is to use a prewash of water, or water plus chlorine, followed by a second wash with SOPP. A simple and low-cost recommendation resulting from these studies would be to reverse the procedure and prewash the fruit with detergent (such as SOPP), and follow this by a wash of chlorine with approximately 45 second contact time on the fruit. Based on the results of these experiments this process will more effectively reduce survival of Xcc and other contaminants on fruit after passing through the packing line. USDA APHIS developed a new Pest Risk Assessment based on this work, proposed a new rule for shipping fruit potentially infected Xcc. The rule has passed and will be implemented during the 2009-10 fruit season, greatly expanding markets for Florida citrus and other citrus infected areas worldwide. Three additional months of data have been now been collected to compare the production of bacteria from foliage, stem and fruit lesions. Early in the season, all sources of inoculum (foliage and fruit) produced copious quantities of Xcc. However, interestingly fruit lesion bacterial populations appear to be declining as the season progress and the fruit mature; apparently substantiating further the reduced numbers of Xcc produced from mature fruit compared to immature. The resulting publication served as the justification for USDA’s APHIS to promulgate a new regulation (7 CFR Part 301, FR Doc E9-15508 ‘Citrus Canker; Movement of Fruit from Quarantined Areas). This rule eliminates the requirement that fruit lots be inspected at the packinghouse and found to be free of canker symptoms and reduces industry costs by nearly $15 million annually.
This is the report for the last quarter of year 1. Sensory impacts and flavor and aroma changes in HLB fruit: Critical juice aroma volatiles were lower in symptomatic (SYM) compared to healthy (H) juices. Static headspace solid phase microextraction volatile concentration and GC-MS analyses of juice from H, asymptomatic (ASYM) and SYM juices were carried out in triplicate for both pasteurized and non-pasteurized Hamlin orange juices. Of the 67 volatiles measured and identified, 20 volatiles from the SYM juices were 25-400% lower than H. Some of these were known aroma impact volatiles such as: linalool, ethyl butanoate, ethyl hexanoate, and E2-hexenal. In contrast, only 15 volatiles were lower in ASYM juices compared to H. Pasteurization decreased total juice volatiles more than 80% on average for all sample types. Two fruit samples from SYM, ASYM and H were used for studies described below. After squeezing, juice was strained and half of each sample was pasteurized, resulting in six test groups for each sampling date. Juice analysis included % oil, Brix, acid, color and ratio determination. No significant differences were seen between pasteurized and fresh juice. Juice from SYM fruit had lower ratios than ASYM or H fruit due to consistently higher acid levels. Consumer sensory analysis was conducted on juice. No clear preferences for pasteurized vs non-pasteurized (juice), but significant differences in preference, sweetness orange intensity were noted among the juice samples from SYM, ASYM and H fruit. Preference was related to brix/acid ratio levels, with higher ratios associated with greater preference. Physiological changes in HLB fruit: An iodine-starch test on fruit flavedo revealed high starch content in healthy (HFF) but not in symptomatic mature green and mature flavedo (SYMFF). Flavedo starch content from zinc deficient and water stressed trees was similar to HFF. Quantification showed that starch was 70 and 90 % reduced in SYMFF compared with HFF in mature green and full mature fruit, respectively. Starch accumulation in ASYMFF was less than HFF. Microscopic observation confirmed the above. Four starch biosynthetic genes were down regulated, and three of four starch degradation genes were up regulated in ASYMFF and SYMFF compared with HFF. Mature fruit drop in SYM trees reached 18% after monitoring trees 20 days, whereas no drop was evident in ASYM or H. Fruit detachment force significantly decreased in SYM and abscission marker genes increased compared with H. Although whole SYM fruit produced less ethylene, 5- and 2-fold more ethylene was produced in the stem-end area of SYM and ASYM, respectively, compared with H. At this time of year (April-June), there was no significant difference in % leaf drop or leaf detachment force in SYM and ASYM leaves after 20 days compared with H leaves. Expression of abscission marker genes in SYM and ASYM leaf abscission zones slightly increased compared with H, indicating potential for leaf drop to occur. Yield, peel color, fruit size and seed abortion in HLB fruit: Ten SYM trees and 10 H trees were harvested from two commercial citrus groves (1 Hamlin, 1 Valencia) to characterize and quantify yield changes (pieces of fruit, fresh weight and fruit size) due to HLB infection. SYM trees had significantly smaller average fruit size and fewer fruit per tree than H trees. Further analysis ongoing. Extension and education: 1) Aroma Differences between HLB/greening Symptomatic and Control ‘Valencia’ Orange Juice. Dagulo, Danyluk, Goodrich, Sims, Valim, Spann, Rouseff; Florida State Horticultural Society, June 8, 2010. 2) Dagulo, Danyluk, Spann, Valima, Goodrich-Schneider, Sims, Rouseff. 2010. Chemical characterization of orange juice from trees infected with citrus greening (Huanglongbing). J. Food Sci. 75: C199-C207.
The purpose of this project is to preserve citrus germplasm in Florida that is threatened by loss due to huanglongbing (HLB) and citrus canker. More experiments are underway to evaluate different antibiotics and temperatures to eliminate graft transmissible prokaryotes from bud sources. In Riverside we are using stubborn, caused by Spiroplasma citri, infected material grafted onto rootstock liners and the tomato psyllid yellows disease causal bacterium, Candidatus Liberibacter psyllaurous grafted onto tomato as model systems. In addition to combinations of antibiotics, treatments include heat treatment and prolonged chilling of the buds. Initial results look promising for prolonged chilling treatments. Accessions established at ARS, Ft. Pierce are being moved to Gainesville for shoot tip grafting by using budsticks in culture media. Experiments are underway to determine how best and at what age the shoot tip grafted material may be safely shipped to Riverside. Once received in Riverside, the material would be established in quarantine, then subjected to biological and laboratory indexing. The Florida accessions already established in Riverside are undergoing therapy and biological and laboratory indexing for release from quarantine status.
Deliverables are summarized below (research results are being compiled and will be published in the near future): Task 1 (CuSiNP/NG design, synthesis, characterization and protocol optimization): A series of copper loaded silica nanoparticle (NP)/nanogel (NG) formulations (CuSiNG 100, CuSiNG 200, CuSiNG 300, CuSiNP 100 and CuSiNP 200) were synthesized during protocol optimization process. CuSiNP 200 and CuSiNG 300 are two optimized nanoformulations (objectives) that were studied extensively. Silica nanogel (SiNG) without copper was synthesized as negative control. Systematic materials characterization was performed on two optimized nanoformulations (i.e. CuSiNP 200 and CuSiNG 300). The HRTEM images confirmed formation of crystalline copper oxide nanoparticles (<10 nm size) embedded in silica matrix. HRTEM based elemental mapping confirmed presence of Si, Cu and O, showing that Cu is distributed uniformly throughout the matrix. The X-ray diffraction (XRD) study showed that the material is overall amorphous. The presence of ultra-small (<10 nm) crystalline copper oxides could not be detected within the resolution of XRD. X-ray photoelectron spectroscopy (XPS) indicates the co-existence of Cu(I) and Cu(II) within the same silica matrix. HRTEM study (measurement of crystallite line spacing) indicates formation of Cu(I) oxide nanocrystals, thus supporting XPS results. The BET measurements (based on nitrogen adsorption/desorption isotherms) was done for CuSiNG300 material. Data showed that the material is highly porous (pore size 7.4 nm and pore volume 1.023 cc/g) and possess enormous surface area (336.1 m2/g). We expect similar porosity and surface area will be obtained for other formulations as their HRTEM based morphologies are similar. FT-IR studies were performed on Cu salt, SiNG, CuSiNP and CuSiNG. Characteristic FT-IR peak shift in the frequency range between 800 and 1200 cm-1 indicated binding of Cu to silica. EDTA (a strong Cu chelator) extracted all Cu (amorphous and crystalline) from the CuSiNP and CuSiNG materials when treated overnight (confirmed by the HRTEM study). Atomic absorption spectroscopy (AAS) was performed (instead of ICP and UV-VIS) to estimate Cu loading in the formulations. The maximum Cu loading in the optimized formulations, CuSiNG 300 and CuSiNP200 was found to be ~33 w/w % and 45 w/w% respectively. The copper ion release from CuSiNG and CuSiNP was tested for a period of four weeks in rain water and DI water. No detectable amounts of copper ion release from silica matrix was observed by AAS, indicating that CuSiNP/CuSiNG material is likely interacting directly with the bacteria cell surface. The strong adherence property of nanoformulation was assessed at the microscopic level using fluorescently labeled SiNG material (FLSiNG, resistant to 16 mins continuous wash under running water due to high surface area and strong intermolecular forces). The viscosity of the CuSiNG 300 (20-22 cps) and CuSiNP 200 (11-13 cps) was measured using a Brooke field viscometer. Task 2 (Assessment of the antimicrobial activity of the CuSiNP/NG nanoformulation): The anti-bacterial efficacy of all the CuSiNG/NP formulations was evaluated with E. coli as well as Xanthomonas Alfalfea (a citrus canker surrogate) as model systems using a disc diffusion assay. The CuSiNG/NP formulations exhibited reproducible and improved results over the positive controls, Kocide 3000 and Copper sulfate against both E. coli and X. alfalfae. In collaboration with James H. Graham, preliminary field trials were conducted last season with CuSiNG 100, CuSiNG 200 and CuSiNP100 nanoformulations (Kocide 3000 and SiNG served as controls). Improved protection against canker incidence was obtained from all nanoformulations. Field trial is being continued this year. Task 3 (Develop large-scale nanoformulation production set-up): All formulations were synthesized both in small scale (up to 1.0 L volume) and large scale (up to 4.5 gallons). We have also established a synthesis set-up with 20 gallons capacity.
The purpose of this project is to preserve citrus germplasm in Florida that is threatened by loss due to huanglongbing (HLB) and citrus canker. Accomplishments thus far and areas of work: ‘ Using input from stakeholders and scientists from a meeting in June 2009, a priority list has been made of citrus germplasm in Florida which needs to be recovered, and new accessions are being added as time goes on. ‘ Fifty unique accessions from around the world, believed only to be at the USHRL Farm in Ft. Pierce, and exposed there to HLB, have been propagated in a secure greenhouse for conservation. ‘ Eleven accessions collected from Florida are currently nearing the final stages of therapy and indexing in the Riverside facility and another five accessions are about mid-way through therapy for release from quarantine. Once released from quarantine, they can be returned to Florida using the ‘Citrus Passport’ protocol. ‘ Antibiotic treatments are under investigation for rescue of breeding material threatened from HLB. Buds from HLB+ trees have been tested with three levels of an antibiotic cocktail for 2 time periods and were grafted onto clean rootstock liners. Treatment with the highest antibiotic level for the longest duration reduced take and growth of scion shoots, but lower levels were not different from controls. Las evaluations via qPCR will be conducted in the next quarter. Experiments will be extended to include vacuum-infiltration of antibiotic into buds prior to propagation. In Riverside, similar experiments have been conducted using Spiroplasma citri (causal agent of stubborn) and Candidatus L. psyllaurous in tomato as model systems. In addition to antibiotics, treatments include prolonged heat treatment of budwood stocks under different temperature regimes, different antibiotics, and prolonged chilling of budwood. ‘ An additional series of propagations were made using 22 plant genotypes which were Las infected; 32% of the resulting propagated plants were Las- using the Wenbin Li primers/APHIS diagnostic protocol. ‘ Additional priority hybrids from the breeding program, found only at the USHRL Farm in Ft. Pierce, and exposed there to HLB, were identified and budwood with or without antibiotic treatment used to propagate in the greenhouse for conservation and further evaluation. ‘ In a project led by Yongping Duan on which we collaborated, a system was developed for testing antibiotics for elimination of Las using an infected periwinkle rooting model system. A method was verified to root HLB-infected citron cuttings, and the strategy previously used with periwinkle is being extended to Citrus using easily these easily-rooted citron. Effects of antibiotic treatments on Las titer will be determined using this in-vitro system in the next quarter. ‘ Several antibiotics and other anti-microbial compounds were tested in-vitro for their ability to suppress growth of two close relatives of Liberibacter (Sinorhizobium and Agrobacterium). In vitro results differed markedly from results using the periwinkle bioassay, suggesting that antibiotics may also differ in their ability to move systemically, or their ability to remain active in the living plant tissue. ‘ Shoots from priority accessions held in a secure greenhouse at Ft. Pierce will be taken to Gainesville for shoot tip grafting (STG). Experiments are providing data on the optimal age/conditions to ship recently STG material from Gainesville to Riverside for indexing.
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, funds were transferred for collaboration, plot maintainence and experimental conduct. We have obtained permission to use an IAPAR farm, in Xambr’, Parana state, located 350 km west from Londrina and 250 km west from Maring’ where replicated field plotshave been be established. The cultivar used is P’ra on Rangpur lime, two years of age at the beginning of the experiment. Windbreaks within the plots have now been completed and plants will be establishein Mid April 2010. The following treatments will be 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 will be estimated on multiple branches on each tree treated as the number of leaves per branch infected. We anticipate running these plots for 2-3 more years. The development of the Programmable leaf wetness controller (PLWC) software is written, debugging is complete and the control program is working well. New leaf wetness sensors were designed and constructed and are in the final stages of calibration. A final circuit to control fans that facilitate wind generation in ambient environments has now been completed and being tested and calibrated. Initial trials trials have been designed to examine the survival characteristics of bacterial pathogens under field conditions and are just beginning this first quarter of 2010.
To determine the effect of prewashing on Xcc survival on asymptomatic lemons, apparently healthy fruit were harvested from infected trees in an affected orchard in Tucum’n, Argentina, and grapefruit from Florida. Replications of 5 fruit were harvested for each of the following treatments: 1) non-treated check, 2) chlorine for 2 min, 3) chlorine for 2 min followed by detergent (Neutro Deter N Sinner) for 20 seconds, 4) prewash with water followed by chlorine for 2 min, 5) prewash with water followed by chlorine for 2 min followed by detergent for 20 seconds. To determine the population of canker bacteria the wash solution was assayed by injection-infiltration of the suspension into two immature leaves on greenhouse grown grapefruit seedlings via needle-less syringe into 8 sites on the surface of each leaf. At 14 days after inoculation, the total number lesions per leaf were counted from all injection sites. There were no significant differences among the treatments, but the lesion counts were low (maximum of 3) and the frequency of zero lesions relatively high across all treatments. Nonetheless, there was a trend suggesting that chlorine treatment slightly reduced the number of lesions recovered, and chlorine treatment after prewashing the fruit, with or without detergent was beneficial in reducing the number of Xcc recovered from the fruit. The effectiveness of prewashing fruit with detergent at the same time or immediately after chlorine appears to be beneficial. Results from the two prewash trials, showed that chlorine as a disinfestant alone did not greatly reduce surface bacterial populations. However when chlorine, detergent, or detergent plus chlorine was added as a prewash, followed by a wash usually with SOPP (a disinfestant with detergent activity), there was a reduction in surface bacterial populations. The effect of a prewash was most apparent when SOPP/detergent was included. Prewashing of the fruit, especially with detergent, effectively wets the surface by lowering surface tension, which in turn allows the chlorine greater access to surface Xcc, as well as removing debris such as dirt, sooty mold, and scale insects from the fruit surface that could tie up the chlorine and/or SOPP which potentially reduce the effectiveness of the disinfestation treatment. By removing surface contaminants, the fruit surface was more exposed and, therefore, more effectively disinfested by the chlorine and/or SOPP that followed in the subsequent fruit wash. Currently, in citrus packing lines, the normal procedure is to use a prewash of water, or water plus chlorine, followed by a second wash with SOPP. A simple and low-cost recommendation resulting from these studies would be to reverse the procedure and prewash the fruit with detergent (such as SOPP), and follow this by a wash of chlorine with approximately 45 second contact time on the fruit. Based on the results of these experiments this process will more effectively reduce survival of Xcc and other contaminants on fruit after passing through the packing line. USDA APHIS developed a new Pest Risk Assessment based on this work, proposed a new rule for shipping fruit potentially infected Xcc. The rule has passed and will be implemented during the 2009-10 fruit season, greatly expanding markets for Florida citrus and other citrus infected areas worldwide. Three additional months of data have been now been collected to compare the production of bacteria from foliage, stem and fruit lesions. Early in the season, all sources of inoculum (foliage and fruit) produced copious quantities of Xcc. However, interestingly fruit lesion bacterial populations appear to be declining as the season progress and the fruit mature; apparently substantiating further the reduced numbers of Xcc produced from mature fruit compared to immature. The resulting publication served as the justification for USDA’s APHIS to promulgate a new regulation (7 CFR Part 301, FR Doc E9-15508 ‘Citrus Canker; Movement of Fruit from Quarantined Areas). This rule eliminates the requirement that fruit lots be inspected at the packinghouse and found to be free of canker symptoms and reduces industry costs by nearly $15 million annually.
(863) 956-8817
(863) 956-5894
700 Experiment Station Road
Lake Alfred, FL 33850
Email Us
