Under Objective 1 (Confirm biofilm formation by X. citri subsp citri [Xcc]) strains of Xcc and X. alfalfae pv. citrumelonis (Xac), the cause of citrus bacterial spot, were transformed with non-stable GFP and used to identify in situ their ability to survive and persist on leaf and fruit surfaces. When suspensions of the non-stable GFP strains were sprayed on leaf and fruit surfaces, bacterial aggregates were observed under confocal laser microscopy. Persistence of fluoresce in these aggregates confirmed the presence of metabolically active cells, but these cells had limited survivability without osmotic protection from desiccation by phosphate buffer. After inoculation of bacteria in water, scattered cells of the non-stable strain on the leaf or fruit surfaces rapidly lost fluorescence, i.e. were non-viable. Only cells in aggregates continued to fluoresce, i.e. maintained viability. These aggregates were restricted to sites on the fruit or the leaf surfaces that conserved moisture. Aggregates on leaves or fruits were located primarily in depressions where water availability was higher. Phosphate buffer acted to mitigate cell desiccation by buffering the matric and osmotic potential stresses on the plant surfaces. The aggregates resembled what has previously been described as biofilms. Under Objective 2 (Assess Xcc survival and biofilm formation under different environmental conditions and after disinfectant and bactericide treatments of leaf and fruit surfaces) persistence of metabolically active cells in biofilms explains the occasional isolation of viable Xcc cells from surfaces of asymptomatic fruits infested with Xcc even after rigorous disinfection treatments with chlorine and sodium-ortho-phenyl-phenate (SOPP). Recently, Gottwald et al. 2009 demonstrated that pre-washing with a detergent was the most effective treatment for disinfection of fruit infested with Xcc. Detergents may disrupt the aggregates, exposing Xcc to the disinfectants such as chlorine. Further investigations, now in progress, will clarify the effect of surfactants and bactericidal treatments on survival of Xcc in biofilms.
The citrus leafminer, Phyllocnistis citrella Stainton (Lepidoptera: Gracillariidae), is a major worldwide pest of citrus. Larval feeding by this insect facilitates proliferation of citrus bacterial canker, Xanthomonas axonopodis pv. citri. Most recently, we have achieved season-long disruption of P. citrella with a newly developed, emulsified wax dispenser of pheromone (SPLAT-CLM). A formulation containing a 3:1 blend of (Z,Z,E)-7,11,13-hexadecatrienal:(Z,Z)-7,11-hexadecadienal at a 0.2% loading rate of AI by weight and deployed twice per season (24 wk total) at 490g of formulation/ha caused season-long disruption of male moth catch in pheromone traps as well as reduced leaf infestation. Analysis of pheromone release from dispensers by gas chromatography revealed that effective disruption of P. citrella occurred at a deployment rate of 126 .g of (Z,Z,E)-7,11,13-hexadecatrienal/ha/h. Direct observation of moth behavior in the field suggested that disruption by this formulation occurred by a non-competitive mechanism. A formulation of the 3:1 attractive blend at a 0.02% pheromone loading rate caused only 2-6 weeks of disruption per deployment and did not reduce leaf infestation during mid- and end of the season evaluations. A formulation containing 0.2% of (Z,Z)-7,11-hexadecadienal alone and deployed at 490g/ha caused 6-7 wk of moth disruption to pheromone traps and did not prevent leaf infestation, while an identical formulation loaded with 0.02% (w/w) of (Z,Z)-7,11-hexadecadienal alone had no effect on P. citrella orientation to pheromone traps. The SPLAT formulation evaluated herein appears to be an excellent release device for (Z,Z,E)-7,11,13-hexadecatrienal given that approximately 100 d of steady release occurred following an initial brief (ca. 7 d) burst of higher release. The advantages of SPLAT as a formulation for P. citrella disruption include low cost of manufacturing, biodegradable and weather resistant characteristics, and flowability allowing machine application. Of the SPLAT treatments evaluated, the formulation containing a 3:1 blend of the two-component attractive P. citrella blend was the only one to cause season-long disruption as well as reduce leaf infestation. However, it required deploying 490g/ha of the SPLAT formulation containing 0.2% AI by weight twice per season, which amounts to deploying 2 g of P. citrella pheromone per ha per season. Although reduced P. citrella larval injury should contribute to reduced incidence of citrus canker infection, we were unable to document this hypothesis because the disease did not appear to be present in the citrus orchard where experiments were conducted. Mating disruption should be an effective alternative to insecticides for management of P. citrella and may reduce the incidence of citrus canker. Our ongoing investigations will continue to develop mechanized sprayers for optimizing deployment of this product. ISCA Technologies is in contact with the Florida citrus industry and working to get their mating disruption product registered for use in Florida citrus. Also, ISCA continues work on decreasing the cost of synthesizing the pheromone to make it more economically accessible. Although it is difficult to predict how long the registration process will take, we are hopeful that this product will be available for commercial use soon.
The Citrus Greening Biographical Database, found at [ http://swfrec.ifas.ufl.edu/hlb/database/ ], went live in January 2009 in cooperation with the Center for Library Automation at the University of Florida. The database now includes approximately 1,631 references with over 675 linked to the original source. All have been cross-referenced for accuracy. The database includes entries from refereed and non-refereed publications, presentations, websites, proceedings, grant reports, and book chapters. Entries have been collected from citrus producing and research areas around the world. Ninety-five percent of the entries are in English, the remaining 5% are in Spanish, Portuguese, Afrikaans, Japanese, Chinese, French, German, and Hebrew. All consern various aspects related to Huanglongbing (HLB), the associated bacteria (Candidatus liberibacter spp.), the vectors [Diaphorina citri Kuwayama, Trioza erytrea (Del Guercio)], effects on the tree health, management techniques, economic effects of the disease, etc. Access to the database and associated links is available to all users at no cost. Users are invited to contribute by providing relevant information from their own research. During the 0 months since the database went live, we have accumulated close to 4,000 visits from all over the world. This number indicates that the database has become an important source of information for a growing number of researchers, growers, and other interested clientele within the US and abroad. We are continuing to add unique references and links to the current entries. The popularity among researchers, students, and growers of this database has surpassed our expectations and we hope to maintain this important source of information for years to come, adding new references, more links to original publications, and updating for the benefit of the citrus community. However, this will require new funding for the 2010-2011 fiscal year and beyond. Presentations related to the citrus greening database database – Arevalo. H. A. and P. A. Stansly. 2009. Monitoreando el Psillydis Asiatico de los Citricos en el Campo y el Internet (Monitoring the Asian Citrus Psyllid in the field and the Internet). XIII Simposio Internacional de Citricultura. CD Victoria, Tamaulipas, Mexico. – Arevalo, H. A., A. B. Fraulo, and P. A. Stansly. 2009. Update on the Citrus Greening Bibliographical Database. (Poster). Florida Entomological Society Annual Meeting. Ft. Myers, FL July 2009 – Arevalo, H. A., G. Snyder, and P. A. Stansly. 2008. The citrus greening Bibliographical Database, a New Tool for Researchers, Students and Growers. (Poster). International Research Conference on Huanglongbing. Orlando, FL December 1-5, 2008 Known outside links to the database. – FCPRAC request for proposals 2009 [http://www.fcprac.com/proposals-2009.html] – Florida Entomological Society. Lutz, FL. [http://www.flaentsoc.org/] – The grower. The Grower’s Citrus Greening Resource Center. Lincolnshire IL ] [http://www.growermagazine.com/CitrusGreeningResearchCenter/tabid/89/Default.aspx] – University of Florida Entomology and Nematology Pest Alert. Gainesville FL. [http://www.entnemdept.ufl.edu/pestalert/] – University of Florida- IFAS- Extension CREC. Lake Alfred, FL [http://www.crec.ifas.ufl.edu/extension/greening/links.htm]
PROJECT PARTICIPANTS: Ronald P. Muraro, Robert Allen Morris and Fritz M. Roka This project is to develop an economic model to evaluate emerging management solutions to HLB/citrus greening as they are developed by other HLB researchers or industry representatives. An Excel citrus investment model developed by UF/IFAS that uses the income approach to asset valuation is being modified to give it flexibilities to cover a range of potential variables needed to evaluate emerging HLB management technologies. The economic model has been designed so that cost, yield data, reset-replant strategies and annual tree loss rates specific to a particular grove situation can be entered by the user and override the default values of the Excel model. Our initial focus of this project has been to update all the default cost, yields and tree lost rates of the Excel model. A user’s guide has been written and should be available by September. The model in its current version has been provided to a few growers to field test. Work has begun in modifying the citrus investment model so that it will adjust yields of remaining trees according to the number of resets that are replanted after trees are removed ( HLB, blight/decline, etc.). The approach is to allow remaining trees to grow and increase tree yields based on the total trees removed within the three tree age groups: 1-3 years old; 4-10 years old; and greater than 11 years old. Also, we are looking at an older UF/IFAS Excel citrus reset model to see if this could also be a tool for growers to use when deciding to replant trees in an HLB problem block. Our next work on the Excel citrus investment model will be to add the ability to evaluate the new HLB management strategies being developed by other researchers. High density plantings can now be evaluated; what is needed is to decide how to incorporate the various psyllid control methods being field tested and the different tree nutritional programs being evaluated.
The Southern Gardens Diagnostic Lab started receiving grower samples on October 31, 2006. Through July 13, 2009, the lab has processed and tested 96,866 grower samples. In addition to the grower samples, an additional ~18,000 internal Southern Gardens samples have been processed over the same period. Although sample volume varies by month due to the temporal variability in symptom expression, monthly sample volume has steadily increased. For the period of time from January 1, 2008 through June 30, 2009, the lab received 70,959 outside grower samples or an average of 3,942 samples a month. During that same period of time, the lab analyzed 71,993 outside grower samples or an average of 4,000 samples a month. Therefore on the whole, the lab capacity met the demand. However, since the sample volume was not constant over the period, there were extended periods of time where the sample volume greatly exceeded the lab capacity resulting in extended turn-around times between sample receipt and the lab analysis. For example, during the period of time from August, 2008 through January, 2009, the average number of grower samples received per month was 5,606 samples a month (exceeded capacity by 1,600 samples a month) resulting in extended turn-around times in the spring. The current level of staffing is 2.5 FTE but additional funding has been received from FCPRAC to increase staffing in order to more adequately meet the expected sample volume going forward. Samples have been received from over 224 individual entities and over 1700 different groves (and many more individual blocks). As noted earlier, there is some seasonality in the submission of samples that is also accompanied by a similar seasonality of the frequency of detection. The months with the highest frequency of detection were August through January (mean = 81% of samples positive) whereas the months with the lowest frequency of detection were March through June (mean=50% of samples positive). The highest frequency of infection has been found in sweet orange and grapefruit samples whereas the lowest frequency of infection has been found in Sunburst, Murcott and Orlando Tangelo. With respect to tree age and size, the highest frequency of infection has been found in trees 6-9 years of age and trees 6-9 ft tall. The lowest level of infection has been found in very young trees (<2years old) and in very large trees (>14ft tall). Based on row and tree data supplied by the growers, trees at the edge of the groves (both row ends and outer rows) appear to be more likely to become infected or express symptoms. The lab has received visitors from multiple states and countries and has provided formal training to researchers from California, Florida, Argentina, Belize, Brazil, Costa Rica, and Mexico. Improvements continue to be made in the methodology resulting in more efficiency in the laboratory and reduced costs. Beginning in June, 2008, the SGDL has begun receiving psyllid samples from growers with the aim of gathering statewide temporal psyllid HLB incidence data for use in the development of control programs.
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 meeting was held at the University of Florida (UF)’s Citrus Research and Education Center, Lake Alfred on June 30, 2009 with the purpose of discussing and identifying what citrus germplasm needs to be rescued from Florida and to prioritize the order of rescue. The participants of the meeting include members of the Crop Germplasm Committee for the USDA ARS National Clonal Germplasm Repository for Citrus and Dates, Riverside with additional participants invited to provide better representation of Florida needs and information as to current status of Florida germplasm. Attending the meeting from Florida: L. Williams, W. Dixon, P. Chaires, F. Gmitter, M. DuBois, W. Castle, E. Stover, N. Jameson, M. Kesinger, J. Grosser, J. Burns, and G. McCollum; from California: R. Lee, R. Krueger, M. Roose (Phone), L. Rose, T. Kahn, M.L. Polek, G. Vidalakis, K. Kosta (phone); G. Wright from Arizona; G. Wisler (USDA ARS), K. Williams (USDA ARS), and E. Rudyj (USDA APHIS) from Maryland. Lists of germplasm already at Chiefland and varieties currently undergoing clean up at Winter Haven were provided by M. Kesinger and P. Sieburth, Florida Citrus Budwood Registration Bureau. The protocol to be used for recovery, therapy, and release from quarantine of the selected germplasm was discussed, as well as the differing needs of the USDA ARS and the UF citrus breeding groups. Preliminary results of L. Williams using standard citrus protocols for thermotherapy (Frison, E.A. and Taher, M.M. (eds.). 1991. FAO/IBPGR Technical Guidelines for the Safe Movement of Citrus Germplasm. Food and Agriculture Organization of the United Nations, Rome/International Board for Plant Genetic Resources, Rome.) suggest that maybe only a small percentage, depending on variety, may be recovered free of HLB. Stover and Lee have been developing alterative approaches to therapy by use of heat and antibiotics, and the group encouraged further research in this direction. It was agreed that interested parties would each assemble a list of candidate germplasm to be considered for recovery. This list would include comments on ‘Level of risk of loss’, ‘Is the selection currently protected elsewhere?’, ‘Stage of development’ (as relates to breeding or commercial release), ‘Patented?’, ‘IP protected’, ‘Value to research and industry’, ‘Current disease status’, ‘Allelic diversity’, ‘Positive horticultural characteristics’, ‘Uniqueness’, and the ‘Sponsor’. The lists will be forwarded via e-mail at the end of August, and circulated among the group for consideration and prioritization. The Repository, Riverside, has received 17 accessions from the USHRL, Ft. Pierce, and four from DPI, Winter Haven. These accessions are currently going through therapy and indexing. A follow-up meeting to assess progress and discuss varieties to be prioritized for the coming year will be held in January 2010 in conjunction with the Plant and Animal Genome Workshop in San Diego, CA.
More field data were collected from HLB infected and healthy trees using the four-band active optic sensor that was developed during last year. Five different classification algorithms (support vector machine, k-nearest neighbors, Neural networks, Logistic regression, and Decision tree) were applied to the data. In the classification each measurement consisted of four reflectance values at 570, 670, 800, and 900 nm. In addition, 12 indices were computed based on these four numbers. Therefore, each measurement was finally represented with 16 different numbers. This was done for all classification methods except for the support vector machines (SVM) method; for SVM, only the original reflectance values were used to represent each measurement and no indices were computed. The best classification methods were support vector machine and k-nearest neighbors. Overall, the best results (less classification error) were obtained when three measurements from each tree were presented to the classifier. In this case, the final prediction was decided on a majority basis, i.e. if the classifier classified two or three of the measurements as HLB, then the final prediction was HLB, otherwise the final prediction was non-HLB. This result indicate that the sensor needed to collect at least three reading from each branch to do a good classification. A paper is written based on this results and is going through internal review. Also another four-band sensor was purchased. An additional four-band sensor will help in covering more area of the canopy during field data collection. A retractable mast was purchased to be installed on a moving plat form for the field data collection. Initial work was started on the collection and identification of volatiles to see if unique chemicals are associated with HLB. A preliminary collection using SPME fibers (pink label) exposed for one hour on the upper surfaces of healthy and HB leaves indicated that there were some differences in the volatile profiles. Tree condition and sampling were not completely controlled so further efforts will be needed. Some possible variants include upper versus lower leaf surface, more gas exchange occurs from the lower surface where the stomata are present, and the time of day, more volatiles are likely produced at higher temperatures but some metabolic pathways may be altered depending on the leaf temperature. These variables will be tested as well as two sources of SPME fibers in order to maximize volatile collection. After this information is obtained, another healthy versus HLB trees test will be run. All data is being compared by gas chromatography and the healthy versus HLB comparisons also will be run on the lab bench volatile detection unit in California.
The goal of this study is to apply nanotechnology for developing Cu based silica nanoparticle/nanogel (CuSiNP/NG) formulation (nanoformulation) for effectively treating citrus canker. By design, this nanoformulation will be unique and expected to become superior to the traditional Cu based formulation in at least two aspects: sustained Cu release mechanism and better adherence properties, allowing long-term prevention of the citrus canker disease. As proposed, we have accomplished the following tasks. Task 1 (CuSiNP/NG design, synthesis, characterization and protocol optimization): During this quarterly reporting period, we have synthesized a series of CuSiNG formulations by varying three experimental parameters: de-ionized water to 95%ethanol (V/V) ratio, amount of copper sulfate pentahydrate (CuSO4.5H2O) and amount of silane precursor (tetraethylorthosilicate, TEOS). The objective is to maximize the Cu loading per gm of CuSiNG nanomaterial. (i) The amount of water in ethanol was varied from 30% to 70% by volume to determine an optimal mixed solvent composition that spreads the CuSiNG formulation uniformly on to the leaf surface. The DI water to ethanol ratio of 1.2 was found to be the optimal solvent composition. The maximum solubility of Cu-sulfate in this solvent mixture was determined to be 4.55 mg/mL. (ii) Keeping the Cu sulfate concentration fixed at 4.55 mg/mL in water/ethanol mixture, we increased the concentration of TEOS from 0.187 gm/mL to 2.61 gm/mL. The optimal concentration of TEOS was determined on the basis of its adherence property to the leaf surface. The CuSiNG with TEOS concentration of 1.31 mg/mL showed the best adherence property to the leaf surface. We also determined that 1.31 mg/mL TEOS was enough to trap all the Cu ions in the SiNG material. (iii) We have performed characterization of a few CuSiNG samples using UV-VIS, TEM, EDAX, XPS and FTIR techniques. The data is being analyzed. (iv) We have sent some nanoformulation to our collaborator, Dr. James H. Graham (CREC, Lake Alfred, FL) to conduct preliminary field experiments. Future Plan: We will report the data on CuSiNP in the next reporting period. We will also report data on CuSiNG characterization. Task 2 (Assessment of the antimicrobial activity of the nanoformulation): We have performed disk-diffusion assay to evaluate the anti-bacterial efficacy of a series of experimental CuSiNG samples (synthesized in the Task 1) using E. coli as a model system. We used Cu-sulfate and Kocide’ 3000 as controls. We are in the process of analyzing the data. We have also received Xanthomonas Alfalfea (a citrus canker surrogate) from our collaborator, Dr. James H. Graham (CREC, Lake Alfred, FL). We have plans to perform similar experiments on Xanthomonas Alfalfea in the near future. Future Plan: We will report the disk diffusion assay data on CuSiNP in the next reporting period. Task 3 (Develop large-scale nanoformulation production set-up): We are in the process of setting up 20 gallon batch synthesis setup for the CuSiNP/NG formulation.
Sensory impacts and flavor and aroma changes in HLB fruit: Two Valencia fruit samples (early April and late May), including symptomatic, asymptomatic and control fruit, were used. A fruit subsample was obtained for juicing. Juice was strained and half of each sample pasteurized, resulting in six test groups for each sampling date. Juice analysis included Brix, color and ratio determination. No significant differences were seen between pasteurized and fresh juice, or between juices collected in early April, while ratios from symptomatic fruit were significantly lower than asymptomatic or control juice collected in late May. Most pasteurized juices were analyzed using headspace SPME GC-MS. Ninety-eight volatile component peak areas were quantified using internal standards. All samples will be analyzed in triplicate and about 50 major volatiles will be identified and employed in the final data set for statistical evaluation. Consumer panel analysis of the corresponding juices will be conducted in summer 2009 and will evaluate hedonic ratings of overall acceptance, sweetness, fresh orange intensity and bitterness across each sample set for each harvest date. Difference testing will be performed on matched samples to determine if differences exist between pasteurized and non-pasteurized juices if stored frozen. Phytohormone changes in HLB fruit: Fruit subsamples both harvest dates were used for ethylene, auxin (IAA) and abscisic acid (ABA) determination. Ethylene production was more than two times lower in symptomatic fruit as compared with healthy fruit. Asymptomatic fruit ethylene production was intermediate between healthy and symptomatic fruit. ACC content followed similar trends. Likewise, similar trends in fruit flavedo ACS1, ACS2, and ACO gene expression were found; ACS genes encode protein products that are key rate controlling steps in ethylene biosynthesis. Expression of ethylene perception genes is being determined. IAA content was twice as high in flavedo from symptomatic fruit when compared with healthy or asymptomatic fruit. No difference in IAA content was measured in juice vesicles from healthy, symptomatic or asymptomatic fruit. ABA content in flavedo and juice vesicle from symptomatic Valencia fruit were significantly higher than in healthy and asymptomatic tissues. Yield, peel color, fruit size and seed abortion in HLB fruit: Fruit collected for yield and size determination were cut in half, and juice from half oranges was collected. Juice was analyzed for Brix, color and ratio determination. Ten HLB symptomatic trees and 10 healthy trees were harvested from four commercial citrus groves (2 Hamlin, 2 Valencia) to characterize and quantify yield changes (pieces of fruit, fresh weight and fruit size) due to HLB infection. Subsamples of fruit from each tree harvested were taken for standard juice analysis. HLB symptomatic trees have significantly smaller average fruit size and fewer fruit per tree than healthy trees. Juice analysis has indicated that only symptomatic fruit from infected trees have altered juice quality, asymptomatic fruit from infected trees have similar juice quality to fruit from healthy trees. Minolta color analysis indicated that symptomatic Valencia fruit had significantly lower a/b ratio (greener), weight and equatorial diameter compared to healthy and asymptomatic fruit. Seed abortion percentage was higher in symptomatic Valencia fruit (12%) than in healthy or asymptomatic fruit (3 and 5%, respectively). Extension and education: Four PI’s (Burns, Rouseff, Sims and Spann) will present work related to this funding at the 2009 International Citrus and Beverage Conference (ICBC) in September 2009. The ICBC generally has an audience of 375-425 technical and managerial personnel from major juice and beverage companies, and businesses that support them, and provides an excellent forum to disseminate information to the citrus industry. Spann, Oswalt, Schumann, Danyluk. 2009. Fruit Size, Yield and Quality in HLB Infected Trees. 2009 FL Citrus Growers Institute Greening Symposium, Bartow, FL
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. Earlier this year we have established replicated field plots in a commercial orchard located at Ourizona, western Paran‡, about 100 km south of S‹o Paulo state, Brazil in commercial citrus. The cultivar used is Pra on Rangpur lime, two years of age at the beginning of the experiment. We are currently working on establishing windbreaks. A specific cooperative agreement has now been established with the Brazilian cooperator, and the funds transferred there. Windbreaks and citrus trees have now been purchased and are being installed. Once the plots are completed, treatments will be 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 fro 2-3 more years. The development of the Programmable leaf wetness controller (PLWC) is nearing completion. The hardware issues that have plagued the initial stages of development have been corrected, and the software that will collect and analyze the data while also controlling the external fans and sprinklers is coming along. There were some issues with calibration procedures for the different sensors (temperature, light, RH and leaf wetness), but most of these have been overcome. Testing was concluded and the best material and arrangement for the leaf wetness sensors, as well as a method to calibrate the light sensors was determined. The base software (control) is complete, and a user interface control has been added. The system should be ready for field trials in 1-3 months to examine the survival characteristics of bacterial pathogens under field conditions.
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 (Xcc) on fruit, 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. The seedlings were returned to the greenhouse and the inoculated foliage covered with a plastic bag for 48h. 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 has written a new Pest Risk Assessment based on this work and subsequently a new proposed rule for shipping fruit potentially infected Xcc. The rule is presently in the comment period. If it become regulation, it would potentially greatly expand markets for Florida citrus and other citrus infected areas worldwide.
The primary hypothesis guiding this project is that a highly simplified and field deployable nucleic acid sample preparation system can be realized using low cost and easily used lateral flow chromatography technologies. The proposed device will enable highly sensitive and accurate pathogen detection and identification under field conditions. Toward this end we are developing and testing lateral flow chromatographic immuno-capture methods, novel passive fluid exchange systems, and integration of these systems to form the basis of a field deployable diagnostic platform for citrus pathogen detection in trees and insect vectors. In this period of performance, we have demonstrated methods for the affinity isolation of nucleic acids from crude plant tissue extracts using a proprietary thin layer chromatography method. These studies have shown: 1) that integration of our passive buffer exchange method with a nucleic acid (NA) affinity matrix enables washing and buffer replacement to attain NA in amplification compatible buffers, and 2) that the isolated NAs are suitable for polymerase chain reaction and isothermal amplification of target sequences. Taken together with our prior work demonstrating immuno-affinity capture of pathogens for enrichment from complex sample matrices, these more recent findings suggest that integration of immuno-affinity enrichment with a subsequent bulk nucleic acid isolation will allow efficient sample processing under field conditions with minimal user intervention. Additionally, the ability to directly isolate NA without reliance upon immuno-affinity enrichment ligands will enable the use of the test device for the detection of pathogens for which immuno-affinity reagents are unavailable. Concomitant with the development and optimization of nucleic acid isolation methods, we are developing approaches to the integration of sample preparation, isothermal amplification and detection that are consistent with the realization of a low cost, easily used field deployable test device. Toward this end, we have initiated an engineering effort to prototype and test varying integration approaches. These efforts are now focused on two integration approaches that will be subjected to laboratory testing and comparative analysis. As these efforts mature, we anticipate the development of a first generation prototype that will allow NA isolation and amplification to take place without user intervention following the initial introduction of crude sample.
Under Objective 1 (Characterization of hypersensitive resistance in kumquat and the cybrid of rough lemon 8166 with maternal ‘Meiwa’ kumquat.), a comparative study of grapefruit (C. paradisi) cv. Duncan, a very susceptible host, and two resistant cultivars of kumquat (Fortunella spp.), ‘Meiwa’ and ‘Nagami’, evaluated the mechanisms involved in the resistance of kumquat to the citrus canker. Xcc inoculum densities of 104 to 108 cfu/ml were infiltrated into immature leaves in the greenhouse (in planta) and into detached leaves incubated on water agar plates (in vitro). At higher bacterial inoculum density, kumquat cultivars developed a hypersensitive (HR)-like reaction in the infiltrated area, within a period of 72 h in vitro, and 96-168 h in planta. No symptoms or a few small necrotic spots developed in kumquats at the lower inoculum density. Susceptible grapefruit infiltrated with the same inoculum densities produced no visible tissue alterations at 72 h after inoculation and required 120 h or longer to develop water-soaking, hypertrophy and hyperplasia typical of canker lesions in compatible hosts. Phenotype of the lesions, bacterial population growth, anatomical changes in the infiltrated tissue and early expression of genes related to programmed cell death are indicative of HR that reduces growth of Xcc in the inoculation site and the further development of disease. A study is underway to characterize the responses in the somatic cybrid of rough lemon 8166 with maternal ‘Meiwa’ kumquat.
Under Objective 1 (Confirm biofilm formation by X. citri subsp citri [Xcc] in comparison with other bacteria that are well known to form biofilms) the following strains were compared for their ability to form biofilm: Xcc stain A (X306 Brazil), Xcc strain A* (X407 Iran), X. fuscans subsp. aurantifolii strain B (X69 Argentina), X. fuscans subsp. aurantifolii strain C (X341 Brazil), X. campestris pv. campestris (1469 Spain). Preliminary results show differences in biofilm formation on abiotic surfaces among the Xanthomonas strains analyzed as well as effects of different growth media: Nutrient Broth (NB), Yeast Medium (Y) and Luria-Bertani (LB), supplemented with or without 2% glucose. X. campestris has the greatest and most rapid biofilm formation. Among canker bacteria, C strain produces more biofilm than the A or B strains. Differences in production depending on the medium confirms that nutrient environment plays a role in biofilm formation. Addition of glucose to the medium does not generally increase biofilm formation but sometimes reduces it.
Citrus canker is a serious disease of most commercial citrus cultivars in Florida. Citrus canker is considered a quarantine pest due to the potential threat to citrus production in citrus producing areas without this disease. Thus, citrus canker has a significant impact on national and international agricultural markets and trade. The goal of the proposed research is to identify and characterize novel and critical genes involved in pathogenicity and copper resistance present in Xanthomonas 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. a. 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. Major achievements: Currently, five Xac related strains are being sequenced, which includes Xac Aw and A* strains which have restricted host range compared to the A type strain, X. axonopodis pv. citrumelo strains (copper resistant and non-copper resistant), and Argentinian strain (copper resistant). Both 454 Titanium and illumina (solexa) methods are being used. Currently, a draft genome sequence is done for Xac Aw strain. Titration run was performed for the rest four strains. Comparative genomics analysis is being performed to identify novel genes which may contribute to host-species specificity and copper resistance. In order to identify potential virulence factors, a X. axonopodis pv. citri mutant library mutant library constructed using EZ-Tn5ª
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