Objective 1: Strains of Xanthomonas citri subsp citri (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. The aggregates resembled what has previously been described as biofilms. In two in vitro assays, biofilm formation by canker strains Xcc strain 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) was compared as well as the effects of different growth media with or without glucose. Biofilm staining with crystal violet showed biofilm formation on abiotic surfaces differed among culture media used to grow the bacteria as well as among Xanthomonas strains. X. campestris had the highest and most rapid biofilm formation. Among canker strains, C strain produce more biofilm than A and B strains. Differences in biofilm production depending on the culture medium confirmed that nutrient environment can alter biofilm formation e.g. addition of glucose to the medium did not increase but sometimes reduced biofilm formation. Next experiments will evaluate expression of genes involved directly in biofilm formation (gumD) or associated phenomena (quorum sensing, motility) for biofilm induced bacteria. Objective 2: 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 biofilms, exposing Xcc to disinfectants, bactericides, osmotic stress agents such as SOPP, chlorine, copper sulfate and high levels of salts. Biofilm formation was evaluated for strains 306 and 341 after 24, 48 and 96 h exposure to sub-lethal concentrations of CuSO4, NaOCl and NaCl. Exposure to sub-lethal concentrations did not reduce, and sometimes increased biofilm formation as a result of the stress condition. Survival of strain 306 that occurred after exposure to SOPP at concentrations less than 2% strain 306 was confirmed by membrane integrity assay, detection of GumD mRNA, and growth in culture medium. Next experiments will utilize the labile gfp marked strain to assess the viability of Xcc in preformed biofilms in vitro and in vivo on plant surfaces as affected by detergents and toxic compounds.
During this reporting period we have accomplished the following. Task 1 (CuSiNP/NG design, synthesis, characterization and protocol optimization): (A) Completed additional CuSiNG characterization as summarized below: (i) CuSiNG synthesis protocol has been optimized; (ii) CuSiNG synthesis in 110 mL (laboratory scale) and 4.5 gallon (large scale) has been successfully tested; (iii) Metallic Cu loading in CuSiNG was about 33 wt% as determined by the atomic absorption spectroscopy (AAS); (iv) X-Ray electron diffraction (XRD) data showed that CuSiNG is amorphous within the sensitivity limit of the technique. However, high-resolution transmission electron microscopy (HRTEM) images confirmed that CuSiNG is loaded with numerous Cu oxide nanocrystals (< 5 nm size) throughout the nanogel matrix; (v) HRTEM elementary mapping confirmed presence of silicon (most abundant), oxygen (most abundant), copper (abundant), and sulfur (least abundant). Presence of sulfur indicates that trapping of sulfate ions occurred within the nanogel matrix along with ionic copper. (B) Performed CuSiNP synthesis and characterization as summarized below: (i) CuSiNP synthesis protocol has been optimized with respect to Cu loading; (ii) AAS study estimates metallic Cu content to be ~45 wt%; (iii) HRTEM data confirmed formation of Cu oxide nanocrystals (2 nm ' 10 nm size range) that remain uniformly distributed throughout the matrix; Optimized CuSiNP formulation showed significant gel like morphology under HRTEM; (iv) Elementary mapping confirmed presence of silicon (most abundant), oxygen (most abundant), copper (abundant), and sulfur (abundant). Presence of sulfur indicates that trapping of sulfate ions occurred within the matrix along with ionic copper; (v) Like CuSiNG material, CuSiNP demonstrated extremely strong adherence property to citrus leaf surface. Future Plan: We have completed optimization of CuSiNP and CuSiNG synthesis protocols as proposed. Further materials characterization of CuSiNP is in progress. Cu release study is also in progress. Task 2 (Assessment of the antimicrobial activity of the CuSiNP/NG nanoformulation): We have systematically performed disk-diffusion assay (DDA) to evaluate the anti-bacterial efficacy of several CuSiNG as well as CuSiNP samples. CuSiNG material exhibited improved anti-bacterial properties in comparison to CuSiNP as well as Kocide 3000 and Cu-sulfate controls. Antibacterial efficacy of CuSiNG is improved by 2 times over Kocide 3000 when tested against Xanthomonas Alfalfae (canker surrogate). We have performed appropriate statistical analysis. We also have observed that CuSiNG material has the ability to spread out on agar. Future Plan: Efficacy of CuSiNP against E. coli is in progress. We will also perform preliminary studies to evaluate direct interaction of CuSiNG material with the bacteria to better understand improved Cu bioavailability. Task 3 (Develop large-scale nanoformulation production set-up): We have synthesized and characterized a few 4.5 gallon batches of CuSiNG as well as CuSiNP materials. Characterization data showed no difference in the quality when compared to the data of 110 mL laboratory scale preparation.
The purpose of this project was to develop pheromone based control strategies for this pest that will serve as alternatives to insecticides and that should be comparable or better than insecticides in terms of efficacy and cost. We have successfully developed two effective pheromone-based technologies for control of citrus leafminer (CLM). Most recently, we developed and evaluated an attract-and-kill formulation, termed MalEx, for control of CLM. MalEx is a viscous paste with UV-protective properties that is dispensed as small (50 .l) droplets using custom-made calibrated pumps. MalEx is manufactured by a company based in New York State called Alpha Scents. The attract-and-kill formulation is applied to tree foliage as small droplets, which release pheromone that is highly attractive to males. Attract-and-kill formulations work by attracting insects to small droplets of the formulation. As the pest insects touch the droplets, they obtain a lethal dose of toxicant upon contact and die. A formulation containing the CLM pheromone and 6% permethrin was found to suppress CLM populations in the field. Continuous treatment of 1.2 acre blocks of citrus with MalEx over the course of 112 days reduced larval infestation of new leaf flush by 3.6-7.2 fold. Complementary laboratory investigations confirmed that 100% of males contacting MalEx droplets, aged up to 35 d in the field, were killed within 24 h. Direct observation of male CLM behavior in the field confirmed that attracted males made contact with droplets. We have also recently shown that the insecticide, imidacloprid, may be more effective than permethrin, extending the longevity of effectiveness of the formulation. Control of CLM with MalEx was more effective than with comparable insecticides and does not effect beneficial insects. This formulation selectively kills CLM with no adverse effects on other insects. Targeted deployment of insecticide with MalEx should decrease the harmful impact of current pest management practices on the wide complex of natural enemies that are known to limit CLM population growth. Large scale commercial use of this formulation will likely mandate the development of specialized mechanical applicators for rapid deployment of material in the field. The company Alpha Scents is looking into the process of registering the product for Florida citrus. The second technology we have successfully finished developing is the SPLAT-CLM mating disruption product manufactured by ISCA Technologies. We have determined the most effective dosage of pheromone in the formulation required for optimal efficacy, the optimal deployment density and distribution of pheromone dispensers within the grove, and the exact blend of pheromone components that are optimal for CLM control. We have also developed several prototype mechanical application devices that we are still in the process of optimizing. This work was completed with USDA-ARS collaborators in Fort Pierce. Also, all of the work was conducted with an industry collaborator (ISCA Technologies, Inc.) who is developing and registering this pheromone mating disruption product named SPLAT’ for CLM management in Florida. This has effectively completed our research and development work on optimizing this product. The SPLAT-CLM formulation consistently reduces leafminer infestation and population densities. We have also observed that deployment of SPLAT-CLM reduces the incidence of citrus canker. Our ongoing investigations will continue to optimize mechanized sprayers and longevity of efficacy for the 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.
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 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 were used. Currently, a draft genome sequence is done for Xac Aw strain. Titration run was performed for the rest four strains. 1/8 plate of Titanium production run was completed. Illumina sequencing was completed. We are assembling all the data using the Xac A strain as the reference genome. We will decide whether further sequencing is needed based on the assembly. Primer walking is being used to fill the gaps. 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-Tn5′
The Southern Gardens Diagnostic Lab started receiving grower samples on October 31, 2006. Through September 30, 2009, the lab has processed and tested 106,329 grower samples, 15,327 internal leaf samples and 3,107 psyllid samples for a total of 124,763 samples for all sources combined. Through the third quarter of 2009, the lab has received 30,185 grower samples and has run and reported on 36,462 grower samples. For the same period of 2008 the lab had received and run 29,881 and 30,735 grower samples, respectively. The discrepancy between the number received and the number run for 2009 is due to the backlog of samples that the lab experienced during late 2008 that were carried over to 2009. Although the number of samples through the 3rd quarter of 2009 is similar to that of 2008, based on the sample volume for the latest 3 months it appears that more samples will be received during 2009 than during 2008. The average number of grower samples received per month during the third quarter of 2008 averaged 3,231/month whereas the number of grower samples received during the third quarter of 2009 was 4,843/month or a 50% increase over the third quarter in 2008. Compared to 2008, the lab has become more efficient during 2009 with the same number of personnel. The average number of grower samples processed per month through the first three quarters of 2008 was 3,415 where as for the same three quarters of 2009 the average number of samples processed was 4,051, or a increase of 19%. A new biologist has recently been hired on FCPRAC funds that will allow us increase the maximum capacity of the lab and to close the gap between the volume of samples received and the number of samples run. In addition to the standard plant samples received from the growers, a number of growers have started to send psyllid samples in to the lab. We are developing standard laboratory data handling procedures that will facilitate the submission of insect samples and the data reporting of results from psyllid samples. Although results from plant samples are kept confidential (with respect to source and results), it is expected that the data from the psyllid samples will be made available to the industry (with respect to region, % infection, and seasonality) so that growers can better make control decisions. The laboratory has also provided training to laboratory personnel from a number of other states and countries. Training has been provided to personnel from California, Florida, Argentina, Belize, Costa Rica, the Dominican Republic, and Mexico.
The citrus agents have been actively involved in the development and conducting the ‘Low Volume Sprayer Rodeo’ in Lake Placid (Sept. 15) and Haines City (Sept. 16). The purpose of the event was to allow growers the opportunity to determine if equipment currently being used effectively meets the 90 micron size for sprayer droplet size distribution as indicated on selected product labels. Equipment could be adjusted and retested as needed during the event. These events allow more than 45 different pieces of equipment to be tested during the two-day event. USDA faculty from Texas assisted in conducting the event using their equipment. This event was free to participating growers. Data collected from the events will be presented at upcoming programs by the citrus agents in October. Agents have been active in making, conducting and/or providing presentations at local grower meetings and the Citrus Expo. These meetings are to enhance knowledge in one or more of the areas regarding: a) field surveying for greening; b) psyllid management; c) grower experiences in greening and psyllid management; d) conducting trials; e) Master Gardener training including greening and canker; and, f) presenting at and attending professional meetings. Additionally, 68 grower visits were conducted to discuss various management practices along with numerous citrus newsletters being distributed all aimed at enhancing practice adoption and knowledge gain. To enhance knowledge and make information readily available, the agents effective utilize their ‘citrus agents’ website as a tool to post presentations for greater access and dissemination to a wider audience. During the last three (3) months their newsletters have had 27,281 downloads, greening symposium had 11,362 views and 100,089 website hits.
The overall goal of this project is to apply nanotechnology to develop Cu loaded silica nanoparticle/nanogel (CuSiNP/NG) formulation that will improve Cu bioavailability and longevity and prevent citrus canker disease infection in long-term. During this reporting period we have accomplished the following. Task 1 (CuSiNP/NG design, synthesis, characterization and protocol optimization): (a) Performed systematic characterization of CuSiNG material (optimized nanoformulation) using high-resolution transmission electron microscopic (HRTEM) technique. We discovered that Cu is present in two different forms, amorphous and crystalline. The selected area electron diffraction (SEAD) and X-ray diffraction (XRD) analysis confirmed that the CuSiNG material is primarily amorphous in nature. Detailed investigation of the crystalline form of Cu by the HRTEM revealed that they are nanocrystalline Cu(I) oxide (crystal size 1-3 nm). X-ray photoelectron spectroscopic (XPS) study confirmed the presence of Cu both in Cu (II) and Cu(I) forms. Cu(II) ions are complexed with the amorphous silica matrix (characteristic blue color appearance). (b) Performed optimization of CuSiNP synthesis protocol with the focus on Cu loading. Cu-sulfate amount was varied from 236 mg to 1900 mg keeping the tetraethyl orthosilicate (TEOS, a silane precursor of silica nanoparticle/nanogel matrix) amount fixed at 0.659 gm. Optimum Cu loading was quantified by the atomic absorption spectroscopy (AAS). Sample preparation for the AAS involved extraction of Cu from the sample using EDTA chelates. The Cu loading was 401 ppm as measured by AAS. (c) Studied adherence property of CuSiNP nanoformulation. Unlike CuSiNG, CuSiNP formulation is 100% water based. When sprayed on the citrus plants (variety – orange and grapefruit; potted) using a regular spray-bottle, the CuSiNP nanoformulation formed droplets on to the citrus leaf surface (as expected). Once droplets dried up it formed whitish-blue spots of CuSiNP material on the leaf surface. Plants were them placed in open air for about a weak that received afternoon rain showers (up to 1 inch of rain fall). We clearly observed the characteristic spots of CuSiNP material, showing that CuSiNP adhered well onto the leaf surface. Future Plan: We have completed first round optimization of CuSiNP and CuSiNG synthesis protocols. We will continue materials characterization of CuSiNP/NG as proposed. Cu release study will be performed as well. Task 2 (Assessment of the antimicrobial activity of the CuSiNP/NG nanoformulation): We have performed preliminary disk-diffusion assay (DDA) to evaluate the anti-bacterial efficacy of several CuSiNG as well as CuSiNP samples (synthesized in the Task 1) using E. coli as well as Xanthomonas Alfalfea (a citrus canker surrogate) as model systems. Both the CuSiNG and CuSiNP formulations reproducibly exhibited improved anti-microbial activity with respect to Kocide 3000 as well as Cu-sulfate (positive controls). Note that metallic Cu concentration in all the samples and controls were identical. As expected, SiNP/SiNG material without Cu did not show any anti-microbial property. The anti-microbial activity of CuSiNP/NG was comparable for both E. coli and X. alfalfea. Future Plan: We will repeat DDA to obtain statistically significant results. Task 3 (Develop large-scale nanoformulation production set-up): Large-scale synthesis setup (up to 20 gallons) of CuSiNP/NG formulation is ready. Future Plan: We will synthesize a batch of CuSiNG as well as CuSiNP materials using this setup and characterize them.
The purpose of this project is to preserve citrus germplasm in Florida that is threatened by loss due to huanglongbing (HLB) and citrus canker. A priority list of germplasm is being made following a meeting that 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 list is being developed considering the following factors: ‘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’. Results obtained which suggest that conventional thermotherapy methods for citrus may not be effective in eliminating HLB have resulted in exploration of alternative methods of therapy. In Riverside, treatments including antibiotics and heat treatment of budwood are being evaluated for their ability to eliminate Candidatus Liberibacter psyllaurous in tomato and citrus stubborn, which is caused by Spiroplasma citri, a phloem limited prokaryote similar to the bacteria associated with HLB. At the Repository, eleven accessions from Florida have completed the cool temperature and warm temperature biological indexing and are entering the final laboratory indexing for release from quarantine. Five additional accessions from Florida are beginning the cool temperature biological index for possible release from quarantine within a year. Eight varieties have been shoot tip grafted, two Florida varieties have successfully completed 16 weeks of thermotherapy and two additional varieties are beginning thermotherapy.
At the request of the CRB, as of 10-1-11, Grafton-Cardwell and Morse merged their core entomology research efforts under a single project, 5500-501 (Morse’s portion of the project is 5500-501b and was funded in the amount of $89,795 for 2011-12 replacing his previous CRB project 5500-118). We have always coordinated our research efforts but this arrangement formalizes the situation. This report summarizes the Morse lab’s recent research within this coordinated project. Morse presented a summary of recent research on Fuller rose beetle at the Korea Export meeting held in Tulare the morning of Sept. 4 and two talks, one on bean thrips and a second on mite contaminants of export citrus, during the Sept. 4 afternoon Australia / New Zealand Export meeting. These presentations will be posted shortly on the CCQC web site. Recent field research with Fuller rose beetle has confirmed Grafton-Cardwell’s laboratory working showing that carbaryl (Sevin) and thiamethoxam (Actara) are two of the more effective foliar treatments that can be used to reduce levels of Fuller rose beetle adults already in trees (most of this year’s soil emergence has already occurred). This late in the season, thiamethoxam is probably the better choice because the allowable MRL for carbaryl in Korea (0.5 ppm) is quite a bit less than the 10 ppm U.S. tolerance for this product that the 5 day label PHI is based on. In contract, the thiamethoxam MRL in Korea and Japan is higher than the U.S. tolerance. We have been working on a second method of controlling Fuller rose beetle, i.e. using trunk sprays with bifenthrin applied to skirt-pruned trees. It appears that 3-4 hours after adults cross the trunk spray they become paralyzed, drop out of the tree, and lie on their back for 2-3 days before they eventually recover (where one assumes predators would eat many of them). This method is modeled after Australian trunk sprays using lambda-cyhalothrin. In our evaluations, bifenthrin appears more effective than lamda-cyhalothrin. Citrus thrips resistance to Delegate has been confirmed in the San Joaquin Valley and we will soon be investigating reports of abamectin resistance in the Coachella Valley. As a result, a spring citrus thrips study was conducted evaluating the efficacy of two new products which are nearing registration for citrus thrips control – Bexar and Closer. We plan to take fruit scarring evaluations soon to compare how these products performed in comparison to standards of Delegate and abamectin.
In the last quarterly report, two harvests of fruit from 15 trees of Hamlin in February and Valencia in April were analyzed for chemical and sensory traits. Now we add information on a June Valencia harvest from 15 trees. For these samples, the Brix ranged from 10.6 to 12.2, acid from 0.50% to 0.58 %, ratio from 20.8 to 21.2 and oil from 0.014% to 0.028 % (highest for symptomatic fruit). For the February Hamlin samples, there were no major differences in Brix, acid, ratio and oil content, but for Valencia fruit from the first harvest (April), the juice from symptomatic HLB fruit was high in acid and oil content compared to the controls, and the asymptomatic HLB fruit juice was in between symptomatic and control values. For the second (June) Valencia harvest, the main difference was that the oil content was higher in asymptomatic and symptomatic fruit juice, possibly due the action of the extractor on fruit of smaller size. Juice samples from all harvests were frozen for other chemical analyses (individual sugars and acids, vitamin C, limonin, nomilin and other secondary metabolites as well as aroma volatiles) for later analysis by HPLC and GC, and samples for gas chromatography-olfactometry (GC-O) work were prepared for Russ Rouseff at the CREC. Individual sugar, acid and total ascorbic acid analyses are now done on all three harvests (1 Hamlin and 2 Valencia). Hamlin and the April Valencia samples were higher in citric and lower in malic acids for both asymptomatic and symptomatic HLB fruit juice compared to healthy, while there were no differences for the June Valencia samples. Hamlin and the April Valencia harvest were often lower in fructose and/or glucose for asymptomatic and sucrose as well for symptomatic HLB juice samples, but again, there were no differences for the June Valencia samples. For the descriptive sensory data, there were differences in Hamlin juice and in the juice of the April Valencia harvest for many descriptors (reported for last quarter), however for the June Valencia harvest, there were no differences in aroma (smell) descriptors , and this time control juice was lower in orange and fresh flavor compared to asymptomatic HLB juice, and higher in the paint descriptor (unlike the previous Hamlin and April Valencia harvests). Symptomatic HLB juice was higher in sour/fermented, peppery/musty and salty/umami descriptors than asymptomatic HLB juice, but not different from control juice. For flavor (taste and smell) of the June Valencia samples, HLB juice from asymptomatic fruit was not significantly different in flavor from control juice in the difference from control tests, and even a trained panel could find little difference between these two types of juices (reflecting the lack of differences in the chemical analyses). The electronic tongue and nose were tested to see if these instruments could discriminate between healthy, asymptomatic and symptomatic HLB juice samples collected in 2008 and 2009. The e-tongue sensors could clearly differentiate between juice from HLB symptomatic fruit, and juice from healthy trees. Juice from asymptomatic HLB fruit was classified between these two samples in 2008 and undifferentiated from the healthy samples in 2009, reflecting the sensory data for those years. The electronic nose could also separate juice from symptomatic fruit in 2008. The use of electronic nose or tongue would be of interest to industry to screeni juice for HLB-induced off-flavors. A postdoc is currently conducting threshold tests for the worst tasting symptomatic juice in normal juice and determining off-flavored compounds for individual thresholds in normal juice.
In the last quarterly report, two harvests of fruit from 15 trees of Hamlin in February and Valencia in April were analyzed for chemical and sensory traits. Now we add information on a June Valencia harvest from 15 trees. For these samples, the Brix ranged from 10.6 to 12.2, acid from 0.50% to 0.58 %, ratio from 20.8 to 21.2 and oil from 0.014% to 0.028 % (highest for symptomatic fruit). For the February Hamlin samples, there were no major differences in Brix, acid, ratio and oil content, but for Valencia fruit from the first harvest (April), the juice from symptomatic HLB fruit was high in acid and oil content compared to the controls, and the asymptomatic HLB fruit juice was in between symptomatic and control values. For the second (June) Valencia harvest, the main difference was that the oil content was higher in asymptomatic and symptomatic fruit juice, possibly due the action of the extractor on fruit of smaller size. Juice samples from all harvests were frozen for other chemical analyses (individual sugars and acids, vitamin C, limonin, nomilin and other secondary metabolites as well as aroma volatiles) for later analysis by HPLC and GC, and samples for gas chromatography-olfactometry (GC-O) work were prepared for Russ Rouseff at the CREC. Individual sugar, acid and total ascorbic acid analyses are now done on all three harvests (1 Hamlin and 2 Valencia). Hamlin and the April Valencia samples were higher in citric and lower in malic acids for both asymptomatic and symptomatic HLB fruit juice compared to healthy, while there were no differences for the June Valencia samples. Hamlin and the April Valencia harvest were often lower in fructose and/or glucose for asymptomatic and sucrose as well for symptomatic HLB juice samples, but again, there were no differences for the June Valencia samples. There was no significant pattern for total ascorbic acid. For the descriptive sensory data, there were differences in Hamlin juice and in the juice of the April Valencia harvest for many descriptors (reported for last quarter), however for the June Valencia harvest, there were no differences in aroma (smell) descriptors , and this time control juice was lower in orange and fresh flavor compared to asymptomatic HLB juice, and higher in the paint descriptor (unlike the previous Hamlin and April Valencia harvests). Symptomatic HLB juice was higher in sour/fermented, peppery/musty and salty/umami descriptors than asymptomatic HLB juice, but not different from control juice. For flavor (taste and smell) of the June Valencia samples, HLB juice from asymptomatic fruit was not significantly different in flavor from control juice in the difference from control tests, and even a trained panel could find little difference between these two types of juices (reflecting the lack of differences in the chemical analyses). The electronic tongue and nose were tested to see if these instruments could discriminate between healthy, asymptomatic and symptomatic HLB juice samples collected in 2008 and 2009. The e-tongue sensors could clearly differentiate between juice from HLB symptomatic fruit, and juice from healthy trees. Juice from asymptomatic HLB fruit was classified between these two samples in 2008 and undifferentiated from the healthy samples in 2009, reflecting the sensory data for those years. The electronic nose could also separate juice from symptomatic fruit in 2008. The use of electronic nose or tongue would be of interest to industry to screeni juice for HLB-induced off-flavors. A postdoc is currently conducting threshold tests for the worst tasting symptomatic juice in normal juice and determining off-flavored compounds for individual thresholds in normal juice.
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. 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 plots will be established. 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 been established with the Brazilian cooperator, and the funds transferred there. Windbreaks and citrus trees have now been purchased for the plot. 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) software is written, debugging is complete and the control program is working well. New leaf wetness sensors have been designed and constructed and are in the final stages of calibration. One final circuit to control fans that facilitate wind generation in ambient environments is being completed and needs to be tested. We anticipate trials to begin next month to examine the survival characteristics of bacterial pathogens under field conditions.
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 with approximately 20X coverage. Titration run was performed for the rest four strains. The production run was also finished for two of the four strains with more than 15X coverage for each of the strains. Comparative genomics analysis is being performed to identify novel genes which may contribute to host-species specificity and copper resistance. Some candidate genes which might contribute to virulence, host specificity, avirulence have been identified. Functional and genetic analysis of those genes are under way. Among the genes that have been identified, two non-mucoid mutants affected in galU of Xac (F6 and D12), were further characterized. The loss of growth of the galU mutants indicates that the galU gene could be a potential target to develop bactericide. The paper describing our findings on galU has been submitted for publication consideration at AEM.
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 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. This information may help in the development of evidence needed to change international fruit shipping regulations and open additional markets for Florida fruit.
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. Microarray produced differential expression of genes in grapefruit and Nagami kumquat inoculated with Xcc. In comparison kumquat expression changes were more marked than those of grapefruit. 172 kumquat and 140 grapefruit ESTs were sequenced and 52.5% of the sequences in the EST library were up regulated at 4 hrs in KN that shared significant sequence similarity with putative senescent related protein, DNA damage related kinases (CHKI), chloroplast binding proteins, chlorophyll carrying proteins, lipoxygenases, multiple complex forming proteins (SKP1), membrane associated peptidases, detoxification related enzymes (such as glutathione S-transferase, ribonucleases, and metallothionein-like protein), transposase, ethylene synthesis and chlorophyll related genes. In DG, sequence identities included major facilitator superfamily (MFS-1) phosphorylases and transferase, lectin related protein precursors, aspartyl tRNA synthethase, phosphatase 2A inhibitor, NADH dehydrogenase, catalase, chloroplast heat shock proteins. Genes associated with hypersensitive responses found in the citrus EST database, like lipoxygenase, superoxide dismutase (SPD), and glutatione peroxidase (GLP), were expressed early in kumquats, but not in grapefruit. A study is underway to characterize the responses in the somatic cybrid of rough lemon 8166 with maternal ‘Meiwa’ kumquat.
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