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 produced 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% 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.
The purpose of this project was to develop pheromone-based control strategies for citrus leafminer (CLM) to serve as alternatives to insecticides and that would be comparable or better than insecticides in terms of efficacy and cost. All of the scientific objectives of this research were met. We successfully developed two effective pheromone-based technologies for control of CLM. We helped develop 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. 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. Having obtained funding from USDA sources to follow-up on this research, we are building appropriate mechanical applicators for this formulation and technology. The company Alpha Scents is currently registering the product for Florida citrus. Fairly large-scale trials will be conducted in the summer of 2010 in Florida. This research has been published in a peer-reviewed scientific journal (Stelinski and Czokajlo 2010, Entomologia Experimentalis et Applicata, Vol 134: 69-77). The second technology we have successfully finished evaluating and co-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 collaborator, Dr. Stephen Lapointe, in Fort Pierce. Also, all of the work was conducted with an industry collaborator (ISCA Technologies, Inc.) who is registering this pheromone mating disruption product named SPLAT’ for CLM management in Florida. Following registration, ISCA will produce and distribute this product in Florida. This has effectively completed our research and development work on optimizing use of this product for CLM control in Florida. The SPLAT-CLM formulation consistently reduces leafminer infestation and population densities. ISCA Technologies is working to get their mating disruption product registered for use in Florida citrus. A portion of this research has also been recently published in two peer-reviewed scientific journal articles: 1) Stelinski, Lapointe, and Meyer 2010, Journal of Applied Entomology, In press and available online; and 2) Lapointe, Stelinski et al. 2009, Journal of Chemical Ecology, Vol 35: 896-903.
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 flexabilities 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 was to update all the default cost, yields and tree lost rates of the Excel model and to develop a user’s guide for the citrus investment model. The citrus investment model was modified to a adjust yields of remaining trees according to the number of resets that are replanted after trees are removed ( HLB, blight/decline, etc.). The approach allows 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. This preliminary tree yield expansion is being discussed with citrus horticultural scientists as to its ‘real world’ application. Many growers have been exploring a ‘foliar nutrient cocktail’ spray program as an alternative approach to managing HLB-citrus greening with hope that it will extend the productive life of an HLB infected tree. Under the foliar nutrient program, a psyllid control spray-pesticide program is included but there are cost savings from no HLB scouting, no HLB tree removal and no resetting young trees. The complete foliar nutrient cocktail mixed costs about $600 per acre. We are modifying citrus investment model and an older UF/IFAS Excel citrus reset model to compare the ‘foliar nutrient’ management program to the traditional HLB management program (scouting HLB tree removing along with or without reset trees). A preliminary discounted-present value comparison of traditional HLB management program and an alternative foliar nutrient HLB program was presented at the 2010 Citrus Show. Work has begun to estimated the optimum replacement time for a citrus grove-block. Also, we are continuing to work on the Excel citrus investment model to add the ability to evaluate any new HLB management strategies being developed by other researchers. As new revisions are made, we will ask growers to field test the investment model.
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 (SGDL) started receiving grower samples on Oct. 31, 2006. Through Dec. 2009, the lab has received and has run 124,204 and123,872 grower samples, respectively. In addition the lab has run tests on 3,678 psyllid samples and 16,825 samples for Southern Gardens for a total of 144,375 samples tested by real-time PCR since the inception of the SGDL. For the calendar year 2009, 48,700 grower samples were tested versus 40,640 for 2008. This represents a throughput increase of 20%. The total number of grower samples received for 2009 was 44,856 versus 49,802 for 2008. This represents a decrease in sample volume of 10%. The lab is now fully staffed and the current capacity is approximately 1,600 samples per week. With the increased capacity, we are continuing the development of additional process improvements to increase the capacity and accuracy of the laboratory. Included in these process improvements are the use of nested PCR in psyllid testing, the use of alternate primers to double check questionable results, and the development of procedures to automate the extraction of DNA. As the procedures are developed they are incorporated into the routine operation of the lab only after successful long-term use in parallel with the already proven and validated protocols. The lab continues to provide training to personnel from other states and countries and also has served as a reference laboratory for laboratories that are just now coming online.
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 plotshave been be established. The cultivar used is P’ra on Rangpur lime, two years of age at the beginning of the experiment and will be planted in early Feburary 2010. We are currently continuing work on establishing windbreaks which should be completed within 45-60 days. A specific cooperative agreement has been established with the Brazilian cooperator, and the funds transferred there. 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 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 will commence within the 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. Te 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.
The purpose of this project is to preserve citrus germplasm in Florida that is threatened by loss due to huanglongbing (HLB) and citrus canker. Using input from stakeholders and scientists, a priority list has been made of citrus germplasm in Florida which needs to be recovered. At Ft. Pierce, 50 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 the greenhouse for conservation. An additional series of propagations were made on 22 Las+ genotypes and likely due to differential Las populations, 32% of the resulting plants were Las- using the Wenbin Li primers/APHIS diagnostic protocol. At the Repository, eleven accessions from Florida have completed the cool temperature and warm temperature indexing and need only to complete the laboratory viroids testing for release from quarantine. Five additional accessions from Florida are currently included in the cool temperature biological index. Twelve varieties have just completed various stages of therapy for cleanup and are ready for biological indexing. Antibiotic treatments are under investigation for rescue of threatened breeding material from HLB. At Ft. Pierce, buds from HLB positive trees have been tested with three levels of an antibiotic cocktail for two time periods and were grafted onto clean rootstock liners. The highest antibiotic concentrations for the longest duration reduced take and growth of scion shoots, but lower concentrations were not different from controls. Las evaluations via PCR will be conducted in the next quarter. Experiments will be extended to include vacuum-infiltration of antibiotic(s) into buds prior to propagation. 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 these easily-rooted citron. Effect of antibiotics on Las 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). Results in-vitro 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. At the Repository, temperature treatments of budwood and different antibiotic cocktails have been tested for recovery of germplasm using citrus stubborn (Spiroplasma citri) in citrus and using Candidatus Liberibacter psyllaurous in tomato as model systems. Using information developed by the testing, additional priority hybrids from the breeding program, found only at the USHRL Farm in Ft. Pierce, and exposed there to HLB, are being identified, and budwood will be both treated and left non-treated with the antibiotic cocktail to propagate in the greenhouse for conservation and further evaluation.
Sensory impacts and flavor and aroma changes in HLB fruit: Harvested symptomatic, asymptomatic and health Hamlin fruit from this season are being juiced and samples collected for analysis and sensory impact. Data from last harvest indicated that esters responsible for the fruity, sweet aroma in OJ were significantly lower in symptomatic juice. Esters such as ethyl butyrate and ethyl-2-methyl butyrate were 20-50% lower in symptomatic juice compared to control juice. Important aldehydes such as hexanal were 50% lower in symptomatic fruit. Alcohols on the other hand were generally higher in symptomatic fruit. Linalool, responsible for the fruity, citrus aroma in OJ was 45% higher in symptomatic fruit. Although there was little difference in juice flavor volatiles between healthy or asymptomatic fruit, aroma volatiles in juice from symptomatic fruit were out of balance compared to that of healthy juice. Flavanone glycoside concentrations were 35% higher in HLB symptomatic juice. Control juice generally had the lowest level of FG concentrations for the three major FG’s in OJ. Thermal pasteurization did not make a consistent difference in FG concentrations when compared to the unpasteurized juice. Six major methoxylated flavones were determined using HPLC. Concentration patterns mirrored that of the flavone glycosides; symptomatic fruit contained higher concentrations than control fruit and HLB asymptomatic juice concentrations were generally intermediate. Pasteurization did not appear to significantly alter methoxylated flavone concentrations. Phytohormone changes in HLB fruit: A third and fourth biological replicate data set for ethylene production in immature Valencia fruit were obtained. Production of ethylene by healthy fruit was higher than in symptomatic (SYM) and asymptomatic (ASYM) fruit. Contrary to mature fruit, juice vesicle ethylene production from healthy, asymptomatic and symptomatic immature fruit was not different. Yield, peel color, fruit size and seed abortion in HLB frut: Preliminary transcriptomic work indicated Hamlin fruit had numerically greater but similar changes compared to Valencia. In Valencia, over 1100 (3.6%) and 1900 (6.3%) ESTs in vascular tissue (VT) below the calyx abscission zone and fruit flavedo (FF), respectively, significantly changed in SYM and ASYM tissues compared with healthy tissues in response to HLB infection. When SYM and ASYM tissues were compared, only 186 (0.6%) and 239 (0.8%) ESTs were differentially expressed in VT and FF, respectively. Compared with healthy juice vesicle (JV), 1701 (5.6%) ESTs changed in SYM JV, while only 18 (0.06%) ESTs changed in ASYM JV. 1425 (4.7%) ESTs expressed differently between SYM and ASYM JV. The results show that HLB causes significant changes in SYM and ASYM VT and FF. Development of SYM JV was impacted by infection while only minor changes were measured in ASYM JV. These results are in line with field observations; Hamlin is more reactive to HLB than Valencia, and ASYM juice quality is not significantly impacted. We reported last quarter that iodine staining revealed high starch content in flavedo from healthy fruit but not in SYM or ASYM fruit. Additional biological replicates this quarter confirmed this result. Electrolyte leakage and lipid peroxidation increased 2-fold in SYM FF compared with healthy FF. Fruit nutrient analysis was performed on Valencia and Hamlin tissues. Data are currently being compiled. Results from two groves sites indicated that size distribution of fruit from HLB-infected and healthy trees are normally distributed, but those from HLB-infected trees are skewed to the smaller sizes. Extension and education: We prepared and presented an extension tabletop display at the Citrus Processors and Subtropical Technology Conference in Lake Alfred on October 22, 2009, and presented HLB fruit size data at Ryan Atwood and Oswalt’s OJ meetings on October 27, 2009 and January 6, 2010.
Objective 1: 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 confirm whether similar responses occur in the somatic cybrid of rough lemon with maternal ‘Meiwa’ kumquat. Objective 2: Validate the inheritance or resistance for cybrids of susceptible Red grapefruit. The in vitro assay was used to evaluate the leaf resistance of 22 cybrids of Ruby red grapefruit with ‘Meiwa’ kumquat compared to grapefruit and kumquat parents. ‘Meiwa’ reduced lesions per leaf 88% and populations of Xcc were 7 log cfu per inoculation site for grapefruit compared to 2.5 log units for kumquat. All cybrids significantly reduced lesion number compared to grapefruit. The range of reduction varied widely from 20% to 88%. The most resistant cybrid produced an equivalent reduction in lesions produced by HR-resistant ‘Meiwa’. Populations of Xcc in cybrids also varied widely from 7 to 2.2 log units. Thus, resistance in cybrids may be inherited at different levels depending which sets of genes for resistance/HR contained in the mitochondrion or chloroplatst genomes are transferred to grapefruit in the cybridization process. In year 2 of the project, Cybrids of early oranges and more Cybrid lines of red grapefruit will be constructed and analyzed for phenotypic and genotypic inheritance of resistance using in vitro assay and HR and mitochondrion gene markers. The current set of Ruby red grapefruit cybrids will be grown out for observation of true-type-morphology and planted in canker-affected locations for observation of vigor and fruiting characteristics, as well as resistance to canker.
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.
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