To determine the effect of prewashing on Xcc survival on asymptomatic lemons, apparently healthy fruit were harvested from infected trees in an affected orchard in Tucum’n, Argentina, and grapefruit from Florida. Replications of 5 fruit were harvested for each of the following treatments: 1) non-treated check, 2) chlorine for 2 min, 3) chlorine for 2 min followed by detergent (Neutro Deter N Sinner) for 20 seconds, 4) prewash with water followed by chlorine for 2 min, 5) prewash with water followed by chlorine for 2 min followed by detergent for 20 seconds. To determine the population of canker bacteria the wash solution was assayed by injection-infiltration of the suspension into two immature leaves on greenhouse grown grapefruit seedlings via needle-less syringe into 8 sites on the surface of each leaf. At 14 days after inoculation, the total number lesions per leaf were counted from all injection sites. There were no significant differences among the treatments, but the lesion counts were low (maximum of 3) and the frequency of zero lesions relatively high across all treatments. Nonetheless, there was a trend suggesting that chlorine treatment slightly reduced the number of lesions recovered, and chlorine treatment after prewashing the fruit, with or without detergent was beneficial in reducing the number of Xcc recovered from the fruit. The effectiveness of prewashing fruit with detergent at the same time or immediately after chlorine appears to be beneficial. Results from the two prewash trials, showed that chlorine as a disinfestant alone did not greatly reduce surface bacterial populations. However when chlorine, detergent, or detergent plus chlorine was added as a prewash, followed by a wash usually with SOPP (a disinfestant with detergent activity), there was a reduction in surface bacterial populations. The effect of a prewash was most apparent when SOPP/detergent was included. Prewashing of the fruit, especially with detergent, effectively wets the surface by lowering surface tension, which in turn allows the chlorine greater access to surface Xcc, as well as removing debris such as dirt, sooty mold, and scale insects from the fruit surface that could tie up the chlorine and/or SOPP which potentially reduce the effectiveness of the disinfestation treatment. By removing surface contaminants, the fruit surface was more exposed and, therefore, more effectively disinfested by the chlorine and/or SOPP that followed in the subsequent fruit wash. Currently, in citrus packing lines, the normal procedure is to use a prewash of water, or water plus chlorine, followed by a second wash with SOPP. A simple and low-cost recommendation resulting from these studies would be to reverse the procedure and prewash the fruit with detergent (such as SOPP), and follow this by a wash of chlorine with approximately 45 second contact time on the fruit. Based on the results of these experiments this process will more effectively reduce survival of Xcc and other contaminants on fruit after passing through the packing line. USDA APHIS developed a new Pest Risk Assessment based on this work, proposed a new rule for shipping fruit potentially infected Xcc. The rule has passed and will be implemented during the 2009-10 fruit season, greatly expanding markets for Florida citrus and other citrus infected areas worldwide. Three additional months of data have been now been collected to compare the production of bacteria from foliage, stem and fruit lesions. Early in the season, all sources of inoculum (foliage and fruit) produced copious quantities of Xcc. However, interestingly fruit lesion bacterial populations appear to be declining as the season progress and the fruit mature; apparently substantiating further the reduced numbers of Xcc produced from mature fruit compared to immature. The resulting publication served as the justification for USDA’s APHIS to promulgate a new regulation (7 CFR Part 301, FR Doc E9-15508 ‘Citrus Canker; Movement of Fruit from Quarantined Areas). This rule eliminates the requirement that fruit lots be inspected at the packinghouse and found to be free of canker symptoms and reduces industry costs by nearly $15 million annually.
The citrus Extension agents are actively involved in conducting educational opportunities to assist Florida citrus growers with citrus greening and canker issues and topics. During the first quarter of 2010 the agents collectively visited 88 grove locations to specifically address grower concerns regarding greening and or canker. In addition to local growers, 3 international grower groups were hosted to discuss citrus issues and Florida citrus production practices. In addition to one-on-one visits, 9 different citrus production school, seminars, OJ meetings or types of local citrus grower programs were conducted to provide specific information related to citrus greening or canker. Citrus agents were also involved with 14 additional programs to provided educational resources support or information on topics ranging from safety, pesticide license renewal, CEUs or the development of urban videos related to abandoned groves or other related urban topics. Citrus newsletters are sent via both electronic and printed media. The third in the series of ‘low volume’ sprayer calibration events was held in Ft. Pierce to continue addressing and educating growers in regards to the particle size being developed by these sprayers. These events were promoted and have exceeded 70 different machines being tested. In addition to local programs, agents are involved in broadcasting other citrus related events via the UF polycom system to the local Extension offices. These polycom sessions enhance and broaden topics presented on the local level. These polycom events can save not only mileage but time to attend these meeting and at little cost to the system. Citrus agents were actively involved with the three local Master Gardener groups in providing sessions on citrus related topics during this quarter.
Mid Florida Citrus Foundation (MFCF) currently supports the research efforts of scientists from the University of Florida, USDA and private industry. The majority of research projects are conducted by UF scientists at the approximately 100 acre citrus grove. The MFCF supports citrus research through the employment of a full time grove manager whom works closely with researchers to ensure that their projects are handled properly and that the grove is an excellent condition. The management of this grove requires extra financial commitment as grove care cost tend to be higher than a commercial grove due to the nature of many of the research projects. Current projects being conducted at the MFCF are Asian citrus psyllid (ACP) pesticide evaluation control trials, low volume applicator trials, windbreak evaluation, HLB nutritional programs, new and existing herbicide trials, variety and rootstock evaluation trials. The MFCF is in the process of expanding grove to allow more research work to be conducted. This was made possible due to the fact that the MFCF received an EQIP grant from the Natural Resources and Conservation Service (NRCS) and matching funds from the FCPRAC. These federal NRCS dollars are for the implementation of windbreaks around existing or planned citrus groves and for the conversion of water guns to micro-sprinklers. Expansion projects include an economic study of high density citrus rooted cuttings for early fruit production in the presence of greening, remedial and preventive tests for HLB infection and new herbicide trials. Future expansion plans (by spring 2011)if continuing to receive matching FCPRA support includes new plantings for ACP control trials.
Sensory impacts and flavor and aroma changes in HLB fruit: We began to coordinate metabolite and gene expression information to evaluate potential targets for mitigating HLB juice changes. The gene expression for metabolic biosynthesis that reflected flavor and aroma compound changes we previously reported are mentioned here. Several genes regulate linalool biosynthesis, such as GGPP synthase, geranyltranstransferase, terpene synthase and S-linalool synthase, were 1.5 to 5-fold up-regulated in symptomatic juice vesicles (SYMJV) compared with healthy juice vesicles (HJV) in ‘Hamlin’ and ‘Valencia’. Naringenin-chalcone synthase expression, which regulates flavanone glycoside synthesis, was 12-fold higher in SYMJV compared with HJV. The lipoxygenase 2 which regulates hexanal biosynthesis was 0.6-fold down regulated in SYMJV compared with HJV. Yield, peel color, fruit size and seed abortion in HLB frut: We worked with cooperators to identify the specificity of the iodine-starch reaction and starch quantification for detection of HLB in flavedo (FF) of ‘Valencia’ with verified zinc deficiency and water stress. No significant difference in starch accumulation was measured in FF of symptomatic versus healthy fruit. Our previous work showed that healthy FF accumulated starch, but like in roots, progressive starch loss was seen as fruit went from asymptomatic to symptomatic condition. Four starch biosynthetic genes (glucose-1-p adenylytransferase, starch synthase, 1,4-alpha-glucan branching enzyme, and isoamylase) were progressively down-regulated in ASYM and SYM ‘Hamlin’ and ‘Valencia’ fruit, but three of four starch degradation genes were up-regulated in HFF. There was only 10% reduction of starch biosynthetic gene expression in immature HFF (that is, green but mature) compared with fully mature HFF, whereas expression of degradation genes was higher in immature HFF compared with mature HFF. To complement work on starch content, we measured sucrose content in flavedo of H, ASYM and SYM Valencia fruit at immature, green mature and full mature stages. SYM and ASYM fruit accumulated less sucrose in flavedo compared with H flavedo. To study the involvement of IAA in the abnormal growth of SYM fruit, we analyzed its content in flavedo removed from misshapen sections and compared with normal shaped sections of the same fruit or healthy fruit. IAA content in normal sections of SYM fruit was similar to that in flavedo from ASYM and healthy fruit, but is significantly higher (2X) in flavedo from the misshape sections of SYM fruit. Our study with ‘Valencia’ fruit at the green mature stage indicated 10-fold higher fruit drop in HLB-infected trees compared with healthy trees. The fruit detachment force (FDF) was reduced 75% in SYM fruit when compared with healthy fruit. The expression of abscission-induced genes such as 1-aminocyclopropane-1-carboxylate synthase (CsACS1), cellulose-a1 (CsCel-a1), polygalacturonase (CsPG), phospholipaseA2. (CssPLA2.) and PhospholipaseA2. (CsPLA2.) were 17-, 7.4-, 531-, 1.4-, and 1.6-fold induced in SYM fruit abscission zones (SYMFAZ) compared with healthy fruit abscission zones (HFAZ), respectively. There was no difference of FDF between ASYM fruit and healthy fruit. However, expression of CsACS1 and CsCel-a1 were 5-fold increased and CsPG was 150-fold increased in asymptomatic fruit abscission zones (ASYMFAZ) compared with HFAZ. Bacterial titer was higher in SYMFAZ than ASYMFAZ. The titer of bacteria in SYMFAZ was over 2,000 fold higher compared with SYM midribs (including leaf abscission zones). The titer of bacteria was correlated with percentage of fruit drop. These results confirm that abscission is induced prematurely in ASFAZ and SFAZ through induction of abscission cell wall hydrolase genes known to be associated with organ loosening. An additional Hamlin harvest was performed and a Valencia harvest is planned. Analyses of these 2010 harvests and will be available for the next report.
This study was conducted to see if differences could be found for juice from healthy versus HLB oranges, representing larger sample sizes (15+ trees/sample), which would better represent the commercial situation. In 2009, there were 2 Hamlin and 2 Valencia harvests for healthy and HLB asymptomatic (AS) and symptomatic (S) samples. 500 fruit/sample were juiced using a JBT extractor and pasteurized. Sensory analyses was done using ‘difference from control’ (DFC) tests and trained descriptive (TD) panel. For chemical analyses, titratable acidity (TA), ‘Brix and % oil were determined by the processor. Individual sugars and acids (HPLC), aroma and flavor (electonic nose and tongue, GC-MS and GC-olfactometry) and leaf analyses (LC-MS) were performed by USDA-ARS and UF. Results for the DFC tests showed no difference between healthy and HLB-AS fruit juice for the first Hamlin and both Valencia harvests, but did show a difference for the last Hamlin harvest for HLB-AS juice (taste), and HLB-S juice (taste and smell). Quality data showed that HLB-S juice was sometimes lower in ‘Brix, higher in TA, lower in ratio and higher in oil content. TD sensory analysis showed differences for HLB-AS and HLB-S juices from healthy juice for smell and taste, especially for HLB-S juice and for taste. Results for other chemical analyses showed that sugars were often lower and acids and bitter liminoids higher in HLB juices compared to healthy controls. The electronic nose (e-nose) and tongue (e-tongue) studies showed that the e-nose differentiated between healthy, HLB-AS and HLB-S samples based on volatiles, as well as between samples from different harvest dates. The e-tongue also separated samples based on non-volatile compounds. For GC-olfactometry (GC-O), early season HLB-S fruit had less overall aroma intensity than control (healthy). However, in more mature fruit there was little difference in overall intensity. The samples were also analyzed by GC-MS to identify and confirm the GCO data. The data showed that the vast majority of aroma active volatiles were very similar between HLB-S and control juices. However, a few volatiles were found either exclusively or at higher aroma intensity in certain sample types. In the case of Valencia juices, 2-methylbutanol (minty, green) and decanal (citrus, fatty) were found primarily in HLB pasteurized juices. ‘-myrcene (geranium, earthy, grass) and octen-3-one, 1 (mushroom, metallic) were identified in both HLB and control unpasteurized juices, but with a higher intensities in HLB juice. For leaf analyses, the data has consistently shown that the alkaloid exhibiting a mass fragment at 188 amu and the compound, feruloyl putrescine, occur at much higher concentrations in HLB affected leaves compared to healthy control leaves as well as several of the polar hydroxycinnamates. Several flavone glycosides occur at lower concentrations in HLB leaves than in healthy leaves. The main flavanone, hesperidin, does not appear to be affected by HLB, but in several cases, HLB leaves contained higher limonin glucoside levels than controls. To conclude, there were minimal if any differences in flavor between healthy and HLB-AS juice for DFC tests with the large sample size, so the average consumer would not be able to tell a difference. Trained panelists picked up more differences in TD analysis, mostly for taste and mostly between healthy controls and HLB-S juice. The GC-O study found generally less aroma intensity in HLB-S fruit juice. There was variation, however, by season, with later season Valencia fruit showing less difference in flavor. Chemically, there were differences in ‘Brix, TA, individual sugars, individual acids, as well as the bitter compounds limonin and nomilin. Generally HLB juice, especially HLB-S, was lower in sugar, sometimes higher in acids, generally higher in limonin and nomilin. The latter two compounds, however, were generally below reported tastes thresholds. This data confirms earlier studies that flavor problems are more likely to come from HLB-S fruit juice. For the second year, thresholds for HLB off-flavored juice will be established in normal juice as well as for HLB off-flavored compounds, which will be identified.
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 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.
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