The overall goal of this project is to characterize the virulence mechanisms of Candidatus Liberibacter asiaticus, the citrus Huanglongbing (HLB) pathogen, thus to come up with new management strategies by genome sequencing and functional genomics approaches. The original goal of the proposed research is to further complete the genome sequencing of Candidatus Liberibacter asiaticus, for which a draft sequence is available. The goal was modified to meet the current progress in genome sequencing of Ca. L. asiaticus with the advice and permission from program manager of FCPRAC. The tile has been changed to the following to better suit the goal: Understand the virulence mechanism of Ca. L. asiaticus by genome sequencing and functional genomics approaches. In addition to previous report, the following progress has been made recently. To understand how Ca. L. asiaticus make a living including nutrients acquisition and overcoming of plant defense in citrus, we first analyzed its metabolic capability and nutrient requirement by conducting curated metabolic reconstruction. Our data indicated that Ca. L. asiaticus could rely only on aerobic respiration rather than on anaerobic respiration. Ca. L. asiaticus could not synthesize 12 amino acids and needs external folate, quinines, biotin, thiamine, pantothenate, and pyridoxine for its growth. By analyzing the metabolic pathway, we also identified 5 genes that were missed in current genome sequence. The work is in progress. The transcriptome study is in progress.
Beneficial microorganisms have been shown in previous studies to have the capacity to control plant diseases by accelerating seedling emergence, promoting plant growth and development, and preventing the invasion of plant pathogens. Characterization of the beneficial microorganisms associated with citrus in the local environment might identify beneficial bacteria for practical use to control HLB. Major achievements: This research has resulted in four publications (three published, one in preparation). 1. A comprehensive study of the bacterial diversity associated with healthy and HLB diseased citrus indicated that Candidatus Liberibacter asiaticus as the pathogen responsible for HLB disease in Florida. Phytoplasma was not found in any of the samples collected from Florida (Sagaram et al. 2009). 2. We characterized the effect of HLB on the bacterial community associated with citrus roots. This research has been summarized in the following publication ‘Huanglongbing, a systemic disease, restructures the bacterial community associated with citrus roots’ (Trivedi et al. 2010) which has been published on AEM. 3.Development of a proper in vitro screening system that provides repeatable and reliable results in shorter periods of time is an important step for isolation of efficient bacterial antagonists. We have developed a method to screen antagonistic bacteria against uncultured HLB pathogen. The method uses the discrimination of live-dead cells by EMA and speed and sensitivity of QPCR. 4.Isolation of plant growth promoting bacteria from potential escape citrus. Isolation of bacteria with the potential of plant growth promoting and biological control potential might reveal innovative ways controlling the HLB disease. We specifically focused on delineation of the cultivated endophytic bacterial isolates and characterization of their salient metabolic features. A total of 227 and 159 morphologically distinct colonies were isolated individually from uninfected and infected samples, respectively and were taken for further analysis. We used a detailed approach for screening novel plant growth promoting (PGP) isolates by conducting qualitative, quantitative and PCR based assays for traits related to mineral nutrition [Phosphate (P) solubilization, siderophore production, Nitrogen (N) fixation], development [Indole acetic acid (IAA) synthesis], health [production of antibiotic and lytic enzymes (chitinase)], induction of systemic resistance [salicylic acid (SA) production], stress relieve [production of 1-amino-cyclopropane-1-carboxylate (ACC) deaminase] and production of quorum sensing [N-Acyl Homoserine Lactones (AHL)] signals. A total of 39 bacterial isolates showing at-least 5 beneficial traits were further taken up for quantitative estimation of PGP activities. The selected isolates belonged to 12 different genera mostly belonging to species of genus Serratia, Pantoea, Pseudomonas, Bacillus Burkholderia. Several promising strains in our culture collection represent the first isolates of bacterial groups that have only been detected based on culture-independent methods. From the culture collection six bacteria isolates were found to reduce the number of viable Ca. L asiaticus cells by EMA-qPCR based method.
The goal of the proposed research is to understand how Candidatus Liberibacter asiaticus causes Huanglongbing (HLB) disease on citrus. Citrus HLB is the most devastating disease on citrus. There are very few options for management of the disease due to the lack of understanding of the pathogen and citrus interaction. Understanding the citrus and citrus HLB pathogen interaction is needed in order to provide knowledge to develop sustainable and economically viable control measures. Major achievements: 1. Microarray analysis of host response of sweet orange to Las infection in greenhouse. The results have been published in the following paper: Kim, J., Sagaram, U.S., Burns, J. K., and Wang N*. 2009 Response of sweet orange (Citrus sinensis) to Candidatus Liberibacter asiaticus infection: microscopy and microarray analyses. Phytopathology 2009 99:50-7. 2. We are currently assessing citrus genes modulated by Las infection in 1) citrus stems and roots, 2) citrus grove, 3) citrus varieties that show tolerance, and 4) at different infection stages. Using Affymetrix microarray analysis, we detected a total of 2,795 and 1142 probe sets with significantly (p< 0.05) altered expression levels in stems and roots of Valencia sweet orange (Citrus sinensis Osbeck), respectively. At a cutoff point of 1x log fold change (logFC), a total of 580 transcripts were significantly up-regulated and 350 down-regulated in stems. A relatively lower number was up-regulated (58) and down-regulated (58) in roots. Different sets of plant genes including those related to response to biotic or abiotic stress, transcriptional factors, transport, cell wall re-modeling and biogenesis were represented in both sets. Highly up-regulated genes (>3x logFC) in stems included 2OG-Fe(II) oxygenase family protein, WAK-like kinase, Lectin-related protein precursor, Cu-Zn superoxide dismutase, Zinc transporter protein ZIP1, many of which are involved in oxidative stress that produce highly toxic reactive oxygen species (ROS). Homologs of nucleotide binding and Leucine-rich repeat (NB-LRR) domain containing proteins involved in gene-for-gene resistance were down regulated in both tissues with some such as TIR-NBS-LRR proteins being re-pressed in stems only. Suppression subtractive hybridization analysis of RNA from Las-infected Mandarin line (Citrus x limonia Osbeck) showed up-regulation of pathogenesis/resistance, biotic stress related, cell wall re-modeling gene groups and transcriptional factors and down regulation of NB-LRR domain containing proteins. Zinc is a cofactor in many redox reactions and zinc deficiency-like in plants have been attributed to damage by ROS. This suggests that the zinc-pattern-deficiency symptoms associated with HLB is caused by ROS generated by citrus plants in response to Candidatus Liberibacter infection.
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. The genome sequences are completed for Xac Aw strain and Xanthomonas axonopodis pv. citremelo. The papers are in preparation. Aw strain: Complete De novo genome sequencing of Xanthomonas axonopodis pv. citri strain Aw was done. 454 Titanium paired end reads were used for making contigs and scaffolds using Newbler with 24X coverage. It was then assembled and closed by using contigs made from Illumina 75bp paired end reads using CLC Bio with about 300X coverage. Finishing was done using Opgen MapSolver. Plasmids for the strain were obtained by reference sequencing using only the Illumina 75bp paired end reads. The size for genome was 5.34 Mb and for plasmids pAw1 and pAw2 was 29 and 60 Kb approximately. The plasmid sequences need further confirmation due to the repeat regions. The genome sequence has been compared to the reference strain 306 using Mavue and global rearrangements were observed. In detail analysis of T3SS revealed presence of AvrGf1 and absence of HpaA gene. Further analysis of other secretion systems, LPS, EPS, motility clusters and other regions different form reference strain is currently in progress. Currently, we are also working on closing the gaps of the other three strains XAC A*270, XAC A44 and XACM 1381.
This project has been revised extensively to take into consideration of suggestions from reviewers and CRDF council. The title has been revised as: Control of citrus Huanglongbing by screening small molecules which are antimicrobial against Candidatus Liberibacter asiaticus. Specifically, the target has been suggested to be SecA for the first year. Protein secretion in bacteria is a critical and complex process. SecA is the protein translocase ATPase subunit and a superfamily 2 RNA helicase, which involves in pre-protein translocation across and integration into the cellular membrane in bacteria. Identification of small molecule inhibitors that intrude the function of SecA could lead to potential antimicrobial agent. In order to find the novel inhibitory structures we followed the below steps in the current design. 1) Identification/Build the 3D protein structure & optimization 2) Pharmacophore design based on ATP binding site 3) Virtual screening of commercial databases 4) Generation of a small subset of structures 5) Molecular docking studies & filtration of the structures 6) Selection of best candidates by scoring functions & chemical intuition 7) Biological activity studies against SecA Several 3D structures of SecA protein have been reported in Protein Data Bank (PDB), nevertheless we used the best resolution (<2.0Ao) and ATP bound model 2FSG.pdb in our study. Maestro module of Schrodinger software was used to add hydrogen's, electro static potential charges to the protein and optimized the structure with molecular minimization by using AMBER force field. Then identified the pharmacophores at ATP binding site and subjected these to virtual screening with the Lead Like structures from commercially available ZINC databases. Further with the best hits from database structures we build a small set of ~5000 structures as subset and all these structures were docked at ATP binding site to evaluate the docking scores and their molecular poses at the active region. Based on the dock scores we filtered ~4500 structures and selected ~500 (10%) for further docking & minimization studies and evaluated the scoring functions. Based on scoring functions, physicochemical properties and our chemical intuition we have chosen 50 structures (10%) for biological activity studies. The activity studies against SecA are in progress and will be presented within couple of months. All the molecular modeling studies have been performed on Linux system by using Schrodinger suite programs.
We have been able to detect Candidatus Liberibacter asiaticus (LAS) in biofilms on the surface of alimentary canals of psyllids (Diaphorina citri) reared on citrus infected with huanglongbing in insectaries or collected from citrus groves with a history of having huanglongbing. The alimentary canals were dissected from psyllids, suspended in a buffered dye solution on microscope slides, and LAS was observed on the canals using epifluorescence microscopy. The DNA-binding fluorochrome, Syto 13, was used as the dye. The percentage of psyllids from which LAS could be detected in this manner varied from about 0 to 30%. The percentage was consistently less in July and August than it was earlier in the year. Thus, it has become more difficult to find enough psyllids carrying LAS for use as a source of inoculum to test different media for their ability to support growth of LAS. To help overcome this limitation, we have been washing individual alimentary canals with medium, examining a sample of this medium for LAS, and using those suspensions containing LAS as inoculum. We have been able to detect LAS cells in inoculated media by epifluoresence microscopy. By counting LAS cells, we should be able to detect changes in population size that might indicate growth of LAS in the media. Although we have not detected definite growth of LAS in any medium formulation as of yet, some formulations appear to support detectable levels of the bacterium for several weeks after inoculation while other formulations do not.
Objective 1: We conducted tests with nutrient analyses of leaf samples collected from HLB-infected and healthy trees to establish relationships that could be used for (early) diagnosis of HLB. Starch accumulation in symptomatic leaves can significantly alter the interpretation of nutrient status when diagnosed on a dry weight concentration basis. When corrected for starch accumulation by using specific leaf area, Ca, Mg and B are consistently lower and K is higher in HLB-infected trees compared to healthy trees under the same fertilization program. These changes exist in both symptomatic and asymptomatic leaves from infected trees. Similarly, separation between asymptomatic healthy leaves and asymptomatic infected leaves could be achieved using the Diagnosis and Recommendation Integrated System (DRIS), particularly when assessing the Dry Mass Index (DMI) generated by the method. DMI integrates the imbalances caused by nutrient losses and starch accumulation in HLB-infected leaves, thus could indicate infection early on despite the absence of visible symptoms. Leaf samples have been collected from trees infected with CTV, blight and phytophthora to determine if these changes are HLB-specific or are common changes to all diseases. Those samples undergoing testing for nutrient, starch and disease status. We are working to sample other diseases not currently present in Florida as well (e.g. CVC). Due to stress-related abscission of HLB-symptomatic leaves from the exterior of infected canopies for much of the year, we are often forced to sample symptomatic leaves from within the canopy and asymptomatic leaves from the outside. We tested the effect of this unavoidable sampling position difference on the measured leaf nutrients analyzed in healthy trees and found no significant differences in 11 elements measured. In year two we will continue or research efforts to determine which, if any, of these nutrient changes is a unique response to HLB infection. Objective 2: Five field studies have been established to evaluate the effects of various foliar nutrient applications on the expression of HLB in infected trees by evaluating tree nutrient status, growth, yield and visual tree appearance through photographic documentation. The first trial is a survey-type trial to monitor the health and yield of trees in Maury Boyd’s grove in Felda. We have harvested the same Hamlin and Valencia trees for two seasons in tThe first of these trials is in a heavily infected mature Hamlin grove in south Florida. Since the initiation of the project the trees in this study have received six foliar applications of one of nine different treatments. Untreated trees serve as controls. The trees were harvested in December 2009. This was the first harvest since the beginning of the trial and did not reveal any significant differences among treatments; however, that is not unexpected following only 1-year of treatment. The second study is in a young (3-5 years old) commercial Valencia grove in Haines City. Treatments in this study have been underway for approximately 7 months and include fertigation in addition to foliar nutrient sprays. Our initial efforts at this site have been to demonstrate the ability to raise the levels of specific nutrients involved in plant defenses within trees. Since treatments began, B levels have been successfully raised to near toxic levels within infected and healthy trees using both foliar and fertigation applied B. This has demonstrated that good nutrient uptake can be achieved through the treatment methods in a relatively short period of time. Analysis of the large quantity of these studies have generated is still being analyzed as of the writing of this report. As soon as the analyses are complete information will be passed along to the Florida citrus community. Two additional field studies are currently being started for this season. Objective 3: A hydroponics system has been constructed in an HLB approved greenhouse at the CREC. We previously reported that trees were being established in this system; however, for undetermined reasons those trees did not perform well in the hydroponics system. The project has been revised and new trees are being established in sand culture instead of liquid hydroponics. All of the plants will be maintained with a complete nutrient solution until HLB symptoms develop on the inoculated trees. Previous experience with greenhouse graft inoculation indicates that this will take about 90 days. At that time, healthy and infected trees will be subjected to various changes in the nutrient solution composition. Based on the data from objective 1, the levels of Ca, Mg, and B and combinations of these three nutrients will be changed to determine plant response with and without HLB. Trees remaining in complete nutrient solution will serve as controls. This experiment will allow us to begin to separate nutrient and HLB effects on plant growth and development.
By piecing together the information collected during the last two years of this project, we have developed, tested, and delivered a comprehensive plan for ACP management that is being widely adopted by our clientele. The 4-pillared plan includes: (1) dormant insecticidal sprays which we have nurtured into a successful annual area-wide cooperative program with leadership from Golf Citrus Growers Association (GCGA), and in cooperation with from DPI-CHRP and Hendry County Cooperative Extension service (2) efficient methods for testing effectiveness of insecticides in lab and field including recurrent low-volume applications of horticultural oil, (3) a rapid and reliable psyllid monitoring system to guide insecticidal control during the growing season, and (4) strategies to conserve and augment biological control agents (see below). Independent information from aerial applicators showed that over 70,000 acres were sprayed by air least once during 2008-09 and 2009-10 dormant season, many more than once as part of the Southwest Florida Cooperative Area-wide Management program. Although our records for ground applications are less accurate, survey results indicate that over 30,000 acres were also sprayed each winter by ground. To our knowledge, all groves in the area have at least conducted one dormant spray last winter. Field counts provided by growers and FDOACS-DPI using personnel we trained show that five months after the dormant sprays, adult ACP populations in 2010 were still less than during the beginning of the dormant season and only a third of what they were the same time in 2009, thus showing an area wide decrease. An article is being submitted this month to Citrus Industry as a follow-up to one published there in 2009 on the area wide program. We are also promoting the use of scouting reports to make informed decisions to use insecticides only when needed during the growing season and an article we wrote on the subject appeared in the April issue of Citrus industry (see below). Also, an extension document on psyllid monitoring methods was submitted to the Electronic Data Information Source (EDIS) of the University of Florida. Survey results indicate that the stem tap method for monitoring psyllid population has become the standard in the region. The cost of monitoring psyllids this way was estimated at about $25 per block up to 100 acres so could be done 20 times per year for as little as $5/ac. and could thus avoid unnecessary sprays during the growing season that may cost up to $50/ac and cause secondary pest outbreaks of mites, scales and leafminers. (3) During this quarter, we have conducted 2 trials testing foliar insecticides (18 treatments) one drench trial (12 treatments), and are extensively testing a new systemic insecticide from Dupont, cyazapyr, for ACP control that could be a critical addition to neonicotinoid insecticides for protection of young trees. Since 2007 we have published and posted on our website http://www.imok.ufl.edu/entlab/pubs/sta_rpts/ 18 reports of insecticide trials in citrus. Last year we conducted a survey of local managers of 106,148 acres of commercial citrus in SW Florida who indicated that there has been widespread adoption of these management strategies. We are presently conducting a new survey to evaluate the changes and tendencies in adoption of ACP management strategies. After only a week, we have already received 11 surveys covering 28,379 acres, 40% of the total number received last year. Preliminary results indicate that 100% of the respondents are satisfied or very satisfied with their management program, that only 63% report increases in secondary pest pressure compared with 92% last year, and that there is strong support for including biological control in the area wide management program. – Stansly, P., A. Arevalo and J. Qureshi. 2010. Monitoring methods for Asian citrus psyllid. Citrus Industry 91(4):20-22. – Ortez M. J., H. A. Arevalo, and P. A. Stansly. 2010. Alternative Methods to Collect Tamarixia radiata (Waterston) (Hymenoptera: Eulophidae) From Mass Rearing Colonies. (Poster) Jupiter, FL July 2010
During the first year of this project, we showed that an already low psyllid population could be reduced further using applications of 435 horticultural oil at 2 gallons per acre (GPA) applied every 2 to 4 weeks, depending on infestation. Better results compared to a modified London Fogger 18-20 were obtained with a Proptec rotary atomizer (spinning disk) P400D machine that propels the spray mixture into the canopy using four hydraulically operated fans. However, we wanted to find a heavy infestation of psyllids to provide a more rigorous test, so we chose a 40-acre block of ‘pineapple’ oranges in Glades County that had not received a dormant spray and consequently had high psyllid populations. In addition, we altered the protocol, increasing volume through the Proptec to 10 GPA to accommodate a modified mixture of micronutrients that has shown promise to mitigate symptoms of greening in another project. Two gallons of oil plus the micronutrient mix are being applied every two weeks in a randomized complete block (RCB) design with four replications. Asian Citrus Psyllid (ACP) populations are being monitored on alternate weeks and leaf samples have been submitted to the HLB laboratory at the Southwest Florida Research and Education Center (SWFREC) for PCR analysis. Contrary to expectations, ACP populations have been low, although the accumulated number of adults is less in the treated plots (5.1′ 1.5 ACP x day) than in untreated plots (8.5’1.9 ACP x day). In another trial employing an RCB design with three replicates, we are evaluating the effects of frequent oil applications in an 85-acre block of organic ‘Valencia’ oranges in Charlotte County. Trees in the treated plots are being sprayed every two weeks with 2 GPA of horticultural oil using a conventional air blast sprayer at 100 GPA. Again, contrary to expectation, ACP populations have been low with no significant differences between treatments. In a third trial recently completed, psyllid suppression with a low volume (LV) application zeta-cypermethrin as a dormant spray was compared to a high volume (HV) application of spirotetramat alone or in combination with early or late soil applications of aldicarb. All treatments including the LV dormant season spray by itself averaged overall adult psyllid populations significantly lower (0.36 ‘ 0.18 ACP x day) than the untreated control (2.19 ‘ 1.19 ACP x day). Results were reported by Ph.D. graduate student Scott Croxton (see below) who won 3rd prize at the annual meeting of the Florida Entomological Society. A fourth trial was recently initiated in a 16-acre mature block of Valencia orange in Collier County to evaluate coverage, deposition, and absorption of foliar nutrients applied with the LV ‘ Proptec rotary atomizer P400D sprayer @ 10 GPA every two weeks compared to an air blast sprayer applying 100 GPA applying the same total amount of active ingredient three times a year on the summer, fall, and spring flush. The trial is designed as a RCB with three treatments and three replications. First applications have been made and leaf samples taken from 16 locations in the canopy of three randomly chosen trees per plot collected before and 3 h after treatment. Scott Croxton, Mike Edenfield and Phil Stansly. 2010. Timing of Temik and Movento Applications for Control of Asian Citrus psyllid (ACP) Diaphorina citri. Florida Entomological Society Annual Meeting. Jupiter, FL. July, 2010
The third season (2010) of the trials with treatments of foliar applied nutritional sprays has begun with trees responding with vigorous vegetative growth and strong flowering resulting in impressive fruit set. Trees in decline at the start of experiments in spring 2008 have regrown canopy on the exterior of the trees leaving dead and declined wood in the tree interior. Our approach of arranging the components of the Boyd cocktail into groups of micronutrients (Mg, Zn, Mn, Mo, B), SARs ( Salicylic acid & Serenade), Phosphite, hydrogen peroxide, and other macronutrients is beginning to identify those components most important for maintaining trees with respect to yield and fruit quality. Although we did experience cold damage to fruit in the January 6 & 11, 2010 freezes with some drying in fruit, our yield and fruit quality data appear sound. In a 30-acre commercial block of 8-year-old Valencia trees the highest yield was with the complete nutrients plus Phosphite (93 lbs/tree) with and (82 lbs/tree) without the SARs. Statistically the addition of hydrogen peroxide in the mix did not improve yield (70 lb/tree). The treatments without the micronurients were among the lowest yields (61 to 42 lbs/tree) as was the treatment with micronutrients without the phosphite (57 lbs/tree). Juice quality among treatments from fruit harvested in April from the 30-acre commercial block of Valencia was generally not different except for Brix and pound solids/box. Percentage juice, acid, and Brix/acid ratio were not generally significant among treatments. Brix were highest for the complete nutrient cocktail without SARs (10.5) and the complete cocktail with SARs (10.4). Brix were generally lowest for treatments without micronutrients (9.7 to 9.9). Pound solids per box were highest for the complete nutrient cocktail without SARs (5.1) and the complete cocktail with SARs (5.0). Pound solids per box were lowest for treatments without micronutrients (4.5 to 4.6). We are in the third year of a replicated experiment in a 12-acre experiment commercial block of 8-year-old ‘Valencia’ oranges on ‘Swingle’ to test effects of micronutrients + systemic acquired resistance inducers, and Asian citrus psyllid (ACP) chemical control on ACP populations on Can. Libericacter asiaticus (CLas) titer, and plant yield. After two dormant sprays (Dimethoate @ 1pt in Dec and Danitol @ 12 oz/ acre in Jan) 2 more sprays, Delegate @ 5 oz/ac in May and Lorsban at 3pt/acre in July, were needed to maintain the ACP population below a threshold of 0.2 adults/tap in treated plots. Trees in plots where ACP was managed using insecticides produced significantly more Lb-solid per tree (5.76’0.36 Lb-solid/tree) than trees in untreated plots (4.17’0.65 Lb-solid/tree), continuing a trend seen last year. Also, trees treated with Micro+SAR produced more (5.26’0.55 Lb-solid/tree) compared with untreated trees (4.55’0.64 Lb-solid/tree), again following trends seen from the first harvest in the 2008-2009 season although differences this year were not significant. Although, neither the insecticide treatment nor the nutrients have slowed down the spread of HLB (now virtually 100%), Treatments where ACP is being managed have had consistently lower titer over the two year study, except in May 2010 where titers were higher than previously and not significantly different among treatments. Arevalo, H. A., and P. A. Stansly. 2010. Effects of Diaphorina citri management on Can. Liberibacter asiaticus titer and fruit yield in Valencia oranges. Florida Entomologist Society. Jupiter, FL. July 2010
During the first quarter of funding, Core Citrus Transformation Facility (CCTF) continued to process the orders for transgenic Citrus material. Demand for genetically transformed citrus plants stayed high resulting in influx of new orders listed here by names of genes of interest or plasmids: p6; p33; p7; p10; pMOG8000; pAS7; pAS13*; pNAC1; pMKK7; pMOD1; and pSucNPR1. The work also continued on the old orders that were previously partially completed. Considering that transgenic Duncan plants carrying NPR1 gene exhibited significant resistance to Citrus canker, CCTF received order for production of commercially important Flame grapefruit cultivar transformed with the same NPR1 gene (order completed-NPR1 gene: 10 plants). A gene thought to be superior to NPR1 (so-called superNPR1) was introduced into Duncan grapefruit (superNPR1 gene: 12 plants). Introduction of NPR1 into Hamlin orange cultivar and superNPR1 into Flame is half completed. Work on the order pAS7 that is associated with HLB tolerance/resistance is half done (5 Duncan plants). Order that included use of pLC plasmid is also completed (8 Hamlin orange plants). Ten Mexican limes transformed with gene in pHK plasmid were produced, but satisfaction of this order will include production of more plants per client’s request. Five plants of Mexican lime transformed with p33 gene were also produced. CCTF produced more plants for the old orders: N1* gene: 3 Duncan; C5*: 3 Duncan; CL1 gene: 2 Valencia, 3 Duncan; CL2 gene: 1 Duncan; PiTA gene: 1 Valencia; CIT108p: 1 Flame. About thirty more soil-adapted plants will be submitted to the PCR testing as a secondary proof of their transgenicity before delivery to clients. Some of the funds from the grant were used to hire additional help through the summer resulting in seasonal increase of CCTF capabilities. Please be informed that the person directly managing the CCTF (and co-PI) is Dr. Vladimir Orbovic.
The objective of this project was to investigate three questions: 1) what is the seasonal pattern of Ca. Liberibacter asiaticus prevalence in leaf tissue on a grove scale; 2) what are the flushing patterns of citrus and whether these flushing patterns affect the prevalence of Ca. Liberibacter asiaticus in Diaphorina citri or citrus leaves; and 3) what is the prevalence of Diaphorina citri carrying Ca. Liberibacter asiaticus on a grove scale and how does it compare the results from the citrus trees in the same grove. In 2008 and 2009 Ebert and Rogers demonstrated that the prevalence of Ca. Liberibacter asiaticus in the Asian citrus psyllid varied seasonally but the pattern between seasons was not consistent. It was suggested that perhaps the reason for the differences between the years related to the flushing patterns of citrus and the prevalence of the bacterium in the leaves where psyllids are feeding. This project aims to determine if there is a relationship between the frequency of disease on branches and psyllids Three locations of low to high disease levels have been selected. Leaves and psyllids are being collected from the sites. Samples are being processed for disease detection via qPCR. The post-doc that will be working on this project has been hired and is expected to begin working at the beginning of September.
Citrus extension agents have been actively involved in assisting Florida citrus growers and others in increasing their knowledge to adopt practices for maximizing the control of the Asian citrus psyllid and management of citrus greening or HLB. The citrus extension agents have used a variety of programs to achieve these educational goals. Major programs conducted by citrus agents are as follows: 1) ‘Low Volume Technology for Citrus Pests’ program series was conducted in 6 locations to discuss application methods and pesticides for optimum control of psyllids. This series reached over 300 participants state wide. 2) ‘Low Volume Sprayer Calibration Rodeo’ events were conducted in 3 locations to determine if low volume equipment currently being used meets the 90 micron requirements being required by some pesticide labels. These events evaluated more than 70 different sprayers. This series increased the awareness in the citrus grower community of the need for equipment to spray at the 90 micron particle size. 3) ‘2010 Florida Citrus Growers’ Institute’ reached over 370 participants with timely information on greening, psyllid management and citrus genomics. Twenty speakers from local, state, national and international research and businesses made presentations during the 7-hour program. Archived web presentations of numerous programs conducted by the citrus agents are available on the web at www.citrusagents.ifas.ufl.edu . This site hosts presentations from the Citrus Growers’ Institute (2008, 09 and 10) and citrus agents program series related to low volume spray programs. Total web hits using Urchin 5 web statistics exceed 709,900 for the site and included 3,034 for the 2010 Florida Citrus Growers’ Institute, 9,254 for the Low Volume Application series and 207,495 for all citrus agents’ newsletters. Providing web-based format allows program information to be readily available for those who could not attend programs and serves as a reference source for all to use. Field visits have always been an important aspect of grower education in Florida by the Extension Service. Annually, more than 500 grower visits are conducted by agents to assist growers in making timely decisions, increase knowledge and choose appropriate production practices to achieve necessary control of specific pests/diseases. Citrus newsletters are offered by agents and are distributed electronically or in a printed version. These newsletters reached over 2,500 growers, citrus employees or others involved in the Florida citrus industry. Timely and unbiased information is essential to maximize production and enhance pest control strategies. Polycom broadcasts have been received in selected counties to ensure citrus related seminars and special programs are offered to ‘local’ growers without having to drive to a single site. These programs save considerable time and money while providing timely information to all growers. Polycom broadcasts included: citrus black spot and seminars broadcast from research and education centers on timely topics focused at effective and timely information dissemination. Urban audiences and Master Gardener trainings have been conducted to ensure that these groups are aware of the risks of growing citrus as well as disease and insect issues. Over the grant period, more than 500 Master Gardeners and urban clientele have participated in one or more of the citrus agent instructed programs. While many of the programs have been in central and south Florida, programs have also been offered in Okaloosa and Jackson Counties in west Florida. Presentations have also been provided by citrus agents at regional, national and international meetings to effectively communicate the latest production and field findings to as wide of an audience as possible. When battling greening and psyllids, agents must reach beyond their local regional area to gain and present information.
The citrus extension agents have been actively involved in educating citrus growers and homeowners about greening (HLB) and other disease issues (citrus black spot) around the state. Major programs where citrus agents were involved include: Citrus Growers Institute ‘ this program was conducted by the citrus extension agents in Avon Park at the South Florida Community College campus. A total of 376 participants attended the full-day program where 20 speakers presented timely information related to greening and/or psyllid management. Program speakers included international, national and in-state experts. The program was free to attend with some program costs being offset by the FCPRAC grant and lunch was sponsored by 7 chemical companies. All presentations were recorded and currently available on the citrus agent’s web site at www.citrusagents.ifas.ufl.edu . The site received 3,034 hits to access presentation information in May and June of 2010. This site also has the presentations from the 2008 and 2009 Institute programs that discussed various aspects of greening. To view or download the presentations, the above site is accessible to anyone, regardless of location around the world and provides timely and historical information in combatting this major disease. Citrus Black Spot ‘ A program was hosted by various county offices or research centers around the state to provide timely information related to citrus black spot. By hosting the polycom broadcast, growers were able to receive timely information with little travel time as compared to attending a central state-wide meeting. Master Gardener Trainings ‘ programs were conducted in Collier, Okaloosa and Jackson counties where over 220 participants received information on citrus and its diseases, including greening and citrus canker. Educating urban audiences is also important in combatting citrus diseases state-wide. Presentation of research findings on the use of Remedy (triclopyr) to control citrus sprouts was presented at the Florida State Horticultural Society annual meeting. Citrus sprout control is essential when removing greening infected trees as a large percentage (up to 80%) of sprouts emerging from stumps of removed trees have been shown to be positive for the disease within 9 months of tree removal. Grove visits by agents during this quarter exceeded 79 field visits in addition to 31 office visits to address greening issues. In addition to field and office visits, agents received a significant number of phone calls and have highlighted greening management topics and issues in their citrus newsletters. Grower educational programs were also conducted or assisted with by agents in Arcadia, Avalon, Dade City, Ft. Pierce, Immokalee, Myakka City, Sebring and Wauchula. These programs highlighted greening, citrus black spot and/or area-wide management areas.
Two experiments were initiated at the Southwest Florida Research and Education Center (SWFREC)T to assess impacts of insecticidal control of ACP on non target pests and beneficial insects. The experimental block consisted of 15-yr-old sweet orange ‘Valencia’ trees planted on double-row raised beds at a density of 132 trees/acre. For first experiment, trees were hedged so new shoots attractive to ACP and citrus leafminer (CLM) and beneficial insects were plentiful. Nine treatments (435 Oil, Movento 240 SC + 435 Oil, NAI-2302 + 435 Oil, Agriflex (MK 936) + 435 Oil, Micromite 80 WGS + 435 Oil, low and high rates of Belt 4SC + Induce, M-Pede + Addit, Portal 0.4EC) all intended to control ACP and an untreated control were randomly distributed across 4 replicates in a RCB design. Treatments were applied on 14 May using a Durand Wayland 3P-10C-32 air blast speed sprayer operating at 1500 RPM with 2 stainless steel T-Jet # 5 nozzles per side delivering 65 gpa and evaluated at 3, 10 and 17 days after treatment (DAT). Ten randomly selected shoots per plot were collected and examined under a stereomicroscope in the laboratory to count ACP nymphs. Three fully expanded leaves on each shoot were examined to count CLM larvae and mines. Density of ACP adults and beneficial insects was estimated from three central trees in each five-tree plot by counting the insects falling on a clipboard covered with an 8 . . 11 inch laminated white sheet placed under randomly chosen branches which were then struck 3 times with the PVC pipe to make a count for one ‘tap’ sample. Four tap samples were conducted per tree. All treatments provided significant reduction in psyllid populations compared to control for up to 17 days. Significant reduction in number of CLM larvae compared to the control was observed in all treatments through 10 DAT except 435 Oil alone at 3 DAT, Portal at 10 DAT and M-Pede + Addit at both observations. CLM larvae or mines were not significantly less than the control in any treatment except for Movento + 435 Oil, Micromite + 435 Oil, Portal and M-Pede + Addit, an effect not observed against ACP. Such increase in CLM population could be due to reduced number of CLM natural enemies in those treatments. No spiders were observed in the Micromite treatment and most were observed in the 435 oil alone treatment. Numbers in the treated and untreated trees did not differ statistically except 435 oil alone and control. Only 9 adults or larvae of Olla v-nigrum and Harmonia axyridis ladybeetles were observed in samples, 4 in the control, 3 in low rate of Belt and one each in 435 oil alone and M-Pede + Addit. In the 2nd experiment treatments of 435 Oil alone, Delegate 25 WG + 435 Oil, Provado 1.6 F + Induce, Movento 240 SC + 435 Oil and three rates of an experimental product from Bayer CropScience with or with 435 Oil or NIS were applied on 12 July and evaluated at 3, 10, 17 and 24 DAT. Application and evaluation procedures were same as above except application volume of 150 gpa. Compared to control all treatments provided significant reduction in ACP populations through 24 DAT. CLM larvae were reduced by all treatments at 3 and 10 DAT but only Movento, Delegate, and low and medium rates of experimental product with 435 Oil were effective at 17 DAT. CLM mines did not differ between treated and untreated trees at 3 DAT, but were less at 10 DAT in treatments of 435 Oil alone, Delegate + 435 Oil, and experimental product + 435 Oil or NIS. At 17 DAT all treatments except Provado and experimental product alone had fewer mines than control. CLM larvae or mines did not increase in any treatment compared to control. Spiders were the only beneficial arthropods observed during sampling and treatment effect was not significant. Overall, treatment effects against CLM were less marked and more short lived than against ACP. Increase in CLM populations in some treatments indicate possible negative impacts on their natural enemies. This year survey of grove managers by SWFREC and IFAS extension was distributed. So far, increase in citrus leafminer, mites, and scales under current ACP management was reported by 50%, 30% and 20%, respectively.
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