The objective of the proposed research is to develop genetically engineered (GE) citrus founder lines containing a platform that will allow the precise insertion of desired traits. In addition to allowing the targeted integration of transgenes, the proposed system also enables the removal of unneeded sequences such as antibiotic resistance marker genes (Wang et. al., 2010), allowing the generation of ‘clean’ (marker-free) GE citrus plants and or fruit. The targeting construct (or TAG) containing the recombinase platform has been completed and consists of the recombinase recognition sites for integration upstream of the positive/negative selection genes, CodA::kan. Kan is used for positive selection of transgenic citrus production while the codA gene will be used to detect successful recombination events. Down stream of the selection system are recognition sites for recombinase-mediated excision. Also included between the recombinase recognition sites is a minimal promoter GUSPlus gene. This gene will allow detection of the TAG construct inserting into hyperactive chromosomal expression loci. This type of loci is desirable for high expression of transgenes. Due to the placement of the minimal promoter GUSPlus gene between the recombinase recognition sites it will be removed during recombinase-mediated targeting. More recently a second version of the TAGII construct has been completed with an improved selectable expression cassette. Efforts are now underway to complete the agro-bacterium based pEXCH vector, which will be used to deliver both recombinases (for RMCE) and genes of interest (for genomic insertion). The Thomson lab has sent the CTAG constructs to the Stover lab. Transformation of citrus uses the disarmed A. tumefaciens strain EHA-105 harboring a binary vector CTAG vector. Six independent transformation events were performed for each genotype (Carrizo and Hamlin), subjected over 2,000 epicotyls and 200 cotyledons to pCTAGII vector. Putative transformed shoots, 157 from Carrizo and 149 from Hamlin, were obtained after 45 days. All putative transformed shoots have being micro-grafted. To date 19 of the initial 25 plants screened have been shown positive for the pCTAGII site by PCR. Two independent transformation events with pCTAGV-KCN3 vector. This newest CTAG vector contains DSred for non-destructive visible selection as well as an anti-apoptotic gene. The anti-apoptotic gene was included to help transformation efficiency. Both genes will be removed (along with the CodA::Kan marker) during the RMCE process. DSRed expression has been observed during co-cultivation of citrus tissues. Well-developed shoots were induced after 35 days in selection medium. This is an improvement of 10 days over the previous transformation efforts. 57 explants from Carrizo and 48 explants from Hamlin have been obtained. It is anticipated that a no-cost extension will be requested to extend the second year of funding to mid-April 2014, permitting a full year of effort on the initial funding.
We applied six therapeutic and two control treatments to infected and healthy Valencia orange on Kuharske Carrizo rootstocks at Florida. These eight treatments each contained 10 healthy and 10 HLB-infected trees growing in a greenhouse. Citrus rootstock trees used in these experiments were HLB inoculated using a standard inverted ‘T’ budding techniquewith bark with pieces from citrus infected trees. The trees were kept in the greenhouse under natural light conditions at 17-25’C until the HLB infection was confirmed by quantitative real-time PCR (qRT-PCR). The six therapeutic treatments included: 2, L- Arginine, 2, gibberellin in combination with 6-benzyl adenine (BA), and 2, atrazine in combination with sucrose. The surfactant Silwet, LK-phite and LDKP3XTRA were added to all treatments. All treatments were sprayed on the citrus foliage; the volume sprayed per tree was enough to wet both the upper and lower leaf surfaces just to the point of runoff. Phosphites were added to our therapeutic compound mixtures, since our data mining of HLB infected citrus suggests the presence of genes associated with phosphate deficiency, the symptoms include starch accumulation and collapse of healthy root function. Our rationale is that by eliminating these deficiencies, we will be able to better evaluate if our therapeutic treatments enhance citrus response to HLB, prolong the life of HLB-infected plants, reduce the bacterial titer and counteract the detrimental effects of the infection on citrus production. We are planning a second treatment in Feb 2013 on the same trees. For the first treatment we used 10 healthy and 10 HLB-infected trees (1 tree = 1 biological replicate) for 2 different concentrations of L-arginine dissolved in water with Silwet as surfactant and LK-phite and LDKP3XTRA. The controls for this treatment were 10 HLB-infected trees treated with just water containing the surfactant and used as control trees. Also, 10 healthy and 10 HLB-infected trees were treated with a mixture of surfactant, LK-phite and LDKP3XTRA in water and used as a second set of control trees. All HLB-infected treated trees and the healthy control sets were sampled. This treatment provided us 4 treatments x 6 trees x 3 biological replicates X 2 sampling points = 144 samples for analysis (RNA). For the second treatment used 2 different concentrations gibberellic acid in combination with a single 6-benzyladenine concentration, the solutions were prepared as explained above. We used 10 healthy and 10 HLB-infected trees for each combination. This experiments provided 2 treatments x 6 trees x 3 biological replicates X 2 sampling points = 72 samples for analysis (RNA). For the third treatment we used 2 different concentrations of atrazine in combination with one concentration of sucrose, solutions prepared as explained above. We use 10 healthy and 10 HLB-infected trees for each combination. This experiments provided us with 2 treatments x 6 trees x 3 biological replicates X 2 sampling points = 72 samples for further analysis (RNA). Three leaves that were mid symptomatic based on observation of blotchiness, mottling and hardening of the veins were collected at 0 time and 3 days after treatment from each individual treated or control tree and processed individually. Leaves were immediately frozen in liquid nitrogen and keep at -80’C until extracted. Total RNA was extracted from half of the leaves the rest were stored at -80’C for extraction later. We have obtained a great deal of variability of RNA quality depending on the severity of the leaf symptoms gene expression is being determined by qRT-PCR.
The purpose of this project was to preserve citrus germplasm in Florida that is threatened by loss due to huanglongbing (HLB) and citrus canker. This project called for close cooperation among the National Clonal Germplasm Repository for Citrus and Dates (Repository), Riverside, CA, the Florida Citrus Germplasm Introduction Program (CGIP), and researchers located at the United States Horticultural Research Laboratory (USHRL), Ft. Pierce, FL. One priority of this project was to identify and prioritize valuable citrus germplasm in Florida that is being threatened and which is not already available in a protected collection. Stakeholders have met a total of three times to identify threatened citrus germplasm, and identified citrus germplasm has been propagated in a secure greenhouse located at the USDA ARS USHRL, Ft. Pierce, FL. Activity in Florida has been directed to cleaning the selected accessions from HLB and citrus canker. Currently 66 accessions are held at Ft. Pierce, FL which test negative for HLB and citrus canker. Additionally there are 25 accessions held in quarantine at the USDA ARS Repository, Riverside, CA. Eleven of these Florida quarantine accessions have been therapied, and all pathogen testing is complete except for viroid indexing from citron on sequential polyacrylamide gels, and this is anticipated to be completed within two months. There is a total of five additional accessions that have been identified and currently held by private owners which will be added to the collection at Ft. Pierce and targeted for clean up. This project has enabled the establishment of a pipeline to allow for collection of accessions in Florida, preliminary clean up in Florida, further therapy and complete testing for all known graft transmissible pathogens of citrus at the Repository, Riverside, CA. Clean accessions are then returned to Florida using the ‘Citrus Passport’ protocol which enables release of the accessions in Florida in less than a year after receipt from the Repository.
Two field experiments were established to conduct research in open hydroponics and high density advanced citrus production systems. They are located near Auburndale in the central Florida ridge region, and near Immokalee in the southwest Florida flatwoods region. The main goal of this research is to develop efficient horticultural systems which can be used for early profitable fruit production and in an IPM approach for managing / mitigating the most serious Florida citrus diseases of HLB and canker. Immokalee experiment: Results to date indicate higher NH4-N, NO3-N, P and K content in the irrigated than non-irrigated zone of drip hydroponics (DOHS). Root distribution also followed a similar pattern for DOHS, showing greater intensity (60-80%) in the irrigated than non-irrigated zone (20-40%). The nutrient concentrations and root length densities of CGP were lower compared with both OHS treatments and were uniformly distributed around the tree. Nutrient (NH4-N, NO3-N, P and K) leached was very low for all the fertigation methods. Trees grew faster under DOHS than CGP as indicated by canopy volume and trunk cross-sectional area measurements (20 to 25%). The leaf N, P and K concentration were adequate according to UF/IFAS recommendations for all treatments. However, there was greater N accumulation with DOHS (~114 kg N ha-1) than CGP (~79 kg N ha-1) supporting greater N uptake efficiency for DOHS treatments. Phosphorus and K accumulation were similar for DOHS and CGP indicating adequate applications of these two nutrients. Soil moisture was adequate in the irrigation of DOHS and grower practice falling between 7-15% on volume/volume basis suggesting that water was non-limiting in all the three irrigation methods. However, measured cumulative and daily water uptake were greater with DOHS than CGP though the crop coefficients were similar for the three irrigation practices indicating greater water use efficiency. Yield after four years was 85% greater for DOHS treatment compared with CGP. Auburndale experiment: The ACPS proved very successful for growing citrus on the Ridge, essentially reducing the time to production by 1-2 years. Those years represent real gains in early return on investment, and, more tangibly, they also represent direct savings in 1-2 years of the cost of production. The best treatment tested in this experiment consisted of the high planting density (363 trees/acre), C-35 rootstock, and the intensive drip open hydroponics (daily) fertigation system. In year 3 the yield of this treatment was an average 222 boxes/acre of ‘Hamlin’ fruit, compared to 90 boxes/acre in the conventionally grown treatment. Expected yields for year 4 in this best treatment are 500-600 boxes/acre. Unfortunately due to rapidly increasing HLB infections in this block (8.5% in year 3), the sustained yield increased seen in the first 3-4 years are unlikely to sustain in years 5 onwards because HLB infection rates are expected to exceed 50% by year 6. In November during the third year, a field day was held in the ACPS field experiment near Auburndale, attracting about 200 citrus growers, researchers and supporting industries. A comprehensive ACPS web site was developed in the first year and continues to serve visitors with timely updates on the ACPS research effort (http://128.227.177.113/ACPS/)
Genome of Candidatus Liberibacter asiaticus (CLas) reveals the presence of luxR that encodes LuxR protein, one of the two components cell-to-cell communication system. But the genome lacks the second components; luxI thar produce Acyl-Homoserine Lactone (AHL) suggesting that CLas has a solo LuxR system. We confirmed the functionality of LuxR by expressing in E. coli and the acquisition of different AHLs we detect AHLs in the insect vector (psyllid) heathy or infected with CLas but not in citrus plant meaning that Insect is the source of AHL. Using different bacterial biosensor, we partly identify these AHLs (number of Carbon). Forming CLas biofilm on the surface of insect Gut confirm the presence of cell to cell communication in insect while the planctonic state of CLas in plant indicate the absence of this communication. In plant, we found molecules that bind to LuxR but inactive its function (plant defence). we try now to characterize these molecule and study their effect on biofilm formation inside insect. we use purified molecule to feed infected insect through artificial diet system. the main aim of this project is to express molecules in plant that interfere the growth of CLas in insect by feeding.
During July and August of 2012, the citrus agents were actively involved in the continued education of citrus growers in the identification of HLB and practices to suppress and/or control psyllids. Educational activities included: grower programs (Citrus Expo and local and regional grower meetings), newsletters, consultations (phone and email), and grove visits. In some parts of the industry, smaller growers are still struggling with the proper identification of HLB, even with the disease being present in Florida since 2006. While no part of the state is HLB free, the incidences of HLB continues to increase impacting all locations where citrus is produced both commercially and in the urban landscape with greater frequency. Agent’s involvement also includes continued support of citrus health management areas (CHMAs) and activities to broaden participation in area sprays by both large and small growers. The successes obtained by CHMAs are directly related to getting as many growers to participate in the coordinated sprays as possible targeting psyllids. To increase grower participation, agents must keep HLB and psyllid information constantly in front of all citrus growers, both small and large. To document the progress of HLB in symptomatic trees, the agents post photos from known positive HLB trees in their area on a monthly basis to the ‘citrus agents’ web site (http://citrusagents.ifas.ufl.edu/hlb_photos/index.htm). This site has monthly photos going back 2 years and easily tacks the progression of the disease in symptomatic trees. The surprising thing about this photo series is that some trees have not declined as earlier expected and still have acceptable crop loads at harvest time. Most of the sites where photos are collected are on some form of enhance nutritional programs to try to maintain tree health as long as possible. The education of homeowners that grow citrus in the urban landscape is also important. Many landscape citrus trees are now becoming HLB positive and citizens are asking for information on the protection of young citrus trees and/or options for mature trees. To broaden our ability to educate these clients, the agents assist with Master Gardener training to educate the new trainees as well as offer assistance to county extension service offices to support their education on identification and control options.
Transcription factors: In order to study the effects of the identified chemicals on CLIBASIA_01180 in a living system, Sinorhizobium meliloti has been utilized a surrogate host. To this end, a mini-transposon mutant of the homolog of CLIBASIA_01180 in S. meliloti was obtained. The transposon is inserted after the coding sequence of the MarR, which would still be functional. Thus, the mutant can be used as a reporter due to the presence of a gus gene in the transposon. Using this strain and the wild type, we did a toxicity test, and found the three of the chemicals previously described were highly toxic while the others were not even at high concentrations. Using lower and up to the highest non-lethal concentrations of chemicals supplemented in the growth media, Gus activity was measured. This activity will reflect the chemical’s ability to enter the cell as well as its ability to affect the DNA-binding activity of the MarR in vivo. Some small differences were found, but because so many of the chemicals will toxic at low concentrations, it was difficult to measure their effect on the activity of the MarR. Because our surrogate also infects plants, we wanted to examine if the homolog of CLIBASIA_01180 in S. meliloti plays a role in the infection of legumes. For this purpose, mini-transposon mutants that would generate an inactive MarR were utilized in comparison to the wild type in infections of Medicago sativa and Medicago truncatula. These mutants also contained a gus reporter gene, making it possible to see if the MarR is expressed in the nodule, as well. The results suggest that this MarR is not essential for nodule formation or function, as the wild type and mutants had similar patterns of nodule formation. However, the Gus staining does show that the MarR is expressed in the nodules, indicating that it can play an important role in plant-bacteria relationship. In order to better understand the effect disrupting CLIBASIA_01180 will have on Ca. L. asiaticus, we are attempting to identify its natural ligand(s). A KEGG small molecule thermal melting screen identified several putative hits that are plant signaling molecules, as well as other biologically relevant ligands. The next step is to validate these hits using gel shift assays. If this regulator is sensing a plant signaling molecule, it would provide a very interesting target to prevent the bacteria from responding to its environment during infection. ZnuA purification: ZnuA as well as its closest homologous (Clibasia_02120 ) were successfully purified. The ongoing assays are directed to enhance ZnuA solubility. We expect to improve the protein recovery by removing the amino terminal sequence of the protein. This highly hydrophobic amino terminal region (30 to 40 aminoacids) hooks the protein to the bacterial membrane. Working with D30ZnuA will allow us to reach the necessary concentration of soluble (functional) protein to perform the biochemical assays proposed.
We successfully synthesized the pheromone methyl (E)-3-(2-hydroxyethyl)-4-methyl-2-pentenoate at USHRL, Ft. Pierce for use in behavioral assays and field trials. Two-way and wind tunnel olfactometer trials were initiated to determine the response of male and female weevils of varying age and mating status to the male-produced pheromone. We confirmed positive female response in a two-way olfactometer to the pheromone compound and no male response. A trial was conducted to test possible effect of rearing conditions and prior exposure on female response to pheromone. We were concerned that continuous exposure of females to male odors in crowded rearing conditions modifies behavior in bioassays designed to test response to those same male odors. Field-collected female Diaprepes weevils were caged in isolation from males and male odors or caged with rubber septa loaded with methyl (E)-3-(2-hydroxyethyl)-4-methyl-2-pentenoate for 24 hours prior to being used in a 2-way olfactometer. Females caged in isolation from males or male odors responded by walking toward synthetic pheromone more often compared with females previously caged with rubber septa emitting the synthetic pheromone. This information will be used to improve experimental design of behavioral assays. Female response to carvacrol was demonstrated in an olfactometer. Carvacrol is a common plant compound that is absent from citrus but present in the gut of male and female Diaprepes adults and appears to be functioning as a pheromone in this species. A multvariate screen is being conducted to identify principal drivers in blends of pheromone and host plant volatiles. Pupae, teneral adults and adult males and females of varying age were dissected and extracts of their hind guts and mid guts were analyzed to determine the location and timing of pheromone and carvacrol production. Results are being collated and prepared for publication.
The purpose of this project is to preserve citrus germplasm in Florida that is threatened by loss due to huanglongbing and citrus canker. Using input from stakeholders and scientists, threatened germplasm has been identified, collected, and established at Ft. Pierce. Presently 76 accessions are being held at Ft. Pierce. Additional nucellar, old line, and unique accessions have been identified which represent germplasm not generally available or in protected collection, and we are adding these accessions to the recovery collection. The new accessions will be antibiotic treated and subjected to clean up. Additional testing is planned in collaboration with the Florida Citrus Germplasm Introduction Program. Twenty five accessions are presently being held in Riverside in various stages of clean up for release from quarantine status.
The purpose of this project is to determine methods to easily eliminate Candidatus Liberibacter asiaticus (Las), the bacterium associated with huanglongbing (HLB) in Florida, from citrus with emphasis on cryotherapy, and to determine the effectiveness of using young indicator plants for biological indexing to verify elimination of graft transmissible pathogens. In this quarter, we have forwarded material to Ft. Collins, CO for treatment by cryotherapy. The methodology for cryotherapy to remove pathogens is being developed. Following cryotherapy treatment, we wait about 12 weeks before testing the surviving buds to give time for any pathogen that may have survived to multiply to the point where it may be detected using laboratory diagnostic methods. In Riverside, the evaluation of the use of very young plants for biological indexing of Citrus tatterleaf virus, citrus vein enation, Citrus psorosis virus, Citrus concave gum disease, and citrus viroids is being continued with the results obtained from using young indicator plants being compared to the conventional method of biological indexing for these pathogens.
This year we have broadened our bioinformatics efforts to include not only Liberibacter genome analysis but also the bioinformatic characterization of other organisms relevant to citrus greening biology. Liberibacter: The CG-HLB Genome Resources Website (http://citrusgreening.org/) hosts a genome browser to aid users in searching and identifying genome properties of sequenced Liberibacter strains. Sequences for CLas psy62, CLso ZC-1 and the phage regions of CLas UF506 are presently available. We are assisting in genome comparisons with an additional CLas strain for inclusion upon public release. Submission of the genome sequence for a cultural Liberibacter, L. crescens BT-1, was reported at the recent Citrus Health Research Forum. Inclusion in the genome viewer will allow visualization of the regions of difference, highlighting genetic features related to citrus pathogenicity and to the ability to grow in culture. Asian Citrus Psyllid (ACP) endosymbionts: Biology of ACP and its bacterial endosymbionts is a focus of intense interest owing to growing interest in exploiting vector biology for control of citrus greening. To corroborate candidate endosymbionts previously identified by rDNA amplification, raw reads from the ACP metagenome sequence were mapped to reference genome sequences. Results of the read mapping provided the most support for Wolbachia and an enteric bacterium most similar to Salmonella. Wolbachia-derived reads were extracted using the complete genome sequences for four Wolbachia strains. Reads were assembled into a draft genome sequence, and the annotation assessed for the presence of features potentially involved in host interaction. Candidate host interaction factors encoded by the ACP Wolbachia genome include 54 ankyrin repeat-containing proteins, a Type IV secretion pathway, and a bacterioferritin gene linked to iron homeostasis in the host. Several metabolic capabilities were identified in the Wolbachia strain that are absent from CLas. Genome alignment with the complete sequences reveals membership of ACP Wolbachia in supergroup B, further supported by phylogenetic analysis of FtsZ. FtsZ and Wsp phylogenies additionally indicate that the Wolbachia strain in the Florida ACP isolate falls into a sub-clade of supergroup B, distinct from Wolbachia present in Chinese ACP isolates, supporting the hypothesis that the ACP introduced into Florida did not originate from China. A manuscript describing this work has recently been accepted for publication by PLoS ONE. The draft genome sequence for ACP Wolbachia has been submitted to Genbank under Bioproject PRJNA29451, and the annotated draft is currently being uploaded onto the CG-HLB Genome Resources genome viewer for easy visualization by researchers interested in exploiting Wolbachia for ACP control. Asian Citrus Psyllid (ACP): As part of a larger effort on ACP sequencing, we have characterized repeat sequences in the current ACP genome assembly and recently submitted transcriptome. Repeats play an important role in genome structure and regulation, and characterization is critical to accurate genome assembly. We have also mined the ACP transcriptome for osmoregulatory transporter genes on behalf of Angela Douglas (Cornell) to use in RNAi-based psyllid control.
Over the course of this funding period, the CG-HLB Genome Resources Website (http://citrusgreening.org/) was developed to serve as a hub for genome resources of Ca. Liberibacter and other organisms impacting the transmission and development of citrus greening. Initially hosting materials for viewing the Ca. L. asiaticus draft genome, the site now hosts a GBrowse based genome viewer on which genome properties of CLas psy62, CLso ZC-1 and phage regions of CLas UF506 can be viewed and searched, a major advantage of the GBrowse model being its ability to hyperlink to diverse outside resources including gene characterization at NCBI, 3rd party sites, and resources funded by CRDF such as the Grishin lab structural analyses. Multiple analyses have been conducted in house for CLas psy62 including screening the sequence for compositional variation suggestive of horizontal transfer and for repeated elements having potential application to diagnostics. Gene regulation is of particular interest given its importance in mediating adaptation of the organism to its host and vector, and predictions of regulatory motifs have also been generated using experimentally determined features from free-living Liberibacter relatives as models. These include rpoH, associated with induction of genes associated with survival at high temperatures, and rirA, a protein known to regulate uptake and metabolism of iron. Results of these analyses are available on the CG-HLB Genome Resources Website. Although the original grant emphasized genome resources for Liberibacter, the biology of the psyllid vector (ACP) and its bacterial endosymbionts became an increasing focus owing to the potential for exploitation of vector biology for disease control, and the apparent need for bioinformatically skilled analyses. To corroborate candidate endosymbionts previously identified by rDNA amplification, raw reads from the ACP metagenome sequence were mapped to reference genome sequences. Results of the read mapping provided support for Wolbachia and an enteric relative of Salmonella. Wolbachia-derived reads were extracted and assembled into a draft genome sequence. The annotation was assessed for features potentially involved in host interaction. Candidate host interaction factors include 54 ankyrin repeat-containing proteins, a Type IV secretion pathway, and a bacterioferritin gene linked to iron homeostasis in the host. Several metabolic capabilities were identified in the Wolbachia strain that are absent from CLas. Genome alignment with the complete sequences reveals membership of ACP Wolbachia in supergroup B, further supported by phylogenetic analysis of FtsZ. FtsZ and Wsp phylogenies additionally indicate that the Wolbachia strain in the Florida ACP isolate falls into a sub-clade of supergroup B, distinct from Wolbachia present in Chinese ACP isolates, supporting the hypothesis that the ACP introduced into Florida did not originate from China. A paper describing this work has been published in PLoS ONE. Contig sequences are available at NCBI, and the annotated draft genome sequence for ACP Wolbachia can be accessed in the supplemental information of the publication as well as in the CG-HLB Genome Resources genome viewer. Project personnel additionally participated in the characterization of repeat sequences in the ACP transcriptome. Repeats play an important role in genome structure and regulation, and their characterization is critical to accurate genome assembly. This work has been submitted for publication in PLoS ONE. Our work on Liberibacter and ACP was presented at, among others, the HLB/Zebra Chip meeting (November 2009), APS (August, 2010), HLB Conference (January 2011), and Citrus Health Research Conference (September,2012).
Citrus canker is an economically important bacterial disease of most commercial citrus cultivars resulting in significant losses worldwide. Spread of citrus canker has been a severe problem to the citrus industry of Florida. How bacteria escape from infected plants is underexplored. Understanding the molecular determinants of lesion rupture, how Xcc survives in the intercellular spaces, and how Xcc releases from lesions of host plants will provide many fundamental and practical benefits. Despite the tremendous effort to eradicate citrus canker, the pathogen has spread to most citrus production areas in Florida and continues to spread. Understanding the genetic mechanism of release of Xanthomonas axonopodis pv. citri (Xac) from citrus canker lesions will help develop effective control and containment strategies to stop citrus canker pathogen from spreading. The goal of the proposed research is to understand the genetic mechanism of release of Xac from citrus canker lesions. The specific objectives are to: 1. characterize critical genes involved in release of X. axonopodis pv. citri from citrus canker lesions; 2. understand the release mechanism by studying the host response of citrus upon infection by Xac wild-type strain and mutant strain(s) affected in release from citrus canker lesions. We have identified 12 EZ-Tn5 transposon mutants of Xac with reduced capacities of release from citrus canker lesions. The insertion sites of the 12 mutants have been identified with insertions in 11 different genes. Currently, complementation analysis of the mutants is underway. Bacterial growth assays of the mutants and the wild type strain in grapefruit ‘Duncan’and sweet orange ‘Valencia’ have been conducted. Three mutants were tested for affect in release from citrus canker lesions (dispersal assay) on grapefruit for 5, 7, 10 and 14 days as compared to wild type strain. The preliminary assay showed differences in growth rate (total count) and dispersal rates (surface count). The dispersal will thus be calculated according to percentage point difference to negate any effect due to growth changes. Pectate lyase assay, proteinase assay and motility tests, and EPS assay have been completed for all the mutants. Further characterization of the mutants on LPS, capsule and biofilm formation is in progress. Also we are investigating the release process by studying the host response of citrus upon infection by Xac wild-type strain and mutant strain(s) affected in release from citrus canker lesions.
The funds for this project were recently released and data was collection soon after during the week of 13 August, 2012. We have already improved the capabilities of laser induced breakdown spectroscopy (LIBS) system by adding multiple detectors in the range 200-1,000 nm. Meanwhile, at the Southwest Florida Research and Education Center, several citrus trees are being treated with multiple nutrient compositions to induce differences in the foliar nutrient concentrations. Leaves from these trees will be collected and analyzed using the LIBS system, followed by chemical analysis.
As a proof of concept, we initially tried to find out if Laser Induced Breakdown Spectroscopy (LIBS) technique can differentiate the healthy citrus leaves from symptomatic stressed leaves. We collected LIBS spectra from abiotic stressed leaves that included magnesium, manganese, iron and zinc deficient leaves, and biotic stressed leaves that included canker and HLB-infected leaves. The classification studies indicated that the nutrient deficient and diseased (symptomatic) leaves could be easily categorized from the healthy ones with more than 90% accuracy. The next step is to perform peak analysis to identify the peaks representing these variations. This step is currently under investigation. We also started the next step of the project in which we will try to evaluate the ability and accuracy of LIBS technique in quantifying the amount of nutrients in the leaves and compare that with currently used analytical approach. We have collected the first set of data. Five six-month-old leaves were sampled from individual trees in two citrus greening studies. The data collected from mature Valencia trees at the Southwest Florida Research and Education Center (SWFREC) and mature Valencia trees in a commercial grove north of Immokalee in Hendry County. The leaves were refrigerated prior to being shipped overnight to the Citrus Research and Education Center (CREC) for spectral analysis using LIBS system. A total number of LIBS spectra collected were about 1250 (about 125 samples with 10 replicates each). The LIBS spectra were collected based on the previously established procedure. After LIBS data collection, the leaves were returned to SWFREC where the samples were dried and ground. The ground samples were analyzed for total nitrogen by combustion using the NA2500 C/N Analyzer (Thermoquest CE Instruments) and for P, K, B, Ca, Cu, Fe, Mg, Mn and Zn by Inductively Coupled Plasma (ICP) after ash digestion. Currently, data analysis protocols are being developed to allow qualitative and quantitative analysis based on the nutrient concentrations.
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