Volatile organic compounds (VOC)-based HLB detection: We have completed the study of the correlation of health status of a plant with its released VOC ‘fingerprint profile’ for the HLB pathogen. It is demonstrated that an early (even at asymptomatic stage), rapid and non-invasive means of detecting the HLB pathogen is possible via analysis of the VOCs emitted by citrus plants as measured using a portable Gas Chromatography/Differential Mobility Spectrometer (GC/DMS) due to their metabolism changes post-infection. Application of GC/DMS allowed us to longitudinally monitor VOC ‘fingerprint profile’ differences in-vivo throughout an entire year to account for seasonal variations of the disease and plant’s life cycle. A mathematical model was developed to classify the diseased and control groups based on GC/DMS data, with discrimination accuracy of 80-90% and close to 100% under optimal conditions. However, while using conventional GC with either flame ionization (FI) or mass spectrometry (MS) detectors, we were unable to consistently differentiate volatiles, due to very low concentration levels of leaf volatiles. Initial testing of SPME fibers with exposure times from 1-8 hrs lead to some identifications but tree to tree variation was as big as healthy to HLB. Purge and trap devices employing Tennax and combinations with charcoal improved sample amounts, but we were never successful in getting consistent results due to sample carryover and apparent breakdown volatiles from the trap itself. Sampling volumes ranged from 12-48 liters of air sampled within 5 mm of the leaf surface. The major limitation was the inability to use the purge and trap samples on the MS detector. We were only set up to use FI detection with these collection tubes. HLB detection using optical sensors: With the aerial hyperspectral images acquired in December 2011, a novel method named ‘extended spectral angle mapping (ESAM)’ was developed to detect the citrus greening disease. Firstly, Savitzky-Golay smoothing filter was applied to remove spectral noise within the data. Then support vector machine was used to build a mask to segment tree canopy from the other background. Vertex component analysis was chosen to extract the pure endmembers of the masked dataset. Then spectral angle mapping (SAM) was applied to classify healthy and HLB infected areas in the image. Finally, red edge position was used to filter out false positive detections. The experimental results were compared with other methods, Mahalanobis distance and K-means. The ESAM performed better with a detection accuracy of 86% than those two methods which yielded accuracies of both 64%. In regard to the ground-based optical sensing, several methods such as visible-near infrared spectroscopy (Vis-NIR), mid-infrared (MIR) spectroscopy, fluorescence spectroscopy, thermal imaging, and laser-induced breakdown spectroscopy (LIBS) were tested. Each of these methods showed unique capabilities, having their own advantages and limitations. Although MIR spectroscopy and LIBS are destructive methods that require leaves to be removed from the trees; these methods provide chemical signature that are robust and remains unaffected by the incident light source. Moreover, MIR spectroscopy could be used for detecting starch accumulation, which begins even before the symptoms appears in the leaves. Similarly, Vis-NIR spectroscopy, fluorescence spectroscopy and thermal imaging are non-destructive methods and can be applied for aerial sensing as well. Based on the evaluation studies, we achieved a classification accuracies of 90% and higher. Total number of peer-reviewed publications: 12+, Presentations: 10+, Patents: 1.
Potential effectors of HLB engineered into Citrus Tristeza Virus (CTV) vector have been transferred into citrus as outlined in the previous research updates and are being screened for their response to HLB pressure. They include the following; CLIBASIA_05165, CLIBASIA_05605, CLIBASIA_01555, CLIBASIA_05195, CLIBASIA_05200, CLIBASIA_05620, CLIBASIA_05635, CLIBASIA_05665, CLIBASIA_05130, CLIBASIA_05155, CLIBASIA_05265, CLIBASIA_05560, CLIBASIA_05150, CLIBASIA_05180, CLIBASIA_05245, CLIBASIA_02250, CLIBASIA_03020, CLIBASIA_03025, CLIBASIA_02090 and CLIBASIA_02905. We are also testing full-length Flagellin gene of CLas and bacterial Flagellin for possible effector function in citrus because of our earlier observation of the Pathogen-associated Molecular Patterns (PAMP) activity associated with Flagellin domain of HLB. We use Citrus macrophylla which is a preferred host for CTV for expression of putative effectors and subsequently transferred into sweet orange and /or grapefruit citrus varieties by graft inoculations. These plants are being challenged inoculated with HLB by exposing them to infected psyllids. Additionally, we have used a faster approach using Potato virus x (PVX) vector system to screen for virulence genes of HLB. Transient expression of prophage-related gene cluster of HLB in PVX vector did not induce obvious phenotype changes in tobacco plant. However, transient expression of HLB-flagellin, and flg22, HLB flagellin domain, induced cell death and callose deposition in N. benthamiana. Substitution experiments revealed that the 38th and 39th amino acid residues were essential for callose induction. However, the synthetic peptide (DRVSSGLRVSDAADNAAYWSIA) could not induce cell death, but retained the ability to induce callose deposition at the minimal concentration of 20 ‘M. These results demonstrated that Flg22Las has PAMP activity, and may play an important role in determining citrus resistance to HLB. These results have been submitted for publication in PLoS One. Progress is also being made in cloning some these potential CLas effectors into a universal secretion study vector with MYC tag to determine if any of these effectors are secreted using western analysis and eventually study the citrus plant response to the secreted proteins.
To date, 716 culture medium formulations have been tested in attempts to grow Liberibacter asiaticus in axenic culture. Some attempts looked promising. The bacterium was still detected in one medium after five transfers and a total of 50 days after the culture was initiated. Efforts to further optimize this medium are under way. Although genomic information indicates that the bacterium has a transport mechanism for zinc, addition of zinc to the culture medium appears to inhibit growth. Similar results were obtained for betaine and taurine. Fetal bovine serum appears to be inhibitory at 30% in the medium, but at 15% or less results have been uncertain. Promising results have been obtained under both aerobic and microaerophilic conditions, but not under anaerobic conditions. Some medium formulations support the development of biofilms of the bacterium on the alimentary canal of Diaphorina citri in culture but not planktonic growth of the bacterium, while other formulations appear to support both. Efforts to culture the bacterium continue.
Diagnostic service for growers for detection of Huanglongbing to aid in management decisions, July 2012. This report covers the entire period that Huanglongbing Diagnostic Laboratory has been in service. The objectives for the funding were to provide continued, uninterrupted diagnostic services to growers while expanding our ability to provide diagnostics quickly and assist with research efforts. The HLB Diagnostic Laboratory has been operational at UF-IFAS-SWFREC since February 2008. Since the opening of the lab, there has been continued development of techniques, protocols and efficiency. The lab has been in operation for nearly four years, we have processed approximately 31,000 grower samples Additionally, more than 40,000 samples have been received for research. Only the equipment is used for the research samples because PI are paying for the cost of materials and personnel on a per charge basis to keep separate from the grower’s service. Techniques, Protocols and Research For DNA extractions, the final protocol was as follows using the magnetic particle based system, which has proved both reliable and fast. Current methods of sample processing have become streamlined and therefore seen little change. We used the TaqMan Fast Advanced MasterMix for real-time-PCR reactions as this is more economical and has shown comparable-to-superior amplification and detection of gene(s)-of-interest when compared to the TaqMan Fast Universal PCR MasterMix. We now lyophilize all plant samples prior to BeadBeating, which enables superior sample maceration when compared to use of liquid nitrogen. Protocol for the detection of HLB in Asian Citrus Psyllid was validated, including quantification of HLB in both plant and psyllid samples, with the primary goal of serving research projects within the entomology and plant pathology departments that also contribute funds from their research grants to support the labor and supply costs for research samples. The protocol established in 2010 for the quantification of the HLB bacteria in both the psyllid and host tissue using a standardize curve was being used for research and extension samples. The basic diagnostic service remains available to growers, researchers, extension faculty and dooryard citrus growers. However, we expanded the data analysis of PCR processed samples to include data from individual groves that consented to have their data used. In conjunction with an epidemiologist and computer mathematician, the spread of the disease will be modeled. These studies are not supported by lab funds but are an offshoot of the database collection.
We are continuing to examine the interactions between the psyllid, the plant, and the greening bacterium. We are examining the disease epidemic under confined conditions. We have developed a containment plant growth room to examine natural infection of citrus trees by psyllid inoculation. We have made several significant observations: First, we have found that the time period between when plants first become exposed to infected psyllids and the time that new psyllids can acquire Las is much shorter that we expected. In our population of psyllids in the containment room, the proportion of infected psyllids born on newly inserted healthy plants starts increasing after about 30 days suggesting that the receptor plants begin becoming donors at about that time. We are examining this process in more detail now. It is clear that psyllids reproduce on new flush, but they also feed on older leaves. We are examining whether and how well the psyllid can transmit the disease when feeding on non-flush leaves. We also have developed methods to greatly speed up results of field tests for transgenic or other citrus trees or trees being protected by the CTV vector plus antibacterial or anti-psyllid genes. In order to interpret results of a field test, most control trees need to become diseased. Under natural field pressure in areas in which USDA APHIS will allow field tests, this level of infection could take 2-3 years. By allowing the trees to become adequately inoculated by infected psyllids in a containment facility, we can create the level of inoculation that would naturally occur in the field within 2-3 years in 2-5 months in the containment room, after which the trees are moved to the field test site. Trees are not being examined in the field that first were maintained under heavy inoculation pressure by infected psyllids for several months. Other peptide protected plants are being prepared for field testing. Another objective is to provide knowledge and resources to support and foster research in other laboratories. A substantial number of funded projects in other labs are based on our research and reagents. We supply infected psyllids to Mike Davis’s lab for culturing of Las and plants containing potential anti-psyllid genes for Kirsten Pelz-Stelinski’s lab and for Bob Shatters et al. lab in Fort Pierce. We routinely screen citrus genotypes or transgenic citrus for other labs for tolerance or resistance to greening or psyllids. We have found poncirus/sweet orange hybrids that are tolerant to HLB and are looking at possibilities of quickly getting sources of trees that can be productive in the field in the presence of HLB.
This is a project to find an interim control measure to allow the citrus industry to survive until resistant or tolerant trees are available. We are approaching this problem in three ways. First, we are attempting to find products that will control the greening bacterium in citrus trees. We have chosen initially to focus on antibacterial peptides because they represent one of the few choices available for this time frame. We also are testing some possible anti-psyllid genes. Second, we are developing virus vectors based on CTV to effectively express the antibacterial genes in trees in the field as an interim measure until transgenic trees are available. With effective antibacterial or antipsyllid genes, this will allow protection of young trees for perhaps the first ten years with only pre-HLB control measures. Third, we are examining the possibility of using the CTV vector to express antibacterial peptides to treat trees in the field that are already infected with HLB. With effective anti-Las genes, the vector should be able to prevent further multiplication and spread of the bacterium in infected trees and allow them to recover. We have completed several large screenings of antibacterial peptides against Las in sweet orange trees. About 50 different antibacterial constructs are being tested in trees. We have found only two peptides that appear to give some protection sweet orange trees from HLB. We are examining whether they can be used to treat infected trees. We are preparing inoculum for a field test. We continue screening for better genes that will more effectively control HLB and can be approved for use in a food crop. We also are improving the CTV-based vector to be able to produce multiple genes at the same time. This could allow expression of genes against HLB and canker or multiple of genes against HLB. Another major goal is to do a field test of the CTV vector with antibacterial peptides, which is an initial step in obtaining EPA and FDA approval for use in the field. This USDA and EPA approved field test is underway.
The objective of this project is 1) to complete the Las genome sequence and conduct comparative genomics studies on the Liberibacter species; 2) to explore the potential role of the microbial community and genetic diversity of Las bacteria in HLB development; 3) to confirm if Las bacteria are seed-transmissible and their role in HLB development. A complete circular genome of Candidatus Liberibacter asiaticus (Las) was obtained using a metagenomics approach and published in MPMI 22:1011-1020, 2009. In collaboration with Dr. Hong Lin at the USDA-ARS in Parlier, California, we have obtained and published a complete genome sequence of Ca. L. solanacearum and published in PLoS ONE 6(4): e1913, 2011. All BAC clones of Las were sequenced, and sequence analyses revealed a potential mechanism of GENOME REDUCTION. Based on the variations within the Las prophages, FP1 (CP001677.5) and FP2 (JF773396.1), twelve (A to H) different populations (genotypes) have been identified. Type A and B are located in FP1 and FP2, respectively. Typing revealed A, B and C as the three most abundant groups in libraries from psyllid, citrus and periwinkle, although psyllid contained much more type A sequence than the plant hosts. Typing also revealed periwinkle and dodder contained all population types, but psyllids did not. They had low to no titers of four types, including type D. In citrus a high titer of type D was associated with typical blotchy mottle symptoms. We are continuing to studies the different population types and their correlations with HLB phenotypes and disease severity. We have characterized the ATP translocase from Las and proved its function using a heterologous E. coli system (J. Bacteriol. 192:834-840, 2010). We are currently developing an antibody-based “drug” to target this protein, aimed at disrupting ATP import, which may be important for its survival. Seed transmission of Las was tested in grapefruit, sweet orange, sour orange and trifoliate orange. A very low titer of Las was detected from the embryos and seedlings using nested PCR and real-time PCR. Most, if not all the seedlings did not show typical HLB symptoms and contained a relatively low Las bacterial titer for HLB, even in the three to four year old seedlings. The results indicated that the seed-transmitted Las could not cause typical HLB disease by themselves, which suggested “Detection of Las was NOT necessarily equal to the presence of “HLB disease” in plants.” Psyllid transmission study on the Las-positive seedlings was performed. High percentage of psyllids acquired Las bacterium but did not have the same bacterial levels as those from HLB-affected citrus plants. However, it is FIRST TIME that ONE SEED-TRANSMITTED HLB SEEDLING was confirmed by PCR using several Las-specific primer sets. Graft transmission of the cutting from this HLB plant confirmed this seed-transmitted HLB. Progress on culture of Las bacterium in vitro has been made. The Las bacterial growth reached Stationary Phase and Death Phase in 48-72 hours in the liquid cultures. We are looking into factors affecting further growth. Fifty-one BAC clones with overlapped Wolbachia endosymbiont of Diaphorina citri (wDia) genome sequences were screened using wDia specific primers from BamHI BAC library and were sequenced. The average size for the 51 clones was 85.4kb with 95-100% coverage and the average GC content is 34.3%. Assembly results indicated that due to large amount of repeat elements, such as transposase, only 13 BAC clones were able assembled into 1 scaffold. We are conducting the gap closing for each BAC clone and hope to get the full wDia genome soon.
The objective of this project is to characterize the hypI (renamed as hyvI) gene and determine its effects on insect transmission and/or virulence in host plants. Transient expression with alternative expression systems and RT-qPCR, etc., will be used to elucidate the function of the hypI (hyvII) gene of Las and shed light on the molecular mechanism of this “phase variation” phenomenon; thereby developing a novel control strategy for citrus HLB. In addition, antibodies and probes along with standardized protocols developed during this project can be applied for better detection and differentiation of the HLB bacteria. The hyvI and hyvII within two Las prophages were further characterized and some of the results were published in Applied Environmental Microbiology 77:6663-6673, 2011. “Diversity and Plasticity of the Intracellular Plant Pathogen and Insect Symbiont “Candidatus Liberibacter asiaticus” as Revealed by Hypervariable Prophage Genes with Intragenic Tandem Repeats”. We have developed an improved real-time PCR using SYBR Green 1 (LJ900fr) and TaqMan’ (LJ900fpr) protocols with primers and probe targeting the nearly identical tandem repeats of 100bp hyvI and hyvII. The results were published in Molecular and Cellular probes, 26:90-98, 2012. Monoclonal antibodies against the partial HyvI protein (only one repeat) were generated, and their sensitivity and specificity were evaluated for the detection of HyvI protein expressed in E. coli and HLB-infected citrus and psyllids. All antibodies were able to recognize the E. coli expressed HyvI antigen, but were not able to detect the HyvI antigen from HLB-infected plants and psyllids. To determine the cellular localization of the HyvI protein in plant cells and the role of the two putative NLSs in hyvI gene, full-length hyvI and C-terminal region including two putative NLSs were amplified and cloned into pCX-DG vector with GFP driven by CaMV35S promoter. The results indicate that the HyvI protein did not target in plant nucleus but located in cytoplasm (possible in organelle) when transient expression in tobacco. We determined that HyvI targeted tobacco mitochondria by transient expression and MitoTracker Mitochondria-selective probe staining. When the full length of hyvI gene was cloned , and replicated in heterologous hosts, the repeat number of hyvI gene remained the same in E. coli, but varied in Xanthomonas citri. Clones of X. citri containing the hyvI gene displayed different degree of growth retardation, indicating potential toxic effect of hyvI gene to X. citri. HyvI C- terminus and full length HyvII were fused to GFP and expressed in E. coli driven by T7 promoter. Confocal microscopy results show both proteins are localized to the bacterial poles. Protein localization, sequence analysis and protein structure prediction suggest both protein belong to autotransporter family. HyvI and II are unique autotransporters because the non-conserved translocator domain can export both its natural passanger domain and also the GFP fusion domain to E.coli’s cell surface. The protein was determined to localize at cell surface by dot blot, and furthermore the protein surface localization was proved by protease treatment of intact bacterial cells. The immunofluorescence assay to confirm the surface localization of the HyvI protein was unsuccessful. The proteins appear to be on the cell surface but are not folded properly.
We found that the camera’s automatic gain control (AGC) function is designed to maintain image’s intensity and contrast consistently regardless of the illumination conditions and this function would prevent us from identifying the effect of starch accumulation in the images. In order to resolve this problem, a two-step calibration was performed using the background pixel values. At the first step, all the images in each specific illumination and filter direction situation were calibrated separately. Also the actual background’s reflectance in each situation was measured using a portable spectrophotometer and the ratios between them were used for the second step of the calibration. Using this approach the effect of AGC was cancelled and the real reflectance measurement was calculated. Using the image acquisition system, two datasets each including both healthy and HLB infected leaves were created. A 4-class (healthy leaf, healthy vein, HLB leaf and HLB vein classes) training set of 25 healthy samples and 25 HLB samples were selected from one of the datasets. Analyzing the histogram of this training dataset showed that the healthy leaf and HLB leaf classes could be distinguished in the images captured with 591 nm illumination and perpendicular filters. In addition, the healthy vein class is very similar to the healthy leaf class, and the HLB vein class is very similar to the HLB leaf class. This can mean that although healthy vein has a color similar to HLB leaf and vein, however healthy vein has different pixel value in 591 nm with perpendicular filters because there is no starch accumulation in healthy veins. Both datasets were classified using this training set and a linear classifier. The classifier was able to identify the HLB samples with 100% accuracy, but it also misclassified 28% of healthy samples of the first dataset and 36% healthy samples of the second dataset in the HLB class. This might be due to either the classification error or the healthy samples actually being infected by the disease. Furthermore, a dataset containing 4 classes of different kinds of yellow leaves (HLB, young leaves, zinc deficient and unknown symptoms) plus a healthy class as the control class was created to find out the difference between HLB yellowish symptoms and yellowish symptoms with other reasons. The analysis of variance (ANOVA) results showed that all five classes were significantly different. In order to find the effect of starch on polarized light, the absorbance of four levels of starch solutions plus pure water were measured using the spectrophotometer and in three conditions: no filter, parallel filters and perpendicular filters. The results showed that the absorbance increases as the starch concentration increases in the solution. However the Min/Max ratio (the ratio between absorbance data using perpendicular filters to parallel filters) didn’t show any reasonable results.
Citrus huanglongbing (HLB) is associated with three species of Liberibacter’Candidatus Liberibacter asiaticus (Las), Ca. L. americanus, and Ca. L. africanus. The majority of the testing in Florida is focused on detection of Las, the only bacterium known to be associated with HLB in Florida to date. Over the past four years with funding from Citrus Research Board, we have conducted regular surveys of citrus and citrus relatives in Florida, from various germplasm collections, backyard plants, native and cultivated trees, testing for tolerance to HLB. We have focused on plants showing HLB symptoms but testing negative by standard qPCR tests. A small selected set of symptomatic, qPCR negative samples were analyzed for detection of other genomic regions of Liberibacters by conventional PCR (cPCR), cloning and sequencing. This study confirmed the presence of Liberibacter variants not detectable by standard assays. The purpose of this project is to conduct further research on variants of Liberibacters from citrus and citrus relatives and to develop rapid methods for detection of these variant populations. We also will study the biology of the variants under greenhouse conditions, determine the changes in Liberibacter populations within individual trees over time from analysis of DNA extractions we have made over the past 5 years, and determine if there are interactions among populations of Liberibacter variants which may ameliorate/enhance the symptoms of HLB. Understanding of HLB disease complex caused by all variants of Liberibacters will be useful for developing novel disease management strategies. Research on this project this first quarter has concentrated on the development of a method to ‘trap’ Liberibacter and Liberibacter-like pathogens so that Liberibacter-enriched DNA extractions may be made to facilitate the molecular characterization of the Liberibacter and Liberibacter-like genomes. We anticipate receiving funds soon which will permit hiring of personnel and facilitate the research.
The objective was to evaluate the efficacy of copper loaded silica nanogel (CuSiNG) material against citrus canker. Two CuSiNG formulations (Cankicide pH 7.0 and Cankicide pH 4.0) were prepared at UCF and delivered to Fort Pierce for the 2012 field trial at Vero Beach. The efficacy is being evaluated against a number of commercially available Cu compounds including Kocide 2000, Kocide 3000, Cuprofix Ultra 40, Kentan DF, Badge X2, NuCop WP, Nordox 75G and Magna-Bon. Laboratory research is being continued on CuSiNG materials for testing formulation stability (Shelf-life) and for improving Cu bioavailability. To date, our research data suggest that Cankicide pH 4.0 (highly-transparent) as-synthesized formula is stable for more than 12 months (no settling observed). In contrast, Cankicide pH 7.0 formula is opaque and settling was observed within a couple of weeks. Data from 2011 field trial showed that Cankicide pH 4.0 formula caused very minor injury to plant tissue (but not significant with respect to controls) whereas Cankicide pH 7.0 formula was non-phytotoxic. This is attributed to the availability of some free ionic Cu in the Cankicide pH 4.0 formula. To understand the Cu oxidation states in CuSiNG material, X-ray Photoelectron Spectroscopy (XPS) technique was used for material characterization. XPS data suggest that mixed valence states of Cu exist in CuSiNG material. Improved Cu bioavailability of the CuSiNG material could be correlated to the presence of such mixed valance states. Further studies are in progress to understand the role of mixed Cu valence states in CuSiNG material. To minimize the metallic Cu amount per spray application, we have prepared a novel core-shell nanoparticle and performed systematic laboratory research. The CuSiNG material was coated over the pure silica nanoparticle (that served as non-Cu containing inactive delivery system), thus forming a core-shell nanoparticle with silica core and CuSiNG composite shell. A peer-reviewed research paper entitled, ‘Novel copper loaded core-shell silica nanoparticles with improved copper bioavailability: synthesis, characterization and study of antibacterial properties’ have been published in the Journal of Biomedical Nanotechnology 2012, 8(4), 558-566. Significant improvement of antibacterial efficacy in comparison to Kocide 3000 control was attributed to improved Cu bioavailability in the core-shell nanoparticle. To characterize adherence property of the silica nanogel (SiNG) material to plant tissue, the SiNG material was labeled successfully with fluorescent dyes. However, background fluorescence signal of the plant tissue and the photobleaching of organic fluorescent dyes severely limited our ability to conduct experiments reliably. In an alternative approach, a stable inorganic based near infra-red (NIR) emitting fluorescent label is being developed to address the above limitations. We will disseminate research results on the NIR imaging of CuSiNG materials and their adherence properties to citrus plant surface in future CRDF reports and peer-reviewed journal publications.
The specific project goals were: 1. Cloning of previously identified early/late gene promoter regions fused with lacZ as a reporter. 2. Cloning and expression of both Las and the Lam repressors and determining responsiveness of the lacZ reporter. 3. Cloning and expression of all 4 Las and the one possible Lam anti-repressors, and determining responsiveness of the reporter and clones from Milestone 2. 4. Development of a chemical assay for Las-responsive SOS. Goal 1 is partially completed and continuing. The project start date was 5/1/12. The pUC19 cloning vector was modified to remove the promoter of lacZ and replace it with a PCR product in a single step, using UDG cloning. In this manner, the intergenic region between the early and late genes of Las phage SC1 and SC2 (regions of both phage between locus tags gp120 and gp125) were cloned in both directions upstream of the lacZ reporter gene in E. coli. When the putatively bidirectionally active SC1 and SC2 promoters were fused with the lacZ gene such that they replaced the early genes in constructs pSZ68 and pSZ64, respectively (early gene direction), both the SC1 and SC2 constructs performed the same and both resulted in light blue color reactions. Conversely, when these promoters were fused with the lacZ gene such that they replaced the late genes (late gene direction), both the SC1 clone (pSZ67) and SC2 clone (pSZ62) resulted in no detected color reaction. These results indicated that, as expected, the early genes of both SC1 and SC2 are constitutively on and the late genes are constitutively off. However, when the bidirectional promoter region was cloned in the late direction, such that it drove expression of the repressor gp125 of SC1, together with the lacZ reporter (in pSZ63), a light to medium blue color reaction was observed. This indicated that gp125 (annotated as a phage C2-like repressor) may be an activator, since it appeared to stimulate its own expression. Alternatively, part of the late gene promoter region may be contained within the gp125 locus. We are testing this idea. In addition, the Las repressor, SC1 gp125 (annotated as a phage C2-like repressor) was cloned into shuttle vector pUFJ5 (Bordatella replicon), forming pUFZ3-4. Finally, the annotated Las anti-repressor (SC1_gp200) was cloned into shuttle vector pUFR047 (forming pSZ77), such that gp200 was constitutively expressed from the lacZ promoter in pUFR047 (oriW). Both the putative repressor and anti-repressor constructs are compatible with pUC19 in the same E. coli cell.
Previously, we sequenced and genotyped Las isolates from four major citrus production countries, USA, Brazil, China and India based on the five virulence-related gene loci, ftn (CLIASIA 03035), phoU (CLIASIA 02950), flgH and two genes homologs to pilus assembly proteins (CLIASIA 03575 and CLIASIA 03545). During this report period (April-July, 2012), we extended our sequencing analyses of Las isolates to wider geological locations including Vietnam, Cambodia, Japan, Taiwan, and Thailand. Single nucleotide polymorphism (SNP) in coding regions could result in a change in the amino acid if such a change alters the codon for the amino acid (non-synonymous) or have no effect on the amino acid due to degenerate codon usage (synonymous). Analysis of ftn gene (CLIASIA 03035) showed that while overall sequence similarities among all isolates are 97-98%, one single nucleotide polymorphism (SNP) was consistently identified in Japan and Indian isolates where nucleotide ‘G’ was substituted with ‘C’. This SNP results in the alternation of amino acid from histidines to glutamine. Sequence alignment of pilus assembly gene (CLIBASIA_03075) showed that isolates from Vietnam, Cambodia, Japan, Taiwan, and Thailand have ‘GT’ instead of ‘AC’ which is found most in isolates from USA (Florida), Brazil, Indian, and China, respectively. The switch of GT to AC results in changes of amino acids from serine to alanine and threonine to valine. For pilus assembly gene (CLIBASIA_03045), the DNA sequencies of most isolates are identical except isolates from Cambodia where 24% of them have SNPs for ‘AG’ or ‘GG’ instead of ‘TC’. Such alternations resulted in the changing of amino acid from serine to glycine and arginine to glycine. Analysis of flgH gene sequences indicated that 45% isolates from Cambodia have ‘A’ instead of ‘G’ while 25% isolates from Vietnam have SNP with ‘G’ instead of ‘T’. Nucleotide substitutions result in amino acid changes from proline to leucine and threonine to proline, respectively. Compiling the genotyping of this data with our previous results, we constructed a Las genotyping database based on five putative virulence gene loci. Clearly, that non-synonymous SNPs identified within a gene’s coding region led to the alternation of amino acid sequences that would have potential effects on protein folding, functionality and cellular response to the environment. To further characterize and determine the relationship between the genotype vs. the phenotype, in vitro orthologous gene replacement approach was used. Two pilus genes were first selected to evaluate the possible functions in Las. Comparative analysis of Las and Xylella fastidiosa (XF) genomes revealed that the Las possessed pilus gene that was homologous to the pilus gene of Xf which is a xylem-limited pathogenic bacterium causing citrus and grape diseases. It was reported that the pilus assembly protein accounts for twitching motility in XF which is essential for virulence. Like other intracellular pathogenic bacteria, ability of movement is necessary for Las to causes a systemic infection. To test the function of pilus genes in Las, we created pilus mutant XF by a site specific gene knock-out technique. The DNA sequences (with their own promoters) encoded for two pilus assembly homologue proteins were amplified from different SNP types of Las isolates. The amplified genes were cloned into a protein expression vector which was then transformed into pilus-deficient mutants of Xf via electroporation. The colonies grown from selected medium will be identified and further tested by PCR to confirm successful replacement of Las pilus gene. Currently, we are developing a bioassay to evaluate the mobility of complementary Xf strains.
The main objective of this proposal is to develop a novel method for inoculation of experimental citrus plants with HLB that would provide an excellent alternative to the existing methods by overcoming the disadvantages of the latter approaches and allowing high throughput inoculations with a greater certainty of pathogen transmission for various research purposes. We are using a Pulse Micro Dose Injection System (PMDIS) to develop a new method for rapid and efficient inoculation of plants with HLB. A positive outcome of our preliminary experiments suggested a feasibility of further adaptation of the PMDIS system for HLB inoculations. This is a new project and the funds for this project are currently being released. The research in in progress. We already have designated personal who would conduct the proposed research. Plant material that will be used in this project is being prepared. Using plant material that is already available (existing HLB-infected plants that are used as inoculum source and healthy plants that are used for inoculations) we are setting up initial trial inoculations.
This is a project to find an interim control measure to allow the citrus industry to survive until resistant or tolerant trees are available. We are approaching this problem in three ways. First, we are attempting to find products that will control the greening bacterium in citrus trees. We have chosen initially to focus on antibacterial peptides because they represent one of the few choices available for this time frame. We also are testing some possible anti-psyllid genes. Second, we are developing virus vectors based on CTV to effectively express the antibacterial genes in trees in the field as an interim measure until transgenic trees are available. With effective antibacterial or antipsyllid genes, this will allow protection of young trees for perhaps the first ten years with only pre-HLB control measures. Third, we are examining the possibility of using the CTV vector to express antibacterial peptides to treat trees in the field that are already infected with HLB. With effective anti-Las genes, the vector should be able to prevent further multiplication and spread of the bacterium in infected trees and allow them to recover. We have completed several large screenings of antibacterial peptides against Las in sweet orange trees. About 50 different antibacterial constructs have been tested in trees. We have found only two peptides that appear to give some protection sweet orange trees from HLB. We continue screening for better genes that will more effectively control HLB and can be approved for use in a food crop. We also are improving the CTV-based vector to be able to produce multiple genes at the same time. This could allow expression of genes against HLB and canker or multiple of genes against HLB. Another major goal is to do a field test of the CTV vector with antibacterial peptides, which is an initial step in obtaining EPA and FDA approval for use in the field. After some delays, we have received permission for USDA APHIS and are now establishing the field test.
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