We were able to close the L. crescens BT-1 genome without the use of pcr. The final genome is 1,504,659 bp long. Genome alignments between BT-1 and Liberibacter asiaticus and Liberibacter solanacearum showed low overall macrosynteny. There was a high degree of gene-sequence conservation but gene order was very divergent. Further genome comparisons of the Liberibacter species have yielded primarily regulatory differences. Attempts to compensate for biosynthetic inadequecies in L. asiaticus through media additives have been unsuccessful. It does not seem that any single metabolic deficiency is responsible for unculturability in L. asiaticus. Two prophage regions were identified in L. crescens. These regions appear to be unrelated to those in L. asiaticus and L. solanacearum.
We have done initial studies on purifying Liberibacter cells from psyllid nymphs and adult psyllids. Our initial attempts to purify Liberibacter cells from nymphs were not productive. Based on qPCR data, the calculated population of Liberibacter cells in a representative portion of the nymphs was much lower than for an equivalent number of adults. Fractionation of Percoll gradients loaded with extracts from nymphs and analysis of the Liberibacter content of the fractions indicated lower than desired recovery of Liberibacter cells from nymphs. Our initial results using adult psyllids were more promising. A sampling of the adults showed a much higher level of Liberibacter than in the nymphs. The Liberibacter numbers from Percoll gradient fractions indicated that Liberibacter cells purified from adult psyllids band much higher up in the gradient than when extractions are made from plant tissue. Fluorescence In Situ Hybridization (FISH) microscopy performed on these fractions indicate that Liberibacter cells are embedded in a matrix which likely is remnant insect tissue. This suggests that the current buffer extraction does not effectively dislodge Liberibacter cells from insect membranes. We will continue working with adult psyllids and will modify the extraction buffers to include different non-ionic detergents as a means of dislodging bacteria from insect tissues and increasing the recovery of “free” bacterial cells. If the detergents act as desired, we expect to see a greater banding of Liberibacter cells further down in the Percoll gradient, indicating an increased number of “free” bacterial cells.
Data generated by Proteomics-based Change Mediated Antigen Technology were finalized to identify the pathogenesis-associated proteins. A total of 155 biomarkers were identified exclusively in the infected citrus phloem. 74 biomarkers were found within the green plant database. 55 biomarkers were found in the bacterial database. More importantly, 26 biomarkers were found in the Candidatus Liberibacter asiaticus database. The discovery of these citrus greening-specific biomarkers will be useful to design future experiments on citrus greening diagnosis, such as generating antibodies to detect citrus greening disease.
The visiting scientist who began work with the scFv library made with the earlier grant from CRDF has been making excellent progress. We had previously used the scFv when expressed as part of the M13 phage vector particle in ELISA and dot blot formats. Our efforts in the past quarter have built on that work, and now we are using the scFv alone in tissue print assays of citrus plants to detect ‘Ca. Liberibacter asiaticus’. scFv are expressed and purified from from E. coli cells using a 6X His tag incorporated in the scFv protein. Study of the literature showed that the media used to grow the E. coli and other details of the culture conditions greatly influence the yield of scFv obtained from culture lysates. We have found that a very rich and buffered medium, (2X yeast extract Tryptone broth with phosphate buffer) works best. The medium is supplemented with glycine, sucrose and IPTG at various stages of the expression protocol. With this protocol we can produce purified scFv at concentrations in the mg/ml range. The tissue print assays are being done on nitrocellulose membranes. A range of parameters have been optimized, including the concentration of non fat milk used as blocking protein, duration of blocking and wash steps, the dilution and concentration of the scFv antibodies used for the detection of the ‘Ca. Liberibacter asiaticus’ in the tissue print, and the concentration of the detection antibody, which is a commercially available monoclonal antibody that recognizes the 6X His Tag and is labeled with alkaline phosphatase. Cross reactions with healthy plant tissue can be a problem, especially if the concentration of scFv is too high. Best results to date are with petioles. We have established tissue printing protocols for 18 scFv, that were selected to bind to proteins expressed on the surface of ‘Ca. Liberibacter asiaticus’. These targets include an ATPase associated with the type IV pilus, a pilus assembly protein, two flagellar proteins, the major outer membrane protein OmpA, and the efflux protein TolC. We have systematically sampled leaves from chronically infected rough lemon trees grown in our greenhouse. Petioles are sliced by hand for tissue prints and alternate slices are set aside for DNA extraction and qPCR to estimate the quantity of ‘Ca. Liberibacter asiaticus’ in the sections used for tissue printing. Interestingly, petioles from leaves with blotchy mottle and low qPCR values (= high concentrations of ‘Ca. Liberibacter asiaticus’ DNA) give poor results by tissue printing, but sections of petioles from asymptomatic leaves from the same branch produce higher qPCR values (fewer bacteria) but better tissue printing results. This is consistent with the concept that the Liberibacter cells present in phloem of severely symptomatic leaves are dead, and that therefore the proteins on their surface have been degraded and are no longer recognized by the scFv. Tissue prints typically show strong color development in the phloem ring. Tissue prints from healthy control plants are clear. In some tissue prints, color development is observed in discrete spots outside of the phloem cylinder. We will carry out further experiments with the scFv using the FLAG epitope for detection of the scFv. The scFv will also be used to label sections for examination by fluorescence and electron microscopy. We have provided several scFv to cooperating researchers at USDA ARS Ft. Pierce.
This project is a continuation of a previous project #95 “PREPARATION OF ANTIBODIES AGAINST CANDIDATUS LIBERIBACTER ASIATICUS”. Progress reports for the previous project are on file. The reimbursable agreement with CRDF was established on September 5, 2012. Efforts have been underway during this quarter to recruit a visiting scientist to work on this project. We continue to study the literature to identify vectors to use for a future scFv library made as part of this project. The goal is to find a suitable vector that is not encumbered by intellectual property and patent issues. We are also optimizing the cloning strategies that will be used to move already selected scFv into transgenic plants. We have identified a suitable candidate for the visiting scientist position and the visa process is moving along. Related research with the existing scFv is underway on project 551.
Understanding foamy bark rot of Fukumoto navel: Preliminary results support the hypothesis that foamy bark rot is initiated by scion-rootstock incompatibility, resulting in nutritional and/or physiological abnormalities, which provide opportunity for infection by certain pathogen(s) and foam production is possibly due to yeast activities. Inoculations with fungi (Fusarium solani, Fusarium sp. and Botryosphaeria stevensii) obtained from declining Fukumoto show that most of these fungi are pathogenic on Fukumoto. Bacterial inoculations produced no obvious symptoms on Fukumoto. Analysis of soil samples indicated no significant correlation between foamy bark rot and pH, total N, C, Olsen-P, K, Na, Ca, Mg, and lime. In vitro fungicide screens are underway to determine the most efficacious products in managing these fungal canker pathogens. Multiple Botryosphaeria species causing ‘dothiorella’ gummosis in citrus: In this survey across six counties, 11 different species of Botryosphaeriaceae, comprising five genera (Diplodia, Dothiorella, Lasiodiplodia, Neofusicoccum and Neoscytalidium), were found associated with cankers of multiple citrus varieties and different ages. Detached shoot assays showed all isolates tested were in fact pathogenic on citrus and further tests on one-year old citrus plants showed the ability of most isolates to produce symptoms of gumming within one to two weeks post inoculation. A manuscript is in preparation for these results and will be submitted to a peer-reviewed journal. Efforts are being made to evaluate fungicides currently available for use as pruning protectants. The alliance between Fusarium solani and other factors in citrus dry root rot: Results from this study indicate that in addition to F. solani and two species of Phytophthora (P. citrophthora P. nicotiana), F. oxysporum and a new Fusarium sp. are major organisms associated with DRR. Although there was no correlation between season and isolation of Fusarium spp., there was correlation between seasons and the isolation of Phytophthora spp. No correlation between nutrient content and DRR was found. A rootstock susceptibility screen is underway to determine the tolerances of 33 rootstocks to infection by F. solani as well as other Fusaria identified from this study. Investigating Branch Canker and Dieback in Lemon and Grapefruit in the California Desert Region: Surveys conducted across Riverside, Imperial and San Diego counties in Southern California show the presence of Neoscytalidium dimidiatum, Eutypella citricola, E. microtheca and Eutypella sp. associated with citrus branch canker and dieback. Pathogenicity tests with detached green shoots show both E. microtheca and Eutypella sp. are pathogenic on citrus, however these fungi appear to have a low to moderate virulence. Preliminary results from an in vitro fungicide screen show there are some products currently available that have the potential for use as pruning protectants.
Substantial progress in the detection of Candidatus Liberibacter asiaticus (CLas) in citrus tissues and psyllid vector using non radioactive digoxigenin labeled probes during the tenure of this project. We developed and used both digoxigenin-labeled Plabeled PCR probes as well as RNA probes. We produced digoxigenin-labeled PCR products using specific primers for the OMP, RNA polymerase beta subunit, DNA polymerase region, the r-DNA region, and the 23S and 16S ribosomal RNA intergenic regions. Citrus plants from the green house infected with HLB pathogen were used as the source plants for obtaining the tissue for the blots and to isolate total nucleic acids template necessary for the amplification of CLas specific DNA. Primers were designed based on the CLas sequence information and amplified specific amplicons from the DNA isolated from HLB infected plants, and amplification was not observed from the healthy control plants grown under similar conditions. In the initial tissue blot experiments we did not observe hybridization signals specific for HLB. The reason probably is the low titer of pathogen and/or the non uniform distribution of the pathogen in the infected tissues. It is also possible that the PCR probes failed to detect low titer of the pathogen. Therefore, the amplified regions of Las were also cloned in the transcription vector, and digoxigenin labeled strand specific RNA probes were generated by transcription and the probes generated substantially improved detection of Las in citrus tissues. In addition to tissue blots of the stem sections, we have used the midrib and petiole region and whole leaf and branch containing multiple leaves from healthy and infected citrus on nylon membranes. The hybridization observed with the midrib imprints and whole leaves showed clear signals compared to stem imprints. In other experiments we used imprints of the leaf midrib, petiole and stem from new flushes of infected citrus since the psyllids preferentially feed and acquire the pathogen from such tissues. We also imprinted on the membrane the inside surface of the bark which contains phloem tissue in which the HLB pathogen is located. This procedure, was observed to be to be very useful to the determine the distribution of CLas in infected tissue. The second area of our focus is on the detection of Las in psyllid vector by tissue blots (squash blot) on nylon membranes. The procedure for isolating the nucleic acid from single psyllid was optimized, and we have been able to amplify CLas specific amplicons from single infected psyllids using pairs of Las specific primers. Conditions of amplifications were optimized with different primer pairs and now we have been able to amplify HLB specific amplicons without non-specific bands in PCR. In initial studies of whole psyllid tissue blots, hybridization signal was also observed with healthy psyllids (psyllid colony from the healthy psyllid containment facility). However use of specific primer pair corresponding to the EFTU gene of CLas has been promising and we will use the probe generated for this gene in tissue blots of psyllids henceforth. Success of this work would benefit the citrus industry by providing simple, sensitive and rapid detection method for large-scale detection of HLB under field conditions. Tissue imprints, once on the membrane could be stored for later use, can be shipped easily and generally are stable. The modified form of tissue blot, the ‘squash-blot’, for psyllids developed will be useful to understand the epidemiology of HLB. Two manuscripts have been submitted and have come back for revisions (one in Molecular and Cellular Probes and the second in the Journal of Bioscience).
Research results are very similar to those reported in the September 2012 quarterly report. We ran additional experiments similar to those described in the previous quarterly report. Because of the season, seeds were mature and more difficult to homogenize completely with or without enzyme treatment. We continued investigation with two experimental approaches: 1) enzyme treatment without subsequent physical pulverization of the tissue and 2) enzyme treatment followed by physical pulverization of the tissue. Fractionation on Percoll gradients yielded results similar to previous. Percoll fractions with the lowest Ct values (most bacteria) were analyzed by Fluorescence In Situ Hybridization (FISH) microscopy. The most intense fluorescence was observed with spherical bodies and a few bacilliform shapes. Because of the manner in which the experiments have to be performed, contamination is possible though precautions are take to minimize this occurrence. As before, the numbers of cells calculated from qPCR values for the Percoll and preceding fractions indicated more cells should be present than were observed in the sample observed by microscopy. We know that the disruption of the vascular bundles is not complete since filtration prior to Percoll fractionation traps tissue fragments that contain Liberibacter DNA; and, most likely Liberibacter cells. We have expanded our isolation work to include adult psyllids and psyllid nymphs. Psyllids are softer bodied and preliminary experiments suggest they homogenize more efficiently than plant tissues. Real-time PCR analysis of fractions from Percoll gradients suggest Liberibacter cells band differently when extracted from psyllids. Currently, fractions are being analyzed by FISH.
This is a project to continue one of the most fruitful leads that accidentally resulted from our previously funded work. We have found that citrus becomes a source of Huanglongbing (HLB) inoculum for spreading the disease to other plants much earlier than previously thought. The working hypothesis is that the female psyllid finds an area of new flush to lay her eggs. As she is laying eggs, she probes the phloem to feed and transfers Candidatus Liberibacter asiaticus (Las) to the tree. As the eggs develop into nymphs, Las begins to multiply in that localized area of the plant, where the new nymphs then feed and acquire Las. Thus, infection of only a micro area of flush tissue where the nymphs develop is sufficient for the first generation of psyllids to become infected and to be vectors to spread the disease to other trees. Thus, the time-period after a tree becomes infested by infected psyllids until it is a donor for other trees could be as short as 15-30 days or less. The limitation is actually the time for the second generation of psyllids to develop. We are continuing experiments to find ways to quickly detect psyllid reproduction as a method to detect early infections.
Goals 1, 2 and 3 are partially completed and continuing. We have cloned three predicted “late gene” promoter regions with promoter activity from SC1. The first (promoter P0) is located between locus tags gp125 and gp130 and includes a portion of the region annotated as gp125. This region was cloned in both directions upstream of the lacZ reporter gene in E. coli. In the early gene direction, this promoter region gave a light blue color reaction that we regard as a moderately constitutive promoter activity. In the late gene direction, this promoter region gave a medium blue color reaction that we regard as a stronger constitutive promoter activity. We subsequently cloned two additional potential promoters from the late gene region of SC1, the first located between gp120 and gp125 (promoter P1) and the second between gp115 and gp120 (promoter P2). Both of the promoters were cloned in the late gene direction. Only the construct (forming pSZ81) of P2 fused with the lacZ gene showed a medium blue color reaction equivalent to that of P0. Promoter P1 was not active. We then cloned the SC1 gp125 (annotated as a phage C2-like repressor) into pUFJ5, forming pUFZ3-4, and transformed into the strain containing pSZ81. No change in color reaction was observed, indicating that either the pUFZ3-4 clone was not functional, or the gp125 locus didn’t function as a repressor of P0. We then searched the Las genome (psy62) and found that besides gp125, there was only one additional locus annotated as a repressor in the genome, CLIBASIA_01645. This protein homologue is also present in Lso (CKC_01785) and Lam (LAMF_00067) and all three have a predicted Peptidase S24 LexA-like protein domain at their its 3’ends. LexA is a repressor of genes involved in the cellular SOS response to DNA damage. We designed primers to clone this C1 repressor from Las strain UF506 based on the pys62 genome sequence. The PCR product sequence revealed that the corresponding ORF in UF506 was truncated and missing the Peptidase S24 LexA-like protein domain that is predicted in Las strain psy62, Lso and Lam. This truncated UF506 ORF was cloned into pUFR047, forming pSZ83, and transformed into the strain containing pSZ81. We also cloned the nontruncated Lam homolog (LAMF_00067) into pUFR047, forming pSZ84, and transformed this clone into the strain containing pSZ81. Again, no change in color reaction was observed, indicating that neither functioned to repress the late gene promoter regions P0 or P2. In an effort to determine if the promoter regions examined were really in the ‘late’ region, we used semi-quantitative, real time PCR assays to determine relative expression levels of several different genes on both SC1 and SC2. Relative expression levels of SC2-gp095 (peroxidase), SC2-gp100 (glutathione peroxidase), SC1-gp110 (‘holin’) although nominally placed in the ‘late’ gene regions of the phage, were much higher (>100X) than expression levels of SC1-gp025 (‘tail fiber’). These results indicated that the promoter regions P0, P1 and P2 that we examined as predicted ‘late gene’ promoters, are likely not late gene promoters, nor repressed. Since repressors are known to work at multiple promoter sites, we are cloning additional predicted promoter regions, this time from genes including SC1-gp025, now experimentally shown to be expressed as a late gene.
The data analysis involved validation of LIBS spectral data with nutrient profiles. Each nutrient (N, P, K, C, B, Ca, Cu, Fe, Mg, Mn, Zn) was independently analyzed with the spectral data to verify whether the LIBS spectral data can be associated with nutrient concentration. Several statistical models such as Partial Least Square Regression (PLSR) and Support Vector Machine were tested on the processed LIBS spectral signals. Some of the LIBS data processing procedure which were performed were baseline correction, wavelet-based denoising for noise removal, resampling of LIBS spectra to reduce the number of spectral features, peak selection and alignment for extracting important peaks, etc. Although, the PLSR model showed a relationship between the LIBS spectra and nutrient concentrations showed good correlation between the actual and predicted values for various nutrients, the model could not be validated with an independent dataset. The major reason for this could be the low variation in the nutrient concentrations among the samples. For example, most nutrients, such as nitrogen, phosphorus, potassium, iron and manganese had nutrient concentrations in optimal or high ranges. Similarly, nutrients such as calcium and copper were either optimal or high/excess, respectively. A few other challenges that were identified in the process were: (i) several LIBS spectra replicates were collected per leaf sample, however, a minimum quantity of leaves (4-5 leaves) are required for chemical analysis, which made the comparison challenging; (ii) the LIBS spectra had to be averaged to compensate for limitations in chemical analysis results, which reduced the number of spectra to about 115 samples. A larger dataset may increase the robustness of the developed models; and (iii) the LIBS spectra have more than 6000 features. The tested feature selection methods were not be robust in representing the entire data; however using all spectral features will also not be feasible. Therefore, our future research will involve the following: (i) collecting a larger dataset, possibly including samples that represent different ranges (low, optimum and high) in order to increase the prediction/classification efficiency, (ii) collecting LIBS spectra from soil to verify if the system is more applicable for soil nutrient analysis, and (iii) improving the statistical analysis to develop a robust model with the right processing tools.
In this project we are developing a new method for rapid and efficient inoculation of plants with HLB based on a Pulse Micro Dose Injection System (PMDIS). In our preliminary experiments we have had some success in inoculation of Periwinkle plants using this technique, which suggested a feasibility of further adaptation of the PMDIS system for HLB inoculations. Our goal now is to optimize the protocol for PMDIS-based plant inoculation. We are conducting experiments in order to 1) identify what types of tissue within an infected citrus plant can serve as a good resource of the HLB bacteria for preparation of the inoculum by comparing extracts from stems, leaves and seed coats as inoculum sources; 2) examine whether HLB-infected psyllids can be utilized for preparation of the inoculum suspension; 3) optimize the composition of the extraction buffer used for preparation of the bacterial suspension and the extraction conditions, so they would support high efficiency of the PMDIS-mediated transmission of the pathogen; 4) optimize the parameters of injection. We are also evaluating how age of receptor plants, types of citrus varieties used as HLB bacterium donors as well as for plants being inoculated, types of flushes being inoculated affect efficiency of inoculation. Several sets of plants have been already injected using PMDIS. Those are being maintained in the greenhouse and monitored for the disease development.
The overall goal of this project is to improve the efficacy of the copper loaded silica nanogel (CuSiNG) formulation for preventing citrus canker disease incidence. 2012 field trial data has been compiled. The data show that CuSiNG pH7 opaque formula demonstrated superior efficacy against preventing incidence of young canker lesions, scab and melanose. The formula exhibited no Cu toxicity. The overall efficacy of the CuSiNG pH7 formula was comparable and in some case better than a number of leading commercial products (at 1.0 lb/acre metallic Cu content). The efficacy was not compromised even at lower metallic Cu concentration of the formula when compared to other commercially available products. Field trial was also conducted on CuSiNG pH 4 which is a transparent water-soluble formula. The metallic Cu content was 0.2 lb/acre in CuSiNG pH 4 formula. Results indeed show that the formula was effective in preventing canker incidence. However, the overall efficacy was not as good as CuSiNG pH 7 formula. This suggests that Cu rate at 0.2 lb/acre was not adequate to fully protect against canker incidence. CuSiNG pH 4 formula did not exhibit any phytotoxicity. Future studies/trials will focus on transparent CuSiNG pH 4 formula to improve its efficacy without increasing metallic Cu content. We also plan on conducting trial in the upcoming season using mixed-valence CuSiNG formulations. Preliminary results were encouraging as improved efficacy was observed in laboratory conditions. Due to high surface area of the silica nanogel material, improved adherence to citrus plant is expected. We have conducted adherence studies using surface modified yellow-emitting CdS:Mn/ZnS quantum dots (a photostable semiconductor fluorescent nanocrystals of 3.5 nm size). Results indeed show that the silica nanogel material itself (in absence of Cu) strongly adhere to the plant surface. We are also conducting adherence studies using red-emitting (Cu insensitive) Qdots where Qdots are embedded in CuSiNG materials. Results from this study will directly demonstrate the adherence property of the CuSiNG nanoformulations.
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 third quarter has involved molecular characterization of samples collected in October in Southern and Central Florida. Research has included development of a unique methodology to selectively prepare DNA from Liberibacter and Liberibacter-like pathogens for genome sequencing. Observations are being made of symptoms produced on citrus in the field and greenhouse conditions.
The 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. At this stage we are studying the interaction of the plants expressing HLB effectors and their response to HLB infection through psyllids. A potential useful outcome of this project is the finding that CTV-vector can be used as an RNAi vector. This has been demonstrated to silence citrus endogenous Phytoene desaturase (PDS), delta amino levulinic acid dehydratase (d ALA dehydaratase) genes, and has also be used to potentially silence endogenous genes of psyllids (vecotr of HLB) in co-operation with scientists at CREC and USDA, Fort Pierce. We have positive feedbacks from these groups on the successful silencing of psyllid endogenous genes. This line of research potentially holds great promise in mitigating the spread of HLB by psyllid vector. An oral presentation based on this investigation will be presented in the upcoming 2013 HLB conference in Orlnado. Shubash Hajeri, Choaa El-Mohtar, William O. Dawson and Siddarame Gowda. Citrus tristeza virus-based RNA-interference (RNAi) vector and its potential in combating citrus Huanglongbing (HLB).
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