A comparative field trial involving two copper loaded silica nanogel (CuSiNG) formulations (Cankicide-opaque and Cankicide-transparent) and a series of commercially available Cu biocides (such as Kocide 2000, Kocide 3000, Champ 30DP, Cuprofix Ultra 40, Kentan DF, Badge X2, NuCop HB, Nordox and Magna-Bon) has been initiated by James H. Graham group (CREC, Lake Alfred, FL). During this trial period, data are being collected to evaluate canker incidence and retention of copper residue on fruit surface. Additional particle size characterization was conducted using Atomic Force Microscopy (AFM). AFM data showed the formation of ultra-small particles as small as 10 nm. These particles were interconnected to form “gel-like” micron-size structures. The AFM data matches with high-resolution transmission electron microscopy (HRTEM) and dynamic light scattering (DLS) data as reported earlier. The antibacterial efficacies of the two formulations (transparent and opaque) were tested against X. alfalfae (a citrus canker surrogate), in nutrient broth under laboratory conditions. Kocide 3000 was used as the positive control and silica nanoparticle/nanogel (SiNP/NG) as the negative control. The metallic copper concentration was varied from 866 microgram to 43.3microgram per milliliter. The minimum inhibitory concentration (MIC) of metallic copper was estimated to be 100 microgram per milliliter. Additional antibacterial tests and fluorescence imaging study are being conducted to evaluate the interaction of the cankicide material with bacteria cells.
The goal of this project is the in vitro culture of Ca. Liberibacter asiaticus (LAS) associated with the Citrus greening syndrome. The strategy consists of primo-cultures of the bacteria in insect cells cultures used as feeder cells. Co-culture and axenisation LAS/Drosophila co-cultures: LAS can be maintained in co-culture with drosophila cells but are quickly lost due to the high growth rate of drosophila cell lines used. We previously observed that sodium pyruvate and ethanol amine had a positive effect on LAS fitness and a negative effect on insect cells growth. We tested those additives on drosophila cells: sodium pyruvate, at the concentration tested, had a lethal effect on drosophila cells but ethanol amine had a less deleterious effect and we are currently monitoring this cell culture before inoculation with LAS. LAS/Aedes co-cultures: Different co-cultures were continuously grown over several months with successive transfers. After successive dilutions we could get rid of the Aedes cells without losing the LAS detection. To verify that the culture was axenic, we checked if LAS was the only bacteria in our cultures. Other bacteria strains were found in co-culture with LAS (Delftia acidovorans, actinobacteria). Antibiotic selection was applied to get rid of the contaminants but resulted in the loss of the LAS signal as a consequence. Enforced antibiotic selection and additional sterilization steps were applied to the infected plant material surface for our new inoculations and the co-cultures are currently monitored. LAS/ Diaphorina citri co-cultures: Two Diaphorina citri cell lines from the Keyhani’s lab are now stably maintained. LAS was detected after 3 transfers but not more. D. citri cells are progressively adapted to an alternative medium that gave good results for LAS/ Aedes co-cultures. Medium improvement and LAS fitness: To improve LAS fitness in culture, we complemented the primo-cultures with various nutrients based on data from healthy and HLB infected Citrus phloem or on potential metabolic pathways deduced from the LAS genome sequence available. Several additives were selected that improved LAS growth in co-cultures. We did inoculations of LAS in medium with different combinations of those additives, with and without Aedes cells. LAS could be detected no more than 4 transfers in medium only but still much less than in conjunction with Aedes cells. A solid version of the medium was tested to try to isolate LAS clones with no success. Citrus inoculation The very next step is to inoculate the cultured bacteria into healthy Citrus in order to verify its pathogenicity and re-isolate it (Koch’s postulates). We set up a protocol of mechanical inoculation of healthy citrus and will test further the diffusion in the citrus phloem with a fluorescent dye. Another way to inoculate the pure LAS culture would be by acquisition through membrane by the insect vector and inoculation of healthy citrus via the infected insect. We are setting up experiments of acquisition through membranes. Our co-cultures conditions prove to be very reproducible in getting LAS in culture and our major line of improvement is to get no ‘contaminant’ bacteria along with liberibacter.
The multilocus simple sequence repeat (SSR) marker system effectively detects and differentiates ‘Candidatus Liberibacter asiaticus’ strains from various geographic and environmental locations worldwide. This genotyping system has improved resolution capabilities in fingerprinting genetically close ‘Ca. L. asiaticus’s strains. HLB-associated ‘Ca. L. asiaticus’ strains vary in pathogenicity. Various types and severities of HLB symptom were observed both in field and greenhouse grown citrus plants. Some of these strains cause only mild symptom while others show more severe symptom. These differences were found even within the same variety of citrus plants, suggesting that the genetic variations of the pathogen could play important roles in these host-pathogen interactions. The objective of this study is to extend from DNA-based genotyping to functional phenotyping. The goal of this research is to develop a gene-based marker system that links to phenotypes of ‘Ca. L. asiaticus’s strains. In silico genome analyses revealed that ‘Ca. L. asiaticus’ contained a number of genes associated with pathogenicity. Some of these genes seem to be of phage origin, suggesting that the bacterium has been actively involved in multiple phage integration events via the horizontal gene transfer. We have identified the SSR loci in this bacterial genome. Some of SSR loci were located in prophage gene sequence regions. Given the fact that the Las has a significantly reduced genome (1.2 Mbp), many of these repetitive regions located within or near coding or regulatory regions could have profound effects on gene expression and function. It has been reported that the repeat number variation in SSR seems to be intimately related to modulation of the expression of virulent factors. Based on the repetitive regions in Las genome, we identified pathogenic genes that are located in or near these regions. These pathogenic genes were classified into four groups based their putative functions: 1) Phage-derived genes which are often involved in pathogenesis through their transfer of genes and/or genetic rearrangement. 2) Genes defined as two component response regulators. This group of genes is important for sensing environmental signals and producing a transcriptional response from those signals. 3) Genes identified with hydrolase activity. One of the interesting genes in this group is salicylic acid hydrolase which could play an important role in hydrolysis of salicylic acid, a signaling molecule expressed in host in defense responses pathogen attack. However, to confirm its hydrolase function, the substrate for this gene-coded enzyme needs to be determined. 4) Zinc binding proteins, znuABC genes identified as putative high-affinity zinc uptake system that belongs to the ATP-binding component of ABC transporter protein. Zn uptake system ZnuABC is required for obligate bacterial zinc homeostasis in intracellular environments which contributes to the virulence of Las in citrus plants. Twenty primer pairs were designed that flank on the upstream and downstream of ORF to produce a full length for each gene. Ten HLB-affected samples representing virulent and weak virulent strains of ‘Ca. L. asiaticus’s were selected and amplified during this period of study. Research is in the process of conducting sequence association analyses to determine the linkage disequilibrium of the gene-based markers associated with the virulent traits of Las strains.
Citrus greening disease also known as Huanglonbing (HLB) is caused by a phloem-limited Gram-negative alpha proteobacterium, Candidatus Liberibacter asiaticus (CLas). It is the most destructive disease of citrus that could potentially compromise the productivity of Florida industry. In general phytopathogenic bacteria and alter the physiology of the host by injecting effector proteins into host cells to establish infection. . The sequence information has identified number of unique features of the CLas and most relevant to this proposal identification of number of potential effectors as part of hypothetical proteins which do not have homologues in other bacterial systems. Based on this supposition and as elucidated in the previous progress report we have cloned number of these effcetor genes into Citrus Tristeza Virus (CTV) vector to express the in the phloem of citrus plants which is the natural niche of the pathogen. The transfer of effectors using CTV vector is facilitated through prior agroinoculation of Nicotiana benthamiana and purification of CTV virions from agroinoculated N. benthamiana carrying CLas effetors before inoculation of citrus. This has been the most time consuming part of research till now. However, we have made good progress and several effectors have been ready for inoculation of citrus. One of the important effectors or virulence factors is hemolysin, a 50 kDa protein secreted by type I secretion system. The hypothesis is that the hemolysin might interfere with metal ion transportation leading to host metabolic imbalance potentially resulting in disease symptoms. Although it is identified as a toxin, it does not induce any visible phenotypic symptoms in N. benthamiana. We had submitted an abstract entitled ‘Functional studies of hemolysin (exotoxin) of ‘Candidatus Liberibacter asiaticus’expression in citrus using Citrus Tristeza Virus vector’ to the 2011 American Phytopathological Society conference. We plan to do the transcriptome analysis of the citrus following its transfer to citrus to understand the potential role as CLas toxin. We also tried to introduce another potential toxin, Serralysin, but did not succeed to clone into CTV mainly because of it s size. But we will make further attempts to introduce this potentially important toxin to citrus. We are also using a different approach to screen for virulence genes of HLB using Potato virus x (PVX) vector system which has been used extensively to screen for virulence genes herbaceous hosts. We have noticed that in the genome of CLas, there is only one copy of flagellin-encoding gene in contrast to four copies of them in Sinorhizobium meliloti. Transient expression of the Las-flagellin using PVX expression vector induces an immunity response with cell death in Nicotiana benthamiana. Although a full flagellum is not observed with HLB, rudimentary flagellar apparatii exist in CLas based on the sequence information. We have submitted an abstract identifying such genes in CLas and inoculating tobacco plants. We are towards the end of year one of this reserach and we have really made good progress which will be presented in the annual report and make a request for extension for the second year.
In the present study, performed in collaboration with Dr. Nelson Wulff at Fundecitrus, we have continued sequencing a fosmid DNA library of Ca. L. americanus (Lam) strain ‘S’o Paulo’ isolated from infected periwinkle from Brazil. Nearly all contigs have now been organized on a predicted “scaffold” and gaps continue to be closed by PCR. The overall gene organization and structure of the Lam genome is more similar to Ca. Liberibacter solanacearum (Lso) than to Las. Lam phage sequences corresponding to both SC1 and SC2 of Las were found, although SC1 (and not SC2 as in Las) appears to replicate as an integrative plasmid prophage. Las phage were found to become lytic in plant infections (Zhang et al. 2011); Lam appears to have same potential (ie., Las and Lam both carry “suicide program” or lytic cycle genes). SC2 in Lam Sao Paulo has clearly degenerated to a nearly nonfunctional state (in terms of lytic cycle, but its replication was detected in circular form. SC1 in Lam appears to more assume the role played by SC2 in Las, since its replication is high, and it carries two peroxidase genes that are likely to be needed for pathogenicity of plants and possibly transmission in psyllids. Notably, 2 of 4 peroxidase genes were missing on the SC2 phage, as well as 2 adhesins (surface anchor proteins).
Thus far, 496 medium formulations have been tested for support of Candidatus Liberibacter asiaticus ( Las). None have supported continuous axenic culture of the bacterium. A basal medium developed with the aid of the bacterium from mountain papaya or babaco that is closely related to Las has been modified in attempts to grow Las. different complex ingredients, such as bovine heart infusion, have been tested as replacements for the brain heart infusion broth in the medium. Supplementing the medium with different concentrations of insect prepared culture media, such as TMN-FH medium, CMRL 1066 medium, medium 199, and DS2 medium, has been tried. Replacing the ACES buffer with HEPES buffer to facilitate testing the effects of higher pH (from pH 7.2 to pH 7.8) than the usual pH that has been used (pH 6.8 to pH 7.0), and pH 7.4 was selected for further tests. Various supplements, such as mucin, zinc sulfate, potassium nitrate taurine, betaine, folic acid, catalase, yeastolate, cyclic AMP, arabinose, sodium pyruvate, and lactoalbumin hydrolysate, have been tried. Some of the medium modifications appear to promote Las biofilm formation around the psyllid alimentary tracts that have been used as a source of inoculum, but planktonic growth has been minimal. Attempts to culture Las continue.
The main objective of the project is to select single chain variable fragment antibodies (scFv) against Huonglongbing (HLB), Citrus Verigated Chlorsis (CVC) and the development of immunoassays that incorporate these scFvs. We have successfully constructed one scFv library from chicken spleens that were immunized with bacterial proteins. The immunization protocol and delivery of spleens were performed in 100 days, and mRNA was immediately extracted to construct the library, using recombinant PCR, into an M13 vector. To validate the library, we have selected through Phage Display several antibodies with high immunoreactivity to their antigen targets for three pathogens: Xylella fastidiosa, Agrobacterium tumefaciens and Xanthomonas axonopodis. For pre-validation of selections, we have performed ELISA assays for each of the selected recombinant antibodies and on the basis of this have selected 3 candidate scFvs. Since these scFv’s were selected using intact whole bacteria we attempted to identify the candidate outer membrane proteins (OMPs) whose epitopes form the basis of their reactivity. We isolated and purified the proteins present in the membrane fraction of the Xylella and Xanthomaonas pathogens and performed cross-reaction analysis of selected scFvs against these proteins, which confirmed three highly reactive clones: the Xf.02, Xf.03 and Xf.05. We then sequenced the genes that encode these three scFvs by sequencing the DNA from the M13 vectors, and performed bioinformatic analysis for structural prediction of the fragment antibodies. The corresponding scFv protein were expressed and secreted in culture in a TOP10 vector, and HPLC-purified for further validation with ELISA and Western blots. All three antibodies strongly recognize a 42 kDa MopB protein but via different epitopes (we speculate). Interestingly, the same antibodies could also recognize the related protein in a related pathogen Xanthomonas axonopodis OMPs, providing further evidence that these antibodies might detect the MopB in CVC and HLB. The detection of MopB in Xylella was comparable with the reactivity of a polyclonal antibody against purified Xylella MopB (made by Prof. Bruening). Proteomic analysis was carried out on the reactive protein band by excising it out of the gel, treating it with trypsinization and then functional identification of the protein by sequencing fragments on a ESI:MS:MS machine. Additional evidence of scFv reactivity was obtained through fluorescence microscopy showing the binding of the selected scFvs to the Xylella surface. We hypothesize that one of these antibodies recognizes multiple epitopes with common motifs and may become a general antibody for citrus bacterial pathogen detection. We have established a very simple conventional ELISA with the antibodies against bacterial OMPs, and the assay parameters will be translated to a lateral flow and fluorescence spectroscopy assays in the near future to improve sensitivity of the test.
The main objective of the project is to select single chain variable fragment antibodies (scFv) against Huonglongbing (HLB), Citrus Verigated Chlorsis (CVC) and the development of immunoassays that incorporate these scFvs. We have successfully constructed one scFv library from chicken spleens that were immunized with bacterial proteins. The immunization protocol and delivery of spleens were performed in 100 days, and mRNA was immediately extracted to construct the library, using recombinant PCR, into an M13 vector. To validate the library, we have selected through Phage Display several antibodies with high immunoreactivity to their antigen targets for three pathogens: Xylella fastidiosa, Agrobacterium tumefaciens and Xanthomonas axonopodis. For pre-validation of selections, we have performed ELISA assays for each of the selected recombinant antibodies and on the basis of this have selected 3 candidate scFvs. Since these scFv’s were selected using intact whole bacteria we attempted to identify the candidate outer membrane proteins (OMPs) whose epitopes form the basis of their reactivity. We isolated and purified the proteins present in the membrane fraction of the Xylella and Xanthomaonas pathogens and performed cross-reaction analysis of selected scFvs against these proteins, which confirmed three highly reactive clones: the Xf.02, Xf.03 and Xf.05. We then sequenced the genes that encode these three scFvs by sequencing the DNA from the M13 vectors, and performed bioinformatic analysis for structural prediction of the fragment antibodies. The corresponding scFv protein were expressed and secreted in culture in a TOP10 vector, and HPLC-purified for further validation with ELISA and Western blots. All three antibodies strongly recognize a 42 kDa MopB protein but via different epitopes (we speculate). Interestingly, the same antibodies could also recognize the related protein in a related pathogen Xanthomonas axonopodis OMPs, providing further evidence that these antibodies might detect the MopB in CVC and HLB. The detection of MopB in Xylella was comparable with the reactivity of a polyclonal antibody against purified Xylella MopB (made by Prof. Bruening). Proteomic analysis was carried out on the reactive protein band by excising it out of the gel, treating it with trypsinization and then functional identification of the protein by sequencing fragments on a ESI:MS:MS machine. Additional evidence of scFv reactivity was obtained through fluorescence microscopy showing the binding of the selected scFvs to the Xylella surface. We hypothesize that one of these antibodies recognizes multiple epitopes with common motifs and may become a general antibody for citrus bacterial pathogen detection. We have established a very simple conventional ELISA with the antibodies against bacterial OMPs, and the assay parameters will be translated to a lateral flow and fluorescence spectroscopy assays in the near future to improve sensitivity of the test.
With GC/DMS, SPME GC/MS, and Twister GC/TOFMS, we are carrying out a year-long (2010-2011) sampling plan for the HLB study with collaborators at Lake Alfred, FL to account for weather and seasonal (trees blossoming, fruit harvest, etc.) variations, with four trips having been made in 11/2010, 12/2010, 01/2011 and 02-03/2011. For a CTV study, using the same instruments, we have collected 5 batches of the samples in Lincove and Pauma Valley CA and the USDA facility in Beltsville, MD. Meanwhile, we have also carried out an analysis of PCR positive sweet orange infected with CVC strains 9a5c and 104 also at the USDA facility in Beltsville, MD. Data obtained from all of these studies was subjected to a variety of data mining and signal processing methods to define the accuracy of disease incidence predictions: (1) Student’s t-test selected separable pixels (signals) of the HLB DMS data, we obtained a diagnosis accuracy of 71% with a PLS model; (2) Applying wavelet transformation to the HLB DMS data, we increased the HLB diagnosis accuracy up to 82% and more interestingly the signal within the first 3 minutes yielded an equally good result to the whole time domain; (3) Similarly, by applying a PLS model to the wavelet coefficients of the CTV DMS data, the cross-validation accuracies are 81% for Healthy vs. severe-CTV, 74% for Healthy vs. mild-CTV and 80% for severe-CTV vs. mild CTV; (4) By developing a 3-class classification model for the wavelet coefficients of the CVC DMS data, we systematically generated the following accuracies: 86% for healthy, 46% for mild-CVC, and 97% for severe-CVC. To identify biomarkers associated with specific diseases, our typical results include: (1) For HLB Twister GC/TOFMS data, we detected 62 potential biomarker and obtained a diagnosis accuracy of 94% for HLB using a 4 latent variables based PLS model; and (2) By developing a genetic algorithm oriented searching process which was coupled with a classification model to evaluate the fitness of the selected biomarkers, we not only reduced the number of pertinent biomarkers from 62 to 14 but also increased the diagnosis up to 98%; (3) Based on the HLB SPME GC/MS data, we located 9 potential biomarkers for HLB diagnosis and obtained a diagnosis accuracy of 83%; (4) Based on the CTV Twister GC/TOFMS data, we detected 41 potential biomarker for CTV diagnosis with a diagnosis accuracy of 86%. After narrowing the potential biomarker set down to 31 biomarkers, we generated an accuracy of 85% for a more complex separation between healthy and two CTV related categories: CTV and CTV + ‘stubborn’, indicating the robustness of the selected biomarkers. We are also analyzing gene expression data on HLB and CTV infected plants and found gene based biomarkers focusing on sugar metabolism and soucrce-sink mediated gene regulation. Overall, the results of citrus disease diagnosis and biomarker detection are promising. With more samples to be collected and analyzed and the exploration on machine learning strategies, we are expecting a robust diagnosis model and a set of pertinent biomarkers for each citrus disease in this study.
We have completed the target selection and design of MOLigo probes for detection of Candidatus Liberibacter, Xylella fastidiosa, Xanthomonas axonopodis pv. citri, and citrus tristeza virus. Testing has begun on characterization probes for Liberibacter and CTV using synthetic DNA targets, and Liberibacter assays show promise for being able to detect and perform species designation. Probes for CTV detection and discrimination between mild and severe clades are also showing promise on synthetic templates. Because there was recently a large influx of new Liberibacter sequence data since probes were designed, we performed an in silico evaluation of all probes and determined they are still valid for detecting all isolates for which sequence data has been reported. Dr. John Hartung of USDA ARS in Beltsville, MD has provided material for testing Xylella and Xanthomonas, and probes for detecting these pathogens are being tested initially on synthetic targets. Targets for Xylella have been selected as well, and design and testing of these additional probes will be initiated shortly. Once these probes show promise on synthetic targets we will begin testing sample material from Dr. Hartung’s lab. Dr. Bill Schneider of USDA ARS Ft. Detrick has agreed to provide nucleic acids from plants infected with CTV and Liberibacter for the validation panels. We will continue to gather and assemble these samples into our validation panels for testing. We focused our early efforts on developing a full detection and species discriminating assay for Liberibacter due to the urgent need to provide tools for management of HLB. The current assay looks promising when synthetic templates are used, and we look forward to testing them on sample material. We have discussed with Luminex their plans to discontinue in January 2012 their non-magnetic beads that we are using to develop these assays. We plan to continue to develop assays using non-magnetic beads, and will then transfer the assays to magnetic beads once they are performing as expected. We have been assured that Luminex or one of its partner companies will be able to provide xTAG non-magnetic beads into the foreseeable future, but we will evaluate the assays on magnetic beads once development is complete. Retail pricing for their MagPix instrument is currently $50K, and their Luminex 100/200 instruments retail for $75K. A higher throughput instrument (FlexMap 3D) capable of up to 500-plex is also available. Additional accomplishments from computational team: 1) Designed overlapping oligos to generate double-stranded targets. The overlapping oligo pairs will be annealed at 55 degree C and extended with a DNA polymerase to generate double-stranded templates. 2) Universal primers were shortened with reduced TM (45 C) to improve sensitivity and reduce background signal in the overall assay. 3) Redesigned MOLigo probes for targeting negative strands of the 4 target pathogens. 4) Identified signatures specific for the virulent strains of Xylella fastidiosa based presence of certain virulence factors such the gene for pili (XF29-31) and fimbriae (X78, XF80) in the virulent strains. We also identified the SNPs that are capable of distinguishing virulent strains from non-virulent strains.
Over the past two seasons, a total of nine experiments were conducted on ‘Fallglo’, ‘Sunburst’, ‘Ruby’ red grapefruit, and navel oranges evaluating the effect of different washing methods on inhibition of degreening and ease of grading out fruit with canker lesions. The results will likely be applicable to other grade or quarantine defects as well. The washing methods tested included: 1) full wash (brush bed + high-pressure wash [HPW]) plus waxing, 2) full wash (brush bed + HPW), 3) HPW only, 4) HPW without the normal brushes, 5) brush wash only, 6) only running over a PVC roller conveyer, or 7) an untreated control. All wash treatments were conducted on commercial wash lines with wash durations of approximately 70 sec. on the brush bed, and 20 sec. on the HPW. Normal brush rotation speed was ~100 rpm. After washing, early season fruit were degreened under simulated commercial conditions (5 ppm ethylene, 85F, 95% RH) and peel color of the fruit was measured almost daily. The fruit were subsequently stored under ambient conditions (~70-75F) and evaluated weekly for the development of decay and physiological disorders. Results showed that with more brushing on the packingline, the greater the inhibition of degreening. Specifically, degreening was most inhibited by fruit receiving the full wash, followed by brush wash or HPW only, and least in fruit that passed under the HPW nozzles without being brushed. Fruit color development was almost completely inhibited if the fruit were also waxed after washing, whereas fruit that were not washed at all, but simply run on a PVC roller conveyor experienced almost no inhibition of degreening. The method of washing had no significant effect on the development of postharvest decay or disorders. These results were observed on all citrus varieties tested. As expected, the method of washing affected how clean the fruit became and how well graders could identify blemishes on the fruit. On a 1 to 9 scale, with higher scores indicating fruit that were easier to grade, fruit receiving the full wash were the easiest to grade, whereas unwashed fruit (the control) were the most difficult. An experiment with 2400 fruit and another utilizing commercial graders evaluating fruit as it was run over a grading line gave similar results. However, the benefits of washing on gradeability was not significant when the fruit were naturally clean at harvest, i.e., early in the season. Such fruit could be adequately graded even before washing. Therefore, if fruit need to be pre-graded for defects of quarantine significance (i.e., citrus canker, citrus black spot, etc.) early in the season when the fruit is relatively clean, very minimal, if any, washing is needed for adequate grading. Grading the fruit without washing would result in minimal effects on degreening. As the season progresses, fruit become dirtier, but the fruit have much better natural color, and pre-washing can be initiated and intensified as needed to allow adequate pre-grading without adversely affecting color.
In order to fulfil Koch’s postulate we wanted to obtain an axenic culture of Ca. Liberibacter asiaticus (LAS) )- Vietnamese strain maintained in greenhouse on Citrus spp. and periwinkles – from primo-cultures of the bacteria in insect cell cultures used as feeder cells. An optimized protocol of bacteria extraction from infected citrus trees or periwinkles has been set up to inoculate various insect cell cultures, in various culture media. Antibiotics were used to select for Gram negative bacteria. Among nine different cell lines tested so far, we selected two Drosophila and one Aedes cell lines that were able to sustain LAS survival and growth the most consistently. About LAS/Drosophila co-cultures: the major challenge remains to decrease the insect cell population. We tested new culture media to contain their growth. Two media were selected for their ability to contain drosophila cells proliferation. However they didn’t give satisfactory primo-cultures. We are now looking into specific molecules that will affect drosophila cells growth without affecting LAS. LAS primocultures in Aedes cells were obtained at a low insect cell concentration. Different co-cultures were continuously grown over several months with successive transfers (transfers periods of~10 days). One of these cultures could be maintained over 10 months. We succeeded in axenising the cultures from the insect cells after 10 successive transfers (the presence of insect cell was checked by light microscopy and by PCR using primers within the cytochrome oxidase gene). A peak of concentration of 1.106 cells/ml was obtained at transfer 16 (as quantified by qPCR). To verify that the LAS culture was really axenic, we checked if LAS was the only bacteria in our cultures. Other bacteria were found in co-culture with LAS (i.e. a Delftia acidovorans or an actinobacteria strain). Enforced antibiotic selection has been applied to get rid of gram positive contaminants and we are looking into a solid medium formulation to isolate LAS clones. To improve LAS fitness in culture, we complemented the primo-cultures with various nutrients based on data from healthy or infected Citrus phloem. In parallel we analyzed metabolic pathways potentially encoded by the released Liberibacter genome sequences to define limiting factors and/or growth inhibitors. We reached a bacteria titer of ~1.107cells/ml in a LAS/Aedes co-culture complemented with proline and ethanolamine. We are also testing other insect cell lines. Two Diaphorina citri cell lines produced by the Keyhani’s lab are now routinely maintained in our lab and we are analyzing their capacity to assist LAS growth. The very next step is to inoculate the cultured bacteria into healthy Citrus in order to verify Koch’s postulates.
‘Candidatus Liberibacter asiaticus’ (CLas), the causal agent of citrus greening also known as Huanglongbing (HLB) is a fastidious, Gram-negative, phloem-limited, alpha-proteobacterium, is one of the most destructive diseases of citrus worldwide. Based on the sequence information number of putative effectors unique to HLB present in prophage related gene clusters and/or pathogenicity islands have been identified and are believed to contain bacterial pathogenicity, multiplication and virulence determinants. The present proposal envisages the expression of these putative effectors in the phloem of citrus, the site of the pathogen’s natural habitat, using citrus tristeza virus (CTV) vector. As outlined in our progress report of January 2011, we have cloned number of putative effectors as additional cistrons between the two coat proteins in the CTV vector under a heterologous, beet yellows virus (BYV) coat protein promoter and agro-inoculated leaves of Nicotiana benthamiana. Partially purified virions of recombinant CTV containing HLB effectors from the infiltrated leaves and /or the systemic leaves of N. benthamiana were used to inoculate citrus plants. Thus, we have inoculated citrus with 7-8 HLB effectors and the remaining are at different stages of transfer to citrus. We have also cloned some of the effectors into the 3′ end of CTV between p23 ORF and the 3′ non-translated region of CTV based on the recent finding of augmented expression of an inserted foreign gene. One of the important effectors or virulence factors is hemolysin, a 50 kDa protein secreted by type I secretion system. The hypothesis is that the hemolysin might interfere with metal ion transportation leading to host metabolic imbalance potentially resulting in disease symptoms. We have submitted an abstract entitled ‘Functional studies of hemolysin (exotoxin) of ‘Candidatus Liberibacter asiaticus’ expression in citrus using Citrus Tristeza Virus vector’ to the 2011 American Phytopathological Society meeting. Additionally, we are also using a much faster approach to screen for virulence genes of HLB. Potato virus x (PVX) vector system has been used extensively to screen for virulence genes in herbaceous hosts and this system can be used much faster to screen for virulence genes of HLB. We have noticed that in the genome of CLas, there is only one copy of flagellin-encoding gene in contrast to four copies of them in Sinorhizobium meliloti. Transient expression of the Las-flagellin using PVX expression vector induces an immunity response with cell death in Nicotiana benthamiana. The flagellin consists of a 22 amino acids near N-terminus (-DRVSSGLRVSDAADNAAYWSIA-), sharing the conserved Flg22 domain. By comparison with the nonfunctional homologue from S. meliloti, three divergent amino acids were identified. This suggests that these three amino acids may be required for Candidatus Liberibacter asiaticus flagellin to induce cell death in tobacco plants. To test potential interaction between Candidatus Liberibacter asiaticus and citrus, 40 ‘M commercial Flg22 (RP19986, GenScript) and FLg22-Las were infiltrated into young tissue of citrus, respectively, and was observed to induce hypersensitive cell death in citrus leaves. An abstract entitled ‘Characterization of the host defense response induced by the flagellin protein of Candidatus Liberibacter asiaticus’ has been submitted detailing these findings to the American Phytopathological Society conference to be held at Hawaii in August 2011. Thus we are making progress in identifying the genes responsible for virulence of HLB and eventually developing strategies towards its control.
‘Candidatus Liberibacter asiaticus’ (CLas), the causal agent of citrus greening, also known as Huanglongbing (HLB) was successfully detected from citrus tissues harboring the pathogen using PCR probes. Thirty gene specific primer-pairs for the t-RNA methyltransferase, elongation factor (EF-TU) proteins, outer membrane protein (OMP), RNA polymerase .-subunit, DNA polymerase region, the rDNA region, and the 23S and 16S ribosomal RNA intergenic regions etc., were developed. Of these 30 primer pairs, 18 primer pairs amplified specific amplicons from the DNA isolated from HLB infected plants and no products were amplified from the healthy plants. We used these primer pairs to prepare non-radioactive Digoxigenin (DIG) labeled DNA probes. Additionally, based on the Ca. L. asiaticus sequence we also identified several putative effector genes encoding hypothetical proteins present as part of pathogenicity island gene clusters that probably are involved in HLB pathogenesis . These effector genes are highly specific to Ca. L. asiaticus and putatively encode proteins which have not been annotated to any specific function. Non-radiactive probes were developed for these amplicons and tested for the detection of HLB infected citrus from the green house. Different tissue parts of citrus trees by tissue blotting on the nylon membrane, and psyllids, and psyllid nymphs as ‘squash blots’ were examined. Results indicated that hybridization observed with probes for EFTU, were much greater than any other probe tested in detecting HLB even in non-symptomatic branches with in an infected tree. Probes developed for putative effector genes were not optimal for detection of HLB since they are present in very low copy numbers in the HLB genome compared to EFTU or the outer membrane proteins. We are in the process of confirming these observations with symptomatic and asymptomatic field samples in new flush during spring and summer months. The results of these observations are being prepared for publication.
The Southern Gardens Diagnostic Laboratory (SGDL) has been in operation since October 2006. To date, the SGDL has received and assayed 168,469 grower samples, and approximately 35,600 additional samples from various sources (research samples, psyllid samples, and samples from Southern Gardens Citrus) for a total of over 204,000 samples since the SGDL began receiving samples. For the period of time covered by this report (July 1, 2010 to March 31, 2011), a total of 25,247 grower samples, 3,337 psyllid samples and 9,588 research and Southern Gardens samples were received and analyzed for a total of 38,172 samples for the period. Based on the number of samples received to date, it is expected that the sample load will approach the budgeted sample load of 45,000 allocated for the period of the funding. Although the total sample volume may not be changing drastically, one thing that is changing, however, is the mix of sample type/sample submitter. Although the number of grower samples is declining slightly, the number of research samples is increasing. The research samples are being submitted by private, state, and federal researchers in support of their research work. The research samples tend to be from large-scale field trials, survey projects, and from chemical/nutritional management projects. We expect this trend to continue. Among the reasons for this are continuity in testing and the fact that the cost of testing is being supported by the industry. Progress has been made in automating a portion of the DNA extraction process. Programming of the liquid handling robot has largely been completed and several hundred samples have been run using the automated extraction process and compared to the standard extraction protocol. Although generally the results from both methods are similar, we have had some problems with consistency using the automated method. Improving the consistency will be the major focus going forward. Plans are being made for the national psyllid ring test. It is hoped that the samples will be sent out during the final quarter of the funding period. It is expected that a large portion of the US labs currently doing HLB testing will participate as well as labs in Mexico and possibly elsewhere.
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