Early detection using cost-effective surveillance techniques is crucial to successfully fighting the spread of HLB. The strategy of early detection of HLB focuses on the analysis of host VOC responses that are triggered early in the infection cycle as part of the plant innate immune responses. Based on previous CRB-funded effort, there is strong evidence that VOC analysis of citrus trees can lead to early detection of the HLB and other citrus diseases. VOC field testing is performed using EZKnowz’ instruments supplied by Applied Nanotech, Inc. (ANI). The EZKnowz’ trace chemical analyzer uses a gas chromatograph (GC) combined with a differential ion mobility spectrometer (DMS). Our effort in this program is the following: ‘ ANI delivered the 1st non-radioactive analyzer to UC Davis in Nov. 2013. They are optimizing the protocol for sampling and analysis before putting the tool into the CRF at UC Davis. Extensive dialog between ANI and UC Davis has lead to improvements in the next deliverable due Sept. 2014. ‘ ANI has improved the handheld sampler, adding battery power for over 8 hours of runtime, added airflow and air volume control. We are working to improve cleaning and conditioning of VOC traps. We have produced about 30 demountable traps to date. Initial data using the handheld sampler was collected in South Texas and compared to our standard instruments, indicating where the improvements are needed to bring the new instrument up to expected standards. ‘ Researchers at UC Davis are developing a graphical user interface for the Yuma that will be used for analysis at point of operation. Expected Outcomes and/or functional product/solution The potential value of this early-detection solution on the citrus industry is tremendous. By ‘flagging’ infected trees at the asymptomatic stage, eradication would be both more effective, and kept to the minimum necessary, since it would take place well before other trees become infected and enter the latent period. This would interrupt the deadly infestation cycle at the source, significantly reduce the heavy costs of losing trees and citrus produce for a period of three to five years and cut down the costs of planting new trees.
Early detection using cost-effective surveillance techniques is crucial to successfully fighting the spread of HLB. The strategy of early detection of HLB focuses on the analysis of host VOC responses that are triggered early in the infection cycle as part of the plant innate immune responses. Based on previous CRB-funded effort, there is strong evidence that VOC analysis of citrus trees can lead to early detection of the HLB and other citrus diseases. VOC field testing is performed using EZKnowz’ instruments supplied by Applied Nanotech, Inc. (ANI). The EZKnowz’ trace chemical analyzer uses a gas chromatograph (GC) combined with a differential ion mobility spectrometer (DMS). Our effort in this program is the following: ‘ ANI delivered the 1st non-radioactive analyzer to UC Davis in Nov. 2013. They are optimizing the protocol for sampling and analysis before putting the tool into the CRF at UC Davis. Extensive dialog between ANI and UC Davis has lead to improvements in the next deliverable due Sept. 2014. ‘ ANI has improved the handheld sampler, adding battery power for over 8 hours of runtime, added airflow and air volume control. We are working to improve cleaning and conditioning of VOC traps. We have produced about 30 demountable traps to date. Initial data using the handheld sampler was collected in South Texas and compared to our standard instruments, indicating where the improvements are needed to bring the new instrument up to expected standards. ‘ Researchers at UC Davis are developing a graphical user interface for the Yuma that will be used for analysis at point of operation. Expected Outcomes and/or functional product/solution The potential value of this early-detection solution on the citrus industry is tremendous. By ‘flagging’ infected trees at the asymptomatic stage, eradication would be both more effective, and kept to the minimum necessary, since it would take place well before other trees become infected and enter the latent period. This would interrupt the deadly infestation cycle at the source, significantly reduce the heavy costs of losing trees and citrus produce for a period of three to five years and cut down the costs of planting new trees.
Down regulation of phloem specific Callose synthase and phloem protein genes of citrus host of HLB by the demonstrated silencing ability of CTV are the objectives of this research. Citrus plants (Citrus macrophylla) potentially expressing he dsRNA for Callose synthase 7 and Phloem Proteins B8 and B14 are in the green house. We are going to analyze them for dsRNA expression soon by checking for positive ELISA for CTV. As soon as they are positive for CTV, we will multiply these Citrus macrophylla plants and inoculate with HLB by grafting and/or exposure to psyllids positive for Candidatus Liberibacter asiaticus. Previously we have shown that psyllids acquire dsRNA from citrus plants while feeding. We will use this strategy to simultaneously target both vector and host genes for silencing. Towards this end we have ligated in-tandem truncated genes for psyllid Awd and troponin, and citrus endogenous callose and phloem protein genes into CTV vector and will inoculate citrus. The goal is to silence the vector and the host genes simultaneously and mitigate the effects of HLB.
We have made progress with the scFv library made with the earlier grant from CRDF. 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. We have produced purified scFv at concentrations in the mg/ml range. Differences are observed among different scFv clones. Results from SDS-PAGE gels are consistent with post translational folding being problematic for some scFv as has been reported in the literature. The tissue print assays continue on nitrocellulose membranes. Color development is observed in the vascular cylinder (phloem) of HLB infected petioles but not in comparable petioles from healthy trees. In some tissue prints, color development is observed in discrete spots outside of the phloem cylinder. Similar results are obtained with all 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. These results have been attributed to non specific cross reactions of the commercial monoclonal antibody directed at the 6X His TAG. The His tag will not be very useful for tissue printing. In continuing work, we have recently made tissue prints with a modified technique, using ‘Super block’, a commercial product used in Northern and Southern blotting on nitrocellulose membranes and detection with a monoclonal antibody directed at the FLAG epitope on the scFv. This protocol produces remarkably sharper tissue prints with dramatically reduced background, and color tightly focused as a ring in the phloem cylinder of HLB infected, but not healthy petioles. These results vary by manufacturer of the monoclonal anti-FLAG antibody conjugate. We have identified the best supplier. We have had unexpected but very interesting results with the negative controls in these assays: When the secondary anti-FLAG monoclonal is used alone, without any scFv, it produces color tightly focused as a ring in the phloem cylinder of HLB but not healthy citrus. Interestingly, the anti-FLAG monoclonal will give similar results with tissue prints from trees infected with CTV and other plant viruses. Thus the anti-FLAG monoclonal alone detects the presence of disease in mature fully expanded leaves but not in young flush, and may be useful as a general diagnostic. We have also prepared rabbit polyclonal antibodies against the major outer membrane protein (OmpA) and detected them with alkaline phosphatase labeled goat anti rabbit monoclonal antibody. The polyclonal antibodies produce distinct spots of color corresponding to individual phloem cells infected with ‘Ca. Liberibacter asiaticus’. This is a useful assay for ‘Ca. Liberibacter asiaticus’, and we are using it to describe the distribution of ‘Ca. Liberibacter asiaticus’ in infected citrus trees, fruit and seed. We have continued work with these antibodies applied to tissue printing of plant samples during the current reporting period. Two manuscripts on the original scFv antibodies have been submitted and two manuscripts on the tissue printing assays are in preparation. The latter manuscripts compare the scFv-based and polyclonal antibody based tissue prints with FLAG based detection and detailed high resolution anatomical and spatial distribution of CaLas within infected citrus plants.
One of the primary goals of this work is to identify a small molecule treatment that can be used to activate the phage lytic cycle genes encoded by Las prophage, thus bringing about the death of Las bacteria carrying these prophage. All Las bacteria examined to date have been found to carry prophages in their genomes. In periwinkles, but not in citrus, lytic phage particles are formed and can be visualized. We previously reported that relative mRNA expression levels of prophage late genes SC2-gp095 (“peroxidase”), SC2-gp100 (“glutathione peroxidase”) and particularly SC1-gp110 (‘holin’) were much higher in periwinkle than in citrus. These results were confirmed and now extended, with identification and cloning of several late gene promoter region fragments. To functionally confirm lytic cycle genes, we cloned and expressed the annotated prophage holin (SC1_gp110) and endolysin (SC1_gp035), as well as a gene outside of the prophage region annotated as an endolysin, CLIBASIA_04790. Functional expression of SC1_gp110 in pET27B in Escherichia coli revealed bacteriostatic activity characteristic of holins. Expression of SC1_gp035, but not CLIBASIA_04790, in pET27B led to lysis of E. coli, confirming SC1_gp035 as an active endolysin. Four potential SC1_gp110 promoter regions were fused with a lacZ reporter; none exhibited LacZ activity in E. coli. One of these promoter regions was also fused with a uidA reporter and exhibited strong GUS activity in Liberibacter crescens, indicating strong promoter activity in L. crescens. Surprisingly, the GUS activity was suppressed by 1:100 diluted, crude, cell-free leaf extracts from citrus, eggplant, tobacco and periwinkle in a dose-dependent manner. Notably, inhibition of GUS activity was not observed following heat inactivation of the leaf extracts. Additional experiments are in progress to determine if a similar effect is noted using psyllid extracts, as well as attempts to identify the specific mechanism of transcriptional repression/activation. The fact that a promoter from a holin gene with demonstrated anti-bacterial activity is functionally active in L. crescens grown in culture and suppressible with plant cell extracts has implications for culturing L. asiaticus (Las). It is likely that this same holin gene is activated in Las when attempts are made to grow Las in culture. Such activation could be expected to halt Las bacterial growth, regardless of whether complete phage particles are formed or not. Further, the fact that plant leaf exacts can suppress this promoter activity indicates that the addition of plant cell extracts to the culture medium might allow additional growth of Las cells and increased overall titers in culture.
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. 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. I have written twice to a laboratory in Germany which has published results with a suitable vector but have had no reply. We are also optimizing the cloning strategies that will be used to move already selected scFv into transgenic plants. We have obtained the vector, pUSHRL-26, to be used for plant transformation of the scFv constructs from Ed Stover at Fort Pierce and the plasmid has been purified. We have purchased the restriction enzymes and designed primers to be used for PCR to amplify the cloned scFv encoding inserts from vector pKM19. The cloned inserts will be sequenced to confirm that they are correct and then cloned into the transformation vector. The scFv have been modified by the addition of a four amino acid leader sequence (KDEL) and both Sma I and Spe I cloning sites. The KDEL sequence is expected to stabilize the concentration of scFv in phloem cells by facilitating proper folding of the protein in the microtubules and thereby protecting the ScFv from proteolytic digestion. Eleven scFv inserts have been sequenced to be sure that the expected sequences are correct, and five ScFv sequences have been successfully cloned into the recombinant vector pUSHRL-26 for transformation of citrus rootstocks. These inserts include three different scFv that bind to the protein InvA and two that bind to the protein TolC. The protein InvA is produced by CaLas and secreted into the host to prevent the infected host cells from entering into apoptosis, and the protein TolC targeted by the scFv, is in the external membrane and is essential for the removal of antimicrobial substances produced by the plant. The vector is designed to direct expression of the scFv into the phloem cells of citrus, where CaLas grows, and the vector encoding the scFv genes is being introduced into rootstock varieties by Agrobacterium mediated transformation.
The present project investigates timeline of the HLB infection in citrus, as reflected by emission of volatile organic compounds (VOCs) for advanced disease diagnostics. We have completed the preliminary arrangements at the Containment Research Facility (CRF) for the longitudinal study. We have developed experimental plans for plants testing that include testing schedule, randomization of plant position in green house, and work flow (sequence of testing steps with different methods). Supplies and parts necessary for the study have been arranged. An overhaul of instrumentation has been conducted for the study. We have conducted all the necessary personnel hire and training and established schedule tables for the rotation at the facility to ensure complete use of available time at the facility. We have conducted multiple sets of preliminary experiments to develop optimized protocols for the VOC testing using gas chromatography/differential mobility spectrometry (GC/DMS). The current protocol utilizes PTFE bags to envelop the tested branch and purge with dry air. The first sets of data using the developed protocols have been collected. 200 samples for GC/DMS have been collected so far. The data analysis revealed satisfactory data quality for the first sample collected, but poor data quality for consecutive replicate samples. Further improvements to the protocol are currently being considered based on the collected body of data. The improvements include changes in purge with the makeup air and different sample intake time. In parallel, we have conducted development of experimental protocols for mass spectrometry analysis. Specifically, we have conducted sampling using solid-phase microextraction (SPME) sorbents and stir bar sorptive extraction (SBSE). We have conducted sampling of trees in the laboratory conditions, in the greenhouse and at the CRF facility for the gas chromatography/mass spectrometry (GC/MS) analysis. Both the sampling protocols and the GC/MS protocols have been optimized. The collection of volatiles was conducted using active sampling device developed under previous CRF-funded effort. We have discovered that SBSE sampling resulted in large variability of the data. Currently we are investigating the ways to mitigate such variability by modifying sampling procedure. We have received a future-generation GC/DMS device for in-field use from the industry partner, Applied Nanotech Inc. and have been adopting the unit for the greenhouse testing. We have conducted, along with ANI, troubleshooting and modifications of the device. We have also compiled the set of guidelines/recommendations from the end user perspective for further improvements of the design and interface. Glenn Sellar from NASA JPL has visited UC Davis. We have installed the rig for the hyperspectral measurements of citrus canopy at the CRF facility and conducted training of all UC Davis personnel involved in sample collection to assure that we will be able to collect hyperspectral measurements independently as a part of multi-method rotation. Current data analysis is focused on ensuring consistent data are being collected and therefore is focused on unsupervised analysis to observe variations within the collected data. We have presented the VOC study aspect of the collaborative project of early HLB detection at the 2014 Citrus Showcase in Visalia, CA.
The present project investigates timeline of the HLB infection in citrus, as reflected by emission of volatile organic compounds (VOCs) for advanced disease diagnostics. We have completed the preliminary arrangements at the Containment Research Facility (CRF) for the longitudinal study. We have developed experimental plans for plants testing that include testing schedule, randomization of plant position in green house, and work flow (sequence of testing steps with different methods). Supplies and parts necessary for the study have been arranged. An overhaul of instrumentation has been conducted for the study. We have conducted all the necessary personnel hire and training and established schedule tables for the rotation at the facility to ensure complete use of available time at the facility. We have conducted multiple sets of preliminary experiments to develop optimized protocols for the VOC testing using gas chromatography/differential mobility spectrometry (GC/DMS). The current protocol utilizes PTFE bags to envelop the tested branch and purge with dry air. The first sets of data using the developed protocols have been collected. 200 samples for GC/DMS have been collected so far. The data analysis revealed satisfactory data quality for the first sample collected, but poor data quality for consecutive replicate samples. Further improvements to the protocol are currently being considered based on the collected body of data. The improvements include changes in purge with the makeup air and different sample intake time. In parallel, we have conducted development of experimental protocols for mass spectrometry analysis. Specifically, we have conducted sampling using solid-phase microextraction (SPME) sorbents and stir bar sorptive extraction (SBSE). We have conducted sampling of trees in the laboratory conditions, in the greenhouse and at the CRF facility for the gas chromatography/mass spectrometry (GC/MS) analysis. Both the sampling protocols and the GC/MS protocols have been optimized. The collection of volatiles was conducted using active sampling device developed under previous CRF-funded effort. We have discovered that SBSE sampling resulted in large variability of the data. Currently we are investigating the ways to mitigate such variability by modifying sampling procedure. We have received a future-generation GC/DMS device for in-field use from the industry partner, Applied Nanotech Inc. and have been adopting the unit for the greenhouse testing. We have conducted, along with ANI, troubleshooting and modifications of the device. We have also compiled the set of guidelines/recommendations from the end user perspective for further improvements of the design and interface. Glenn Sellar from NASA JPL has visited UC Davis. We have installed the rig for the hyperspectral measurements of citrus canopy at the CRF facility and conducted training of all UC Davis personnel involved in sample collection to assure that we will be able to collect hyperspectral measurements independently as a part of multi-method rotation. Current data analysis is focused on ensuring consistent data are being collected and therefore is focused on unsupervised analysis to observe variations within the collected data. We have presented the VOC study aspect of the collaborative project of early HLB detection at the 2014 Citrus Showcase in Visalia, CA.
The ultimate utility of airborne detection and mapping of HLB infection depends to some degree on the time lag between HLB infection and the development of a detectable reflectance signature. In Year 2 (FY 2014) JPL will participate in the longitudinal study proposed to CRB by UC Davis. UC Davis will provide citrus specimens, some of which will be inoculated with HLB in the UC Davis contained research facility. JPL will lead an effort to measure the reflectance spectral of these specimens with weekly frequency as the disease progresses. The following milestones have been achieved: * Design of a test station incorporating a reflectance spectrometer with spectral performance similar to AVIRIS-ng, an appropriate light source (simulating solar illumination), and calibration standards. * Fabrication of the test station. * Development of the experiment procedure. * Testing of the apparatus at JPL. * Delivery and installation of the test station at UC Davis. * Training of UC Davis personnel in the experiment procedure.
This project provides the infrastructure to support eight CRB-funded projects investigating detection of the putative causal agent of huanglongbing (HLB), Candidatus Liberibacter asiaticus (CLas) in citrus prior to symptom development. The 8 projects are using a systems approach that includes transcriptomics, proteomics, and metabolomics to develop early detection methodology. The researchers are all using the same cohort of plants, so that results of the various methods are comparable. The plants used to initiate the studies were grown and grafted using plant material certified to be free of pathogens by the Citrus Clonal Protection Program. The scions Washington navel, Tango mandarin, and Lisbon lemon were grafted onto Carrizo rootstock to produce 48 Washington navel plants, 42 Tango mandarin plants, and 54 Lisbon lemon plants that were delivered to the Contained Research Facility (CRF) on July 31, 2013. These scion/rootstock combinations are common in California citrus production. The plants were placed in a greenhouse at the CRF to allow the newly grafted buds to grow. Of these plants, 113 plants were available for the first round of experiments and the remaining plants were used to establish plants from which scion material could be harvested to continue to produce more experimental plants. Carrizo seed was obtained and the rootstock for the next cohort of experimental plants (approximately 175 plants ) is being grown in the CRF. The CLas (California Hacienda Heights strain) was obtained from USDA, ARS ‘ Beltsville as infected plant material on August 23, 2013, and grafted into Volkameriana citrus to initiate a culture at the CRF on August 27, 2013, using leaf-patch grafts and T-bud grafts. Once these plants became infected, buds from these plants were used to infect two Washington navels, two Tango mandarins, and two Lisbon lemons as CLas source plants for the experiments that began March 3, 2013. Samples of leaf tissue were taken from the source plants and sent to the Citrus Research Board Laboratory in Riverside for CLas testing. The source plants were PCR-positive for CLas and had CT values between 24.0 ‘ 30.17; and the control (not infected) source plants had CT values greater than 40. Establishment of a CLas-positive Asian citrus psyllid (ACP) colony began in January 2014, and ACP adults and nymphs from this colony will be tested for the presence of CLas in early April. This colony will be used to infect citrus plants in experiments in the future. This project provides addition support by providing the conference call line, and compiling minutes of the research group monthly meeting. The minutes and other pertinent materials for the group are archived on a secure website.
Present proposal involves the down regulation of phloem specific Callose synthase and phloem protein genes involved in phloem plugging in citrus infected with Huanglongbing (HLB) pathogen. There is substantial increase in the accumulation of the transcripts and proteins corresponding to these in infected plants. Down-regulation of these over-expressed genes responsible for phloem-plugging potentially would negate the disease severity. Towards this goal we have isolated and cloned Calloase-7 (CsCal-7) and Phloem proteins B-8 and B-14 (Cs PP2) from sweet orange total RNA. A truncated version of these genes have been cloned into the CTV silencing vector. We have also engineered these genes individually, in tandem and individually in the CTV vector to evaluate their potential silencing ability of endogenous genes of CsCal-7 and CsPP2. We agro-inoculated these constructs into Nicotiana benthamiana and isolated CTV virions carrying truncated genes of CsCal-7 and CsPP2 and have inoculated citrus plants for subsequent evaluation.
The insect-spread bacterial infection known as citrus greening or Huanglongbing (HLB) is a very destructive citrus disease and has caused massive losses in Florida’s citrus industry. Early, easy, and less expensive HLB detection based on particular symptoms, such as starch accumulation in citrus leaves, would increase the chance of preventing the disease from being spread and causing more damage. The ability of narrow-band imaging and polarizing filters in detecting starch accumulation in symptomatic citrus leaf was evaluated in this project. Two custom-made HLB detection systems were developed for this purpose. In the first detection system, leaf samples were illuminated with polarized light using narrow-band high-power LEDs, and the reflectance was measured by two monochrome cameras. Two polarizing filters were mounted in perpendicular directions in front of the cameras so that each camera acquired an image with reflected light in only one direction (parallel or perpendicular to the illumination polarization). Four groups of textural features were extracted and ranked using several feature selection methods. Seven classifiers were evaluated, and the best classifiers and sets of features were selected based on their accuracies. Leaf samples were collected from the ‘Hamlin’ and ‘Valencia’ varieties of citrus. Overall average accuracies of 93.1% and 89.6% in HLB detection were obtained for the ‘Hamlin’ and ‘Valencia’ varieties, respectively. The second detection system was designed as a vision sensor for real-time HLB detection purpose which could work under field conditions. The sensor was first tested and calibrated in a simulated in-field condition in a laboratory. Then it was examined under a real situation in a citrus grove. Two datasets for the in-lab experiment and one dataset for the in-field experiment were collected which contained HLB negative, HLB positive, and zinc deficient samples. The HLB statuses of all the samples were confirmed by a qrt-PCR test. The HLB detection accuracies ranging from 95.5% to 97% were achieved for the in-lab and in-field experiments. Compared to the results of the first image acquisition system, the HLB detection accuracy also increased significantly in both zinc and non-zinc deficient classes. The results of this study showed that the starch accumulation in HLB-symptomatic leaves rotated the polarization planar of light, and this property can be effectively used in a fast and inexpensive HLB detection system.
One of the primary goals of this work is to identify a small molecule treatment that can be used to activate the phage lytic cycle genes encoded by Las prophage, thus bringing about the death of Las bacteria carrying these prophage. All Las bacteria examined to date have been found to carry prophages in their genomes. In periwinkles, but not in citrus, lytic phage particles are formed and can be visualized. We previously reported that relative mRNA expression levels of prophage late genes SC2-gp095 (“peroxidase”), SC2-gp100 (“glutathione peroxidase”) and SC1-gp110 (‘holin’) were much higher in periwinkle than in citrus. These results were confirmed and now extended, with identification and cloning of several late gene promoter region fragments. These fragments are currently being screened for activity using a promoter probe vector in Liberibacter crescens with a beta-glucuronidase (GUS) reporter. A secondary goal of this work is to test the hypothesis that the prophage genomes found in Las contribute to Las pathogenicity to citrus and that this is the reason that these prophage are conserved in the Las genome. A corollary hypothesis is that any Las strains that are missing these genes should be less pathogenic than normal Las. Las strain B430, which is capable of colonizing citrus but does not cause HLB disease symptoms (Bill Schneider, personal communication), was closely examined for phage gene content. Conventional PCR was carried out with primer sets based on the psy62 genome and with additional Las genomic DNA samples used as positive controls. The following phage genes were not detected in strain B430: SC1_gp025 (tail fiber), SC1_gp035 (endolysin), SC2_gp095 (peroxidase), SC2_gp100 (glutathione peroxidase), SC1_gp110 (holin), and SC1_gp165 (DNA primase). Four additional phage regions normally found in both SC1 and SC2 phage were absent from Las strain B430. Chromosomal gene fragments flanking both sides of the phage insertion site of Las strain psy62 were cloned and sequenced and found to be 100% identical to Las strain psy62. These results may indicate that the phage region carries lysogenic conversion genes that contribute to HLB symptoms in citrus, and that a method of curing the phage might help control HLB. In an effort to functionally confirm predicted lytic cycle genes from the prophage region, two putative lytic cycle genes (SC1_gp035, annotated as a hypothetical protein, possible endolysin, and SC1_gp110, annotated as a hypothetical protein, possible holin; Zhang et al. 2011) were cloned separately into expression vector pET27b and transformed into Escherichia coli strain BL21(DE3). Heterologous expression of both the predicted holin and the predicted endolysin genes revealed strong bacteriolytic activity of both clones that was entirely consistent with the annotation. Attempts to artificially inoculate marked BT-1 strains into tobacco, citrus and periwinkle are currently in progress.
The non-PCR based 10-plex QuantiGene assay for RNA pathogens of citrus such as Citrus tristeza virus, Citrus leprosis virus, and citrus viroids has been developed and optimized during the first year of this project. During this reporting period the RNA assay was evaluated with field samples and the use of crude and nucleic acid extracts. We have confirmed that crude extracts from citrus samples provide comparable results to nucleic acid extracts. This was achieved by using the Affymetrix Inc proprietary lysis buffer ‘Homogenizing Solution’ and proteinase K. Because of the costly nature of the proprietary buffer, we are currently experimenting with less expensive alternatives. This approach was also evaluated by members of the Citrus Pest Detection Program of the Central California Tristeza Eradication Agency (CCTEA). The detection capacity of the 3-plex QuantiGene assay for DNA pathogens of citrus was improved during this reporting period. The previous 3-plex assay provided detection and quantification for only two citrus pathogens: C. Liberibacter asiaticus (associated with Huanglongbing) and X. axonopodis pv. citri (Citrus canker) and one citrus gene as internal control (Nad5). Three new DNA targets, one for C. Phytoplasma aurantifola (Witches’-broom disease of citrus) and two for Xylella fastidiosa subsp. pauca (Citrus variegated chlorosis), have been added to the QuantiGene assay increasing the capacity of the assay to reliably detect four distinct citrus bacterial pathogens. QuantiGene probes were developed for Spiroplasma citri (Stubborn) and C. Liberibacter asiaticus but detection still remains inconsistent. It is possible that the uneven distribution of the pathogens in the citrus hosts is affecting the sensitivity and Median Fluorescence Intensity (MFI) values of the developed methods. More experimentation is under way to confirm the sample collecting and extraction methods as well as the efficacy of the designed probes in order to improve MFI values. Preliminary data from the use of a sonicator for sample preparation revealed that higher amplitude and longer duration resulted in greater MFI values. More specifically, citrus samples sonicated at 80% amplitude for 10 or 20 seconds (with 10 second intervals) generated higher MFI values than samples sonicated at lesser amplitude and duration as well as the non-sonicated controls. These results were promising however, further testing is required to refine and standardize the sonication treatment for citrus pathogens. Moving forward we are targeting a 10-plex assay for DNA pathogens similarly to RNA. The final DNA assay will have one probe for Nad5 (internal control) and the pathogens of citrus canker, and witches’-broom; two probes for the pathogens of stubborn and citrus variegated chlorosis; and three probes for the pathogen associated with Huanglongbing. In addition, more samples will be used for validation of the DNA and RNA panels. Finally, we will continue working on the technology transfer of the 10-plex RNA assay to the CCTEA and initiate the transfer to the Citrus Research Board Jerry Dimitman Laboratory.
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 working with a group in the Math Department of UF to develop a model of spread of HLB in new planting of citrus. A manuscript is being prepared reporting these results. We are using this system to screen RNAi constructs and different peptides against psyllids. Preliminary results are encouraging. We also are attempting to adapt the system into a method to screen peptides against HLB more quickly. We are using this rapid screen to determine whether a peptide can inhibit Las multiplication within 60 days instead of approximately one year. However, this rapid screen measures resistance, but not tolerance. We are still screening using infected psyllids to inoculate plants, but this information allows us to know which plants are inoculated with Las and is greatly improving those assays also. This work is continuing as described above.
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