In the present study, performed in collaboration with Dr. Nelson Wulff at Fundecitrus, we have now closed the circular genome of Ca. L. americanus (Lam) strain ‘S’o Paulo’ isolated from infected periwinkle from Brazil. A draft circular genomic DNA sequence of Ca. L. americanus (Lam) strain ‘S’o Paulo’ isolated from infected periwinkle (Vinca) in Brazil has been completed. The genome size is 1,195,167. The circular genome has been extensively confirmed by PCR. All weak assembly areas, predicted truncated or incomplete proteins and pseudogenes are currently being verified by PCR. The average GC content of Lam is 31.12%. So far, 1,071 genes have been called, with 1,015 encoding predicted proteins, and 56 encoding rRNA genes and 45 encoding tRNAs. Of the protein coding genes, 839 (78.34%) have a predicted function, while 176 (16.43%) have no predicted function. The overall gene organization and structure of the Lam genome is more similar to Lso than to Las. There are 845 genes common to Lam, Lso and Las, 26 genes found in Lam and Lso but not Las, and only 6 genes common to Lam and Las but not found in Lso. The latter included a DNA uptake competence gene. As with both Liberibacter asiaticus (Las) and L. solanacearum (Lso), two prophage were found. As with Las, two circular forms were confirmed in Lam, with Lam-SC1 being 39,941 bp and Lam-SC2 being 16,398 bp in size. SC1 (and not SC2 as in Las) appears to replicate as an integrative plasmid prophage. This stable plasmid lysogen can be useful for lysogenic conversion (host range and pathogenicity enhancing), and indeed lysogenic conversion genes are found on these plasmid prophage—specificially peroxidases and adhesins. 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 a “suicide program” or lytic cycle genes). In the stable “lysogenic” state, the phage keeps its lytic genes dormant by its use of gene repressors. To ensure its own survival, however, phage can also carry anti-repressors that sense host cell stress and then activate the phage “lytic” cycle genes, thereby forming phage particles, killing the host cell and liberating infectious phage. Some phage hijack available host cell repressors, such as LexA, to maintain a stable lysogenic state by repressing phage antirepressors. Some phage also hijack available host cell anti-repressors to assist sensing of host stress, including host heat shock and other SOS responses. Externally applied chemicals, as well as heat shocks have been used in other bacterial systems to activate the SOS response and derepress various phage lytic cycle genes. Lam is heat sensitive and carries a unique cold shock gene; Las is missing this gene, is not heat sensitive, and encodes two likely lytic cycle repressors and four anti-repressors. Lam encodes only one putative repressor and a rearranged anti-repressor. Las and Lam both are missing LexA, found in Lso. Experiments designed to exploit these genomics findings and determine what stresses can activate the Las lytic response are currently underway.
The purpose of this project is to develop a robust ELISA-based detection method for the citrus stubborn disease. We used a comparative proteomic approach using mass spectrometry (MS) from which the proteins secreted from the causal agent, Spiroplasma citri, to citrus phloem were identified. The secreted protein profiles from S. citri cell cultures with or without the induction of citrus phloem extracts were determined by MS and several potential secreted proteins were identified. To facilitate with the analysis on the MS data, we obtained an additional funding from an UC Discovery Grant to fully sequence the genome of S. citri strain S616 using next-generation Illumina sequencing. Using these sequencing data, we were able to match most of the peptides from the MS analysis to the S. citri genome sequences and obtained the complete gene sequences. The deduced amino acid sequences of many of the identified proteins contain potential N-terminal secretion signals indicating that they are secreted in a Sec-dependent manner. Through these approaches, two suitable proteins (CAK99824 and CAK98563) were chosen for further detection marker development. CAK99824 and CAK98563 are being evaluated for their suitability as detection markers according to the following criteria: 1) high abundance and dispersal in phloem; 2) wide distribution in representative S. citri isolates. By comparing the genome sequences of S. citri strain S616 from our own sequencing and the available genome sequence of another S. citri strain, GII3, we confirmed that the selected protein candidates are present in both genomes with almost identical DNA sequences indicating that they are conserved genes in S. citri. In addition, none of these proteins have close homologs in other organisms according to a search in the Genbank database, suggesting that they are also unique for S. citri. These characteristics make them excellent detection markers for citrus stubborn disease. Polyclonal antibodies have been generated for CAK99824 and CAK98563, as well as for spiralin. Spiralin is a highly abundant membrane protein and will be used as a control for the presence of S. citri. Using Western blots, the antibodies were tested on S. citri cultures grown in the presence of citrus phloem extracts. CAK99824 and CAK98563 were present in both the cell pellets and the cell-free supernatants. Furthermore, both proteins were induced by the phloem extracts, which is evident by increased amounts in the supernatant of the induced samples. The antibodies are now being tested on samples from trees grafted with S. citri infected buds and from field trees confirmed to be infected with spiroplasma. Preliminary results indicate that the CAK98563 antibody is capable of detecting spiroplasma within the leaf tissue of the graft tree and within the phloem tissues dissected from the leaf, branch and fruit of the field trees. It has not been possible to detect CAK99824 using any citrus tissue. Further work is necessary to optimize the methods of extracting proteins from plant tissues and on the detection of the spiroplasma proteins.
As concern regarding the spread of citrus greening or Huanglongbing (HLB) and other agricultural diseases has increased, the need for more accessible, easily used and rapid point-of-use diagnostic tests has become more critical. Molecular diagnostic tests (i.e. DNA and RNA-based assays) for citrus diseases potentially offer the most sensitive and specific means of detecting viral and bacterial infections, often capable of detecting the presence of a pathogenic agent prior to the overt exhibition of visually detectable symptoms. Unfortunately, existing molecular diagnostics rely on instrumentation dependent laboratory manipulations requiring highly trained technicians for execution. These constraints often limit the accessibility and utility of molecular methods due to the infrastructure, costs and complexities associated with their use. The reliance of molecular techniques on laboratory instrumentation and personnel must ultimately be eliminated to broaden the utility of such tests and enable their execution under field conditions. In past work, MTI developed a highly simplified means of purifying nucleic acids (NAs) from plant tissue and contaminating pathogens without human intervention or machine-based automation. The resulting technology enables the isolation of amplification ready NA in less than 15 minutes using a completely disposable device. To make use of this rapid and simple NA isolation technology to enable field deployable molecular diagnostics, we have focused our efforts in this period on the development of extremely low cost yet precise temperature control systems to support robust amplification of nucleic acids in a disposable test device. To support the thermal requirements of NA amplification reaction strategies within the context of a self-contained and disposable diagnostic device, we developed low cost microheaters capable of supporting rapid PCR (and reverse transcriptase PCR for RNA targets) as well as isothermal amplification methods. The design employs an inexpensive microcontroller to modulate temperatures in multiple low thermal mass reaction chambers interfaced to the microheater elements. The use of multiple independently controlled reaction chambers allows parallel amplification of multiple targets thus reducing the burden inherent to multiplexing a single amplification reaction while retaining the capacity to amplify multiple pathogen NA signatures in a single device. Detection of amplification reaction products can be accomplished in minutes using lateral flow microarrays, a rapid and inexpensive yet sensitive detection technology developed by the PI while at Los Alamos National Laboratory. The combination of instrumentation independent sample preparation technology with low cost battery operated microheater mediated amplification and lateral flow microarray-based detection offers a means to overcome the major hurdles facing field deployable molecular testing. We are now working to incorporate our innovative sample preparation, amplification and detection subsystems into a single low cost disposable device that is both easy to use and capable of providing molecular diagnostic data within one hour of sample introduction.
Multiple Botryosphaeria species causing ‘dothiorella’ gummosis in citrus: Dothiorella gregaria was long believed to be the pathogen causing Dothiorella gummosis but recent data showed that this may not be valid. Our objectives were to (a) determine the Botryosphaeria species causing branch canker and dieback on citrus and their geographical distribution in California. In 2010, cankered branches were collected in six citrus growing counties in California, namely ‘ Fresno, Riverside, San Diego, San Luis Obispo, Tulare, and Ventura. The combination of morphology with three molecular markers (ITS, Beta Tubulin, and Elongation Factor) helped to distinguish among closely related isolates. Hence, 18 isolates were fully identified and tested indicating that multiple species of Botryospharia, including B.australis, B. iberica, B. lutea, B. species, B. parva, B. stevensii, B. viticola, and Neofusiccocum mediterraneum, were causing symptoms on citrus that were previously believed to be caused by Dothiorella gregaria (B. ribis). These multiple species were widely distributed within counties. To our knowledge, this is the first comprehensive study of Botryosphaeria gummosis on citrus. The alliance between Fusarium solani and other factors in citrus dry root rot: The main pathogen causing dry root rot (DRR) is Fusarium solani but its interactions with Phytophthora spp. and other biotic and abiotic factors is an unclear dynamic in DRR. We are studying the distribution, seasonal occurrence, and survival of F. solani and its interaction with P. nicotianae, P. citrophthora, other biotic (other Fusarium spp), and abiotic (soil) factors on citrus in California. Microorganism isolations were made from infected tree samples and soil were collected and analyzed across the state. About 60 Fusarium isolates and 18 Phytophthora isolates have been identified across the state. Detailed identification was done using molecular methods with 3 primers (ITS, BT, and TEF). Preliminary results indicate that in addition to F.solani and Phytophthora spp., F. proliferatum, and F. oxysporum are major organisms associated with DRR. Pathogenicity tests are currently being conducted for these specific isolates to be sure of their roles in DRR. Results will help towards better management of DRR. Understanding foamy bark rot of Fukumoto navel: Fukumoto navel orange has many desirable attributes but ‘foamy bark rot’ is causing tree decline. The objective of this study was to isolate fungal and bacterial pathogens associated with foamy bark rot. Several bacterial and fungal isolates have been collected from infected trees in Fresno, Kern, and Tulare counties and identified using molecular methods. 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. At a UC-Riverside greenhouse, Carrizo and Volkameriana rootstocks are almost ready for pathogenicity tests with the identified microorganisms. These experiments will fulfill the principle of Koch’s postulates and help to determine the pathogenicity and aggressiveness of the isolated organisms on Fukumoto navel. Findings will provide a baseline for the management of foamy bark rot.
Multiple Botryosphaeria species causing ‘dothiorella’ gummosis in citrus: Dothiorella gregaria was long believed to be the pathogen causing Dothiorella gummosis but recent data showed that this may not be valid. Our objectives were to (a) determine the Botryosphaeria species causing branch canker and dieback on citrus and their geographical distribution in California. In 2010, cankered branches were collected in six citrus growing counties in California, namely ‘ Fresno, Riverside, San Diego, San Luis Obispo, Tulare, and Ventura. The combination of morphology with three molecular markers (ITS, Beta Tubulin, and Elongation Factor) helped to distinguish among closely related isolates. Hence, 18 isolates were fully identified and tested indicating that multiple species of Botryospharia, including B.australis, B. iberica, B. lutea, B. species, B. parva, B. stevensii, B. viticola, and Neofusiccocum mediterraneum, were causing symptoms on citrus that were previously believed to be caused by Dothiorella gregaria (B. ribis). These multiple species were widely distributed within counties. To our knowledge, this is the first comprehensive study of Botryosphaeria gummosis on citrus. The alliance between Fusarium solani and other factors in citrus dry root rot: The main pathogen causing dry root rot (DRR) is Fusarium solani but its interactions with Phytophthora spp. and other biotic and abiotic factors is an unclear dynamic in DRR. We are studying the distribution, seasonal occurrence, and survival of F. solani and its interaction with P. nicotianae, P. citrophthora, other biotic (other Fusarium spp), and abiotic (soil) factors on citrus in California. Microorganism isolations were made from infected tree samples and soil were collected and analyzed across the state. About 60 Fusarium isolates and 18 Phytophthora isolates have been identified across the state. Detailed identification was done using molecular methods with 3 primers (ITS, BT, and TEF). Preliminary results indicate that in addition to F.solani and Phytophthora spp., F. proliferatum, and F. oxysporum are major organisms associated with DRR. Pathogenicity tests are currently being conducted for these specific isolates to be sure of their roles in DRR. Results will help towards better management of DRR. Understanding foamy bark rot of Fukumoto navel: Fukumoto navel orange has many desirable attributes but ‘foamy bark rot’ is causing tree decline. The objective of this study was to isolate fungal and bacterial pathogens associated with foamy bark rot. Several bacterial and fungal isolates have been collected from infected trees in Fresno, Kern, and Tulare counties and identified using molecular methods. 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. At a UC-Riverside greenhouse, Carrizo and Volkameriana rootstocks are almost ready for pathogenicity tests with the identified microorganisms. These experiments will fulfill the principle of Koch’s postulates and help to determine the pathogenicity and aggressiveness of the isolated organisms on Fukumoto navel. Findings will provide a baseline for the management of foamy bark rot.
We are investigating volatile organic compound (VOC) based detection diagnostics with two major citrus diseases: HLB and CTV. We performed simultaneous biomarker extraction using cutting-edge analytical chemistry approaches with off-gassed chemicals in field studies to evaluate disease detection based on these gaseous chemical species using a portable differential mobility spectrometer (DMS) system. The main idea is to combine data of individual compounds to develop an integrated panel of markers to obtain a more robust means of early detection for these diseases. The CTV sampling was done at the Lincove Station, CA (summer 2010) and Pauma Valley, CA (Feb 2011). DMS measurements of healthy, mild and severely CTV infected Valencia citrus was performed. In addition to the mobile VOC analysis, we collected leaf samples from all three groups for later real time PCR gene expression analysis, (15 leafs per tree, 6 trees per class, 3 class in total). We will analyze for genes that seem to be strong host biomarkers of early infections. We also collected whole-leaf metabolite extracts that will be subjected GC-TOF/MS analysis to investigate the secondary metabolism of host plant in response to CTV infection. Our initial results indicate a set of 18 VOC compounds (with distinct chemical identifications) that appear associated with milder forms of the CTV disease. We found that co-infection with stubborn also produced a distinct VOC metabolite profile, and we can still identify a clear ‘CTV’ signal even in the presence of the confounding co-infection factor. We have acquired additional VOC samples from severely CTV infected group only, in order to investigate if the same volatiles are up-regulated in these phenotypically distinct trees. Future work would include a return visit to Puma Valley to increase the number of DMS measurements, and a return visit to Lindcove (if new infections are discovered). Additional sampling in other regions of CA will help to quantify how geographical variation may (or may not) affect the detection of disease specific biomarkers. We are also investigating VOC diagnostics for HLB detection. Prior to in-field sampling, we completed preliminary studies including sample collection protocol optimization from greenhouse citrus on the UC Davis campus. The in-field testing was carried out with collaborators at Lake Alfred, FL throughout this year (2010-2011) to account for weather and seasonal (trees blossoming, fruit harvest, etc.) variations. Currently, 3 trips were made in November 2010, December 2010 and January 2011. Sample collection was carried out in parallel using polymer-based absorption devices (SPME and Twister), as well as two portable GC/DMS analysis units. Approximately, 80 samples were collected up to date using SPME, and analyzed using GC/MS. Approximately 100 individual GC/DMS runs were recorded for the same pair-matched trees used for the SPME sample collection. In addition, approximately 160 samples were taken using Twister devices (December 2010), and the GC/MS analysis of these samples is on-going. For all of the above experiments, trees of one varietal (Hamlin) were included. Half of all samples were taken for HLB-infected and presumed healthy trees selected by scouts at the CREC facility in Lake Alfred FL. A number of trees with symptoms varying from relatively mild to severe were included in the study. For the next stage, we will include PCR-positive symptomatic trees, PCR-positive asymptomatic trees and PCR-negative presumably asymptomatic trees. This will allow us to assess feasibility of the DMS-based chemical sensing for early-stage asymptomatic disease detection. The collected data are being analyzed and a mathematical model is currently being developed for the differentiation of HLB-sick and healthy trees based on our data.
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. Characterization probes for Liberibacter have also been designed, and testing of all Liberibacter probes will begin this week using synthetic DNA targets. Because there has been a large influx of new Liberibacter sequence data since probes were designed, we have initiated an in silico evaluation of all probes to determine if they will detect and characterize the new isolates. We met with Dr. John Hartung of USDA ARS in Beltsville, MD and Dr. Bill Schneider of USDA ARS Ft. Detrick to discuss samples for the validation panels. Both have agreed to send purified nucleic acids from infected plants, insect vectors, and healthy plants. We will assemble these samples into our validation panels for testing once the assays appear to be working properly on synthetic targets. We recently learned that Luminex plans to discontinue in January 2012 the type of non-magnetic beads that we are using to develop these assays. Because we plan to transfer the technology to service labs, we need to either locate another source of non-magnetic beads, or move our development efforts to the magnetic beads. We have contacted people at Luminex and Radix, a Luminex partner about the availability of tagged, non-magnetic beads past January 2012. We will proceed with development on the type of bead that will be available for the foreseeable future. The advantage of the magnetic beads is that they perform as well or better than the non-magnetic beads, and are the only beads compatible with Luminex’s new, less expensive machine. This machine only has a 50-plex capacity, but costs around $35K, making it feasible to deploy our citrus assays to additional labs at lower startup costs. Our concern is that the magnetic beads will cost more, which will increase the cost of each assay. Still, it is anticipated that the total assay cost will still be a fraction of the cost per marker as compared to RT-PCR assays currently in use. We will continue to pursue options for tagged, non-magnetic beads. Additionally, we have moved the oligo design software to a new server, which is online and accessible to us and the public at http://moligodesigner.lanl.gov . We are in the process of automating the multiplex function of the design tool, which will facilitate probe selection and assay design. This will make the iterative re-design of probes and assays should we encounter some probes that do not perform according to our specifications.
The project main objective is to select antibody fragments type single chain fragment variable (scFv) against Huonglongbing (HLB), Citrus Verigated Chlorsis (CVC) and Citrus Stubborn Disease the latter as a proof-of-concept, and the development of immunoassays that incorporate these scFvs. We have requested Stubborn samples from many collaborators, but they are not available yet as a pure culture. However, during this time, we have constructed one scFv library from chicken spleens that were immunized with bacterial proteins isolated in Fort Detrick from cultures of Candatus Libribacter asiaticus (CaLas). The immunization protocol and deliver 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 performed a Phage Display selection of specific scFvs against Xyllela OMPs. Three highly reactive fragment antibodies, from 12 reactive clones, were further secreted in culture in a TOP10 vector, and HPLC-purified for further validation with ELISA and western blots. All three antibodies strongly recognized the expected 42 KDa MopB protein. Fluorescence microscopy analysis of scFvs in Xyllela cells is under investigation. Interestingly, the same antibodies could recognize the related protein in a related pathogen Xanthomonas juglandis OMPs giving confidence that these antibodies should detect the mopB in CVC. The detection of mopB in Xylella were comparable with a polyclonal antibody against Xylella MopB developed by Dr. Bruening. Additionally, we have also started working with fluorescence spectroscopy based detection. Besides training and calibration of equipment, we have established the parameters to work with specific range of wavelengths for our reactions with quantum dots (q-dots). In order to work under field conditions, we decide to use q-dots with the excitation at 480nm and emission in the spectrum of 500-900 nm. Protocols for antibodies conjugation to q-dots are under development. Tests of antibody stability are also under way. In conclusion, we have established a very simple conventional ELISA with the antibodies against bacteria OMPs, and the assay parameters will be translated to fluorescence spectroscopy in the near future to improve sensitivity of the test.
The recently published Ca. Liberibacter asiaticus (Las) strain psy62 genome, derived from a psyllid, revealed a prophage-like region of DNA in the genome, but phage have not been associated with Las to date. In the present study, shotgun sequencing and a fosmid DNA library of curated Las strain UF506, originally derived from citrus symptomatic for HLB, revealed two largely homologous, circular phage genomes, SC1 and SC2. SC2 encoded putative adhesin and peroxidase genes that had not previously been identified in Las and which may be involved in lysogenic conversion. SC2 also appeared to lack lytic cycle genes and replicated as a prophage excision plasmid, in addition to being found integrated in tandem with SC1 in the UF506 chromosome. By contrast, SC1 carried suspected lytic cycle genes and was found in nonintegrated, lytic cycle forms only in planta. Phage particles associated with Las were found in the phloem of infected periwinkles by transmission electron microscopy. The average phage head size was ~21.30 nm and the average neck length was ~ 5.03 nm; based on the relatively short neck length, the observed phage particles belong to the Podoviridae. In psyllids, both SC1 and SC2 were found only as prophage. Similar phage DNA sequences (corresponding to both SC1 and SC2 and including putative lysogenic conversion genes) were found in Ca. L. americanus (Lam) strain ‘S’o Paulo’ isolated from infected citrus in Brazil in collaboration with Dr. Nelson Wulff at Fundecitrus. This Lam genome is about one year from completion. It currently has a total of 1,203,790 bps on 355 contigs, with the avg. length of the contigs at 3,390 bps. In planta, including citrus, both SC1 and SC2 can enter a phage-programmed cell death cycle called the lytic replication cycle. Once the lytic cycle is engaged, cell lysis is assured by a variety of SC1-encoded genes. Surprisingly, a potential colicin IA and potential cognate colicin immunity protein were identified on SC1 and SC2, respectively. Colicins are anti-bacterial proteins that kill any competitor cells lacking the colicin immunity protein. The fact that the SC2 replicon carries a putative immunity protein would render Las cells with SC2 resistant to any colicins produced by SC1, and also would prioritize SC2 preservation in a given Las strain. Assuming infectious capability of both phages, SC2 might infect a Liberibacter strain and confer a selective advantage by virtue of its lysogenic conversion genes (confering major pathogenic advantages), while SC1, by itself, is predicted to be lethal to any strain it can infect (unless it was previously or simultaneously infected with SC2). The hypothesized lethality of SC1 would confer a selective advantage to strains that shed infectious SC1 particles by virtue of its colicin gene alone, which could eliminate potential competition by similar bacteria in the phloem cell or in the psyllid alimentary canal. This mechanism might also ensure essentially monoculture infections of citrus by only a single Las strain (with SC1 and SC2) at a given time. Over time in a grove setting, this could lead to survival of only the fittest of multiple Liberibacter strains in a case of competition by two or more populations. In Brazil, Lam used to be the predominant form of predominant form of Liberibacter causing HLB, but has been replaced by Las as the predominant species. It is possible that the Las colicin kills Lam. Further investigation of Liberibacter colicins and activation of the phage lytic cycle seems warranted as potential control strategies for Liberibacters generally.
The overall objectives of this project were: 1) Development of rapid regeneration of HLB-affected periwinkle by optimizing growth conditions for the diseased cuttings; 2) Screening and evaluating anti-microbial molecules for suppression of Liberibacter in periwinkle. In the past three months, the research was mainly focused on optimizing a citrus grafting system for screening chemical compounds in HLB control. 1. Developing an optimized citrus grafting system for screening chemical compounds in HLB control The HLB-affected scions from different phenotypic types (yellow shoot and Blotchy mottle) and different cultivars (lemon and grape fruit) were grafted onto the Las-free citrus rootstocks (sour orange and grapefruit). The results showed that more than 60% of HLB-affected lemon scions survived and 70% of the inoculated rootstocks were infected at 6 months after grafting with high Las bacterial titers of HLB-affected scions. If the Las bacterial titers in the grafted scions were low (less than 1000), all grafted scions survived and grew better, and less than 30% rootstocks were infected with low bacterial numbers. The results also showed that plant growth regulators (PGRs) increased the survival rates of the grafted scions, but had no effects on the infection rates. 2. Evaluation of chemical compounds against Las bacteria using the optimized grafting systems HLB-affected lemon branches were sampled and soaked in the PS (penicillin and streptomycin), MDL (metronidazole), Kasumin and DBNPA (2, 2-dibromo-3-nitrilopropionamide) solutions overnight, cut into 3 buds for scions (S1, S2 and S3) and then grafted onto the Las-free grapefruit (R). More than 80% of the scions treated with PS survived and grew better than the untreated controls, and no rootstocks and scions tested positive by qPCR for Las bacteria. When the HLB-affected scions were treated with MDL, more than 60% of the scions survived, but more than 95% rootstocks and grown scions were infected by Las bacteria. DBNPA-treatment partly inhibited the Las bacteria with 40% of infection rates in the grafted scions and rootstocks. Only 4.4% HLB-affected scions survived when treated with kasumin at 1mg/ml, showing the phytotoxic effects. 3. Continue to monitor the effect of antibiotics on HLB-affected citrus trees in the field In June of 2010, the screened antibiotic combinations (PS and KO) have been applied to the HLB-affected citrus in the field by trunk-injection. The primarily results showed that the antibiotic combinations decreased the Las bacteria titer in the treated citrus as compared to controls. In the past three months, the trunk injection of antibiotic combination was continued in Oct and Dec. The Las bacterial number was quantified by qPCR. Two abstracts were submitted in 2nd International HLB meeting, one manuscript has been accepted in phyopathology.
Fluorescence and visible-near infrared spectrometry is currently under investigation for detecting HLB. The Multiplex’ fluorescence data were collected from 30 healthy, nutrient-deficient, and HLB infected trees (Hamlin and Valencia) under field and laboratory conditions from CREC groves. In addition, fluorescence and visible-near infrared data from about 498, 53, and 549 corresponding citrus leaves were collected from different varieties of citrus trees (Southern Garden groves) under laboratory conditions. The PCR analysis of the leaf samples from CREC groves has been performed. Preliminary analysis on the fluorescence data (21 features) indicated that yellow fluorescence (green excitation) and red fluorescence (UV) could indicate stressed leaf condition; while blue-to-red fluorescence (UV) could indicate HLB infection under laboratory conditions. Similarly, fluorescence excitation ratios (red & UV) and yellow fluorescence (red) could indicate stress- and HLB detection under field condition, respectively. Further analysis is ongoing to evaluate the fluorescence features that can be used as a predictor for stressed-condition and HLB infection in citrus leaves. Aerial images (color,multispectral and hyperspectral cameras) were acquired from a grove near Clewiston in Hendry County and CREC groves on Dec. 3, 2010. Reflectance data from healthy and infected canopies (with RTK-GPS recorded coordinates) were measured. The pre-processing and analysis of these aerial images are ongoing. Analysis procedure and results for previous set of data (2007, 2009) have been reported to a journal article, ‘Citrus greening disease detection using aerial hyperspectral and multispectral imaging techniques’, submitted to the Journal of Applied Remote Sensing. In this paper, based on the calibration set data, a spectral library was created for healthy and infected canopies, and some of the HLB detection algorithms that were tested using ENVI hyperspectral imaging software are spectral angle mapping (SAM), linear spectral unmixing, mixture tuned matched filtering, and spectral feature fitting. Comparing the ground-truth data, SAM with multispectral data yielded a detection accuracy of 87%. Currently, support vector machine and neural networks in addition to the previously-tested algorithms are being evaluated to validate the results with the new data set. Based on the completed preliminary studies on data acquisition and sample collection protocols optimization, field data from Hamlin trees (CREC groves) were collected in Nov. and Dec. 2010 for the estimation of citrus volatiles. The sample collection was carried out using the SPME (for GC/MS analysis), Twister devices and two portable gas chromatography-differential mobility spectrometry (GC/DMS) units. Approximately 80, 100 and 160 samples were collected using SPME, GC/DMS, and Twister devices with about equal number of samples from HLB-infected and healthy trees. Preliminary data analysis on the volatiles data collected using the GC/DMS units using principal component analysis (PCA) indicated that the first two PCs presented a clear separation between HLB and healthy samples. Using the leave-one-out validation strategy, the principal component regression yielded a classification accuracy of 83% on this sample set. These results show a very promising direction of using portable GC/DMS unit for field detection. At CREC, leaf volatiles from an HLB-infected tree leaf (new leaves) were collected at times 1, 4, 8, and 16 h and temperatures range from 47-75’F with a 1 cm tri-phase SPME fiber. Volatiles collected were very localized and tend to vary depending on leaves and branches sampled. The whole day sampling appears to collect more volatiles than overnight sampling, presumably due to higher daytime temperatures. We are continuing to sample positional differences in greening trees and comparing them to control trees to determine branch to branch and tree to tree variation.
At least a couple of most promising silica nanogel based copper (Cu) formulations (which demonstrated superior biocidal efficacy in laboratory based testing) will be systemically evaluated in the field in the upcoming season. The Co-PI (Jim Graham at the CREC, Lake Alfred, FL)and his team will conduct this trial at the Fort Pierce, FL facility. Santra group at the UCF have synthesized the materials for this upcoming trial. Material characterization is currently on-going to check product quality. A number of commercial Cu products including Kocide will be used as positive control whereas pure silica nanogel (without Cu) will be used as negative control. Laboratory data successfully demonstrated improved biocidal efficacy against X. Alfalfae (indicative of improved Cu bioavailability) and strong adherence properties to citrus leaf surface (indicative of improved longevity). Preliminary field trail data collected so far indicate that nanogel based Cu formulations are quite effective against citrus canker. Details will be available in future reports.
The HLB disease causing ‘Candidatus Liberibacter asiaticus’ (CLas) pathogen was successfully detected using PCR probes. The digoxigenin (DIG) based DNA-PCR probes showed promising results compared to riboprobes (RNA probes). Though highly specific, RNA probes did not improve hybridization of the tissue bolts and occasionally non-specific hybridization was observed in healthy tissue blots. Of all the DNA-PCR probes, EF ‘Tu probes were much promising in their specificity to detect the ‘CLas’ pathogen (both in HLB infected citrus plants as well as HLB positive psyllids). These EF ‘Tu probes were also effective in detecting CLas’ pathogen even in the non-symptomatic tissues and in different developmental stages of ‘CLas’ pathogen carrying psyllids. The sensitivity of EF ‘Tu probes against detecting HLB infected citrus plants directly from the field was also tested. The EF ‘Tu DIG labeled tissue blot hybridization was shown to be positive for the field sampled HLB diseased plants and we did not find any hybridization signal for the healthy plants. Tissue imprints, once on the membrane can be stored for later use, can be shipped easily and generally are stable. The results of these observations were presented as a poster presentation in the 2nd International HLB Conference at Orlando. Salient feature of this presentation included results of hybridization of a branch containing symptomatic and asymptomatic leaves which showed strong hybridization signal in the leaf midrib, veins, petiole and the main branch stem. These results confirmed hybridization observed with detached plant parts. We are in the process of doing tissue-blots of very young leaves and actively growing shoot tips which have been shown to contain high HLB concetrations. Real time PCR (qPCR) evaluations of different probes employed in this study were performed and results suggested that the hybridization signals by the probes in the tissue imprints were directly correlated with the copy number in CLas genome.
The research proposal involves expression of putative effectors of ‘Candidatus Liberibacter asiaticus’ (CLas) in citrus using Citrus tristeza virus in an effort to understand if the putative effectors contain determinants for pathogenicity to citrus. CLas, is a fastidious, Gram-negative, phloem-limited, alpha-proteobacterium is the causative agent of Huanglongbing (HLB) also known as citrus greening, and is one of the most destructive diseases of citrus worldwide. It is believed that many pathogenicity islands and prophage related gene clusters observed in the CLas genome contain many hypothetical proteins unique to CLas which may function as putative bacterial effectors required for pathogen virulence, multiplication etc., and insect transmission determinants. Towards this end, as a first step we have amplified at least 20-25 hypothetical protein genes by polymerase chain reaction (PCR) using specific primers designed based on the CLas sequence, and the total nucleic acids isolated from HLB infected citrus as template. Amplification was not observed with the template from healthy citrus which indicated the specificity of the putative effector amplicons. The genes encoding putative effector proteins were cloned into citrus tristeza virus vector, behind heterologous beet yellows virus CP sub-genomic RNA controller element engineered between the CPm and CP genes of the CTV vector in a binary vector. By the use of citrus tristeza virus (CTV) vector, we could express putative effectors of the CLas directly inside the phloem of citrus. Initially, the CTV vector with engineered putative HLB effectors was used to transform Agrobacterium (EHA 105) and agro-infiltrate Nicotiana benthamiana seedlings for transient expression in inoculated leaves and subsequent systemic spread. The expectation is that the expression of the putative effectors of CLas in N. benthamiana would induce a phenotypic response. We are using N. benthamiana as an intermediate host since the direct agroinfiltration of citrus has not been possible. The CTV virions containing HLB effectors from N. benthamiana will be purified on sucrose step gradient and will be used to inoculate citrus plants by bark-flap inoculation. The resulting systemic spread and expression of the putative effectors throughout citrus trees will enable us to understand the role of the putative effectors in disease induction. We are at a stage to transfer the CTV virions from the N. benthamiana seedlings to citrus. Recently, early infection of N. benthamiana seedling (6- 8 weeks as opposed to 10- 12 weeks) with CTV vector was observed (Choaa and Dawson, personal communication) when a foreign was engineered under CTV CP promoter between the p23 ORF and the 3′ non-translated region, and we plan to use this vector in future experiments in addition to the expression of HLB effectors in citrus using as originally proposed.
To date, 400 different media formulations have been tested for their ability to support growth of Candidatus Liberibacter asiaticus. Inoculum was prepared by washing the bacteria from biofilms formed on the surface of alimentary canals of the psyllid, Diaphorina citri, which were reared on citrus plants infected with huanglongbing. Two days ago, a four to six fold increase in the number of the bacterium was detected in one medium formulation (BM397) five days after it was inoculated. Several other medium formulations exhibited lesser degrees of increased numbers. The culture in medium BM397 was transferred to fresh medium and will be monitored for further growth. New cultures in medium BM397 and the other media that had promising results are being established to determine if the these results are repeatable. Medium BM 397 has an extremely complex formulation that will need to be optimized if it continues to support growth of Liberibacter asiaticus. In summary, our approach to culturing Liberibacter asiaticus appears to be working working.
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