This is a project to continue one of the most fruitful leads that accidentally resulted from our previously funded work. We have found that citrus becomes a source of Huanglongbing (HLB) inoculum for spreading the disease to other plants much earlier than previously thought. The working hypothesis is that the female psyllid finds an area of new flush to lay her eggs. As she is laying eggs, she probes the phloem to feed and transfers Candidatus Liberibacter asiaticus (Las) to the tree. As the eggs develop into nymphs, Las begins to multiply in that localized area of the plant, where the new nymphs then feed and acquire Las. Thus, infection of only a micro area of flush tissue where the nymphs develop is sufficient for the first generation of psyllids to become infected and to be vectors to spread the disease to other trees. Thus, the time-period after a tree becomes infested by infected psyllids until it is a donor for other trees could be as short as 15-30 days or less. The limitation is actually the time for the second generation of psyllids to develop. We are continuing experiments to find ways to quickly detect psyllid reproduction as a method to detect early infections. We are developing this system into a method to screen RNAi constructs against psyllids. Preliminary results are encouraging. We also are attempting to adapt the system into a method to screen peptides against HLB more quickly.
The objective of this project is 1) to complete the Las genome sequence and conduct comparative genomics studies on the Liberibacter species; 2) to explore the potential role of the microbial community and genetic diversity of Las bacteria in HLB development; 3) to confirm if Las bacteria are seed-transmissible and their role in HLB development. A complete circular genome of Candidatus Liberibacter asiaticus (Las) was obtained using a metagenomics approach and published in MPMI 22:1011-1020, 2009. In collaboration with Dr. Hong Lin at the USDA-ARS in Parlier, California, we have obtained and published a complete genome sequence of Ca. L. solanacearum in PLoS ONE 6(4): e1913, 2011. All BAC clones of Las were sequenced, and sequence analyses revealed a potential mechanism of genome reduction. Based on the variations within the Las prophages, FP1 (CP001677.5) and FP2 (JF773396.1), twelve (A to H) different populations (genotypes) have been identified. Type A and B are located in FP1 and FP2, respectively. Typing revealed A, B and C as the three most abundant groups in libraries from psyllid, citrus and periwinkle, although psyllid contained much more type A sequence than the plant hosts. Variations of Las populations existed not only in different varieties, but also in different individuals of the same variety. We have characterized the ATP translocase from Las and proved its function using a heterologous E. coli system (J. Bacteriol. 192:834-840, 2010). We have also revealed only one of the two putative znu operons is responsible for zinc uptake. The results were published in PLoS ONE 7(5): e37340. doi:10.1371/journal.pone.0037340,2012. In addition, we have demonstrated Las encoded a functional flagellin characteristic of a Pathogen-Associated Molecular Patterns (PAMP). The results were published in PLoS ONE 7(9): e46447. doi:10.1371/journal.pone.0046447, 2012. Seed transmission of Las was tested in grapefruit, sweet orange, sour orange, lemon and trifoliate orange. A very low titer of Las was detected from the embryos and seedlings using nested PCR and real-time PCR. Most, if not all the seedlings did not show typical HLB symptoms and contained a relatively low Las bacterial titer for HLB, even in the three to four year old seedlings. The results indicated that the seed-transmitted Las could not cause typical HLB disease by themselves, which suggested “Detection of Las was NOT necessarily equal to the presence of “HLB disease” in plants.” Psyllid transmission study on the Las-positive seedlings was performed. High percentage of psyllids acquired Las bacterium but did not have the same bacterial levels as those from HLB-affected citrus plants. However, it is first time that one seed transmitted plant was confirmed by PCR using several Las-specific primer sets. Graft transmission of the cutting from this HLB plant confirmed this seed-transmitted HLB. Of the 50,000+ available operational taxonomic units (OTUs, “Species”) on PhyloChip’ G3, 7,028 known OTUs were present in citrus leaf midribs. These OTUs were from 58 phyla, and five, Proteobacteria (44.1%), Firmicutes (23.5%), Actinobacteria (12.4%), Bacteroidetes (6.6%) and Cyanobacteria (3.2%), contained 100 or more OTUs. The results illustrate that the low Las level was seen as a both a seasonal fluctuation, part of the bacterial population dynamics, and as a response to the antibiotic treatments, The results are going to be published in BMC Microbiology. Using Las-specific molecular markers, we are able to differentiate the Las populations with different pathogenicity, and differentiate psyllid-transmissible and non-transmissble populations.
The objective of this project was to characterize the hypI gene and determine its effects on insect transmission and/or virulence in host plants. Transient expression in alternative expression systems, RT-qPCR, and other molecular tools were used to elucidate the function of the hypI gene of Las. HypI has been redesignated as LasAI. A second prophage gene, hypII (now LasAII), was identified and both genes were characterized in global Las isolates. This revealed sequence conservation within the individual repeats but an extensive variation regarding repeat numbers, their rearrangement, and the sequences outside of repeat region. Detailed results have been published in Applied Environmental Microbiology, 2011: 6663-6673. We developed real-time PCR protocols using SYBR Green 1 (LJ900fr) and TaqMan’ (LJ900fpr) with primers and probe targeting nearly identical tandem repeats of 100 bp within the two Las prophage genes. Because of the higher copy number of the tandem repeats per bacterial genome, these methods significantly improved the detection capacity for the HLB bacterium, especially for the detection of extreme low titer. The results were published in Molecular and Cellular probes, doi:10.1016/j.mcp.2011.12.001. Working with Mesa Tech International, Inc., we have also developed a rapid and simple HLB detection kit (tree-side detection kit) for field-testing, which requires less than 30 minutes. we have characterized two novel autotransporter proteins of ‘Candidatus Liberibacter asiaticus’ (Las), and redesignated them as LasAI and LasAII in lieu of the previous names HyvI and HyvII. Bioinformatic analyses revealed that LasAI and LasAII share the structural features of an autotransporter family containing large repeats of a passenger domain and a unique C-terminal translocator domain. When fused to the GFP gene and expressed in E. coli, the LasAI C-terminus and the full length LasAII were localized to the bacterial poles, similar to other members of autotransporter family. Despite the absence of the signal peptide, LasAI was found to localize at the cell surface by immuno-dot blot using a monoclonal antibody against the partial LasAI protein. Its surface localization was also confirmed by the removal of the LasAI antigen using a proteinase K treatment of the intact bacterial cells. When co-inoculated with a P19 gene silencing suppressor and transiently expressed in tobacco leaves, the GFP-LasAI translocator targeted to the mitochondria. This is the first report that Las encodes novel autotransporters that target to mitochondria. Using the LasAI-specific marker, we were able to associate better insect transmission and pathogenicity with wild-type LasAI, but variations with LasAI mutants. Using transient expression of the LasAI in tobacco leaf, the dissociation of the cell wall and cytoplasm in affected cells was observed under electron-microscope. Further characterization of the LasAI and LasAII are underway so as to have a better understanding of the pathogenesis of this intracellular ‘energy parasitic’ bacterium, and to control HLB by blocking the functions of the LasAI and LasAII.
We are developing a new method for rapid and efficient inoculation of plants with HLB based on a Pulse Micro Dose Injection System (PMDIS). In our preliminary experiments we have had some success in inoculation of Periwinkle plants using this technique, which suggested a feasibility of further adaptation of the PMDIS system for HLB inoculations. Our goal now is to optimize the protocol for PMDIS-based inoculation of citrus hosts with HLB. We are conducting experiments in order to 1) identify what types of tissue within an infected citrus plant can serve as a good resource of the HLB bacteria for preparation of the inoculum by comparing extracts from stems, leaves and seed coats as inoculum sources; 2) examine whether HLB-infected psyllids can be utilized for preparation of the inoculum suspension; 3) optimize the composition of the extraction buffer used for preparation of the bacterial suspension and the extraction conditions, so they would support high efficiency of the PMDIS-mediated transmission of the pathogen; 4) optimize the parameters of injection. We are also evaluating how age of receptor plants, types of citrus varieties used as HLB bacterium donors as well as for plants being inoculated, types of flushes being inoculated affect efficiency of inoculation. Several sets of plants have been already injected using PMDIS. Those are being maintained in the greenhouse and monitored for the disease development. Some successful infections of citrus plants using PMDIS were achieved, however infection rates were less than those seen upon graft-inoculation of plants with HLB-containing tissue. Currently we are working on improvement of PMDIS-based inoculation procedure. During this funding period we added another direction. In collaboration with Dr. Dean Gabriel we are testing our inoculation approach using the culture of Liberibacter crescens, a bacterium that belongs to the same genus as HLB causing agent, Candidatus Liberibacter asiaticus. We believe that this would allow us to better make necessary improvements to the inoculation conditions and to assess whether citrus could be a host for this bacteria. Successful inoculation of Liberibacter crescens into citrus would suggest that this bacterium could be used as a model in various experiments to better understand the biology of HLB agent.
Citrus huanglongbing (HLB) is associated with three species of Candidatus Liberibacter: Ca. Liberibacter asiaticus (Las), Ca. L. americanus (Lam), and Ca. L. africanus. The majority of the testing in Florida is focused on detection of Las as this is the only bacterium known to be associated with HLB in Florida to date, although recently Lam has been reported in Texas detected only from psyllids, not plants. In October 2012, we conducted a wide survey of citrus and citrus relatives in the landscape and in collections in Central and South Florida. The DNA extracts were forwarded to Riverside and analyzed. Based on these results, 12 specific plants that had Ca. Liberibacter-like infections were identified and recollected in March 2013. These specific isolates were graft inoculated into four plants each in an isolated greenhouse at Ft. Pierce. One of these isolates originated from pummelo and has undergone treatment to eliminate Las. These plants will be tested in late May ‘ early June to determine if the HLB-like pathogen has been established, and the isolates that have become established will be utilized to establish trials to establish ‘cross protection’ trials. The DNA extracts collected from samples collected in March 2013 are being analyzed by the macro array qPCR.
Goals 1, 2 and 3 were recently completed. These were: 1) cloning of previously identified early/late gene promoter regions fused with lacZ as a reporter; 2) cloning and expression of both Las and the Lam repressors and determining responsiveness of the lacZ reporter constructs, and 3) cloning and expression of all 4 Las and the one possible Lam anti-repressors, and determining responsiveness of the reporter and clones from Milestone 2. None of the genes annotated as repressors or anti-repressors in the Las or Lam genomes functioned in these assays as repressors or as activators. The primary goal was Goal 4: development of a chemical assay for Las-responsive SOS. In order to accomplish this, we developed and tested an entirely different assay approach; instead of using artificial promoter and reporter constructs in E. coli, we used semi-quantitative, real time PCR (qRT-PCR) assays to determine levels of expression of the Las SC1 late genes in citrus and periwinkle. The qRT-PCR methodology turned out to be a surprisingly successful approach, and obviated the need for E. coli reporter constructs. In citrus, relative expression levels of SC2-gp095 (peroxidase), SC2-gp100 (glutathione peroxidase), SC1-gp110 (‘holin’) although nominally placed in the ‘late’ gene regions of the phage, were much higher than expression levels of SC1-gp025 (‘tail fiber’). These results, initially performed using cyber green, were repeated and confirmed in citrus using gene-specific probes, and indicated that the promoter regions P0, P1 and P2 that we examined as predicted ‘late gene’ promoters, are likely not late gene promoters, nor repressed. These results were expanded to include the late gene SC1-gp035 (“endolysin”), heat treatment of Las infected citrus, and Las-infected periwinkle. In citrus, expression was monitored before and after heat treatment at 42’C for two days, a level demonstrated to cure plants of Las infection. The relative expression of three of the three lytic cycle genes examined, SC1_gp025, SC1_gp110 and SC2_gp095, were much more highly expressed in periwinkle than citrus. However, heat treatment of citrus leaves failed to increase expression of these genes, indicating that lytic cycle induction of these prophage is not tied into a presumed heat stress response of Las in citrus. In addition to use of qRT-PCR in Las-infected citrus and periwinkle, we began to develop Liberibacter crescens as a model Liberibacter species. To determine if L. crescens might be tractable for functional genomics studies, the minimum inhibitory concentrations (MICs) of several antibiotics commonly used for plasmid selection was determined. BT-1 was found to be quite sensitive to: chloramphenicol, < 4 mg/L; gentamycin, < 1 mg/L; kanamycin, <2.5 mg/L, and tetracycline, < 0.3 mg/L. Both the repW (on pUFR071) and Bordatella replicons (pUFJ05, derived from pBBR1MCS5) were transformed by electroporation at high frequencies into BT-1. Stability of pUFR071 was evaluated; this plasmid was >95% stable, without selection, when grown in BM7 medium for over 20 generations. pUFR071 was extracted from BT-1, retransformed into E. coli and appeared from restriction analysis to be unchanged. Attempts to artificially inoculate marked strains into tobacco, citrus and periwinkle are currently in progress, as well as attempts to reconstruct the replicating form of phage SC2 in L. crescens, for use in chemical treatment assays.
The objective of this project is to improve the bioavailability of copper loaded silica nanogel (CuSiNG) material, as well as increase retention of the spray formulation to plant surface. Our studies have shown that both Cu(I) and Cu(II) co-exist in CuSiNG material. The presence of mixed-valence Cu has been correlated to improved antimicrobial efficacy of CuSiNG material over Kocide 3000 and Cu-sulfate controls based on both laboratory based test results and field trials. To further confirm the role of mixed-valence Cu in CuSiNG material, we have synthesized a series of CuSiNG nanoformulations with varying Cu(I)/Cu(II) ratios. Our laboratory test results have shown that a higher ratio of Cu(I)/Cu(II) improved antimicrobial efficacy in comparison to our previous formulations. Characterization of the material was done using HRTEM, XPS, UV-VIS and DLS. The two most promising CuSiNG mixed-valence formulations with increased Cu(I) amount over Cu(II) were prepared and delivered for 2013 field trial. We have also conducted an adherence property study of CuSiNG material using CuInS2/ZnS red-emitting quantum dots (Qdots) as planned and discussed in our previous reports. The adherence study was carried out using Hamlin Orange leaves and red-emitting Qdots. Red-emitting Qdots were quite visible when exposed to a hand-held 366 nm multi-band UV excitation source and easily seen after being applied to the plant surface. After washing and comparing with controls, more Qdots remained on the plant surface that was treated with CuSiNG material. This study suggests that the CuSiNG material adheres well to the citrus leaf surface. In future reports, we will include phytotoxicity results, antimicrobial studies from mixed-valence CuSiNG materials along with SEM material characterization data.
The following effectors of HLB were engineered into Citrus Tristeza Virus (CTV) vector have been in Citrus macrophylla and are being screened for their response to HLB pressure by Mike Irey.They include the following; CLIBASIA_05165, CLIBASIA_05605, CLIBASIA_01555, CLIBASIA_05195, CLIBASIA_05200, CLIBASIA_05620, CLIBASIA_05635, CLIBASIA_05665, CLIBASIA_05130, CLIBASIA_05155, CLIBASIA_05265, CLIBASIA_05560, CLIBASIA_05150, CLIBASIA_05180, CLIBASIA_05245, CLIBASIA_02250, CLIBASIA_03020, CLIBASIA_03025, CLIBASIA_02090 and CLIBASIA_02905. We are almost in the final stages of this project and interacting with Mike Irey at U S Sugar Corporation on the effect of HLB effectors on disecase mitigation in citrus. A major useful outcome of this project is the finding that CTV-vector can be used as an Virus Induced Gene Silencing (VIGS) vector. This has been demonstrated to silence citrus endogenous Phytoene desaturase (PDS), and has also be used to potentially silence endogenous genes of psyllids (vecotr of HLB) in co-operation with scientists at CREC and USDA, Fort Pierce. We have positive feedbacks from these groups on the successful silencing of psyllid endogenous genes. This line of research potentially holds great promise in mitigating the spread of HLB by psyllid vector. We presented an oral report based on this investigation in the 2013 International HLB conference in Orlnado. Shubash Hajeri, Choaa El-Mohtar, William O. Dawson and Siddarame Gowda. Citrus tristeza virus-based RNA-interference (RNAi) vector and its potential in combating citrus Huanglongbing (HLB). Additionally, CTV-RNAi vector, engineered with truncated abnormal wing disc (Awd) gene of D. citri, on upon replication in citrus generated RNA silencing triggers, which induced altered Awd expression when ingested by feeding D. citri nymphs. Decreased Awd in nymphs resulted in malformed wing phenotype in adults and increased adult mortality. This impaired the ability of D. citri to fly, would potentially limit the successful vectoring of CLas bacteria between citrus trees in the grove. CTV-RNAi vector would be relevant for fast-track screening of candidate sequences for RNAi-mediated pest control. A full report of this investigation has been formatted into publication and will be submitted shortly to Plant Biotechnology, entitled; RNA virus-based plant-mediated RNAi induces silencing in phloem-sap sucking insect. Shubash Hajeri, Nabil Killiny, Choaa El-Mohtar, William O. Dawson and Siddarame Gowda. This line of research was conducted with the co-operation of Dr. Nabil Killiny, Vector Entamologist at CREC.
The manuscript (which was written for ASABE transaction journal) was revised several times and the final revision was submitted in March, 2013. The effect of random selection of 5 folds in the cross validation process was evaluated and the results were added to the manuscript during the revision. As explained in the previous report, a new dataset of 96 samples containing 20 healthy, 20 magnesium deficient, 20 zinc deficient, 20 HLB infected, and 16 HLB infected zing deficient samples in Valencia variety were collected from the CREC grove. The results of starch measurement for the second dataset were received in January 2013. Based on the measured amount of starch in the samples, all healthy and magnesium deficient samples (based on the crop scouting) contained less than 5 ‘g/mm2 of starch, and so they were considered as healthy. Also the amount of starch in all HLB infected samples exceeded 5 ‘g/mm2, and so they were considered as HLB symptomatic. Interestingly, only two out of 20 samples in the zinc deficient class and two out 16 samples in the HLB infected zinc deficient class contained the amount of starch below the threshold, and so all other 32 samples in both classes were considered HLB infected based on the starch measurement results. The samples were also sent for the PCR test in January, 2013 and the results were received in March, 2013. PCR results confirmed all the starch measurement results for healthy, magnesium deficient, and HLB infected samples. However, those four samples which were considered HLB infected based on starch measurement in zinc deficiency class were actually healthy based on the PCR results. The PCR results also showed that there were six other samples in the zinc deficiency class which were considered healthy based on starch measurement experiment. The samples images were calibrated to cancel the automatic gain control (AGC) effect as explained in the previous reports and the textural features including Gray, GLCM, LBT, and LSP were extracted from the calibrated dataset. Further data analysis will be carried out soon. An on-the-go HLB detection system was designed according to the third objective of the project which is to commercialize the developed prototype system that can be easily used by the growers for an efficient HLB management. Some experimental images acquired using a color digital camera (Canon) in the field to determine the required specification for the imaging system. Based on these images, a highly sensitive camera was chosen and a high luminance illumination system was designed to make sure the camera sensor will receive enough light which will be reflected from the canopy. The imaging system is being assembled and it will be tested first in the on-campus citrus grove to check the functionality and then in the CREC grove for a final test.
The Main goals of this project are: (i) to detect citrus pathogens from insect and plant samples (ii) to perform pre-symptomatic diagnosis of citrus diseases caused by various pathogens. Currently, the primary focus is on Candidatus Liberibacter that is transmitted by psyllid vector and causes Huanglongbing (HLB). It is extremely important to develop the capability for pre-symptomatic diagnosis of HLB because the disease symptoms appear years after initial Liberibacter exposure. This project is a collaboration between Los Alamos National Laboratory (LANL) and Sharp Laboratories of America (SLA). LANL is responsible for discovery and validation of pathogen and pre-symptomatic biomarkers whereas SLA is responsible for the design of impedance biosensor for pathogen detection and pre-symptomatic diagnosis. LANL-SLA will jointly design the assays for pathogen detection and pre-symptomatic diagnosis. During the first two months of this project, we carried out the following tasks. Task 1. We primarily focused on detection of nucleic acids (DNA and RNA). For this, we measured the change in impedance upon the binding of oligonucleotide probe complementary to target DNA or RNA. In this detection scheme, the specificity is determined by the perfect complementarity of the probe to the target sequence. We further enhanced the specificity of detection by measuring the melting curve (i.e., the melting temperature and the first derivative of melting) for probe-target binding. The notion behind this is that the pattern of impedance change and melting curve would be specific for the perfect complementarity between a given target and its probe. Task 2. Typically, probes are immobilized on the gold electrodes of the chip by thiol chemistry, which often does not guarantee uniform immobilization of the probes. To remedy that, we developed an alternative method in which we immobilized biotinylated poly-G tract on the gold electrode and constructed probes with poly-C tails. Thus all probes were immobilized with the same G-C pairing and therefore, we eliminated the problem of probe non-uniformity due to non-uniform thiol chemistry. Task 3. We now have the access to two different Liberibacter asiaticus genome sequences (due to Duan Ping; and Hong Lin and Cliff Han) and two Liberibacter americanus genome sequences (due to Dean Gabriel; and and Hong Lin and Cliff Han). We identified genetic markers that are unique and common to them. We designed appropriate probes for the SLA impedance biosensor. We will soon test the presence of these plus the reference markers used by the CRB Riverside Laboratory in the psyllid samples.
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 EZDiagnostix (EZDx), the sensor commercialization arm of 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: ‘ Reduce sampling and analysis time from 10 minutes (currently) to < 1 minute: Several GC columns have been tested. We believe we can achieve 2 minute analysis time but we are still targeting 1 minute. ' Develop a VOC sampling method to collect VOCs from a significant portion of the tree. We have separated the VOC collection from the analysis, which will lead to reduction in time as collection and analysis can be done in parallel. A hand-held sniffer prototype is completed. ' Develop an algorithm for identification of HLB (Year 1) based on the modified tool: A tool with the modifications above is being completed and is the platform on which to make a final decision on what the configuration will be for the device deliverables. Once the modifications on the tool are complete, we will begin testing to develop a library based on the modified tool. ' Develop software to implement the disease detection algorithms: Analysis is intended to be directly on the device for rapid feedback. We have purchased a Trimble YUMA-2 which will be the platform on which analysis will be performed in the field. This will be the interface to the analyzer, the sniffer and the operator. The UC Davis team is starting to work on the scripts that will perform the algorithm analysis in the field. We continue to test the VOC algorithm using standard devices in orchard fields in south Texas. We are expanding the libraries of citrus varieties beyond sweet orange, and are testing grapefruit now. 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.
Without the ability to culture Candidatus Liberibacter asiaticus (CLas) in vitro, the pathogen can only be studied within the Asian citrus psyllid vector or in the citrus or other host plants. CLas DNA in citrus tissue can be detected with various highly sensitive and robust PCR protocols, however, these methods do not reveal if the DNA target is from living, and pathogenic cells, from dead cells, of from extracellular CLas DNA that may be excreted by the pathogen. Treatment of bacterial cells with DNA intercalating dyes prior to qPCR has promise for distinguishing between live and dead CLas cells in citrus tissues; however, because CLas resides in citrus phloem there are obstacles to this approach. The overall goal of this project is to extend previous findings regarding the use of DNA intercalating dyes and optimize them for quantification of live CLas cells in citrus. During months 1-4 of the project our objectives were to: 1) Determine suitability of PMA-qPCR for distinguishing between living and dead CLas cells in citrus; and 2) validate and compare results of PMA-qPCR with EMA-qPCR. We have made significant progress towards meeting each of these objectives. Specificity and efficacy of EMA- and PMA-qPCR were determined using both purified plasmid DNA containing the CLas DNA target sequence and E. coli cells transformed with the same plasmid. Results with this model system confirm that both EMA and PMA treatments are specific for the CLas target sequence. Amplification of plasmid DNA in qPCR was inhibited 100% by both EMA and PMA. Estimates of live cells using E. coli with EMA or PMA gave similar results of ca. 10% live cells. If cells are heat killed prior to dye treatment, amplification is inhibited 100% . In the course of these experiments we also optimized variables in the protocol to give greatest sensitivity in the assay and the widest working range. We have conducted preliminary tests of EMA- and PMA-qPCR for distinguishing between live and dead cells in citrus seed coat vascular bundles, a tissue known to contain high titers of CLas, and in citrus leaves, both from CLas-inoculated trees in the greenhouse and from HLB symptomatic trees in the field. DNA extracted from seed coat vascular bundles that had been treated with EMA prior qPCR protocol showed about 25% of the CLas copy number of that in DNA from non-treated seed coat vascular bundles. We compared results of EMA- and PMA-qPCR with citrus leaf samples. We used leaves that expressed a range of HLB symptoms for these experiments. Samples were collected both from the greenhouse and from the field. Estimates of the number of live CLas cells in leaves treated with EMA were typically less than those obtained using PMA. Over a range of total CLas titers, estimates of live cells averaged 15% based on EMA-qPCR and 50% based on PMA-qPCR. Experiments during the remainder of this project will focus on validating preliminary results.
The L. crescens BT-1 genome was shared with Chris Henry at Argonne National Laboratory. He and his group are using this information along with the culturing medium formulation provided by Dr. Michael Davis to optimize the metabolic reconstruction of Liberibacter asiaticus. This has been hindered slightly by the absence of a defined growth medium for L. crescens. We have begun development of a defined version of the standard L. crescens culture medium developed by Dr. Davis. Manual curation of the L. crescens genome annotation and comparison of this with the genomes of L. asiaticus and L. solanacearum continues. The RAST annotation is the starting point for this comparison. In this system the gene functions are divided into 28 different metabolic subsystems. Of these subsystems the greatest differences between L. crescens and L. asiaticus were observed in: ‘amino acids and derivatives’ with 154 genes in L. crescens vs. 69 in L. asiaticus; ‘membrane transport’ with 26 in L.c. and 18 in Las; ‘motility and chemotaxis’ with 39 in Lc vs. 14 in Las; ‘stress response’ with 40 in Lc vs. 28 in Las; and ‘virulence, disease, and defense’ with 28 genes in Lc and only 19 in Las. These differences have implications in both culturing and disease development.
Primers to amplify the pathogenesis-specific genes are in hand and being used to amplify those genes in infected citrus. The search for the full length genes that are pathogenesis-specific in citrus was hampered by the lack of availability of citrus genome sequences. We hope that these genomes will be made available soon.
DNA from a close cultured relative to Liberibacter asiaticus, strain BT-1, was given to our group by Dr. Michael Davis. In Dr. Davis’ studies strain BT-1 showed 92% 16S rRNA gene homology with Liberibacter americanus. From this DNA we obtained 16S sequences that matched most closely to Liberibacter solanacearum at 95% DNA sequence homology. Based on this close relationship to the Liberibacter genus we have begun sequencing the genome of strain BT-1 on the Illumina GaIIx platform. This genome being from a close cultured relative to Liberibacter asiaticus is anticipated to greatly improve metabolic models and aid in the development of a L. asiaticus growth medium.
(863) 956-8817
(863) 956-5894
700 Experiment Station Road
Lake Alfred, FL 33850
Email Us
