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.
Additional sequencing is needed to close the current Bt-1 draft genome. Two 454 mate-pair libraries were prepared with 3 and 8kb fragment length. PacBio and IonTorrent shotgun libraries were also sequenced. Using this data the draft genome has been reduced from 109 to 6 contigs as of June 1st. With the aid of the optical map we are able to order these remaining contigs and are attempting to close the remainder of the genome with both targeted Sanger sequencing and bioinformatic methods. All genomic and physicological data has lead us to believe that the babaco bacterium BT-1 is the closest cultured relative to Liberibacter asiaticus and Liberibacter solanacearum. We plan to propose the name Liberibacter crescens for strain BT-1 and will refer to it as such in all subsequent reports.
An analysis of the candidate pathogenesis-specific proteins was made to determine which ones would serve as good candidates for protein purification. The citrus genome was not yet publicly available so it was impossible to design primers that could amplify the host genes that appear to be preferentially expressed during pathogenesis. Primers are being designed for the 26 Liberibacter proteins that are expressed exclusively during pathogenesis.
Multiple medium formulations based on the metabolic reconstruction of Liberibacter asiaticus have yielded no positive results. More information on the intracellular environment in the plant and in the psyllid is needed. Additional Liberibacter genomes will also increase the specificity of these metabolic models. We are attempting to isolate the undescribed beta-proteobacterial endosymbiont of the psyllid gut. Correlations between Liberibacter asiaticus populations in the psyllid gut with this unknown beta-proteobacterium were found in our 16S study. If isolated this bacteria may serve as a partner for L. asiaticus in culture. Only three isolates have been obtained thus far and none proved to be the beta-proteobacterium based on 16S sequences.
An analysis of the pathogensis-specific Liberibacter proteins was made. Most are not obvious drug targets but one certainly is, topoisomerase IV subunit A. This enzyme is the target of quinolones. We also expect quinolones to be pholoem mobile. All that remains is whether quinolones such as can inihibit a Liberibacter infection in planta. In the meantime, we will determine whether quinolones inhibit the colosely related babaco bacterium.
A fatty acid profile of the babaco bacterium isolate BT-1 was generated at OpGen. This profile could not be compared to members of the Liberibacter genus as they are not currently cultured; therefore placement of BT-1 in the Liberibacter genus was inconclusive. A comparison of Liberibacter asiaticus genome and the draft BT-1 genome suggested several potential inadequecies in L. asiaticus. Based on these findings, additions to L. asiaticus media preparations were suggested to Dr. Michael Davis. The transcriptome of L. asiaticus in culture media may provide insight into metabolic insufficiencies and is anticipated to improve the metabolic model. We have begun working toward the sequencing of the L. asiaticus transcriptome across time in Dr. Davis’ static cultures as well as in removed psyllid midguts. Preliminary RNA extractions had insufficient yields for transcriptome sequencing.
Since the completion of the metabolic model of L. crescens we have been focusing on the development of a genetic system in this Liberibacter model. Specific genes involved in carbon metabolism, regulation, and cell wall recycling have been selected as targets for knockout in L. crescens. These genes are present in L. crescens but not L. asiaticus and through the development of knockouts in L. crescens we hope to better understand the constraints on L. asiaticus growth in culture. Electroporation using several plasmids / cosmids, such as p15TV-L, pLAFR1, pGS9, pHRGFPGUS, and pUFR071, were performed to test the transformation efficiency on L. crescens. It was found that only the vector pUFR071 was able to transform and replicate in L. crescens, which leads to an assumption that plasmids of smaller or similar size of pUFR071 should be able to electroporated into L. crescens. The gene targetted for knockout will be cloned into a suicide vector (such as p15TV-L) with a kanamycin cassette inserted in the middle of the gene and transformed into Escherichia coli DH5a. The construct will be confirmed by sequencing and the development of kanamycin resistance. The final construct will be electroporated into L. crescens. Chromosomal crossover will occur and mutants will be selected for kanamycin resistance. The knockout mutant will be confirmed by sequencing of the target gene.
Media development based on the Liberibacter asiaticus metabolic reconstruction continues. This process is slowed by an incomplete annotation and the lack of a closely related bacteria with an established growth medium. It was suggested that Liberibacter asiaticus may be dependent on other microbes in the psyllid gut and the citrus phloem. The bacterial diversity in citrus phloem was determined previously (Tyler et al 2009). The microbial communities of the psyllid will be elucidated through targeted 16S amplicon sequencing. If a relationship is discovered a co-culture methodology will be explored further. To this end DNA was obtained from psyllids and their Liberibacter asiaticus titer was evaluated with qPCR. A universal bacterial 16S rRNA gene primer set was then used to amplify this gene from any bacteria present in the psyllids.
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