Thus far, 646 different media formulations have been tested to support growth of Candidatus Liberibacter asiaticus under various culture conditions. None of the media supported planktonic growth of the bacterium. Biofilm formation was observed on the surface of the alimentary canals of Diaphorina citri used as a source of inoculum. These biofilms were more extensive in some media than in others. The addition of mixtures of essential and of non-essential amino acids appeared to promote biofilm formation. Biofilm formation was more evident under aerobic and in microaerophilic conditions than under anaerobic conditions.
Discovery of causal agents is important to prevent disease dissemination. Pathogens evolve continuously and a large number of graft-transmissible citrus diseases indicate that viruses and viroids the most likely causative agents. In California, Citrus Clonal Protection Program (CCPP) offers quarantine disease testing and budwood certification services. Quality control using traditional immunological or nucleic acid based methods are great, but are limited to only known pathogens and diseases. Now, the major concern is to develop protocols to identify and catalog previously uncharacterized viruses and viroids to achieve the primary goal of CCPP to provide disease free citrus cuttings to the industry and farmers. My lab has been successfully developed a novel method known as vdSAR to identify viruses from insects and plants (Wu et al., 2010). This approach is based on the fact that virus-specific small interfering RNAs (viRNAs) produced in infected plant and insect cells are overlapping and can be assembled back into large fragment of the invading viral genomes. Thus, deep sequencing and assembly of total small RNAs from the disease samples of citrus will likely identify previously unknown viruses. Viroids are a distinct class of free RNA pathogens that cause diseases in citrus and other plant species. However, current approaches for viroid discovery are time-consuming and technically demanding so that only a small number of viroid species have been identified in the last 40 years. It is therefore likely that some of the unknown graft-transmissible citrus diseases are caused by viroids. Thus, we have focused on the development of a deep sequencing-based approach for viroid discovery in the last year. We found that viroid-specific siRNAs accumulated in infected plants are also overlapping and that full-length circular viroid genomic RNA can be assembled by a new computational algorithm we have developed (Wu et al., in preparation). We have tested this approach for viroid discovery using a published small RNA library made from Italian grapevine and discovered a new viroid that shares no significant homology to any known viroid. We have obtained nine graft-transmissible citrus disease samples from our collaborator Dr. Vidalakis laboratory, UCR. In the first batch, we have completed small RNA library construction for four samples: Yellow Vein and Vein Enation, Fukumoto decline on trifoliate and Beck. Using our approach, we predicted two candidate viroids (Vd-1 and Vd-2) from the yellow vein small RNA library. Based on the predicted sequence, we were able to amplify and clone Vd1 (276 nt) and Vd2 (234 nt) from Yellow Vein using RT-PCR and to show that Vd1 existed as a circular molecule. We are in the process of testing the infectious nature of Vd1 in plants. In the second batch, we have constructed small RNA libraries for five more samples: Concave Gum, Cristocortis and Fatal Yellows (CCPP, UCR), Bahia scale bark and Leprosis (nuclear type) from Brazil. Deep sequencing of small RNAs is in progress. Once the data is received, we will adapt our vdSAR strategy to discover viruses and viroids from these samples.
Detection of genetic diversity within pathogen populations is fundamental for ecological and epidemiological studies of a disease. The genetic structure within a given pathogen population is an indispensable prerequisite for determining sources of infection and risk management of the disease. The microsatellite markers developed in this project provided a useful tool for detection and genetic diversity study of ‘Ca. L. asiaticus’ isolates for global populations and the epidemiology of HLB. A panel of seven polymorphic microsatellite markers was developed. Microsatellite analyses amongst 134 Las isolates from Asian and American continents showed that the genetic diversity of ‘Ca. L. asiaticus’- is higher in Asia than diversity in the Americas. UPGMA and STRUCTURE analyses identified three major genetic lineages of ‘Ca. L. asiaticus’ worldwide. Isolates from India were most genetically distinct compared to other countries. East-southeast Asian and Brazilian isolates were generally included in the same group, few members of this group were identified in Florida, but the majority of the isolates from Florida were clustered in a separate group. Isolates from Brazil showed similar genetic makeup with east-southeast Asian dominated groups, suggesting the possibility of a common origin. In contrast, most of the isolates from Florida were clustered in a separate group. While the actual sources of the dominant ‘Ca. L. asiaticus’ strains in Florida are still in question, the genetic analysis from this study suggests that the introduction of less- pervasive strains may have been introduced directly from Asia or via Brazil based on their genetic similarity. However, the recent outbreak of HLB in Florida is predicted to have occurred from multiple introduction events. The multilocus microsatellite marker system developed in this study provides adequate discriminatory power for the identification and differentiation of closely-related ‘Ca. L. asiaticus’ strains, as well as for population genetic studies of the HLB-associated Liberibacters. Based on the reference genome of Florida isolates, we identified pathogenic genes that are located in or near these regions. The gene-based of new molecular markers were thus designed and classified into four major catalogues: 1) Phage-derived genes which are often involved in pathogenesis through their transfer of genes and/or genetic rearrangement. 2) Genes defined as two component response regulators. This group of genes is important for sensing environmental signals and producing a transcriptional response from those signals. 3) Genes identified with hydrolase activity. One of the interesting genes in this group is salicylic acid hydrolase which functions as hydrolysis of salicylic acid, a signaling molecule expressed in host associated with defense response to pathogen attack. However, to confirm its hydrolase function, the substrate for this gene-coded enzyme needs to be determined. 4) Zinc binding proteins, znuABC genes identified as putative high-affinity zinc uptake system that belongs to the ATP-binding component of ABC transporter protein. Zn uptake system, znuABC is required for obligate bacterial zinc homeostasis in intracellular environments which contributes to the virulence of Las in citrus plants. To provide insight of genetic variation of Las at whole genome level, more recently, we successfully obtained the whole genome sequence of a Chinese Las strain using Illumina sequencing technology. Preliminary genome analyses revealed that sequence variations were observed between Florida and Chinese strains. Research is in process toward the completion of sequence contig validation and connection. Comparative genomic analyses will provide critical information about the evolutionary history of genetic relationships between Asian and American strains. The availability of additional HLB-associated Liberibacter genomes will also facilitate development of gene-based markers that can be used for strain characterization.
We have made substantial progress in the first two objectives of the proposal and the citrus plants containing some of HLB effectors are being challenged inoculated with HLB through psyllids which is the third objective. Based on the sequence information number of putative effectors unique to Candidatus Liberibacter asiaticus, the causal agent of citrus Huanglongbing (HLB), present in prophage related gene clusters and/or pathogenicity islands have been identified and are believed to contain bacterial pathogenicity, multiplication and virulence determinants. We have cloned most of the effectors into binary vector based citrus tristeza virus (CTV) vector, between CP and CP ORFs as a separate cistron under an heterologous beet yellows virus coat protein controller element and were agro-inoculated into leaves of Nicotiana benthamiana as outlined in earlier progress reports. Partially purified virions of recombinant CTV containing HLB effectors from the infiltrated leaves and /or the systemic leaves of N. benthamiana have been used to inoculate Citrus macrophylla plants. 15 individual HLB effector genes are in citrus ready for challenge inoculation with HLB and many more in N. benthamiana at different stages of transfer to citrus. Observations of Mike Irey of US Sugar have indicated that sweet orange is the preferred host for screening with HLB as against Citrus macrophylla. Thus, we will use sweet orange citrus cultivar for primary inoculations from N. benthamiana. We have also cloned some of the effectors into the 3′ end of CTV between p23 ORF and the 3′ non-translated region of CTV based on the recent finding of augmented expression of an inserted foreign gene. One of the important effectors or virulence factors is hemolysin of HLB is a 50 kDa protein secreted by type I secretion system. The hypothesis is that the hemolysin might interfere with metal ion transportation leading to host metabolic imbalance potentially resulting in disease symptoms. An abstract entitled ‘Functional studies of Hemolysin of ‘Candidatus Liberibacter asiaticus’ in citrus using citrus tristeza virus vector’ was presented at the 2011 American Phytopathological Society meeting and we plan to publish together with results of HLB challenge inoculation. Additionally, we are using a faster approach to screen for virulence genes of HLB using Potato virus x (PVX) vector system which has been used extensively to screen for virulence genes in herbaceous hosts. Agrobacterium-mediated transient expression of 34 genes derived from CLas prophage-related gene cluster into PVX vector, did not induce obvious phenotype changes in tobacco plant. However, transient expression of CLas-flagellin, flg22Las induced cell death and callose deposition in N. benthamiana, while the transcription of Bak1 and SGT1 associated with plant innate immunity were up-regulated. Substitution experiments revealed that the 38th and 39th amino acid residues were essential for callose induction. The synthetic flg22Las peptide (DRVSSGLRVSDAADNAAYWSIA) could not induce cell death, but retained the ability to induce callose deposition at the minimal concentration of 20 ‘M. These results demonstrated that Flg22Las has PAMP activity, and may play an important role in determining citrus resistance to CLas. These results were presented at the 2011 American Phytopathological Society meeting and a manuscript will be submitted for publication in Phytopatholgy.
This research agreement became effective 1-Jun-11. Funds were provided in the grant for hiring a GS5-7 biological support technician as an ARS employee. The recruitment agreement was drawn up, applicant names have been provided to the PI by ARS Human Resources and candidates for interviews have been selected. Preliminary results of foliar and seed coat tissue fractionation and filtration experiments were obtained both before and after the effective date of the Research Agreement. Analysis suggests that Liberibacter cells reside in a matrix within the phloem sieve elements and that a large portion of this matrix is composed of bacterial DNA. Results from microscopy with Fluorescence In Situ Hybridization (FISH) indicated that individual bacteria or clumps of bacteria could be liberated by mechanical pulverization of source tissues, but numbers of cells were less than expected based on real time PCR (qPCR) data, and cell morphology appeared to be altered. Experiments are continuing to generate additional data.
Transcription factors: The second transcription factor, CLIBASIA_01510 was successfully purified. This protein was purified in sufficient amounts (yield = 1.18 mg/ ml) to carry out the chemical library screening. Thermal denaturation kinetics indicated that the protein is highly sensitive to heat; protein denaturation was obtained at 37 ‘C. The optimization assays to perform thermal melting screening are being currently evaluated. The previously purified CLIBASIA_01180 was successfully used in thermal melting assays; three chemicals were selected to perform further studies. Examination of the genomic environment of CLIBASIA_01180 suggests it may be involved in regulation of CLIBASIA_01185, which putatively encodes a delta-aminolevulinic acid dehydratase. This enzyme is part of the biosynthesis of tetrapyrroles, such as heme. This may provide an interesting target to affect the viability of Ca. Liberibacter asiaticus, as heme is an essential enzyme cofactor. Enzymes: The attempts carried out in our laboratory to overproduce and purify CLIBASIA_ZnuA using Escherichia coli as a host were unsuccessful. Several genetic strategies were designed to overcome solubility problems. ZnuA was cloned in different expression vectors in order to be produced with the yeast Pichia pastoris. The gene of interest was successfully cloned in three different genetic backgrounds (WT, ‘SP, ’21). The ZnuA gene was genetically modified according to the expression background to purify a full length protein and a chimerical protein with a deletion of the first 21 amino acids. A combination of three vectors (pPICZ-A, pPICZ-B, and pPICZ.-A) was used to clone both ORFs. Transformed yeasts were selected for on YPDS/Zeocin plates. Mut phenotyping was carried out on MMH and MDH plates to determine the site of recombination (AOX1 locus or the 3’AOX1 region); this information is critical to optimize the overproduction process. ZnuA overproduction using the 12 different clones integrated into the P. pastoris chromosome is currently being evaluated. The overproduction will be optimized using a combination of different culture conditions (factorial design). The second strategy selected in order to increase the yield of the recombinant proteins (including transcription factors) is cloning of the genes into expression vectors to be transformed into Sinorhizobium meliloti. The targets (CLIBASIA_00835, CLIBASIA_03370, CLIBASIA_01510, and CLIBASIA_02535) have been ligated into the pBBR1-MCS5 vector. This broad host range vector will allow for expression of these proteins in S. meliloti. This bacterium is closely related to Ca. Liberibacter asiaticus, and we may be able to obtain soluble protein if the protein is expressed constitutively at low levels.
LEAF VOLATILE ANALYSIS: A total of 31 one hour leaf air samples were collected and analyzed from two HLB and two control trees at CREC. Each sample was analyzed for approximately 45 volatiles. Major volatiles were characterized as terpenes, aldehydes and linear alkane acids. There was no statistical difference between any of the major volatiles except for a single volatile organic acid which was twice as high in HLB leaf volatiles than in healthy control leaf volatiles. There were two minor peaks of interest. One was found only in 8 of the 16 HLB samples and none of the healthy samples. Another was found only in 12 of the 15 healthy samples and 5 of the 16 HLB samples. In addition, researchers from University of California-Davis are continuing their yearly study with monthly/bimonthly sampling trips to CREC facility for the collection of SPMEs, twisters (GC/MS) and DMS data on HLB-infected and healthy control trees. Two additional trips were made since July, 2011, and currently the second to last trip (month 11) is being conducted. It was determined that sampled orchard trees are co-infected with CTV, therefore, additional studies were carried out in parallel using CTV-free greenhouse seedling trees at CREC (both SPME and DMS). The results from the data analysis carried out so far on the existing data (month 1-10) are highly encouraging in both GC/MS and DMS studies; high classification accuracies have been achieved. For DMS, accuracies vary from month to month, but typically exceed 80%. For GC/MS, identification of a set of discriminating biomarkers is currently underway. HYPERSPECTRAL ANALYSIS: Several classification and spectral mapping methods were conducted to evaluate their applicability for HLB detection using aerial images. For the 2010 hyperspectral images, with better ground truth records, more precise libraries for HLB infected and healthy canopies were collected, and a higher classification accuracy was achieved. Simpler classification methods such as minimum distance (64% accuracy) and Mahalanobis distance (76% accuracy) showed more stable and balanced performance between the training and validation sets, thus those two methods were highly recommended in future studies. Disease infection density maps were created and most of the methods were able to detect the severely infected areas in the density maps, and their similar infection trend with ground truthing could provide a promising way to assist more efficient citrus greening disease management. For ground-based hyperspectral studies, hyperspectral and thermal data were collected using a multispectral and thermal camera from the top of 30 healthy and 30 HLB-infected citrus canopies. In addition, aerial hyperspectral images were collected from a distance of about 100 m using a multi-rotor remote sensing platform (microcopter). Two multispectral cameras, each comprising of six spectral bands in the visible-near infrared region (440-900 nm) were used for data collection; while thermal camera provided the spectral reflectance values in the thermal infrared region (7.5-13.5 ‘m). Preliminary evaluation of thermal images indicated that the average pixel values were 15-20% higher in HLB-infected samples than those of healthy samples. Presently, we are continuing to work on the spectral data analysis. Publications/Presentations: > Li, X., W. S. Lee, M. Li, R. Ehsani, A. Mishra, C. Yang, and R. Mangan. 2011. Spectral difference analysis and airborne imaging classification for citrus greening infected trees. Computers and Electronics in Agriculture. In review. > Sankaran, S., and R. Ehsani. 2011. Detection of Huanglongbing disease in citrus using fluorescence spectroscopy. Transactions of the ASABE. In review.
The goal of this project is the in vitro culture of Ca. Liberibacter asiaticus (LAS)- Vietnamese strain maintained in greenhouse on Citrus spp. and periwinkles – associated with the Citrus greening syndrome. The strategy consists of primo-cultures of the bacteria in insect cell cultures used as feeder cells. An optimized protocol of bacteria extraction from infected citrus trees or periwinkles has been set up to inoculate various insect cell cultures, in various culture media. Selective pressure, stress protectants, and nutrients allowing and/or improving the bacteria survival and growth in co-cultures were selected. Various cell lines were tested and a couple selected for further studies. Different co-cultures were continuously grown over several months with successive transfers. A publication describing this work is in preparation. We obtained LAS cultures cured from insect cells. The presence of insect cell DNA was checked by microscopy and by PCR using primers within the insect cell cytochrome oxidase gene. We verified whether LAS was the only bacteria in our cultures with broad-range PCR based on bacterial 16S rDNA. Other bacteria were found in co-culture with LAS (i.e. a Delftia acidovorans or an actinobacteria strain). Enforced antibiotic selections have been applied to successfully get rid of contaminants and we are monitoring their effects. We tried to isolate LAS colonies on a solid version of the culture medium used in the co-cultures. However, so far this approach was not successful in getting any bacterial colonies. The very next step is to inoculate the cultured bacteria into healthy Citrus in order to verify whether after a period of in vitro culture, it keeps its pathogen properties, and if we are able to re-isolate it (Koch’s postulates). We set up a protocol of mechanical inoculation in healthy citrus. By microscopy, we verified with a fluorescent tracer that we had circulation of the inoculated material throughout the plant via the citrus phloem. We are monitoring several LAS co-cultures and we will inoculate them to healthy citrus after several transfers.
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. psyllaurous in PLoS ONE 6(4): e19135. doi:10.1371/journal.pone.0019135. All BAC clones of Las were sequenced, and sequence analyses revealed genetic diversity and a potential mechanism of genome reduction. We have characterized the ATP translocase from Las and proved its function using a heterologous E. coli system. This data was published in J. Bacteriol. 192:834-840, 2010. We are currently developing an antibody-based “drug” to target this protein, aimed at disrupting ATP import, which may be important for its survival. Three peptides screened from a phage display library showed inhibition to the cell growth of Las bacteria in vitro. We have also characterized the individual genes of two putative zinc operons in Las, confirming only one operon responsible for zinc uptake. Each gene of the ZnuABC operon has been characterized, and their expression associated with Zinc metabolism is under investigation. Seed transmission of Las was tested in grapefruit, sweet orange, sour orange and trifoliate orange. Relatively high titers of Las were detected from both seed coats and inner seed coats collected from HLB-affected citrus plants. 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 Candidatus Liberibacter asiaticus 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 THREE SEED-TRANSMITTED HLB SEEDLINGS were confirmed by PCR using several Las-specific primer sets. Graft transmission of the cutting from this HLB plant confirmed these seed-transmitted HLB. Efforts to reveal the difference between seed-transmitted and field infected Las populations are underway. Progress on culture of Las bacterium in vitro has been made. Up to 1,000,000 to 100,000,00 cells/ml were obtained within 24 hrs based on qPCR estimation. The Las cells number in the cultures were staggering and decline when they reached to Ct value 21.00 to 22.00 (16S rDNA-based RT-PCR) in liquid media. We are looking into factors affecting further growth.
The objective of this project is to characterize the hypI (renamed as hyvI) gene and determine its effects on insect transmission and/or virulence in host plants. Transient expression with alternative expression systems and RT-qPCR, etc., will be used to elucidate the function of the hypI (hyvII) gene of Las and shed light on the molecular mechanism of this “phase variation” phenomenon; thereby developing a novel control strategy for citrus HLB. In addition, antibodies and probes along with standardized protocols developed during this project can be applied for better detection and differentiation of the HLB bacteria. The hyvI and hyvII within two Las prophages were further characterized. Sequencing analysis of the hyvI/hyvII genes from 40 Las DNA samples collected globally revealed sequence conservation within the individual repeats but an extensive variation regarding repeat numbers, their rearrangement, and the sequences outside of repeat region. These differences are found not only in samples with distinct geographical origins but also from a single origin and even from a single Las-infected sample. The Florida isolates contain both hyvI and hyvII while all other global isolates contain only one of the two. Interclade assignments of the putative HyvI/II proteins from Florida isolates with other global isolates in the phylogenic trees imply a multi-source introduction of the Las bacterium into Florida. A manuscript regarding these results has been published in Applied Environmental Microbiology, 2011: 6663-6673. We have developed a real-time PCR using SYBR Green 1 (LJ900fr) and TaqMan’ (LJ900fpr) protocols with primers and probe targeting nearly identical tandem repeats of 100bp hyvI and hyvII. Because of higher copy number of the tandem repeats per bacterial genome, these methods significantly improved detection capacity of the HLB bacterium. In comparable samples relative to 16S HLBaspr real-time PCR, these new methods reduced the relative detectable threshold by approximate 9 and 3 cycles for LJ900fr and LJ900fpr, respectively. From HLB samples with extreme low titer of Las bacterium, both LJ900fr and LJ900fpr detected Las from otherwise non-detectable samples by APHIS standard, HLBaspr. To determine the cellular localization of the HyvI protein in plant cells and the role of the two putative NLSs in hyvI gene, full-length hyvI and C-terminal region including two putative NLSs were amplified and cloned into pCX-DG vector with GFP driven by CaMV35S promoter, and transformed Agrobacterium tumefaciens strain GV 2660 with the pCX-DG-hyvI constructs. The results indicate that the HyvI protein did not target in plant nucleus in tobacco. Truncated HyvI was expressed better than the full length HyvI when fussed with GFP and mixed with gene silencing suppressor. RT(Reverse transciptase) PCR confirmed the hyvI gene expression both in the host plant and psyllids though the HyvI protein was not detected with Western blot. The obstacles for detection of the antigen from HLB-infected plants remains to be further investigated. When the full length (12 full repeats) of hyvI gene was cloned into pUF047plasmid, and replicated in heterologous hosts, the repeat number of hyvI gene remained the same in E. coli, but varied in Xanthomonas citri (citrus canker bacterium), ranging from 1.0 to 10.0 full repeats. Clones of X. citri containing the hyvI gene displayed different degree of growth retardation, indicating potential toxic effect of hyvI gene to X. citri.
To evaluate the adherence property of copper loaded silica nanogel (CuSiNG) formula to citrus leaf/stem/fruit surface, we have prepared silica nanogel material co-loaded with copper and a fluorescent probe. In the presence of Cu, fluorescence of the dye is ‘turned off’ due to heavy metal (Cu) induced fluorescence quenching phenomenon. Once Cu is released from the silica nanogel material, probe fluorescence is restored (‘turned on’). This fluorescence sensing based assay is expected to provide valuable information related to Cu release rate and retention of silica nanogel to citrus leaf/stem/fruit surface. Atomic Absorption Spectroscopy (AAS) is considered as a standard technique to determine residual Cu on citrus plant surface. This technique, however, could not be reliably used for the assessment of residual silica nanogel material, in particular, on fruit surface. Additional fluorescence imaging based studies are on-going to understand the interaction of CuSiNG material with the bacterial cells. We will report fluorescence assay results as well as our 2011 field trial data in coming months.
Diagnostic service for growers for detection of Huanglongbing to aid in management decisions, October 2011. As we did in previous reports, this update covers the entire period that Huanglongbing Diagnostic Laboratory has been in service because one of the objectives for the funding for continued, uninterrupted diagnostic services to growers while expanding our ability to provide diagnostics quickly and assist with research efforts. The HLB Diagnostic Laboratory has been operational at UF-IFAS-SWFREC since February 2008. Since the opening of the lab, there has been continued development of techniques, protocols and efficiency. The lab has been in operation for fifteen months, and as the first week of June, 2011, we have processed more than nearly 27,000 grower samples, with approximately 1000 arriving since the last report, June 2011. Additionally, approximately 18,000 samples have been received for research, of which more than 2300 were within that same time frame. Techniques, Protocols and Research For DNA extractions, we continue to use the magnetic particle based system, which has proved both reliable and fast. Current methods of sample processing have become streamlined and therefore seen little change. We have recently introduced the use of TaqMan Fast Advanced MasterMix for real-time-PCR reactions as this is more economical and has shown comparable-to-superior amplification and detection of gene(s)-of-interest when compared to the TaqMan Fast Universal PCR MasterMix. Protocol for the detection of HLB in Asian Citrus Psyllid has been validated, including quantification of HLB in both plant and psyllid samples, with the primary goal of serving research projects within the entomology and plant pathology department that also contribute funds from their research grants to support the labor and supply costs for research samples. The protocol established in 2010 for the quantification of the HLB bacteria in both the psyllid and host tissue using a standardize curve is being used for research and extension samples. The basic service remains the available to growers, researchers, extension faculty and dooryard citrus growers. However, we are also expanding the data analysis of PCR processed data from individual groves that consented to have their data used. In conjuction with an epidemiologists and computer mathematician, the spread of the disease will be modeled. This studies are not supported by lab funds but are an offshoot of the database collection. The intent is to have additional tools for looking at the spread of HLB in sites where incidence is still relatively low.
For the quarter beginning July 1, 2011, the SGDL has processed 11,866 samples. The breakdown of the samples is as follows: growers 6,810, psyllid samples 2,162, research samples 2,486 and 408 for Southern Gardens. Included in the research samples are samples that were part of a cooperative project between USDA-ARS (Dr. David Hall), CRB (Citrus Research Board in CA, Dr. MaryLou Polek) and SGDL. The purpose of the study was to determine what storage methods should be used for psyllids being tested for the presence of CLas. Although many labs throughout the world are using psyllid testing in advance of the discovery of HLB symptoms, there are essentially no studies to document how the psyllids should be stored prior to testing in order to provide the most accurate data possible. Thus this study will provide valuable documentation for future and past HLB testing efforts. Preliminary results were presented at the Second Annual Citrus Health Response Plan meeting in Denver, October 2011 and a publication in a refereed journal will be forthcoming. As mentioned in the previous report, the SGDL was in the process of validating a robotic DNA extraction procedure. This validation is now complete and all DNA extractions (vegetative material and of psyllids) are now being done robotically. This will result in a lowering of cost, more efficient operations in the lab, and hopefully a more robust assay.
During this period, we continued working on a gene-based marker system that links to phenotypic characteristics of ‘Ca. L. asiaticus’s strains. In silico genome analyses grouped these selected genes into four categories; 1) Phage-derived genes which are often involved in pathogenesis through their transfer of genes and/or genetic rearrangement. 2) Genes defined as two component response regulators, important for sensing environmental signals and producing a transcriptional response to those signals. 3) Genes identified with hydrolase activity. 4) Zinc binding proteins, znuABC genes identified as putative high-affinity zinc uptake system. Thirty five primer pairs designed for these sequencing loci were used to amplify 16 selected Las strains collected from Florida, China, India and Brazil. The Amplified products were then cloned into pGEM vectors. ABI 3130 sequence analyzer was used to obtain amplicon sequences. Sequence alignments were then performed to identify the deletion, insertion or nucleotide substitution among geographic strains in these gene loci. The loci that are variable amongst different strains had been selected for further tests. To confirm polymorphism in these gene-based sequencing loci, we conducted the second round test by including more Las isolates. This work is still in process. Recently, we successfully sequenced the whole genome of a Chinese Las strain by Illumina sequencing technology. De novo assembly and BLASTn analyses identified 31 contigs with 18 -30 X coverage representing 1.2 Mbp of the whole genome. A series of comparative genome analyses were conducted by comparing available reference Las genome with this new Las genome to understand the genome conservations and variations between Las strains derived from different geographic origins. These analyses will provide critical information about evolutionary selection, adaptation and genetic signatures of Las at a genomic level. The availability of additional HLB-associated Liberibacter genomes will also facilitate genome-wide searches and identifications of potential gene targets for development gene-based markers tightly linking to phenotypic characteristics.
The Southern Gardens Diagnostic Laboratory (SGDL) has been in operation since October 2006. To date, the SGDL has received and assayed 176,403 grower samples, and approximately 43,000 additional samples from various sources (research samples, psyllid samples, and samples from Southern Gardens Citrus) for a total of over 219,403 samples since the SGDL began receiving samples. For the period of time covered by this report (July 1, 2010 to June 30, 2011), a total of 27,971 grower samples, 4,354 psyllid samples and 12,690 research and Southern Gardens samples were received and analyzed for a total of 45,015 samples for the period. The budgeted amount of samples for the period was 45,000. Although the total sample volume may not be changing drastically, one thing that is changing, however, is the mix of sample type/sample submitter. Although the number of grower samples is declining slightly, the number of research samples is increasing. The research samples are being submitted by private, state, and federal researchers in support of their research work. The research samples tend to be from large-scale field trials, survey projects, and from chemical/nutritional management projects. We expect this trend to continue. Among the reasons for this are continuity in testing and the fact that the cost of testing is being supported by the industry. One major accomplishment for the period was the conversion of the DNA extraction procedure from a completely manual process to a semi automated process. Samples are still put into tubes manually but all of the DNA extraction procedure has been automated using a Qiagen BioSprint 96 robot and Qiagen MagAttract chemistry. This has resulted in an increase in efficiency (resulting in faster turn around times for the samples) and a slight decrease in the cost of the assay. The procedure has been validated internally for both tissue and psyllid samples.
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