Progress 08/15/05 to 08/14/08
Outputs
OUTPUTS: In the first year of this project several novel genomic DNA markers that represent potential candidates for distinguishing members of four plant pathogenic bacterial genera at the sub-species level were identified through computational methods. Preliminary experimentation identified the dnaA marker as the optimal genetic marker. Because the dnaA gene encodes the "replication initiator factor" protein, the resulting marker was named RIF. This year the primers for three of the genera were redesigned in order to create more uniform sequence data, and signature sequences for the RIF marker were generated for 731 of the most diverse accessions representing four genera (Clavibacter, Erwinia, Ralstonia and Xanthomonas) present in the Alvarez library of phytopathogenic bacteria. These sequences were used to build a separate framework of reference sequences for each genus. PARTICIPANTS: The overall supervision of this project was the responsibility of the PI Gernot G. Presting. Kevin Schneider is a graduate student in the Presting laboratory and conducted the computational analysis required for marker identification as well as the laboratory work required to generate the large number of DNA sequences for each of the markers. Co-PI Anne Alvarez provided access to her extensive collection of plant pathogenic bacteria, as well as taxonomic expertise and unknown samples. Technicians Asoka S. De Silva and Wendy Kaneshiro (both Alvarez lab) validated cultures within the Alvarez collection. TARGET AUDIENCES: The target audiences for this work are researchers in the field of epidemiology and plant pathogenic disease, as well as users of diagnostic methods for identifying plant pathogenic bacteria. We have reached a large proportion of this group effectively by presenting this work at the 2008 annual meeting of the American Phytopathological Society in Minneapolis. PROJECT MODIFICATIONS: Nothing significant to report during this reporting period.
Impacts
The sequence frameworks derived from 731 bacterial accessions can be used for 1) the identification of unknowns, 2) the detection of annotation problems in the existing culture collection and 3) the comparison of members from different culture collections without requiring the transport of specimens. We have used our frameworks to identify 10 unknowns from a recent outbreak of Clavibacter michiganense within 48 hours of receiving the DNA samples. We also were able to use the RIF sequence frameworks to correct annotation errors in our collection. Most importantly, we have been able to use the RIF sequence frameworks of Xanthomonas and Ralstonia to begin to understand the genetic relationships between species, pathovars and races of these two genera. This work was presented at the annual meeting of the American Phytopathological Society, where over thirteen attendees requested copies of the poster. The request that we recently received from a major US seed company to share sequence and accession data indicates that this work may mediate a major shift in the diagnostic field from antibody-mediated to DNA sequence-based pathogen detection and identification.
Publications
- Schneider K, Alvarez A, Presting G. 2008. DNA markers for identification of the bacterial phytopathogens Clavibacter, Erwinia, Ralstonia, and Xanthomonas. American Phytopathological Society Centennial Meeting, July 26-30, Minneapolis, MN, USA.
- Schneider K, Alvarez A, Presting G. 2008. DNA markers for identification of four phytopathogenic bacteria. CTAHR Symposium, April 11-12, Honolulu, HI, USA.
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Progress 08/15/06 to 08/14/07
Outputs
Objective: Identification and testing of different DNA regions for development of a DNA barcode in four genera of phytopathogenic bacteria (Clavibacter, Erwinia Ralstonia and Xanthomonas = CERX). Criteria for a DNA barcode region are 1) single copy region with 2) a low rate of horizontal gene transfer, 3) containing conserved regions flanking 4) a sufficiently variable sequence. Twenty-five of the 48 genes identified as potential DNA barcodes in year 1 contain conserved regions suitable for the development of a single primer pair for all four genera. However, the primers were too degenerate to yield single PCR amplicons, and this approach was discontinued. In an alternative approach, analysis of 6 complete Xanthomonas genomes (2 X. oryzae pv. oryzae, 2 X. campestris pv. campestris, 1 X. citri, and 1X. campestris pv. vesicatoria) revealed 3 potential DNA barcode regions: acr, an acriflavin resistance gene, dnaA, a DNA replication initiation gene, and trmu, a tRNA
methyltransferase gene. Primers for each genus were developed based on the dnaA gene of sequenced strains. The internal transcribed spacer (ITS), between the 16S and 23S rDNA, a commonly used region for bacterial identification, was used as a standard to assess the resolution of new marker regions. The acr gene was eliminated from further consideration when it was discovered to be absent in the gram-positive Clavibacter. DNA sequences for ITS, dnaA and trmu were determined for a total of 333, 264 and 192 CERX isolates, respectively. Success rates for PCR amplification and DNA sequencing varied for different genera and genes. The trmu marker yielded inconsistent phylogenetic profiles, which may be indicative of horizontal transfer among related species, a small amplicon in Clavibacter, and lower sequencing success. The best DNA barcode identified in this work is the dnaA gene as evidenced by its high sequence diversity, low likelihood of horizontal transfer between species, the highest
rate of sequencing, and amplicons of approximately the same size. The dnaA barcode resolves pathovars into distinct clades more often than does the ITS region.
Impacts
The collection of 264 dnaA sequences obtained from 4 genera of plant pathogenic bacteria provides a framework to which unknowns can be compared in a DNA-based identification system. The use of this DNA barcode requires a single set of genus-specific primers for identification at species or pathovar level. The framework contains sequence data for accessions from 4 genera collected at different locations, times, and from different hosts, and may help elucidate how pathogens arrived in Hawaii. The dnaA marker derived in this work distinguishes the select agent Ralstonia solanacearum race 3 biovar 2 from other races and biovars, and resolves two pathovars of X. campestris (X. c. pv. campestris and X. c. pv. vesicatoria). Furthermore, the dnaA barcode supports the recent separation of Erwinia into the genera Pectobacterium and Dickeya. Taken together, these data indicate that the newly derived dnaA marker is the best marker currently available for the identification of the
four different genera of phytopathogenic bacteria.
Publications
- No publications reported this period
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Progress 08/15/05 to 08/14/06
Outputs
Objective in year 1: Identify potential DNA barcode regions in four phytopathogenic bacteria. In order to be useful for genomic DNA barcoding, the target region has to be present in all target species and sufficiently variable in sequence to allow distinction between closely related organisms. The identification of a suitable region has been the first objective of this work. A comparison of the widely used rDNA region from 3 species of Xanthomonas has revealed insufficient levels of sequence diversity to distinguish between pathovars. We have used two bioinformatics approaches to identify potential barcode regions. In the first approach we examined the nucleotide sequence of 5 plant pathogens to identify conserved sequences present in all of five different plant pathogens. The complete genomes of five pathogenic bacteria (Clavibacter michiganensis, Erwinia carotovora, Ralstonia solanacearum, Xanthamonas citri and Xanthamonas campestris) were compared with a suffix
tree algorithm. These comparisons have led to the identification of a gene that is shared by all of these species and presumably can be used as a barcode region. This gene region can be amplified in all five species using a degenerate primer set and is currently being tested for level of sequence divergence in closely related species. In the second approach we examined the protein sequences from the completed genomes of fifteen bacterial species, including several plant pathogens, to identify proteins that are present a) as a single copy gene, b) in all organisms and c) contain sufficient phylogenetic signal to distinguish close relatives. Using this method we were able to identify 47 of the 4,427 Xanthomonas genes as potential barcode regions for plant pathogens.
Impacts
High levels of commercial traffic and concerns about bioterrorism make identification, detection and tracking of plant pests a priority. Our long-term objective is to develop a DNA-based diagnostic barcode, which uniquely identifies bacterial isolates at the subspecies level and distinguishes among subpopulations of pathogens based on race, host plant and geographic origin. Such a bar-coding system will enable rapid diagnosis and risk assessment of future disease outbreaks. Furthermore, this system will make detailed epidemiological studies of these pathogens possible and provide a comprehensive method for measuring genome evolution (e.g. determination of which genome adaptations increase virulence of a pathogen following introduction to Hawaii). Traditional methods of plant pathogen identification involve diagnostic tests that can take several days. The introduction of a genetic barcode test for phytopathogenic bacteria will reduce this period to a few hours,
allowing rapid testing of plant or seed shipments. Immunological tests can be used to identify several plant pathogens at the subspecies level, but different sets of antibodies need to be carried used for every group of pathogens. The major advantage of DNA barcodes over these immunological tests is the universal applicability of the former.
Publications
- No publications reported this period
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