Nucleic Acid Probes for Identification of Phytobacteria: Identification of Genus-Specific 16s rRNA Sequences

DeParasis, J

doi:10.1094/phyto-80-618

Cited by 29 publications

(11 citation statements)

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“…The sensitivity of the probe derived from unique 16S rRNA sequences is greatly enhanced, because of the presence of large number of copies in a bacterial cell. A DNA sequence based on the 16S rRNA which hybridized only with plant pathogenic pathovars of Xoo could be identified (De Parasis and Roth 1990).…”

Section: Nucleic Acid Hybridization Methodsmentioning

confidence: 99%

“…A sequence of rRNA gene of X. oryzae pv. oryzae that specifically hybridized only with this pathovar was identified and employed for its detection (De Parasis and Roth 1990). Likewise, a probe specific for P. glumae causing bacterial grain rot of rice was designed and hybridization between the DNA of all strains of P. glumae and the probe occurred, but not with X. oryzae pv.…”

Section: 3 Detection Of Bacterial Pathogens In Seeds and Planting Mamentioning

confidence: 99%

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Detection of Bacterial and Phytoplasmal Pathogens

Narayanasamy¹

2010

Microbial Plant Pathogens-Detection and Disease Diagnosis:

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Bacterial pathogens classified as prokaryotes are much simpler in structure and smaller in size and they have less complex structural features compared to fungal plant pathogens. As the morphological characteristics of bacterial cells are less variable, biological, biochemical, physiological, immunological and genomic characteristics have to be determined for reliable identification and meaningful classification of bacterial pathogens. Detection and identification of bacterial plant pathogens present in whole plants and in propagative plant materials have been possible by employing isolation on cultural media and metabolic fingerprinting methods. But they are labor-intensive and require long time. Results often are inconclusive. Isozyme analysis, direct colony thin layer chromatography and gel electrophoresis techniques have been successfully applied for the detection of some bacterial pathogens. Immunoassays and nucleic acid-based assays have become widely accepted techniques, providing more sensitive and specific detection and quantification of bacterial pathogens affecting a wide range of host plant species. However, the major limitation of the molecular techniques is their inability to discriminate living cells from the dead ones. Attempts have been made to overcome this limiting factor by using DNA binding dyes and estimating pathogen RNAs as an indicator of cell viability. The diagnostic methods have both merits and demerits and hence, appropriate method has to be selected based on cost-effectiveness and possibility of obtaining reliable results rapidly to suit the requirements of the investigation concerned.Phytoplasmas are cell wall-less, nonculturable bacteria belonging to Mollicutes. Lack of cultural characteristics has made obligatory to study the morphological characteristics of phytoplasmas present in the phloem cells of infected plant hosts by observing under electron microscopes. As most of the phytoplasmas look alike in the ultrathin sections, the morphological characters have no diagnostic value. Histochemical methods using DNA binding dyes have been employed to localize the phytoplasmas in the host cells. Application of immunoassays and nucleic acidbased techniques has been effective in providing information for the identification and differentiation of phytoplasams. Polyclonal and monoclonal antibodies have been generated for detection of phytoplasmas infecting several plant species. Universal and species-specific primers and probes have been designed based on

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Section: Nucleic Acid Hybridization Methodsmentioning

confidence: 99%

Section: 3 Detection Of Bacterial Pathogens In Seeds and Planting Mamentioning

confidence: 99%

Detection of Bacterial and Phytoplasmal Pathogens

Narayanasamy¹

2010

Microbial Plant Pathogens-Detection and Disease Diagnosis:

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“…In the case of bacteria and fungi in particular, identification of discriminating sequences in a relatively large genome can be difficult. One strategy which can make the search easier is to concentrate on regions known to be variable, such as parts of the rRNA coding genes (DeParasis & Roth, 1990;Moukhamedov et al, 1994).…”

Section: Nucleic Acid Based Diagnosticsmentioning

confidence: 99%

Plant pathogen diagnostics: present status and future developments

O'Donnell

1999

Potato Res

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“…In the last two decades, molecular methods, especially 16S rRNA gene sequencing, have been a reliable aid to the identification of diverse bacteria. Although the 16S rRNA method has served as a powerful tool for finding phylogenetic relationships among bacteria (24), because of its molecular clock properties and the large database for sequence comparison, the molecule is too conserved to provide good resolution at the species and subspecies levels (2,4,9,20,23). The relationship between 16S rRNA gene similarity and percent DNA-DNA reassociation is a logarithmic function in which the sequence similarity within a species (Ͼ70% DNA relatedness) is expected to be Ͼ98% (3), and the similarity among different species in a genus, e.g., fluorescent Pseudmonas spp., is 93.3 to ϳ99.9% (11).…”

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confidence: 99%

Bacterial Species Determination from DNA-DNA Hybridization by Using Genome Fragments and DNA Microarrays

Cho

Tiedje

2001

Appl Environ Microbiol

192

101

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Whole genomic DNA-DNA hybridization has been a cornerstone of bacterial species determination but is not widely used because it is not easily implemented. We have developed a method based on random genome fragments and DNA microarray technology that overcomes the disadvantages of whole-genome DNA-DNA hybridization. Reference genomes of four fluorescent Pseudomonas species were fragmented, and 60 to 96 genome fragments of approximately 1 kb from each strain were spotted on microarrays. Genomes from 12 well-characterized fluorescent Pseudomonas strains were labeled with Cy dyes and hybridized to the arrays. Cluster analysis of the hybridization profiles revealed taxonomic relationships between bacterial strains tested at species to strain level resolution, suggesting that this approach is useful for the identification of bacteria as well as determining the genetic distance among bacteria. Since arrays can contain thousands of DNA spots, a single array has the potential for broad identification capacity. In addition, the method does not require laborious cross-hybridizations and can provide an open database of hybridization profiles, avoiding the limitations of traditional DNA-DNA hybridization.Bacterial identification methods currently used include analysis of morphological, physiological, biochemical, and genetic data. In the last two decades, molecular methods, especially 16S rRNA gene sequencing, have been a reliable aid to the identification of diverse bacteria. Although the 16S rRNA method has served as a powerful tool for finding phylogenetic relationships among bacteria (24), because of its molecular clock properties and the large database for sequence comparison, the molecule is too conserved to provide good resolution at the species and subspecies levels (2,4,9,20,23). The relationship between 16S rRNA gene similarity and percent DNA-DNA reassociation is a logarithmic function in which the sequence similarity within a species (Ͼ70% DNA relatedness) is expected to be Ͼ98% (3), and the similarity among different species in a genus, e.g., fluorescent Pseudmonas spp., is 93.3 to ϳ99.9% (11). Considering the high sequence conservation and relative standard errors at 98 and 90% sequence similarities of 19 and 8%, respectively (5), 16S rDNA analysis results on closely related strains could be inaccurate and inconsistent with the results obtained by other methods. Incongruity between genome structure and 16S rDNA sequence similarity was also reported (8). Since many important ecological and clinical characteristics of bacteria, such as pathogenicity, competitiveness, substrate range, and bioactive molecule production, vary below the species level, methods with higher resolution than that of 16S rDNA sequencing are needed.DNA-DNA hybridization is one method that provides more resolution than 16S rDNA sequencing, and the 70% criterion (22) has been a cornerstone for describing a bacterial species. In spite of these values, the method is not popular. Major disadvantages are the laborious nature of pairwise cross-hyb...

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Nucleic Acid Probes for Identification of Phytobacteria: Identification of Genus-Specific 16s rRNA Sequences

Cited by 29 publications

References 0 publications

Detection of Bacterial and Phytoplasmal Pathogens

Detection of Bacterial and Phytoplasmal Pathogens

Plant pathogen diagnostics: present status and future developments

Bacterial Species Determination from DNA-DNA Hybridization by Using Genome Fragments and DNA Microarrays

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