Use of Polymerase Chain Reaction to Detect Pathogenic Strains ofAgrobacterium

Dong, Lu; Sun, C.-W.; Thies, Karen L.; Luthe, D. S.; Graves, C. H.

doi:10.1094/phyto-82-434

Cited by 38 publications

(23 citation statements)

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“…Polymerase chain reaction (PCR) assay was applied for the detection of 34 strains of Agrobacterium tumefaciens (At) infecting Vitis spp. The results of PCR assay were corroborated by the conclusions from pathogenicity tests and DNA slot blot hybridization technique (Dong et al 1992). By employing two PCR primers designed from the sequences of virD2 and ipt genes, a wide range of pathogenic strains of At were detected.…”

Section: Polymerase Chain Reactionmentioning

confidence: 66%

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: Polymerase Chain Reactionmentioning

confidence: 66%

Detection of Bacterial and Phytoplasmal Pathogens

Narayanasamy¹

2010

Microbial Plant Pathogens-Detection and Disease Diagnosis:

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“…The introduction of the polymerase chain reaction (PCR) in plant pathology (Louws et al, 1999) opened up new possibilities for rapid detection and identification of Agrobacterium in agriculturally important plants. First studies were started in the early 90s (Dong et al, 1992;Schulz et al, 1993). Primers designed for the amplification of pathogenic strains have been based on specific chromosomal (Ponsonnet and Nesme, 1994;Eastwell et al, 1995;Szegedi and Bottka, 2002), or Ti plasmid sequences including the vir-region (Ponsonnet and Nesme, 1994;Haas et al, 1995;Sawada et al, 1995) or T-DNA (Dong et al, 1992;Schulz et al, 1993;Haas et al, 1995;Kauffmann et al, 1996;Pulawska and Sobiczewski, 2005).…”

Section: Diagnostic Methodsmentioning

confidence: 99%

“…First studies were started in the early 90s (Dong et al, 1992;Schulz et al, 1993). Primers designed for the amplification of pathogenic strains have been based on specific chromosomal (Ponsonnet and Nesme, 1994;Eastwell et al, 1995;Szegedi and Bottka, 2002), or Ti plasmid sequences including the vir-region (Ponsonnet and Nesme, 1994;Haas et al, 1995;Sawada et al, 1995) or T-DNA (Dong et al, 1992;Schulz et al, 1993;Haas et al, 1995;Kauffmann et al, 1996;Pulawska and Sobiczewski, 2005). In order to increase the sensitivity of detection PCR methods were combined with serological techniques ('immunocapture', Kauffmann et al, 1996).…”

Section: Diagnostic Methodsmentioning

confidence: 99%

Agrobacterium: A disease-causing bacterium

Otten

Burr

Szegedi³

2008

Agrobacterium: From Biology to Biotechnology

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“…2). Suzaki et al, 2004）；2） Ti プラスミド特異的プライマー（Cubero et al, 2006Haas et al, 1995;Picard et al, 1992;Pionnat et al, 1995;Pulawska and Sobiczewski, 2005;Puopolo et al, 2007;Sobiczewski et al, 2005）；3）Ri プラスミド特異的プライマー（伊予住・市川，1999）；4）特定の系統（オパイン型など）の病原性プラスミドのみを対象としたプライマー（Bini et al, 2008Dong et al, 1992;Eastwell et al, 1995;Kaufmann et al, 1996;Szegedi and Bottka, 2002;Tan et al, 2003;Weller and Stead, 2002;Yakabe et al, (Table 2) to determine which pathogenic plasmid respective strains carry and which pathogenic state they belong to. Amplification of the DNA fragments (780-784 bp) from the 16S rRNA gene (16S rDNA) (internal control) of the respective strains indicated that the respective PCR reactions were completed properly.…”

Section: Te Buffer 340unclassified

Simultaneous plasmid profiling of phytopathogenic Rhizobium species (former Agrobacterium species) using multiplex colony-direct PCR.

宏之

浩一

章

2016

Jpn. J. Phytopathol.

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(2016). Simultaneous plasmid profiling of phytopathogenic Rhizobium species (former Agrobacterium species) using multiplex colony-direct PCR. Jpn. J. Among Rhizobium species, six species include plant pathogens as a member: R. radiobacter species complex (including R. nepotum and R. pusense), R. rhizogenes, R. vitis, R. rubi, R. larrymoorei, and R. skierniewicense. Based on their pathogenic states, the members belonging to the six species can be divided into three types, namely, crown gall bacteria carrying Ti plasmid, hairy root bacteria carrying Ri plasmid, and nonpathogenic bacteria carrying neither pathogenic plasmids. In this study, we developed a plasmid-profiling method using a multiplex colony-direct PCR to identify any pathogenic plasmid present in the respective strains and thus determine their pathogenic state. For that purpose, a universal primer set for both pathogenic plasmids and a specific primer set for the Ri plasmid were designed based on the sequences of virC1 and rolC, respectively. The universal primer set for 16S rRNA gene (16S rDNA), chosen as an internal control, was also added to the PCR mix to readily judge the success of the reaction and exclude false-negative reactions. Reliability of the method as a plasmid-profiling tool was then validated using the Rhizobium species containing plant pathogens and their relatives of 383 strains in total. As a result, two DNA fragments, one from virC1-specific amplification (278 bp) and the other (780-784 bp) from 16S rDNA as the internal control, were stably obtained from all crown gall bacteria used. For hairy root bacteria, three fragments derived from virC1 (278 bp), rolC (438 bp) and 16S rDNA were always observed. On the other hand, only a fragment derived from 16S rDNA was amplified from the other nonpathogenic bacteria. Therefore, this method is useful for rapid plasmid profiling of the Rhizobium species containing plant pathogens, which will improve the efficiency in areas such as surveillance and diagnosis of crown gall and hairy root diseases and epidemiological and ecological studies of the pathogens.

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Use of Polymerase Chain Reaction to Detect Pathogenic Strains ofAgrobacterium

Cited by 38 publications

References 0 publications

Detection of Bacterial and Phytoplasmal Pathogens

Detection of Bacterial and Phytoplasmal Pathogens

Agrobacterium: A disease-causing bacterium

Simultaneous plasmid profiling of phytopathogenic <i>Rhizobium</i> species (former <i>Agrobacterium </i>species) using multiplex colony-direct PCR.

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