title: Molecular detection of bacteria in plant tissues, using universal 16S ribosomal DNA degenerated primers.
abstract: Highly specific, sensitive and rapid tests are required for the detection and identification of covert bacterial contaminations in plant tissue cultures. Current methods available for this purpose are tedious, time consuming, highly error prone, expensive, require advanced technical expertise and are sometimes ineffective. We report here the development of a sensitive polymerase chain reaction (PCR) based method for the rapid detection and identification of bacteria occurring in plant tissue cultures. A total of 121 16S ribosomal DNA (rDNA) coding regions from 14 different groups of bacteria, algae and plants, available in theGene Bank/European Molecular Biology Laboratorydatabases, were aligned and several conserved DNA sequences of bacterial origin were identified. From those, five degenerated primers were designed in order to amplify only the bacterial DNA present in mixed plant/bacteria genomic DNA extracts. A known amount of bacterial suspension of either covertPseudomonasor covertBacilluswere added toin vitroplant leaves and total plant/bacterial DNA extracted using three different methods to determine the lowest number of bacteria required to be present in order to allow their detection. The highest sensitivity of the bacterial cell detection was 2.5 × 106cells of bothBacillusandPseudomonasinoculums, using template DNA prepared by theMiniPrepmethod. Generation of PCR amplification fragments was achieved only for the 16S rDNA bacterial gene by using four combinations of degenerated primers. Successive sequence analysis of these amplified fragments led to the rapid detection and molecular identification of bacteria covertly associated with plants.
authors.affiliation: Department of Agricultural Sciences, Imperial College at Wye, University of London , Ashford , Kent , UK ; Department of Crop Production, School of Agriculture, Alexander Technological Educational Institute of Thessaloniki , Thessaloniki , Greece.
authors.forename: Georgios
authors.initials: G
authors.lastname: Tsoktouridis
authors.affiliation: Department of Agricultural Sciences, Imperial College at Wye, University of London , Ashford , Kent , UK ; Department of Environmental and Natural Resources Management, University of Western Greece , Agrinio , Greece.
authors.forename: George
authors.initials: G
authors.lastname: Tsiamis
authors.affiliation: Department of Crop Production, School of Agriculture, Alexander Technological Educational Institute of Thessaloniki , Thessaloniki , Greece.
authors.forename: Nikolaos
authors.initials: N
authors.lastname: Koutinas
authors.affiliation: Department of Agricultural Sciences, Imperial College at Wye, University of London , Ashford , Kent , UK ; Nakhlatec International Development Advisors , Gödelöv , Genarp , Sweden.
authors.forename: Sinclair
authors.initials: S
authors.lastname: Mantell
keywords: 16S rDNA; PCR; bacteria; degenerated primers; diagnostics; plant tissues