title: Molecular detection of a bacterial contaminant Bacillus pumilus in symptomless potato plant tissue cultures.
abstract: An aberrant random amplified polymorphic DNA (RAPD) marker in genomic DNA of tissue culture plantlets was frequently observed during a comparison of DNA fingerprints derived from potato germplasm grown in tissue culture and the field. The RAPD marker was cloned, sequenced and determined to be of bacterial origin. A bacterial contaminant was isolated from the tissue culture plants and identified as a Bacillus pumilus. A set of sequence characterised amplified region (SCAR) primers were designed from the sequence of the cloned fragment and tested for the specific detection of B. pumilus. Polymerase chain reaction-restriction fragment length polymorphisms (PCR-RFLPs) were also used to generate B. pumilus profiles specific to our isolate in order to test and confirm the sequence homology of amplified markers generated from a range of DNA samples isolated from tissue culture plants and pure isolates of B. pumilus-like bacteria.
authors.affiliation: Institute for Horticultural Development, Agriculture Victoria, Knoxfield, Private Bag 15, 3180, Ferntree Gully Delivery Centre, Victoria, Australia. daniel.isenegger@nre.vic.gov.au
authors.forename: D A
authors.initials: DA
authors.lastname: Isenegger
authors.forename: P W J
authors.initials: PW
authors.lastname: Taylor
authors.forename: K
authors.initials: K
authors.lastname: Mullins
authors.forename: G R
authors.initials: GR
authors.lastname: McGregor
authors.forename: M
authors.initials: M
authors.lastname: Barlass
authors.forename: J F
authors.initials: JF
authors.lastname: Hutchinson
mesh_terms: D001407:Bacillus / Q000235:genetics* / Q000302:isolation & purification; D001483:Base Sequence; D046508:Culture Techniques; D004269:DNA, Bacterial / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification; D008969:Molecular Sequence Data; D010935:Plant Diseases / Q000382:microbiology*; D016133:Polymerase Chain Reaction; D012150:Polymorphism, Restriction Fragment Length; D012336:RNA, Ribosomal, 16S / Q000235:genetics; D019105:Random Amplified Polymorphic DNA Technique / Q000379:methods; D012680:Sensitivity and Specificity; D017422:Sequence Analysis, DNA; D011198:Solanum tuberosum / Q000235:genetics / Q000382:microbiology*
chemical_list: D004269:DNA, Bacterial; D012336:RNA, Ribosomal, 16S