Molecular detection of a bacterial contaminant Bacillus pumilus in symptomless potato plant tissue cultures.
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.
Isenegger
D A
DA
Institute for Horticultural Development, Agriculture Victoria, Knoxfield, Private Bag 15, 3180, Ferntree Gully Delivery Centre, Victoria, Australia. daniel.isenegger@nre.vic.gov.au
Taylor
P W J
PW
Mullins
K
K
McGregor
G R
GR
Barlass
M
M
Hutchinson
J F
JF
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*
D004269:DNA, Bacterial; D012336:RNA, Ribosomal, 16S