Skip to main content
SpringerLink
Log in

Molecular detection of a bacterial contaminant Bacillus pumilus in symptomless potato plant tissue cultures

  • Biotic and Abiotic Stress
  • Published:
Plant Cell Reports Aims and scope Submit manuscript

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.

This is a preview of subscription content, log in via an institution to check access.

Access this article

Log in via an institution

Price excludes VAT (USA)
Tax calculation will be finalised during checkout.

Instant access to the full article PDF.

Institutional subscriptions

Fig. 1.
Fig. 2.
Fig. 3.
Fig. 4.
Fig. 5.

Similar content being viewed by others

Abbreviations

PCR-RFLP :

Polymerase chain reaction-restriction fragment length polymorphism

RAPD :

Random amplified polymorphic DNA

SCAR :

Sequence characterised amplified region polymorphism

References

  • Ashmore SE (1997) Status report on the development and application of in vitro techniques for the conservation and use of plant genetic resources. International Plant Genetic Resources Institute, Rome, Italy

  • Benjama A, Charkaoui B (1997) Control of Bacillus contaminating date palm tissue in micropropagation using antibiotics. In: Cassells AC (ed) Pathogen and microbial management in micropropagation. Kluwer, Dordrecht, pp 237–244

  • Buckley PM, DeWilde TN, Reed BM (1995) Characterization and identification of bacteria isolated from micropropagated mint plants. In Vitro Cell Dev Biol Plant 31:58–64

    Google Scholar 

  • Cassells AC (1991) Problems in tissue culture: culture contamination. In: Debergh PC, Zimmerman RH (eds) Micropropagation technology and application. Kluwer, Dordrecht, pp 31–44

  • Cassells AC (1997) Pathogen and microbial contamination management in micropropagation—an overview. In: Cassells AC (ed) Pathogen and microbial management in micropropagation. Kluwer, Dordrecht, pp 1–14

  • Debergh PC, Vanderschaeghe AM (1988) Some symptoms indicating the presence of bacterial contaminants in plant tissue cultures. Acta Hortic 225:77–81

    Google Scholar 

  • Falkiner FR (1997) Antibiotics in plant tissue culture and micropropagation-what are we aiming at? In: Cassells AC (ed) Pathogen and microbial management in micropropagation. Kluwer, Dordrecht, pp 155–160

  • Fellner M, Kneifel W, Gregorits D, Leonhardt W (1996) Identification and antibiotic sensitivity of microbial contaminants from callus cultures of garlic Allium sativum L. and Allium longicuspis Regel. Plant Sci 113:193–201

    Article  CAS  Google Scholar 

  • Gaëta BA (1997) ANGIS bioinformatics handbook. CSIRO, Australia

  • Gaëta B (2000) blast on the web. Biotechniques 28:436–439

    Google Scholar 

  • Granada GA, Sequeira L (1983) A new selective medium for Pseudomonas solanacearum. Plant Dis 67:1084–1088

    Google Scholar 

  • Heid CA, Stevens J, Livak KJ, Williams PM (1996) Real time quantitative PCR. Genome Res 6:986–994

    CAS  PubMed  Google Scholar 

  • Herman EB (1999) New method for detecting latent contaminants in plant tissues. Agricell Rep 33:21

    Google Scholar 

  • Isenegger DA, Taylor PWJ, Ford R, Franz P, McGregor GR, Hutchinson JF (2001) DNA fingerprinting and genetic relationships of potato cultivars (Solanum tuberosum L.) commercially grown in Australia. Aust J Agric Res 52:911–918

    Article  CAS  Google Scholar 

  • Kamoun R, Lepoivre P, Boxus P (1997) Evidence for the occurrence of endophytic prokaryotic contaminants in micropropagated plantlets of Prunus cerasus cv. Montmorency. In: Cassells AC (ed) Pathogen and microbial management in micropropagation. Kluwer, Dordrecht, pp 145–148

  • Legatt IV, Waites WM (1988) Characterisation of micro-organisms isolated from plants during micropropagation. Acta Hortic 225:93–102

    Google Scholar 

  • Leifert C, Cassells AC (2001) Microbial hazards in plant tissue and cell cultures. In Vitro Cell Dev Biol 37:133–138

    Google Scholar 

  • Leifert C, Woodward S (1997) Laboratory contamination management; the requirement for microbiological quality assurance. In: Cassells AC (ed) Pathogen and microbial management in micropropagation. Kluwer, Dordrecht, pp 237–244

  • Martin RR, James D, André Lévesque C (2000) Impacts of molecular diagnostic technologies on plant disease management. Annu Rev Phytopathol 38:207–239

    Article  CAS  PubMed  Google Scholar 

  • Paran I, Michelmore RW (1993) Development of reliable PCR-based markers linked to downy mildew resistance genes in lettuce. Theor Appl Genet 85:985–993

    CAS  Google Scholar 

  • Poussier S, Luisetti J (2000) Specific detection of biovars of Ralstonia solanacearum in plant tissues by nested-PCR-RFLP. Eur J Plant Pathol 106:255–265

    Article  CAS  Google Scholar 

  • Reed BM, Tansprasert P (1995) Detection and control of bacterial contaminants of plant tissue cultures: a review of recent literature. Plant Tissue Cult Biotechnol 1:137–142

    Google Scholar 

  • Reeves JC (1997) Molecular diagnostics for pathogen detection in seeds and planting material. In: Cassells AC (ed) Pathogen and microbial management in micropropagation. Kluwer, Dordrecht, pp 83–96

  • Seal S (1997) Cost analysis of detection of bacteria and phytoplasmas in plant tissue cultures by PCR. In: Cassells AC (ed) Pathogen and microbial management in micropropagation. Kluwer, Dordrecht, pp 123–130

  • Stead DE, Hennessy J, Wilson J (1997) Modern methods for identifying bacteria. In: Cassells AC (ed) Pathogen and microbial management in micropropagation. Kluwer, Dordrecht, pp 61–74

  • Taylor PWJ (1997) In vitro germplasm conservation of sugarcane cultivars, basic sugarcane species and related genera. In: Razdan MK, Cocking EC (eds) Conservation of plant genetic resources in vitro. Science, USA, pp 243–256

  • Towner P (1991) Isolation of DNA by SDS-proteinase K treatment. In: Brown TA (ed) Essential molecular biology, vol 1. A practical approach. IRL Press, Oxford University Press, UK, pp 52–53

  • Tsoktouridis G, Mantell S, Bantinaki E, Thangavelu M (1997) DNA profiling and strategies for in planta localization of bacteria intimately associated with Billbergia magnifica ssp. acutisapalia. In: Cassells AC (ed) Pathogen and microbial management in micropropagation. Kluwer, Dordrecht, pp 115–122

  • Wang X, Fang Z, Huang S, Sun P, Liu Y, Yang L, Zhuang M, Qu D (2000) An extended random primer amplified region (ERPAR) marker linked to a dominant male sterility gene in cabbage (Brassica oleracea var. capitata). Euphytica 112:267–273

    Article  CAS  Google Scholar 

  • Weisberg WG, Barns SM, Pelletier DA, Lane DJ (1991) 16S ribosomal DNA amplification for phylogenetic study. J Bacteriol 173:697–703

    CAS  PubMed  Google Scholar 

  • Weller SA, Aspin A, Stead DE (2000) Classification and identification of plant-associated bacteria by fatty acid profiling. Bull OEPP/EPPO Bull 30:375–380

    Google Scholar 

  • Zijlstra C (2000) Identification of Meloidogyne chitwoodi, M. fallax and M. hapla based on SCAR-PCR: a powerful way of enabling reliable identification of populations or individuals that share common traits. Eur J Plant Pathol 106:283–290

    Article  CAS  Google Scholar 

Download references

Acknowledgments

We thank Horticulture Australia Ltd, the Australian Potato Industry Council, AusIndustry and Department of Natural Resources and Environment for financial support. We also would like to thank IHD-Knoxfield colleagues Dr. Jo Luck and Dr. Brendan Rodoni for guidance with bacterial taxonomy, Dr. Nancy Jensen from Food Science Australia for technical advice and Dr. Ric Cother from NSW Agriculture for the supply of bacterial cultures.

Author information

Authors and Affiliations

  1. Institute for Horticultural Development, Agriculture Victoria, Knoxfield, Private Bag 15, 3180, Ferntree Gully Delivery Centre, Victoria, Australia

    D. A. Isenegger, G. R. McGregor, M. Barlass & J. F. Hutchinson

  2. BioMarka, School of Agriculture and Food Systems, Institute of Land and Food Resources, The University of Melbourne, 3030, Victoria, Australia

    P. W. J. Taylor

  3. Boulevarde Nurseries, 3498, Irymple, Victoria, Australia

    K. Mullins

Authors
  1. D. A. Isenegger
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. P. W. J. Taylor
    View author publications

    You can also search for this author in PubMed Google Scholar

  3. K. Mullins
    View author publications

    You can also search for this author in PubMed Google Scholar

  4. G. R. McGregor
    View author publications

    You can also search for this author in PubMed Google Scholar

  5. M. Barlass
    View author publications

    You can also search for this author in PubMed Google Scholar

  6. J. F. Hutchinson
    View author publications

    You can also search for this author in PubMed Google Scholar

Corresponding author

Correspondence to D. A. Isenegger.

Additional information

Communicated by P. Debergh

Nucleotide sequence 2-10500 is Genbank accession number AF396672. Nucleotide sequence for the almost-complete 16S rDNA is Genbank accession number AY112667.

Rights and permissions

Reprints and permissions

About this article

Cite this article

Isenegger, D.A., Taylor, P.W.J., Mullins, K. et al. Molecular detection of a bacterial contaminant Bacillus pumilus in symptomless potato plant tissue cultures. Plant Cell Rep 21, 814–820 (2003). https://doi.org/10.1007/s00299-003-0583-z

Download citation

  • Received:

  • Revised:

  • Accepted:

  • Published:

  • Issue Date:

  • DOI: https://doi.org/10.1007/s00299-003-0583-z

Keywords

Search

Navigation