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Research Article Open access CC BY 3.0

Improved Sample Preparation for PCR-Based Assays in the Detection of Xanthomonads Causing Bacterial Leaf Spot of Tomato

E. R. Mbega, J. Adriko, C. N. Mortensen, E. G. Wulff, O. S. Lund, R. B. Mabagala

Biotechnology Journal International · pp. 556–574 · Published 27 Aug 2013

10.9734/BBJ/2013/3810

Abstract

Aims: To develop a sampling procedure for PCR-based screening of bacterial leaf spot (BLS)-causing xanthomonads without DNA extraction from infected tomato plants. Place and Duration of Study: University of Copenhagen, Denmark and Sokoine University of Agriculture, Morogoro, Tanzania between July 2008 and November 2010. Methodology: Flinders Technology Associates (FTA®) plant cards and Chromatography paper or Whatman® paper strips (WPS) were spotted with bacterial suspensions from 24-h-old cultures from reference strains of BLS-causing xanthomonads, or sap obtained by grinding or hand maceration of plant tissue, were used as templates in PCR reactions or isolation of live bacterial cells on Nutrient agar (NA) media. Samples were tested by PCR with Xan 7 genus/-specific Xanthomonas primers or in multiplex with 26S rRNA primers. Isolation of bacteria was done by streaking aliquots of 75 µl of a suspension from a disc (2-mm-punch by Harris Micro Punch®) in triplicate, removed from each of the FTA plant card and WPS onto NA media.  Results: The FTA plant card spotted with pure cultures of reference strains of xanthomonads and sap from grinding or direct maceration of plant tissue resulted in more clear PCR bands (402 bp) and (594 bp of rRNA gene in multiplex) than the WPS samples. Sensitivity of detection by the FTA paper-based PCR was ≈ 5.0 x 102, while that of the WPS was > 1.0 x 103 CFU/ml. The WPS (but not the FTA) was proved to be useful for saving living bacteria cells for up to one week of storage at ambient temperatures. Conclusion: Both FTA plant card and WPS can be used for PCR detection of BLS-causing xanthomonads in tomato. However, the FTA plant card is recommended as it produced clearer PCR products than WPS. WPS is recommended for experiments requiring isolation of live bacterial cells on NA media.

FTA Whatman diagnosis bacteria tomato

References (29)

  1. 1 Methods for the diagnosis of bacterial diseases of plants
  2. 2 Reclassification of the Xanthomonads Associated with Bacterial Spot Disease of Tomato and Pepper [DOI]
  3. 3 Collecting, archiving and processing DNA from wildlife samples using FTA® databasing paper [DOI]
  4. 4 Culturable Leaf-Associated Bacteria on Tomato Plants and Their Potential as Biological Control Agents [DOI]
  5. 5 Improved Template Preparation for PCR-Based Assays for Detection of Food-Borne Bacterial Pathogens [DOI]
  6. 6 Application of FTA technology for sampling, recovery and molecular characterization of viral pathogens and virus-derived transgenes from plant tissues [DOI]
  7. 7 Tween Media for Semiselective Isolation ofXanthomonas campestrispv.vesicatoriafrom Soil and Plant Material [DOI]
  8. 8 Characterization and PCR-based Typing of Xanthomonas campestris pv. vesicatoria from Peppers and Tomatoes in Serbia [DOI]
  9. 9 Identification and Characterisation of Xanthomonas campestris pv. campestris Strains from Tanzania by Pathogenicity Tests, Biolog, rep-PCR and Fatty Acid Methyl Ester Analysis [DOI]
  10. 10 Development and Evaluation of PCR-Based Diagnostic Assays for the Bacterial Speck and Bacterial Spot Pathogens of Tomato [DOI]
  11. 11 Physiological, Chemical, Serological, and Pathogenic Analyses of a Worldwide Collection ofXanthomonas campestrispv.vesicatoriaStrains [DOI]
  12. 12 Evaluation of the Biolog GN MicroPlate System for Identification of Some Plant-Pathogenic Bacteria [DOI]
  13. 13 Evidence for the Preemptive Nature of Tomato Race 3 ofXanthomonas campestrispv.vesicatoriain Florida [DOI]
  14. 14 Detection ofXanthomonas campestrispv.vesicatoriaAssociated with Pepper and Tomato Seed by DNA Amplification [DOI]
  15. 15 Inactivation, Storage, and PCR Detection of Mycoplasma on FTA®Filter Paper [DOI]
  16. 16 Detection of Plant Genes Using a Rapid, Nonorganic DNA Purification Method [DOI]
  17. 17 Optimization of FTA technology for large scale plant DNA isolation for use in marker assisted selection
  18. 18 Application of FTA® technology to extraction of sperm DNA from mixed body fluids containing semen [DOI]
  19. 19 Application of FTA®Cards to Sample Microbial Plant Pathogens for PCR and RT‐PCR [DOI]
  20. 20 Bacterial spot ( Xanthomonas campestris pv. vesicatoria ) of tomato and sweet pepper in Tanzania [DOI]
  21. 21 OCCURRENCE OF XANTHOMONAS CAMPESTRIS PV. RAPHANI ON TOMATO PLANTS IN THE RUSSIAN FEDERATION [DOI]
  22. 22 A New Disease, Arboricola Leaf Spot of Bell Pepper, Caused by Xanthomonas arboricola [DOI]
  23. 23 Development of a diagnostic DNA probe for xanthomonads causing bacterial spot of peppers and tomatoes [DOI]
  24. 24 Application of FTA� sample collection and DNA purification system on the determination of CTG trinucleotide repeat size by PCR-based southern blotting [DOI]
  25. 25 PCR‐genotyping of barley seedlings using DNA samples from tissue prints [DOI]
  26. 26 Current status of bacterial -speck and -spot diseases of tomato in three tomato-growing regions of Tanzania [DOI]
  27. 27 A Medium for Differentiating Tomato and Pepper Strains of Xanthomonas Campestris pv.Vesicatoria [DOI]
  28. 28 Molecular characterisation of cowpea breeding lines for Striga resistance using FTA® technology.
  29. 29 Serotypes of Xanthomonas vesicatoria Unrelated to Its Pathotypes [DOI]

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