Rapid Method for Isolation of PCR Amplifiable Genomic DNA of Ralstonia solanacearum Infested in Potato Tubers

Abstract

The aim of the present study was to develop a very fast and simple genomic DNA isolation method for Ralstonia solanacearum which infest potato tubers. One hundred potato tubers were collected and ten composite samples were prepared having 10 tubers each. Four different DNA isolation methods were used for bacterial genomic DNA isolation present in tubers. PCR with R. solanacearum specific primers and pathogenicity tests were performed. Out of four methods two gave PCR amplifiable DNA. The simplest method was boiling the cell lysate for 5 min, vortexing for 2 min then extraction with phenol chloroform method. This method provides significant amount of DNA which is free from contaminants thus rendering the DNA amicable to PCR amplification. The developed method would be useful for quick and sensitive detection of this pathogen in seed potatoes and would be beneficial to stop the further spread of pathogen.

Share and Cite:

A. Grover, S. K. Chakrabarti, W. Azmi and S. M. P. Khurana, "Rapid Method for Isolation of PCR Amplifiable Genomic DNA of Ralstonia solanacearum Infested in Potato Tubers," Advances in Microbiology, Vol. 2 No. 4, 2012, pp. 441-446. doi: 10.4236/aim.2012.24056.

Conflicts of Interest

The authors declare no conflicts of interest.

References

[1] http://www.commodityindia.com/templates/ more_articles.aspx?gid=All&fn=Potato031111
[2] A. C. Hayward, “Biology and Epidemiology of Bacterial Wilt Caused by Pseudomonas solanacearum,” Annual Review of Phytopathology, Vol. 29, 1991, pp. 65-87. doi:10.1146/annurev.py.29.090191.000433
[3] G. S. Shekhawat, A. V. Gadewar and S. K. Chakrabarti, “Potato Bacterial Wilt in India,” Technical Bulletin, Shimla, Vol. 38, 2000, p. 56.
[4] A. V. Gadewar, V. Sunaina and S. K. Chakrabarti, “Bacterial Diseases of Potato and Their Management,” In: S. M. P. Khurana, J. S. Minhas and S. K. Pandey, Eds., The Potato: Production and Utilization in Sub-Tropics, Mehta Publishers, New Delhi, 2003.
[5] M. I. Siri, A. Sanabria and M. J. Pianzzola, “Genetic Diversity and Aggressiveness of Ralstonia solanacearum Strains Causing Bacterial Wilt of Potato in Uruguay,” Plant Disease, Vol. 95, No. 10, 2011, pp. 1292-1301. doi:10.1094/PDIS-09-10-0626
[6] A. Grover, W. Azmi, A. V. Gadewar, D. Pattanayak, P. S. Naik, G. S. Shekhawat and S. K. Chakrabarti, “Genotypic Diversity in a Localized Population of Ralstonia Solanacearum as Revealed by Random Amplified Polymorphic DNA Markers,” Journal of Applied Microbiology, Vol. 101, No. 4, 2006, pp. 798-806. doi:10.1111/j.1365-2672.2006.02974.x
[7] K. Anitha, S. K. Chakrabarty, G. A. Girish, R. D. V. J. P. Rao and K. S. Varaprasad, “Detection of Bacterial Wilt Infection in Imported Groundnut Germplasm,” Indian Journal of Plant Protection, Vol. 32, 2004, pp. 147-148.
[8] J. Schonfeld, H. Heuer, J. V. Elsas, K. Smalla and J. D. van Elsas, “Specific and Sensitive Detection of Ralstonia solanacearum in Soil on the Basis of PCR Amplification of Flic Fragments,” Applied and Environmental Microbiology, Vol. 69, No. 12, 2003, pp. 7248-7256. doi:10.1128/AEM.69.12.7248-7256.2003
[9] A. Grover, W. Azmi, S. M. P. Khurana and S. K. Chakrabarti, “Multiple Displacement Amplification as a Pre-Polymerase Chain Reaction (Pre-PCR) to Detect Ultra Low Population of Ralstonia solanacearum (Smith 1896) Yabuchi et al. (1996),” Letters in Applied Microbiology, Vol. 49, No. 5, 2009, pp. 539-543. doi:10.1111/j.1472-765X.2009.02687.x
[10] K. H. Pastrik and E. Maiss, “Detection of Ralstonia solanacearum in Potato Tubers by Polymerase Chain Reaction,” Journal of Phytopathology, Vol. 148, No. 11-12, 2000, pp. 619-626. doi:10.1046/j.1439-0434.2000.00567.x
[11] S. Poussier, J. J. Cheron, A. Couteau and J. Luisetti, “Bacterial Wilt Disease and the Ralstonia solanacearum Species Complex,” APS Press, Saint Paul, 2005.
[12] J. Sambrook, E. F. Fritsch and T. A. Maniats, “Molecular Cloning: A Laboratory Manual,” Cold Spring Harbor Laboratory Press, New York, 1989.
[13] J. Murmur, “A Procedure for the Isolation of Deoxyribo-nucleic Acid from Micro-Organisms,” Journal of Molecular Biology, Vol. 3, 1961, pp. 208-218. doi:10.1016/S0022-2836(61)80047-8
[14] F. H. Ausbel, R. Brent, R. E. Kingston, D. D. Moore, J. G. Seidman, J. A. Smith and K. Struth, “Current Protocols in Molecular Biology,” John Wiley and Sons, Hoboken, 1994.
[15] S. E. Seal, L. A. Jackson, J. P. W. Young and M. J. Daniels, “Differentiation of Pseudomonas solanacearum, Pseudomonas syzygii, Pseudomonas pickettii and Blood Disease Bacterium by Partial 16S rRNA Sequencing: Construction of Oligonucleotide Primers for Sensitive Detection by Polymerase Chain Reaction,” Journal of General Microbiology, Vol. 139, No. 7, 1993, pp. 1587-1594. doi:10.1099/00221287-139-7-1587
[16] A. Kelman, “The Relationship of Pathogenicity of Pseudomonas solanacearum to Colony Appearance in a Tetrazolium Medium,” Phytopathology, Vol. 44, 1954, pp. 693-695.
[17] L. Overbeek, J. H. W. Bergervoet, F. H. H. Jacobs and J. Elsas, “The Low-Temperature-Induced Viable-but-Non-culturable State Affects the Virulence of Ralstonia solanacearum Biovar 2,” Phytopathology, Vol. 94, No. 5, 2004, pp. 463-469. doi:10.1094/PHYTO.2004.94.5.463
[18] A. C. Hayward, “Characteristics of Pseudomonas solanacearum,” Journal of Applied Bacteriology, Vol. 27, No. 2, 1964, pp. 265-277. doi:10.1111/j.1365-2672.1964.tb04912.x
[19] L. He, L. Sequeira and A. Kelman, “Characteristics of Strains of Pseudomonas solanacearum from China,” Plant Disease, Vol. 67, 1983, pp. 1357-1361. doi:10.1094/PD-67-1357
[20] N. N. Winstead and A. Kelman, “Inoculation Techniques for Evaluating Resistance to Pseudomonas solanacearum,” Phytopathology, Vol. 42, 1952, pp. 628-634.
[21] F. Niepold, “A Simple and Fast Extraction Procedure to Obtain Amplifyable DNA from Ralstonia (Pseudomonas) solanacearum and Clavibacter michiganensis ssp Sepedonicus Inoculated Potato Tuber Extracts and Naturally Infected Tubers to Conduct a Polymerase Chain Reaction (PCR),” Journal of Phytopathology, Vol. 147, 1999, pp. 249-256. doi:10.1046/j.1439-0434.1999.147004249.x
[22] D. N. Miller, J. E. Bryant, E. L. Madsen and W. C. Ghiorse, “Evaluation and Optimization of DNA Extraction and Purification Procedures for Soil and Sediment Samples,” Applied and Environmental Microbiology, Vol. 65, No. 11, 1999, pp. 4715-4724.
[23] H. J. Rogers, N. A. Burns and H. C. Parkes, “Comparison of Small Scale Methods for the Rapid Extraction of Plant DNA Suitable for the PCR Analysis,” Plant Molecular Biology Reporter, Vol. 14, No. 2, 1996, pp. 170-183. doi:10.1007/BF02684906
[24] G. C. Allen, M. A. Flores-Vergara, S. Krasynanski, S. Kumar and W. F. Thompson, “A Modified Protocol for Rapid DNA Isolation from Plant Tissues Using Cetyltrimethylammonium Bromide,” Nature Protocols, Vol. 1, No. 5, 2006, pp. 2320-2325. doi:10.1038/nprot.2006.384
[25] K. A. Lutz, W. Wang, A. Zdepski and T. P. Michael, “Isolation and Analysis of High Quality Nuclear DNA with Reduced Organellar DNA for Plant Genome Sequencing and Resequencing,” BMC Biotechnology, Vol. 11, No. 54, 2011, pp. 1-9. doi:10.1186/1472-6750-11-54
[26] D. W. Burden, “Guide to the Homogenization of Biological Samples,” Random Primers, Vol. 7, 2008, pp. 1-14.
[27] S. Bereswill, P. Bugert, I. Bruchmueller and K. Geider, “Identification of the fire Flight Pathogen Erwinia Amylovora by PCR Assay with Chromosomal DNA,” Applied and Environmental Microbiology, Vol. 61, 1995, pp. 2636- 2642.
[28] S. Priou, L. Gutarra and P. Aley, “An Improved Enrich- ment Broth for the Sensitive Detection of Ralstonia solanacearum (Biovars 1 and 2A) in Soil Using DAS- ELISA,” Plant Pathology, Vol. 55, 2006, pp. 36-45. doi:10.1111/j.1365-3059.2005.01293.x

Copyright © 2024 by authors and Scientific Research Publishing Inc.

Creative Commons License

This work and the related PDF file are licensed under a Creative Commons Attribution 4.0 International License.