Skip to main content
SpringerLink
Log in

Detection of Ralstonia solanacearum from Asymptomatic Tomato Plants, Irrigation Water, and Soil Through Non-selective Enrichment Medium with hrp Gene-Based Bio-PCR

  • Published:
Current Microbiology Aims and scope Submit manuscript

Abstract

Bacterial wilt of tomato caused by Ralstonia solanacearum (Smith) Yabuuchi et al. (Microbiol Immunol 39:897–904, 1995) is a serious disease, which causes losses up to 60 % depending on environmental conditions, soil property, and cultivars. In present investigation, nucleotide sequences of virulence, hypersensitive response and pathogenicity (hrp) gene were used to design a pair of primer (Hrp_rs 2F: 5′-AGAGGTCGACGCGATACAGT-3′ and Hrp_rs 2R: 5′-CATGAGCAAGGACGAAGTCA-3′) for amplification of bacterial genome. The genomic DNA of 27 isolates of R. solanacearum race 1 biovar 3 & 4 was amplified at 323 bp. The specificity of primer was tested on 13 strains of R. solanacearum with other group of bacteria such as Xanthomonas oryzae pv. oryzae, X. campestris pv. campestris, and X. citri subsp. citri. Primer amplified DNA fragment of R. solanacearum at 323 bp. The sensitivity of the primer was 200 cfu/ml and improved further detection level by using non-specific enrichment medium casamino acids-pepton-glucose broth followed by PCR (BIO-PCR). Out of 130 samples of asymptomatic tomato plants, irrigation water, and soil collected from bacterial wilt infested field in different agro-climatic regions of India, R. solanacearum was detected from 86.9, 88.5, and 90.9 per cents samples using BIO-PCR, respectively. The primer was found specific for detecting viable and virulent strains of R. solanacearum and useful for the diagnosis of R. solanacearum in tomato seedlings and monitoring of pathogen in irrigation water and soil.

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

Similar content being viewed by others

References

  1. Ayers SH, Rupp P Jr, Johnson WT (1919) A study of the alkali-forming bacteria in milk. U S Dep Agri Bull 782:1–139

    Google Scholar 

  2. Berg T, Tesoriero L, Hailstones DL (2005) PCR–based detection of Xanthomonas campestris pathovars in Brassica seed. Plant Pathol 54:416–427

    Article  CAS  Google Scholar 

  3. Brumbley SM, Carney BF, Denny TP (1993) Phenotype conversion in Pseudomonas solanacearum due to spontaneous inactivation of PhcA, a putative LysR transcriptional regulator. J Bacteriol 175:5477–5487

    CAS  PubMed Central  PubMed  Google Scholar 

  4. Elphinstone JG, Hennessy J, Wilson JK, Stead DE (1996) Sensitivity of different methods for the detection of Ralstonia solanacearum in potato tuber extracts. Bull OEPP/EPPO Bull 26:663–678

    Article  Google Scholar 

  5. George MLC, Quinto V, Villamayor M, Nelson RJ (1996) A simple and rapid method for isolation of bacterial genomic DNA. Inter Rice Res Notes 21(2/3):84

    Google Scholar 

  6. Hayward AC (1994) Characteristics of Pseudomonas solanacearum. J Appl Bacteriol 27:27–265

    Google Scholar 

  7. Ito S, Ushijima Y, Fujii T, Tanaka S, Kameya-Iwaki M, Yoshiwara S, Kishi F (1998) Detection of viable cells of Ralstonia solanacearum in soil using a semi-selective medium and a PCR technique. J Phytopathol 146:379–384

    Article  Google Scholar 

  8. Kang MJ, Lee MH, Shim JK, Seo ST, Shrestha R, Cho MS, Hain JH, Park DS (2007) PCR- based specific detection of Ralstonia solanacearum by amplified of cytochrome c1 signal peptide sequences. J Microbiol and Biotechnol 17(11):1765–1771

    CAS  Google Scholar 

  9. Lin CH, Hsu ST, Tzeng KC, Wang JF (2009) Detection of race 1 strains of Ralstonia solanacearum in field samples in Taiwan using a BIO-PCR method. Eur J Plant Pathol 124:75–85

    Article  CAS  Google Scholar 

  10. Murray MG, Thompson WF (1980) Rapid isolation of high molecular weight DNA. Nucleic Acids Res 8:4321–4325

    Article  CAS  PubMed Central  PubMed  Google Scholar 

  11. Pradhanang PM, Elphinstone JG, Fox RTV (2000) Sensitive detection of Ralstonia solanacearum in soil: a comparison of different detection techniques. Plant Pathol 49:414–422

    Article  Google Scholar 

  12. Schaad NW, Jones JB, Chun W (2001) Laboratory guide for identification of plant pathogenic bacteria, 3rd edn. APS, St. Paul, p 245

    Google Scholar 

  13. Schonfeld J, Heuer H, Van Elsas JD, Smalla K (2003) Specific and sensitive detection of Ralstonia solanacearum in soil on the basis of PCR amplification of flic C fragments. Appl Environ Microbiol 69:7248–7256

    Article  CAS  PubMed Central  PubMed  Google Scholar 

  14. Seal SE, Jackson LA, Young JPW, Daniels MJ (1993) Differentiation of Pseudomonas solanacearum, Pseudomonas syzygii, Pseudomonas pickettii and the blood disease bacterium by partial 16S rRNA sequencing: construction of oligonucleotide primers for sensitive detection by polymerase chain reaction. J Gen Microbiol 139:1587–1594

    Article  CAS  PubMed  Google Scholar 

  15. Singh D, Dhar Shri (2011) Bio-PCR based diagnosis of Xanthomonas campestris pathovars in black rot infected leaves of crucifers. Indian Phytopath 64(1):7–11

    CAS  Google Scholar 

  16. Singh D, Sinha S, Yadav DK, Sharma JP, Srivastava DK et al (2010) Characterization of biovar/races of Ralstonia solanacearum, the incitant of bacterial wilt in solanaceous crops. Indian Phytopath 63(3):261–265

    CAS  Google Scholar 

  17. Tamura K, Peterson D, Peterson N, Stecher G, Nei M, Kumar S (2011) MEGA5: molecular Evolutionary Genetics Analysis using Maximum Likelihood, Evolutionary Distance, and Maximum Parsimony Methods. Mol Bio Evol 28:2731–2739

    Article  CAS  Google Scholar 

  18. Van der Wolf JM, Vriend SGC, Kastelen P, Nijhuis EH, Van Bekkum PJ, Van Vuurde JVL (2000) Immunofluorescence colony- staining (IFC) for detection and quantification of Ralstonia ( Pseudomonas) solanacearum biovar 2 (race 3) in soil and verification of positive results by PCR and dilution plating. Eur J Plant Pathol 106:123–133

    Article  Google Scholar 

  19. Yabuuchi E, Kosako Y, Yano I, Hotta I, Nishiucliy Y (1995) Transfer of two Burkholderia and an Alcaligenes species to Ralstonia General Nov: proposal of Ralstonia pikettii (Ralston, palleroni and Doudoroff 1973) Comb. Nov, Ralstonia solanacearum (Smith1896) Comb. Nov. Microbiol Immunol 39:897–904

Download references

Acknowledgments

The authors are grateful to The Director, IISR, Calicut and Indian Council of Agricultural Research, New Delhi for providing financial support under outreach Project on “Phytophthora, Fusarium and Ralstonia Diseases of Horticultural and Field Crops” to conduct various experiments. The authors are also thankful to Dr. R. K. Jain, Head, Division of Plant Pathology, IARI New Delhi for his keen interest and help throughout the course of these investigation.

Author information

Authors and Affiliations

  1. Division of Plant Pathology, Indian Agricultural Research Institute, New Delhi, 110012, India

    Dinesh Singh, Shweta Sinha, D. K. Yadav & Garima Chaudhary

Authors
  1. Dinesh Singh
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. Shweta Sinha
    View author publications

    You can also search for this author in PubMed Google Scholar

  3. D. K. Yadav
    View author publications

    You can also search for this author in PubMed Google Scholar

  4. Garima Chaudhary
    View author publications

    You can also search for this author in PubMed Google Scholar

Corresponding author

Correspondence to Dinesh Singh.

Rights and permissions

Reprints and permissions

About this article

Cite this article

Singh, D., Sinha, S., Yadav, D.K. et al. Detection of Ralstonia solanacearum from Asymptomatic Tomato Plants, Irrigation Water, and Soil Through Non-selective Enrichment Medium with hrp Gene-Based Bio-PCR. Curr Microbiol 69, 127–134 (2014). https://doi.org/10.1007/s00284-014-0566-z

Download citation

  • Received:

  • Accepted:

  • Published:

  • Issue Date:

  • DOI: https://doi.org/10.1007/s00284-014-0566-z

Keywords

Search

Navigation