Skip to main content

Molecular detection and genotyping of Fusarium oxysporum f. sp. psidii isolates from different agro-ecological regions of India

Journal of Microbiology volume 51, pages 405–412 (2013)Cite this article

Abstract

Twenty one isolates of Fusarium oxysporum f. sp. psidii (Fop), causing a vascular wilt in guava (Psidium guajava L.), were collected from different agro-ecological regions of India. The pathogenicity test was performed in guava seedlings, where the Fop isolates were found to be highly pathogenic. All 21 isolates were confirmed as F. oxysporum f. sp. psidii by a newly developed, species-specific primer against the conserved regions of 28S rDNA and the intergenic spacer region. RAPD and PCR-RFLP were used for genotyping the isolates to determine their genetic relationships. Fifteen RAPD primers were tested, of which five primers produced prominent, polymorphic, and reproducible bands. RAPD yielded an average of 6.5 polymorphic bands per primer, with the amplified DNA fragments ranging from 200–2,000 bp in size. A dendrogram constructed from these data indicated a 22–74% level of homology. In RFLP analysis, two major bands (350 and 220 bp) were commonly present in all isolates of F. oxysporum. These findings provide new insight for rapid, specific, and sensitive disease diagnosis. However, genotyping could be useful in strain-level discrimination of isolates from different agro-ecological regions of India.

This is a preview of subscription content, access via your institution.

Access options

Buy single article

Instant access to the full article PDF.

43,34 €

Price includes VAT (Finland)
Tax calculation will be finalised during checkout.

References

  • Abd-Elsalam, K.A., Schnieder, F., Khalil, M.S., Aly, A.A., and Verreet, J.A. 2003. Genetic variation at the intra- and interspecific level in Fusarium spp. associated from Egyptian cottons. J. Plant Dis. Prot. 110, 46–53.

    CAS  Google Scholar 

  • Achenback, L.A., Patrick, J.A., and Gray, L.E. 1997. Genetic homogeneity among isolates of Fusarium solani that cause soybean sudden death syndrome. Theor. Appl. Genet. 95, 474–478.

    Article  Google Scholar 

  • Annamalai, P., Ishii, H., Lalithakumari, D., and Revathi, R. 1995. Polymerase chain reaction and its applications in fungal disease diagnosis. J. Plant Dis. Protect. 102, 91–104.

    CAS  Google Scholar 

  • Appel, D.J. and Gordon, T.R. 1996. Relationships among pathogenic and nonpathogenic isolates of Fusarium oxysporum based on the partial sequence of the intergenic spacer region of the ribosomal DNA. Mol. Plant Microbe Interact. 9, 125–138.

    PubMed  Article  CAS  Google Scholar 

  • Assigbetse, K.B., Fernandez, D., Doubios, M.P., and Geiger, J.P. 1994. Differentiation of Fusarium oxysporum f. sp. vasinfectum races on cotton by random amplified polymorphic DNA (RAPD) analysis. Phytopathol. 84, 622–626.

    Article  CAS  Google Scholar 

  • Bruns, T.D., White, T.J., and Taylor, J.W. 1991. Fungal molecular systematics. Ann. Rev. Ecol. Sys. 22, 525–564.

    Article  Google Scholar 

  • Bryan, G.T., Daniels, M.J., and Osbourn, A.E. 1995. Comparison of fungi within the Gaeumannomyces-Phialophora complex by analysis of ribosomal DNA sequence. Appl. Environ. Microbiol. 61, 681–689.

    PubMed  CAS  Google Scholar 

  • Cenis, J.L. 1992. Rapid extraction of fungal DNA for PCR amplification. Nucleic Acids Res. 20, 2380.

    PubMed  Article  CAS  Google Scholar 

  • Chattapadhyay, S.B. and Bhattachariya, S.K. 1968. Investigations on the wilt disease of guava (Psidium guajava L.) in West Bengal I. Ind. J. Agri. Sci. 38, 65–72.

    Google Scholar 

  • Danaldson, G.C., Ball, L.A., Axelrood, P.E., and Glass, N.L. 1995. Primer sets developed to amplify conserved genes from filamentous ascomycetes are useful in differentiating Fusarium species associated with conifers. Appl. Environ. Microbiol. 61, 1331–1340.

    Google Scholar 

  • Edel, V., Steinberg, C., Avelange, I., Lagurre, G., and Alabouvette, C. 1995. Comparison of three molecular methods for the characterization of Fusarium oxysporum strains. Phytopathol. 85, 579–585.

    Article  CAS  Google Scholar 

  • Guadet, J., Julien, J., Lafay, J.F., and Brygoo, Y. 1989. Phylogeny of some Fusarium species, as determined by large-subunit rRNA sequence comparison. Mol. Biol. Ecol. 6, 227–242.

    CAS  Google Scholar 

  • Gupta, V.K. 2012. PCR-RAPD profiling of Fusarium spp. causing guava wilt disease in India. J. Environ. Sci. Health Part B. 47, 315–325.

    Article  CAS  Google Scholar 

  • Hyun, J.W. and Clark, C.A. 1998. Analysis of Fusarium lateritium using RAPD and rDNA RFLP techniques. Mycol. Res. 102, 1259–1264.

    Article  CAS  Google Scholar 

  • Ibrahim, G. and Nirenberg, H.I. 2000. Recent studies on Fusarium vascular wilt of cotton at the Fedral Biological Center for Agricultural and Forstery (BBA), Berlin. — Mitt. Biol. BundAnst Ld- u.Forstwirtsch H. 377, 87–88.

    Google Scholar 

  • Jana, T.K., Sharma, T.R., Prasad, D., and Arora, D.K. 2003. Molecular characterization of Macrophomina phaseolina and Fusarium species by single primer RAPD technique. Microbiol. Res. 158, 264–274.

    Article  Google Scholar 

  • Jurgenson, J.E., Zeller, K.A., and Le slie, J.F. 2002. Expanded genetic map of Gibberella moniliformis (Fusarium verticillioides). Appl. Environ. Microbiol. 68, 1972–1979.

    PubMed  Article  CAS  Google Scholar 

  • Leong, S.K., Latiffah, Z., and Baharuddin, S. 2009. Molecular characterization of Fusarium oxysporum f. sp. cubense of banana. Ameri. J. Appl. Sci. 6, 1301–1307.

    Article  CAS  Google Scholar 

  • Manulis, S., Kogan, N., Reuven, M., and Benyephet, Y. 1994. Use of the RAPD technique for identification of Fusarium oxysporum f. sp. dianthi from carnation. Phytopathol. 84, 98–101.

    Article  Google Scholar 

  • McDonald, B.A. 1997. The population genetics of fungi: tools and techniques. Phytopathol. 87, 448–453.

    Article  CAS  Google Scholar 

  • Migheli, Q., Briatore, E., and Garibaldi, A. 1998. Use of random amplified polymorphic DNA (RAPD) to identify races 1, 2, 3 and 8 of Fusarium oxysporum f. sp. dianthi in Italy. Eur. J. Plant Pathol. 104, 49–57.

    Article  CAS  Google Scholar 

  • Misra, A.K. 2006 Wilt of guava-a disease of national importance. Ind. Phytopathol. 59, 269–280.

    Google Scholar 

  • Misra, A.K. and Pandey, B.K. 1996. Wilt of guava and associated pathogens. Ind. J. Mycol. Plant Pathol. 22, 85–86.

    Google Scholar 

  • Misra, A.K. and Pandey, B.K. 2000. Progressive natural wilting of guava plants during different months. Indian Phytopathol. 53, 423–427.

    Google Scholar 

  • Mishra, R.K., Pandey, B.K., Muthukumar, M., Pathak, N., and Zeeshan, M. 2013. Detection of Fusarium wilt pathogens of Psidium guajava L. in soil using culture independent PCR (ciPCR). Saudi J. Biol. Sci. 20, 51–56.

    PubMed  Article  CAS  Google Scholar 

  • NHB. 2012. Indian horticulture database. p. 4.

    Google Scholar 

  • Nicholson, P., Simpson, D.R., Weston, G., Rezanoor, H.N., Lees, A.K., Parry, D.W., and Joyce, D. 1998. Detection and quantification of Fusarium culmorum and Fusarium graminearum in cereals using PCR assays. Physiol. Mol. Plant Pathol. 53, 17–37.

    Article  CAS  Google Scholar 

  • O’Donnell, K., Cigelnik, E., and Nirenberg, H.I. 1998a. Molecular systematic and phylogeography of the Gibberella fujikuroi species complex. Mycologia 90, 465–493.

    Article  Google Scholar 

  • O’Donnell, K., Kistler, H.C., Tacke, B.K., and Casper, H.H. 2000. Gene genealogies reveal global phylogeographic structure and reproductive isolation among lineages of Fusarium graminearum, the fungus causing wheat scab. Proc. Natl. Acad. Sci. USA 97, 7905–7910.

    PubMed  Article  Google Scholar 

  • Prasad, N., Mehta, P.R., and Lal, S.B. 1952. Fusarium wilt of guava (Psidium guajava L.) in Uttar Pradesh, India. Nature 169, 753.

    PubMed  Article  CAS  Google Scholar 

  • Rajan, S., Yadava, L.P., Kumar, R., and Saxena, S.K. 2007. GIS based diversity analysis of guava growing distribution in Uttar Pradesh. Acta Horti. 735, 109–113.

    Google Scholar 

  • Rohlf, F.J. 1998. NT-SYS-pc: Numerical Taxonomy and Multivariate Analysis System. Version 2.11V, Exteer Software, Setauket, N.Y., USA.

    Google Scholar 

  • Samuels, G.J. and Seifert, K.A. 1995. The impact of molecular characters on systematics of filamentous ascomycetes. Ann. Rev. Phytopathol. 33, 37–67.

    Article  CAS  Google Scholar 

  • Schroers, H.J., Geldenhuis, M.M., Wingfield, M.J., Schoeman, M.H., Yen, Y.F., Shen, W.C., and Wingfield, B.D. 2005. Classification of the guava wilt fungus Myxosporium psidii, the palm pathogen Gliocladium vermoesenii and the persimmon wilt fungus Acremonium diospyri in Nalanthamala. Mycologia 97, 375–395.

    PubMed  Article  CAS  Google Scholar 

  • Voight, K., Schleier, S., and Bruckner, B. 1995. Genetic variability in Gibberella fujikuroi and some related species of the genus Fusarium based on random amplification of polymorphic DNA (RAPD). Curr. Genet. 27, 528–535.

    Article  Google Scholar 

  • White, T.J., Bruns, T., Lee, S., and Taylor, J.W. 1990. Amplification and direct sequencing of fungal ribosomal RNA genes for phylogenetics, pp. 315–322. PCR Protocols: A Guide to Methods and Applications. In Innis, M.A., Gelfand, D.H., Sninsky, J.J., and White, T.J. (eds.) Academic Press, Inc., New York, N.Y., USA.

    Google Scholar 

Download references

Author information

Affiliations

  1. Molecular Plant Pathology Laboratory, Central Institute for Subtropical Horticulture, Lucknow, 227107, India

    Rupesh Kumar Mishra, Brajesh Kumar Pandey, Vijai Singh & Amita John Mathew

  2. Department of Biotechnology, Integral University, Lucknow, 226026, India

    Rupesh Kumar Mishra, Neelam Pathak & Mohammad Zeeshan

Authors
  1. Rupesh Kumar Mishra
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. Brajesh Kumar Pandey
    View author publications

    You can also search for this author in PubMed Google Scholar

  3. Vijai Singh
    View author publications

    You can also search for this author in PubMed Google Scholar

  4. Amita John Mathew
    View author publications

    You can also search for this author in PubMed Google Scholar

  5. Neelam Pathak
    View author publications

    You can also search for this author in PubMed Google Scholar

  6. Mohammad Zeeshan
    View author publications

    You can also search for this author in PubMed Google Scholar

Corresponding authors

Correspondence to Rupesh Kumar Mishra or Vijai Singh.

Rights and permissions

Reprints and Permissions

About this article

Cite this article

Mishra, R.K., Pandey, B.K., Singh, V. et al. Molecular detection and genotyping of Fusarium oxysporum f. sp. psidii isolates from different agro-ecological regions of India. J Microbiol. 51, 405–412 (2013). https://doi.org/10.1007/s12275-013-2638-3

Download citation

  • Received:

  • Accepted:

  • Published:

  • Issue Date:

  • DOI: https://doi.org/10.1007/s12275-013-2638-3

Keywords

  • Psidium guajava L.
  • RAPD
  • RFLP
  • Fusarium oxysporum f. sp. psidii
  • diagnostic
  • genotype
  • ITS