Skip to main content
Log in

Exploration of D1/D2 domain of large-subunit ribosomal DNA for specific detection of Sclerotium rolfsii by polymerase chain reaction assay

  • Published:
European Journal of Plant Pathology Aims and scope Submit manuscript

Abstract

Collar rot disease of Amorphophallus paeoniifolius caused by Sclerotium rolfsii is an important disease existing in all Amorphophallus growing areas. The pathogen propagules present in soil and planting material form key basis of inoculum. This study presents the aptness of D1/D2 domain of large-subunit ribosomal DNA (rDNA-LSU) for PCR based detection of S. rolfsii. The detection limit of conventional PCR was 10 pg and that of nested PCR was 100 fg of pathogen DNA. The designed primer was found to be highly specific and could be used for accurate identification of pathogen up to the species level. The protocol was standardized for detection of the pathogen in artificially and naturally infected field samples. The PCR-based method developed here could be used for both disease diagnosis and pathogen monitoring, as well as in guiding plant disease management.

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

Access this article

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

  • Aycock, R. (1966). Stem rot and other diseases caused by Sclerotium rolfsii or the status of Rolf’s fungus after 70 years. North Carolina Agricultural Experiment Station Technical Bulletin, 174, 202.

    Google Scholar 

  • Cilliers, A. J., Herselman, L., & Pretorius, Z. A. (2000). Genetic variability within and among mycelial compatibility groups of Sclerotium rolfsii in South Africa. Phytopathology, 90, 1026–1031.

    Article  CAS  PubMed  Google Scholar 

  • Cullen, D. W., Lees, A. K., Toth, I. K., & Duncan, J. M. (2002). Detection of Colletotrichum coccodes from soil and potato tubers by conventional and quantitative real-time PCR. Plant Pathology, 51, 281–292.

    Article  CAS  Google Scholar 

  • De Koker, T. H., Nakasone, K. K., Haarhof, J., Burdsall, H. H., & Janse, B. J. H. (2003). Phylogenetic relationships of the genus Phanerochaete inferred from the internal transcribed spacer region. Mycological Research, 107, 1032–1040.

    Article  PubMed  Google Scholar 

  • Dey, Y. N., De, S., Ghosh, A. K., Gaidhani, S., Kumari, S., & Jamal, M. (2011). Synergistic depressant activity of Amorphophallus paeoniifolius in Swiss albino mice. Journal of Pharmacology and Pharmacotherapeutics, 2(2), 121–123.

    Article  PubMed Central  PubMed  Google Scholar 

  • Goud, J. C., & Termorshuizen, A. J. (2003). Quality of methods to quantify microsclerotia of Verticillium dahliae in soil. European Journal of Plant Pathology, 109(6), 523–534.

    Article  Google Scholar 

  • Hall, T. A. (1999). Bioedit: a user friendly biological sequence alignment editor and analysis program for Windows 95/98/NT. Nucleic Acids Symposium Series, 41, 95–98.

    CAS  Google Scholar 

  • Harlton, C. E., Levesque, C. A., & Punja, Z. K. (1995). Genetic diversity in Sclerotium (Athelia) rolfsii and related species. Phytopathology, 85, 1269–1281.

    Article  Google Scholar 

  • Hassouna, N., Michot, B., & Bachellerie, J. P. (1984). The complete nucleotide sequence of mouse 28S rRNA gene. Implications for the process of size increase of the large subunit rRNA in higher eukaryotes. Nucleic Acids Research, 12, 3563–3583.

    Article  PubMed Central  CAS  PubMed  Google Scholar 

  • Hayden, K. J., Rizzo, D., Tse, J., & Garbelotto, M. (2004). Detection and quantification of Phytophthora ramorum from California forests using a real-time polymerase chain reaction assay. Phytopathol, 94, 1075–1083.

    Article  CAS  Google Scholar 

  • Hillis, D. M., & Dixon, M. T. (1991). Ribosomal DNA: molecular evolution and phylogenetic inference. Quarterly Review of Biology, 66, 411–453.

    Article  CAS  PubMed  Google Scholar 

  • Ippolito, A., Schena, L., & Nigro, F. (2002). Detection of Phytophthora nicotianae and P. citrophthora in citrus roots and soils by nested PCR. European Journal of Plant Pathology, 108, 855–868.

    Article  CAS  Google Scholar 

  • Iwen, P. C., Hinrichs, S. H., & Rupp, M. E. (2002). Utilization of the internal transcribed spacer regions as molecular targets to detect and identify human fungal pathogens. Medical Mycology, 40, 87–109.

    Article  CAS  PubMed  Google Scholar 

  • Jayaraman, A., Kunga, M. R., Ulaganathan, P., & Poornima, R. (2010). Antioxidant potential of Amorphophallus paeoniifolius in relation to their phenolic content. Pharmaceutical Biology, 48(6), 659–665.

    Article  Google Scholar 

  • Jeeva, M. L., Sharma, K., Mishra, A. K., & Misra, R. S. (2008). Rapid extraction of genomic DNA from Sclerotium rolfsii causing collar rot of Amorphophallus. Genes, Genomes and Genomics, 2(1), 60–62.

    Google Scholar 

  • Jeeva, M. L., Mishra, A. K., Vidyadharan, P., Misra, R. S., & Hegde, V. (2010). A species-specific polymerase chain reaction assay for rapid and sensitive detection of Sclerotium rolfsii. Australasian Plant Pathol, 39, 517–523.

    Article  Google Scholar 

  • Khan, A., Moizer, R., & Islam, M. S. (2008). Antibacterial, antifungal and cytotoxic activities of amblyone isolated from Amorphophallus campanulatus. Indian Journal of Pharmacology, 40(1), 41–44.

    Article  PubMed Central  CAS  PubMed  Google Scholar 

  • Larsson, K. H., Larsson, E., & Koljal, G. U. (2004). High phylogenetic diversity among corticioid homobasidiomycetes. Mycological Research, 108, 983–1002.

    Article  CAS  PubMed  Google Scholar 

  • Madhavi, G. B., & Bhattiprolu, S. L. (2011). Integrated disease management of dry root rot of chilli incited by Sclerotium rolfsii (Sacc.). International Journal Plant Animal Environmental Science, 1(2), 31–37.

    Google Scholar 

  • Martin, R. R., James, D., & Lévesque, C. A. (2000). Impacts of molecular diagnostic technologies on plant disease. Annual Review of Phytopathology, 38, 207–239.

    Article  CAS  PubMed  Google Scholar 

  • McCartney, H. A., Foster, S. J., Fraaije, B. A., & Ward, E. (2003). Molecular diagnostics for fungal plant pathogens. Pest Management Science, 59, 129–1472.

    Article  CAS  PubMed  Google Scholar 

  • Mishra, A. K., Sharma, K., & Misra, R. S. (2008). Rapid and efficient method for the extraction of fungal and oomycetes genomic DNA. Gene, Genome and Genomics, 2, 57–59.

    Google Scholar 

  • Misra, R. S. (1997). Diseases of tuber crops in Northern and Eastern India, CTCRI Technical Series (Vol. 22, p. p.27). Thiruvananthapuram: CTCRI.

    Google Scholar 

  • Okabe, I., & Matsumoto, N. (2003). Phylogenetic relationship of Sclerotium rolfsii (teleomorph Athelia rolfsii) and S. delphini based on ITS sequences. Mycological Research, 107, 164–168.

    Article  CAS  PubMed  Google Scholar 

  • Peterson, S. W. (2000). Phylogenetic relationships in Aspergillus based on rDNA sequence analysis. In R. A. Samson & J. I. Pitt (Eds.), Integration of modern taxonomic methods for Penicillium and Aspergillus classification (pp. 323–355). Amsterdam: Harwood Academic Publishers.

    Google Scholar 

  • Punja, Z. K. (1985). The biology, ecology and control of Sclerotium rolfsii. Annual Review of Phytopathology, 23, 97–127.

    Article  CAS  Google Scholar 

  • Punja, Z. K. (1988). Sclerotium (Athelia) rolfsii, a pathogen of many plant species. Adv in Plant Pathol, 6, 523–534.

    Google Scholar 

  • Punja, Z. K., & Damiani, A. (1996). Comparative growth, morphology, and physiology of three Sclerotium species. Mycologia, 88, 694–706.

    Article  Google Scholar 

  • Sambrook, J., Fritsch, R. F., & Maniatis, T. (1989). Molecular cloning: A laboratory manual. New York: Cold Spring Harbor Press.

    Google Scholar 

  • Vilgalys, R., & Hester, M. (1990). Rapid genetic identification and mapping of enzymatically amplified ribosomal DNA from several Cryptococcus species. Journal of Bacteriology, 172, 4239–4246.

    Google Scholar 

  • Wang, H., Qi, M., & Cutler, A. J. (1993). A simpe method of preparing plant samples for PCR. Nucleic Acids Research, 21, 4153–4154.

    Article  PubMed Central  CAS  PubMed  Google Scholar 

  • Weiss, M., & Oberwinkler, F. (2001). Phylogenetic relationships in Auriculariales and related groups - Hypotheses derived from nuclear ribosomal DNA sequences. Mycological Research, 105, 403–415.

    Article  CAS  Google Scholar 

  • White, T. J., Bruns, T., Lee, S., & Taylor, J. (1990). Amplification and direct sequencing of fungal ribosomal RNA genes for phylogenetics. In M. A. Innis, D. H. Gelfand, J. J. Sninsky, & T. J. White (Eds.), PCR protocols—a guide to methods and applications (pp. 315–322). San Diego: Academic Press.

    Google Scholar 

  • Zhang, Z., Schwartz, S., Wagner, L., & Miller, W. (2000). A greedy algorithm for aligning DNA sequnces. Journal of Computational Biology, 7, 203–214.

    Article  CAS  PubMed  Google Scholar 

  • Zhang, Z. G., Zhang, J. Y., Wang, Y. C., & Zheng, X. B. (2005). Molecular detection of Fusarium oxysporum f.sp. niveum and Mycosphaerella melonis in infected plant tissue and soil. FEMS Microbiology Letters, 249, 39–47.

    Article  CAS  PubMed  Google Scholar 

Download references

Acknowledgments

The funding provided for research work by the National Fund for Basic Strategic and Frontier Application Research in Agricultural Sciences (NFBSFARA), ICAR, New Delhi, India, is gratefully acknowledged. The authors thank The Director, Central Tuber Crops Research Institute, Thiruvananthapuram for providing the infrastructure facilities. We are also grateful to the Indian Institute of Spices Research (Calicut, India) for providing the Phytophthora cultures and the College of Agriculture (Vellayani, India) and CTCRI (Sreekariyam, India) for providing the other fungal and bacterial cultures.

Compliance with ethical standards

The authors declare no conflict of interest. This research work does not include any animal studies.

Author information

Authors and Affiliations

Authors

Corresponding author

Correspondence to M. L. Jeeva.

Rights and permissions

Reprints and permissions

About this article

Check for updates. Verify currency and authenticity via CrossMark

Cite this article

Pravi, V., Jeeva, M.L. & Archana, P.V. Exploration of D1/D2 domain of large-subunit ribosomal DNA for specific detection of Sclerotium rolfsii by polymerase chain reaction assay. Eur J Plant Pathol 142, 557–565 (2015). https://doi.org/10.1007/s10658-015-0633-8

Download citation

  • Accepted:

  • Published:

  • Issue Date:

  • DOI: https://doi.org/10.1007/s10658-015-0633-8

Keywords

Navigation