European Journal of Plant Pathology

, Volume 142, Issue 3, pp 557–565 | Cite as

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

  • V. Pravi
  • M. L. Jeeva
  • P. V. Archana


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.


Early detection Molecular diagnosis Phytopathogen Elephant foot yam 



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.


  1. 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
  2. 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.CrossRefPubMedGoogle Scholar
  3. 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.CrossRefGoogle Scholar
  4. 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.CrossRefPubMedGoogle Scholar
  5. 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.CrossRefPubMedCentralPubMedGoogle Scholar
  6. 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.CrossRefGoogle Scholar
  7. 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.Google Scholar
  8. Harlton, C. E., Levesque, C. A., & Punja, Z. K. (1995). Genetic diversity in Sclerotium (Athelia) rolfsii and related species. Phytopathology, 85, 1269–1281.CrossRefGoogle Scholar
  9. 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.CrossRefPubMedCentralPubMedGoogle Scholar
  10. 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.CrossRefGoogle Scholar
  11. Hillis, D. M., & Dixon, M. T. (1991). Ribosomal DNA: molecular evolution and phylogenetic inference. Quarterly Review of Biology, 66, 411–453.CrossRefPubMedGoogle Scholar
  12. 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.CrossRefGoogle Scholar
  13. 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.CrossRefPubMedGoogle Scholar
  14. 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.CrossRefGoogle Scholar
  15. 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
  16. 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.CrossRefGoogle Scholar
  17. 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.CrossRefPubMedCentralPubMedGoogle Scholar
  18. Larsson, K. H., Larsson, E., & Koljal, G. U. (2004). High phylogenetic diversity among corticioid homobasidiomycetes. Mycological Research, 108, 983–1002.CrossRefPubMedGoogle Scholar
  19. 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
  20. Martin, R. R., James, D., & Lévesque, C. A. (2000). Impacts of molecular diagnostic technologies on plant disease. Annual Review of Phytopathology, 38, 207–239.CrossRefPubMedGoogle Scholar
  21. McCartney, H. A., Foster, S. J., Fraaije, B. A., & Ward, E. (2003). Molecular diagnostics for fungal plant pathogens. Pest Management Science, 59, 129–1472.CrossRefPubMedGoogle Scholar
  22. 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
  23. 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
  24. 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.CrossRefPubMedGoogle Scholar
  25. 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
  26. Punja, Z. K. (1985). The biology, ecology and control of Sclerotium rolfsii. Annual Review of Phytopathology, 23, 97–127.CrossRefGoogle Scholar
  27. Punja, Z. K. (1988). Sclerotium (Athelia) rolfsii, a pathogen of many plant species. Adv in Plant Pathol, 6, 523–534.Google Scholar
  28. Punja, Z. K., & Damiani, A. (1996). Comparative growth, morphology, and physiology of three Sclerotium species. Mycologia, 88, 694–706.CrossRefGoogle Scholar
  29. Sambrook, J., Fritsch, R. F., & Maniatis, T. (1989). Molecular cloning: A laboratory manual. New York: Cold Spring Harbor Press.Google Scholar
  30. 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
  31. Wang, H., Qi, M., & Cutler, A. J. (1993). A simpe method of preparing plant samples for PCR. Nucleic Acids Research, 21, 4153–4154.CrossRefPubMedCentralPubMedGoogle Scholar
  32. Weiss, M., & Oberwinkler, F. (2001). Phylogenetic relationships in Auriculariales and related groups - Hypotheses derived from nuclear ribosomal DNA sequences. Mycological Research, 105, 403–415.CrossRefGoogle Scholar
  33. 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
  34. Zhang, Z., Schwartz, S., Wagner, L., & Miller, W. (2000). A greedy algorithm for aligning DNA sequnces. Journal of Computational Biology, 7, 203–214.CrossRefPubMedGoogle Scholar
  35. 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.CrossRefPubMedGoogle Scholar

Copyright information

© Koninklijke Nederlandse Planteziektenkundige Vereniging 2015

Authors and Affiliations

  1. 1.Central Tuber Crops Research InstituteThiruvananthapuramIndia

Personalised recommendations