Exploration of D1/D2 domain of large-subunit ribosomal DNA for specific detection of Sclerotium rolfsii by polymerase chain reaction assay
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.
KeywordsEarly 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.
- 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
- 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
- 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
- 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
- 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
- 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. (1988). Sclerotium (Athelia) rolfsii, a pathogen of many plant species. Adv in Plant Pathol, 6, 523–534.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
- 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