Article
Species-Specific Detection of the Maize Pathogens Sporisorium reiliana and Ustilago maydis by Dot Blot Hybridization and PCR-Based Assays
Plant Disease
(1999)
Abstract
Head smut of maize, caused by Sporisorium reiliana, may substantially reduce grain yield. The objective of the present study was to develop a highly specific and sensitive DNA-based assay for detection of S. reiliana and its differentiation from Ustilago maydis, a maize fungus inducing the symptomatically similar common smut disease. Plasmid libraries of S. reiliana and U. maydis were constructed using a shotgun cloning procedure. Clones containing strongly hybridizing species-specific DNA were selected by screening libraries with their own labeled genomic DNA, followed by cross-hybridization with genomic DNA of maize and other maize-pathogenic fungi. The selected clones were used to generate subclones with short insert fragments to facilitate PCR amplification for labeling and primer design for a PCR assay. Using Dig-dUTP labeled inserts, detection of less than 0.16 ng of fungal DNA was possible by dot blot hybridization. Sequences of insert fragments were determined to design primer pairs for a PCR-based assay. Primer pairs SR1 and SR3 are species-specific for S. reiliana, and UM11 is species-specific for U. maydis. The PCR-based assays can detect fungal DNA of less than 1.6 pg using SR1 and SR3, and 8 pg using UM11, irrespective of the presence of maize DNA. Use of SR1 and SR3 allowed detection of S. reiliana in the extracts of pith, node, and shank from S. reiliana-infected plants, but not in leaves. Thus, both the dot blot hybridization and the PCR-based assays provide a highly sensitive and reliable tool for detection and differentiation of corn smut caused either by S. reiliana or by U. maydis.
Disciplines
Publication Date
April, 1999
DOI
10.1094/PDIS.1999.83.4.390
Publisher Statement
This article is published as Xu, M. L., A. E. Melchinger, and T. Lübberstedt. "Species-specific detection of the maize pathogens Sporisorium reiliana and Ustilago maydis by dot blot hybridization and PCR-based assays." Plant Disease 83, no. 4 (1999): 390-395.Xu, M. L., A. E. Melchinger, and T. Lübberstedt. "Species-specific detection of the maize pathogens Sporisorium reiliana and Ustilago maydis by dot blot hybridization and PCR-based assays." Plant Disease 83, no. 4 (1999): 390-395. 10.1094/PDIS.1999.83.4.390. Posted with permission.
Citation Information
Thomas Lubberstedt, M.L. XU and A.E. Melchinger. "Species-Specific Detection of the Maize Pathogens Sporisorium reiliana and Ustilago maydis by Dot Blot Hybridization and PCR-Based Assays" Plant Disease Vol. 83 Iss. 4 (1999) p. 390 - 395 Available at: http://works.bepress.com/thomas-lubberstedt/6/