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.
Sensitive and Specific Detection ofMycoplasma speciesby Consensus Polymerase Chain Reaction and Dot Blot Hybridization