Species-specific detection of the maize pathogens Sporisorium reiliana andUstilago maydis by dot blot hybridization and PCR-based assays

Head smut of maize, caused by Sporisorium reiliana, may substantially reduc e grain yield. The objective of the present study was to develop a highly s pecific and sensitive DNA-based assay for detection of S. reiliana and its differentiation from Ustilago maydis, a maize fungus inducing the symptomat ically similar common smut disease. Plasmid libraries of S. reiliana and U. maydis were constructed using a shotgun cloning procedure. Clones containi ng strongly hybridizing species-specific DNA were selected by screening lib raries with their own labeled genomic DNA, followed by cross-hybridization with genomic DNA of maize and other maize-pathogenic fungi. The selected cl ones were used to generate subclones with short insert fragments to facilit ate 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 det ermined 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 u sing SR1 and SR3, and 8 pg using UM11, irrespective of the presence of maiz e 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 leave s. Thus, both the dot blot hybridization and the PCR-based assays provide a highly sensitive and reliable tool for detection and differentiation of co rn smut caused either by S. reiliana or by U. maydis.