RESEARCH ARTICLE Use of PCR to detect infection of differentially susceptible maize cultivars using Ustilago maydis strains of variable virulence Received: 15 January 2003 / Accepted: 27 February 2003 / Published online: 24 May 2003 Ó Springer-Verlag and SEM 2003 Abstract Ustilago maydis was specifically detected in infected maize plants by means of the polymerase chain reaction (PCR) using oligonucleotides corresponding to a specific region downstream of the homeodomain of the bE genes of the pathogen. The reaction gave rise to amplification of a ca. 500-bp product when tested with U. maydis DNA, but no amplification was detected with DNA from fungi not related to U. maydis. Using these primers, U. maydis was detected in infected maize plants from differentially susceptible cultivars as early as 4 days after inoculation with strains of variable degrees of vir- ulence. Detection of U. maydis at early stages of infec- tion, or in asymptomatic infected plants should assist in studies on plant–pathogen interactions. Keywords PCR detection Æ b locus Æ Ustilaginales Æ Plant infection Æ Mating type Introduction The basidiomycete Ustilago maydis (DC.) Cda. is the smut pathogen of maize (Zea mays L.) with worldwide distribution. During the saprophytic phase, the fungus is haploid and grows as budding yeasts (sporidia). Mating of compatible sporidia leads to the formation of a dikaryotic mycelium, which invades the plant and eventually induces formation of tumors full of diploid teliospores. Teliospores germinate forming a promyce- lium in which meiosis occurs giving rise to the meiotic segregants, which multiply by budding and thus com- plete the life cycle of the fungus [2, 15]. Different events in the life cycle of U. maydis are regulated by the mating-type loci a and b. The a locus is required for cell-to-cell recognition during the mating process [5] and for the maintenance of filamentous growth [3]. The a locus has two idiomorphs, a 1 and a 2 , and both have been cloned. Each idiomorph encodes a pheromone and a receptor for the pheromone synthe- sized by the compatible partner strain [5]. The b locus regulates the steps in sexual development that occur after fusion of haploid cells. The b locus has at least 25 alleles at each of two genes, bE and bW. Different a and b alleles in mating partners are necessary to trigger mating, fila- mentous growth, and tumor induction [10, 16]. The polymerase chain reaction (PCR) has been used to detect a number of fungal plant pathogens based on the internal transcribed spacer (ITS) regions of ribosomal DNA (rDNA) or by sequences specific to the pathogen [1, 11, 17]. This technique is useful in plant infection studies when no external or characteristic symptoms of disease are apparent. For our studies on the course of experimental infection of maize plantules by virulent and avirulent strains of U. maydis obtained in the laboratory, we utilized the PCR method described below to detect the pathogen in infected symptomatic and asymptomatic plants. Primers whose sequence corresponds to a con- served region downstream of the homeodomain of the bE genes of U. maydis [1, 13] were used to amplify the corresponding fragment as a diagnostic tool. Materials and methods Fungal strains The following haploid strains of Ustilago maydis were used in this study: wild-type strains FB1 (a 1 b 1 ) and FB2 (a 2 b 2 ) (provided by Flora Banuett, University of California, San Francisco), BX27 Int Microbiol (2003) 6: 117–120 DOI 10.1007/s10123-003-0117-0 Alfredo D. Martı´nez-Espinoza Claudia G. Leo´ n-Ramı´rez Æ Nisha Singh Jose´ Ruiz-Herrera A. D. Martı´nez-Espinoza Æ C. G. Leo´n-Ramı´rez J. Ruiz-Herrera (&) Departamento de Ingenierı´a Gene´tica, Unidad Irapuato, Centro de Investigacio´ n y de Estudios Avanzados del Instituto Polite´ cnico Nacional, Apartado Postal 629, 36500 Irapuato, Gto., Me´ xico E-mail: jruiz@ira.cinvestav.mx Tel.: +52-4626239600 Fax: +52-4626245849 N. Singh Botany Department, University of Durban-Westville, South Africa Present address: A. D. Martı´nez-Espinoza University of Georgia, Griffin, Georgia, USA