Abstract Monospore isolates of Mycosphaerella grami- nicola considered to originate from one ascus were ana- lysed by the polymerase chain reaction (PCR) with 32 RAPD primers. Eighteen of these revealed three classes of polymorphisms, which enabled a RAPD-based tetrad anal- ysis. Four pairs of isolates resulting from a single diploid nucleus were determined. A procedure to cross these iso- lates was developed to investigate the mating system. Three of six crosses were successful, and the segregation of mating types in accordance with the tetrad analysis strongly points to a bipolar heterothallic mating system in M. graminicola. Random ascospore progenies from the successful crosses, each comprising 54 isolates, were stud- ied with three primers to determine the mode of inheritance of the RAPD markers. Mendelian segregation and recom- bination of RAPD markers was observed in all progenies. Key words Crossing procedure · Epidemiology · Genetic variation · Heterothallism · Mating system · Septoria tritici Introduction Septoria tritici leaf blotch is an important fungal disease of bread wheat and durum wheat, Triticum aestivum L. and T. turgidum (L.) Thell. ssp. durum L., respectively, in re- gions with a temperate climate, and is becoming increas- ingly important in north-western Europe (Polley and Thomas 1991; Van Ginkel and Rajaram 1993). The disease is caused by the ascomycete Mycosphaerella graminicola (Fuckel) J. Schröt. in Cohn (anamorph: Septoria tritici Rob. ex Desm.), which was described for the first time in New Zealand by Sanderson (1972). Since then, M. grami- nicola has also been reported in several other wheat-pro- ducing areas of the world (Madariaga 1986; Garcia and Marshall 1992; Scott et al. 1988). The air-borne ascospores are considered to be the most important source of primary inoculum that establishes the disease, whereas splash-dis- persed pycnidiospores are believed to be the major propa- gules for the progression of the disease during the grow- ing season (Sanderson et al. 1985; Scott et al. 1988; Shaw and Royle 1989, 1993; Royle 1994). The genetic variation of several markers in M. grami- nicola is vast. McDonald and Martinez (1990 a) identified 22 haplotypes among 93 isolates from a single wheat field, and Boeger et al. (1993) identified different genotypes even within one lesion, whereas different conidia from the same pycnidium had identical genotypes. Kema et al. (1996 a, b) recently described genetic variation for virulence among 80 isolates and provided evidence of specificity for host species and host cultivars. The large degree of genetic variation among M. grami- nicola isolates points to a more important role of ascos- pores in the epidemiology of septoria tritici leaf blotch than hitherto believed (Shaw and Royle 1993; McDonald et al. 1995). Nevertheless, both the mating system and the role of the sexual cycle for generating genetic variation in the pathogen remain to be resolved. We report a procedure to cross selected M. graminicola isolates, characterize the mating system and analyse segregation of molecular and morphological markers in the progeny. Materials and methods Pathogen isolation procedure. Dutch farmers’ fields in Wageningen and Kraggenburg, which were planted with the major wheat cv Vi- vant, were sampled between July 1994 and August 1995 for diseased leaves, wheat debris (stubble and leaves), and infected volunteer plants in order to investigate the occurrence of M. graminicola. Col- Curr Genet (1996) 30: 251 – 258 © Springer-Verlag 1996 Received: 14 February / 10 May 1996 Gert H. J. Kema · Els C. P. Verstappen Maria Todorova · Cees Waalwijk Successful crosses and molecular tetrad and progeny analyses demonstrate heterothallism in Mycosphaerella graminicola ORIGINAL PAPER G. H. J. Kema () · E. C.P. Verstappen · C. Waalwijk DLO-Research Institute for Plant Protection (IPO-DLO), P.O. Box 9060, 6700 GW Wageningen, The Netherlands M. Todorova Plant Protection Institute, 2230 Kostinbrod, P.O. Box 238, Sofia, Bulgaria Communicated by P. J. G. M. de Wit