Mol Gen Genomics (2003) 269: 1–12 DOI 10.1007/s00438-002-0795-x ORIGINAL PAPER S. B. Goodwin Æ C. Waalwijk Æ G. H. J. Kema J. R. Cavaletto Æ G. Zhang Cloning and analysis of the mating-type idiomorphs from the barley pathogen Septoria passerinii Received: 15 April 2002 / Accepted: 5 December 2002 / Published online: 11 March 2003 Ó Springer-Verlag 2003 Abstract The genus Septoria contains more than 1000 amplified polymorphic DNA markers revealed that each species of plant pathogenic fungi, most of which have no isolate had a unique genotype. The common occurrence known sexual stage. Species of Septoria without a known of both mating types on the same leaf and the high levels sexual stage could be recent derivatives of sexual species of genotypic diversity indicate that S. passerinii is almost that have lost the ability to mate. To test this hypothesis, certainly not an asexual derivative of a sexual fungus. the mating-type region of S. passerinii, a species with no Instead, sexual reproduction probably plays an integral known sexual stage, was cloned, sequenced, and com- role in the life cycle of S. passerinii and may be much pared to that of its close relative S. tritici (sexual stage: more important than previously believed in this (and Mycosphaerella graminicola). Both of the S. passerinii possibly other) ‘‘asexual’’ species of Septoria. mating-type idiomorphs were approximately 3 kb in size and contained a single reading frame interrupted by one Keywords Cochliobolus Æ Evolution Æ (MAT-2)ortwo(MAT-1) putative introns. The putative Loculoascomycetes Æ Multiplex PCR Æ Mycosphaerella products of MAT-1 and MAT-2 are characterized by graminicola alpha-box and high-mobility-group sequences, respec- tively, similar to those in the mating-type genes of M. graminicola and other fungi. The mating-type genes of Introduction S. passerinii and M. graminicola are evolving rapidly, approximately ten times faster than the internal tran- Many species of fungi have different binomial names for scribed spacer region of the ribosomal DNA, and are not their sexual and asexual life stages. Septoria is a genus closely related to those from Cochliobolus or other lo- that includes the asexual stages of more than 1000 species culoascomycetes in the order Pleosporales. Therefore, the of plant pathogenic fungi. Many economically important class Loculoascomycetes may be polyphyletic. Further- diseases are caused by species of Septoria, including cel- more, differences between the phylogenetic trees may ery leaf spot (caused by S. apiicola), azalea leaf scorch indicate separate evolutionary histories for the MAT-1 (S. azaleae), hard rot and leaf spot of gladiolus (S. glad- and MAT-2 idiomorphs. A three-primer multiplex-PCR ioli), and tomato leaf spot (S. lycopersici). Most species technique was developed that allowed rapid identifica- have a limited host range, but the genus as a whole infects tion of the mating types of isolates of S. passerinii.Both plants ranging from primitive (e.g., ferns and lycopods) mating types were present in approximately equal fre- to advanced (e.g., composites, grasses, and orchids) quencies and often on the same leaf in fields in Minnesota types, and with both herbaceous and arboreal growth and North Dakota. Analyses with isozyme and random forms. Most species have no known sexual stage. How- ever, for those species in which one has been identified, Communicated by E. Cerda´-Olmedo the sexual stage is placed in the genus Mycosphaerella (Hawksworth et al. 1995), another large and economi- S. B. Goodwin (&) Æ J. R. Cavaletto Æ G. Zhang cally important group of plant pathogenic fungi. To- USDA-ARS, Department of Botany and Plant Pathology, gether, the genera Septoria and Mycosphaerella contain Purdue University, 915 West State Street, more than 1500 species that infect most, if not all, of the West Lafayette, IN 47907-2054, USA E-mail: [email protected] major plant families. Fax: +1-765-494-0363 One species with no known sexual stage is Septoria C. Waalwijk Æ G. H. J. Kema passerinii, the cause of speckled leaf blotch of barley Plant Research International B.V., (Hordeum vulgare and closely related species). This P.O. Box 16, 6700 AA Wageningen, The Netherlands pathogen was first described more than 120 years ago, 2 but has never been associated with a sexual stage type sequences of M. graminicola could be used to clone (Cunfer and Ueng 1999). One possible explanation for the homologous region(s) from closely related species this could be that the sexual stage was lost during the such as S. passerinii. evolutionary process and no longer exists. If this is The purpose of this research was to use both direct correct, then the proliferation of the pathogen now oc- and indirect approaches to test the hypothesis that curs solely by asexual reproduction. Another possibility S. passerinii is a recently derived, asexual relative of is that the sexual structures may be small, ephemeral, or M. graminicola. A secondary goal was to estimate the produced only under specific conditions and, because of rates of change in the mating-type genes compared to this, have simply been overlooked. According to this the internal transcribed spacer (ITS) region of the rib- scenario the pathogen should undergo cycles of sexual osomal DNA, in order to test the hypothesis that the reproduction that have remained unobserved. mating-type genes in the Mycosphaerellaceae evolve Recent phylogenetic analyses of S. passerinii revealed rapidly, as they do in the genus Cochliobolus (Turgeon a close evolutionary relationship with the septoria tritici 1998). The final goal was to design primers for multiplex leaf blotch pathogen of wheat, Mycosphaerella gra- PCR that could be used to quickly determine the mating minicola (Goodwin and Zismann 2001). The asexual types of large numbers of isolates of S. passerinii. stage of M. graminicola, S. tritici, is quite similar to S. passerinii morphologically (Goodwin and Zismann 2001). However, the sexual stage of S. tritici has been Materials and methods known since 1972 (Sanderson 1972), and it appears to play an important part in the life cycle of this pathogen Fungal isolates and culture methods (Hunter et al. 1999; Kema et al. 1996; Shaw and Royle Diseased leaves were sampled from one barley field in Minnesota 1989; Zhan et al. 1998). If S. passerinii is truly asexual, it and three in North Dakota, by David Long (USDA-ARS, Cereal could be a recent derivative from the same evolutionary Disease Laboratory, St. Paul, Minn.) during 1995 (Table 1). Leaf lineage as M. graminicola. pieces were surface-sterilized by immersion in a 0.5% solution of In the absence of a known sexual stage, several ap- sodium hypochlorite for 30 s, then placed on glass microscope slides in petri dishes. The petri dishes were lined with filter paper proaches can be used to test for evidence of sexual re- saturated with sterile distilled water to provide humidity and were production (Milgroom 1996). Indirect methods analyze incubated at 20°C for 2–4 days to induce sporulation of the fungus. the observed levels of genotypic diversity. Populations When spore masses became visible under a dissecting microscope, with regular cycles of sexual reproduction should have the conidia were diluted in sterile distilled water and transferred to potato dextrose agar (PDA, Difco Laboratories, Detroit, Mich.) many more genotypes, with consequently higher levels plates. After a few days, single germinated conidia were transferred of genotypic diversity, compared to those with only to fresh PDA plates. At least three germinated conidia were asexual reproduction. This type of genetic structure is transferred from different asexual spore structures on each leaf. seen in most populations of M. graminicola worldwide Following isolation, the cultures were maintained on yeast-malt agar (YMA) plates (4 g yeast extract, 4 g malt extract, 4 g sucrose, (McDonald et al. 1995). Other methods to test for the 15 g agar per liter, with 50 lg kanamycin per ml added after possibility of sexual reproduction involve assaying the autoclaving) at room temperature (22–24°C). Three additional occurrence, frequency, or expression of the mating-type isolates were purchased from the American Type Culture Collec- genes directly. Populations of parasitic fungi in which tion (Accession Nos. 22585, 26515, and 26516). For long-term mating occurs by direct contact on the host substrate are storage, cultures were kept on lyophilized filter-paper pieces at )80°C. expected to have both mating types in approximately Mycelia for isozyme and random amplified polymorphic DNA equal frequencies (Milgroom 1996). In contrast, asexual (RAPD) analyses were grown on YMA plates for 12 days at room fungi often have only one of the two mating types re- temperature. For medium-scale DNA extraction, mycelium was quired to initiate sexual reproduction (Christiansen et al. grown in yeast-malt broth on a shaking platform at room temperature, then harvested, lyophilized and stored as described 1998; Sharon et al. 1996), although some exceptions are previously (Goodwin et al. 2001a). known (Yun et al. 1999, 2000). A direct assay for possible sexual reproduction of species of Septoria became possible recently when both DNA extraction, library construction and Southern analyses mating-type genes were cloned from M. graminicola (Waalwijk et al. 2002). This species has an outcrossing Medium-scale DNA extraction from lyophilized mycelia was car- ried out as described by Biel and Parrish (1986), with major modi- mating system (Kema et al. 1996) with two mating-type fications. One gram of mycelium was frozen in liquid nitrogen and genes designated MAT1-1 and MAT1-2 (Waalwijk ground to a fine powder with a mortar and pestle. Approximately et al. 2002). Because only one mating-type locus is pre- 500 mg of the powder was transferred to a microcentrifuge tube, sent, these can be referred to as MAT-1 and MAT-2 suspended in 1 ml of lysis buffer (50 mM TRIS-HCl pH 7.5, 5 mM EDTA, 2% SDS, and 250 lg proteinase K per ml), and incubated at according to the informal nomenclature of Turgeon and 37°C for 1 h.