Fusarium: more than a node or a foot-shaped basal cell.

Recent publications have argued that there are potentially serious consequences for researchers in recognising distinct genera in the terminal fusarioid clade of the family Nectriaceae. Thus, an alternate hypothesis, namely a very broad concept of the genus Fusarium was proposed. In doing so, however, a significant body of data that supports distinct genera in Nectriaceae based on morphology, biology, and phylogeny is disregarded. A DNA phylogeny based on 19 orthologous protein-coding genes was presented to support a very broad concept of Fusarium at the F1 node in Nectriaceae. Here, we demonstrate that re-analyses of this dataset show that all 19 genes support the F3 node that represents Fusarium sensu stricto as defined by F. sambucinum (sexual morph synonym Gibberella pulicaris). The backbone of the phylogeny is resolved by the concatenated alignment, but only six of the 19 genes fully support the F1 node, representing the broad circumscription of Fusarium. Furthermore, a re-analysis of the concatenated dataset revealed alternate topologies in different phylogenetic algorithms, highlighting the deep divergence and unresolved placement of various Nectriaceae lineages proposed as members of Fusarium. Species of Fusarium s. str. are characterised by Gibberella sexual morphs, asexual morphs with thin- or thick-walled macroconidia that have variously shaped apical and basal cells, and trichothecene mycotoxin production, which separates them from other fusarioid genera. Here we show that the Wollenweber concept of Fusarium presently accounts for 20 segregate genera with clear-cut synapomorphic traits, and that fusarioid macroconidia represent a character that has been gained or lost multiple times throughout Nectriaceae. Thus, the very broad circumscription of Fusarium is blurry and without apparent synapomorphies, and does not include all genera with fusarium-like macroconidia, which are spread throughout Nectriaceae (e.g., Cosmosporella, Macroconia, Microcera). In this study four new genera are introduced, along with 18 new species and 16 new combinations. These names convey information about relationships, morphology, and ecological preference that would otherwise be lost in a broader definition of Fusarium. To assist users to correctly identify fusarioid genera and species, we introduce a new online identification database, Fusarioid-ID, accessible at www.fusarium.org. The database comprises partial sequences from multiple genes commonly used to identify fusarioid taxa (act1, CaM, his3, rpb1, rpb2, tef1, tub2, ITS, and LSU). In this paper, we also present a nomenclator of names that have been introduced in Fusarium up to January 2021 as well as their current status, types, and diagnostic DNA barcode data. In this study, researchers from 46 countries, representing taxonomists, plant pathologists, medical mycologists, quarantine officials, regulatory agencies, and students, strongly support the application and use of a more precisely delimited Fusarium (= Gibberella) concept to accommodate taxa from the robust monophyletic node F3 on the basis of a well-defined and unique combination of morphological and biochemical features. This F3 node includes, among others, species of the F. fujikuroi, F. incarnatum-equiseti, F. oxysporum, and F. sambucinum species complexes, but not species of Bisifusarium [F. dimerum species complex (SC)], Cyanonectria (F. buxicola SC), Geejayessia (F. staphyleae SC), Neocosmospora (F. solani SC) or Rectifusarium (F. ventricosum SC). The present study represents the first step to generating a new online monograph of Fusarium and allied fusarioid genera (www.fusarium.org).

New endemic Fusarium species hitch-hiking with pathogenic Fusarium strains causing Panama disease in small-holder banana plots in Indonesia.

Fusarium species are well known for their abundance, diversity and cosmopolitan life style. Many members of the genus Fusarium are associated with plant hosts, either as plant pathogens, secondary invaders, saprotrophs, and/or endophytes. We previously studied the diversity of Fusarium species in the Fusarium oxysporum species complex (FOSC) associated with Fusarium wilt of banana in Indonesia. In that study, several Fusarium species not belonging to the FOSC were found to be associated with Fusarium wilt of banana. These Fusarium isolates belonged to three Fusarium species complexes, which included the Fusarium fujikuroi species complex (FFSC), Fusarium incarnatum-equiseti species complex (FIESC) and the Fusarium sambucinum species complex (FSSC). Using a multi-gene phylogeny that included partial fragments of the beta-tubulin (tub), calmodulin (cmdA), translation elongation factor 1-alpha (tef1), the internal transcribed spacer region of the rDNA (ITS), the large subunit of the rDNA (LSU), plus the RNA polymerase II large subunit (rpb1) and second largest subunit (rpb2) genes, we were able to identify and characterise several of these as new Fusarium species in the respective species complexes identified in this study.

Phylogeny and genetic diversity of the banana Fusarium wilt pathogen Fusarium oxysporum f. sp. cubense in the Indonesian centre of origin.

Fusarium oxysporum f. sp. cubense (Foc), the causal agent of Fusarium wilt or Panama disease on banana, is one of the major constraints in banana production worldwide. Indonesia is the centre of origin for wild and cultivated bananas, which likely co-evolved with Foc. This study explored the widest possible genetic diversity of Foc by sampling across Indonesia at 34 geographically and environmentally different locations in 15 provinces at six islands. This resulted in a comprehensive collection of ∼200 isolates from 40 different local banana varieties. Isolates were identified and assessed using sequence analysis of the translation elongation factor-1alpha (tef1), the RNA polymerase II largest subunit (rpb1), and the RNA polymerase II second largest subunit (rpb2). Phylogenetic analyses of these genes allowed the identification of 180 isolates of Fusarium oxysporum f. sp. cubense (Foc), and 20 isolates of the Fusarium fujikuroi species complex (FFSC), the Fusarium incarnatum-equiseti species complex (FIESC), and the Fusarium sambucinum species complex (FSSC). Further analyses, incorporating a worldwide collection of Foc strains, revealed nine independent genetic lineages for Foc, and one novel clade in the Fusarium oxysporum species complex (FOSC). Selected isolates from each lineage were tested on the banana varieties Gros Michel and Cavendish to characterise their pathogenicity profiles. More than 65 % of the isolates were diagnosed as Tropical Race 4 (Foc-TR4) due to their pathogenicity to Cavendish banana, which supports the hypothesis that Foc-TR4 is of Indonesian origin. Nine independent genetic lineages for Foc are formally described in this study. This biodiversity has not been studied since the initial description of Foc in 1919. This study provides a detailed overview of the complexity of Fusarium wilt on banana and its diversity and distribution across Indonesia.

A SIX1 homolog in Fusarium oxysporum f.sp. cubense tropical race 4 contributes to virulence towards Cavendish banana.

The fungus Fusarium oxysporum f.sp. cubense (Focub) causes Fusarium wilt of banana. Focub strains are divided into races according to their host specificity, but which virulence factors underlie these interactions is currently unknown. In the F. oxysporum f.sp. lycopersici (Fol)-tomato system, small secreted fungal proteins, called Six proteins, were identified in the xylem sap of infected plants. The Fol Six1 protein contributes to virulence and has an avirulence function by activating the I-3 immune receptor of tomato. The Focub tropical race 4 (TR4) genome harbors three SIX1 homologs: SIX1a, b and c. In this study, the role of Focub-SIX1a in pathogenicity was evaluated since this homolog is present in not only TR4 but also in other races. A deletion mutant of the SIX1a gene from Focub TR4 strain II5 was generated (FocubΔSIX1a) and tested in planta. Mutants were found to be severely compromised in their virulence. Ectopic integration of the Focub-SIX1a gene in the FocubΔSIX1a strain restored virulence to wild type levels. We conclude that Focub-SIX1a is required for full virulence of Focub TR4 towards Cavendish banana.

Agrobacterium tumefaciens-mediated transformation of Lasiodiplodia theobromae, the causal agent of gummosis in cashew nut plants.

Lasiodiplodia theobromae is a major pathogen of many different crop cultures, including cashew nut plants. This paper describes an efficient Agrobacterium tumefaciens-mediated transformation (ATMT) system for the successful delivery of T-DNA, transferring the genes of green fluorescent protein (gfp) and hygromycin B phosphotransferase (hph) to L. theobromae. When the fungal pycnidiospores were co-cultured with A. tumefaciens harboring the binary vector with hph-gfp gene, hygromycin-resistant fungus only developed with acetosyringone supplementation. The cashew plants inoculated with the fungus expressing GFP revealed characteristic pathogen colonization by epifluorescence microscopy. Intense and bright green hyphae were observed for transformants in all extensions of mycelium cultures. The penetration of parenchyma cells near to the inoculation site, beneath the epicuticle surface, was observed prior to 25 dpi. Penetration was followed by the development of hyphae within invaded host cells. These findings provide a rapid and reproducible ATMT method for L. theobromae transformation.

First Report of Fusarium oxysporum f. sp. cubense Tropical Race 4 Associated with Panama Disease of Banana outside Southeast Asia.

Fusarium wilt or Panama disease of banana, caused by Fusarium oxysporum f. sp. cubense (Foc), is among the most destructive plant diseases (3). Race 1 ravaged 'Gros Michel'-based export trades until the cultivar was replaced by resistant Cavendish cultivars. However, a new variant of Foc, tropical race 4 (TR4), was identified in Southeast Asia in 1992 and has spread throughout the region (3). Cavendish clones, which are most important in subsistence and export production, are among the wide range of cultivars that are affected, and there is a huge concern that TR4 will further disseminate in Africa since its presence was announced in November 2013 and move into Latin America, thereby threatening other vital banana-growing regions. In Jordan, Cavendish bananas are produced on 1,000 to 1,500 ha in the Jordan Valley (32°N, 35.5°E). In 2006, symptoms of Fusarium wilt were observed and sampled for the isolation of Foc. On half-strength PDA amended with 100-ppm streptomycin sulfate, pale salmon-colored colonies with floccose mycelia developed consistently from surface-disinfested xylem. Single microconidia from these colonies were transferred to half-strength PDA, and conidia and mycelia from these monospore colonies were stored at -80°C in 15% glycerol. On banana leaf agar (Co60-irradiated leaf tissue on water agar), isolates resembled F. oxysporum phenotypically by producing infrequent three- to five-celled macroconidia, copious, usually aseptate microconida on monophialides, and terminal and intercalary chlamydospores after 2 weeks (2). With nitrate-nonutilizing (nit) mutants and testers for different vegetative compatibility groups (VCGs), each of seven examined monospore isolates were placed in VCG 01213, which contains only strains of TR4 (3). Total DNA was extracted from six isolates and PCR analyses, which confirmed their identity as TR4 (1). Subsequently, one of the isolates (JV11) was analyzed for pathogenicity. Inoculum production and inoculation were according to (1) by dipping (30 min) root-wounded 10-week-old plants of the Cavendish cv. Grand Naine in 2 liters of spore suspension (1.0 × 106 spores/ml). Inoculated plants were then placed in sand in 3-liter pots under 28°C, 70% relative humidity, and a 16/8-h light/darkness photoperiod. Sets of three plants were each treated with either JV11 or two TR4 controls (isolate II-5 and a strain isolated from an affected Cavendish plant in Mindanao, Philippines, both of which were diagnosed as TR4 by PCR and pathogenicity analyses). Control sets were either treated with race 1 originating from Cruz das Almas, Bahia, Brazil (1), or water. After 2 weeks, plants inoculated with JV11 and TR4 controls produced typical symptoms of Fusarium wilt. After 4 weeks, tissue was collected from all plants and plated on Komada's medium. TR4 was directly confirmed by PCR (1), either directly from symptomatic plants (JV11 and TR4 controls), or from isolates that were recovered from these plants. Nothing was re-isolated from race 1 inoculated plants and water controls, which remained asymptomatic. This is the first report of TR4 affecting Cavendish outside Southeast Asia, is its northernmost outbreak, and represents a dangerous expansion of this destructive race. Currently, 80% of the Jordan Valley production area is affected by Fusarium wilt, and 20 to 80% of the plants are affected in different farms. References: (1) M. A. Dita et al. Plant Pathol. 59:348, 2010. (2) J. F. Leslie and B. A. Summerell. The Fusarium Lab Manual. Blackwell, Ames, 2006. (3) R. C. Ploetz. Phytopathology 96:653, 2006.

Zymoseptoria gen. nov.: a new genus to accommodate Septoria-like species occurring on graminicolous hosts.

The Mycosphaerella complex is both poly- and paraphyletic, containing several different families and genera. The genus Mycosphaerella is restricted to species with Ramularia anamorphs, while Septoria is restricted to taxa that cluster with the type species of Septoria, S. cytisi, being closely related to Cercospora in the Mycosphaerellaceae. Species that occur on graminicolous hosts represent an as yet undescribed genus, for which the name Zymoseptoria is proposed. Based on the 28S nrDNA phylogeny derived in this study, Zymoseptoria is shown to cluster apart from Septoria. Morphologically species of Zymoseptoria can also be distinguished by their yeast-like growth in culture, and the formation of different conidial types that are absent in Septoria s.str. Other than the well-known pathogens such as Z. tritici, the causal agent of septoria tritici blotch on wheat, and Z. passerinii, the causal agent of septoria speckled leaf blotch of barley, both for which epitypes are designated, two leaf blotch pathogens are also described on graminicolous hosts from Iran. Zymoseptoria brevis sp. nov. is described from Phalaris minor, and Z. halophila comb. nov. from leaves of Hordeum glaucum. Further collections are now required to elucidate the relative importance, host range and distribution of these species.

Variable number of tandem repeat markers in the genome sequence of Mycosphaerella fijiensis, the causal agent of black leaf streak disease of banana (Musa spp).

We searched the genome of Mycosphaerella fijiensis for molecular markers that would allow population genetics analysis of this plant pathogen. M. fijiensis, the causal agent of banana leaf streak disease, also known as black Sigatoka, is the most devastating pathogen attacking bananas (Musa spp). Recently, the entire genome sequence of M. fijiensis became available. We screened this database for VNTR markers. Forty-two primer pairs were selected for validation, based on repeat type and length and the number of repeat units. Five VNTR markers showing multiple alleles were validated with a reference set of isolates from different parts of the world and a population from a banana plantation in Costa Rica. Polymorphism information content values varied from 0.6414 to 0.7544 for the reference set and from 0.0400 and 0.7373 for the population set. Eighty percent of the polymorphism information content values were above 0.60, indicating that the markers are highly informative. These markers allowed robust scoring of agarose gels and proved to be useful for variability and population genetics studies. In conclusion, the strategy we developed to identify and validate VNTR markers is an efficient means to incorporate markers that can be used for fungicide resistance management and to develop breeding strategies to control banana black leaf streak disease. This is the first report of VNTR-minisatellites from the M. fijiensis genome sequence.

Evidence for Natural Selection in the Mitochondrial Genome of Mycosphaerella graminicola.

ABSTRACT Pathogenicity assays were combined with restriction fragment length polymorphism (RFLP) markers in the mitochondrial and nuclear genomes to compare Mycosphaerella graminicola populations adapted to bread wheat (Triticum aestivum) and durum wheat (T. turgidum) in the Mediterranean Basin. The majority of isolates had unique nuclear DNA fingerprints and multilocus haplotypes. Only six mitochondrial DNA (mtDNA) haplotypes were identified among 108 isolates assayed. There were minor differences in frequencies of alleles at nuclear RFLP loci between the two host-adapted populations, but differences in the frequencies of mtDNA haplotypes were highly significant (P < 0.0001). mtDNA haplotype 1 dominated on the isolates adapted to bread wheat, and its frequency was twice as high as for the isolates adapted to durum wheat. mtDNA haplotype 4, which contained a unique approximately 3-kb insertion, was detected only in isolates showing specificity toward durum wheat and was the dominant haplotype on this species. We propose that the low mitochondrial diversity in this pathogenic fungus is due to a selective sweep and that differences in the frequencies of mtDNA haplotypes between the two host-adapted populations were due to natural selection according to host species.

Cloning and analysis of the mating-type idiomorphs from the barley pathogen Septoria passerinii.

The genus Septoria contains more than 1000 species of plant pathogenic fungi, most of which have no known sexual stage. Species of Septoria without a known sexual stage could be recent derivatives of sexual species that have lost the ability to mate. To test this hypothesis, the mating-type region of S. passerinii, a species with no known sexual stage, was cloned, sequenced, and compared to that of its close relative S. tritici (sexual stage: Mycosphaerella graminicola). Both of the S. passerinii mating-type idiomorphs were approximately 3 kb in size and contained a single reading frame interrupted by one (MAT-2) or two (MAT-1) putative introns. The putative products of MAT-1 and MAT-2 are characterized by alpha-box and high-mobility-group sequences, respectively, similar to those in the mating-type genes of M. graminicolaand other fungi. The mating-type genes of S. passerinii and M. graminicolaare evolving rapidly, approximately ten times faster than the internal transcribed spacer region of the ribosomal DNA, and are not closely related to those from Cochliobolusor other loculoascomycetes in the order Pleosporales. Therefore, the class Loculoascomycetes may be polyphyletic. Furthermore, differences between the phylogenetic trees may indicate separate evolutionary histories for the MAT-1 and MAT-2 idiomorphs. A three-primer multiplex-PCR technique was developed that allowed rapid identification of the mating types of isolates of S. passerinii. Both mating types were present in approximately equal frequencies and often on the same leaf in fields in Minnesota and North Dakota. Analyses with isozyme and random amplified polymorphic DNA markers revealed that each isolate had a unique genotype. The common occurrence of both mating types on the same leaf and the high levels of genotypic diversity indicate that S. passerinii is almost certainly not an asexual derivative of a sexual fungus. Instead, sexual reproduction probably plays an integral role in the life cycle of S. passerinii and may be much more important than previously believed in this (and possibly other) "asexual" species of Septoria.

Distribution of mating type alleles in the wheat pathogen Mycosphaerella graminicola over spatial scales from lesions to continents.

A total of 2035 Mycosphaerella graminicola strains collected from 16 geographic locations on four continents were assayed for the mating type locus. RFLP fingerprints were used to identify clones in each population. At the smallest spatial scale analyzed, both mating types were found among fungal strains sampled from different lesions of the same leaf as well as from different pycnidia in the same lesion. At larger spatial scales, the two mating types were found at equal frequencies across spatial scales ranging from several square meters to several thousand square kilometers. Though the absolute frequencies of the two mating types sometimes varied for different sampling units within the same spatial scale in the hierarchy (plots within a field, fields within a country, or different continents of the world), none of the differences were statistically significant from the null hypothesis of equal frequencies for the two mating types. The evolutionary forces likely to maintain the even distribution of the two mating types in this pathogen were discussed.

DNA Fingerprint Probe from Mycosphaerella graminicola Identifies an Active Transposable Element.

ABSTRACT DNA fingerprinting has been used extensively to characterize populations of Mycosphaerella graminicola, the Septoria tritici blotch pathogen of wheat. The highly polymorphic DNA fingerprints of Mycosphaerella graminicola were assumed to reflect the action of transposable elements. However, there was no direct evidence to support that conclusion. To test the transposable element hypothesis, the DNA fingerprint probe pSTL70 was sequenced, along with three other clones from a subgenomic library that hybridized with pSTL70. Analysis of these sequences revealed that pSTL70 contains the 3' end of a reverse transcriptase sequence plus 29- and 79-bp direct repeats. These are characteristics of transposable elements identified in other organisms. Southern analyses indicated that either the direct-repeat or reverse-transcriptase sequences by themselves essentially duplicated the original DNA fingerprint pattern, but other portions of pSTL70 contained single-copy DNA. Analysis of 60 progeny from a sexual cross between two Dutch isolates of Mycosphaerella graminicola identified several new bands that were not present in the parents. Thus, the putative transposable element probably is active during meiosis. Tests of single-spore isolates revealed gains or losses of one or more DNA fingerprint bands in 4 out of 10 asexual lines derived from isolate IPO94269. Therefore, DNA fingerprint patterns produced by the putative transposable element were capable of changes during asexual reproduction of this isolate. Probe pSTL70 did not hybridize at high stringency to genomic DNAs from other fungi related to Septoria and Mycosphaerella. These results indicate that the DNA fingerprint probe pSTL70 most likely identifies a transposable element in Mycosphaerella graminicola that may have been acquired recently, and appears to be active during both sexual and asexual reproduction.

Avirulence in the wheat septoria tritici leaf blotch fungus Mycosphaerella graminicola is controlled by a single locus.

Segregation of avirulence in Mycosphaerella graminicola, a heterothallic ascomycete that causes wheat septoria tritici leaf blotch, was studied in F1, BC1, and F2 populations by inoculation assays on five wheat cultivars in the seedling stage and by amplified fragment length polymorphism and random amplified polymorphic DNA analyses. F1 was generated by crossing isolates IPO323 (avirulent) and IPO94269 (virulent). All F1, BC1, and F2 progeny isolates were virulent on the susceptible check cultivar Taichung 29 and were avirulent on the resistant check cultivar Kavkav-K4500. Avirulence segregation was observed in F1 and in several BC1 and F2 generations on the differential cultivars Shafir, Kavkaz, and Veranopolis at a 1:1 ratio. Avirulence for the three differential cultivars always cosegregated. We conclude that avirulence in isolate IPO323 is controlled by a single, seemingly complex locus.

Genetic Variation for Virulence and Resistance in the Wheat-Mycosphaerella graminicola Pathosystem III. Comparative Seedling and Adult Plant Experiments.

ABSTRACT Fourteen Dutch Mycosphaerella graminicola isolates were studied for their virulence to 22 wheat cultivars in the seedling stage in an experiment set up as a completely randomized block design with three repetitions. Isolate x cultivar interactions were highly significant. Cluster analyses were applied to select three isolates with significantly different virulence characteristics for both disease parameters. These were retested in the seedling stage and used to inoculate two field experiments that were planted according to a split plot design in 1992 and 1995. Overhead inoculations were conducted after flowering to avoid the effects of plant height; hence, these experiments were intended as monocyclic tests for virulence differences between the isolates. Significant isolate x cultivar interactions were detected in each experiment, demonstrating specificity in the wheat-M. graminicola pathosystem in the adult plant stage under field conditions. The reproducibility of the adult plant data was high. Genetic differences among the isolates were additionally demonstrated by randomly amplified polymorphic DNA (RAPD) patterns, which also showed that no significant contamination of the inoculated plots with the natural M. graminicola population had occurred. Rank correlations between seedling and adult plant data were significant for M. graminicola isolates IPO323 and IPO290, but not for isolate IPO001. Hence, evaluation of resistance and virulence may require seedling, as well as adult plant, tests.

First Report of Puccinia striiformis f. sp. tritici on Wheat in South Africa.

During August 1996, stripe (yellow) rust, caused by Puccinia striiformis f. sp. tritici, was observed for the first time on bread wheat (Triticum aestivum) in the Western Cape, South Africa. Ensuing surveys during the growing season indicated that stripe rust occurred throughout most of the wheat-producing areas in the winter rainfall regions of the Northern, Western, and Eastern Cape provinces. The disease was also observed on irrigated wheat in the summer rainfall area south of Kimberley. Stripe rust was most severe in the Western Cape, where prolonged cool and wet conditions favored epidemic development and necessitated extensive and often repeated applications of triazole fungicides. Due to spike infection and destruction of foliage, significant losses in grain quantity and quality occurred in certain fields. Avirulence/virulence characteristics of 32 stripe rust isolates, collected from commercial wheat fields, trap nurseries, and triticale, were determined on 17 standard differential wheat lines and seven supplementary testers supplied by C. R. Wellings, Plant Breeding Institute, Cobbitty, Australia. All isolates were representative of one pathotype, characterized by avirulence to Chinese 166 (Yr1), Vilmorin 23 (Yr3), Moro (Yr10), Strubes Dickkopf, Suwon 92/Omar, Clement (Yr2,9), Triticum aestivum subsp. spelta var. album (Yr5), Hybrid 46 (Yr4), Reichersberg 42 (Yr7), Heines Peko (Yr2,6), Nord Desprez (Yr3), Carstens V, Spaldings Prolific, Heines VII (Yr2), Federation*4/Kavkaz (Yr9), and Avocet-S/Yr15, and by virulence to Kalyansona (Yr2), Heines Kolben (Yr2,6), Lee (Yr7), Compair (Yr8), and Federation 1221. Cultivars Trident (Yr17), Avocet-R (YrA), and Selkirk (YrSk) appeared heterogeneous for stripe rust reaction. The pathotype resembled race 6E16, previously detected in East and North Africa, the Middle East, and western Asia. Pathotype identity was confirmed at IPO-DLO, Wageningen, using one South African isolate of P. striiformis f. sp. tritici. In view of the rapid dispersal of the pathogen during 1996, susceptibility of several high-yielding cultivars, and favorable climatic conditions in many wheat-growing areas, stripe rust is considered potentially damaging to South African wheat production. Field observations and seedling tests have shown, however, that certain cultivars are resistant to the introduced pathotype. At present the genetic basis of this resistance is largely unknown.

Successful crosses and molecular tetrad and progeny analyses demonstrate heterothallism in Mycosphaerella graminicola.

Monospore isolates of Mycosphaerella graminicola considered to originate from one ascus were analysed by the polymerase chain reaction (PCR) with 32 RAPD primers. Eighteen of these revealed three classes of polymorphisms, which enabled a RAPD-based tetrad analysis. Four pairs of isolates resulting from a single diploid nucleus were determined. A procedure to cross these isolates 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 studied with three primers to determine the mode of inheritance of the RAPD markers. Mendelian segregation and recombination of RAPD markers was observed in all progenies.