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Multiple alleles were constantly detected in Alternaria solani isolates by simple sequence repeat (SSR) analysis, and sectors were also observed in their subcultures. These preliminary results and observations point to a possible parasexual cycle in A. solani. In this study, codominant SSR markers were used as molecular markers on the chromosomes of A. solani and single-conidium subculture was used to simulate the mitosis process of A. solani in nature. The number of alleles at locus As-95236 changed from 2 to 1 as a molecular marker for haploidy of parasexuality of A. solani. Fifty monosporic F1 strains were tested. The results showed that two parent strains lost allele with a haploid probability of 38%. For F2 strains, the results showed that all four F1 strains lost allele with a haploid probability of 75%. Since sexual recombination of A. solani has not been found so far, the allele lost in the subcultures of A. solani isolates provides molecular evidence for the existence of parasexual reproduction in A. solani.

Monobasidiospore isolates were prepared from basidiocarps of Stereum sanguinolentum. Five isolates per basidiome were paired with each other and with isolates from the trama. Interbasidiome pairings of the trama isolates and of a selection of single-spore isolates also were performed. Thin sections of the hymenium were stained with DAPI and examined by fluorescence microscopy to study the nuclei in the basidia. Spore prints were stained with DAPI to count the number of nuclei per spore. SEM was used to determine the number of basidiospores per basidium. All intrabasidiome pairings were compatible. In contrast, interbasidiome pairings, except one, were incompatible, independent of whether single-spore or trama isolates were paired. Fertile basidiomes were formed in single-basidiospore cultures. Basidia were regularly four-spored. On average, 5% of the basidiospores possessed one nucleus, 82% two, 2% three and 1% four nuclei. Ten percent of the spores appeared to be empty. Karyogamy, meiosis and postmeiotic mitosis were observed in the basidia. Nuclei resulting directly from meiosis, i.e., without having undergone postmeiotic mitosis, sometimes were observed in the sterigmata or spore primordia. The high number of vegetative compatibility groups (VCG) of S. sanguinolentum observed in this study and earlier studies is difficult to explain without sexual or parasexual recombination. We suppose that the majority of spores with ≥2 nuclei are amphithallic, possessing at least one nucleus of each mating type. Recombination could occur by exchange of nuclei among VCGs via anastomoses between homothallic compartments. Transfer of nuclei from heterothallic to homothallic mycelia or matings between homothallic mycelia, which originate from monokaryotic spores, might be other paths for gene exchange.

Heterokaryotic colonies of Stagonospora nodorum consist of a fine mosaic of individual cells of the two component genotypes that are linked by frequent hyphal anastomosis. The intimate mixing does not extend to the colony edge where 1 mm3 blocks of hyphae all of the same genotype occur. Col- onies developed from single pycnidiospores from heterokaryons were homokaryotic and segregated into the two component genotypes, demonstrating that all the nuclei in a spore are of the same geno- type. Spore progenies from single pycnidia produced by heterokaryons, however, were frequently geneti- cally heterogeneous, indicating that these asexual fruiting structures are not uninucleate in origin. The nuclear ratio in heterokaryons was characteristic of the component genotypes and could deviate mark- edly from 1: 1 with no reduction in growth. Only 1.6% of pairs of physiologically-different nitrate non- utilizing mutants from different strains showed com- plementation, indicating that vegetative incompati- bility is widespread in S. nodorum. The few compati- ble strains had common origins and may be clonally related. Tests on a limited number of combinations suggested that vegetative incompatibility could not be overcome by protoplast fusion. Attempts to isolate so- matic heterozygous diploids and parasexual recom- binants were largely unsuccessful. Heterokaryosis and parasexuality do not appear to be significant sources of natural variation in this fungus.

Averufin is a polyhydroxyanthraquinone first isolated from Aspergillus versicolor (Vuill.) Tiraboschi (Pusey and Roberts, 1963; Holker et al., 1966). This anthraquinone inhibits ATP synthesis in mitochondria by exerting an extremely potent uncoupling effect and a significant depression on mitochondrial respiration (Kawai et al., 1984). Averufin is an intermediate in aflatoxin biosynthesis and has been extensively studied in a blocked mutant of A. parasiticus Speare (Donkersloot et al., 1972; Lin et al., 1973) and has been useful in elucidating the origin of the difuran ring in the polyketide pathway leading to sterigmatocystin and subsequently aflatoxin production (Townsend et al., 1982). Because of its bright orange color, averufin is also a useful marker in parasexual studies of aflatoxigenic fungi (Bennett and Papa, 1988). Aflatoxins are highly toxic secondary metabolites produced by certain strains of A. parasiticus and A. flavus Link (Betina, 1989). Genetic studies of aflatoxin biosynthesis have been hampered by the absence of a sexual (teleomorphic) stage in either aflatoxigenic species. Transformation systems have recently been developed for both species (Woloshuk et al., 1989; Skory et al., 1990) and will facilitate future molecular analyses. Nevertheless, it will require years of research to characterize A. flavus or A. parasiticus to the level of A. nidulans, which has well developed classical and molecular genetic systems (Smith and Pateman, 1977; Timberlake, 1980). A recent paper reports that sterigmatocystin, a late intermediate in aflatoxin biosynthesis, is produced in significant amounts by the "Glas

SUMMARYSix diploids of Aspergillus parasiticus involving 12 genetic markers were analyzed by means of the parasexual cycle. Heterokaryotic heads consisted of homokaryotic and heterokaryotic conidia...

The recovery of mutants impaired in aflatoxin production and the identification of parasexuality in Aspergillus flavus Link ex Fries now make genetic investigations of aflatoxin biogenesis possible. In wildtype strains, aflatoxin B2, the dihydro derivative of B1, generally accumulates in smaller quantities than B1. Isolates accumulating only aflatoxin B2 were reported by van Walbeek et al. (9) in A. flavus var. columnaris and by Schroeder and Carlton (8) in an A. flavus isolate from black pepper. A relatively stable mutant of A. flavus accumulating high levels of aflatoxin B2 (5,000 jug/g mycelial dry wt) and low levels of aflatoxin B1 (600 tsg/g mycelial dry wt) was recently recovered following treatment of conidia with nitrosoguanidine (6). A parasexual analysis of this mutant revealed a genetic locus involved in aflatoxin biosynthesis (7). Heterozygous diploids exhibited the dominant phenotype of B2 B1. No linkage was detected between this gene locus and selected gene markers of the seven genetically identified linkage groups. The present study is a continuation of our genetic analysis of this mutant, including linkage to the histidine locus and the identification of linkage group VIII.

Parasexual crosses involving a mutant of Aspergillus flavus having a high and relatively stable level of aflatoxin B2 relative to aflatoxin B1 have revealed a genetic locus for control of aflatoxin...

Species of Trichoderma and the closely related genus Gliocladium are ubiquitous, active components of the soil microflora. As biocontrol agents, these fungi can protect a wide range of agronomic, horticultural, and ornamental plants against a variety of plant pathogens (6, 13), and a few strains have been patented for commercial use. Some strains enhance plant growth or rooting of cuttings even in the absence of pathogens (4, 14). Trichoderma spp. have also been studied extensively as potential sources of cellulases for utilization of lignocellulosic materials (10, 17). The taxonomy, genetics and population composition of these fungi are poorly understood. Limited knowledge of variability in these fungi and infrequent culturing of their sexual stages make delineation of narrowly defined species difficult (16). Additionally, little is known about such genetic components as ploidy levels, frequency of heterokaryosis, or the prevalence of parasexual events. Finally, methods are lacking to differentiate among strains for patent purposes, or to determine the variability and abundance of strains in natural ecosystems. Isozyme analysis by starch gel electrophoresis is a powerful method for taxonomic, genetic and population studies. Descriptions of general techniques and of numerous enzyme assay systems have been published (9, 12, 18, 20, 21). However, for studies of specific organisms, suitable enzyme assays and corresponding electrophoretic buffers must be determined empirically (8, 12, 18). For an enzyme assay to be useful, enzyme activity must be reliably detected and activity bands must be clearly resolved. In addition, polymorphism must exist among isolates of the study population. Also, the usefulness of an enzyme assay for a particular study often depends on the number of loci detected. Assays detecting products of single loci can be particularly useful because polymorphism among individuals reflects allelic differences, and electrophoretic variants can be scored as allozymes. The genetic basis of an isozyme phenotype usually can be inferred from the complexity of the activity band pattern visualized (9, 12, 18). In this study, 63 enzyme assays were tested in combination with four electrophoretic buffers to assess detection of enzyme activity, resolution of activity bands, and complexity ofphenotypes visualized. This report summarizes the most useful systems for studies of Trichoderma and Gliocladium spp. and identifies those which apparently visualize products of single loci, i.e., allo

(1973). The Parasexual Cycle in Aspergillus Flavus. Mycologia: Vol. 65, No. 5, pp. 1201-1205.