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The D1/D2 regions of the large subunit (LSU) rRNA genes of 65 strains of Monascus and Xeromyces were PCR amplified and sequenced in both directions. Maximum-parsimony analysis produced five most parsimonious trees. The strict consensus tree of these five parsimonious trees clustered M. eremophilus, M. ruber, M. pilosus, M. purpureus, and M. sanguineus in the same clade, reflecting high sequence similarity. M. sanguineus, M. purpureus, M. ruber, and M. pilosus differed in one or two nucleotides. The sequence of M. eremophilus ATCC 62925 isolated from a xerophilic environment differed from M. purpureus in only one nucleotide, despite pronounced morphological and ecological differences when compared with the other species. M. lunisporas, M. floridanus, M. pallens, and X. bisporus were each placed in a separate branch, confirming their taxonomic descriptions as individual species.Maximum-likelihood analysis on the same data set generated a single tree and grouped the species of the first clade in the parsimony analysis into a single clade but placed the rest of the Monascus species and Xeromyces bisporus on different branches. The trees inferred from both analyses revealed a monophyletic relationship between Monascus and Xeromyces, when compared with other related cleistothecial or imperfect genera.

A total of 169 strains of the Aspergillus reference cultures in the Aspergillus flavus group maintained in the American Type Culture Collection (ATCC) were studied for their aflatoxin-producing abilities in rice, peanut and semisynthetic medium, utilizing high pressure liquid chromatography. Fifty-nine of the strains examined were positive for aflatoxin formation. No strains of the food fungi A. oryzae or A. sojae produced detectable levels of aflatoxins, while 33-85% of the strains of A. flavus and A. parasiticus were toxigenic.

The yeast Schwanniomyces occidentalis produces a killer toxin lethal to sensitive strains of Saccharomyces cerevisiae. Killer activity is lost after pepsin and papain treatment, suggesting that the toxin is a protein. We purified the killer protein and found that it was composed of two subunits with molecular masses of approximately 7.4 and 4.9 kDa, respectively, but was not detectable with periodic acid-Schiff staining. A BLAST search revealed that residues 3 to 14 of the 4.9-kDa subunit had 75% identity and 83% similarity with killer toxin K2 from S. cerevisiae at positions 271 to 283. Maximum killer activity was between pH 4.2 and 4.8. Killer yeasts secrete toxins lethal to sensitive yeasts but are immune to their own toxins. Since first discovered in Saccharomyces cerevisiae (2), killer strains have been isolated from several yeast genera, including Candida (46), Cryptococcus (10), Hanseniaspora (33), Kluyveromyces (14), Pichia (27), Torulopsis (7), Ustilago (30), Williopsis (45), and Zygosaccharomyces (32). Based on killing and immunity interactions among killer yeasts, killer phenotypes are classified into at least 10 groups (48) and the responsible genes may be carried on a chromosome (S. cerevisiae KHS, KHR, Williopsis mrakii) (11,12,21), on a double-stranded RNA (dsRNA) (S. cerevisiae K1, K2, K28, Ustilage maydis, Hanseniaspora uvarum) (5,15,22,35,49), or on a linear double-stranded DNA (dsDNA) (Kluyveromyces lactis, Pichia inositovora, Pichia acaciae) (14,16,44).Schwanniomyces occidentalis produces amylolytic enzymes, including ␣-amylase and glucoamylase (8). It is one of the few yeasts capable of completely hydrolyzing soluble starch. Moreover, it can grow to high cell mass by utilizing cheap starch from plants such as cassava, corn, potato, sorghum, and wheat as the carbon source (40). S. occidentalis has been used to produce ethanol and single cell protein from starch fermentation (19,42). S. occidentalis has no detectable extracellular proteases and can secrete large proteins (40). It also has an established transformation system and available inducible promoters, which could make it a commercially important system for the production of heterologous proteins (40). For example, endoglucanase D recently has been successfully expressed and secreted in this system (31).Wild killer yeasts sometimes contaminate cultures of industrial yeasts, resulting in lagging or stopped fermentation and poor product quality (39). To avoid such complications, the use of industrial killer strains as starters has been suggested (17). Commercially interesting killer strains for sake brewing, wine making, alcohol fermentation, and lager, beer, and ale production have been constructed (29,36,39,47). Furthermore, the W. mrakii mycocin expressed by Aspergillus niger can reduce silage and yogurt spoilage caused by yeasts (25).The killer phenotype of S. occidentalis has not been described previously. Thus, our objectives in this study were as follows: (i) to screen killer strains from S. occidentalis for a killer phenotype; (ii...

Restriction fragment length polymorphisms (RFLPs) in two regions of the ribosomal DNA (rDNA) repeat unit were examined in 33 strains representing 18 species of Saprolegnia. The Polymerase Chain Reaction (PCR) was used to separately amplify the 18S rDNA and the region spanning the two internal transcribed spacers (ITS) and the 5.8S ribosomal RNA gene. Amplified products were subjected to a battery of restriction endonucleases to generate various fingerprints. The internal transcribed spacer region exhibited more variability than the 18S rDNA and yielded distinctive profiles for most of the species examined. Most of the species showing 100% similarity for the 18S rDNA could be distinguished by 5.8S + ITS restriction polymorphisms except for S. hypogyna, S. delica, S. lapponica, and S. mixta. The rDNA data indicate that S. lapponica and S. lapponica and S. mixta are conspecific with S. ferax, whereas there is no support for the proposed synonymies of S. diclina with S. delica and of S. mixta with S. monoica. Results from cluster analysis of the two data sets were very consistent and tree topologies were the same, regardless of the clustering method used. A further examination of multiple strains in the S. diclina-S. parasitica complex showed that restriction profiles are conserved across different strains of S. parasitica originating from the U.K. and Japan. HhaI and BsaI restriction polymorphisms were observed in isolates from the U.S. and India. The endonuclease BstUI was diagnostic for S. parasitica, generating identical fingerprints for all stains regardless of host and geographic origin. Except for the atypical strain ATCC 36144, restriction patterns were also largely conserved in S. diclina. Correlation of the rDNA data with morphological and ultrastructural features showed that S. diclina and S. parasitica are not conspecific. Restriction polymorphisms in PCR-amplified rDNA provide a molecular basis for the classification of Saprolegnia and will be useful for the identification of strains that fail to produce antheridia and oogonia.