Twenty-four species assigned to the genus Saccharomyces sensu stricto were examined for deoxyribonucleic acid relatedness. Results with type strains demonstrated the presence of four distinct species: S. cerevisiae, S. bayanus, S . carlsbergensis, and S . kluyveri. S . carlsbergensis NRRL Y-12693T showed intermediate relatedness between S . cerevisiae and S . bayanus and has a genome size approximately 1.5 times those of the last two species. These data suggest S . carlsbergensis to be a partial amphidiploid which may have arisen from natural hybridization between S. cerevisiae and S . bayanus.Numerous studies in recent years have shown that the criteria used in traditional yeast taxonomy are not always satisfactory for delimitation of species. This is partly because many separations are based on the presence or absence of assimilative abilities which are often controlled by a single mutable gene (12, 14, 18,23,25). The species of Saccharomyces sensu stricto (34) provide a good example of this problem because of strain variability in standard carbon assimilation and fermentation tests (5, 16,28,34,35,36). As a result of the uncertainty of these tests, a number of other approaches have been attempted, such as comparisons of cell wall antigens (2, 10, 11,27), proton magnetic resonance spectra of extracted mannans (11,32), and attempted matings between haploid mating types of similar species (3, 21). Requirements for vitamins (9) and amino compounds (7) have also been used for taxonomic separations, as have differences in deoxyribonucleic acid (DNA) base composition (guanine plus cytosine [G + C] contents in moles percent) (36). Finally, numerical analyses (1, 8, 15, 17) by Adansonian principles have been used for species separations. None of these methods, however, seems to have resolved the status of the species investigated (25).More significant information concerning taxonomic relationships among yeasts is obtainable by comparison of their nucleic acids (18,25). In this light, some progress has been made with regard to Saccharomyces spp. Bicknell and Douglas (6) determined DNA relatedness among several species, whereas Rosini et al. (26) concluded from their DNA studies that common wine yeasts comprised at least three Saccharomyces species. In the present study, we measured DNA relatedness among all taxa assigned to Saccharomyces sensu stricto (34,35,36) in an effort to clarify their relationships. MATERIALS AND METHODSMicroorganisms. Twenty-nine strains representing 24 species were examined. Strain designations are given in Table 1. were used to assess DNA purity, as were thermal melting profiles and ultracentrifuge scans.The G + C contents of nuclear DNAs were calculated from buoyant density values in cesium chloride (29, 33) based on three or four separate determinations made with a model E analytical ultracentrifuge (Beckman Instruments, Inc .) equipped with an electronic scanner. Micrococcus luteus (synonym, M . lysodeikticus) DNA was used as a reference; this DNA has a buoyant density of 1.7311 g/ml (25...

A total of 1045 milk samples collected from infected and non infected quarters of 269 cows were investigated. This study showed that 4.7% of samples possessed cells of Prototheca spp. (10(6)cells/ml). The presence of other pathogenic microorganisms was also monitored. Prototheca spp. isolates were classified on the basis of current taxonomic guidelines and identified as P. zopfii. Susceptibility tests carried out in vitro by using 25 antibiotic compounds revealed that the strains of P. zopfii. were susceptible only to nystatin and amphotericin B (58 and 33% of total strains, respectively). The present study represents the first large-scale investigation carried out in Italy on the isolation of this achlorophyllous yeast-like microalga in milk samples produced by dairy herds.

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The yeast flora associated with the surface of various natural materials have been studied on the assumption that individual cells and microcolonies are strongly retained by gummy and mucous secretions. Preisolation treatments based on vigorous shaking, percolation with an excess of water, and the sonication of samples allowed the recovery of a much higher number of species than did traditional isolation procedures. Results confirmed that a sequence of aggressive and disruptive actions on samples, together with an enrichment culture, is necessary to obtain an exhaustive picture of the actual yeast flora.

Deoxyribonucleic acid relatedness among the type strains of all taxa and known anamorphs assigned to the yeast genus Kluyveromyces was assessed by the optical reassociation technique. Three groups of species related at the 95% level or higher were found: (i) K. lactis including K . drosophilarum, K. phaseolosporus, K . vanudeni, and the anamorph Candida sphaerica; (ii) K . marxianus with K. bulgaricus, K. cicerisporus, K . fragilis, K . wikenii, and the anamorphs Candida kefyr, Candida macedoniensis, and Candida pseudotropicalis; (iii) K. thermotolerans with K. veronae and the anamorph Candida dattila. The remaining species, including the recently described K . blattae and K . waltii, are not related to each other or to the members of the above three groups. The nomen nudum K. cellobiovorus is not conspecific with any of the species of the genus. The species assignment obtained by nuclear deoxyribonucleic acid reassociation does not entirely conform with the previously proposed reorganization of the genus Kluyveromyces (J.-P. van der Walt and E. Johannsen, p. 224-251, in N. J. W. Kreger-van Rij, ed., The Yeasts. A Taxonomic Study, 1984).The genus Kluyveromyces was established in 1956 by van der Walt (52) to classify the newly isolated, budding, fermentative yeast species K. polysporus, which produces large, multispored asci containing as many as 70 or more reniform to long oval spores. In the same year, an additional species, K. africanus, forming up to 16 spores per ascus, was included in the genus by van der Walt (53). The salient property of the asci of these two species was their early evanescence at maturity, to release the spores.In 1965, the diagnosis of the genus was emended by van der Walt (54) on the assumption that the species with multispored asci represented only a separate line of development in relation to other species which normally form only four spores. The above approach was based on a series of controversial taxonomic treatments of those yeast species that form kidney-shaped spores (5, 18, 19, 34,60). Van der Walt included in Kluyveromyces all the species formerly classified in the genera Fahospora, Zygofahospora Kudriawzev (19), Dekkeromyces nomen nudum Wic kerham and Burton (59), and Guilliermondella (5). Later, Santamaria and Sanchez (41) proposed to assign the nomen nudum Dekkeromyces to those species of the genus that are unable to form multispored asci (K. dobzhanskii, K. drosophilarum, K . fragilis, K . lactis, K . phaseolosporus, and K . wickerhamii). Van der Walt (55) applied the overall criteria that had suggested the earlier emendation and assigned to the genus 19 species including several recently described new taxa.Several subsequent approaches to the taxonomy of the genus Kluyveromyces were attempted, such as those based on vitamin requirements (9), electrophoretic isoenzyme patterns (45, 46), structure of exo-P-glucanases (23), chemical and immunochemical studies of cell wall components (7,13, 31,40,51), numerical taxonomy (6, 3 3 , ultrastructure of the ascospore wall (2), t...

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The genus Schizosaccharomyces comprises a small, somewhat heterogeneous group of yeast species which have in common a unique mode of vegetative reproduction by cross-wall formation without constriction as well as a certain degree of osmophilia, most strains having been isolated from habitats of high sugar concentration. This study evaluated inter- and intraspecific relationships utilizing nDNA/nDNA optical reassociation and by the analysis of physiological profiles of several strains of each species. Results demonstrate that the genus should be divided into three species: Schiz. japonicus, Schiz. octosporus and Schiz. pombe.