The ability of some groups of closely related yeasts to use potassium nitrate as a source of nitrogen was applied successfully by Stelling-Dekker (1931) to the classification of the sporogenous yeasts. Later, Lodder (1934) added other nitrogen compounds, namely, ammonium sulfate, urea, asparagine, and peptone, in diagnostic tests for her classification of the nonsporogenous, nonfilamentous yeasts. She employed a modification of Beijerinck's (1889) auxanographic technique in the following manner: About 2 ml of a dense suspension of the yeast to be tested were placed in a petri dish. It was assumed on the basis of Wildiers' (1901) work that the use of such a heavy inoculation of cells would provide adequate growth factors. An agar medium consisting of 2 per cent glucose, 0.1 per cent potassium dihydrogen phosphate, 0.05 per cent magnesium sulfate, and 2.0 per cent washed agar was cooled to 40 C and poured into the dish. The medium and yeast were quickly mixed. After the medium had solidified, the plate was placed in an incubator to dry for a few hours at 30 C. Then small portions of the nitrogen-containing compounds were placed on the solid surface of the inoculated agar. On incubation at 25 C, an area of growth was produced around those compounds that were assimilated. Lodder's study disclosed that a majority of the yeasts with which she worked were capable of utilizing all the compounds that she had introduced. However, some species of Torulopsis and all species of Kloeckera failed to assimilate ammonium sulfate, urea, and asparagine. These facts were subsequently incorporated in her descriptions of species and genera. These nitrogen compounds, as well as the use of the auxanographic plate method, were generally adopted for diagnostic purposes by succeeding workers in Europe and South America. Langeron and Guerra (1938) used Lodder's medium and technique for their study of filamentous yeasts belonging to the genus Candida. Because they found that urea diffused through the medium so rapidly that it sometimes overlapped the diffusion zones of other nitrogen sources, only one other test substance was placed in the same plate. Langeron and Guerra found that 2 of their 16 species of Candida, C. pelliculosa and C. zeylanoides, utilized peptone only, but 6 assimilated urea. At variance with these results were the findings of Diddens and Lodder (1942) with respect to the assimilation reactions of the Candida species. They found that C. pelliculosa and C. zeylanoides utilized ammonium sulfate, urea, and asparagine in addition to peptone, and that the species C. tropicalis, C. guil-1 One of the laboratories of the Bureau of Agricultural and Industrial Chemistry, Agricultural Research Administration, U. S. Department of Agriculture.

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Two new species of the yeast genus Pichia were isolated from frass obtained from loblolly pines growing in Mississippi. The new taxa, designated Pichia amylophila and Pichia mississippiensis, are heterothallic, and Candida o btusa var. arabinosa proved to be a haploid member of the latter species. The species show interspecific mating, but ascospores from the crosses are infertile. The nuclear deoxyribonucleic acids from the species differ by 2 mol% in guanine plus cytosine content and exhibit low (25%) base sequence complementarity. Deoxyribonucleic acid relatedness was determined spectrophotometrically, and the methodology is given in detail.During the isolation of yeasts from insect frass collected from pine trees in Mississippi, we recovered strains that were phenotypically similar to Pichia wickerhamii (van der Walt) Kregervan Rij and Pichia rhodanensis (Ramirez et Boidin) Phaff. These strains were heterothallic but failed to conjugate with mating types from either P. wickerhamii or P. rhodanensis. One of the newly isolated cultures differed from the others by its ability to assimilate starch, and genetic crosses between it and the other isolates gave only infertile and generally poorly formed ascospores. In an effort to clarify speciation, we examined the nuclear deoxyribonucleic acid (DNA) base sequence complementarity of the strains. As a result, the isolates proved to be of particular interest because they allowed an examination of species parameters through both genetic crosses and DNA reassociation studies. ilation tests were previously given (21). Single-ascospore isolates were obtained by micromanipulation. MATERIALS AND METHODSThe criteria used for speciation within the genus Pichia were those proposed by Kreger-van Rij (7).DNA purification and determination of base composition. Extraction and purification of DNA were done by a combination of the procedures of Marmur (9) and A~~o / A~w were used to assess DNA purity. Purification was repeated if the preparation deviated more than 0.05 from the ratios AZW/A'W = 1.86 and ABO/AM = 0.5 (11). The quality of the DNA was further assessed from analytical ultracentrifuge scans and from thermal-melt profiles. Hyperchromicity of the preparations ranged from 34.4 to 36.8%.The guanine plus cytosine (G+C) content of the nuclear DNA was calculated from buoyant density values in cesium chloride (16,19) and was based on three or four separate determinations made with a Spinco model E analytical ultracentrifuge equipped with an electronic scanner. Micrococcus lysodeikticus Fleming DNA was used as a reference; this DNA had a buoyant density of 1.7311 g/ml when compared to DNA from Escherichia coli K-12, whose density was taken to be 1.7100 g/ml (16). DNA reannealing reactions.The extent of DNA reassociation was determined spectrophotometrically, essentially by the method reported by Seidler and Mandel(18) and Seidler et al. (17), as described below. DNA was sheared by two passages through a French press at 10, OOO lb/in2 and was recovered as intact double-strand...

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