(1918) described cultures of S. ellipsoideus, Saccharomyces pastorianus, occurring on grapes. Bioletti and Cruess (1912) briefly

Transcription

1 YEASTS OCCURRING ON GRAPES AND IN PRODUCTS IN CALIFORNIA GRAPE E. M. MRAK AND L. S. McCLUNG University of California, Division of Fruit Products, Berkeley, California Received for publication February 27, 1940 The yeasts found on the grapes and in the wines of California have received little attention from the botanical point of view, although the viticultural industries of the area are comparatively old. Holm (1908) incompletely described several yeasts obtained from California grapes designating them by number or as Saccharomyces ellipsoideus (Bioletti I Nov. Var.). He concluded that yeasts found on grapes produced in regions remote from wine-making operations are inactive in respect to the formation of alcohol and that many are detrimental to wine by producing films, turbidity, and unpleasant flavors. According to Bioletti (1911) S. ellipsoideus is the most common wine yeast and Saccharomyces apiculatus the most common pseudoyeast occurring on grapes. Bioletti and Cruess (1912) briefly described the following yeasts obtained from grapes produced in the Davis, Acampo and Contra Costa vineyard areas of California: Saccharomyces apiculatus, Saccharomyces ellipsoideus, "wild yeast (Saccharomyces sp.)," and other "wild yeasts" characterized by film formation and slow fermentation. Cruess (1918) described cultures of S. ellipsoideus, Saccharomyces pastorianus, Willia anomalus, S. apiculatus, Mycoderma and Torula obtained from California grapes but failed to obtain cultures of Saccharomyces ludwigii, Saccharomyces cerevisiae, Saccharomyces malei, or Saccharomyces marxianus. Grapes from the wineproducing area of Ripon, Tulare, Fresno and Contra Costa regions yielded more true yeasts than grapes obtained from the remote areas of Davis and El Centro, and ripe grapes contained greater numbers of yeasts than green grapes. Suminoe (1928, 1930) reported on wine yeast isolated from California wine mash. 395

2 396 E. M. MRAK AND L. B. McCLUNG EXPERIMENTAL PROCEDURE Grapes (at various stages of maturity ranging from firm green to soft ripe, and in a few instances, the raisin stage), grape must, and pomace were collected in various grape and wine-producing areas primarily in Central California. In addition a few samples of cloudy bottled wine, vineyard soil and grape leaves were included. All wine samples were from unsulphited naturally fermented musts. All samples were collected in sterile jars and examined as quickly as possible. The grapes were crushed in the original containers and incubated at room temperature. Sterile 100 Balling grape juice was added if needed. Streaks on wort agar were made as soon as fermentation started, and in some instances after it had ceased. Purification was accomlished by three platings on agar with isolated colonies in each case picked to grape juice. Cultures were identified in accordance with the procedures and systems of Stelling-Dekker (1931) and Lodder (1934). Color comparisons were made with the charts of Ridgway (1921). Carrot, beet, potato and cucumber wedges and Gorodkowa agar slants were used as sporulation media. Other media were used only when spores failed to form on those listed. In a few instances sporulation occurred on cucumber wedges only. The utilization tests with anascosporogenous yeast were conducted with synthetic liquid medium rather than auxanogramic plates because the liquid method expedited the determinations and gave results comparable to those obtained by the auxanogramic method. Considerable difficulty was experienced in the differentiation of the families Rhodotorulaceae and Torulopsidaceae because of the inconclusiveness of the carotene crystallizing experiments. Numerous attempts to obtain crystals from Rhodotorula rubra (Demme) Lodder were often doubtfully positive. Prolonged storage (6 weeks) of the cells in the Molisch solution seemed to give better results. It was necessary to rely to a considerable extent on visible color differentiation as well as on the carotene crystal method to differentiate the two families. A large number of the cultures differed slightly from forms described by others. In view of the recent contributions of

3 YEASTS OCCURRING ON GRAPES 397 Satava (1934), Winge (1934) and Winge and Laustsen (1937, 1938) which have established the occurrence of haploid and diploid generations as well as hybridization in yeast, it is inadvisable to establish new varieties or species unless the differences are considerable. A large number of organisms showing only slight variations from described species have been included in existing species or varieties. Such differences, however, are discussed in the text as a matter of record. RESULTS Sporulating yeasts Genus Saccharomyces: Ninety-five of 118 cultures of Saccharomyces were identified as S. cerevisiae Hansen and only 4 as S. cerevisiae var. ellipsoideus (Hansen) Dekker. Wine yeasts are normally included in the ellipsoideus variety of S. cerevisiae on the basis of cell shape and size, but it is very difficult at times to distinguish the variety on cell morphology alone. Since many of the cultures identified as S. cerevisiae Hansen were isolated as the predominating organism from naturally fermenting musts it is quite possible that they are ecologically different from the type cultures of S. cerevisiae and S. cerevisiae var. ellipsoideus, although the physiological characteristics used in taxonomy are similar. Three cultures (16B1, 37B1, 74A1) were termed S. oviformis Osterwalder, although they differ slightly in slant culture characteristics. S. oviformis is described by Stelling-Dekker as producing smooth, slightly glossy slant cultures with waxy borders. The slant culture of 74A1 is similar to that of S. oviformis but that of 16B1 differs by having a finely contoured surface and lacerate border. The streak of 37B1 has an entire border and a slightly vesicular surface. Physiological characteristics corresponded in detail to those of S. oviformis proper. Fifteen cultures closely resembling S. chodati Steiner were isolated. S. chodati ferments sucrose slowly and has slightly wrinkled slant cultures, whereas the cultures isolated ferment sucrose rapidly and possess smooth slant cultures. Other characteristics were similar.

4 398 E. M. MRAK AND L. S. McCLUNG A single culture (119A1), which corresponded exactly to the description, was identified as S. carlsbergensis Hansen. Subgenus Zygosaccharomyces: Cultures were assigned to Zygosaccharomyces only when conjugation preceded spore formation. In most cases sexuality was observed rather infrequently. In several instances conjugating cells formed only sterile "dumbbells" on beet wedges. These organisms were assigned to the subgenus Saccharomyces rather than the subgenus Zygosaccharomyces although Stelling-Dekker assigned Z. japonicus, which displayed similar abortive sexual characteristics, to the subgenus Zygosaccharomyces. This culture, however, had been maintained in the laboratory for a long period of time. Two cultures (31A3 and 40A1) were identical with Z. priorianus Klocker and 11 were similar. Eight other cultures were included in this species although the vegetative cells were slightly smaller and the slant cultures somewhat more smooth than those of the described culture. Our cultures utilized alcohol slowly, whereas Z. priorianus does not. Cultures 48C2 and 213 have been termed Z. barkeri Saccardo and Sydow although they ferment sucrose and raffinose more rapidly than the organisms described by Stelling-Dekker. Genus Pichia: Four species of Pichia were represented as follows: P. alcoholophila Klbcker (83B1), P. membranaefaciens Hansen (83A1F), P. neerlandica Lodder, and P. belgica (Lindner) Dekker (220). Although culture 83B1 was designated as P. alcoholophila, it varied from the description of this organism by producing a finely wrinkled slant culture and failing to produce definitely allantoid cells. The two cultures of P. neerlandica differed from the organisms described by Lodder in failing to produce slant cultures with serrate edges. Subgenus Zygopichia: Two cultures (represented by 53B3TW) very similar to Z. chevalieri (Guilliermond) Kldcker were isolated from wines. These cultures liberate their ascospores soon after formation so that the sexual character is easily overlooked. A few spores with very small brims have been noticed, but the majority of the spores are hemispherical. Genus Debaryomyces: Relatively few cultures of Debaryomyces

5 YEASTS OCCURRING ON GRAPES 399 were obtained from grapes and grape products although species of this genus have been found commonly in California on other products by Mrak and Bonar (1939). Two cultures (7A1 and 7B1) similar to D. globosus Kl6cker differed by failing to produce a pellicle on alcohol medium. A single culture (50B1) was similar to D. maturuchoti Grigoraki and Peju except for slightly smaller cells and less moist slant culture. Genus Hansenula: Although species of Hansenula have been observed on various food products produced in California, only four were isolated from grape products. One (192) is very similar to H. anomala var. sphaerica (Nageli) Dekker except that the streak culture is slightly punctate rather than mealy. Culture (216-H) is similar to Hansenula pans Castelli except that the, spores are globose rather than hat-shaped, and alcohol and nitrate are not utilized. These differences are sufficient to warrant specific segregation which, however, will not be made here since a study of this entire genus is being made by C. L. Bedford of this Laboratory. Designation of species and varieties of this genus are likewise withheld for the same reason. Genus Torulaspora: A single culture (56B4) of Torulaspora rose Guilliermond was isolated from grapes. This culture is identical with that described by Stelling-Dekker, but is unusual in forming frequently three spores per ascus and numerous rather long copulation trial tubes. Genus Hanseniaspora: This genus is commonly found on grapes and musts in Europe. Bioletti and Cruess (1912) discussed several cultures of Hanseniaspora obtained from California grapes and musts but failed to indicate the specific designation. Eleven cultures were found to be almost identical with H. guilliermondii Pijper. In a few instances slight variations (believed to be insignificant) were observed in alcohol and nitrate utilization. Genus Kloeckeraspora: The validity of this genus is questioned; Niehaus (1932), Castelli (1935a) and Dvornik (1938). It is not our purpose to discuss this question or to interpret the morphological significance of the central bodies termed spores by Niehaus and Dvornik and vacuoles by Castelli. The culture

6 400 E. M. MRAK AND L. S. McCLUNG (95B1) studied was morphologically and physiologically identical with Kloeckeraspora uvorum Niehaus. The spherical bodies, illustrated by Niehaus, were observed in approximately five per cent of the cells from beet and potato wedge cultures stored at room temperature for three weeks. In a few instances rough surfaces were observed on these bodies. Non-8porulating yeasts Less than half of the cultures studied failed to produce, in 60 days, ascospores on various media. The Stantial (1935) procedure was tried and gave negative results when ascospores were not formed on the common sporulation media. Torulop s, Mycoderma, Kloeckera or Candidii were most frequent although species of Rhodotorula, Asporomyces and Schizoblatosporion were found. Difficulty was experienced in identifying the non-sporulating yeasts because of slight variation in physiological and morphological characters. Genus Torulopsis: Culture (98A2-2) is similar to Torulopsis lipofera (den Dooren de Jong) Lodder in all characteristics except that the utilization of urea is somewhat doubtful and the cells are not quite as long as those of the organism described by Lodder. Three cultures (41B3R, 58B1, 116A) were similar to T. pulcherrima (Lindner) Saccardo but formed slant cultures that were smooth and dull rather than shiny. A second group of six cultures, typified by 31A1W, failed to show definite utilization of alcohol and urea. A third group of 10 cultures, typified by 124A, differed from Torulopsis pulcherrima in forming dull smooth slant cultures. Three cultures of non-film-forming budding yeast with globose to spherical cells (represented by 80C2) were assigned to the genus Torulopsis although they were strongly fermentative and in this way distinctly different from any of the described species. These organisms are described as Torulopsis californicus Nov. Sp. A fourth culture (5001) is also strongly fermentative but differs from the aforementioned cultures in alcohol and nitrogen utilization characteristics and the partial fermentation (as determined in the van Itterson-Kluyver apparatus) of raffinose. This culture is described as Torulopsis fermwntans Nov. Sp.

7 YEASTS OCCURRING ON GRAPES 401 Torulopsis californicus Nov. Sp. Cells mostly globose to spherical. Cell size in 24-hour wort culture range ( M) x (4.5-6 M) and average 3.5 x From threeday wort culture range (5.25-7,u) x ( ,A) and average 6 x 6.5 a. Ferments glucose, levulose, maimose, galactose, maltose, and sucrose, not raffinose or lactose. Asparagin, ammonium sulphate, urea and peptone utilized slowly, nitrate not. Good growth when alcohol is the only source of carbon. Slant cultures cartridge buff, smooth to slightly punctate, dull, dry, and pulvinate, with entire to undulate borders. Giant colony cartridge buff, smooth, dull dry, and convex with entire borders. Gelatin not liquefied in 70 days. Toratlopsis fermentans dnov. Sp. Cells globose to spherical. Cell size in 24-hour wort range ( IA) x ( Au) and average 4.5 x 5.75,u. From three-day wort culture range (3.5-7 ti) x ( Au) and average 6 x 7 ju. Ferments glucose, levulose, maniose, galactose, maltose, sucrose and (3 only) raffinose. Lactose not fermented. Peptone and alcohol are utilized. Asparagin, urea, ammonium sulphate, and nitrate not utilized. Slant culture pale pinkish cinnamon, smooth, dry, dull, and raised with entire borders. Giant colonies pale pinkish buff, smooth, dull, dry, flat crateriform cone with entire borders. Gelatin not liquefied in 70 days. Genus Mycoderma: Few organisms corresponding to the genus as defined by Leberle (1909) and Lodder (1934) were found. These were similar to Mycodermna cerevisiae Desm. Several cultures identified as Candida were similar to Mycoderma in all respects except for the formation of pseudomycelia on Rivalier and Seydel slides. Genus Kloeckera: The majority of the 16 cultures, typified by 104A1, belonging to this genus are very similar to Kloeckera lindneri (Kl6cker) Janke. Slight variations were observed in cell size and streak culture characteristics. In a few cases the cell size range varied slightly from those given by Lodder. The streak cultures ranged in color from avelleneous to wood brown and were dry but glistening. Culture 120A1 was comparable to Kloeckera africana (Klocker) Janke in cell size and most other characteristics although it liquefied gelatin slightly in 60 (lays. Genus Asporownyces: According to Cblaborski (1919) this genus

8 402 E. M. MRAK AND) L. S. McCLUNG is characterized by forming abortive conjugation tubes on Gorodkowa agar without producing ascospores. Asporomyces asporous Chaborski was isolated from bananas and was characterized by fermenting glucose, levulose, mannose, sucrose, maltose and raffinose. Unfortunately Chaborski's culture was lost. and hence is not available for comparison with the cultures isolated from grapes. Cultures 96B3W and 96B3R produce abortive conjugation tubes in 10-per-cent sucrose solution and on beet and cucumber sporulation wedges but not on Gorodkowa FI(;. 1. VEGETATIVE CELLS OF ASPOROMYCES UVAE N OV. SP. SHOWINGA-(BORTIvE CONJUGATION TUBES agar slants. If the genus Asporoinyces is acceptable, the substrate in or on which these ab)ortive tubes form should not be limited to the single sporulation medium but should include all sporulation medlia used in the identification of yeast. For this reason the cultures (96B3W and 96B3R) isolated are included in this genus and described as Asporomyces itvae Nov. Sp. Cells showing the abortive conjugation tubes are shown in Figure 1..Asporoinyccs uae Nov. Sp. Cells in young wort culture globose, ellipsoidal, spherical or pyriform. Cell size in 1 and 3-day wort culture, range ( A,) x ( ,u)

9 YEASTS OCCURRING ON GRAPES 403 and average 4.5 x 6 M. Thin pellicle and ring appears on wort culture after 4 days. Cells form abortive conjugation tubes on cucumber and beet wedges. Glucose, fructose, and mannose fermented slowly. Sucrose, maltose, lactose and raffinose not fermented. Utilization of alcohol in synthetic medium doubtful. Peptone, asparagin, ammonium sulfate and urea assimilated, nitrate not. Slant cultures ivory yellow (sometimes with a pinkish tint), smooth, dull, dry, convex with lobatelobulate border. Giant colonies cartridge buff, smooth, moist and glistening in the center, dull and dry at the border, flat with entire border. Gelatin liquefied in 60 days. Genus Schizoblastosporion:1 A single culture was isolated from grape leaves. Bud-fission was quite evident in actively growing wort cultures when the organism was first isolated. After prolonged cultivation (3 years) on wort agar, however, this characteristic was much more difficult to observe. Observations made on an authentic culture of this type species revealed that the bud fission character was likewise not very evident in this culture which had been held in culture for a number of years. The culture isolated differed very slightly from Schizoblastosporion starkeyi-henricii Ciferri (98A1C) in slant and giant colony characteristics and cell size. The streak culture of our culture was relatively dry and dull. The giant colony was not raised in the center, and the cells were slightly larger than those of a type culture. Mycotoruloideae: The taxonomy of the organisms belonging to this subfamily is so confused at present that it is useless to attempt to identify species. Langeron and Guerra (1938) grouped the genera of the subfamily Mycotoruloideae into the single genus Candida, including the various species studied in seven groups. Our cultures fit into group five, characterized by fermenting glucose and levulose only, and group seven, characterized by failing to ferment any sugars. Recently Diddens and Lodder (1939) proposed a scheme for classifying the organisms of the subfamily Mycotoruloideae whereby the subfamily is comprised of the genera Candida Berkh. and Trichosporon Behrend. They divide the genus Candida into four groups. 1 Langeron and Guerra (1938) have recently reduced this genus to synonomy with Candida.

10 404 E. M. MRAK AND L. S. McCLUNG Several of our organisms fit into the Candida albicans group (group 1) characterized by pseudomycelia, varied forms of blastospores, no pellicle formation on wort, creamy or membranaceous appearance of the cultures which are never flat or dull. Most of the cultures, however, fit into the Candida krusei group (group 2) because of the presence of pseudomycelia, characteristic blastospores and pellicle formation, and the fermention of glucose, fructose and mannose, although fermentation has not been ob- TABLE 1 Occurrence of yeast genera in grapes and grape products in California FRES M 8TSAND CLOUDY POMACE TOTAL FRSAPH MNSW WIND BOTTLED LEAVES NUMBER OF GRAPENEW INES WINE OR SOIL CULTURE Saccharomyces Zygosaccharomyces Hanseniaspora Kloeckeraspora Pichia Debaryomyces Hansenula Zygopichia Torulaspora... 1I 1 Torulopsis Mycoderma Kloeckera Rhodotorula Asporomyces Schizoblastosporion 1 1 Candida served in a few cultures. Only one culture possessed the characteristics of Brettanomyces (group 4) characterized by variedshaped blastospores; a creamy appearance which is never dull but develops a brown tinge, and the fermentation of several sugars. Grouping the numerous ill defined genera of the subfamily Mycotoruloideae into one or two well defined genera by the above authors is a desirable step forward in the taxonomy of this group, and their suggested changes should be adopted to facilitate the establishment of a reliable system for specific taxonomy. Specific

11 YEASTS OCCURRING ON GRAPES designation of our cultures is withheld pending publication of a monograph on the subfamily Mycotoruloideae by Diddens and Lodder. Genus Rhodotorula: Six cultures of red-colored non-sporulating yeast were isolated. It cannot be stated definitely, however, that any of these organisms belong to the genus Rhodotorula because of the difficulty experienced in obtaining reproducible results with the Molisch technique for the crystallization of carotene, as previously mentioned. Further consideration is being given this particular problem. In view of these difficulties the cultures designated as Rhodotorula have been judged so on the basis of morphological and physiological characteristics and by comparison with an authentic culture. Three of the organisms, similar to 56B2S, are similar to R. sanniei (Ciferri and Redaelli) Lodder except that the cells of our cultures are slightly smaller and the slant culture is smoother than those of R. sanniei. The other 3 cultures, represented by 114A3, are similar to R. glutinis (Fres) Harrison as described by Lodder. DISCUSSION 405 The 241 cultures of yeasts fell into the genera presented in table 1. The genus Saccharomyces was obtained most frequently, and the next most common group consisted of the apiculate yeasts, Kloeckera, Kloeckeraspora, and Hanseniaspora. The latter genera, however, were obtained more commonly from grapes than from musts or wines. Cultures of Torulopsis and Candida were obtained commonly from grapes. This distribution is in general agreement with the findings of De Rossi (1935), Castelli (1935b, 1938), and Verona and Luchetti (1936) in the wine-producing regions of Europe. The organism most commonly obtained from cloudy bottled wine was Saccharomyces cerevisiae although occasional cultures of Candida, Pichia, Zygopichia, and Torulopsis were isolated. No definite correlation between the distribution of any genera or species with a particular district has been observed. Cultures of the organisms, especially described in this report,

12 406 E. M. MRAK AND L. S. McCLUNG have been retained by the authors and have been deposited in the Centraalbureau voor Schimmelcultures and the American Type Culture Collection. SMUMARY Two hundred and forty-one cultures of yeasts were isolated from California grapes and grape products. The 159 ascospore forming yeasts included: Saccharomyce8 cerevisiae Hansen, S. cerevsiae var. ellipszodeu (Hansen) Dekker, S. oviformis Osterwalder, S. chodati Steiner, S. carlsbergersis (Hansen); Zygosaccharomyces priorianus Kl6cker, Z. barker Saccardo; Pichia alcoholophilakl6cker, P. membranaefaciens Hansen, P. neerlandica Lodder, P. belgica (Lindner) Dekker; Zygopichia chevalieri (Guilliermond) Kl6cker; Debaryomyces globosus Klocker, D. matruchoti Grigoraki and Peju; Hansenula anomala var. sphaerica (Nageli) Dekker; Torulaspora rosei Guilliermond; Hanseniaspora guilliermondii Pijper and Kloeckeraspora uvorum Niehaus. The 82 imperfect yeasts included: Torulopsis lipofera (den Dooren de Jong) Lodder, T. pulcherrima (Lindner) Saccardo; Mycoderma cerevisiae Desmazieres; Kloeckera lindneri (Klocker) Janke, K. africana (Kl6cker) Janke; Schizoblastosporion starkeyi-henricii Ciferri; Rhodotorula sanniei (Cif. and Red.) Lodder, R. glutinis (Fres) Harrison and several species of Candida. Two new species of Torulopsis were described as: Torulopms californicus Nov. Sp. and Torulopsis fermentans Nov. Sp. Two new cultures of the lost genus Asporomyces were isolated and described as Asporomyces uvae Nov. Sp. REFERENCES BIOLETn, F. T The principles of wine making. Calif. Agr. Expt. Sta. Bull., No BIOLETi, F. T., AND CROIESS, W. V Enological investigations. Calif. Agr. Expt. Sta. Bull., No CASTELLI, T. 1935a Sur la sporulation du Pseudowaccharomyces apiculatmu. Boll. sez. ital., Soc. intern. microbial. Fasc. 4 XXXX. CASTELLI, T. 1935b Stu alcuni blastomiceti dei most umbri. Boll. ist. sieroterap. milan., 14, Fasc. 9, 3-8. CASTELLI, T Nuovi blastomiceti isolati da mosti del Chianti e zone limitrofe. Arch. Mikrobiol., 9, CHABORSKi, G Recherches sur les levures thermophiles et cryophiles. Bull. Soc. Botan. Geneve, 11,

13 YEASTS OCCURRING ON GRAPES 407 CRUMBS, W. V The fermentation organisms of California grapes. Univ. Calif. Pub. Agr. Sci., 4, No. 1, pp DE Rossi, G I lieviti della fermentazione vinaria della regione Umbra. Relaz. Quarto Congr. Intern. Vigna e Vino Lausanne. (Thru Zentr. Bakt. Parasitenk., II Abt., 98, 469, 1938.) DIDDENS, H. A., AND LODDER, J An appeal for unification of the generic taxonomy in the Mycotorldeae. Mycopathologia, 2, 1-6. DVORNIK, R tiber die Sporulation der Apiculatushefen. Zentr. Bakt. Parasitenk., II Abt., 98, HoLM, H. C A study of yeasts from California grapes. Univ. Calif., Pub. Coll. Agr., LANGERON, M., AND GuERRA, P Nouvelles recherches de zymologie m6dicale. Ann. parasitol. 16, 36-84, , , LEBERLE, H Beitrige zur Kenntnis der Gattung Mycoderma. Inaugural Dissertation. Minch., LODDER, J Die Hefesammlung des Centraalbureau voor Schimmelcultures. II Teil, Die anaskosporogenen Hefen, Erste H~lfte, Verhandel. Akad. Wetenschappen Amsterdam 2nd Sect., Deel 32, MRAK, E. M., AND BONAR, L Film yeasts from pickle brines. Zentr. Bakt. Parasitenk., II Abt., 100, NIzHAus, C. J. G Untersuchungen fiber Apiculatushefen. Zentr. Bakt. Parasitenk., II Abt., 87, RIDGWAY, R Color Standards and Color Nomenclature. Washington, D. C. SATAVA, M Les formes sexuelles et asexuelles des levures et leur pouvoir fermentatif. III Congres International Technique et Chimique des Industries Agricoles, 3, 1-8. STANTIAL, H The sporulation of yeast. Trans. Roy. Soc. Can., Ser. 3, 29, Section III, STELLING-DEKKER, N. M Die Hefesammlung des Centraalbureau voor Schimmelcultures. I. Teil, Die Sporogenen Hefen, Verhandel. Akad. Wetenschappen 2nd Sect., Deel 28, SUMNOE, K On species of the wine yeasts isolated from Californian wine mash. Part I. J. Agr. Chem. Soc. Japan 4, 247. SumINoE, K On species of the wine yeasts isolated from Californian wine mash. Part II. J. Agr. Chem. ooc. Japan 6, 70. VERONA, O., AND LUCETm, G Richerche Microbiologiche su di aliuni vin: Ed. alcuni uve delle Marche. Boll. ist. super. agrar. Pisa, 12, WINGE, On haplophase and diplophase in some Saccharomycetes. Compt. rend. trav. lab. Carlsberg, S6r. physiol., 21, WINGE, O., AND LAUSTsEN, O On two types of spore germination and on genetic segregations in Saccharomyces through single-spore cultures. Compt. rend. trav. lab. Carlsberg, S6r. physiol. 21, WINGE, O., AND LAUST5EN, O Artificial species hybridization in yeast. Compt. rend. trav. lab. Carlsberg, S6r. physiol. 22,