YEASTS OCCURRING IN SOURING FIGS. formed. At full maturity the flesh of each floret becomes juicy, forming an ideal

YEASTS OCCURRING IN SOURING FIGS E. M. MRAK, H. J. PHAFF, R. H. VAUGHN AND H. N. HANSEN Univer8ity of California, Berkeley, California' Received for publication, February 26, 1942 Figs frequently undergo an internal yeast fermentation while maturing on the tree. This is possible because botanically the fig is a syconium; a more or less hollow receptacle the inner walls of which are lined with flowers when immature and by seed-like fruits when ripe. It has an opening at the flattened end which is closed by overlapping scales during the early stages of development. After the fruit begins to ripen these scales loosen and an opening termed the "eye" is formed. At full maturity the flesh of each floret becomes juicy, forming an ideal medium for the growth of microorganisms carried into the fruit by various agencies, particularly by insects entering through the "eye". Beneficial as well as harmful insects may enter the fruit since the fig "wasp" (Blastophaga psenes L.) is necessary for pollination of the Calimyrna variety. This insect, however, as well as the undesirable ones may be responsible for infection of the fruit with destructive microorganisms. Fungi, other than yeasts and bacteria, causing fig spoilage have been studied extensively by Caldis (1927), Smith and Hansen (1931) and Hansen and Davey (1932). Most authors discussing fig spoilage refer to yeasts in connection with a type of deterioration termed "souring"; a spoilage involving the production of acid and a vinegar-like odor. Smith and Hansen (1927) described souring as a form of spoilage causing the contents of ripe figs to ferment and sour with subsequent dripping of liquid from the "eyes". Condit (1941) stated in regard to souring; "this is the cause of an immense loss of figs every year, especially in the Adriatic and Calimyrna varieties. Souring is caused by the action of specific yeasts and bacteria on the internal saccharine juice of the fig. Investigation has shown that figs are internally sterile until they are entered by insects, after which they commonly become infected with yeasts, molds, and bacteria." Caldis (1930) indicated that fig souring is primarily an alcoholic fermentation but subsequent changes may take place, the commonest being that brought about by the action of acetic acid bacteria on the alcohol with the production of acetic acid. Caldis isolated three types of yeasts, two of which produced typical souring when inoculated into ripe figs; the third type produced a different form of disease. The first two were termed Mycoderma and Apiculata and the third Torula. Davey and Smith (1933) included Mycoderma, Pseudosaccharomyces, Hansenia, and Pichia in the true souring yeasts of figs. The nonsouring yeasts included those forming membranous, wrinkled, dry, surface growth on solid media. No other publications are available concerning the yeasts occurring in "souring" figs. It is apparent that the information available on these yeasts is meager and useless from the standpoint of the currently accepted taxonomy of yeasts. 1 Part of the non-technical assistance was supplied by W.P.A. Project No. 65-1-08-91, Unit B-5. 441

442 E. M. MRAK, H. J. PHAFF, R. H. VAUGHN AND H. N. HANSEN EXPERIMENTAL PROCEDURE Thirty samples, each containing several souring figs were obtained in 1938 and 1939 from trees growing in widely separated areas in California. Most of the samples were collected in 10 different fig-producing districts located in the San Joaquin Valley, although some were obtained from an isolated orchard in Diablo Valley and others from three remotely located trees in the Santa Clara Valley. The latter two valleys are about 30 and 100 miles distant, respectively, from the centers of commercial production in the San Joaquin Valley. The varieties collected included the Calimyrna which is insect pollinated and the Adriatic and Kadota which require no pollinization. Isolation was accomplished by direct plating from the interior of infected fruits, all of which yielded yeasts. Identification procedures were similar to those employed by Mrak, Phaff, and Vaughn (1943). Qualitative tests for acid formation were made by observing for the clarification of slants of yeast infusion agar containing 2 per cent of chalk, and of slants of 100 Balling fig extract agar also containing 2 per cent chalk. For the quantitative determination of acid production 50 ml. of 200 Balling extract of dried figs were sterilized in 4 oz. bottles, then inoculated and stored 10 days at room temperature. Analyses were then made for total and volatile acids by the methods of the Association of Official Agricultural Chemists (1936). To determine the tolerance to high concentration of sugars all yeasts isolated were inoculated in 300 Balling fig syrup. If growth occurred the cultures were transferred subsequently to 400 and finally 500 Balling syrup. EXPERIMENTAL RESULTS One hundred and fifteen isolates of yeast were obtained from 30 samples of souring figs. Sixty-four were sporulating and 51 non-sporulating forms. Most isolates proved to be species of Saccharomyces or Candida. Several isolates of Pichia, Hanseniaspora, Kloeckera and Torulopsis were also found. Of Zygosaccharomyces, Zygopichia, Hansenula and Debaryomyces, only single representatives were obtained. Sporulating yeasts. Genus Saccharomyces: Thirty-six out of 37 of these organisms isolated were members of the subgenus Saccharomyces s.s. and only 1 of the sub-genus Zygosaccharomyces. Twenty-five of the isolates of Saccharomyces were S. cerevisiae Hansen, 6 S. tubiformis Osterwalder, 2 S. fragilis J6rgensen and 1 each of S. cereviseae var. ellipsoideus (Hansen) Dekker, S. carlsbergensis var. monacensis (Hansen) Dekker, and S. carlsbergensis var. polymorphus Dekker. The isolation of a single culture of S. cerevisiae var. ellipsoideus as compared with 25 of S. cerevisiae compares well with the results of Mrak and McClung (1940) in a study of yeasts occurring on California grapes. The six isolates of S. tubiformis fermented maltose so slowly that gas formation could be determined only by use of the van Iterson-Kluyver fermentometer. Durham tubes and modified Durham tubes using 25 mm test tubes and 5 ml vials as suggested by Henrici (1941) gave negative results. S. fragili& J6rgensen is a lactose-fermenting yeast that has been isolated com-

YEASTS OCCURRING IN SOURING FIGS 443 monly from dairy products but not previously from fruits. The isolates defined as S. fragilis differ from the type species in the three following respects: 1. Lactose is fermented only after adaptation to this sugar and then at a slower rate than by the type species; 2. Adaptation occurs only when the cells multiply in the presence of lactose and not simply by standing, while in a nonproliferating condition, in a medium containing this sugar; 3. One of the isolates was able to respire and assimilate maltose but could not ferment it; a phenomenon discussed in detail bykluyver and Custers (1940). The metabolic activities of these organisms and of the type species of S. fragilis were compared by use of Durham fermentation tubes, van Iterson- Kluyver fermentometers and the Warburg manometric technique. Multiplication of cells is facilitated in Durham tubes because of aerobic conditions. Adaptation occurs during this multiplication, hence the cells will ferment lactose irrespective of the medium on which previously grown. The rate of fermentation, however, is faster if the cells are taken from yeast infusion lactose slants. In the vaniterson-kluyver fermentometer, multiplication of cells is practically absent because of the strictly anaerobic conditions. In view of this fact, scarcely any fermentation is observable if the cells are taken from malt agar slants. Cells taken from yeast infusion lactose slants show a slow fermentation requiring about 60 hours for completion. The type species on the other hand ferments a 4 per cent lactose solution to completion in 12-18 hours affected only slightly by the medium on which the cells were previously grown. These results show that if the fermentometer is to be used to test for the fermentation of specific sugars, the yeast should also be grown in a medium containing the sugar to be tested as the sole source of carbon. The results obtained by use of the Warburg manometric technique were similar but more exact from a quantitative point of view. The cultures isolated from figs showed neither respiration nor fermentation of lactose when grown on malt agar plates. When the cultures tested were grown on yeast-infusion lactose agar plates for 48 hours at 30 C. the rate of fermentation of the type species of S. fragilis was about five times that of the cultures isolated from figs. The rate of respiration on the other hand was of the same order of magnitude for all three cultures. The ability to ferment lactose is possessed by relatively few yeasts. To our knowledge no other species have been reported to require lactose adaptation before being able to ferment this sugar. One of the lactose-fermenting yeasts isolated from figs showed the ability to assimilate maltose on an auxanogram plate. This was checked quantitatively by use of the Warburg technique. Anaerobic fermentation did not occur but there was appreciable respiration and slight aerobic fermentation (fermentation in the presence of air) of this sugar. The second culture isolated from figs as well as the type species was unable to attack maltose. Kluyver and Custers made a similar observation with S. fragilis. Other examples of this type of respiration and assimilation of disaccharides by yeasts which do not ferment these sugars have been given bykluyver and Custers (1940) and Mrak etal. (in press). Kluyver and Custers explain this phenomenon by assuming a reversible inactivation of the hydrolases for certain disaccharides, under anaerobic conditions. They reject the possibility of direct respiration of the disaccharide.

444 E. M. MRAK, H. J. PHAFF, R. H. VAUGHN AND H. N. HANSEN Although the morphological characters of all these cultures are similar, the physiological differences between those isolated from figs and the type species of S. fragilis might appear to warrant the specific or varietal segregation of these organisms. However, cultures of S. fragilis sporulate freely and rapidly with subsequent discharge of the spores by rupturing of the ascus. This permits the mixing and fusion of spores from different cells with possible development of homozygous and heterozygous strains which may account for the physiological differences discussed above. In view of this it is advisable to term the isolates S. fragilis until single spore culture studies are made to determine whether or not the differences in fermentation and respiration result from hybridizations in nature. The isolate of S. carlsbergensis var. monacensis agrees with the description of Stelling-Dekker (1931) but the variety polymorphus differs by forming slant cultures with entire rather than hairy borders. This species which ferments raffinose completely has been isolated in most instances from juices and fermenting liquors although it has been obtained occasionally from California grapes, dates, figs and prunes. The subgenus Zygosaccharomyces was represented by a single culture of Z. globiformis Kroemer and Krumbholz which was similar in all respects to one previously isolated from California dates (Mrak et al.). The isolation of a large number of Saccharomyces and only 1 Zygosaccharomyces from figs differs markedly from the proportion of these yeasts found on dates by Mrak et al. and on dried prunes by Mrak and Baker (1939). The yeast flora of figs resembles more closely that of fresh grapes, (Mrak and McClung, 1940). The different fruits mentioned above vary considerably in sugar content. Fresh figs contain 20 to 35 and grapes 20 to 25 per cent of sugar. Dates, on the other hand, contain 65 to 70, dried figs 50 to 65, and dried prunes 40 to 50 per cent of sugar. Since most species of Zygosaccharomyces can grow in the presence of higher concentrations of sugar than other yeasts, the sugar content of the fruit is undoubtedly a factor influencing the relative prevalence of these 2 subgenera. Genus Pichia: Sixteen isolates were included in the subgenus Pichia s.s. and 1 in Zygopichia. Fourteen of these were similar to P. kluyveri, an organism recently described by Bedford (1941). It is characterized by the fermentation of glucose, fructose and mannose, poor growth in synthetic medium, inability to utilize asparagin, ammonium sulfate, urea and nitrate, and by poor growth in alcohol medium without film formation, but with ester production. P. kluyveri differs from P. fermentans Lodder by failing to utilize asparagine, ammonium sulfate and urea; by the production of esters and by the absence of a pellicle in alcohol medium. The production of esters is typical for the genus Hansenula, while unknown for species of Pichia until the isolation of Pichia kluyveri. It may be well to discuss in some detail the gradually fading borderline between the genera Hansenula and Pichia. Until the publication of Stelling-Dekker's monograph in 1931 the former genus differed from the latter by ascospore characteristics, its fermentative power, the formation of esters, the ability to split esculin and to utilize nitrate as the single source of nitrogen.

YEASTS OCCURRING IN SOURING FIGS 445 To differentiate the genera Pichia and Hansenula, the older literature laid considerable stress on spore morphology. Kloecker (1923), for example, defines the genus Pichia as having spherical, hemispherical, irregular or angular spores and Hansenula (Willia) as having hat- or saturn-shaped spores. However, Stelling- Dekker (1931) stated that both genera have hat-shaped spores in common, which also has been observed by the writers although the detailed morphology is somewhat different. Pichia spores usually have an oil droplet, but so have the spores of Hansenula saturnus. The authors are disinclined to believe that the spore morphology forms a strict character for generic differentiation. In 1932 Lodder published the description of Pichia fermentans, which broke down the fermentation point of difference. In 1941 Bedford described Pichia kluyveri, able to produce at least as much ester as some Hansenula species and also able to split esculin. This left as the only point of difference the utilization of nitrate, because morphologically the two genera are very similar. Bedford (1941) and also Mrak et al. found a species of Hansenula, termed H. subpelliculosa, which does not attack nitrate in a completely synthetic medium in contrast to all other Hansenula species. The auxanogram plate is positive probably because the heavy inoculum introduces enough growth substances to permit development at the expense of the nitrate added. To differentiate two genera on a single physiological character of a nature as described seems unsound to us. It may be advisable in the near future to form one genus, containing subgenera of the nature of Pichia, Zygopichia, Hansenula and Zygohansenula. A single organism was considered to be P. fermentans Lodder although there are differences in film, slant culture and spore characteristics. The film produced is thin and smooth, the slant culture dull, and ascospores spherical rather than hat-shaped. Such differences might be considered sufficient for species segregation but in our experience these characters show considerable variation in the genus Pichia. Slant cultures and films frequently undergo variations in response to the particular conditions of growth and ascospores may vary in shape from spherical to hemispherical or be slightly hat-shaped in the same species. One isolate of Pichia belgica (Lindner) Dekker was found, which agrees quite well with the description of the type species by Stelling-Dekker. In view of the fact that this worker was unable to obtain ascospores with the type species present in the collection of the Centraalbureau voor Schimmelcultures at Baarn, Holland, it is worthwhile mentioning that our culture sporulated abundantly on any type of medium. The morphology of the spores is similar to the original description and drawings given by Lindner (1909), that is, indistinctly hatshaped with a short brim. The isolate of Zygopichia is similar to Z. chevalieri (Guilliermond) Dekker. The occurrence of fermenting species of Pichia has been reported relatively few times. Aside from P. fermentans Lodder all isolations of fermenting species of Pichia have been made in California. P. kltuyveri has been obtained from figs and pickled olives, P. chodati var. fermentans from dates, and P. fermentans from figs. Genus Hanseniaspora: Eight isolates, similar in all respects to H. melligeri

446 E. M. MRAK, H. J. PHAFF, R. H. VAUGHN AND H. N. HANSEN Lodder, were obtained from eight different fig samples. This is one of the commonest species of Hanseniaspora in California. Besides Melliger's isolate (1931) from Egyptian dates, we have obtained this organism from California dates, figs, prunes and apples from widely separated areas. Except for the description by Lodder (1932) no other reports of the occurrence of this organism have been found. Genus Hansenula: A single isolate of H. anomala var. sphaerica (Naegeli) Dekker was obtained. Relatively few cultures of Hansenula have been reported from California fresh fruits. Several cultures, on the other hand, have been isolated in California from beverages, concentrates, pickles and stored foods such as dried fruits. Genus Debaryomyces: The single isolate obtained differs from described species in its ability to ferment maltose, growth in alcohol medium and in cell size. The fermentation of maltose is slow but is easily detectable in Durham tubes. The culture isolated from figs is described as D. dekkeri. Debaryomyces dekkeri nov. sp. Cells spherical to globose in 1, 3, and 40 day liquid wort cultures. Cells in 1 and 3 day liquid wort cultures range (2.4-3.6,u) x (2.4-3.6,u) and average (3 x 3A). No pellicle formation in 40 days but a narrow ring appears in 5 days. In young cultures cells single or in pairs; clusters present in 40 day cultures. Budding on all sides. Ascospore formation follows iso. or heterogamic conjugation. Ascospores spherical and rough with centrally located oil droplet. One or 2 ascospores form in one of the conjugating cells. Ascospore size averages (2.9 x 2.9M). Cells sometimes form abortive conjugation tubes. Ferments glucose, fructose, mannose, sucrose, maltose slowly and 1 of raffinose. Does not ferment galactose or lactose. Utilizes asparagin, ammonium sulfate, urea and peptone but not nitrate. A good growth in alcohol medium. Slant culture pale olive buff, smooth to slightly verrucose, glistening, and convex with entire borders. Species of Debaryomyces apparently seldom occur on fruits or fruit products but commonly on pickles and meats (Mrak and Bonar 1938, 1939). Non-sporulating yeasts. The majority of the non-sporulating yeasts were species of Candida, Kloeckera and Torulopsis. Genus Kloeckera: Of 13 isolates belonging to this genus 12 were K. lindneri (Kloecker) Janke and 1 K. africana (Kloecker) Janke. The isolates of K. lindneri differed slightly from the organism described by Lodder (1934) in cell size and consistent inability to liquefy gelatin. The culture of K. africana produced esters and an incomplete pellicle of loose islets in wort. These characteristics apparently do not occur in the type culture of K. africana. Sporulating as well as non-sporulating apiculate yeasts have been isolated from fresh fruits in California but only the sporulating genus Hanseniaspora from dried fruits. The concentration of sugar present in dried fruits may be considered a factor favoring sporulation or inhibiting the growth of Kloeckera. However, the isolates of Kloeckera and Hanseniaspora from figs showed no difference in tolerance to high concentration of sugar. Both grew in 400, but not in 50 Balling fig syrup. Cultures of Hanseniaspora isolated from dates on the other hand grew well in 500 Balling date syrup.

YEASTS OCCURRING IN SOURING FIGS It is possible that the isolates of Kloeckera are in reality imperfects of Hanseniaspora that have lost their ability to sporulate. If this is true it may be assumed that the isolates of K. lindneri are non-sporulating forms of H. melligeri. The consistent ability of H. melligeri and inability of K. lindneri to liquefy gelatin might be explained by the observation of Beijerinck (1898), that sporulating cells of certain yeasts liberate the protoplasm not involved in spore formation, soon after this process has taken place. The liberated protoplasm can then cause liquefaction as soon as it comes in contact with the gelatin. According to Beijerinck, protoplasm is not liberated by non-sporulating strains until the cells die. Since this occurs more slowly than sporulation, a much longer time is required for the non-sporulating strain to liquefy gelatin if liquefaction occurs at all. Genus Torulopsis: Six isolates of T. stellata (Kroemer and Krumbholz) Lodder were obtained from samples collected in four different districts. As far as can be determined this organism was previously isolated only in Germany, from the juice of partially vine-dried grapes (Trockenbeerenauslese). Genus Candida: Thirty-three isolates of Candida were obtained, twenty-six of which were C. krusei (Castellani) Berkhout as described by Langeron and Guerra (1938). Six were considered to be C. chalmersi Castellani although maltose and raffinose were fermented slowly and detectable only by use of the van Iterson-Kluyver fermentometer. The maltose auxanogram was definitely positive. C. chalmersi, according to Langeron and Guerra, ferments neither maltose nor raffinose but utilizes maltose in an auxanogram plate. One isolate of Candida differs from the described species although it possesses characteristics of the guilliermondi group. It differs from C. guilliermondi by forming a dry, wrinkled rather than slimy film on wort, forming a film on alcohol medium and failing to ferment maltose, although utilizing it on an auxanogram plate. This isolate differs from C. chalmersi in pellicle formation on wort and alcohol medium and by fermenting galactose, sucrose, and slowly, i of the raffinose. This organism is not an imperfect form of Hansenula because of its inability to utilize nitrate, nor of Endomycopsis because of the absence of true septa. However, until Diddens and Lodder complete their monograph of the genus Candida this yeast will be included in the group guiuiermondi, without specific designation. The isolation of such large numbers of Candida from California figs, dates and grapes indicates that these organiss are rather wide-spread in nature. The isolates from figs and dates have been in most instances C. krusei and C. chalmersi. Relatively few or none of the other species have been found on these fruits. It is of interest that thus far C. albicams has not been encountered in our yeast flora studies. Distribution. The commonly isolated yeasts such as S. cerevisiae, H. melligeri, C. krusei and C. chalmersi were obtained from all varieties of figs tested. The more common yeasts were found in all of the important fig producing areas. S. cerevisiae was found in all and C. krusei in 8 of the 10 districts in which samples were collected. The single isolates of H. anomala var. sphaerica and Z. globiformis were from fruit grown in Santa Clara Valley which is located about 447

448 E. M. MRAK, H. J. PHAFF, R. H. VAUGHN AND H. N. HANSEN 100 miles from the center of fig production. Candida species, on the other hand, were not obtained from figs produced in this area even though several samples were collected at various times during two seasons. All of the commonly isolated yeasts were obtained in both 1938 and 1939. Tolerance to high concentrations of sugar. Most of the yeasts isolated from figs grew in 400 but not 500 Balling fig syrup. Thirteen cultures of S. cerevisiae, 7 of C. krusei and 1 each of Zygopichia chevalieri and P. belgica failed to grow in 400 Balling fig syrup. The cultures of C. chalmersi and Z. globiformis were the only organisms showing even slight growth in 500 Balling fig syrup. These yeasts are considerably less tolerant to high concentrations of sugar than those found on dates. Acid production. Soured figs have been given this designation primarily because of the presence of a distinct acetic acid taste and odor. In fact, an or- ORGANISM TABLE 1 Acid produced by different yeasts in fig extract* NUMBER OF ACID PRODUCED IN 10 ML. AS ML. O0 N/10 NaOH ISOLATES TESTED Total Volatile Non-volatile P. kluyveri... 11 2.83-5.50 0.24-0.60 2.59-9.40 H. melligeri... 8 3.18-5.30 1.25-2.65 1-93-2.65 C. chalmersi... 6 0.69-1.88 0.0-0.05 0-69-1.83 K. lindneri... 3 3.78-4.03 1.30-3.02 2.48-1.01 C. krusei... 2 0.0-1.97 0.0-0.05 0.0-1.92 S. cerevisiae... 2 3.94-3.95 0.25-1.11 2.84-3.69 T. stellata... 2 3.18-4.63 0.29-0.44 2.89-3.19 K. africana....... 1 3.77 1.25 2.52 H. anomala var sphaerica... 1 8.60 6.30 2.30 * When determinations were made with more than 1 culture of a given species the range of acid production is given. ganoleptic procedure is used by dried fig inspectors to determine the presence or absence of souring. As indicated in an earlier paragraph, many of the authors discussing fig spoilage have indicated the direct or indirect connection of yeasts with fig souring. These circumstances prompted the present authors to culture the yeasts on Custer's yeast-water-glucose-chalk and fig-infusion-chalk agar slants. Thirty-six isolates showed at least a slight clearing of these chalk containing media. H. anomala var. sphaerica and K. africana clarified the slants much more rapidly than any of the others. Quantitative tests were made for volatile acid and fixed acid production with the thirty-six isolates causing clarification of the chalk agar. The results are summarized in table 1. It is apparent from the data given that volatile and fixed acid production was low in most instances and not sufficient to cause the spoilage termed souring. In addition, members of the two genera Saccharomyces and Candida which were represented most abundantly produced least acid of all, while H. anomala and K. africana were only represented by single isolates. Experiments with acetic acid bacteria

YEASTS OCCURRING IN SOURING FIGS isolated from figs showed that these organisms alone produce appreciable quantities of fixed acid but only traces of volatile acid in fig infusion medium which cannot account for the acetification observed in fig spoilage, whereas heterofermentative lactic acid bacteria produced appreciable quantities of volatile acid. However, only a few cultures of Lactobacillus and Leuconostoc were encountered whereas cultures of Acetobacter were isolated from all figs examined. Most of the fig souring apparently results from the associative action of yeasts and acetic acid bacteria. SUMMARY One hundred and fifteen yeasts were isolated from 3 varieties of souring figs Most of the yeasts isolated were species of Saccharomyces or Candida. The species of Saccharomyces in order of importance were S. cerevisiae (25), S. tubiformis (6), S. fragilis (2) and 1 each of S. cerevisiae var. ellipsoideus, S. carlsbergensis var. monacensis and S. carlsbergensis var. polymorphus. The Candida species were C. krusei (26), C. chalmersi (6), and an unidentified species. Other organisms were Pichia kluyveri (14), P. fermentans (1), P. belgica (1), Zygospichia chevalieri (1), Hanseniaspora melligeri (8), Kloeckera lindneri (12), K. africana (1), and Torulopsis stellata (6), and single isolates of Zygosaccharomyces globiformis, Hansenula anomala var. sphaerica, and a new species of Debaryomyces. The sugar tolerance of the organisms isolated was low; most of them growing in 400 but not 500 Balling fig syrup. The production of volatile and fixed acids was low and not sufficient to cause the spoilage termed souring. Evidence is presented to show that adaptive lactase formation occurs in organisms termed S. fragilis. REFERENCES Association of Official Agricultural Chemists 1936 Official and Tentative Methods of Analyses. Washington, D. C. 4th ed. BEDFORD, C. L. 1941 Genus Hansenula Sydow. Morphological and Physiological Studies. Ph.D. Thesis, University of California at Berkeley, 1-128. BEIJERINCK, M. W. 1898 Sur la r6g6neration de la facult6 de produire des spores chez des levures en voie de la perdre. Arch. n6erland. sci. Serie II, Tome II. CALDIS, P. D. 1930 Souring of figs by yeasts and the transmission of the disease by insects. J. Agr. Research, 40, 1031-1051. CALDIS, P. D. 1927 Etiology and transmission of endosepsis (internal rot) of the fruit of the fig. Hilgardia 2, 287-328. CONDIT, IRA J. 1941 Fig culture in California. Calif. Agr. Ext. Circ., 77, 1-67. CUSTERS, M. TH. J. 1940 Onderzoekingen over het gistgeslacht Brettanomyces. Diss. Delft. DAVEY, A. E., AND SMITH, RALPH E. 1933 The Epidemiology of Fig Spoilage. Hilgardia, 7, 523-551. HANSEN, H. N., AND DAVEY, A. E. 1932 Transmission of smut and mold in figs. Phytopathology, 22, 247-252. HENRICI, ARTHUR T. 1941 The yeasts: genetics, cytology, variation, classification and identification. Bact. Revs., 5, 97-179. KLOCKER, ALB. 1924 Die Giirungsorganismen. Dritte Aufi. Urban & Schwarzenberg, Berlin-Wien. 449

450 E. M. MRAK, H. J. PHAFF, R. H. VAUGHN AND H. N. HANSEN KLUYVER, A. J., AND CUSTERS, M. TH. J. 1940 The suitability of disaccharides as respiration and assimilation substrates for yeasts which do not ferment these sugars. Antonie van Leeuwenhoek, 6, 121-162. LANGERON, M., AND GUERRA, P. 1938 Nouvelles recherches de zymologie medicale. Ann. parasitol., 16, 36-84, 162-179, 481-525. LINDNER, R. 1909 Mikroskopische Betriebskontrolle in den Garungsgewerben. Fuinfte. Aufl., Paul Parey, Berlin. LODDER, J. 1932 ttber einige durch dass "Centraalbureau voor Schimmel-cultures" neuerworbene sporogene Hefearten. Zentr. Bakt. Parasitenk., II, 100, 227-253. MELLIGER, R. 1931 Contribution A l'6tude des ferments figur6s et les fermentations de la datte. These No. 893. Geneve. MRAK, E. M., AND BAKER, E. E. 1939 Sugar tolerant yeast causing the spoilage of dried fruits. Third International Congress for Microbiology. New York, 707-708. MRAK, E. M. AND BONAR, LEE 1938 A note on yeast obtained from slimy sausage. Food Research 3, 615-618. MRAK, E. M. AND BONAR, LEE 1939 Film yeast from pickle brines. Zentr. Bakt. Parasitenk, II, 100, 289-294. MRAK, E. M. AND MCCLUNG, L. S. 1940 Yeasts occurring on grapes and in grape products in California. J. Bact., 40, 395-407. MRAK, E. M., PHAFF, H. J. AND VAUGHN, R. H. 1942 Yeasts occurring on dates. J.Bact., 43, 689-700. SMITH, RALPH E. AND HANSEN, H. N. 1927 The improvement of quality in figs. Univ. Calif. Agr. Expt. Sta. Circ., 311, 1-23. SMITH, RALPH E., AND HANSEN, H. N. 1931 Fruit spoilage diseases of figs. Calif. Agr. Expt. Sta. Bull., 506, 1-84. STELLING-DEKKER, N. M. 1931 Die Hefesammlung des "Centraalbureau voor Schimmelcultures." 1 Teil. Die Sporogenen Hefen. Verhandel. Akad. Wetenschappen Amsterdam. Afdeel. Natuurkunde. 2 sectie. Deel 28 No. 1. Downloaded from http://jb.asm.org/ on November 10, 2018 by guest