3671

Mycopathologia vol. 55, 1, pag. 29-34, 1975

SCANNI G ELECTRON "nCROSCOPY OF ASCOSPORES OF DEBARYOMYCES AND SACCHAROl\-fYCES c. P. KURTZMAN. M. J. SMILEY & F. L. BAKER

onhern Regional Research Laboratory. Agricultural Research Service. U.S. Department of Agriculture. Peoria. Illinois 61604

Keywords: Ascospores. Debaryomyces, Saccharomyces

Abstract Kawakami & Nehira (1959) examined thin sections by transmission electron microscopy (TIM) and reported Ascospores from species of Debaryomyces and the Torula­ that warts on ascospores of Debaryomyces hansenii (Zopf) spora-group of Saccharomyces were examined by scanning Lodder & Kreger-van Rij, D. nicotianae Giovannozzi, D. electron microscopy. Ornamentation on ascospores of D. kloeckeri Guillierrnond & Peju, D. marama di Menna, and hansenii varied from short to long interconnected ridges D. globosus Klocker formed a 'wavy' pattern, whereas or broad based. elongated conical protuberances. A spiral those of D. subglobosus (Zach) Lodder & Kreger-van Rij ridge system was detected on the ascospores of D. marama, had a 'gear' type of pattern. but wart-like protuberances occurred on those of D. can­ Kreger-van Rij (1964, 1969) also using TEM observed tarellii, D. castellii, D. coudertii, D. formicarius, D. phalfii, the ascospore walls of Debaryomyces to be made up of two D. vanriji and D. yarrowii. Ascospores of D. halotolerans layers. The outer layer was thin and electron-dense, but did not have protuberances and the species appears to be the inner wall was thicker, less dense and formed the identical with Pichia farinosa. Wart-like protuberances wart-like protrusions. While the Torulaspora-like species also were found on ascospores of S. delbrueckii, S. micro­ Saccharomyces fermentati (Saito) Lodder & Kreger­ ellipsodes, S. rosei. S. inconspicuus, S. fermentati, S. mon­ van Rij; S. rosei (Guilliermond) Lodder & Kreger-van Rij; tanus and S. L'afer, but the ascospore surface of S. pre­ S. pretoriensis van der Walt & Tscheuschner [= D. fran­ toriensis was covered by tine ridges. Short tapered ridges ciscae (Capriotti) Kodama et aI.]; S. microellipsodes Oster­ covered the ascospores of S. kloeckerianus. walder var. microellipsodes [= D. nilssonii (Capriotti) Kodama et al.J; and S. kloeckerianus van der Walt (= D. Wartiness of the ascospore wall is considered an im­ globosus) had an appearance generally similar to Debaryo­ portant criterion in separating Deharvnml'ces from certain myces, the inner, less dense, layer of the ascospore wall other nitrate-negative ~east genera (Kre~er-van Rij. 1970). was divided into two parts by an electron-dense line. The Torulaspora-group of Saccharomyces. which ferments Clearly, much useful information has been obtained by sugars more vigorously than species of Debaryomyces and examining thin sections of ascospores by TEM, and these forms protuberances that may function as conjugation studies seem to separate Debaryomyces further from the tubes, also produces roughened ascospores although an Torulaspora-group of Saccharomyces. What cannot be electron microscope is frequently needed to perceive this perceived from this type ofstudy is the overall surface-fine surface ornamentation (Kodama et aI., 1964a. 1964b; structure of the ascospores and the actual form taken by Kreger-van Rij, 1969). Consequently, the roughness of the ornamentation. The scanning electron microscope the ascospores of these two possibly related genera has (SEM) by virtue of its great depth of tield and apparent caused much confusion resulting in some species being three dimensional imagery is ideally suited to gather in­ transferred from one genus to the other. formation about surface-tine structure. Kurtzman et at. (1972) demonstrated the complexities of ascospore orna­ Accepted for publication: 1. L1973 mentation in Schwanniomyces by SEM, and Belin (1973)

29 Purchased by Agricultural Rsscarch Sor'Ii\:~ U. S. Department of Aaricultura For Official Usa showed its value in studying the budding process of Sac­ titter with the glossy side down was placed over the spread charomyces uvarum Beijerinck. The present work was ascospore suspension and the filter unit assembled. undertaken to determine the pattern formed by protrusions The filter unit was attached to a syringe and the asco­ on the surface of ascospores of Debar.vomyces and the spores were washed with 10 ml of a 0.3 o/~ solution of the Torulaspora-group of Saccharomyces and to see if this laboratory detergent Haemo-Sol (Haemo-Sol. Inc., Balti­ surface-fine structure might have any taxonomic implica­ more, Md.) followed by 10 ml of distilled water. After tions. removal from the titter unit, the filters were kept together and placed between two aluminum plates of 3-rnm thick­ ness and centered on I-em holes that were drilled in both Materials and Methods plates. The plates, held together by two screws, have two parallel rows of four holes thus allowing eight ascospore Cultures and conditions for sporulation preparations to be processed at once. The holder was All strains of Debaryomyces and Saccharomyces used are passed at lO-min intervals through a graded acetone maintained in the ARS Culture Collection at the Northern dehydration series (30~~, 50~~, 70%, 3 changes lOO%) Regional Research Laboratory. Conditions for sporula­ and placed in the pressure bomb of a Denton DCP-I tion varied considerably, and the culture media included critical point drying apparatus (Denton Vacuum. Inc., yeast-malt agar (Wickerham, 1951), restricted growth agar Cherry Hill, N.J.) where it was slowly flushed with liquid (Herman, 1971) and Gorodkowa agar (van der Walt, carbon dioxide for 1 hour. After this treatment. the bomb 1970a). Generally, the best temperature for sporulation was sealed and heated in a 50.JC water bath until maximum was 15°C, but the time of sporulation varied from I week pressure was reached, which must be in excess of 1072 psi to several months. (Anderson, 1951); then the pressure was slowly (3()-4Q minutes) released. Upon removal of the filters from the Preparation of cultures for SEi\;[ holder, the upper filter (uncoated) was peeled ofT and A 2-mm loopful of cells from sporogenous cultures was discarded. The lower filter was cemented at the periphery placed in 0.1 ml of the enzyme preparation G1usulase* to an SEM specimen stage (stub) with silver electrical (Endo Laboratories. Inc.. Garden City. N.Y.). and the conductive paint and vacuum coated to a thickness of mixture was incubated at 25 .JC until approximately three­ about 15 om with gold-palladium alloy. Cementing of the fourths of the ascospores were released (ca. 1-1/2 hours). filters must be with rather thick conductive paint; other­ The enzyme-cell mixture was added to 5 ml of distilled wise, the solvent will flow across the filter and ruin the water and mixed, and then the cells were removed by preparation. centrifugation in a Clay-Adams table-top centrifuge. The The preparations were examined in a Cambridge cells were washed three additional times in this manner, Stereoscan Mark II scanning electron microscope at an and the final pellet was resuspended in 0.2 ml of distilled accelerating voltage of 20 kv; the final aperature was water. 200 .urn, and the specimen stage was inclined 45'" to the Because of adhering debris and the tendency to collapse incident beam. when air dried, the ascospores had to be detergent washed and subjected to crhical point drying before viewing. uclepore filters (General Electric, Irradiation Processing Results Center, Vallecitos uclear Center, Pleasanton, Calif.) of 13 mm diam and 1.0 .urn pore size were vacuum coated on Debaryomyces hansenii is collected from a variety of the dull side of the filter with gold-palladium alloy to a habitats and, partly owing to variability in carbon as­ thickness of 15 nm. A coated titter was then placed over similation reactions and pellicle formation, has a large the support-containing half of a Swinnex filter unit (Milli­ number of synonyms (Kreger-van Rij, 1970). SEM shows pore Corp., Bedford, Mass.) and a small drop of the spore the type strain NRRL Y-7426 (fig. 1) predominantly forms }. suspension was spread over the surface. An uncoated ascospores with ridges and blunt protuberances as is also true for Y-1458 (= D. lyroco/a Konokotina, fig. 2) and the slightly more ridged strain Y-7268 (fig. 3). The orna­ • The mention of firm names or trade products does not imply that they are endorsed or recommended by the Department of mentation is still more ridged for Y-1454 (= D. nicotianae, Agriculture over other firms or similar products not mentioned. fig. 4) and shows an intertwining in Y-1455 (= D. mem-

30 Figs. 1-6. Scanning electron micrographs of ascospores from Figs. 7-12. Scanning electron micrographs of ascospores from different strains of Debaryomyces hansenii. I. NRRL Y-7426. species of Debaryomyces. 7 & 8. D. marama NRRL Y-2171. type strain. 2. RRL Y·1458. 3. RRL Y·7268. 4. RRL 9. D. marama NRRL Y-7427. 10. D. cantarellii RRL Y-7421. Y-1454.5. RRL Y·1455. 6. RRL Y-I778. 11. D. castellii NRRL Y·742J. 12. D. coudertii NRRL Y-5984. branaefaciens aganishi, fig. 5). A pattern of ridges and Dekker). tapered projections was the predominant form of Y-1778 Debaryomyces marama fonns up to four oval spores (= D. marylandii, fig. 6). per ascus and so differs from other species of Debaryo­ While differences do exist in ascospore ornamentation myces, which usually fonn a lesser number of round spores. among species now considered synonyms of D. hansenii, Kawakami (1958) observed warts on the ascospore wall the figures illustrate the predominant fonn for each strain, of D. marama by TIM and because of this, and the similar­ and nearly all strains showed sufficient variability that each ity of the species to D. hansenii in carbon assimilation also formed ascospores with ornamentation similar to that reactions and cell morphology, Kreger-van Rij (1970) of the other strains. The type strain Y-7426 had ascospores retained it in the genus Debaryomyces. SEM shows that that were predominantly like those shown in Figure 1, but the ascospore ornamentation of Y-2171 consists of spiral forms such as those in figure 5 were also noted. Similarly, ridges (figs. 7 and 8). It appears that the ridge is not always the system of tapered projections typical of Y-1778 (fig. 6) continuous between the two applanate sides of the asco­ was occasionally observed in most of the other strains. spore but that sometimes two ridges are present. Rarely, Additional strains of D. hansenii examined included one of these ridges was a ring rather than a spiral. Another YB-4482, Y-I449 (both fonnerly D. kloeckeri), YB-331l isolate of D. marama, Y-7427, had a less extensive spiral (= D. miso Mogi), Y-1476, Y-2417, Y-1452 (= D. macru­ ridge system, and the ascospore surface was covered with choti Grigoraki et Peju) and Y-l448 (= D. guilliermondii many minute round protuberances (fig. 9).

31 The ascospores of D. cantarellii Capriotti (Y-7421); D: reactions of these strains were typical of Pichia farinosa castellii Capriotti (Y-7423, type strain); D. coudertii Saez (Lindner) Hansen; consequently, the species should be (Y-5984, Y-7425, type strain); D. phaffii Capriotti considered a synonym of P. farinosa. Debaryomyces (YB-5161, Y-7429); D. vanriji (van der Walt & Tscheusch­ nepalensis Goto & Sugiyama Y-7108 fonned ascospores ner)Abadieetal. (Y-6664, Y-i430, type strain and Y-7431); with ornamentation intermediate to that shown in figures D. formicarius Golubev et Bab'eva (Y-7533, type strain); land 2, and also appears identical to D. hansenii in respect and D. yarrowii Santa Maria & Aser (Y-7535, type strain) to morphology and physiology. were essentially identical having numerous wart-like pro­ Saccharomyces kloeckerianlfs. formerlv {)fhnrl'nmw'es tuberances (figs. 10-16). Occasionally, two of the pro­ globosus the type species of Debaryomyces, produced asco­ tuberances appeared to fuse and form a short ridge (fig. 12). spores with a series ofridges similar to those ofsome strains In contrast to D. Izansenii, all strains ofeach species within of D. hansenii. The strains examined were Y-2021 (fig. 17) this group showed remarkable similarity of ascospore and Y-776 X Y-1982. Ascospores of Saccharomyces del­ ornamentation. brueckii Lindner (Y-866); S. microellipsodes (Y-6687) ; Three strains of a yeast received as Debaryomyces halo­ S. rosei (YB-4770. Y-6000); S. inconspicuus van der Walt tolerans Sasaki & Yoshida (Y-7478, Y-7479 and Y-7480) (Y-7435, type strain); and S. vafer van der Walt (Y-7432, formed ascospores that were smooth. In our examination, type strain) are seen in figures 18-22. These species, which the morphology and carbon assimilation and fermentation are from the Torulaspora-group of Saccharomyces, have

Figs. 13-18. Scanning electron micrographs of ascospores from Figs. 19-24. Scanning electron micrographs of ascospores from species of Oebaryomyces and Saccharomyces. 13. O. phaffii species of Saccharomyces. 19. S. rosei NRRL YB~770. 20. NRRL Y-7429. 14. O. vanriji RRL Y-7430. 15. O. formicarius S. vafer 'RRL Y-7432. 21. S. microellipsodes NRRL Y-6687. NRRL Y-7533.16. O. yarrowii NRRL Y-7535. 17. S. kloeckeria· 22. S. inconspicuus NRRL Y·7435, type strain. 23. S. pretoriensis nus RRL Y-2021. 18. S. delbrueckii NRRL Y-866. NRRL Y-6686. 24. S. pretoriensis NRRL YB-4104.

32 wart-like protuberances and consequently show much D. castellii and D. phaffii have been described. Still similarity to most species of Debaryomyces. Some fusion separating the groups. however, is the occurrence of of the wart-like protuberances into ridges was especially evaginations on the cells of the Tontlaspora-group and evident on ascospores of S. delbrueckii (fig. 18) and S. differences in ascospore wall structure. The dark line wfer (fig. 20). The ornamentation on ascospores of S. dividing the inner ascospore wall of S. kloeckerianus fermentati (Y-856) was much like that shown for S. rosei (= D. globosus). S. preroriensis (= D.franciscae), S. micro­ and the ascospores of S. montanus Phaff, Miller & Shifrine ellipsodes (= D. nilssonii). S. fermenrari and S. rosei was (Y-7434, type strain) are similar to those ofS. inconspicuus. not found in species of Debar,vomyces (Kreger-van Rij, Ascospores of S. pretoriensis van der Walt & Tscheusch­ 1969). Moreover, the DNA base composition of the ner differed from the other species examined. Strain Torulaspora species has a higher GC ratio than that of Y-6686 (fig. 23) had a network of fine ridges, but this Debaryomyces species (Meyer & Phaff, 1969, 1970; ornamentation was barely perceptible in another isolate, Nakase & Komagata, 1971a, 1971b). YB-4I04 (fig. 24). Isolates with smooth- and rough-walled ascospores have been detected in S. pretoriensis by TIM. and van der Walt (1970b) summarizes the data in his discussion ofvariability Discussion encountered among strains ofthis species. We also detected rough and relatively smooth-walled ascospores in strains The variability in carbon assimilation pattern and other of S. pretoriensis (figs. 23 & 24) and suggest that the charaCteristics found among isolates of D. hansenii seems difference may be a matter of degree of roughness since also manifested in surface-fine strUCture of the ascospores. even the nearly smooth ascospores of YB-41 04 have a faint While the predominant ascospore morphology of such ridge-like pattern. synonyms of D. hansenii as D. tyrocola. D. nicotianae, D. Although this study has provided much information on membranaefaciens and D. marylandii tends to be distinct, ascospore surface-fine structure, most of the species each occasionally forms ascospores with morphology examined do not have ascospores with sufficiently distinct typical of the other species. Whether this variation can be ornamentation to allow species separation or certain accounted for by differences in maturity, culture nutrition, generic assignment. Studies of other genera may. however, or through genetic segregation of the genes controlling reveal whether ascospore morphology is an indicator of ascospore morphology is unknown, but Kreger-van Rij's relatedness between genera. (1970) decision to consider these species as synonyms of D. hansenii seems sound. A surprising finding in our work was that ascospores Summary of D. marama differed markedly from other species of Debaryomyces. The variation in ascospore shape found Surface ornamentation on ascospores of Debaryomyces among species of Pichia may be sufficient to allow the hansenii was shown by SEM to vary from short-to-Iong inclusion of D. marama in this genus as Phaff (1956) h~d interconnected ridges or to consist of broad-based elon­ suggested previously but for different reasons. gated conical protuberances. While the majority of asco­ Ascospores of D. coudertii, D. vanriji, D. formicarius, spores from a particular strain had the same type of orna­ D. yarrowii, D. phaffii, D. castellii and D. cantarellii look mentation, a few ascospores with the the other types of much alike, and Kreger-van Rij (1970) pointed out other ornamentation also were present, and strain separation similarities among the last three species. There appears based on ascospore surface structure was not possible. The to be the possibility that certain of these species should apparent warts observed on ascospores of D. marama by be combined. More definitive information may be obtained TEM were shown actually to form a spiral ridge system. By through either nucleic acid homology studies or from contrast, discrete wart-like protuberances occurred on the attempts to obtain interspecific hybridizations. ascospores of D. cantarellii, D. castellii, D. couderrii, D. Almost all species of the Torulaspora-group of Sac­ formicarius. D. phaffii, D. vanriji and D. yarrowii. charomyces have ascospores with wart-like protuberances Wart-like protuberances also were found on the asco­ similar to the majority of species in Debaryomyces. The spores of Saccharomyces delbrueckii, S. microellipsodes, distinCtion between these two groups is less clear-cut now S. rosei, S. inconspicuus, S. fermentati, S. monranus and that the more rapidly fermenting species D. cantarellii. S. vafer. Ascospores of these species were quite similar to

33 those from most species of Debaryomyces. The ascospores akase, T. & K. Komagata. 1971 b. Significance of DA base of S. pretoriensis were covered by a network of fine ridges; composition in the classification ofyeast genus Saccharomyces on one strain these were barely perceptible and made the J. Gen. Appl. Microbia!. 17: 227-238. Phatr. H. 1. 1956. A proposal for amendment of the diagnosis ascospores appear almost smooth. In view of these obser­ of rhe genus Pichia Hansen. Antonie van Leeuwenhoek 22: vations, waniness of the ascospore wall does not appear 113--116. to be a criterion specific enough for the taxonomic separa­ van der Walt, J. P. 1970a. Cnteria and methods used in tion ofthe above two genera or the species assigned to them. classification. In: J. Lodder (ed.), 'The Yeasts,' 2nd ed., pp. 34-113. North Holland Publishing Co., Amsterdam. van der Walt, J. P. 1970b. Saccharomyces Meyen emend. Reess. In: J. Ladder (ed.), 'The Yeasts,' 2nd ed.. pp. 555-718. .~ References North Holland Publishing Co., Amsterdam. Wickerham, L. 1. 1951. Taxonomy of yeasts. U.S. Dept. Agr., Anderson. F. 1951. Techniques for the preservation of three­ T. Tech. Bull. No. 1029. dimensional structure in preparing specimens for the electron

microscope. Trans. N.Y. Acad. Sci. 13: 130-134. I • Belin, J. M. 1972. A study of the budding of Saccharomyces uvarum Beijerinck with the scanning electron microscope. Antonie van Leeuwenhoek 38: 341-349. Herman, A. r. 1971. Mating responses in the yeast Hansenula holstii. Antonie van Leeuwenhoek 37: 275-280. Kawakami, N. 1958. Electron-microscopy of fungi (V). The morphological study of the spore of non-pellicle group in the genus Debaryomyces and the classification of the genus Debaryomyces. Electron Microsc. 7: 235-237. Kawakami, . & T. Nehira. 1959. Electron-microscopy of fungi (IX). Intracellular structures of Rhodotorula glutinis and 1 adsonia fulvescens and their relation to the physiological characters and taxonomic affinity. J. Ferment. Technol. 37: 125-132. Kodama, K., T. Kyono, H. Naganishi & Y. Takahara. I964a. Taxonomic studies on the genus Debaryomyces (III). De­ baryomyces franciscae (Capriotti) nov. comb. J. Ferment. Techno!' 42: 1-6. Kodama, K., T. Kyono, H. aganishi & Y. Takahara. I964b. Taxonomic studies on the genus Debaryomyces (IV). De­ baryomyces nilssoni (Capriotti) nov. comb. J. Ferment. Techno!. 42: 655-660. Kreger-van Rij, . J. W. 1964. A taxonomic study of the yeast genera Endomycopsis, Pichia and Debaryomyces. Thesis. Leiden. Kreger-van Rij, . J. W. 1969. Some features of yeast asco- spores observed under the electron microscope. Proc. Int. Symp. Yeasts 2: 169-176, Bratislava, 1966. Kreger-van Rij, ". J. W. 1970. Debaryomyces Lodder et Kreger­ van Rij nom. conserv. In: J. Lodder (ed.), 'The Yeasts,' 2nd ed.. pp. 129-156. North Holland Publishing Co., Amster­ dam. Kurtzman, C. P., M. J. Smiley & F. L. Baker. 1972. Scanning electron microscopy of ascospores of Schwanniomyces. 1. Bact. 112: 1380-1382. Meyer, S. A. & H. J. Pharr. 1969. Deoxyribonucleic acid base composition in yeasts. J. Bact. 97: 52-56. Meyer, S. A. & H. J. Pharr. 1970. Taxonomic significance of the DNA base composition in yeasts. In: D. G. Ahearn (ed.), .Recent Trends in Yeast Research,' pp. 1-29. Spectrum Monograph Series. Georgia State University, Atlanta. Nakase, T. & K. Komagata. 1971a. Significance of DA base composition in the classification of yeast genus Debaryomy­ ces. 1. Gen. Appl. Microbiol. 17: 43-50.

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