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.