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Cephaloascus Albidus, a New Heterothallic Yeastlike Fungus 1

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Cephaloascus Albidus, a New Heterothallic Yeastlike Fungus 1 4 02 9 "Purchased by the U.S. Department of Agriculture, for official use." Reprinted from MYCOLOGIA, Vol. LXIX, No.3, pp. 547-555, May-June, 1977 Printed in U. S. A. CEPHALOASCUS ALBIDUS, A NEW HETEROTHALLIC YEASTLIKE FUNGUS 1 c. P. KURTZMAN Northern Regional Research Center} Agricultural Research Service} U. S. Department of Agriculture} Peoria} Illinois 61604 SUMMARY The new species Cephaloascus albidus was isolated from cranberry / pumace in ova Scotia, Canada. It differs from the only other known species of Cephaloascus} C. jragrans} in assimilating a greater number of compounds and in being nonpigm,ented and heterothallic. In August 1971, an unidentified yeastlike fungus was received from C. L. Lockhart, Canada Department of Agriculture. The fungus had been isolated from cranberry pumace where it caused an odor of isobutyl acetate. Abundant budding cells as well as true hyphae were produced by the culture, but there was no evidence of a sexual stage. Reexami­ nation of the culture after several months storage in a refrigerator showed the presence of ascospores. Asci were formed in brush-like heads on hyaline ascophores that occasionally were erect and stout. This culture, which represents a new heterothallic species of Cephalo­ ascus) shows a clear relationship to such filamentous yeast genera as Saccharomycopsis Schionning. MATERIALS AD METHODS Methods for carrying out fermentation and assimilation tests, and procedures for morphological examination were previously given by Wickerham (1951). Single ascospore isolates were obtained by micro­ manipulation. All cultures used in this study are maintained by the ARS Culture Collection at the orthern Regional Research Center. Mycelium to be examined by transmission electron microscopy was fixed in 1% potassium permanganate for 1 h and dehydrated through a graded ethanol series (25%, 500/0, 75%, 950/0, 1000/0, 100%, 30-min intervals). Fixation and dehydration were carried out at 5 C. Fol­ lowing fixation, the mycelium was embedded in Spurr epoxy resin (Polysciences, Inc., Warrington, Pennsylvania) and thin-sectioned 1 Presented before Mycological Society of America, AIBS Meeting, Tulane University, ew Orleans, Louisiana, May 3O-June 4, 1976. 547 548 MYCOLOGIA, VOL. 69, 1977 with a diamond knife. Thin sections were stained for 2 h in 0.57'0 uranyl acetate (in 7: 3 ethanol-methanol) followed by staining in 37'0 lead citrate solution for 10 min. The preparations were examined in an RCA EMU-3 electron microscope. RESULTS Cephaloascus albidus Kurtzman, sp. nov. FIGS. 1-5, 7 Species heterothallica. Ascospori pileiformes 4 (1.5-2.0 X 2.5-3.0 ,u.m) fiunt per ascum. Asci dehiscentes. Asci (2.5-3.0 X 5.5-6.0 ,u.m) fiunt, alii singulariter, alii catenis brevibus ex ascophoris brevibus non differentiatis, alii in spica terminali penniformi quae oritur ex ascophoro erecto, crasso vitreo. Rare fiunt asci in axil1is hypharum ramosarum. In agare maltoso cellulae solent singulae esse, ellipsoidales-elongatae, saepe paulo fastigatae, 2.5-4.0 X 3.5-10 ,u.m. Cellulae fermentosae gemmantes non raTe fiunt. Blastoconidia supra denticula in lateribus hypharum et in spicis coniophorum simplicium. Hyphae (2.3-4.5 JlI11) pseudohy.phae que abundant. In agare morpho­ logico coloniae fiunt humiles, albidae, pulverosae, medium habentes fuscum et e1atum. Odor adest tenuis at suavis. Lente et languide fermentantur n-glucosum et n-galactosum. Maltosum, sucrosum, lactosum, raffinosum, trehalosum que non fermentantur. Assimilantur n-glucosum, n-galaotosum, L-sorbosum, cellobiosum (latens), trehalosum, melibiosum, raffinosum, n-xylosum, L-arabinosum, ethanolum, glycero­ lum, ribitolum, n-mannitolum, n-glucitolum, salicinum, nL-acidum lacticum (dilutum), acidum succinicum, acidum citricum (dilutum). on assimilantur potassii nitras, sucrosum, maltosum, lactosum, melezitosum, inulinum, amylon solubile, n-arabinosum, n-ribosum, L-rhamnosum, n-glucosaminum· HCl, i-erythri­ tolum, galactitolum, a-methyl-n-glucosidum, potassii-n-gluconas, calcii-2-keto­ gluconas, potassi-5-ketogluconas, potassi-sodii-saccharas, acidum pyruvicum, ethyl­ acetoacetas, et i-inositolum. Augmentum non fit in temperatura 37 C, aut si absint vitamina. Typus. N RRL Y-7343 designat stirpem typicam. Isolata est ab amurca oxycocciana in provincia ova Scotia, Canada. Typi complementarii conj ungentes : NRRL Y-7343-2 et RRL Y-7343-6. Omnes culturae servantur in Collectione Culturarum, Officina Investigationum Tractus Borealis, Peoria, Illinois, U.S.A. Growth on malt extract agar.-After 3 da at 25 C, the yeast cells are mostly single, ellipsoidal to elongate (2.5-4.0 X 3.5-10.0 fLm) and frequently somewhat tapered. Hyphae are abundant and have a diam of 2.3-4.5 JLm. Dolipore septa are not visible with the light microscope. Pseudohyphae are present. Dalmau plate culture on morphology agar.-Colonies are low, white, and powdery with a raised light tannish-gray center. Colony margins are entire with an appressed mycelial fringe. Asexual reproduction.-In addition to proliferation by budding yeast cells (FIG. 1), this species also forms blastospores. Blastospores occur at the ends of pseudohyphal segments and on denticles found singly KURTZMAN: A EW CEPHALOASCUS 549 20-11m 1 '? o FIGS. 1-7. 1-5. Cephaloascus albidus RRL Y-7343. 1. Budding yeast cells. 2. Dentic1es (arrow) on which blastospores form. 3. Simple ascophore bearing asci. 4. Branched ascophore bearing asci. 5. Erect ascophore with terminal asci. 6-7. Cephaloascus fragrans RRL Y-6742. 6. Budding yeast cells. 7. Erect arscopho'fe with terminal asci. 550 MYCOLOGIA, VOL. 69, 1977 on the sides of hyphal cells or in clusters at hyphal tips (FIG. 2). For comparison, budding cells from Cephaloascus fragrans Hanawa are shown in FIG. 6. Sexual reproduction.-Of commonly used media, there was significant sporulation only on YM agar. Ascospores formed at 7 and 15 C, but not at 20, 25, or 28 C. Markedly greater sporulation occurred on the mycelial outgrowth from the culture. Consequently, if an agar slant was point-inoculated at the top of the agar, mycelium grew downward and sporulation occurred, but if the whole slant was inoculated, budding yeast cells predominated and there was little or no sporulation. At 15 C, sporulation began 2-3 wk after inoculation. Asci (2.5-3.0 X 5.5-6.0 fllTI) contain four hat-shaped ascospores (1.5-2.0 X 2.5-3.0 flm) and dehisce upon maturity. Considerable varia­ tion is found in the complexity of the ascophore. Frequently the asco­ phore will be no more than a short hyphal branch bearing one to several asci that may be in short chains (FIG. 3). Asci have occasionally formed at the internodes of branched hyphae. Generally, ascophores produce a terminal head of asci which form in short chains. Smaller heads may occur lower on the ascophore as a result of hyphal branch­ ing (FIG. 4). The greatest degree of development (FIG. 5) is found in occasional stout, erect ascophores that are similar to those of Cephaloascus fragrans. However, these ascophores are smooth and hyaline rather than rough and pigmented reddish brown as with C. fragrans (FIG. 7). Erect ascophores were most frequently found on RG agar (0.2 g yeast extract, 0.2 g peptone, 1 g glucose, 20 agar, 1 liter distilled water). Single ascospores from C. albidus RRL Y-7343 produced asporo­ genous colonies, but appropriate pairings gave cell-to-cell conjugations and sporulation. The most fertile complementary pair was Y-7343-2 X Y-7343-6, but all single spore isolates had mating competence. Previous investigators (Wells, 1954; Schippers-Lammertse and Heyting, 1962) showed C. fragrans to be homothallic and that work was verified in the present study through single ascospore isolations from C. fragrans RRL Y-6741, Y-6742 (type strain), and Y-6743 (type strain of Ascocybe grovesii Wells). Cytological studies of C. fragrans were conducted by Dixon (1959) and Wilson (1961). The growth from two-thirds of the single spore isolates of C. albidus was quite butyrous because of the proliferation of budding cells and low frequency of true hyphae. When incubated at 15 C, these strains produced an extracellular polymer that would extend 1-3 cm when KURTZMAN: A NEW CEPHALOASCUS 551 TABLE I PHYSIOLOGICAL CHARACTERISTICS OF Cephaloascus albidus AND C. fragrans a Growth reaction Growth reaction of: of: Physiological C. albi- C. fra- Physiological C. albi- C.fra- test dus grans test dus grans Fermentation of carbon compounds: D-Glucose Weak, slow Lactose D-Galactose Weak, slow Raffinose Maltose Trehalose Sucrose Assimilation of carbon compounds: D-Glucose + + Ethanol + + D-Galactose + + Glycerol + + L-Sorbose + -, Lb i-Erythritol Sucrose Ribitol + + Maltose -, + Galactitol Cellobiose L D-Mannitol + + Trehalose + + D-Glucitol + + Lactose a-Methyl-D-glucoside Melibiose + Salicin + Raffinose + K-D-Gluconate Melezitose Ca-2-Ketogluconate Inulin K-5-Ketogluconate Soluble starch -,+ K-Na-Saccharate D-Xylose + + Pyruvic acid L-Arabinose + + DL-Lactic acid Weak -, Weak D-Arabinose -,L Succinic acid + Weak D-Ribose Citric acid Weak r.-Rhamnose Ethylacetoacetate D-Glucosamine· HCl i-Inositol Assimilation of potassium nitrate Growth at 37 C Starch formation Growth in vitamin-free medium + Production of esters + + Liquefaction of gelatin Growth in 10% sodium chloride plus 5% glucose in yeast nitrogen base + -, weak a Physiological characteristics of C. albidus are from NRRL Y-7343 and those of C.fragrans are from NRRL Y-6741, Y-6742, and Y-674-3. b Latent; strong positive reaction developing between 3 and 4- wk after inoculation. drawn out with a transfer needle. The remaining single spore isolates were predominantly mycelial and produced proportionally less polymer. Physiological characteristics of C. albidus and C. fragrans are compared in TABLE 1. Source.- RRL Y-7343 is the only known strain of C. albidus. It was isolated from cranberry pumace in Nova Scotia, Canada. 552 M YCOLOGIA, VOL. 69, 1977 , ,. 8<.. 0.5 11m FIGS. 8-9. 8. Cephaloascus albidus RRL Y-7343. Septum with simple central pore separating two hyphal cells.
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