During the Course of a Study on Wild Yeasts in Japan, Five

J. Gen. Appl. Microbiol. Vol. 12, No. 4, 1966

NEW YEASTS, ENDOMYCOPSIS MUSCICOLA AND PICHIA ZAR UENSIS

TAKASHI NAKASE and KAZUO KOMAGATA Central Research Laboratories, Ajinomoto Co., Inc., Kawasaki, Japan Received December 10, 1966

During the course of a study on wild yeasts in Japan, five strains of hitherto undescribed yeasts were isolated from moss (3 strains), exudate of deciduous tree (1 strain), and soil (1 strain). The three strains originated from moss were designated as Endomycopsis muscicola and two strains from exudate and soil were named Pichia zaruensis. This paper describes these two new yeasts.

MATERIALS AND METHODS

Yeasts were isolated by the agar plate method using acidified yeast extract- malt extract agar (pH 5.0) with 0.2% of sodium propionate, and purified by the conventional streaking technique. Determinative procedures were mainly those of WICKERHAM (1), LODDER and KREGER-VAN RIJ (2), and WICKERHAM and BuRToN (3). Vitamin requirements were determined by the procedure of VAN UDEN and FARINHA (4). Maximum growth temperature was confirmed by recording the growth in yeast extract-malt extract broth incubated in water bath at various temperatures under shaking. Growth was recorded daily for a period of 5 days.

RESULTS AND DISCUSSION

1. Endomycopsis muscicola Nakase et Kogagata sp. nov. Strains: M-5-1, M-5-2, and M-5-3 In musto maltato cellulae rotundae, subovoideae, ovoideae, tomaculiformes aut longae, (3-8) x (3.5-11-20) p, singulae, binae aut catenatae. Sedimentum et anulus formantur cultura in agaro maltato (post unum mensem,17°) flavo- albida, glabra aut rugosa, mollis aut rigida, nitida aut non nitida, margine piloso. Pseudomycelium et mycelium formantur. Formantur asci extrans- f ormatione cellularum vegetativarum diploidearum aut con jugatione inter cellulas haploidearum. Ascosporae peleiformes aut rotundae, 1-4 in asco. D-Glucosum et D-galactosum (lente) f ermentantur at non saccharosum, maltosum, lactosum, raffinosum, trehalosum, melibiosum, melezitosum, inulinum nec amylum. In medio minerali D-glucosum, D-galactosum, maltosum (varium), cellobiosum, trehalosum (aut lente), D-xylosum, L-arabinosum, D-ribosum (lente), L-rhamnosum, ethanolum, glycerolum, erythritolum (lente), adonitolum, 347 348 NAKASE and KOMAGATA VoL 12

D-mannitolum, D-sorbitolum, a-methyl-D-glucosidum (lente), salicinum, acidum gluconicum, acidum DL-lacticum, et acidum succinicum assimilantur at non saccharosum, lactosum, L-sorbosum, melibiosum, raffinosum, melezitosum, inulinum, amylum, D-arabinosum, dulcitolum, acidum 2-ketogluconicum, acidum citricum, nec inositolum. Nitras kalicus assimilatur. Arbutinum finditur (lente et exiguum). Biotinae, pyridoxinae et thiaminae necessariae ad crescentiam. Maxima temperatura crescentiar : 40-41°. Isolata ex musco. Growth in malt extract: After 3 days at 25° cells are almost round, short oval to oval, sausage-shaped or elongate, (3-8) x (3.5-11-20) p, and occur singly, in pairs, or in chains. A trace of ring and a sediment are formed. After one month at 17° a trace of ring and a sediment are present. Growth on malt agar: After 3 days at 25° cells are nearly the same as seen in the malt extract. After one month at 17° two types of colonies are observed. One is yellowish gray, raised to pulvinate, smooth, soft, shining, and has a margin fringed with mycelia. The other is rather tough and wrinkled, and the border fringed by abundant mycelia. Slide culture: Ramified true mycelia with many septa develop abundant- ly. Blastospores are short oval to oval, and occur singly, in chains, or in verticals. Besides true mycelia, pseudomycelia are also formed but not abun- dantly. Sporulation : Spores are hat shaped or occasionally seem to be round, (1.5-2.7) x (2.5-4.0), 1-4, usually 4 per ascus. Usually they are liberated at maturation, but those formed in hyphal swellings are not. Most of asci are formed parthenogenetically, but conjugation preceding ascus formation is also observed. Monosporic cultures sporulate readily. 1966 Endomycopsis muscicola and Pichia zaruensis 349

Fermentation: D-Glucose and D-galactose (latent) are fermented. Sac- charose, maltose, lactose, raffinose, trehalose, melibiose, melezitose, inulin, and soluble starch are not fermented. Assimilation of carbon compounds : D-Glucose, D-galactose, maltose (var- iable), cellobiose, trehalose (or latent), D-xylose, L-arabinose, D-ribose (latent), L-rhamnose, ethanol, glycerol, erythritol (latent), adonitol, D-mannitol, D- sorbitol, a-methyl-D-glucoside (latent), salicin, gluconate, DL-lactate, and succinate are assimilated. Saccharose, lactose, L-sorbose, melibiose, raffinose, melezitose, inulin, soluble starch, D-arabinose, dulcitol, 2-ketogluconate, citrate, and inositol are not utilized. Potassium nitrate is utilized as the sole nitrogen source. Arbutin is rather weakly split. Biotin, pyridoxine, and thiamine are essentially required at 25°. No growth occurs in moderate osmotic pressure medium. Maximum growth temperature : 40-41 °. Gelatin is not liquefied. Source : Moss, Tottori Prefecture, Japan, 1963.

This yeast is ascosporogenous and forms abundant true mycelia besides budding cells. These are the typical characters of the genus Endomycopsis DEKKER delimitted by LODDER and KREGER-VAN RIJ (2). On the other hand, this yeast also agrees with Hansenula SYDOW sensu WICKERHAM (1), because it is able to utilize potassium nitrate as the sole nitrogen source. Although general acceptance was not found whether true mycelia formation or nitrate assimilation is taxonomically more significant, the authors tentatively identified this yeast with the genus Endomycopsis. Hitherto described prominent true mycelia-forming, ascosporogenous yeasts, such as E. bispora (5), E. platypodis (6), H. petersonii (7), and H. bimundalis (8), differ from E. muscicola in the following properties: E. bispora does not ferment sugars, assimilates saccharose, melezitose, and citrate but not D-galactose or erythritol, and accord- ing to BECK (5), it produces two spores per ascus; E. platypodis ferments D-glucose weakly but not D-galactose, assimilates saccharose, melezitose, and citrate, but not D-galactose or L-arabinose; H. petersonii ferments saccharose and raffinose but not D-galactose, assimilates saccharose, raffinose, melezitose, inulin, and citrate but not D-galactose, L-arabinose, D-ribose, erythritol, or adonitol; H. bimundalis is heterothallic, does not ferment D-galactose, assimilates saccharose, melezitose, and citrate but not D-galactose, D-ribose, erythritol, or adonitol. The specific epithet is derived from the Latin " muscus " meaning "moss ", and "cob" " meaning "inhabit ".

2. Pichia zaruensis Nakase et Komagata sp. nov. Strains: J-7 and YO-276 In musto maltato cellulae rotundae, subovoideae aut ovoideae, (2.5-7) x (3-8),a, singulae, binae aut catenatae. Sedimentum et anulus formantur. Cultura in agaro (post unum mensem, 17°) glaucof usca, plana, glabra, parum 350 NAKASE and KOMAGATA VOL. 12

nitida, mollis, margine glabra. Pseudomycelium nullum. Plerumque formantur asci ex conjugatione cellularum haploidearum. Ascosporae saturni- formes, 1-3 in asco. D-Glucosum et trehalosum (varium) fermentantur at non D-galactosum, saccharosum, maltosum, lactosum, melibiosum, raffinosum , melezitosum, inulinum nec amylum. In medio minerali D-glucosum, trehalosum, ethanolum, adonitolum (lente), D-mannitolum, D-sorbitolum, acidum DL-lacticum (aut lente) et acidum succinicum (aut lente) assimilantur at non D-galactosum, saccharosum, maltosum, lactosum, L-sorbosum, cellobiosum, melibiosum, raffinosum, melezitosum, inulinum, amylum, D-xylosum, L-arabinosum, D-arabinosum, D-ribosum, L-rhamnosum, glycerolum, erythritolum, dulcitolum, a- methyl-D-glucosidum, salicinum, acidum gluconicum, acidum 2-ketogluconicum, acidum citricum nec inositolum. Nitras kalicus non assimilatur. Arbutinum non finditur. Pyridoxinae et thiaminae necessariae ad crescentiam. Maxima temperatura crescentiar : 34-36°. Isolata ex gummi Querci et ex solo. Growth in malt extract : After 3 days at 25° cells are round, short oval to oval, (2.5-7) x (3-8),~, and occur singly, in pairs, or sometimes in short chains. A ring and a sediment are formed. After one month at 17° a ring and a sediment are present. Growth on malt agar: After 3 days at 25° cells are round or almost round, (3.5-8) x (3.5-8)i, and occur singly, in pairs, or in chains. After one month at 17° the streak culture is grayish brown, flat, smooth, slightly shining, soft, and has an entire margin. Slide culture: No pseudomycelia are formed. Sporulation : Spores are Saturn-shaped or sometimes seem to be round with an oily drop in the middle, (2.0-3.0) x (2.3-3.5)p,1-3, usually 2 per ascus. Spore wall looks smooth. Usually heterogamous conjugation precedes ascus formation, but sometimes an isogamous one. Asci do not rupture at matur- 1966 Endomycopsis muscicola and Pichia zaruensis 351 ation. Abundant sporulation gives the culture a brown color. Fermentation : D-Glucose is fermented. Trehalose is latently or not fermented. D-Galactose, saccharose, maltose, lactose, melibiose, raffinose, melezitose, inulin, and soluble starch are not fermented. Assimilation of carbon compounds : D-Glucose, trehalose, ethanol, adonitol (latent), D-mannitol, D-sorbitol, DL-lactate (or latent), and succinate (or latent) are assimilated. D-Galactose, saccharose, maltose, lactose, L-sorbose, cellobiose, melibiose, rafl'inose, melezitose, inulin, soluble starch, D-xylose, L-arabinose, D-arabinose, D-ribose, L-rhamnose, glycerol, erythritol, dulcitol, a-methyl-D- glucoside, salicin, gluconate, 2-ketogluconate, citrate, and inositol are not assimilated. Potassium nitrate is not utilized as the sole nitrogen source. Arbutin is not split. Gelatin is not liquefied. Starch-like compound is not produced. Pyridoxine (essentially) and thiamine (essentially or stimulatively) are required at 25°. Maximum growth temperature : 34-36°. No growth occurs in moderate osmotic pressure medium. Source : Strain J-7 from exudate of Quercus serrata THUNB., Zaru- ga-take (Mt. Zaru), Yamanashi Prefecture, Japan, May 1965. Strain YO-276 from forest soil, Kamakura City, Kanagawa Prefecture, Japan, May 1965. This yeast belongs to the genus Pichia amended by PHAFF (9), and resembles P. saitoi (10) in the shape of ascospores, and in fermentation and assimilation of carbohydrates. In this yeast, however, heterogamous or isogamous conjugation precedes ascus formation, usually 2 spores are produced in an ascus, and most of asci do not rupture at maturation, while in P. saitoi no conjugation occurs, 2-4, usually 4, spores are formed in an ascus, and asci rupture at maturation. Furthermore, P. zaruensis forms no pellicle in liquid media and assimilates adonitol though latently. The specific epithet " zaruensis " was chosen since it was first isolated at Zaru -ga-take (Mt. Zaru) The authentic cultures of E. muscicola M-5-1 and P. zaruensis J-7 have been deposited with the Institute of Applied Microbiology, University of Tokyo (IAM), and Institute for Fermentation, Osaka (IFO).

SUMMARY

Two new sporogenous yeasts, Endomycopsis muscicola and Pichia zaruensis, were described. The former was isolated from moss, and the latter from exudate of deciduous tree and forest soil.

The authors wish to thank Dr. K. Kodama who kindly supplied the authentic culture of P. saitoi for comparative study, and also to Miss. H. KATO for her tech- nical assistance. 352 NAKASE and KOMAGATA VOL. 12

REFERENCES

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