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Antonie van Leeuwenhoek 43 (1977) 317-322 317
Metschnikowia lunata
sp. nov.
W. I. GOLUBEV
Institute of Biochemistry and Physiology of Microorganisms, USSR Academy
of Sciences, Pushchino, Moscow Region 142 292, U.S.S.R.
GOLUBEV, W. I. 1977. Metschnikowia lunata sp. nov. Antonie van Leeuwenhoek
43: 317-322.
Ascosporulation in the yeast strain designated as Selenotila intestinalis
Krassilnikov was achieved. On the basis of mode of ascus formation and
ascospore morphology it is included in the genus Metschnikowia Kamienski
as a new species, M. lunata.
INTRODUCTION
During the study of the yeast obtained as Selenotila intestinalis Krassilnikov
a few pulcherrima cells were discovered in an old culture. This finding coupled
with other properties of the strain suggested a relationship of this organism
to Metschnikowia Kamienski. To confirm this, successful attempts were made
to induce ascosporulation. The development of asci and ascospores is charac-
teristic of the genus Metschnikowia as currently delimited (Miller and van Uden,
1970). Since the studied organism differs from other known species of this genus,
it is described as a new species, M. lunata.
MATERIALS AND METHODS
Origin of strain. The strain, D-32, of S. intestinalis was obtained from
Dr. Irma P. Bab'eva (Department of Soil Biology, Faculty of Soil Science,
Moscow State University). It had been isolated by A. I. Egorova from flowers
of tufted vetch (Vicia cracca L.) near Novovladimirovka town (Spasski district,
Primorski Territory, U.S.S.R.) during an extensive survey (the period 1965 to
1966) of yeasts associated with the bee (Apis mell!fera L.). This strain together
with other iso!ated yeast strains was preliminary identified by her in coopera-
tion with Dr. Bab'eva and listed as Torulopsis sp. in Table 2 by Egorova and
Bab'eva (1967). Later from its morphology Prof. N. A. Krassitnikov noted
318 W.I. GOLUBEV
that the strain could be assigned to the genus SelenotiIa Lagerheim (1892) and
obviously to the species S. intestinalis described by him (Krassilnikov, 1927)
(In his description Prof. Krassilnikov mentioned the formation of chlamy-
dospores). The strain was deposited as S. intestinal& in the collection of yeast
cultures of the Department of Soil Biology. D. Yarrow obtained it from Dr.
Bab'eva and compared its physiological properties with those of the species
described by him as S. peltata (Yarrow, 1969). He found that contrary to
Krassilnikov's description the strain was able to ferment glucose.
The description of M.lunata is based on the standard techniques currently
employed in yeast taxonomy (van der Walt, 1970). The deoxyribonucleic acid
(DNA) extraction and purification were done by the procedure of Marmur
(1961). The guanine plus cytosine (G + C) content of DNA was determined
using thin-layer chromatography on cellulose.
Metschnikowia lunata Golubev sp. nov.
In aqua glucosum et extractum fermenti et peptonum continente ad 26 C, post 3 dies
cellulae lunatae 2-4 x 5-8 #met ovoideae 2.5-5 x 3.5-8 #m, singulae, binae. Capsula
non
formata. Chlamydosporae absentes. Post unum mensem sedimentum et annulus formantur;
chlamydosporae praesentes, globosae et subgtobosae 8-10.5 x 8-12 #m, interdum duabus
protuberantiis minutis praeditae.
Cultura in medio eodem agaro ad 26 C, post 3 dies alba, mollis, glabra, nitida, post unum
mensem cremea, moltis, glabra, non nitida, margine integra. Pulcherriminum non format.
Pseudomycelium indistinctum adesse potest.
Asci sphaeropedunculati, partes sporiferae 7-10 x 212 #m, pedunculi cylindrici 13-40 x
2-4 #m. Ascosporae aciculares, 1-2 in quoque asco.
Glucosum fermentatur neque galactosum, sucrosum, maltosum, cellobiosum, trehalosum,
lactosum, melibiosum, raffinosum, melezitosum, inulinum, amylum solubile et c~-methyl-
D-glucosidum.
Glucosum, galactosum, L-sorbosum, sucrosum, maltosum, cellobiosum, trehalosum,
melezitosum, D-xylosum (lente), ethanolum (lente), glycerolum, ribitolum (lente), o-gluci-
tolum, D-mannitolum, c~-methyl-o-glucosidum (lente), salicinum, arbutinum, OL-acidum
lacticum, acidum succinicum et glucono-f-lactonum assimilantur neque lactosum, melibio-
sum, raffinosum, amylum solubile, inulinum, L- et D-arabinosum, L-rhamnosum, D-ribosum,
i-erythritolum, acidum citricum, acidum aceticum, calcium 5-ketogluconatum, acidum
glucuronicum et i-inositolum.
Kalium nitricum non assimilatur.
Vitamina addita necessaria.
In 37 C non crescit.
In medio 50~o glucosum aut 10% sodium chloridum continente crescit (lentissime).
Proportio molaris guanidini et cytosini basium in acido DNA 49.2%.
Typus: D-32, e flore Viciae craccae isotatus. In collectione zymotica Centraalbureau
voor Schimmelcultures Delphis Batavorum sub No. 5946 deposita est.
Growth in glucose - peptone water: After 3 days at 26 C the cells are lunate
2-4 x 5-8 #m or rarely oval 2.5-5 × 3.5-8 #m, single or in pairs. A capsule is
not formed. Chlamydospores not present. The percentage of oval cells increases
in aged cultures. On the oval cells budding is multilateral, on the lunate cells
the reproduction occurs by budding more frequently at the extremities of the
METSCHNIKOWIA LUNATA SP. NOV. 319
cells on the convex face. After one month at 20 C there is a sediment and
a ring but no pellicle. Pulcherrima cells present, highly refractile, globose or
subglobose 8-10.5 x 8-12 #m, containing a lipid globule, occasionally with
two small pointed protrusions (Fig. 1).
Growth on glucose-yeast extract-peptone agar: After 3 days at 26 C the
streak culture is white, pasty, smooth, glossy. After one month at 20 C the
culture is cream, pasty, smooth, mat; the border is entire. Pulcherrimin is not
produced.
Rudimentary pseudomycelium is formed,
Asci sphaeropedunculate, spherical part 7-10 x 7-12 #m, peduncles cylin-
drical 2-4 x 13-40 #m, containing 1-2 acicular ascospores (Fig. 2). A suitable
medium for spore-formation is dilute (1:49) V-8 and the temperature is 15 C.
Fig. 1. Chlamydospores of
Metschnikowia lunata.
Material from one month' culture on malt
agar. Phase contrast, 1000 x.
Fig. 2. Ascospores of
Metschnikowia lunata.
Material from a 3-weeks' culture on diluted
(1:49) V-8, 15 C. Phase contrast, 1000 x.
Fermentation :
Glucose + e-Methyl-D-glucoside -
Galactose - Soluble starch -
Sucrose - Inulin -
Maltose - Cellobiose -
Lactose - Melezitose -
Raffinose -- Trehalose -
Melibiose
320 W. 1. GOLUBEV
Assimilation of carbon compounds :
Glucose + Ethanol + (slow)
Galactose + Glycerol +
Sucrose + i-Erythritol -
Maltose + Ribitol + (slow)
Lactose - Galactitol -
L-Sorbose + D-Mamlitol +
Cellobiose + D-Glucitol +
Trehalose + e-Methyl-D-glucoside + (slow)
Melibiose - Salicin +
Raffinose -- Arbutin +
Melezitose + DL-Lactic acid +
Soluble starch - Succinic acid +
Inulin - Citric acid -
D-Xylose + (latent) Acetic acid -
L-Arabinose - 5-Ketogluconate -
D-Arabinose - Glucono-6-1actone +
L-Rhamnose - i-Inositol -
D-Ribose - D-Glucuronic acid -
Assimilation of potassium nitrate: Negative.
Growth on 50~o (w/w) glucose-yeast extract agar: Positive (very slow and
weak).
Maximum concentration of sodium chloride allowing stow and weak
growth: 10~
Growth in vitamin-free medmm: Absent.
Formation of starch-like compounds : Negative.
Growth at 37 C: Absent.
Mol ~ G + C: 49.2.
Etymology:
lunata
from the L. adj. lunatus (crescent, falcate); referring to
the shape of vegetative cells.
Type strain D-32 was isolated by A. I. Egorova from a flower of
Vicia
cracca
L. at Primorski Territory, U.S.S.R. Subcultures of this strain have been
deposited in the collections of 1) the Department of Soil Biology, the Faculty
of Soil Science, Moscow State University, Moscow, U.S.S.R. (MGU 1564);
2) the Institute of Biochemistry and Physiology of Microorganisms USSR
Academy of Sciences, Pushchino, Moscow region, U.S.S.R. (IBPhM Y-652);
3) the Institute of Microbiology USSR Academy of Sciences, Moscow,
U.S.S.R. (BKM Y-1651); 4) the Yeast Division of the Centraalbureau voor
Schimmelcultures, Delft, the Netherlands (CBS 5946); 5) the Northern
Utilization Research and Development Division, U.S. Department of Agricul-
ture, Peoria, Illionois, U.S.A. (NRRL Y-7131); 6) the Institute of Fermentation,
Osaka, Japan (IFO 1605) and 7) at the American Type Culture Collection,
Rockville, Maryland, U.S.A. (ATCC 22033).
METSCHNIKOWIA LUNATA SP. NOV.
321
DISCUSSION
Of the currently recognized species of Metschnikowia the species described
here is most like M. pulcherrima in chlamydospore and ascus morphology
and in G + C-content. The value of the latter for the type strain of M. pul-
cherrima is 48.3 mol~% (Meyer and Phaff, 1969). The striking feature of M.
tunata not exhibited by M. pulcherrima or any other species of this genus, is the
lunate shape of vegetative cells. Such shape of cells is a stable characteristic,
it persists despite this strain having been maintained for more than a decade
in laboratory conditions. It also effects chlamydospore morphology; on the
whole chlamydospores are globose or subglobose but they have often two small
pointed protrusions which have been the sharp ends of lunate vegetative cells
(Fig. 1). Sometimes these protrusions persist on the asci as well (Fig. 2b, e).
Because of the mode of ascus formation and ascospore morphology in the
studied yeast it would fit in the genus Metschnikowia. However, the current
diagnosis of this genus does not include organisms with lunate cells (Miller
and van Uden, 1970). Deviation in this single property can hardly be a reason
to establish a new ascogenous genus. Therefore the diagnosis of the genus
Metschnikowia is extended as follows:
"Cells spheroidal to ellipsoidal, also pyriform, cylindrical or lunate."
Contrary to M. pulcherrima the new species is assumed to be homothallic
as heat-treatment of ascosporic material according to the procedure devised
for the recovery of mating types yielded self-sporulating cultures. The conjuga-
tion was not observed. Using heat treatment, various media for sporulation
and various temperatures it was possible to increase chlamydospore and ascus
production but ascospore differentiation remained low. Most of the resulting
asci was abortive.
In their physiological properties all species of the genus Metschnikowia are
strikingly similar and in this respect M. lunata is no exception. The results of
the assimilation tests are in good agreement with those of Yarrow (1969). The
differences concern only citric acid and lactic acid. Yarrow reported that the
first acid was assimilated latently while the second was not assimilated at all.
The contradiction in the ability to ferment between Krassilnikov's descrip-
tion of S. intestinalis and the strain D-32 found by Yarrow may be due either
to different techniques or also to changes in the physiological patterns as the
result of prolonged maintenance of the latter strain in the laboratory. On the
other hand, the possibility cannot be excluded that the studied strain is the
representative of an other imperfect species although similar to S. intestinalis.
Krassilnikov's original strain of S. intestinalis has not been preserved and
so unfortunately no investigations on its physiological properties are possible.
Obviously the name Selenotila intestinalis Krassilnikov should be regarded as
a nomen dubium.
M. lunata appears to be a very rare species. All our repeated attempts to
322 W.I. GOLUBEV
isolate additional strains from various natural substrates were negative. The
present author is unaware of any reports, besides that mentioned in this paper,
of the isolation of such yeasts. It is not likely that these yeasts with their unique
cell shape would have been overlooked by investigators,
After the preparation of this paper for publication Dr. Irma P. Bab'eva and
Mr. D. Yarrow (the Yeast Division of the Centraalbureau voor Schimmel-
cultures, Delft) drew author's attention to the report of the reidentification of
Schizoblastosporion kobayasii
Soneda et Uchida (1971) as
Selenotita intestinatis
(Yarrow, 1976). The author thanks them for this reference and Dr. Bab'eva
for the information about the origin of studied yeast strain.
Received 9 June
1977
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L,). -- Izvestiya
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Branch of USSR Acad. Sci., Ser. Biol.), No. 10:127 132.
KRASSILNIKOV,
N. A. 1927. A new yeast-like fungus from intestine of butterfly
Brenthis
pales
Schift. -- Mikrobiologitscheskii jurnal (Microbiol. J.) 4: 134-136.
LAGERHEIM, G. 1892. Die Schneeflora des Pichinha.- Ber. Deut. Botan. Gesell. 10: 517-533.
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organisms.- J. Mol. Biol. 3: 208-218.
MEYER, S. A. and
PILAFF,
H. J. 1969. Deoxyribonucleic acid base composition in yeasts. --
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MILLER, M. W. and VAN UDEN, N. 1970.
Metschnikowia
Kamienski, p. 408-429.
In
J. Lodder,
(ed.), The Yeasts. A Taxonomic Study. -- North-Holland PuN, Co., Amsterdam.
SONEDA,
M. and UCmDA, S. 197t. A survey on the yeasts. -- Bull. Nat. Sci. Museum 14:
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VAN DER WALT, J. 1970. Criteria and methods used in classification, p. 34-113.
In
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Selenotita peltata
comb. n. ---- Antonie van Leeuwenhoek 35: 418-420.
YARROW, D. 1976.
In
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