International Journal of Systematic and Evolutionary Microbiology (2013), 63, 372–376
DOI 10.1099/ijs.0.045898-0
Yamadazyma terventina sp. nov., a yeast species of
the Yamadazyma clade from Italian olive oils
Gino Ciafardini,1 Biagi Angelo Zullo,1 Livio Antonielli,2 Laura Corte,2
Luca Roscini2 and Gianluigi Cardinali2
Correspondence
Gianluigi Cardinali
[email protected]
1
Department of Agriculture Food and Environmental Science, University of Molise,
Campobasso 86100, Italy
2
Department of Applied Biology – Microbiology, University of Perugia, Perugia 06123, Italy
During an investigation of olive oil microbiota, three yeast strains were found to be divergent from
currently classified yeast species according to the sequences of the D1/D2 domain of the gene
encoding the rRNA large subunit (LSU) and the internal transcribed spacer region including the
gene for 5.8S rRNA. Phylogenetic analysis revealed that these strains, designated CBS 12509,
CBS 12510T and CBS 12511, represent a novel anascosporogenous species described herein
as Yamadazyma terventina sp. nov; the type strain is DAPES 1924T (5CBS 12510T5NCAIM
Y.02028T). This novel species was placed in the Yamadazyma clade, with Yamadazyma scolyti,
Candida conglobata and Candida aaseri as closest relatives. Y. terventina differs from the abovementioned species in the ability to strongly assimilate DL-lactate and weakly assimilate ethanol.
INTRODUCTION
Olive oil is obtained from the complete crushing of the
fruits and from the successive separation of the oily
fraction through centrifugation. During the crushing of the
olives, micro-organisms present on the olives’ carposphere
migrate into the oil together with suspended material,
consisting of solid particles of fruit and micro-drops of
vegetation water. In the olive oil habitat, the microbiological profile is quite different to that of healthy olive fruit’s
microbiota. In the olive oil, some microbes from the olives’
carposphere succumb for a brief period whereas others,
primarily yeasts, reproduce in a selective way, according to
the chemical composition of the oil, and make up the
typical microbiota of the olive oil (Ciafardini et al., 2004).
In a recent study performed with chromogenic media,
poly-varieties of extra virgin olive oil had up to three
chromogenic yeast groups, while mono-varieties had no
more than one (Zullo & Ciafardini, 2008). Studies have
shown that some yeast species found in olive oil produce
enzymes that may affect the oil’s quality in both positive
and negative ways. The main yeast species isolated from the
Italian extra virgin olive oils are classified as Candida
wickerhamii, Candida boidinii, Candida diddensiae,
Candida guilliermondii, Barnettozyma (Williopsis) californica,
Abbreviations: ITS, internal transcribed spacer; LSU large subunit.
The GenBank/EMBL/DDBJ accession numbers for the ITS1, 5.8S, ITS2
and D1/D2 domains of the 26S rRNA gene sequences of CBS 12511,
CBS 12509 and CBS 12510T are JQ247718, JQ247716 and
JQ247717, respectively.
The MycoBank accession number for Yamadazyma terventina sp. nov. is
MB 800582.
372
Candida parapsilosis and Saccharomyces cerevisiae
(Ciafardini & Zullo, 2002; Ciafardini et al., 2006; Zullo
et al., 2010), while in a turbid Greek virgin olive oil
Candida lusitaniae, Candida famata and Rhodotorula
mucilaginosa (Koidis et al., 2008) were also observed.
Some of these yeast species are considered useful as they
improve the sensory characteristics of the oil during
preservation. In fact, some b-glucosidase- and esteraseproducing strains of C. wickerhamii and S. cerevisiae are
able to hydrolyse oleuropein into simpler, non-bitter
compounds characterized by a high antioxidant activity
(Ciafardini & Zullo, 2002). On the other hand, some
lipase-producing strains of B. californica or S. cerevisiae
can worsen olive oil quality through triglyceride hydrolysis. Cadez and co-workers (Cadez et al., 2012), recently
described two novel yeast species (Candida adriatica and
Candida molendinolei) isolated from olive oil and its byproducts. The present study describes a novel yeast
species represented by three strains isolated from extra
virgin olive oils from two different Italian regions.
METHODS
Yeast isolation. The yeasts studied were dimorphic forms isolated
from commercial extra virgin olive oils during a survey including 23
producers located in Italy as described previously (Zullo et al., 2010).
Three of these yeasts, designated DAPES (Collection of Department of
Animal, Plant and Environmental Sciences) 1916, DAPES 1924 and
DAPES 1931 in the previous study, could not be assigned to any known
yeast species on the basis of the D1/D2 sequence and are described as
representatives of a novel species in the present paper. The yeast strain
DAPES 1916 was isolated from a commercial extra virgin olive oil of
the ‘Taggiasca’ variety produced in Liguria (north Italy), whereas the
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Yamadazyma terventina sp. nov.
DAPES 1924 and DAPES 1931 strains were isolated from commercial
extra virgin olive oil of the ‘Leccino’ variety, produced in the Trivento
area (Molise region) located in the middle of Italy. The yeast strains
were grown 5 days at 30 uC in malt extract-yeast extract-glucosepeptone (MYGP) agar as described by Kurtzman et al. (2011c) and then
stored in 17 % glycerol at 280 uC before any other study.
NaOH (0.2 M aqueous solution). After 5 min of incubation at room
temperature, the yeast cells were pelleted as described previously, then
resuspended in 250 ml sample buffer (0.06 M Tris/HCl pH 6.8, 2 %
glycerol, 2.5 % SDS, 5 % b-mercaptoethanol, 0.003 g bromophenol
blue), vortexed for 1 min and boiled for 5 min. The treated cells were
centrifuged and 20 ml supernatant for each strain was taken for
electrophoretic analysis. Five microlitres of standard weight ladder were
loaded into the first well of a 10 % acrylamide mini-gel (Bio-Rad Mini
Protean cell), followed by the samples, loaded in the subsequent lanes,
and analyzed as described by Laemmli (1970).
Sporulation and physiological tests. Ascospore formation was
examined using McClary’s acetate agar and yeast extract-malt extract
(YM) agar; mating activity was examined using McClary’s acetate
agar. The cultures initially were grown for 5 days at 25 uC to induce
sporulation and then were observed daily for ascospores for 1 week.
The other physiological tests were carried out in accordance with the
methods described in the 5th edition of ‘The Yeasts: a Taxonomic
Study’ (Kurtzman et al., 2011a).
Molecular identification. Genomic DNA was extracted from a
representative of each single morphology (Bolano et al., 2001; Cardinali
et al., 2001). Amplification of the internal transcribed spacer (ITS)1,
5.8S, ITS2 and D1/D2 domains of the 26S rRNA gene was performed
using primer ITS5 (59-GGAAGTAAAAGTCGTAACAAGG) and LR6
(59-CGCCAGTTCTGCTTACC) as indicated by Boekhout et al. (2003)
with EuroTaq enzyme (EuroClone) and a PTC-100 Peltier Thermal
Cycler (MJ Research). Amplicons were purified with the GFX PCR
DNA purification kit (GE Healthcare) while the electrophoresis was
performed on 1.5 % agarose gel (Gellyphor; EuroClone). Amplicons
were sequenced using a ABI PRISM 3730xl with primer ITS1 (59TCCGTAGGTGAACCTGCGG) and NL4 (59-GGTCCGTGTTTCAAGACGG). Electropherogram trimming, contig assembly and BLAST
search were performed with Geneious 5 (Drummond et al., 2011).
Analysis of yeast proteins. Yeast proteins were studied using the
SDS-PAGE technique. The yeast proteins were extracted according to
the Kushnirov protocol (Kushnirov, 2000) with some modifications.
Briefly, yeast strains were grown on MYGP agar medium at 30 uC. After
5 days of growth, 0.12 g (w/w) yeast biomass was scraped off the agar
plate with a bacteriological loop and transferred to 10 ml pyrex tubes
with screw caps, containing 5 ml sterile distilled water. After 10 min of
incubation at room temperature (hereinafter RT), the cells were
centrifuged 5 min at 60006g (5491 r.p.m.) and resuspended in 5 ml
95
99
10
57
Yamadazyma nakazawae EU343867 - U45748 (CBS 6700T)
Yamadazyma philogaea AB054107 - U45765 (CBS 6696T)
85
60
Yamadazyma akitaensis DQ409164 - U45766 (CBS 6701T)
Candida dendronema HQ283365 - U45751 (CBS 6270T)
99
Candida diddensiae AY580315 - U45750 (CBS 2214T)
Candida tenuis HQ283371 - U45774 (CBS 615T)
51
Candida insectorum HQ283372 - U45791 (CBS 6213 T)
Candida aaseri AY821838 - U45802 (CBS 1913T)
53
84
Candida conglobata AJ539370 - U45789 (CBS 2018T)
Yamadazyma terventina JQ247718 (CBS 12511)
53
89
54
Yamadazyma terventina JQ247717 (CBS 12510T)
Yamadazyma scolyti EU343807 - U45788 (CBS 4802T)
Candida buinensis HQ283376 - U45778 (CBS 6796T)
65
95
Yamadazyma terventina JQ247716 (CBS 12509)
Candida friedrichii HQ283377 - U45781 (CBS 4114T)
99
81
Candida membranifaciens AJ606465 - U45792 (CBS 1952T)
Yamadazyma mexicana AB054110 - U45797 (CBS 7066T)
Candida atlantica AJ539368 - U45799 (CBS 5263T)
99
Candida atmosphaerica AJ539369 - U45779 (CBS 4547T)
Yamadazyma triangularis EU343869 - U45796 (CBS 4094T)
Schizosaccharomyces pombe AY046223 - U40085 (CBS 356T)
Fig. 1. Evolutionary relationships of taxa related to Y. terventina. Phylogram of the Yamadazyma clade showing the most
parsimonious tree based on the concatenation of the sequence from the D1/D2 region of the LSU rDNA and the ITS1, 5.8S
ribosomal gene, and ITS2 sequence. The relationships of strains CBS 12509, CBS 12510T and CBS 12511 were
reconstructed using the maximum-parsimony method set with the Subtree-Pruning-Regrafting (SPR) tree inference option.
Gaps and missing data were treated as complete deletion. The percentages of replicate trees in which the associated taxa
clustered together in the bootstrap test (1000 replicates) are shown next to the branches. Type strain sequences were retrieved
from GenBank and CBS databases (first number identifies ITS sequence, the second is the D1/D2 sequence). Nucleotide
sequences were concatenated and then aligned with MUSCLE (Edgar, 2004). Phylogenetic analyses were conducted in MEGA5
(Tamura et al., 2011). Schizosaccharomyces pombe was used as outgroup. Bar, number of changes over the whole sequence.
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373
G. Ciafardini and others
Table 1. Comparison of the assimilation and fermentation profile of selected substrates by species phylogenetically closely related to
Yamadazyma terventina
Strains: 1, Y. terventina CBS 12509; 2, Y. terventina CBS 12510T; 3, Y. terventina CBS 12511; 4, C. aaseri CBS 1913T; 5, C. insectorum CBS 6213T; 6, Y.
scolyti CBS 4802T; 7, C. friedrichii CBS 4114T; 8, C. conglobata CBS 2018T; 9, Y. mexicana CBS 7066T; 10, C. tenuis CBS 615T; 11, C. membranifaciens
CBS 1952T; 12, C. buinensis CBS 6796T; 13, Y. triangularis CBS 4094T. +, Positive; 2, negative; w, weak; d, delayed; ND, not determined. All
physiological data were retrieved from the CBS database (http://www.cbs.knaw.nl/collections/BioloMICS.aspx).
Characteristic
Assimilation of:
L-Sorbose
D-Glucosamine
D-Ribose
D-Arabinose
L-Rhamnose
Methyl a-D-glucoside
Melibiose
Lactose
Raffinose
Glycerol
DL-Lactate
Citrate
Ethanol
Ethylamine
Glucosamine
Growth at/in:
Cycloheximide (0.01 %)
37 uC
42 uC
Glucose fermentation
1
2
3
4
5
6
7
8
9
10
11
12
13
+
2
2
2
+
+,w
2
2
2
+,w
+
+
+,w
2
+
+
+,w
+,w
2
+
+,w
2
2
2
+
+
+
+,w
2
+
+
2
+,w
2
+
+
2
2
2
+,w
+
+
+,w
2
+
d
2
+
2
2
+, d, w
2
+
2
+
2
+
+
+
2
d
2, d, w
+
+, d, w
+
+
2
2, d, w
+, d, w
+
2
+
+
+
2
2
2
+
d
+
+
+
+
+
+
2
+
+
+
+
2
+
+
+
+
w
w
2
w
+
2
2
+
2
2
+
2
+
+, d, w
2
2
2
2
2
+
2
2
+
+
2
2
d
+
d
+
+
2, d, w
+
2, d, w
+
2
+
2
+
+
d
+
+
+
+
+
2
d
2
+
2
+
+
+
2
+
+
+
+
d
+
+
2
+
+
w
+
+
+
2
+
+
2, d, w
+
2
+
2
2
2
d
2
+
+
+
+
2
2
+
2
+
+
2
2
2
+
2
d
+
+
+
2
2
2
+
2
2
2
+
2
2
2
w
+
d
+
2
+
2
+
2
+
2
+
+
+
2
2
2
+
2
+
+
+
2
2
2
+
2
2
2
+
2
2
2
+
2
2
2
w, d
ND
ND
2
Sequence alignment was then carried out by Muscle (Edgar, 2004).
Phylogenetic analysis was conducted in MEGA5 (Tamura et al., 2011).
RESULTS
The large subunit (LSU; 26S) rRNA D1/D2 domain, ITS1,
5.8S rDNA and ITS2 sequences (see Fig. 1) of the abovementioned strains were previously compared in GenBank
by a BLAST search. The similar sequences, previously
identified, were included in the phylogenetic analysis,
which confirmed the currently accepted Yamadazyma clade
composition and structure (Groenewald et al., 2011;
Kurtzman & Suzuki, 2010; Kurtzman et al., 2011b).
According to the phylogenetic reconstruction, the novel
species proposed herein was placed in a well-supported
clade, including members of the genera Yamadazyma and
Candida, globally named the Yamadazyma clade (Fig. 1).
Due to the collocation of the novel species in the
Yamadazyma clade, the novel species has been named
Yamadazyma terventina and not Candida terventina,
according to the ‘one fungus – one name’ recommendation
(Hawksworth, 2011). The closest relatives were Candida
conglobata (42 substitutions equivalent to 3.33 % difference), Candida aaseri (50 substitutions equivalent to
3.97 % difference), a synonym of the butter contaminant
Candida butyri (Pfüller et al., 2011) isolated from butter
and at low frequency in cases of mastitis (Spanamberg et al.,
2008) and Yamadazyma scolyti (61 substitutions equivalent
374
Fig. 2. Light microscopic morphology of cells of Y. terventina CBS
12509, CBS 12510T and CBS 12511. (a) Budding cells; (b)
pseudomycelia. Bars, 5 mm.
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Yamadazyma terventina sp. nov.
to 4.84 % difference). Members of the clade rather distant
to the novel species were Yamadazyma triangularis, a yeast
of clinical relevance, isolated in lung tissue and considered
as a potential opportunistic pathogen (99 substitutions,
equivalent to 7.86 % difference) (Kurtzman et al., 2011a;
Smith & Batenburg-Van der Vegte, 1986) and Yamadazyma
philogaea (73 substitutions, equivalent to 5.79 % difference) (Robiglio et al., 2011; van der Walt & Johannsen,
1975).
The three yeast strains, derived from different extra virgin
olive oils produced both in the north and in the middle of
Italy, were characterized by identical ITS and D1/D2
sequences, but could be differentiated by some physiological traits independently from their geographical origin.
In fact, D-glucosamine was assimilated only by the strain
CBS 12510T, whereas D-ribose was used by CBS 12510T and
CBS 12511 (Table 1). Another difference was that the type
strain could form pseudomycelium on YEPDA (1 % yeast
extract, 1 % peptone, 2 % glucose, 1.8 % agar; Difco),
Dalmau plates and MYGP agar-olive oil medium (O/N
growth), whereas the other two strains produced pseudomycelium only on Dalmau culture or MYGP agar-olive oil
medium after 15 days incubation. It was the presence of
this more complex morphology that induced us to
designate CBS 12510T as type strain. The electrophoretic
profiles of the yeast proteins (SDS-PAGE) extracted from
the three yeast strains showed the same pattern in a range
between 30 and 90 kDa (data not shown), further
supporting the fact that the three strains belong to the
same species. The data reported above indicate that this
species presents some degree of phenotypic variability in
spite of the identity of the three molecular markers that are
currently widely used to differentiate yeasts at the species
level. However, the assimilation and fermentation profiles
of the proposed species differ for several traits from those
of the closest relatives of the clade (Table 1).
Y. terventina does not assimilate D-arabinose (also found
for C. aaseri and Y. triangularis) or ethylamine (also found
for C. friedrichii); the strains give instead variable responses
for the assimilation of D-ribose, which is actively used by
almost all closely related species. Furthermore, Y. terventina
is the only species to assimilate DL-lactate actively.
Description of Yamadazyma terventina
Ciafardini, Zullo, Antonielli, Corte, Roscini &
Cardinali sp. nov.
Yamadazyma terventina (ter.ven9ti.na. N. L. fem. adj.
terventina of or pertaining to Terventum, referring to the
Latin name of the town in the Molise region, middle of
Italy, where the yeasts have been isolated for the first time).
After growth in YM broth at 25 uC for 3 days, the cells are
ellipsoidal (2–464–6 mm) and occur singly or in pairs (Fig.
2a). Asexual reproduction occurs by multilateral budding.
Sediment is not present. After 5 days at 25 uC on YM agar,
streak cultures present round colonies with both sharp and
jagged edges, and a colour varying from glossy to matt white.
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On Dalmau slide cultures with cornmeal agar or rice extract
agar after 5 days at 25 uC, pseudomycelium is formed under
the cover glass and without cover glass (Fig. 2b). Sporulation
does not occur on McClary’s acetate agar and YM agar at
17 uC and 25 uC after 10 days. Glucose is fermented.
Glucose, galactose, L-sorbose, sucrose, cellobiose, a,atrehalose, melezitose, maltose, D-xylose, L-arabinose, Lrhamnose, erythritol, adonitol, D-mannitol, D-sorbitol,
salicin, D-glucono-1,5-lactone, DL-lactate, succinate, citrate,
D-glucosamine, L-lysine, malic acid and N-acetylglucosamine are assimilated. Other carbon compounds tested in
this study, including, D-ribose, ethanol, glycerol, methyl a-Dglucoside and 2-keto-D-gluconate are weakly assimilated. DGlucuronate, myo-inositol, raffinose, D-arabinose, melibiose, lactose, inulin, starch, dulcitol, methanol, nitrate,
nitrite and ethylamine are not assimilated. Growth on 50 %
glucose, 10 % NaCl and 12.5 % NaCl is negative. Growth
occurs on 5 % NaCl, but not in the presence of 0.1 p.p.m.
cycloheximide. Growth occurs at 25 uC but not at 4, 37 or
42 uC. No starch-like substance is produced. Urea hydrolysis
and the Diazonium blue B reaction are negative. Lipase
activity is positive. Proteinase activity verified on skim milk
agar and on growth medium supplemented with azocasein;
in the former all three strains show a positive reaction, while
only the type strain shows proteolytic activity in the latter.
The cultures of the three strains representing the novel
species in this study did not show any mating activity when
mixed in pairs.
The type strain, DAPES 1924T (5CBS 12510T5NCAIM
Y.02028T), was isolated from an extra virgin olive oil
produced in the ‘Trivento’ area (Molise region) located in
the middle of Italy. MycoBank no. is MB 800582. Two
further reference strains of Y. terventina are DAPES 1916
(5CBS 125095NCAIM Y.02027) and DAPES 1931 (5CBS
125115NCAIM Y.02029).
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