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Isolation and characterization of yeasts


associated with plants growing in heavy metal-
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Article in Canadian Journal of Microbiology December 2015


DOI: 10.1139/cjm-2015-0226

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1

ARTICLE
Isolation and characterization of yeasts associated with
plants growing in heavy-metal- and arsenic-contaminated
soils
Juan Ramos-Garza, Rafael Bustamante-Brito, Gabriela ngeles de Paz,
Ma. Gabriela Medina-Canales, Mara Soledad Vsquez-Murrieta, En Tao Wang,
Can. J. Microbiol. Downloaded from www.nrcresearchpress.com by 148.204.163.40 on 03/03/16

and Ada Vernica Rodrguez-Tovar

Abstract: Yeasts were quantied and isolated from the rhizospheres of 5 plant species grown at 2 sites of a
Mexican region contaminated with arsenic, lead, and other heavy metals. Yeast abundance was about 102 CFU/g of
soil and 31 isolates were obtained. On the basis of the phylogenetic analysis of 26S rRNA and internal transcribed
spacer fragment, 6 species were identied within the following 5 genera: Cryptococcus (80.64%), Rhodotorula (6.45%),
Exophiala (6.45%), Trichosporon (3.22%), and Cystobasidium (3.22%). Cryptococcus spp. was the predominant group.
Pectinases (51.6%), proteases (51.6%), and xylanases (41.9%) were the enzymes most common, while poor production
of siderophores (16.1%) and indole acetic acid (9.67%) was detected. Isolates of Rhodotorula mucilaginosa and
Cystobasidium slofae could promote plant growth and seed germination in a bioassay using Brassica juncea.
Resistance of isolates by arsenic and heavy metals was as follows: As3+ 100 mmol/L, As5+ 30 mmol/L, Zn2+
For personal use only.

2 mmol/L, Pb2+ 1.2 mmol/L, and Cu2+ 0.5 mmol/L. Strains of Cryptococcus albidus were able to reduce arsenate
(As5+) into arsenite (As3+), but no isolate was capable of oxidizing As3+. This is the rst study on the abundance and
identication of rhizosphere yeasts in a heavy-metal- and arsenic-contaminated soil, and of the reduction of
arsenate by the species C. albidus.
Key words: yeast, rhizosphere, reduction, arsenic, speciation.
Rsum : On a dnombr et isol des levures de rhizosphres de 5 espces de plantes cultives a` deux emplace-
ments dune zone mexicaine contamine a` larsenic, au plomb et a` dautres mtaux lourds. Labondance des
levures tait denviron 102 UFC/g de sol et on a obtenu 31 isolats. En vertu dune analyse phylogntique de
lARNr 26S et du fragment ITS, on a identi 6 espces appartenant a` 5 genres, a` savoir Cryptococcus (80,64 %),
Rhodotorula (6,45 %), Exophiala (6,45 %), Trichosporon (3,22 %), et Cystobasidium (3,22 %). Cryptococcus spp. tait le groupe
prdominant. Les pectinases (51,6 %), les protases (51,6 %), et les xylanases (41,9 %) taient les enzymes les plus
communes, tandis quon a dtect une faible production de sidrophores (16,1 %) et dacide indole-actique (9,67 %).
Les isolats de Rhodotorula mucilaginosa et de Cystobasidium slofae pouvaient favoriser la croissance vgtale et la
germination des semences dans un bioessai employant Brassica juncea. La rsistance des isolats a` lAs et aux mtaux
lourds tait comme suit : As3+ 100 mmol/L, As5+ 30 mmol/L, Zn2+ 2 mmol/L, Pb2+ 1,2 mmol/L, et Cu2+
0,5 mmol/L. Les souches de Cryptococcus albidus sont parvenues a` rduire larsniate (As5+) en arsnite (As3+), mais
aucun isolat na su oxyder larsnite. Il sagit de la premire tude traitant de labondance et de lidentication de
levures de la rhizosphre provenant dun sol contamin aux mtaux lourds et a` larsenic, et de la rduction de
larsnite par lespce C. albidus. [Traduit par la Rdaction]
Mots-cls : levure, rhizosphre, rduction, arsenic, spciation.

Received 12 April 2015. Revision received 24 November 2015. Accepted 14 December 2015.
J. Ramos-Garza. Laboratorio de Micologa General y Mdica, Departamento de Microbiologa, Escuela Nacional de Ciencias Biolgicas
(ENCB), Instituto Politcnico Nacional (IPN), Prolongacin de Carpio y Plan de Ayala s/n, 11340 Mexico City, Mexico; Laboratorio de
Ecologa Microbiana, Departamento de Microbiologa, ENCB, IPN, 11340 Mexico City, Mexico.
R. Bustamante-Brito and A.V. Rodrguez-Tovar. Laboratorio de Micologa General y Mdica, Departamento de Microbiologa,
Escuela Nacional de Ciencias Biolgicas (ENCB), Instituto Politcnico Nacional (IPN), Prolongacin de Carpio y Plan de Ayala s/n,
11340 Mexico City, Mexico.
G. ngeles de Paz and M.G. Medina-Canales. Laboratorio de Nematologa Agrcola, Departamento de Parasitologa, ENCB, IPN,
11340 Mexico City, Mexico.
M.S. Vsquez-Murrieta. Laboratorio de Microbiologa Industrial, Departamento de Microbiologa, ENCB, IPN, 11340 Mexico City,
Mexico.
E.T. Wang. Laboratorio de Ecologa Microbiana, Departamento de Microbiologa, ENCB, IPN, 11340 Mexico City, Mexico.
Corresponding author: Ada Vernica Rodrguez-Tovar (email: avrodriguez@hotmail.com).

Can. J. Microbiol. 62: 113 (2016) dx.doi.org/10.1139/cjm-2015-0226 Published at www.nrcresearchpress.com/cjm on 21 December 2015.
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2 Can. J. Microbiol. Vol. 62, 2016

Introduction San Luis Potos, Mexico, a traditional mining region with


Arsenic (As) is a common toxic contaminant produced mine tailings, soils, and plants contaminated with high
by industry extraction or anthropogenic input in nature, concentrations of As and HM (Franco-Hernndez et al.
but it is also released as a consequence of rock weather- 2010). To date, no study on microbial diversity and the
ing or volcanic explosions (Donahoe-Christiansen et al. HM and As detoxication potential in this region has
2004; Wang and Mulligan 2006). In different environ- been reported.
ments, As is present as inorganic (As5+ and As3+) or or-
ganic (monomethylarsonic acid, dimethylarsinic acid, Materials and methods
and trimethylarsine oxide) forms, which can be trans- Sampling sites
formed by microbial activities, such as oxidation The soil sampling sites are located in Villa de la Paz in
reduction and methylationdemethylation (Turpeinen the State of San Luis Potos (lat 23.7, long 178.7), a zone
et al. 2002).
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with a mean annual temperature of 18 C and an average


Many bacteria, archaea, and fungi have developed annual precipitation of 486 mm. The 2 sampling sites are
mechanisms for avoiding As toxicity, including the fol- (i) a mine with an altitude of 1557 m above sea level and
lowing: (i) reduction of arsenate (As5+) to arsenite (As3+)
(ii) a natural hill with an altitude of 1830 m above sea
and expelling the latter outside of the cells (Cernansk
level, with a distance of about 5 km between them. In a
et al. 2009); (ii) As complexation with other molecules
previous study, it was reported that this soil is contami-
inside the cells, such as glutathione and vacuolar seques-
nated with risk elements (Franco-Hernndez et al. 2010).
tration (Rosen 2002); (iii) methylation of As to the less
toxic organic forms (Qin et al. 2006); (iv) oxidation of Soil analysis revealed the following metal concen-
arsenite to the less toxic arsenate (Gihring and Baneld trations (mg/kg): in mine tailings As > 3574, lead
2001); and (v) metalloid immobilization by absorption (Pb) > 555.45, copper (Cu) > 2012, and zinc (Zn) > 1337; at
and accumulation in biomass (Wang and Zhao 2009). the hill site As > 1071.50, Pb > 5478.60, Cu > 749.30,
Similar to other microorganisms in the soil and rhizo- and Zn > 1672.85 (data are shown in supplementary
For personal use only.

sphere, yeasts play an important ecological role, such as Table S11). In this work, the physicochemical parameters of
recycling nutriments, aggregating soil particles, assimi- the mine site and the hill site were analyzed using the
lating secondary products from bacteria and other fungi, methods described by Vsquez-Murrieta et al. (2006).
exhibiting different interactions (amensalism, predation,
and competition) with other microorganisms and plants, Plant identication and soil sampling characterization
and possessing versatile metabolisms for utilizing and At both sites, 4 individual plants of the most abundant
transforming nitrogen compounds (Botha 2011). Some plant species were sampled randomly by extracting
yeasts, such as Trichosporon spp., Rhodotorula spp., and plants with their roots together with their soils. The sam-
Candida tropicalis, are known for their ability to resist and ples were maintained in plastic bags, transported imme-
accumulate As and heavy metals (HMs) (Olasupo et al. diately to the laboratory, and stored thereafter at 4 C.
1993; Ilyas et al. 2014; Ilyas and Rehman 2015). The All of the sampled plants were mature and appeared
biotransformation of As is well studied in the yeast healthy without the presence of parasites. The plants
Saccharomyces cerevisiae, and its related pathways and the were identied by botanists in the Plant Ecology Labora-
genes and mutants involved have been characterized tory of the National School of Biological Science of the
(Menezes et al. 2004; Shah et al. 2010; Todorova et al. National Polytechnic Institute in Mexico City, on the
2010). Other yeasts, such as Schizosaccharomyces pombe, basis of typical morphology and taxonomic features
Trichosporon spp., Rhodotorula spp., and Cryptococcus depending on the plant species (McVaugh 1984, 1987;
humicolus have been studied in terms of the accumula-
Caldern de Rzedowski and Rzedowski 2001). Rhizo-
tion, biosorption, and quantication of the oxidation
sphere soils were prepared by brushing off the soils ad-
and reduction process (Button et al. 1973; Salgado et al.
hering to the root surface of the plants, as previously
2012; Ilyas et al. 2014). However, no information exists
described (Trujillo-Cabrera et al. 2013), and these were
about the yeast population associated with HM- and As-
resistant plants or their characterization. used for yeast isolation. Soils sampled together with the
The aim of this work was to evaluate the diversity of plants of each species at the same site were compiled in
rhizospheric yeasts associated with HM- and As-resistant the same volume, which was then employed for analysis
plants and to evaluate their ability to biotransform As. To of the contents of HM, As, and nutrients, as well as
accomplish this, we isolated and characterized the rhi- the physicochemical features, as reported previously
zosphere yeasts associated with 5 plant species grown at (Vsquez-Murrieta et al. 2006; Franco-Hernndez et al.
2 sites in the small town of Villa de la Paz, in the State of 2010).

1Supplementary data are available with the article through the journal Web site at http://nrcresearchpress.com/doi/suppl/10.1139/cjm-
2015-0226.

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Ramos-Garza et al. 3

Yeast isolation nlm.nih.gov/Blast.cgi) were used to extract the sequences


For isolation of yeasts, 0.5 g of rhizospheric soil from for related taxa and were utilized as references for the
each plant species was suspended in 4.5 mL of sterile subsequent phylogenetic analysis. All of the sequences
saline solution (0.85% NaCl). Serial dilutions were pre- obtained in this study were manually edited with the
pared up to 103, and an aliquot of 0.1 mL from each reference alignment editor BIOEDIT and aligned together
dilution was spread on the following media in Petri dishes: with the reference sequences using CLUSTAL X ver-
yeastpeptonedextrose (YPD) agar (yeast extract, 10 g; sion 1.7 software (Thompson et al. 1997). The phylo-
peptone, 20 g; dextrose, 20 g; agar, 15 g; distilled water, genetic tree was constructed by the maximum-likelihood
1 L; supplemented with streptomycin, 20 mg) and Rose method (Guindon and Gascuel 2003) using the best models:
Bengal agar (Difco) (chloramphenicol, 0.1 g; MgSO47H2O, Kimura 2-parameter + I + G for 26S rRNA, and Tamura
0.5 g; KH2PO4,1.0 g; dextrose, 10 g; Rose Bengal, 0.05 g; soy 3-parameter + I + G for ITS fragments, selected by using
peptone, 5.0 g; agar, 15 g; and distilled water, 1 L). The jModelTest version 3.06 software, based on the Akaike
Can. J. Microbiol. Downloaded from www.nrcresearchpress.com by 148.204.163.40 on 03/03/16

inoculated Petri dishes were incubated at 28 C for Information Criterion (Posada 2008). Statistical valida-
7 days. Single colonies were selected according to their tion at each node was determined by 1000 bootstrap
morphology and were puried by repeated cross-striking replicates. A zygomycete, Rhizopus microsporum, was em-
on YPD plates until all of the colonies in the same isolate ployed as an outgroup for tree rooting.
presented similar morphology (Al-gabr et al. 2014). Purity
of the isolates was veried microscopically, and the Determination of enzymatic activities
pure isolates were conserved at 70 C in microtubes The production of the following enzymes was ana-
(1.5 mL) half lled with YPD broth supplied with glycerol lyzed: amylase, pectinase, xylanase, cellulase, and pro-
(50%, m/v). tease. Enzymatic activities were performed by initially
growing the isolates in YPD broth for 24 h at 28 C. Af-
Amplication and sequencing of the 26S rRNA gene and terward, 100 L aliquots were inoculated on the specic
ITS fragment of yeasts
Genomic DNA extracted from each yeast isolate using culture media for the investigation of each enzyme. The
For personal use only.

the protocols of Allers and Lichten (2000) was used as plates were incubated at 28 C for 72 h. Screening for
template to amplify the 26S rRNA and ITS fragments by amylases and pectinases was performed in Castaeda
PCR with a thermocycler (MaxyGene Thermal Cycler medium (Castaeda-Agullo 1956), to which 2% (m/v)
THERM 1061; AxyGen Scientic), using the following pro- starch or 1% (m/v) pectin was added, correspondingly. For
tocol: an initial 10 min denaturing step at 94 C, followed xylanase and cellulase screening, the Congo red medium
by 30 cycles of 1 min at 94 C, 1 min of annealing at 54 C, was used (Hankin and Anagnostakis 1977; Suto et al.
1 min extension at 72 C, and a nal 8 min polymeriza- 2002). For protease detection, skim-milk medium was
tion step at 72 C. The PCR mixture (50 L) contained utilized (Atlas 1997). Positive amylase activity was re-
50100 ng of DNA template, 3 mmol/L MgCl2, 2.5 U vealed by the presence of a clear zone around a colony
of Taq DNA polymerase (Invitrogen, USA), 1 PCR after immersing the yeasts colonies in Lugol solution.
buffer, 20 pmol of each primer for 26S rRNA (NL1 Positive pectinase activity was recognized by the presence
5=-GCATATCAATAAGCGGAGGAAAAG-3=; NL4 5=- of a clear ring around a colony following the immersion in
GGTCCGTGTTTCAAGACGG-3= (Redecker 2000)) and the 5% (m/v) CTAB solution (cetyl trimethyl ammonium bro-
ITS fragment (ITS1 5=-TCCCGTAGGTGACCTGCGG-3=; mide). Positive degradation of cellulose, xylan, or casein
ITS4 5=-TCCTCCGCTTATTGATATGC-3=) under the was identied in the corresponding medium by the pres-
same thermal condition (Gardes and Bruns 1993), and ence of a clear ring around a colony. The enzymatic index
200 mol/L each dNTP. Amplication products were vi- was determined within 72 h of incubation, which was
sualized after electrophoresis in agarose gel (1%, m/v) in expressed as the relationship between the average diam-
0.5 TAE buffer by staining with an aqueous solution of eter of the transparent (degradation) ring and the aver-
ethidium bromide (0.5 g/mL). A PureLink 310002 com- age diameter of the colony (Hankin and Anagnostakis
mercial kit (Invitrogen) was utilized for purifying the 1975).
PCR products, which were then sequenced under Big Dye
Determination of the potential of plant-growth-
terminator cycling conditions with the same primers,
promoting traits for isolates
using the Automatic Sequencer 3730XL in Macrogen Phosphate solubilization capacity was evaluated with
(Korea). Pikovskaya medium (Paul and Sundara Rao 1971) and was
Phylogenetic analysis considered positive if a clear ring appeared around the
The D1/D2 regions of the 26S rRNA sequences obtained colony. Polyamine production was detected using the
and the ITS fragment were employed to estimate the Long Asthon decarboxylase medium (Arena and Manca
taxonomic afliation and genotype designations of the de Nadra 2001), with a change in medium color from
yeast isolates. BLASTn searches for 26S rRNA and ITS se- yellow to red considered as a positive test. Siderophore
quence data in the National Center for Biotechnology production was evaluated according to Schwyn and
Information (NCBI) GenBank database (http://blast.ncbi. Neilands (1987) using Cromo-Azurol S medium, with a

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4 Can. J. Microbiol. Vol. 62, 2016

change in medium color to yellow, orange, or purple imal medium (MBMM) proposed by Rathnayake et al.
around the colony considered as a positive test. Indole (2013). HM and As were supplemented at concentrations
acetic acid (IAA) production was evaluated in YPD broth of 0.05, 0.1, 2.0, 4.0, 10, 20, 30, 40, 50, 60, 70, 80, 90, and
according to Nassar et al. (2005) modied by Limtong and 100 mmol/L in the forms of CuSO4, ZnSO4, Pb(NO3)2,
Koowadjanakul (2012); the appearance of a pink color NaH2AsO4, and NaAsO2. Inoculation and incubation con-
within 30 min after treatment with Salkowski reagent ditions were the same as those for resistance to salinity.
was recorded as a positive test.
Oxidation and reduction of As compounds by the isolates
Quantication of IAA The ability of the yeast isolates to oxidize the arsenite
Quantication of the IAA produced by the yeast isolates or reduce the arsenate was estimated by utilizing the
was carried out using the Salkowski method (Glickmann KMnO4 qualitative test (Salmassi et al. 2002). The yeasts
and Dessaux 1995). One pre-inoculum of each yeast was were cultured in liquid Chemically Dened Medium
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prepared in YPD at 28 C for 24 h. One millilitre of this (Weeger et al. 1999) medium containing NaH2AsO4 or
culture was employed to inoculate a second ask with NaAsO2 at a nal concentration of 100 mg/L with an ini-
YPD medium and was incubated at 28 C with shaking at tial optical density of 0.4 at 600 nm. The culture was
150 rev/min, until reaching about 108 cells/mL, at which incubated for 5 days with shaking (120 rev/min). Then,
time tryptophan 1% (Sigma) was added. Aliquots (1 mL) 20 L of KMnO4 solution (0.01 mol/L) was added to 1 mL of
were taken every 24 h; these were placed in an Eppen- the culture. The generation of a pink color in the broth
dorf tube and centrifuged at 1500 rev/min (151g) for indicates positivity for arsenite oxidation, whereas a yel-
5 min. The supernatant was transferred into a new tube, low color indicates arsenate reduction. The isolates pos-
and the same amount of Salkowski reagent was added to itive in the qualitative test were evaluated quantitatively
this (12 g/L FeCl3, 7.9 mol/L H2SO4), the reaction was left with the molybdenum blue method (Hu et al. 2012).
for 45 min at 28 C under conditions of darkness, and the
reaction was read in a spectrophotometer at 540 nm. The Results
quantity of IAA was determined using a standard curve
For personal use only.

Plant identication and soil characterization


with a range of concentrations of 1, 2, 3, 5, 6, 8, 9, 10, 15, A total of 5 plant species were collected in the region
and 20 g/mL. The standard curve was prepared using a of Villa de La Paz. At the mine tailing site, the plants
stock solution of IAA (JT Baker) with a nal concentra- Sphaeralcea angustifolia, Prosopis spp., Flaveria angustifolia,
tion of 1 mg/mL, effecting proper dilutions in YPD me- and Tithonia diversifolia were the most abundant, while
dium. Each determination was carried out in triplicate. Bahia absinthifolia, Sphaeralcea angustifolia, and Prosopis
Statistical analysis by means of 2-way analysis of variance spp. comprised the main plants on the hill. Soils at both
(ANOVA) was conducted using the Duncan multiple sampling sites were seriously contaminated by As and
range test, with p value of <0.05 indicating a signicant multiple HMs, especially As, Cd, Cu, Mn, Pb, and Zn,
difference. which presented at concentrations (mg/kg) of 3574, 129,
2012, 1382, 555, and 1337 in the mine tailings, and 1071,
Effect of IAA produced by rhizospheric yeast on Brassica juncea
seed germination 62, 749, 3054, 5488, and 1672 on the hill, respectively
Five hundred microlitres of sterile, ltered superna- (detailed information available in supplementary
tants from the cultures described previously and con- Table S11). In addition, greater values related to nutrient
taining 4 g of IAA were allowed to be absorbed onto features were observed in the hill soil, such as cation
Petri dishes containing water agar. Ten seeds of B. juncea exchange capacity, organic carbon, total nitrogen, and
disinfected with 2% sodium hypochlorite were placed in total phosphorous, were 77 cmolc/kg, 7.7%, 0.32 mg/kg,
each Petri dish. The negative control had 10 seeds per and 69 mg/kg, respectively, while the corresponding
Petri dishes without IAA. All Petri dishes were incubated values for the mine tailings were 38 cmolc/kg, 1.9%,
at 28 C and germination percentage was determined at 0.09 mg/kg, and 33 mg/kg.
24 and 48 h. All assays were performed in triplicate.
Yeast isolation and identication
KruskalWallis statistical analysis was performed to
During the isolations, we found that the abundance of
evaluate the germination percentage and the one-way
yeasts in rhizosphere soils was around 200 CFU/g of soil.
ANOVA was conducted to evaluate plant growth. The
In total, 31 rhizospheric yeasts representing different col-
means of each experiment were compared using the
ony morphology were isolated from the sampled plants
Duncan multiple range test. All analyses were carried out
(Table 1). The D1/D2 fragment of the 26S rRNA gene was
using SigmaPlot version 12.0 statistical software (2006;
amplied from all of the isolates, with the expected size
Systat Software, Inc.), and we considered p < 0.05 accept-
of 600 bp. Additionally, the ITS fragment was amplied
able for statistical signicance.
with the same expected size. The nucleotide sequences
Resistance of the isolates to HM and As acquired in this study have been deposited in GenBank
Minimum inhibitory concentration (MIC) was evalu- under accession Nos. KP455389KP455419 for 26S rRNA
ated using resistance to HM and As in MES-buffered min- and KT716400KT716430 for the ITS fragment.

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Ramos-Garza et al. 5

Table 1. Yeast isolates with their relevant information and enzymatic and plant-growth-promoting
activities.
Index of activity for:* Production
Strain IAA
No. Taxonomic afliation Cellulases Xylanases Pectinases Proteases Siderophore (g/mL)
From Tithonia diversifolia in mine tailings
YR1 Cryptococcus albidus ND ND ND ND ND
YR2 Cryptococcus albidus ND ND 2 1 ND
YR3 Cryptococcus albidus ND ND ND 1.33 ND
YR4 Cryptococcus albidus ND ND ND 2 ND
YR5 Cryptococcus albidus ND ND 1 ND ND
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From Flaveria angustifolia in mine tailings


YR6 Cryptococcus albidus ND ND 1.4 1.38 ND
YR7 Cryptococcus albidus ND ND ND ND 1.83
YR8 Cryptococcus albidus ND 1.5 3.91 1.43 ND
YR9 Cryptococcus albidus ND 1.5 2.48 1.52 ND
YR10 Cryptococcus albidus ND 1.18 3.18 1.38 ND
YR11 Cystobasidium slofae ND 1.2 ND ND ND 6.8
From Sphaeralcea angustifolia in mine tailings
YR12 Cryptococcus albidus ND ND ND ND 1
YR13 Cryptococcus albidus ND ND 1.66 1.77 ND
YR14 Cryptococcus uzbekistanensis ND ND 1.29 ND ND
From Prosopis sp. in mine tailings
YR15 Cryptococcus albidus ND ND ND ND ND
For personal use only.

YR16 Cryptococcus albidus ND ND ND ND ND


YR17 Cryptococcus albidus ND ND ND ND ND
YR18 Cryptococcus albidus ND ND ND 1.94 ND
YR19 Cryptococcus albidus ND ND ND 1.52 ND
YR20 Trichosporon sp. ND 1.52 ND ND 1.34
From Bahia absinthifolia at hill site
YR21 Cryptococcus albidus ND 1.5 2.16 1.88 ND
YR22 Cryptococcus albidus ND 2 1.66 1.33 ND
From Sphaeralcea angustifolia at hill site
YR23 Cryptococcus albidus ND ND ND ND ND
YR24 Rhodotorula mucilaginosa 1.5 2.11 3.75 ND 2.53 9.02
YR25 Cryptococcus albidus ND 1.4 3.16 1.63 ND
YR26 Cryptococcus albidus ND 1.63 2.33 2.16 ND
YR27 Cryptococcus albidus ND 1.5 4.66 1 ND
YR28 Cryptococcus albidus ND ND 2.16 2.22 ND
From Prosopis sp. at hill site
YR29 Rhodotorula mucilaginosa 1.75 ND 3.2 ND 1 9.61
YR30 Exophiala pisciphila 1.97 3.13 ND ND 1
YR31 Exophiala pisciphila 2.16 2.38 ND ND ND
Proportion (%) of positive isolates 12.9 41.9 51.6 51.6 16.1
No. of positive isolates 4 13 16 16 5 3
Note: ND, not detected; IAA, indole-3-acetic acid.
*Index of rhizospheric yeast.

Phylogenetic analyses of the D1/D2 sequences grouped YR31, presented 100% coverage and 97% identity with
the isolates within the phyla Basidiomycota (29 isolates) Exophiala pisciphila. Twenty-four isolates were identied as
and Ascomycota (2 isolates), with 25 isolates belonging to Cryptococcus spp. related to the species Cryptococcus albidus,
the genus Cryptococcus (80.64%), 2 to Rhodotorula (6.45%), Cryptococcus kuetzingii, and Cryptococcus adeliensis; YR14 was
2 to Exophiala (6.45%), 1 to Trichosporon (3.22%), and 1 to designated as Cryptococcus uzbekistanensis; isolate YR11 was
Cystobasidium (3.22%) (Fig. 1a). The majority of the isolates identied as Cystobasidium slofae; and nally, isolate YR20
exhibited high sequence coverage and identity (100% and was designated as Trichosporon spp. and was related to the
99%100%) with the dened species (data available as group Trichosporon japonicum, Trichosporon coremiiforme,
supplementary Table S21). Only 2 isolates, YR30 and and Trichosporon insectorum.

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6 Can. J. Microbiol. Vol. 62, 2016

Fig. 1. Maximum-likelihood phylogenetic trees showing the taxonomic afliation of the rhizospheric yeasts isolated from
2 soils seriously contaminated by heavy metals and arsenic. (a) Tree was constructed with a Kimura 2-parameter + I + G model
with the sequences of the 26S rRNA D1/D2 domain. (b) Tree was constructed with a Tamura 3-parameter + I + G model with
sequences of the ITS fragment. Bootstrap values >50% are presented alongside the branch. Isolate numbers are presented in
boldface and sequence accession numbers are indicated in parentheses.
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For personal use only.

The ITS sequences demonstrated the following infor- selected for IAA quantication. Polyamine production and
mation. The Cryptococcus genus was the most common phosphate solubilization were absent in all isolates.
and was represented mainly by the species C. albidus and
C. uzbekistanensis (25 isolates exhibited an identity range IAA quantication of the 2 isolated yeasts
Three yeasts were capable of producing IAA: 2 strains
of 99%100%). The genus Rhodotorula mucilaginosa was
of R. mucilaginosa (YR29 and YR24), producing auxin con-
represented by 3 isolates (YR24, YR29, and YR11). The
Exophiala genus was represented by isolates belonging to centrations of 9.61 and 9.02 mg/mL in 7 days, respec-
the species Exophiala capensis with low identity of 87%. tively, and 1 strain of C. slofae (YR11) producing only
Finally, isolate YR20 was again designated as Trichosporon, 6.8 mg/mL, with IAA production differing signicantly
related to the group T. japonicum, T. insectorum, Trichosporon among strains and among incubation times (p < 0.05). In
faecale, and Trichosporon asteroides, as depicted in Fig. 1b. addition, the concentration of IAA increased as time
passed from incubation (Fig. 2).
Enzymatic activity and potential plant-growth-promoting
features of rhizospheric yeast isolates Germination promotion of B. juncea seed by IAA secreted by
Results of the enzymatic characterization demon- rhizospheric yeasts
strated that 51.6% (16/31), 51.6% (16/31), 41.9% (13/31), 12.9% A signicant increase was observed in seed germina-
(4/31), and 0% of the rhizospheric yeasts exhibited pectin- tion of B. juncea with treatments containing ltrates of
ase, protease, xylanase, cellulase, and amylase activities, rhizospheric yeasts (YR24, YR11, and YR29) compared
respectively (Table 1). For the plant-growth-promoting with the control. Seed germination at 48 h of incubation
features, siderophore and IAA productions were de- increased by >70% in all treatments, including the con-
tected only among some isolates at the proportions of trol. Only the treatment with the YR24 strain ltrate
16.1% (5/31) and 9.67% (3/31), respectively. The species demonstrated a signicant difference (p < 0.05) from the
R. mucilaginosa (2 isolates) and C. slofae (1 isolate) were rest of these, with 96.6% germination (Fig. 3). In all treat-
positive for IAA production, and both genera were ments supplemented with yeast ltrate, seedling growth

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Ramos-Garza et al. 7

Fig. 1 (concluded).
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Fig. 2. Kinetics of indole acetic acid (IAA) production by Fig. 3. Percentage of seed germination at 2448 h of
the yeasts Cystobasidium slofae (YR11) and Rhodotorula incubation in water-agar supplemented with yeast
mucilaginosa (YR24 and YR29). Statistical analysis was by ltrates. Yeast strains: YR24, Rhodotorula mucilaginosa; YR11,
means of 2-way ANOVA with a Duncans multiple range Cystobasidium slofae; and YR29, R mucilaginosa. Bar = 1
test, with signicant difference set at a p value of <0.05. standard error of the mean. Different letters above bars
indicate statistical signicance (p < 0.05).

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8 Can. J. Microbiol. Vol. 62, 2016

Fig. 4. Plant growth in water-agar supplemented with are important because they could affect the bioavailabil-
yeast ltrates. Yeast strains: YR24, Rhodotorula mucilaginosa; ity of HMs and As and their toxicity for plants and micro-
YR11, Cystobasidium slofae; and YR29, R. mucilaginosa. Bar = organisms.
1 standard error of the mean. Different letters above bars Yeasts have been isolated from different environments,
indicate statistical signicance (p < 0.05).
including extreme environments and from the rhizo-
sphere of some plants (Hong et al. 2002, 2006), and are
employed as biocontrol agents or plant growth promot-
ers (El-Mehalawy 2004; Mestre et al. 2011). However, little
information is available on yeasts associated with HM-
and As-resistant plants. Deng and contributors (Deng
et al. 2012) characterized an HM-resistant endophytic
yeast, Cryptococcus sp. strain CBSB78, which was resistant
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to 20 mmol/L Cd2+, 20 mmol/L Pb2+, 10 mmol/L Zn2+, and


7 mmol/L Cu2+, and could improve the growth and HM
extraction of inoculated Brassica plants. Castro-Silva
et al. (2003) isolated and analyzed 12 HM-resistant yeasts
from water, sediment or soil, and plant resources, which
exhibited HM resistance to 1 mmol/L CdCl2 and (or)
120 mmol/L ZnCl2, but the authors did not identify the
isolates.
exhibited a signicant increase compared with the con- Currently, molecular identication employing the
trol (p < 0.05) (Figs. 3 and 4). These results suggest that the large-subunit rDNA D1/D2 domain and ITS fragment se-
IAA produced by the rhizospheric yeasts promotes ger- quence analysis has been utilized to estimate the taxo-
mination and plant growth under the conditions tested. nomic afliation of the yeasts (Gardes and Bruns 1993;
For personal use only.

Kurtzman and Robnett 1998; Linton et al. 2007). In the


HM resistance and As oxidationreduction present study, these techniques could successfully iden-
The isolated yeasts demonstrated high resistance to As
tify the isolates into 5 genera (Figs. 1a and 1b) (Table 1).
(As3+ and As5+) but were sensitive to the remaining HM,
However, species afliations were uncertain in the fol-
especially to Cu. The MIC of the 31 isolates in MBMM
lowing 1 case: the isolate YR20 possessed a sequence
was 05 mmol/L for ZnSO4, 01.2 mmol/L for Pb(NO3)2,
nearly identical to those of T. japonicum, T. insectorum, and
00.5 mmol/L for CuSO4, 030 mmol/L for NaH2AsO4, and
T. coremiiforme on the basis of 26S rRNA analysis and was
0100 mmol/L for NaAsO2 (Table 2). The order of toxicity
related to the group including T. japonicum, T. insectorum,
of risk element to the isolates was Cu > Zn > Pb > As5+ > As3+.
T. faecale, and T. asteroides on ITS. These cases indicated
Higher resistance was observed among yeasts isolated
the inability of 2 molecular markers for differentiating
from the mining tailings. As oxidationreduction assays
showed that 51.6% (16/31) of the rhizospheric yeasts can closely related species; therefore, the exact species
reduce arsenate, but none of these oxidized arsenite; all denition of Trichosporon spp. isolates requires additi-
positive isolates belonged to the species C. albidus and onal molecular analysis, such as multilocus sequencing
exhibited a reduction percentage within the range of (Linton et al. 2007; Bovers et al. 2008; Bernhardt et al. 2015).
10%40% in samples with 0.15 mmol/L As(V) (Table 2). Both the abundance and the taxon number detected in
this study were rather low, which might be related to the
Discussion extreme environmental factors, especially the high con-
In the present study, the rhizospheric yeasts were iso- centrations of HMs and As. Botha (2006) revealed that the
lated and characterized for the rst time (to our knowl- pH and Cu concentration in soil were the principal fac-
edge) from the plants grown in a region that were tors for decreasing yeast number. In our study, Cu con-
seriously contaminated with multiple HMs and As. Of centration reached 2012 and 749 mg/kg soil in the
the 2 sampling sites, both the hill and the mining tailings mining tailings and the hill soil, respectively, much
could be classied as extreme environments based on greater than their MIC values (00.5 mmol/L 032.8 mg/kg)
their low nutrients and high As, Pb, Zn, Mn, Cd, and Cu for the isolates (Table 2). These results suggest a differ-
(see data in supplementary Table S11). The mine tailings ence in the rhizospheric Cu concentration and in the soil
possess high concentrations of As and HMs (As, Pb, Zn, surrounding the plants, suggesting positive interaction
Mn, Cd, and Cu), poor organic matter, low nitrogen, and between yeast and plants. These possible interactions
are slightly salty and slightly acidic to neutral pH, while require further investigation.
the hill site also possesses high concentrations of HM Previously, Cryptococcus, Rhodotorula, and Trichosporon
and As; however, the concentration of nitrogen and or- have been isolated from the phylloplane and rhizo-
ganic matter presented a normal-to-high range and was sphere of sugar cane (Insuellas de Azeredo et al. 1998;
slightly acid to neutral pH. The parameters of these soils Biswas et al. 2001) and from bulk and rhizosphere soils in

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Ramos-Garza et al. 9

Table 2. Minimum inhibitory concentration (MIC) of risk elements, and capacity for oxidizing* or reducing
arsenic of rhizospheric yeasts.
MIC (mmol/L)
Arsenate
Strain Taxonomic afliation Zn2+ Pb2+ Cu2+ As5+ As3+ reduction (%)
From Tithonia diversifolia in mine tailings
YR1 Cryptococcus albidus 0.5 0.6 NG 2 30 10.24
YR2 Cryptococcus albidus 0.5 1.2 NG 1 30 10.24
YR3 Cryptococcus albidus 0.5 1.2 NG NG NG
YR4 Cryptococcus albidus NG 1.2 NG 0.5 1
YR5 Cryptococcus albidus 1 0.6 NG 2 50 23.21
From Flaveria angustifolia in mine tailings
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YR6 Cryptococcus albidus 1 1.2 NG 2 100 30


YR7 Cryptococcus albidus 1 0.6 NG 1 20
YR8 Cryptococcus albidus NG 1.2 NG NG 30 15
YR9 Cryptococcus albidus NG 1.2 NG 0.5 1
YR10 Cryptococcus albidus 1 1.2 0.5 NG 20 20
YR11 Cystobasidium slofae 2 NG NG 2 10
From Sphaeralcea angustifolia in mine tailings
YR12 Cryptococcus albidus 1 1.2 NG 2 30 20
YR13 Cryptococcus albidus 1 1.2 NG 30 100 40
YR14 Cryptococcus uzbekistanensis 2 1.2 0.5 NG NG
From Prosopis sp. in mine tailings
YR15 Cryptococcus albidus 2 0.6 0.5 10 30 16
YR16 Cryptococcus albidus 1 1.2 NG 30 30 15
For personal use only.

YR17 Cryptococcus albidus 1 1.2 NG 4 60 15


YR18 Cryptococcus albidus 0.5 1.2 NG 20 90 30
YR19 Cryptococcus albidus 0.5 1.2 0.5 4 100 20
YR20 Trichosporon japonicum 0.5 NG NG NG 2
From Bahia absinthifolia at hill site
YR21 Cryptococcus albidus 1 1.2 NG 0.5 30 20
YR22 Cryptococcus albidus NG 1.2 NG 0.5 4
From Sphaeralcea angustifolia at hill site
YR23 Cryptococcus albidus 1 1.2 NG 0.5 30 10
YR24 Rhodotorula mucilaginosa 0.5 NG NG NG NG
YR25 Cryptococcus albidus 1 1.2 NG 0.5 30
YR26 Cryptococcus albidus 2 1.2 NG 0.5 30
YR27 Cryptococcus albidus 1 1.2 NG 0.5 20 10
YR28 Cryptococcus albidus 2 1.2 NG NG 20
From Prosopis sp. at hill site
YR29 Rhodotorula mucilaginosa 1 1.2 NG NG 1
YR30 Exophiala pisciphila 5 0.3 NG NG 1
YR31 Exophiala pisciphila 5 1.2 NG NG 1
Range or percentage 0.52 0.61.2 00.5 0.54 1100 51.6%
Note: NG, no growth.
*Oxidation of arsenite was negative for all the isolates.

forest regions (Hong et al. 2002; Mestre et al. 2011). The the most important organism in sandy and desert soils,
isolation of these in the present study further conrmed described as dominant in Mexican soils (Vishniac 2006).
their ubiquitous distribution. However, all 3 species in this clade, that is, C. albidus,
Identication of Cryptococcus as the main group in our C. adeliensis, and C. kuetzingii (synonym for Cryptococcus
study was similar to previous reports that Cryptococcus albidus var. kuetzingii), have rarely been isolated as patho-
comprised the cosmopolitan yeasts in different soils gens (Tintelnot and Losert 2005; Liu et al. 2014).
(Vishniac 1995; Slvikov and Vadkertiov 2003; Slvikov In the past decade, Rhodotorula species, including
et al. 2007) and in rhizosphere and endophytic environ- R. mucilaginosa, which covered 2 isolates in this study,
ments (Insuellas de Azeredo et al. 1998; Mestre et al. 2011; have been found to be ubiquitous in soil and rhizosphere
Deng et al. 2012). The C. albidus clade, which covered the (Hong et al. 2002). Similar to Cryptococcus, Rhodotorula spe-
majority of the isolates obtained in the present study, is cies belong to capsulated yeasts, but present an orange or

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10 Can. J. Microbiol. Vol. 62, 2016

red color. Both capsule and pigments are able to protect 1992; Matthijs et al. 2007; Ali and Vidhale 2013). The 6
these yeasts from different stress conditions, such as ra- siderophore-producing yeasts were dened as members
diation (Molin et al. 2010). of 4 genera and were isolated from the rhizosphere of
The genus Trichosporon contains species that are widely 3 plant species grown at both sites: Cryptococcus spp.
distributed in different environments. It has rarely strains YR6 and YR12 and Trichosporon sp. strain YR20
infected immunocompromised persons as a pathogen from Flaveria angustifolia, Sphaeralcea angustifolia, and
(Colombo et al. 2011). Some of the saprophytic species Prosopis plants, respectively, grown at the mine tailings
possess capacities for bioremediation and biotechnology site; Rhodotorula sp. strain YR24 and Exophiala spp. strains
(Santos et al. 2001). Trichosporon insectorum is a killer yeast YR30 and YR31 from Prosopis plants grown at the hill
isolated from the gut of insects and artisanal cheese site. However, the low proportion (16.1% of the isolates)
(Fuentefria et al. 2008), while T. coremiiforme could de- and the low index implied that siderophore production
grade corncob acid and produce diverse lipids (Huang
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might not comprise determinants for the colonization


et al. 2013). and survival of yeasts in the rhizosphere of plants grown
Previously, little information on Exophiala and in the tested environments.
Cystobasidium from the environments was available. In IAA production was detected in 3 isolates, associated
the present study, 2 isolates were identied as members with 3 plant species grown at the mine tailings site or at
of Exophiala, classied as a dark septate endophyte (DSE); the hill site. These were members of Cystobasidium sp.
there are some reports concerning the extreme cad- strain YR11 (from F. angustifolia, mine tailings site),
mium (Cd) tolerance of Exophiala pisciphila, including Rhodotorula sp. strain YR24 (from S. angustifolia at the hill
metal ion binding and transportation, and organic acid site), and Rhodotorula sp. strain YR29 (from Prosopis sp. at
metabolism (Zhao et al. 2015). Because DSEs are typical the hill site). The genera Rhodotorula and Cystobasidium
root endophytes, their potential effects on host plants produced IAA in amounts of 6.89.61 g/mL, and these
should also be considered. Under HM stress, these fungi concentrations are considered low in comparison with
can affect HM uptake and the tolerance of the host that of other genera in Ascomycota, such as Candida maltosa,
For personal use only.

plants, e.g., Zea mays L. (Li et al. 2011). Cystobasidium is a


Candida glabrata, and Candida tropicalis (Limtong and
genus described as living in different associations with
Koowadjanakul 2012). Their low proportion in this study
other fungi, lichens, and woods; in addition, some iso-
demonstrated that this feature was not selected by the
lates have clinical relevance (Yurkov et al. 2015). To our
rhizosphere in the soils tested. However, IAA production
knowledge, this is the rst time that Cystobasidium yeast
was sufcient for promoting seed germination and plant
has been isolated from the rhizosphere of HM- and As-
growth. Polyamines and inorganic phosphate solubiliza-
resistant plants. Detection of Cystobasidium and Exophiala
tion were not detected among the yeasts isolated in this
in the present study expanded the known diversity of
study, perhaps based upon the same reason estimated
rhizosphere yeasts, especially in HM-contaminated re-
for IAA production.
gions, and implied that they may play some ecological
HM and As resistance has been reported for some
role in HM-contaminated soils.
yeasts (Olasupo et al. 1993; Vadkertiov and Slvikov
In tests for enzymatic and potential plant-growth-
2006). In our work, the yeast isolates demonstrated poor-
promoting activities (Table 1), considerable proportions
to-intermediate resistance to HM and As, compared with
of the isolates presented pectinases (51.6%), proteases
the results of other authors (Vadkertiov and Slvikov
(51.6%), and xylanases (42.9%), but only 12.9% of the iso-
lates presented cellulases. These enzymes are involved in 2006). However, C. albidus-related yeasts exhibited high
the degradation of the plant cell wall (Riou et al. 1991; resistance to As: up to 30 mmol/L for arsenate and
Schulz and Boyle 2005). Therefore, the presence of these 100 mmol/L for arsenite. In general, arsenate is less toxic
enzymes may help these rhizospheric yeasts colonize the than arsenite, and greater resistance to arsenate than
rhizosphere. arsenite has been reported in some bacteria and fungi.
Previously, it was reported that some fungi, including Thus, it could be estimated that these 13 yeast isolates
the Rhodotorula yeast, could produce siderophores (such may possess a different mechanism for arsenite resis-
as rhodotorulic acid) under iron-decient conditions, tance. A mechanism related to this estimation has been
which exerts an antagonistic effect against other well studied in Saccharomyces cerevisiae, in which the arse-
lamentous fungi (Calvente et al. 2001). However, the nate was reduced to arsenite and was expelled from the
soils involved in the present study are iron-rich (about cell (Cernansk et al. 2009). In nature, arsenite can be
35 g Fe/kg soil); thus, siderophore production should not be also detoxied by methylation, which transforms the ar-
related to iron deciency but rather linked with other senite into a volatile form (Su et al. 2011). Further study is
functions. It has been reported that the siderophore required on our isolates to ascertain whether they pos-
could increase producer resistance to HM or metalloids sess the methylation function or another. To date, scarce
(Schalk et al. 2011), increase As (Shah et al. 2010), improve information is available on As transformation by yeasts,
soil fertility, and inhibit fungal pathogens (Shah et al. and there is no report on As transformation by Cryptococcus.

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Ramos-Garza et al. 11

Therefore, to our knowledge, our study was the rst re- tochrome b gene. Int. J. Syst. Evol. Microbiol. 51: 11911199.
port on arsenate reduction by this yeast. doi:10.1099/00207713-51-3-1191. PMID:11411687.
Botha, A. 2006. Yeast in soil. In The yeast handbook. Biodiversity
In conclusion, yeasts associated with the rhizosphere
and ecophysiology of yeast. Edited by C.A. Rosa and G. Peter.
of S. angustifolia, Prosopis spp., T. diversifolia, F. angustifolia, Springer-Verlag, Berlin, Germany. pp. 1100.
and Bahia absinthifolia plants grown in soils seriously con- Botha, A. 2011. The importance and ecology of yeasts in soil. Soil
taminated with As and other HM exhibited low abun- Biol. Biochem. 43: 18. doi:10.1016/j.soilbio.2010.10.001.
dance (102 CFU/g of soil) and low diversity, covering only Bovers, M., Hagen, F., Kuramae, E.E., and Boekhout, T. 2008.
Six monophyletic lineages identied within Cryptococcus
5 species in 5 genera. The Cryptococcus group comprised
neoformans and Cryptococcus gattii by multi-locus sequence typ-
the major yeast found in all 5 plants grown at both sam- ing. Fungal Genet. Biol. 45: 400421. doi:10.1016/j.fgb.2007.12.
pling sites. Many yeast isolates produce pectinases, pro- 004. PMID:18261945.
teases, and cellulases and have a low production of Button, D.K., Dunker, S.S., and Morse, M.L. 1973. Continuous
siderophore and IAA. The majority of the yeasts demon- culture of Rhodotorula rubra kinetics of phosphate-limited
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growth. J. Bacteriol. 113(2): 599611. PMID:4690960.


strated resistance to salinity, alkaline conditions, and
Caldern de Rzedowski, G., and Rzedowski, J. 2001. Flora faner-
high concentrations of multiple HMs and As. Some iso- ogmica del Valle de Mxico. 2nd ed. Instituto de Ecologa,
lates presented higher resistance to arsenite than to ar- A. C. y Comisin Nacional para el conocimiento y uso de la
senate and they reduced arsenate to arsenite. Future biodiversidad, Ptzcuaro (Michoacn). p. 1406.
study to evaluate the capacity for transforming As3+ into Calvente, V., Orellano De, M.E., Sansone, G., Benuzzi, D., and
Sanz de Tosetti, M.I. 2001. Effect of nitrogen source and pH on
methyl compounds and the potential for bioremediation
siderophore production by Rhodotorula mucilaginosa strains
is needed. and their application to biocontrol of phytopathogenic
moulds. J. Indust. Microbiol. Biotechnol. 2: 226229. PMID:
Acknowledgements 11464271.
We thank A. Patio-Siliciano for identifying the plant Castaeda-Agullo, M. 1956. Studies on the biosynthesis of extra-
samples, and Luis Vsquez-Mndez and Enriqueta Amora- cellular proteases by bacteria. I. Serratia marcescens, synthetic
Lazcano for their help in soil analysis. This study was and gelatin media. J. Gen. Physiol. 39: 369375. doi:10.1085/
jgp.39.3.369. PMID:13286455.
funded by SIP (Secretara de Investigacin y Posgraduo)
For personal use only.

Castro-Silva, M.A., De, Souza, Lima, A.O., Gerchenski, A.V.,


Projects 20130722, 20130828, 20140124, and 201511150, Jaques, D.B., Rodrigues, A.L., de Souza, P.L., and Rrig, L.R.
authorized by IPN. JR-G received fellowships from 2003. Heavy metal resistance of microorganisms isolated
CONACyT (Consejo Nacional de Ciencia y Tecnologia) and from coal mining environments of Santa Catarina. Braz. J.
PIFI (Programa Integral de Fortalecimiento Institucional). Microbiol. 34: 4547. doi:10.1590/S1517-83822003000500015.
Cernansk, S., Kolenck, M., evc, J., Urik, M., and Hiller, E.
MSV-M, AVR-T, and ET-W appreciate their fellowships
2009. Fungal volatilization of trivalent and pentavalent arse-
from COFAA (Comisin de Operacin y Fomento de Ac- nic under laboratory conditions. Biores. Technol. 100: 1037
tividades Acadmicas), EDIIPN (Estimulos al Desempeo de 1040. doi:10.1016/j.biortech.2008.07.030.
los Investigadores Instituto Politcnico Nacional), and Colombo, A.L., Padovan, A.C., and Chaves, G.M. 2011. Current
SNICONACyT (Sistema Nacional de Investigadores knowledge of Trichosporon spp. and trichosporonosis. Clin.
Microbiol. Rev. 24: 682700. doi:10.1128/CMR.00003-11. PMID:
CONACyT), respectively. The authors thank Maggie
21976604.
Brunner, M.A., for editing services. The authors have no Deng, Z., Wang, W., Tan, H., and Cao, L. 2012. Characterization
conicts of interest to declare. of heavy metal-resistant endophytic yeast Cryptococcus spp.
CBSB78 from rapes (Brassica chinensis) and its potential in pro-
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12 Can. J. Microbiol. Vol. 62, 2016

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