1. Full Length Article
Characterization of new strains of Hortaea
werneckii isolated from salt marshes of Egypt
Ashraf Elsayed *, Amr M. Mowafy, Hoda M. Soliman, Ahmed Gebreil,
Nada I. Magdy
Botany Department, Faculty of Science, Mansoura University, Mansoura, Egypt
A R T I C L E I N F O
Article history:
Received 28 June 2016
Received in revised form 8
September 2016
Accepted 9 September 2016
Available online
A B S T R A C T
Three new black yeast strains (EGYNDA08, EGYNDA16 and EGYNDA90) of marine origin were
isolated from the Egyptian off shore salt marshes and molecularly identified by ITS1 and
ITS2 5.8S rRNA gene sequencing.The molecular identification showed a high sequence iden-
tity between the two of them and Hortaea werneckii Hw6 strain, while the other strain was
unique. The biochemical characterization using different nutritional media showed differ-
ent growth capabilities and the qualitative enzyme tests (such as catalase, urease, lipase,
proteases, amylases and cellulose) showed different activity levels. The morphological char-
acterization showed different developmental stages of hyphal maturation. The phylogenetic
analysis of the three strains indicated that, two isolates were evolutionary relevant to the
Hw6 strain isolated from Spain and one novel strain was isolated with rather different mo-
lecular and morphological characteristics.
© 2016 Production and hosting by Elsevier B.V. on behalf of Mansoura University. This is
an open access article under the CC BY-NC-ND license (http://creativecommons.org/
licenses/by-nc-nd/4.0/).
Keywords:
Black yeast
Hortaea and salt marshes
1. Introduction
From the taxonomic and phylogenetic point of view,‘Black yeast’
is a polyphyletic group [1,2] of dematiaceous, filamentous [3]
fungi of which several representatives share morphological fea-
tures [4] such as reproducing by unicellular growth [5]. They
thrive in extreme environments characterized by scarce nu-
trients, low oxygen tension, high temperature, harmful UV
radiation, osmotic stress as well as a mixture of these condi-
tions [4].
Hortaea werneckii was identified as an extreme halotolerant
fungus [6] that belongs to the black yeast group and was iso-
lated from different marine habitats. H. wernrckii was reported
to inhibit the afore-mentioned extreme conditions. Hortaea sp.
belongs to the Capnodiales order in the Ascomycota phylum
[7]. Based on the state of the niche (static or dynamic) [5],
H. werneckii switches between two main life phases yeast phase
and hyphal phase [8]. Both phases ended up with sclerotial
bodies under sever environmental conditions [8].
Under light microscope, H. werneckii is a synanomorphic mi-
croorganism which produces buds, conidia, arthrocondia and
* Corresponding author.
E-mail address: ashraf.badawy@yahoo.com (A. Elsayed).
http://dx.doi.org/10.1016/j.ejbas.2016.09.001
2314-808X/© 2016 Production and hosting by Elsevier B.V. on behalf of Mansoura University. This is an open access article under the
CC BY-NC-ND license (http://creativecommons.org/licenses/by-nc-nd/4.0/).
e g y p t i a n j o u r n a l o f b a s i c a n d a p p l i e d s c i e n c e s ■ ■ ( 2 0 1 6 ) ■ ■ – ■ ■
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Available online at www.sciencedirect.com
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2. endoconidia. Buds are enteroblastic annellidic ones [8] and they
emerged as polar, lateral, and bipolar from yeast-like forms [9].
Conidiogenesis and budding mode are the same as both buds
and conidia are released by the complete separation of the de-
veloped abscission line in a septum [3]. When cells are
successively generated from the same locus, the remnants of
the outer layers of the cell wall are arranged consecutively
around the tip, an increase in neck (collars) of the mother cell
results in bottle-shaped cells marked by many rings directed
toward the pole and the number of rings is dependent on the
number of buds or conidia released [8]. The conidia are pro-
duced from the generative apex of annellidophores which are
unbranched intercalary hyphal cells with tapered tips. Conidia
are mainly unicellular; however two-celled conidia separated
by conspicuous dark cross wall were found [3]. H. werneckii pro-
duces hydrophobic dematiaceous septated mycelia [3] which
are formed of coherent thalli and extended from the mother
cell wall [8] and lateral branches sometimes are expanded from
them [9].
Hyphal cells septa have an abscission line separating
between septum sheets. In addition to the simple central pore,
triangular points in mature cells are laying at the anastomo-
sis of inner and outer layer which marks the future points of
separation and the adjacent outer layer remains intact without
a separation line until releasing enteroarthric thin-walled scar-
free cells either in the form of an endogenous conidia from
the yeast-like cells or arthroconidia from the fragmentation
of hyphae by shedding off parts of the outer cell wall layers,
which are easily ruptured [8,9].
Up to our knowledge, isolation and characterization of
H. werneckii from the Egyptian environment was not reported
before. In this survey, the isolated strains of H. werneckii from
Egypt were molecularly, biochemically and morphologically
characterized to focus on differences between them and
between other previously published characterized H. werneckii
strains.
2. Materials and methods
2.1. Sample collection
Water samples from different off shore salt marshes located
after 10 kilometers from west of Gamasa on Damietta road,
were collected at the beginning of December and three samples
in the end of March. Samples were stored in glass sealed jars.
2.2. Isolation and purification of the marine black yeast
One milliliter from the collected samples was inoculated in two
saline enriched liquid medium (0.5% yeast extract without/
with 1% glucose) dissolved in sea water and 100 μg/ml of
streptomycin was added after sterilization in order to inhibit
undesired bacterial growth. The incubation was held at 15 °C,
25 °C and 30 °C in an orbital incubator shaker at 150 rpm for
three weeks [10].
After one week, sub-culturing from the liquid media of tur-
bidity was performed on the same saline enriched agar media.
Temperatures 15 °C, 25 °C and 30 °C were repeated until the
filamentous community invading during the first week and then
developed to unicellular communities appeared on the third
week until the appearance of yeast-like melanized colonies
which are purified by sub-culturing on media containing 10%
NaCl. The pure cultures were preserved in 20% glycerol and
stored at −20 °C.
2.3. Molecular identification of the isolates
Fungal DNA was extracted by employing the FastDNA® Spin
Kit according to the supplier’s instructions.The yield of genomic
DNA was measured by a Nano Drop spectrophotometer by mea-
suring the absorbance at 260 nm. The ITS1 region from DNA
sample extracts was amplified in triplicate using primers with
high specificity for ascomycete fungi (18FITS1 (CTTGGTCATTTAG
AGGAAGTAA) and 18RITS4 (TCCTCCGCTTATTGATATGC). The
PCR reactions were performed in a thermo-cycler at a total
volume of 50 μl using the temperature programs: 94 °C for 5 min,
94 °C for 40 s, 55 °C for 45 s, 72 °C for 1.5 min, 72 °C for 7 min
(35 cycles). The sizes of the PCR products were determined by
electrophoresis on 1.5% agarose gels.The desired products were
excised and purified by the Qiagen II Agarose Gel Extraction
Kit.
The sequencing reactions were performed in a thermocycler
(Master cycler, Eppendorf, Hamburg, Germany) at a total volume
of 10 μl by using the temperature program: 96 °C for 1 min, 96 °C
for 30 s, 60 °C for 10 s, 60 °C for 4 min, 72 °C for 5 min (25 cycles).
The sequencing reaction products were purified by employ-
ing the Dye Ex 2.0 Spin Kit. The purified sequencing reaction
products were dried in vacuum centrifuge and then analyzed
using applied biosystems (ABI PRISM Big Dye Terminator v1.1),
Ready Reaction Cycle Sequencing Kit and employing an ABI
3130 XL Genetic Analyser (Applied Biosystems, Darmstadt) [11].
The obtained sequences were annotated using the
Sequencher™ 4.8 Software. DNA similarity searches were per-
formed using the BlastN program and the databases of
European Molecular Biology Laboratory (EMBL) and GenBank
from the National Center for Biotechnology Information website
(NCBI).
Phylogenetic and molecular evolutionary analyses for 18S
rRNA gene nucleotide sequences were conducted for se-
quence alignments using the computer programs ClustalW and
BioEdit 7.0.5.3 and implement in MEGA software version 5. Phy-
logenetic trees were constructed using the Neighbor-Joining [12]
algorithm method. Distances were generated using the Kimura
Matrix, and the tree stability was supported through Boot-
strap analysis (1000 replications).
2.4. Biochemical characterization
2.4.1. Nutritional characteristics
Different media were tested for supporting the growth of the
isolates such as Malt Extract Agar media (MEA) (2% malt extract
and 1.5% agar) [2], Potato Dextrose Agar media (PDA) (2% dex-
trose 20 g, sliced potato 1.5% agar) and modified Yeast Extract
Peptone Dextrose media (YPD) (2% glucose-1% pepton-1% yeast
extract-1.5% agar) [13]. All media were dissolved in sea water
and cultures were incubated at 25 °C.
ARTICLE IN PRESS
Please cite this article in press as: Ashraf Elsayed, Amr M. Mowafy, Hoda M. Soliman, Ahmed Gebreil, Nada I. Magdy, Characterization of new strains of Hortaea werneckii
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3. 2.4.2. Enzyme tests
2.4.2.1. Catalase test. In room temperature, a drop of diluted
30% Hydrogen Peroxide was placed onto a slide containing a
drop of liquid modified YPD containing tested organisms with
6 days old. Effervescence reaction could be observed in-
stantly as a positive result [14].
2.4.2.2. Urease test. Rustigian and Stuart’s urea broth media
[15] containing urea 20 g, monopotassium phosphate 9.1 g, di-
potassium phosphate 9.5 g and Phenol Red 0.01 g were dissolved
in one liter of distilled water with final pH 6.8, were sterilized
using Millipore filter papers, poured into sterilized tubes, in-
oculated with culture and incubated at 25 °C for five days. The
change in media color from yellow to pink was regarded as a
positive result.
2.4.2.3. Lipase test. Sierra lipase test protocol was done [16]
using Tween-80 (10.0 g) and agar 20.0 g in one liter of sea water
and the final pH was 6. Agar was dissolved in sea water and
autoclaved and Tween was sterilized separately. After inocu-
lation, the plates were incubated at 25 °C for three days. The
observation of white precipitation around the growing
inoculums was considered as a positive result.
2.4.2.4. Proteases test. A modified protocol of 30% skim milk-
agar medium [17] was done by adding 30 ml packed liquid skim
milk (the used skim milk nutrition label on the pack: Fats .44 g,
Carbohydrates: 10.6 g, Protein: 6.9 g, Vitamin: B1 .1 mg and
Vitamin: B2 .14 mg).The formation of transparent zone around
the growing isolates was considered as a positive result.
2.4.2.5. Amylases test. Vedder starch media composed of
soluble starch 10 g, agar 12 g and sodium nitrate 6.5 g were dis-
solved in one liter sea water and adjusted to pH 6. Amylases
activity was detected by flooding the surface of inoculated
media with Grams Iodine [18].
2.4.2.6. Cellulase test. The Carboxymethylcellulose (CMC) media
[19,20], which were composed of: CMC 10 g, Sodium Nitrate 6.5 g,
Potassium Hydrogen Phosphate 6.5 g, Potassium chloride 6.5 g,
Magnesium sulphate heptahydrate 3.0 g and Agar 17.5 g in one
liter with final pH of 6, were used for cellulose activity. The ac-
tivity was visualized by staining the media with Congo red dye
followed by de-staining with Sodium Chloride solution. De-
staining process was repeated continuously until a clear zone
appeared around the growing hyphae.
2.5. Microscopic characterization
Specimens of the three strains of the five days old light green
colonies for yeast phase formation and one month old black
colonies for hyphal phase formation were examined under light
microscope (40×).
3. Results
3.1. Black yeast isolation
The incubational temperature 25 °C and 10% NaCl enhanced
the growth of the black yeast isolates. Two isolates were re-
covered from water samples, EGYNDA08 at the beginning of
December and EGYNDA16 at the end of March, while another
isolate EGYNDA90 was recovered from NaCl crystals of salt
marshes at the beginning of May.
3.2. Molecular identification
From the phylogenetic tree (Fig. 1), three new black yeast strains
(EGYNDA08, EGYNDA16 and EGYNDA90) were visualized. One
of them (EGYNDA90, accession number KU341734) lay in a sepa-
rate branch while the others, EGYNDA08 (accession number
KU341732) and EGYNDA16 (accession number KU341733), were
very close to each other and to Hw6-JN997370.1 which was iso-
lated from Spain. The similarity between EGYNDA16 and the
other ecotypes was 97.4% maximum using Pairwise Sequence
Alignment (Table 1).
3.3. Biochemical characterization
3.3.1. Nutritional characteristics
EGYNDA08 grew on MEA as black small colonies with hyphal
growth stimulation; also it grew on the modified YPD media,
meanwhile, EGYNDA16 and EGYNDA90 grew on YPD media and
they hardly grew on MEA and the media contained 0.5% yeast
extract.
3.3.2. Enzymes tests
3.3.2.1. Catalase test. The effervescence that resulted on a slide
was very weak in case of EGYNDA08, very strong in case of
Fig. 1 – Phylogenetic tree constructed by MEGA using Neighbor-Joining tree, with number of Bootstrap replications 500,
between different Hortaea werneckii strains including the three isolates and Phaeotheca triangularis. T = ex-type strain.
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isolated from salt marshes of Egypt, Egyptian Journal of Basic and Applied Sciences (2016), doi: 10.1016/j.ejbas.2016.09.001
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4. EGYNDA16 and almost intermediate in case of EGYNDA90
(Fig. 2A).
3.3.2.2. Urease test. In case of EGYNDA08, the color change from
yellow to pink was sharper than that in case of EGYNDA90 in-
dicating higher urease activity, while, the lowest activity was
recorded for EGYNDA16 (Fig. 2B).
3.3.2.3. Lipase test. White precipitate appeared around all the
isolates (Fig. 2C) indicating the lipase activity for all.
Table 1 – Sequence homology of the 18S rRNA gene of H. werneckii isolates from the salt marshes with yeast isolates
from public data bases.
Isolate Accession number Closely related yeast Homology (%) Size (bp)
EGYNDA08 KU341732 H. wernechii 6-JN997370 100 501
EGYNDA16 KU341733 H. wernechii 6-JN997370 97.4 471
EGYNDA90 KU341734 H. wernechii 6-JN997370 100 459
Fig. 2 – Enzyme tests results. (A) Positive results of catalase test, (B) positive results of urease test, (C) positive results of
lipase test, (D) positive results of proteases test, (E) positive results of amylases test and (F) positive results of cellulase test.
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5. 3.3.2.4. Protease test. Hydrolysis of proteins around colonies
was achieved by the three isolates (Fig. 2D) indicating prote-
ase activity for all the isolates.
3.3.2.5. Amylase test. Clear zone around colonies was achieved
by the three isolates (Fig. 2F) indicating the ability for all of them
to degrade starch.
3.3.3. Microscopic characteristics
Generally, the isolated strains have all morphological fea-
tures recorded previously to other H. werneckii strains and
mentioned briefly in the introduction. Table 2 summarizes the
morphological differences among the isolated black yeast
strains. EGYNDA08 was studied as a model for the develop-
mental stages of hyphal maturation. Newly developed
hyphae appeared as non-septated coenocytic thin hyphae
(Fig. 3D1).
Polar septum formation started from the older parts of the
filaments at the colony base and continued to the tip of the
filaments and the intercalary cells become wider by age and
followed by conidiogenesis (Fig. 3D2). Two weeks old fila-
ments terminated conidiogenesis and intercalary cells started
to divide leading to the irregular size and shape of cells. Frag-
mentation has also been noticed in old filaments (Fig. 3D3) then
sclerotial elements production was the final step in the cycle
(Fig. 3D4).
Table 2 – Morphological characteristics of H. werneckii strains isolated from salt marshes.
Characteristic Strain
EGYNDA08 EGYNDA16 EGYNDA90
Cell length 5.2–7.8 μm 5.2–46.8 μm 5.2–20.8 μm
Cell width 2.6–5 μm ~2.6 μm 2.6–5.2 μm
Spherical cells Not present 10.4 μm 6.5–7.8 μm
Cell description Two-celled pattern, conspicuous and heavily
melanized thick septum and cell wall. (Fig. 3A).
Melanosomes were low in numbers and large in
size, they were located inside and outside the
cell (Fig. 3B1), anastomosis triangles in septum
are pigmented (Fig. 3B2).
Two shapes of cells: rod and
spherical (Fig. 4A). Cell wall,
septum and melanization were
weak (Fig. 4A). Many fine
melanosomes.
Cell wall and septum were
medium in thickness and
melanization
Melanosomes were many and
fine (Fig. 5A).
Hyphal development Short mucilage Rare, if present, zigzag non-
septated (Fig. 4C).
Zigzag coenocytic (Fig. 5B). Long,
albino aerial mycelium (Fig. 2D).
Budding style Polar (Fig. 3C1), sided polar (Fig. 3C2), bipolar
(Fig. 3C3) and sympodial (Fig. 3C4)
Mainly polar and other styles
were rare
Mainly polar and other styles
were rare
Annellidic ring Few rings max. 3 (Fig. 3D2) Many rings (Fig. 4B1, B2 and B3). Medium number of rings
Fig. 3 – EGYNDA08 isolate under light microscope (40×). (A) The most frequent morphological characteristics (two-celled
pattern, conspicuous septa and melanized cell wall. (B1) Bold arrows showing the cytoplasmic melanosomes aggregated
inside and outside the cell. (B2 and B3) Light arrows referring to the accumulation of melanin position. (C1–C4) Different
styles of budding cells (C1) polar, (C2) sided polar, (C3), bipolar (C4) sympodial. (D1–D4) Filaments at different ages. (D1) Five
days old hyphae, (D2) ten days old hyphae, arrows are pointing to annellidophores, (D3) twenty days old petri-dish bold
arrow pointing to fragmented filament and double-headed arrow is pointing to the same number of vesicles inside two
cells and inside fragmented hyphae, (D4) one month old culture with irregular sclerotial elements.
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Please cite this article in press as: Ashraf Elsayed, Amr M. Mowafy, Hoda M. Soliman, Ahmed Gebreil, Nada I. Magdy, Characterization of new strains of Hortaea werneckii
isolated from salt marshes of Egypt, Egyptian Journal of Basic and Applied Sciences (2016), doi: 10.1016/j.ejbas.2016.09.001
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6. 4. Discussion
The biggest obstacle to the halophilic black yeasts study is not
laying in extreme conditions but in their inability to prohibit
the invasion of other competing microorganisms that is similar
to the behavior of the acidophilic fungi [21]. Additionally, iso-
lating H. werneckii was a big challenge [4] because a successful
purification depends on choosing the unsuitable isolation con-
ditions to other fungi contaminating the sample and in the
same time tolerable by the organism of interest such as high
salt concentration in media [4], high pH >9 and low tempera-
ture <20 °C. Media type, temperature and media state affected
the examined shape and the stages of Hortaea cells under mi-
croscope, even the temperature in which they are growing
affected the relativity between hyphal and yeast phase. Yeast
phase was more frequently observed and could be described
as the dominant phase at low temperatures (15–20 °C), however,
hyphal phase was the dominant phase at high temperatures
(25–30 °C). When a four-day-old colony was picked and exam-
ined by light microscope, it showed the different growth phases
except the sclerotial bodies. On the other hand, the isolates
examined from the liquid media were much more homog-
enous cells with almost the same shape and size.
Morphological differences were observed and recorded by
careful comparison between the three isolates cultivated under
the same laboratory conditions to avoid the gradual change
and loss in the unnecessary morphological characteristics of
the tested lab organisms over the research study period.
The general environmental or controlled conditions sur-
rounding black yeasts are majorly responsible for sculpturing
their morphological identity so culturing the organisms re-
peatedly in controlled conditions probably leads to different
phenotype. As an example, EGYNDA16 was rarely observed to
produce filaments until it was cultured repeatedly on solid
media at which wide spaces between spots were considered
indicating that the laboratory conditions and the cultivation
method forced the cultured organism to produce hyphae which
might be necessary for the organism to obtain nutrients.
The yeast-like pattern gave a wide variety in budding styles:
polar and sided polar budding (Fig. 3C1 and C2) which was trig-
gered by growing on solid media may be when cells on the
surface of colony were budding vertically downward then hori-
zontally outward. Bipolar budding (Fig. 3C3) maybe due to
nuclear division occurrence in one mother cell and each nuclei
ordered its half with budding from each pole before a sepa-
rating septum is formed. Sympodial budding (Fig. 3C4) was
triggered in old cultures. Hyphal branches’ irregularity has been
mentioned as a cause to prolonged culturing of the organism
in liquid culture [22].The characteristic shape of zigzag hyphae
has been noticed in Ashbya gossypii and the dimorphic patho-
genic fungus Histoplasma capsulatum as the studies proved that
the hyphae shape might be affected with calcium availability
in surrounding media [23] or calcium binding protein gene mu-
tation [24].
Fig. 4 – EGYNDA16 isolate under light microscope (40×). (A) Black bold arrows are pointing to big cells while thin arrows are
pointing to long cells. (B1–B3) Annellidic budding and brackets are referring to annellidic conidiogenesis, arrows are
pointing to the original cell (B3) which was potential for shedding off endoconidia. (C) Isolated hyphaea from broth media.
Fig. 5 – EGYNDA90 isolate under light microscope. (A) Black
bold arrow is pointing to a relatively big cell while thin
arrow is pointing to long cells. (B) Zigzag-shaped branched
hyphae growing on the figure after few days; filament is
straightened once it gets septated.
ARTICLE IN PRESS
Please cite this article in press as: Ashraf Elsayed, Amr M. Mowafy, Hoda M. Soliman, Ahmed Gebreil, Nada I. Magdy, Characterization of new strains of Hortaea werneckii
isolated from salt marshes of Egypt, Egyptian Journal of Basic and Applied Sciences (2016), doi: 10.1016/j.ejbas.2016.09.001
6 e g y p t i a n j o u r n a l o f b a s i c a n d a p p l i e d s c i e n c e s ■ ■ ( 2 0 1 6 ) ■ ■ – ■ ■
Q7 Q8
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7. It could be concluded that new black yeast strains have been
isolated and characterized in this study.They all have been iden-
tified as H. werneckii, two of them EGYNDA08 and EGYNDA16
were ecotypes of each other and EGYNDA90 lay in a separate
branch. Up to our knowledge, this is the first report describ-
ing the occurrence of this organism in Egypt.The isolates show
differences in morphological characteristics as described in the
results and the enzymatic assays showed qualitative differ-
ences. Further studies are ongoing to verify the pleomorphic
characterization and the adaptation mechanism of these iso-
lates to the saline habitat they could survive.
Acknowledgments
Many thanks to Prof. Samia Ali Haroun, the former head of
botany department, Faculty of Science, Mansoura University,
Egypt and many thanks to Prof. Heshmat Soliman Aldesuquy
the current head of the department for their valuable help and
encouragement. We thank Dr. Eladl Gala from the same de-
partment for his continuous support and valuable discussions.
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ARTICLE IN PRESS
Please cite this article in press as: Ashraf Elsayed, Amr M. Mowafy, Hoda M. Soliman, Ahmed Gebreil, Nada I. Magdy, Characterization of new strains of Hortaea werneckii
isolated from salt marshes of Egypt, Egyptian Journal of Basic and Applied Sciences (2016), doi: 10.1016/j.ejbas.2016.09.001
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