Aureobasidium pullulans from Juncus trifidus L. roots

Abstract

Studies on endophytes of Juncus trifidus roots have been conducted. An analysis of isolated Aureobasidium pullulans strain is presented. Taxonomical data were confirmed by morphological and molecular data. Sequence of the ITS region has been determined and used for phylogenetic assesment.

Full text

Phytotaxa 266 (2): 125–133
http://www.mapress.com/j/pt/
Copyright © 2016 Magnolia Press Article PHYTOTAXA
ISSN 1179-3155 (print edition)
ISSN 1179-3163 (online edition)
Accepted by Sajeewa Maharachchikumbura: 30 May 2016; published: 22 Jun. 2016
http://dx.doi.org/10.11646/phytotaxa.266.2.6
125
Aureobasidium pullulans from Juncus trifidus L. roots
1*12
1Department of Mycology, W. Szafer Institute of Botany Polish Academy of Sciences, Poland
2Department of Biotechnology, Vienna Institute of Biotechnology (VIBT), University of Natural Resources and Life Sciences, Austria
*e-mail: m.owczarek@botany.pl
Abstract
Studies on endophytes of Juncus trifidus roots have been conducted. An analysis of isolated Aureobasidium pullulans strain
is presented. Taxonomical data were confirmed by morphological and molecular data. Sequence of the ITS region has been
determined and used for phylogenetic assesment.
Key words: endophyte, black yeast, ITS
Introduction
There are several definitions for the term “endophyte”. Within this paper an interpretation of Petrini (1991) will be

symptoms and/or any harm to its host. In this sense also mycorrhizae as a symbiotic association between microorganism
and plant roots should be recognised as a special form of endophytic relation. Most fungal endophytes belong to
teleomorphs and anamorphs of ascomycetes, however some species can be pointed out within basidiomycetous fungi
(Rungjindamai et al. 2008).
Endophytes include inter alia a group of specific fungi called dark septate endophytes (DSE), commonly occurring
in plant roots. Fungi belonging to DSE have been found in roots of about 600 plant species of 320 genera (Sieber &
               

and balanced relation is established, in this case a positive, mutualistic cooperation brings benefits to both partners
(Newsham 2011).
Aureobasidium pullulans Dothideales (Dothideomycetes,
Ascomycota), teleomorph is unknown. A. pullulans belongs to DSE fungi as well as to the heterogenous group of dark
     
from various environments such as air in buildings, stones, bathrooms, wall paintings, food and feeds, aviation fuel,
gasoline car tank, glacier ice, hypersaline waters in salterns, water and air in malt-house, malt, honey and honey-dew,
soil, seeds, rhizosphere (as non-mycorrhizal fungal root endophyte), dead leaves and also as a human opportunistic
pathogen (Andrews et al. 
et al. 2000; Hawkes et al. 2005; Isola et al. 2013, Kita 1986;
et al. et al. 2014,
Punnapayak et al. 2003; Rauch M.E. et al. 2006; Ruibal et al. 2005; Samson et al. 2004; Smyk 1954; Sterflinger et al.
2001; Zarzycka 1958).
Recently, based on molecular data and place of origin four varieties were distinguished – A. pullulans 
. pullulans; A. pullulans . melanogenum Hermanides-Nijhof; A. pullulans var.
subglaciale A. pullulans var. namibiae
 et al. 2008), and in 2014 were elevated to the species level - Aureobasidium melanogenum
     Aureobasidium subglaciale     
    Aureobasidium namibiae     
et al. 2014). A. pullulans is the one most often pointed out as an endophyte.
The aim of the study was to confirm taxonomical identification of Juncus trifidus root DSE endophyte.

ET AL.
126 • Phytotaxa 266 (2) © 2016 Magnolia Press
Material and methods
Isolation and cultivation
Samples of Juncus trifidus 
bags in refrigerator (-20º C) for 6 months. Roots surface was sterilized by suspension in a 96% ethanol for 1 minute
and washing in a 3% chlorox solution for 3 minutes. After rinsing in ethanol for 1 minute, samples were transferred
to a 90 mm Petri plates containing Ferency medium with addition of 5 ml oxytetracycline (100 mg in 50 ml acetone)
and cultivated in an incubator at 10º C. Obtained fungal isolates were transferred to a PDA medium and cultivated in
an incubator at 10º C.
Morphological observations
Colonies parameters such as colour, structure and size were measured. Micro- and macromorphological observations
were done by light microscopes Nikon SMZ 1500 and Nikon Eclipse 80i. Photographs were taken by Digital Sight
DS-Ri1 Nikon camera.
DNA extraction
The strain mycelium was grown on 2% MEA plates for two weeks at room temperature. The mycelium was scrapped

glass bulbs. The probes were disrupted by severe shaking and later incubated for 10 minutes at 65° C. After addition
of chloroform/phenol (Roth), the solution was centrifuged at 14000 rpm. The supernatant was transferred to fresh
tubes and poured with 2 vol. of ice-cold ethanol (70%). The samples were stored for 30 minutes at –20° C for DNA
precipitation, then centrifuged for 5 minutes at 14000 rpm. The resulting pellet was washed in ethanol, dried and
dissolved in 100 µl of ddH2O.
PCR and sequencing
The PCR reaction was performed in 25 µl volume: 1X buffer (with MgCl2), 200 µM dNTP, 5 pmol forward and
              
The sequences were aligned and adjusted using ClustalW. The phylogenetic tree was constructed with Molecular
        et al. 2007), using the neighbour joining
method (Saitou & Nei 1987). Confidence values for individual branches were determined by bootstrap test in which
500 trees were generated (Felsenstein 1985).
Results
Aureobasidium pullulans .
Anamorph, teleomorph unknown.
Juncus
trifidus
Morphology
Colonies on PDA medium pale pink in the centre with darker, greenish margin (fig. 1), smooth and glistening due
to exopolysaccharides (EPS) production. Colonies attain 20–25 mm in diameter after 7 days of cultivation in room
temperature. Dark segments in the margin start appearing after 10 days of growth. No aerial mycelium.
Vegetative hyphae are smooth, hyaline and thin-walled, 2–10 µm wide, transversely septate (fig. 2). Conidiogenous
cells undifferentiated, present intercalary on the hyphae. Hyaline conidia produced synchronously in dense groups,
smooth and ellipsoidal, 8–30 × 3.5–5 µm. At the margin present dark brownish green hyphae, thick-walled, 6–12 µm

of conidia in EPS seen occasionally.

AUREOBASIDIUM PULLULANS Phytotaxa 266 (2) © 2016 Magnolia Press • 127
FIGURE 1. Aureobasidium pullulans 
formed on PDA medium with addition of 5% NaCl.
FIGURE 2. Aureobasidium pullulans
conidia.
Sequence alignment
        A. pullulansA.
subglaciale strain EXF-2479 (vsEXF-2479), A. namibiae      A. melanogenum
     

sequences.
Phylogenetic analysis
In the fig. 4 a phylogenetic tree including 18 fungal species is presented. The obtained results suggest that the analysed
strain belongs to the same cluster as the other isolates of A. pullulans. Results correspond to current taxonomical status
of the Aureobasidium genus. Species that have been previously described as an A. pullulans varietes (var. pullulans,
melanogenum, subglaciale and namibiae), and now elevated to the species level, form separate clusters, close to

is also an Aureobasidium pullulans strain.

ET AL.
128 • Phytotaxa 266 (2) © 2016 Magnolia Press
FIGURE 3. Sequence alignment.
In table 1 are listed sequences used in phylogenetic analysis. All fungi which cluster with studied strain were
isolates from various plant material, an exception is A. pullulans var. pullulans strain TSN-43 which was described
from soil sample, collected in different regions of Europe and Africa.
 
Discussion
Aureobasidium pullulans 
Martini et al. et al. 2011). A. pullulans was noted in all plant organs of sycamore, but diminished in the
A. pullulans are extremely versatile in

AUREOBASIDIUM PULLULANS Phytotaxa 266 (2) © 2016 Magnolia Press • 129
their ecology and variation in production of metabolites (Slepecky & Starmer 2009). Moreover antagonistic effect of
A. pullulans et al. 2011). In our investigation we have observed only single root
colonisation of A. pullulans in J. trifidus among 24 localities. In the same roots of the plant were observed other fungi:
Umbelopsis autotrophica, non-sporulating brown mycelium with dark chlamydospores and Ascochyta sp. (Chlebicki

TABLE 1.
Organism Strain Isolation source 
accession no.
 Collector
Aureobasidium
pullulans
 Slime flux of Quercus sp. FJ150902.1 
near Hamburg
-
Aureobasidium
pullulans
 fruit of Vitis vinifera FJ150906.1 

E.J. Hermanides-Nijhof
Aureobasidium
proteae
CPC 2824 Protea eximia x Protea
susannae cv. Sylvia
JN712491.1 South Africa S. Denman
Aureobasidium
pullulans
TSN-43 soil FR716139.1 Swabian Jura,


-
Aureobasidium
pullulans
 Malus sylvestris, fruit FJ150907.1 - -
Aureobasidium
proteae
CPC 2826 Protea eximia x Protea
susannae cv. Sylvia
JN712493.1 South Africa S. Denman
Aureobasidium
microstictum
 dying leaf of Convallaria
majalis
FJ150903.1  
Aureobasidium
namibiae
 dolomitic marble FJ150875.1 Namibia, Namib
Desert
U. Wollenzien
Aureobasidium
melanogenum
 - FJ150886.1 - -
Aureobasidium
melanogenum
 deteriorated army supplies FJ150885.1 Russia -
Aureobasidium
melanogenum
 - FJ150881.1 - -
Aureobasidium
leucospermi
CPC 15180 leaves of Leucospermum
conocarpodendron
JN712489.1 South Africa F. Roets
Aureobasidium
caulivorum
 Trifolium incarnatum FJ150871.1 U.S.A., Oregon -
Aureobasidium
microstictum
 Hemerocallis sp. FJ150873.1 Netherland -
Aureobasidium
subglaciale
EXF-2481 subglacial ice from sea
water
FJ150895.1 Norway, Svalbard,
Kongsvegen

Aureobasidium
subglaciale
dH 13876 coastal ponds of melted
snow and ice
FJ150892.1 Norway, Svalbard,
Kongsvegen

Aureobasidium
subglaciale
EXF-2479 glacial ice from sea water FJ150893.1 Norway, Svalbard,
Kongsvegen


ET AL.
130 • Phytotaxa 266 (2) © 2016 Magnolia Press
FIGURE 4.
marked with red dot.
  Aureobasidium pullulans isolate from Juncus trifidus, but the first studied with
use of molecular techniques (Chlebicki 2009). First of A. pullulans isolates from J. trifidus was described from culms.
Chlebicki (2009) gives a full list of fungal endophytes noted on J. trifidus. It seems that A. pullulans, including


fungi.
Nowadays the A. pullulans is often recognised as a potentially effective biocontrol agent of phytopathogens with
emphasis on postharvest control. This effect is accomplished by expressing variety of antagonistic strategies including
production of volatile organic compounds (VOCs), lytic enzymes, antimicrobial substances as well as competition for
nutrients (Castoria 2001; Felice et al. et al. 2009; Mari et al. 2012; Rich et al. 2013, Schena et al. 1999,
2003). Thus, the presence of A. pullulans within tissues of plants seems as potentially beneficial in agriculture.
What enables A. pullulans survival in a great variety of habitats is most likely a melanin production. As most of

abilities of microorganisms under stress conditions such as toxic environment or raised UV-radiation levels (Fogarty
& Tobin, 1996). Each variety of the Aureobasidium species differ in culture age in which the production of a melanine
starts, which is the basic macromorphological quality enabling identification (Zalar et al. 2008). A. pullulans shows
the lowest melanin production rates, what can be probably a reason why this variety is isolated more often from living
organisms than an oligotrophic environment. Its tolerance towards some hash conditions is attributed to other qualities
et al. 2014).
As mentioned, what previously was considered as A. pullulans variety is rather rarely given what results sometimes
in difficulties in distinguishing where each variety occurs the most often. Nevertheless, morphological and molecular
differences between varieties of the species have been described (Zalar et al. 2008). Doubtlessly identification to the
species/variety level should be required in all research papers.

AUREOBASIDIUM PULLULANS Phytotaxa 266 (2) © 2016 Magnolia Press • 131
Acknowledgements
This work was supported by the Ministry of Science and Information Society Technologies, Poland (Project No. K118/

References
Acta Mycologica 8: 175–189.
http://dx.doi.org/10.5586/am.1972.012
Aureobasidium pullulans 
postharvest pathogens of fruits: study on its modes of action. Postharvest Biology and Technology 22: 7–17.
http://dx.doi.org/10.1016/S0925-5214(00)00186-1
Ciferri, R., Ribaldi, M. & Corte, A. (1957) Revision of 23 strains of Aureobasidium pullulans Pullularia pullulans). Atti
dell’Istituto Botanico della Università e Laboratorio Crittogamico di Pavia 14: 78–90.
Chlebicki, A. (2009) Some endophytes of Juncus trifidus from Tatra Mts. in Poland. Acta Mycologica 44 (1): 11–17.
http://dx.doi.org/10.5586/am.2009.003

rotation system. Acta Mycologica 36: 241–249.
http://dx.doi.org/10.5586/am.2001.016
Aureobasidium pullulans can lower
ochratoxin A contamination in wine grapes. Phytopathology 98: 1261–1270.
 
Aureobasidium and Hormonema. Antonie
van Leeuwenhoek 65: 41–54.
 

of growth and reproduction of Didymella rabiei under laboratory conditions. Journal of Phytopathology 153: 431–439.
http://dx.doi.org/10.1111/j.1439-0434.2005.00996.x
Felsenstein, J. (1985) Confidence limits on phylogenies: An approach using the bootstrap. Evolution 39: 783–791.
http://dx.doi.org/10.2307/2408678
Fogarty, R.V. & Tobin, J.M. (1996) Fungal melanins and their interactions with metals. Enzyme and Microbial Technology 19 (4): 311–
317.
http://dx.doi.org/10.1016/0141-0229(96)00002-6

International Biodeterioration & Biodegradation 46: 277–284.
http://dx.doi.org/10.1016/S0964-8305(00)00103-7

  Aureobasidium pullulans
varieties: biotechnological potential, stress tolerance, and description of new species. BMC Genomics 15: 549.
http://dx.doi.org/10.1186/1471-2164-15-549
    Fungal
Biology 115 (10): 978–986.
http://dx.doi.org/10.1016/j.funbio.2011.04.004
          
halophilic black yeasts. FEMS Microbiology Ecology 32: 235–240.
Hawkes, M., Rennie, R., Sand, C. & Vaudry, W. (2005) Aureobasidium pullulans infection: Fungemia in an infant and a review of human
cases. Diagnostic Microbiology and Infectious Disease 51: 209–213.
http://dx.doi.org/10.1016/j.diagmicrobio.2004.10.007

Fungi as Degraders of Volatile Aromatic Hydrocarbons. Mycopathologia 175: 369–379.
http://dx.doi.org/10.1007/s11046-013-9635-2
Kita, W. (1986) Mycoflora of phylloplane as a factor protecting the oil sunflower against the diseases caused by fungi, depending on

ET AL.
132 • Phytotaxa 266 (2) © 2016 Magnolia Press
ecological conditions. Acta Mycologica 22: 205–221.
http://dx.doi.org/10.5586/am.1986.024
Kowalski, S. (1980) Studies on the communities od soil fungi in selected mountain stands in southern Poland. Acta Mycologica 16:
55–87.
http://dx.doi.org/10.5586/am.1980.003
Aureobasidium pullulans from bathroom surfaces and their antifungal
activity against some Aspergilli. African Journal of Microbiology Research vol. 3 (5): 253–257.
Picea Excelsa Acta Societatis Botanicorum
Poloniae 27: 45–75.
Aureobasidium pullulans.
Postharvest Biology and Technology 73: 56–62.
http://dx.doi.org/10.1016/j.postharvbio.2012.05.014

fungal endophytes Aureobasidium pullulans and Epicoccum nigrum. Plant Disease 93: 993–998.
http://dx.doi.org/10.1094/PDIS-93-10-0993
Acta Mycologica 27:
121–126.
http://dx.doi.org/10.5586/am.1992.011
Aureobasidium pullulans 
Acta Scientiarum Polonorum 13(3):13–22.
Newsham, K.K. (2011) A meta-analysis of plant responses to dark septate root endophytes. New Phytologist 190: 783–793.
http://dx.doi.org/10.1111/j.1469-8137.2010.03611.x
Petrini, O. (1991) Fungal endophytes in tree leaves. In: Andrews, J.H. & Hrano, S.S (Eds.) Microbial Ecology of Leaves. Springer, New

http://dx.doi.org/10.1007/978-1-4612-3168-4_9
 Aureobasidium pullulans: an endophyte in sycamore and other trees. Transactions of the British
Mycological Society 57 (2): 227–231.
http://dx.doi.org/10.1016/S0007-1536(71)80004-9
Punnapayak, H., Sudhadham, M., Prasongsuk, S. & Pichayangkura, S. (2003) Characterization of Aureobasidium pullulans isolated from
airborne spores in Thailand. Journal of Industrial Microbiology and Biotechnology 30: 89–94.
http://dx.doi.org/10.1007/s10295-002-0016-y

of microbial contamination in United States Air Force aviation fuel tanks. Journal of Industrial Microbiology and Biotechnology
33: 29–36.
http://dx.doi.org/10.1007/s10295-005-0023-x
Enzyme
and Microbial Technology 53: 33–37.
http://dx.doi.org/10.1016/j.enzmictec.2013.03.015
       
Mycological Progress 4 (1): 23–38.
http://dx.doi.org/10.1007/s11557-006-0107-7
               
endophytes isolates from leaves, rachis petioles of the oil palm, Elaeis guineensis, in Thailand. Fungal Diversity 33: 139–161.
Saitou, N. & Nei, M. (1987) The neighbor-joining method: A new method for reconstructing phylogenetic trees. Molecular Biology and
Evolution 4: 406–425.
Aureobasidium
pullulans isolates against postharvest rots. Postharvest Biology and Technology 17: 189–199.
http://dx.doi.org/10.1016/S0925-5214(99)00036-8

endophytic isolates of Aureobasidium pullulans. Postharvest Biology and Technology 30: 209–220.
http://dx.doi.org/10.1016/S0925-5214(03)00111-X

Endophyte Phialocephala fortini s.l.. In:   Microbial root endophytes. 
Heidelberg, pp. 107–132.

AUREOBASIDIUM PULLULANS Phytotaxa 266 (2) © 2016 Magnolia Press • 133
http://dx.doi.org/10.1007/3-540-33526-9_7
Slepecky, R.A. & Starmer, W.T. (2009) Phenotypic plasticity in fungi: a review with observation on Aureobasidium pullulans. Mycologia
101: 823–832.
http://dx.doi.org/10.3852/08-197
Roczniki Nauk Rolniczych, Ser. A 69: 409–470.
Sterflinger K. & Prillinger, H. (2001) Molecular taxonomy and biodiversity of rock fungal communities in an urban environment (Vienna,
Austria). Antonie van Leeuwenhoek 80: 275–286.

Molecular Biology and Evolution 24: 1596–1599.
http://dx.doi.org/10.1093/molbev/msm092
Aureobasidium pullulans
and its varieties. Studies in Mycology 61: 21–38.
http://dx.doi.org/10.3114/sim.2008.61.02
Zarzycka, H. (1958) Mikroflora nasion maku. Roczniki Nauk Rolniczych, Ser. A 78 (2): 309–342.