ISSN 10674136, Russian Journal of Ecology, 2015, Vol. 46, No. 5, pp. 490–493. © Pleiades Publishing, Ltd., 2015.

Original Russian Text © K.A. Kravchenko, A.S. Vlashchenko, O.V. Prilutskii, A.S. Prilutskaya, 2015, published in Ekologiya, 2015, No. 5, pp. 397–400.

SHORT COMMUNICATIONS

A Search for Geomyces destructans, a Dangerous Pathogen of Bats,

in Caves of Eastern Europe
K. A. Kravchenkoa, A. S. Vlashchenkoa, O. V. Prilutskiib, and A. S. Prilutskayaa
a
Feldman Ecopark Bat Rehabilitation Center, Kievskoe sh. 12, Lesnoe, Kharkov oblast, 62340 Ukraine
b
Karazin Kharkov National University, pl. Svobody 4, Kharkov, 61022 Ukraine
email: shusha.kravcheneo@gmail.com
Received March 5, 2014

Keywords: Geomyces destructans, fungi, caves, bats, Ukraine, Russia

DOI: 10.1134/S1067413615050124

In 2006, a pandemic of previously unknown fungal
disease suddenly broke out in caves of North America
(Cohn, 2008; Blehert et al., 2009), affecting bats dur
ing winter hibernation (Gargas et al., 2009; Cryan
et al., 2010). Between 2006 and 2012, six million bats
of ten species died (http://www.batcon.org/index.
php/whatwedo/whitenosesyndrome/subcategory),
and the infection was spreading from the east coast of
the United States into the continent for several hun
dred kilometers per year (Turner, 2011; Raloff, 2011).
The mass mortality of bats from this disease and the
high rate of its spread attracted close attention from
researchers. In 2009, the causative agent of this dis
ease, named White Nose Syndrome (WNS), was iden
tified as Geomyces destructans Blehert et Gargas (Gar
gas et al., 2009), a fungal species previously unknown
to science whose mycelium grows into the surface
body tissues of hibernating bats (Carol et al., 2009).
The main hypothesis concerning the origin of this
pandemic outbreak—that the fungus was introduced
from Europe (Wibbelt et al., 2010; Puechmaille et al.,
2010)—was confirmed in subsequent morphological,
immunological, and genetic studies (Wernecke et al.,
2012). In 2008 to 2011, largescale surveys of under
ground bat winter roosts were conducted in Western
and Central Europe in order to find bats affected by
WNS (Wibbelt et al., 2010; Puechmaille et al., 2011).
The easternmost record of G. destructans was made in
Gumenetskie Shtol’ni, a system of adits in Khmelnitsky
oblast, Ukraine (Puechmaille et al., 2011). Information
on the occurrence of this fungus in eastern and north
ern regions of Europe was absent.
The purposes of our study were as follows: (1) to
evaluate the occurrence of G. destructans in under
ground cavities of Ukraine and European Russia,
(2) to compare the efficiency of its detection by tradi
tional morphological and modern genetic methods,
and (3) to present preliminary data on the mycobiota
of surveyed caves.
Samples for analysis were taken in 2010 and 2010
from seven manmade caves serve as major winter
roosts for bats and are located in four regions of
Ukraine and European Russia (Table 1). Special cen
suses of hibernating bats were taken only if compre
hensive published data were not available. Spores were
trapped on the surface of glass slides (below, referred to
as spore traps) or Petri dishes coated with agar medium
(Flannigan, 1997). In both cases, Sabouraud pep
tone–glucose agar (SAB) and cornmeal agar (CMA)
was used (Bills and Foster, 2004). Spore traps were
fixed on cave walls and exposed for 60 days, while Petri
dishes were exposed for 1–3 h. Both sampling meth
ods were tested in Liptsy1 and Liptsy2 caves in June
to August 2010; samples from other caves were taken
only with Petri dishes.
In Liptsy1 and Liptsy2, ten Petri dishes and ten
spore traps were exposed in each cave, half of them
with CMA and the other half with SAB. Subsequent
samples were taken only with CMA, which proved to
be more effective. Twenty dishes were exposed in caves
of Leningrad oblast (12 in Levoberezhnaya and 8 in
Plyazhnaya); 15 dishes, in Zaporozhye oblast (12 in
Skelyanskie Mines and 3 in Mayachanskaya); and
16 dishes, in Gumenetskie Mines, where samples
(scrapings) were also taken directly from the skin of
affected bats, from patches with white fungal over
growth.
After sampling, Petri dishes were incubated in a
cooler, at 4–10°С, and examined for fungal growth
every 4–5 days, recording characteristics of colonies.
Sporebearing colonies were subcultured to new
dishes with CMA to obtain pure colonies. In some
cases two to three passages were made using stab inoc
ulation (Bilai, 1982).
Skin scrapings were plated in Petri dishes with
CMA and incubated in a cooler. Pure cultures were
examined to record their color, texture, features of
mycelium growth, and spore production. All samples
were identified to the level of genus (in some cases, of

490
 A SEARCH FOR Geomyces destructans, A DANGEROUS PATHOGEN OF BATS 491

Table 1. Characteristics and locations of underground chambers serving as bat winter roosts

Total length Temperature

Location, Type of chamber, Hibernating
of underground and humidity References
coordinates, name rock bat species
tunnels, m in winter

Ukraine, Mine, 5–9°C; M. daubentonii, Vlashchenko

Kharkov oblast, sandstone 90–100% M. dasycneme, and Naglov, 2006
50°12′ N, Pl. auritus
36°22′ E:
Liptsy1 230
Liptsy2 600

Russia, Mine, 3–7°C; M. daubentonii, Strelkov, 1971;

Leningrad oblast, white quartz sand 85–100% M. dasycneme, Chistyakov, 2010
59°40′ N, 30°47′ E: M. nattereri,
M. brandtii,
Levoberezhnaya 5500 E. nilssonii,
Plyazhnaya 100 Pl. auritus

Ukraine, Mine, 9–12°C; Rh. hipposideros, Tishchenko, 2005;

Khmelnitsky oblast, limestone unstable humidity M. myotis, Godlevska et al., 2011
48°45′ N, 26°37′ E: M. daubentonii,
Gumenetskie Mines 29000 M. dasycneme,
M. brandtii,
B. barbastellus,
Pl. auritus

Ukraine, Mine, 11–12°C; M. daubentonii, Own data

Zaporozhye oblast, limestone; 70–85% M. aurascens,
47°26′ N, 35°6′ E: karst cave, Pl. austriacus
Skelyanskie Mines limestone 10000
Mayachanskaya 27

species or a suprageneric taxon) using light micros
copy (Bilai, 1982). Cultures morphologically similar
to G. destructans were sequenced for the same molec
ular marker, the rRNA gene internal transcribed
spacer region.
Genetic identification was performed as described
(Peuchmaille et al., 2010; Wibbelt et al., 2010) in the lab
oratory of Leibniz Institute for Zoo and Wildlife Research
(Berlin, Germany). Cultures from Levoberezhnaya and
Plyazhnaya caves, Skelyanskie and Gumenetskie mines,
and Mayachanskaya ravine were identified by the
Loopmediated Isothermal amplification (LAMP)
method (Niessen and Vogel, 2010) at the Technical
University of Munich (Germany).
As a result, we obtained pure cultures of fungi from
13 genera of 9 families and one fungus classified with
the class Coelomycetes Grove (phyla Ascomycota
Caval.Sm., Basidiomycota Whittaker ex Moore, and
Zygomycota Moreau) (Table 2). A noteworthy fact is
that none of the identified taxa proved to be obviously
dominant in the majority of samples or occur in more
than half of the surveyed caves. This concerns even
representatives of widespread genera that are abun
dant in all land ecosystems, such as Penicillium Link,
Aspergillus P. Micheli ex Haller, and Cladosporium
Link. A probable explanation to this situation is that
continuously low temperatures in the caves have pro
vided favorable conditions for the formation of a com
plex of welladapted stenobiontic fungi, with conse
quent displacement of eurybiontic species. In ecologi
cal terms, most of isolated micromycetes are
saprotrophs, and part of the others are parasites of
insects (Table 2). According to Vanderwolf et al. (2013),
representatives of these genera are widespread in the
mycobiota of caves at temperate latitudes.
None of the cultures from our samples was identi
fied as G. destructans, but representatives of the genus
Geomyces were found in two localities, Gumenetskie
adits and Levoberezhnaya Cave (Sablinskie Mines). In
the former locality, where G. destructans was previ
ously detected visually and its presence on bats was
confirmed (Puechmaille et al., 2011), this species was
not recorded this time, but the samples were found to
contain G. pannorum, a nonpathogenic representative
of soil mycobiota described from underground bat
winter roosts (Hayes, 2012; Vanderwolf et al., 2013).
The species from Levoberezhnaya Mine was not

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492 KRAVCHENKO et al.

Table 2. Micromycetes isolated from surveyed underground bat winter roosts

Identification
Taxon L1, L2 Lb Pl GSh SSh M
method

Acremonium sp. + – – – – – 1
Aspergillus spp. + + – – – – 1
Beauveria cf. bassiana + – – – – – 2
Cladosporium sp. + – – + – + 1, 3
Coelomycetes sp. – + –  – – 1
Eurotium sp. – – – + – – 3
Fusarium cf. sporotrichiella – – – + – – 3
Geomyces cf. pannorum – – – + – – 3
Geomyces sp. – + – – – – 3
Mortierella sp. – + – – – – 1
Nigrospora sp. – – – + + – 3
Oidiodendron sp. – – – – + – 3
Penicillium spp. + + – – + – 1, 3
Scopulariopsis sp. – – + – – – 3
Stachybotrys cf. chartarum – – – + – – 3
Designations: (L1) Liptsy1, (L2) Liptsy2, (Lb) Levoberezhnaya, (Pl) Plyazhnaya, (GSh) Gumenetskie Mines, (SSh) Skelyanskie
Mines, (M) Mayachanskaya karst cave; (1) visual identification by basic morphological structures, (2) PCR, (3) LAMP assay.

described previously but proved to be genetically close
to G. destructans.
ACKNOWLEDGMENTS
The authors are grateful to Dr. Gudrun Wibbelt,
Leibniz Institute for Zoo and Wildlife Research, Ber
lin (http://www.izwberlin.de/pathology.html), and
Dr. Ludwig Niessen, Technical University of Munich
(http://tmw.wzw.tum.de/index.php?id=146), for their
help with genetic analysis of samples; to A.Yu. Akulov
and V. Gar’kavenko for providing facilities are resources
for cultivation and morphological identification of
fungi; and to A.V. Naglov, V.A. Busel, M. Sudakova,
M. Drebet, E. Zajnagutdinova, and E. Aksenov for
their help in organizing field research.
This study was supported by the Youth Activity
Fund of The Explorers Club, project “Bats and Fungi
in Eastern Europe: The First Step for WNS Signs
Search in the Territory,” 2010.
REFERENCES
Bilai, V.I., Metody eksperimental’noi mikologii (Methods of
Experimental Mycology), Kiev: Naukova Dumka, 1982.
Bills, G.F. and Foster, M.S., Formulae for selected materi
als used to isolate and study fungi and fungal allies, in Biodiver
sity of Fungi: Inventory and Monitoring Methods, Mueller, G.M.,
Ed., San Diego, CA: Elsevier, 2004, pp. 595–618.
Blehert, D.S., Hicks, A.C., Behr, M., et al., Bat whitenose
syndrome: An emerging fungal pathogen?, Science, 2009,
vol. 323, no. 5911, p. 227.
Carol, U.M., Buckles, E.L., Blehert, D.S., et al., Histo
pathologic criteria to confirm whitenose syndrome in bats,
J. Vet. Diagn. Invest., 2009, vol. 21, no. 4, pp. 411–414.
Chistiakov D.V., Nikulin A.D., Vlijanije antropogennogo
factora na sostojanije zimovok rukokrylych Lenongradskoj
oblasti (The impact of the anthropogenic factor on the state
of bat hibernaculum in Leningrad region), Abstract of
International Scientific Conference “Speleology ans Spe
leostology: development and cooperation,” 16–20 Nov.,
2010, Nabereznuje Chelny, pp. 320–322.
Cohn, J.P., Whitenose syndrome threatens bats, Bio
Science, 2008, vol. 58, no. 11, p. 1098.
Cryan, P., Meteyer, C., Boyles, J., and Blehert, D., Wing
pathology of whitenose syndrome in bats suggests life
threatening disruption of physiology, BioMed Central Biol.,
2010, vol. 8, pp. 135–143.
Flannigan, B., Air sampling for fungi in indoor environ
ments, J. Aerosol Sci., 1997, vol. 28, no. 3, pp. 381–392.
Gargas, A., Trest, M.T., Christensen, M., et al., Geomyces
destructans sp. nov. associated with bat whitenose syn
drome, Mycotaxon, 2009, vol. 108, pp. 147–154.
Godlevska, O.V., Ghazali, M.A., Tyschenko, V.M., et al.,
Resulats of the Winter Bat Census in Two Bat Sites in the

RUSSIAN JOURNAL OF ECOLOGY Vol. 46 No. 5 2015

 A SEARCH FOR Geomyces destructans, A DANGEROUS PATHOGEN OF BATS
Central Podolia (Ukraine), Vestnik Zoologii, 2011, vol. 45,
no. 1, pp. 34–37.
Hayes, M.A., The Geomyces fungi: Ecology and distribu
tion, BioScience, 2012, vol. 62, no. 9, pp. 819–823.
Niessen, L. and Vogel, R.F., Detection of Fusarium
graminearum DNA using a loopmediated isothermal
amplification (LAMP) assay, Int. J. Food Microbiol., 2010,
vol. 140, pp. 183–191.
Puechmaille, S.J., Verdeyroux, P., Fuller, H., et al., White
nose syndrome fungus (Geomyces destructans) in bat,
France, Emerg. Infect. Dis., 2010, vol. 16, pp. 290–300.
Puechmaille, S.J., Wibbelt, G., Korn, V., et al. PanEuro
pean distribution of whitenose syndrome fungus (Geomy
ces destructans) not associated with mass mortality, PLoS
One, 2011, vol. 6, e19167.
Raloff, J.D., Helping bats hold on, Sci. News, 2011, vol. 180,
pp. 22–55.
Strelkov P.P., Ecological monitoring of bat (Chiroptera,
Vespertilionidae) hibernation in Leningrad oblast), Tr. Zool.
Inst. Akad. Nauk SSSR, 1971, vol. 48, pp. 251–302.
Turner, G.G., Reeder, D.M., and Coleman, J.T.H., A five
year assessment of mortality and geographic spread of
whitenose syndrome in North American bats and a look to
the future, Bat Res. News, 2011, vol. 52, no. 33, pp. 13–27.
RUSSIAN JOURNAL OF ECOLOGY Vol. 46 No. 5
493
Tyshchenko V., Matveev M., and Bovtunova J., On the bat
fauna (Chiroptera) of Khmelnytski Region, Nauch. Byull.
Uzhgorod. Univ., Ser. Biol., 2005, no. 17, pp. 173–183.
Vanderwolf, K.J., McAlpine, D.F., Malloch, D., and
Forbes, G.J., Ectomycota associated with hibernating bats
in eastern Canadian caves prior to the emergence of white
nose syndrome, Northeast. Nat., 2013, vol. 20, no. 1,
pp. 115–130.
Vlaschenko, A.S. and Naglov, A.V., Hibernation of bats
(CHiroptera, Vespertilionidae) in artificial caves of nother
neastern Ukraine, J. Karazin Nats. Univ., Ser. Biol., vol. 729,
no. 3, pp. 168–175.
Warnecke, L., Turner, J.M., Bollinger, T.K., et al., Inocula
tion of bats with European Geomyces destructans supports
the novel pathogen hypothesis for the origin of whitenose
syndrome, Proc. Natl. Acad. Sci. U. S. A., 2012, vol. 109,
no. 18, pp. 6999–7003.
Wibbelt, G., Kurth, A., Hellmann, D., et al., Whitenose
syndrome fungus (Geomyces destructans) in bats, Europe,
Emerg. Infect. Dis., 2010, vol. 16, no. 8, pp. 1237–1243.
Translated by N. Gorgolyuk
2015