Inhibition of Pseudogymnoascus destructans Growth from Conidia and Mycelial Extension by Bacterially Produced Volatile Organic Compounds

Abstract

The recently identified causative agent of white-nose syndrome (WNS), Pseudogymnoascus destructans, has been implicated in the mortality of an estimated 5.5 million North American bats since its initial documentation in 2006 (Frick et al. in Science 329:679-682, 2010). In an effort to identify potential biological and chemical control options for WNS, 6 previously described bacterially produced volatile organic compounds (VOCs) were screened for anti-P. destructans activity. The compounds include decanal; 2-ethyl-1-hexanol; nonanal; benzothiazole; benzaldehyde; andN,N-dimethyloctylamine. P. destructans conidia and mycelial plugs were exposed to the VOCs in a closed air space at 15 and 4 °C and then evaluated for growth inhibition. All VOCs inhibited growth from conidia as well as inhibiting radial mycelial extension, with the greatest effect at 4 °C. Studies of the ecology of fungistatic soils and the natural abundance of the fungistatic VOCs present in these environments suggest a synergistic activity of select VOCs may occur. The evaluation of formulations of two or three VOCs at equivalent concentrations was supportive of synergistic activity in several cases. The identification of bacterially produced VOCs with anti-P. destructans activity indicates disease-suppressive and fungistatic soils as a potentially significant reservoir of biological and chemical control options for WNS and provides wildlife management personnel with tools to combat this devastating disease.

Full text

Inhibition of Pseudogymnoascus destructans Growth
from Conidia and Mycelial Extension by Bacterially
Produced Volatile Organic Compounds
Christopher T. Cornelison •Kyle T. Gabriel •
Courtney Barlament •Sidney A. Crow Jr.
Received: 8 August 2013 / Accepted: 22 October 2013 / Published online: 5 November 2013
ÓSpringer Science+Business Media Dordrecht 2013
Abstract The recently identified causative agent of
white-nose syndrome (WNS), Pseudogymnoascus
destructans, has been implicated in the mortality of
an estimated 5.5 million North American bats since its
initial documentation in 2006 (Frick et al. in Science
329:679–682, 2010). In an effort to identify potential
biological and chemical control options for WNS, 6
previously described bacterially produced volatile
organic compounds (VOCs) were screened for anti-
P. destructans activity. The compounds include dec-
anal; 2-ethyl-1-hexanol; nonanal; benzothiazole;
benzaldehyde; andN,N-dimethyloctylamine. P. de-
structans conidia and mycelial plugs were exposed
to the VOCs in a closed air space at 15 and 4 °C and
then evaluated for growth inhibition. All VOCs
inhibited growth from conidia as well as inhibiting
radial mycelial extension, with the greatest effect at
4°C. Studies of the ecology of fungistatic soils and the
natural abundance of the fungistatic VOCs present in
these environments suggest a synergistic activity of
select VOCs may occur. The evaluation of formula-
tions of two or three VOCs at equivalent
concentrations was supportive of synergistic activity
in several cases. The identification of bacterially
produced VOCs with anti-P. destructans activity
indicates disease-suppressive and fungistatic soils as
a potentially significant reservoir of biological and
chemical control options for WNS and provides
wildlife management personnel with tools to combat
this devastating disease.
Keywords Pseudogymnoascus destructans 
Mycelia VOC White-nose syndrome 
Fungistatsis
Introduction
White-nose syndrome (WNS) was first documented
near Albany, New York, in 2006 [2,15]. Since its
discovery, WNS has caused severe declines in bat
populations in the Eastern United States and Canada
[9,12,23]. Although the exact ecological and
economic impact of this disease has yet to be
determined, many researchers agree that continued
declines in insectivorous bat populations will have a
significant impact on forest management, agriculture
and insect-borne disease [4]. The rapid spread of WNS
and the high mortality rates associated with the disease
[9,13] necessitate the rapid development of disease
management tools. In 2011, the fungus Geomyces
destructans was shown to be the putative causative
agent of WNS [18].
Electronic supplementary material The online version of
this article (doi:10.1007/s11046-013-9716-2) contains supple-
mentary material, which is available to authorized users.
C. T. Cornelison (&)K. T. Gabriel C. Barlament 
S. A. Crow Jr.
Applied and Environmental Microbiology, Georgia State
University, 24 Peachtree Center Ave, Atlanta, GA, USA
e-mail: ctcornelison1@gsu.edu
123
Mycopathologia (2014) 177:1–10
DOI 10.1007/s11046-013-9716-2

Recently, the fungus Geomyces destructans has
been reclassified as Pseudogymnoascus destructans
[19,20]. P. destructans is a psychrophilic ascomycete
with optimal growth at 12.5–15.8 °C[15,23]. Its
psychrophilic nature makes P. destructans ideally
suited for colonization of bats in torpor, when body
temperatures and immune function are greatly
depressed [3,5]. The clinical manifestation of P.
destructans infection is characterized by fuzzy white
growth on the muzzle and wings of hibernating bats
and results in severe physical damage to bat wing
membranes [8]. Due to the recent identification of P.
destructans, many ecological and physiological traits
and their influence on virulence are yet to be
elucidated.
The rapid spread and high mortality rates associated
with WNS make the development of in situ treatment
options for P. destructans a significant objective for
wildlife management agencies. Accordingly, the
development of biological and chemical treatment
options is a priority for State and Federal agencies as
stated in the 2011 National WNS Management Plan
established by the United States Fish and Wildlife
Service (USFWS) [1]. With this goal in mind, 6
previously described bacterially produced antifungal
VOCs [7,11] were assayed for their in vitro potential
to inhibit the growth and proliferation of P.
destructans.
Previous investigations of fungistatic soils were
able to identify bacteria that produced antifungal
VOCs which were later identified via SPME/GC/MS
of cultures and soils. The VOCs were produced by
Pseudomonas and Bacillus spp. and demonstrated
broad spectrum antifungal activity [7,11]. Volatile-
based fungistasis in soils has been observed in
terrestrial environments around the globe [25]. Due
to the biological and chemical complexity of these
environments, the ultimate source of the active VOCs
is often unknown but typically attributed to bacteria
[16,25]. The geology and ecology of soil make the
presence of inhibitory volatiles of particular interest,
as low levels of VOCs are able to inhibit fungal growth
in a dense, compartmentalized, and diverse ecosystem
[10,11,14,16]. Using the soil ecosystem as an ideal
example of naturally occurring biological control of
fungal proliferation, we began to investigate biolog-
ically derived VOCs with known antifungal activity.
The influence of the VOCs on the growth from
conidia and mycelial extension of P. destructans was
evaluated using digital imaging techniques. In an
effort to optimize the efficacy of the VOCs for
potential in situ applications, formulations of VOCs
were evaluated for potential synergistic effects. Com-
binations of two VOCs applied at equal quantities as
individual VOCs revealed several potentially syner-
gistic combinations. Accordingly, these synergistic
blends were used to establish formulations of three
VOCs ultimately yielding highly effective formula-
tions with greatly increased anti-Pseudogymnoascus
activity at relative concentrations of \1 ppm. The
identification of biologically produced inhibitory
volatiles expands the pool of potential biocontrol
agents of P. destructans, and the development of
chemical formulations with significant anti-Pseud-
ogymnoascus activity at low concentrations provides
promising chemical control options for in situ man-
agement of WNS.
Materials and Methods
Culture Acquisition and Maintenance
All P. destructans isolates used in the project were
provided by Kevin Keel through his WNS diagnostic
work at the University of Georgia’s Southeastern
Cooperative Wildlife Disease Study (SCWDS). P.
destructans cultures were maintained on Sabauroud
Dextrose Agar (SDA) or in Sabauroud Dextrose Broth
(SDB) (BD, Maryland) at 4–15 °C. P. destructans
spores were stored in phosphate-buffered saline (PBS)
at -20 °C. Spores were stored no longer than 3 weeks
prior to use.
VOC Exposure Assays and Evaluation
of Bacterially Produced VOCs for Anti-P.
destructans Activity
Volatile organic compounds previously shown to be
produced by bacteria [7,11] were screened for anti-P.
destructans activity via VOC exposure to spores and
mycelial plugs. The VOCs included: decanal; 2-ethyl-
1-hexanol; nonanal; benzothiazole; benzaldehyde; and
N,N-dimethyloctylamine (Sigma-Aldrich, Missouri).
All VOCs were chosen based on their identification in
fungistatic soils and their observed production in
bacteria [7,11]. All VOCs purchased as pure, liquid,
research grade reagents and used directly, without
2 Mycopathologia (2014) 177:1–10
123

modification, in all subsequent assays. A single-com-
partment Petri plate (150 mm 915 mm) was used for
a contained airspace to assess P. destructans growth
characteristics in the presence of fungistatic VOCs. Ten
microliters of P. destructans conidia suspension (10
6
conidia ml
-1
in PBS) was spread onto SDA plates
(35 mm 910 mm). Aliquots of 30, 3.0, or 0.3 llof
each VOC corresponding to maximum possible relative
concentrations ranging from 113 ppm (v/v) to
1.13 ppm (v/v) (Table S1) were pipetted onto a sterile
filter paper disk (12.7 mm) on a watch glass (75 mm).
Each VOC containing disk and watch glass was placed
inside a large Petri plate (150 mm 915 mm) along
with a P. destructans-inoculated SDA plate
(35 mm 910 mm) (Fig. 1). P. destructans mycelial
plugs cut from the leading edge of actively growing
colonies were inserted into fresh SDA plates
(35 mm 910 mm) and placed in large Petri plates
(150 mm 915 mm) with each formulation or pure
VOC containing paper disk and sealed with parafilm M
(Sigma-Aldrich, Missouri). Plates were then incubated
at 15 °C for 21 days. Unexposed cultures and the
addition of activated carbon to exposure assays served
as negative controls for each trial. Anti-P. destructans
activity was scored on a plus/minus scale for conidia-
inoculated plates, and the radial growth from mycelial
plugs was used to determine percent inhibition by
comparing growth area of VOC exposed plugs to
unexposed controls. All assays were performed in
triplicate and averaged.
VOC Formulation Assay for Anti-P. destructans
Activity
VOC formulations utilizing combinations of two pure
VOCs were created with all fifteen possible combina-
tions of the six VOCs by applying volumes corre-
sponding to 2.0 lmol of each VOC to separate
absorbent disks and arranging combinations of two
disks of different VOCs on a single watch glass.
Volumes corresponding to 4.0 lmol of each pure VOC
were used as synergism controls to determine syner-
gism. P. destructans mycelial plugs were harvested
and inserted into fresh SDA plates (35 mm 910 mm)
and sealed with parafilm in large Petri plates
(150 mm 915 mm) with each formulation or pure
VOC. Plates were then incubated at 15 °C for 21 days
as described above. Each test was conducted in
triplicate. Area measurements were conducted every
2 days post-inoculation with the use of digital pho-
tography and computer analysis as described below.
Area Measurement of Radial Growth with Digital
Photography and Open-Source Software
Filamentous fungi grow by hyphae elongation and not
by distinct cellular division. Accordingly, measuring
the difference between the area growth of control
agent-exposed mycelial plugs and control plugs has
been a vetted method for assessing antimicrobial
susceptibility [11,17,22]. The use of a ruler to
measure the area of mycelial growth of filamentous
fungi has its own challenges. Mycelial plugs will often
grow asymmetrically, either naturally or because of
exposure to the compound being tested. To provide
more accurate measurement of mycelial growth, a
digital photography and analysis technique was
developed.
The GIMP (GNU Image Manipulation Program) is
open-source, freely distributed software for image
editing and authoring, compatible with GNU/Linux,
Microsoft Windows, Mac OS X, Sun OpenSolaris, and
FreeBSD operating systems. This software allows for
the direct measurement of the number of pixels in a
given selected area of a photograph. GIMP version
2.8.2 for Microsoft Windows was used at the time of
this writing. A Nikon D3100 digital single lens reflex
camera with an 18–55 mm lens was used to capture
images. A standard three-leg tripod was used for
support during capture.
Fig. 1 Shared airspace assay with previously described bacte-
rially produced VOCs
Mycopathologia (2014) 177:1–10 3
123

The camera was attached to the tripod and aimed
down to a surface to provide a consistent distance from
the lens to the mycelial surface being photographed;
ensuring the same pixel to millimeter ratio was
retained for all acquired images. Images of mycelial
plugs had their corresponding image numbers cata-
logued for later identification. All Petri plate agar
heights were similar to ensure the focal point remained
consistent as well as to retain the same pixel to
distance ratio. Manual focus was activated to retain the
same focal point throughout all image captures, and a
remote shutter release device was used to assure
stable, shake-free images were acquired.
Contrast between the growth medium and myce-
lium was required to obtain an accurate selection for
measurement as well as to be able to discern the
margin of the ruler graduation marks with GIMP.
Therefore, the camera’s white balance, exposure,
f-stop, and ISO were adjusted to retain a consistent
contrast between photograph acquisitions. A photo-
graph of a ruler was used to set the focal point for the
proceeding photographs as well as serving as a
calibration device for determining the length of each
pixel during image analysis.
The ruler tool was used to determine the number of
pixels between two demarcations of a photographed
ruler placed at the level of the agar surface in the Petri
plates. The resulting pixel count was used to determine
the millimeter-to-pixel ratio.
A different set of tools were necessary to measure
the mycelial area. The selection tools were used to
outline the margin of the mycelia. The Histogram tool
was used to determine the number of pixels that
comprised the selected area. The area of the selection
was converted from the number of pixels to mm
2
with
our previously derived number of pixels per mm and
Eq. 1.
Number of pixels in are a
Number of pixels per mm

2
= Area of mycelia in mm2
ð1Þ
Tape Mount Preparation and Microscopic
Evaluation
Pseudogymnoascus destructans cultures with aberrant
phenotypes as compared to control cultures and pub-
lished descriptions [14] were examined microscopically
by tape mount. The adhesive side of standard
transparent packaging tape was gently pressed against
the surface ofplate grown fungal colonies. The resulting
tape-adhered sample was treated with 10 llof70%
ethanol and placed onto a microscope slide with
lactophenol cotton blue dye. Slides were viewed on a
light microscope (Nikon optiphot-2) at 200 9magni-
fication and images captured using a scope mounted
camera (QImaging micropublisher 3.3 RTV).
Results
Anti-P. destructans Activity of Bacterially
Produced Volatiles
Initial investigation demonstrated inhibitory activity
for most VOCs at relative concentrations less than
1 ppm. Decanal; 2-ethyl-1-hexanol; nonanal; benzo-
thaizole; dimethyltrisulfide; benzaldehyde; and N,N-
dimethyloctylamine all demonstrated anti-P. destruc-
tans activity when 30 ll of the respective compound
were placed adjacent to SDA plates inoculated with P.
destructans conidia in a closed system at 15 °C
(Table 1). Control plates containing 1 g activated
carbon showed no inhibition for decanal; 2-ethyl-1-
hexanol; and benzaldehyde, while the remaining
compounds inhibitory activity persisted in the pre-
sence of activated carbon (Table 1). Subsequent
assays with 3 ll of each compound demonstrated
similar results with only N,N-dimethyloctylamine
unable to completely inhibit P. destructans growth
from conidia at 7 days (Table 1). The addition of
activated carbon abolished all inhibitory activity of the
assayed compounds at 3 ll (Table 1). At 11 days of
exposure to 3 ll of each respective compound, only
2-ethyl-1-hexanol, decanal, and nonanal demonstrated
inhibitory activity, with all activated carbon controls
abolishing the inhibitory activity (Table 1). Addition-
ally, P. destructans cultures from conidia exposed to
3ll benzothiazole without activated carbon revealed
unique colony morphology characterized by increased
pigmentation of the underside of the culture and
diffusion of pigment into the growth media as
compared to unexposed cultures and cultures exposed
to benzothiazole in the presence of activated carbon
(Fig. S1).
Assays using mycelial plugs cut from the leading
edge of actively growing P. destructans colonies on
SDA exposed to the previously described bacterially
4 Mycopathologia (2014) 177:1–10
123

produced volatiles at 30, 3, and 0.3 ll of each
respective compound and incubated in a contained
air space at 15 °C gave varied results. At 30 ll, all
compounds completely inhibited the growth of P.
destructans mycelia for up to 9 days (Fig. 2a). At
14 days of exposure, only P. destructans plugs
exposed to decanal showed any radial growth, with
83 % reduction in growth as compared to unexposed
controls (Fig. 2a). At 3 ll of each compound, decanal
and N,N-dimethyloctylamine yielded only minor
reductions in radial growth, whereas the remaining
compounds completely inhibited radial mycelial
growth of P. destructans for up to 14 days (Fig. 2b).
At 0.3 ll of each compound, only benzothiazole
demonstrated significant inhibitory activity with a
60 % reduction in radial growth after 14 days of
exposure (Fig. 2c). Interestingly, at 0.3 ll, N,N-
dimethyloctylamine induced growth as compared to
unexposed controls (Fig. 2c). This result may be due
to hormesis [21].
In order to forecast the in situ efficacy of the VOCs
additional in vitro evaluation was conducted at 4 °Cto
more accurately represent the environmental conditions
of North American hibernacula. Exposure to 30 llor
3.0 ll of each respective VOC completely inhibited
radial growth of P. destructans for greater than 21 days
(data not shown). Exposure to 0.3 ll of each respective
VOC inhibited radial growth for all VOCs except
benzaldehyde (Fig. 2d). The greatest degree of inhibi-
tion was observed with decanal which demonstrated a
greater than 99 % reduction in growth area at 35 days
post-inoculation (Fig. 2d). Based on these initial results,
VOC exposure was standardized to 4.0 lmol per
headspace for subsequent evaluations. In addition to
evaluating individual VOCs, formulations were inves-
tigated for potential synergistic effects.
VOC Formulations Demonstrate Synergistic Anti-
P. destructans Activity
Three VOC formulations comprised of two VOCs
were observed to synergistically inhibit the growth of
P. destructans mycelial plugs, more than the com-
bined inhibition of each of the pure VOCs alone.
Table 1 Evaluation of anti-P. destructans activity of bacterially produced antifungal VOCs with P. destructans conidia
VOC Chemical structure 30 ll30ll
c
3ll
a
3ll
a,c
3ll
b
3ll
b,c
2-ethyl-1-hexanol 21 21 21
Benzaldehyde 21 21 11
Benzothiazole 22 21 11
Decanal 21 21 21
Nonanal 22 21 21
N,N-dimethyloctylamine 22 11 11
Control 11 11 11
?, growth from spores; -, no visible growth
a
7 day exposure
b
10 day exposure
c
Incubated with activated carbon
Mycopathologia (2014) 177:1–10 5
123

Those include 2-ethyl-1-hexanol and benzaldehyde;
2-ethyl-1-hexanol and nonanal; 2-ethyl-1-hexanol and
decanal; and 2-ethyl-1-hexanol and N,N-dimethyloc-
tylamine (Fig. 3a, 3b, 3c, respectively). The greatest
inhibition by the formulation occurred with 2-ethyl-1-
hexanol and nonanal, which demonstrated greater than
95 % reduction in growth as compared to unexposed
controls 14 days post-inoculation (Fig. 3c).
Two VOC formulations comprised of three VOCs
at 1.33 lmol, respectively, were observed to syner-
gistically inhibit the growth of P. destructans mycelial
plugs, more than the combined inhibition of each of the
pure VOCs alone at 4.0 lmol. Those include 2-ethyl-
1-hexanol; benzaldehyde; and decanal; as well as
2-ethyl-1-hexanol; nonanal; and decanal (Fig. 4a, 4b).
Discussion
Since its initial documentation in 2006, P. destruc-
tans has spread to twenty-four states and four
Canadian providences and is implicated in the
mortality of 5.5 million bats [13]. Cave closures
and culling of infected individuals appear to have
little to no impact on the spread and mortality
associated with this devastating disease. Classic
disease management practices applied in agriculture
such as broad spectrum dissemination of antibiotics
are not realistic options for management of disease
in wild, highly disseminated, and migratory animal
populations. Accordingly, the development of novel
treatment options is needed to avert the spread of
0.00
20.00
40.00
60.00
80.00
100.00
Area (mm2)
Days post inoculation
a
Control (No VOC)
Decanal
0.00
20.00
40.00
60.00
80.00
100.00
Area (mm2)
Days post inoculation
b
Control (No VOC)
Decanal
N,N-
dimethyloctylamine
0.00
20.00
40.00
60.00
80.00
100.00
120.00
Area (mm2)
Days post inoculation
c
Control (No VOC)
2-ethyl-1-hexanol
Benzaldehyde
Benzothiazole
Decanal
Nonanal
N,N-dimethyloctylamine
0.00
10.00
20.00
30.00
40.00
50.00
60.00
70.00
80.00
90.00
024681012 024681012
0 2 4 6 8 10 12 0 2 4 6 8 10 12 14 16 18 20 22 24 26 28 30 32 34
Area (mm2)
Days post inoculation
d
Control (No VOC)
2-ethyl-1-hexanol
Benzaldehyde
Benzothiazole
Decanal
Nonanal
N,N-
dimethyloctylamine
Fig. 2 Growth areas of P. destructans mycelial plugs exposed
to bacterially produced VOCs at 15 °Cat30ll(a), 3 ll(b),
0.3 ll(c), respectively. Growth area of mycelial plugs exposed
at 4 °C to 0.3 ll(d) of bacterially produced VOCs. VOCs not
shown in the legend completely inhibited radial growth for the
duration of the experiment
6 Mycopathologia (2014) 177:1–10
123

this disease and reduce the mortality associated with
currently infected hibernacula. To this end, the
evaluation of previously described bacterially pro-
duced antifungal volatiles was conducted to identify
potential chemical control agents as well as identify
potential environmental reservoirs of anti-P. de-
structans activities and expand the pool of potential
biological control agents.
Bacterially derived volatile fungistasis is a well-
documented microbial antagonism and may be com-
mon in terrestrial ecosystems [7,10,11,14,16,25].
Harnessing the potential of these natural antagonisms
is already a powerful tool in the development of highly
effective biological and chemical control options [7,
11,17]. The biological origin of many fungistatic
VOCs lends itself to obtainable inhibitory applications
20.00
40.00
60.00
80.00
100.00
120.00
140.00
160.00
02468101214
Area (mm2)
Days post inoculation
Control
2-ethyl-1-hexanol
Benzaldehyde
2-ethyl-1-hexanol +
Benzaldehyde
a
18.00
38.00
58.00
78.00
98.00
118.00
138.00
Area (mm2)
Days post inoculation
Control
2-ethyl-1-hexanol
Decanal
2-ethyl-1-hexanol + Decanal
b
20.00
40.00
60.00
80.00
100.00
120.00
140.00
02 4 6 8101214
02 4 6 8101214
Area (mm2)
Days post inoculation
Control
2-ethyl-1-hexanol
Nonanal
2-ethyl-1-hexanol + Nonanal
c
Fig. 3 Growth areas of P.
destructans mycelial plugs
exposed to each individual
VOC as well as formulations
at 15 °C. Measurements
taken every 2 days for
14 days. a2-ethyl-1-
hexanol and benzaldehyde,
b2-ethyl-1-hexanol and
decanal, and c2-ethyl-1-
hexanol and nonanal
Mycopathologia (2014) 177:1–10 7
123

due to the typically low level of production in the
natural hosts and the significant antagonistic activity
observed at these low levels [7,10,14,16,21,24]. The
contact-independent activity of antagonistic VOC has
several advantages over topical and oral, contact-
dependent, treatment options that have been shown to
be highly effective at inhibiting the growth of P.
destructans in previous studies [6]. Contact-indepen-
dent antagonisms allow for treatment of many indi-
viduals with a single application and ensure uniform
exposure, avoiding the potential for microbial refugia
on the host that may facilitate re-colonization of the
host once the inhibitory compound has been removed
or degraded. Accordingly, the evaluation of previously
described bacterially produced VOCs reduces the
processing required to identify viable treatment
options, and the contact-independent activity of
antagonistic VOCs has several advantages over topical
and oral antifungal compounds and should be a focus
of studies tasked with identifying novel treatments for
newly emerging fungal diseases.
The coevolution of soil microbiota has produced
antagonisms ideally suited for the complex ecology of
soil. Harnessing these natural antagonisms can be a
powerful tool in combating WNS as many of the traits
of these antagonisms equate favorably with the
ecology of hibernacula and the terrestrial heritage of
the Geomycota and Pseudogymnoascus spp. warrants
their susceptibility. The long-term efficacy of low
quantities of VOCs illustrates the potential of these
compounds for in situ application in the treatment of
WNS. Additionally, the development of synergistic
blends bolsters the appeal of soil-based fungistasis as a
source of potential control agents as VOC mixtures are
likely responsible for the observed fungistatic activity
of repressive soils [14,16,24]. While several pure
VOCs and blends produced significant growth inhibi-
tion, compounds and/or quantities unable to signifi-
cantly inhibit growth caused noteworthy stress to P.
destructans as determined by the abnormal pheno-
types observed under these conditions (Fig. S1). The
evaluation of bacterially derived VOCs has expanded
the pool of potential biological control agents as well
produced several VOC formulations with excellent
anti-Pseudogymnoascus activity. The availability of
volatile formulations for control of P. destructans
15.00
35.00
55.00
75.00
95.00
115.00
135.00
155.00
Area (mm2)
Days Post inoculation
a
Control
2-ethyl-1-hexanol
Benzaldehyde
Decanal
2-ethyl-1-hexanol +
Benzaldehyde +
Decanal
15.00
35.00
55.00
75.00
95.00
115.00
135.00
155.00
0 1 2 3 4 5 6 7 8 9 10 11 12 13 14
0 1 2 3 4 5 6 7 8 9 10 11 12 13 14
Area (mm2)
Days post inoculation
bControl
2-ethyl-1-hexanol
Nonanal
Decanal
2-ethyl-1-hexanol
+ Nonanal +
Decanal
Fig. 4 Growth areas of P.
destructans mycelial plugs
exposed to each individual
VOC as well as formulations
at 15 °C. Measurements
taken every 2 days for
14 days. a2-ethyl-1-
hexanol; benzaldehyde; and
decanal. b2-ethyl-1-
hexanol; nonanal; and
decanal
8 Mycopathologia (2014) 177:1–10
123

growth could prove to be a powerful tool for wildlife
management agencies if appropriate application meth-
ods can be developed.
Current technology for dissemination of VOCs and
essential oils for control of odors and pests in indoor
environments is common with several companies,
including TimeMist
tm
, Aire-Master
tm
, Prolitec
tm
and
Air-Scent
tm
actively marketing these systems. Manu-
facturers claims vary significantly depending on
product with treatment capacities varying from 6,000
to 50,000 ft
3
for a single unit and maintain 1–10 ppm
concentrations in that area based on timed releases
with various product lines. Although these claims are
promising, appropriate scientific validation is lacking.
Fragrance dispenser systems are compatible with a
wide range of VOC and essential oils and their efficacy
claims warrant further investigation as a potential
application method for anti-Pseudogymnoascus VOCs
in the treatment of WNS in hibernating bats.
The ecology of susceptible bat populations makes
treatment of WNS difficult. The highly dispersed,
difficult to access, and potentially hazardous nature of
bat hibernacula require the development of control
options with persistent inhibitory activity at low
levels. Our results indicate a strong inhibitory activity
for several compounds and have provided valuable
leads for identification of potential biological control
agents. The increased inhibitory effect observed in low
temperature (4 °C) exposures is promising for field
application as they represent a promising duration at
the low temperatures associated with hibernacula
during the time of infection and could have a
significant impact on the mortality associated with
infected hibernacula. Currently, the prognosis for
susceptible North American bat populations is bleak at
best. The development of biological and chemical
treatment options must be investigated to provide
wildlife management agencies with tools for control of
P. destructans transmission and infectivity.
This project identified biologically derived chem-
ical control agents that can potentially disrupt trans-
mission by inhibiting growth from conidia as well as
decrease infection/mortality rates in hibernacula cur-
rently infected with P. destructans. Additionally,
several of the compounds evaluated in this study have
the potential for application in captive-recovery
programs purposed in the National Response Plan
[1]. Cumulatively, this study has highlighted potential
control options for further investigation for
application in management of WNS as well as
identified fungistatic soils as a potentially significant
reservoir of biological control agents.
Acknowledgments This work was funded by the Georgia State
University Department of Biology, as well as the Georgia State
University Environmental Research Program. The authors would
like to thank Kevin Keel for providing the fungal cultures used in
this study. The authors would also like to thank Ian Sarad, Blake
Cherney, and Ben Poodiak for their contributions to this effort.
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