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Background The recently identified causative agent of White-Nose Syndrome (WNS), Pseudogymnoascus destructans, has been responsible for the mortality of an estimated 5.5 million North American bats since its emergence in 2006. A primary focus of the National Response Plan, established by multiple state, federal and tribal agencies in 2011, was the identification of biological control options for WNS. In an effort to identify potential biological control options for WNS, multiply induced Rhodococcus rhodochrous strain DAP96253 was screened for anti-P. destructans activity.ResultsConidia and mycelial plugs of P. destructans were exposed to induced R. rhodochrous in a closed air-space at 15°C, 7°C and 4°C and were evaluated for contact-independent inhibition of conidia germination and mycelial extension with positive results. Additionally, in situ application methods for induced R. rhodochrous, such as fixed-cell catalyst and fermentation cell paste in non-growth conditions, were screened with positive results. R. rhodochrous was assayed for ex vivo activity via exposure to bat tissue ex-plants inoculated with P. destructans conidia. Induced R. rhodochrous completely inhibited growth from conidia at 15°C and had a strong fungistatic effect at 4°C. Induced R. rhodochrous inhibited P. destructans growth from conidia when cultured in a shared air-space with bat tissue explants inoculated with P. destructans conidia.Conclusion The identification of inducible biological agents with contact-independent anti- P. destructans activity is a major milestone in the development of viable biological control options for in situ application and provides the first example of contact-independent antagonism of this devastating wildlife pathogen.
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R E S E A R C H A R T I C L E Open Access
A preliminary report on the contact-independent
antagonism of Pseudogymnoascus destructans by
Rhodococcus rhodochrous strain DAP96253
Christopher T Cornelison
1*
, M Kevin Keel
2
, Kyle T Gabriel
1
, Courtney K Barlament
1
, Trudy A Tucker
1
,
George E Pierce
1
and Sidney A Crow Jr
1
Abstract
Background: The recently-identified causative agent of White-Nose Syndrome (WNS), Pseudogymnoascus
destructans, has been responsible for the mortality of an estimated 5.5 million North American bats since its
emergence in 2006. A primary focus of the National Response Plan, established by multiple state, federal and tribal
agencies in 2011, was the identification of biological control options for WNS. In an effort to identify potential
biological control options for WNS, multiply induced cells of Rhodococcus rhodochrous strain DAP96253 was
screened for anti-P. destructans activity.
Results: Conidia and mycelial plugs of P. destructans were exposed to induced R. rhodochrous in a closed air-space
at 15°C, 7°C and 4°C and were evaluated for contact-independent inhibition of conidia germination and mycelial
extension with positive results. Additionally, in situ application methods for induced R. rhodochrous, such as
fixed-cell catalyst and fermentation cell-paste in non-growth conditions, were screened with positive results.
R. rhodochrous was assayed for ex vivo activity via exposure to bat tissue explants inoculated with P. destructans
conidia. Induced R. rhodochrous completely inhibited growth from conidia at 15°C and had a strong fungistatic
effect at 4°C. Induced R. rhodochrous inhibited P. destructans growth from conidia when cultured in a shared
air-space with bat tissue explants inoculated with P. destructans conidia.
Conclusion: The identification of inducible biological agents with contact-independent anti- P. destructans activity
is a major milestone in the development of viable biological control options for in situ application and provides the
first example of contact-independent antagonism of this devastating wildlife pathogen.
Keywords: Pseudogymnoascus destructans, Mycelia, Conidia, Rhodococcus rhodochrous, White-Nose Syndrome,
Biocontrol
Background
The rapid spread and high mortality rates associated with
white-nose syndrome (WNS) make the development of in
situ treatment options for the causative agent, Pseudogym-
noascus destructans [1,2], a significant objective for wild-
life management agencies. Accordingly, the development
of biologically-derived treatment options may have advan-
tages over chemical or physical treatments, since classic
examples of chemical and physical treatments in karst
environments are now a cautionary tale [3]. To this end,
A National Plan for Assisting States, Federal Agencies,
and Tribes in Managing White-Nose Syndrome in Bats
[4] was released in May, 2011. In this plan, significant
focus was placed on the identification and development of
biological control options for WNS.
Rhodococcus rhodochrous strain DAP 96253 is a ubi-
quitous, soil-associated, Gram-positive bacterium with
tremendous metabolic and physiological diversity [5-9].
Rhodococcus rhodochrous has been used extensively in
bioremediation as well as in the production of nitrile-
containing compounds [5-7] and it has demonstrated
delayed fruit ripening activity with climacteric fruits and
* Correspondence: ccornelison1@gsu.edu
Equal contributors
1
Applied and Environmental Microbiology, Georgia State University, 161
Jesse Hill Jr. Dr, Atlanta, GA, USA
Full list of author information is available at the end of the article
© 2014 Cornelison et al.; licensee BioMed Central Ltd. This is an Open Access article distributed under the terms of the
Creative Commons Attribution License (http://creativecommons.org/licenses/by/4.0), which permits unrestricted use,
distribution, and reproduction in any medium, provided the original work is properly credited. The Creative Commons Public
Domain Dedication waiver (http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this
article, unless otherwise stated.
Cornelison et al. BMC Microbiology 2014, 14:246
http://www.biomedcentral.com/1471-2180/14/246
vegetables [8]. Several enzymes have been shown to have
increased activity and prevalence in bacteria induced to
delay fruit ripening and these enzymes may play a role in
the observed antifungal activity [8]. Initial investigation of
the potential antagonism of P. destructans by R. rhodo-
chrous indicated that, when induced under the protocol
outlined in US patents 7,531,343, and 7,531,344 [10,11],
R. rhodochrous strain DAP 96253 demonstrated significant
contact-independent antagonism of P. destructans in vitro.
As a result, the principal objective of this was evalu-
ation of R. rhodochrous induced with urea for potential
in situ application as a biological control agent for
P. destructans.
In addition to the strong evidence established via
in vitro analysis of the observed antagonism, the eva-
luation of the efficacy of induced R. rhodochrous was
pursued in order to establish in vivo efficacy at preven-
ting fungal invasion of bat tissue. This goal was accom-
plished using a bat-skin explant assay. The evaluation of
induced R. rhodochrous to prevent or reduce the infect-
ive potential of P. destructans conidia was demonstrated
by the inhibition of P. destructans growth on living bat
tissue. This is the first example of antifungal efficacy on
living bat skin for any biological control agent of WNS
and represents a major milestone in this effort.
In order to optimize biocontrol efficacy and reduce
potential cross-contamination of karst environments,
various whole- and fixed-cell applications were investi-
gated. The evaluation of various application methods of
induced cells of R. rhodochrous for potential in situ ap-
plication, including whole-cell application, non-growth
fermentation cell-paste, and fixed-cell catalyst [8,12,13],
were conducted. Non-growth fermentation cell-paste de-
monstrated persistent inhibitory activity and represents
the most promising application method evaluated. The
associated cell-paste activity is a significant development
as it represents multiple hallmarks of ideal biocontrol
agents.
Methods
Culture acquisition and maintenance
All P. destructans isolates used in the project were acquired
from the WNS diagnostic lab at The University of Georgia
Southeastern Cooperative Wildlife Disease Study (UGA
SCWDS). Initial investigations have shown very low ge-
netic and physiological variability amongst P. destructans
isolates [14]. Accordingly, all assays were conducted with
a small isolate sample size (n3). P. destructans cultures
were maintained on Sabouraud Dextrose Agar (SDA,
Difco) or in Sabouraud Dextrose Broth (SDB, Difco) at
4°C, 7°C, or 15°C depending on anticipated usage.
P. destructans conidia were harvested from fungal lawns
on SDA plates by adding 10 ml of conidia harvesting solu-
tion (CHS; 0.05% Tween 80, 0.9% NaCl) to the surface of
the plate and gently scrapping with a sterile loop to dis-
lodge conidia. The resulting solution was filtered through
glass wool and centrifuged at 5000 rpm for 10 minutes.
The resulting supernatant was removed and the spore pel-
let washed with 5 mL of sterile phosphate buffered saline
(PBS, pH = 7), re-suspended, and filtered through glass
wool. Conidia were stored in sterile PBS at 20°C. Conidia
were stored no longer than six weeks prior to use based
on in-house assessment of conidial viability under these
conditions (unpublished data). R. rhodochrous strain DAP
96253 cells were maintained as glycerol stock aliquots
(30% v/v) from 10 l fermentations carried out at GSU.
Fresh glycerol stocks were used as the source of cells at
the onset of each assay. The induction process was per-
formed using the addition of urea or urea and cobalt as
described in US patents 7,531,343 and 7,531,344 [8,10,11].
Co-culture assays with R. rhodochrous
A single-compartment Petri plate (150 mm × 15 mm) was
used for a contained air-space to assess P. destructans
growth characteristics in the presence of induced cells of
R. rhodochrous.A10μl inoculum of P. destructans conidia
solution (10
6
ml
1
) in a phosphate buffer solution was
spread onto SDA in Petri plates (35 mm x 10 mm). Multi-
ply induced cells of R. rhodochrous [10,11] were inocu-
lated onto Petri plates (35 mm × 10 mm) containing Yeast
Extract/Malt Extract agar (YEMEA) with or without urea
(7.5 g/l) [8], and cultured in the contained air-space for up
to 30 days. All assays were conducted in triplicate. The
ability of induced R. rhodochrous to inhibit healthy estab-
lished hyphae of P. destructans was assessed using myce-
lial plug assays. A lawn of P. destructans was allowed to
grow for up to 20 days at which time a 5-mm-diameter
transfer tube was used to remove a plug from the mat of
fungus. The plugs were then inserted into a similarly sized
core removed from an uninoculated culture plate. The
plates were co-incubated in a shared air-space as described
previously and radial growth from the plug was assessed
over time.
Induced R. rhodochrous germule suppression assay
Thin layers (~750 μl) of 10% SDA were applied to stan-
dard microscope slides (24.5 × 76.2 mm) and 100 μlof
P. destructans conidia solution (10
6
ml
1
) were spread
across the agar surface. R. rhodochrous-inoculated Petri
plates (35 mm × 10 mm) were placed in larger Petri
plates (150 mm × 15 mm) and sealed with parafilm.
Negative controls consisted of similarly-cultured conidia
with no R. rhodochrous exposure. All trials were con-
ducted in triplicate. At 4 and 7 days post-inoculation,
conidia were observed in a light microscope at 200X
magnification for the presence of germule formation.
Germules were defined as single mycelial extensions em-
anating from conidia with a length equal to or greater
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than the intact conidia. Control and exposed slides were
retained and examined daily for up to 21 days after
germule formation was first observed on control slides.
Recovery of conidia was determined by removing the
R. rhodochrous after 24 hours, 72 hours, and 7 days.
Slides were observed for 21 days after removal of control
agent to assess recovery.
Preparation and evaluation of fixed-cell catalyst and
fermentation cell-paste in non-growth conditions
Immobilization of whole bacteria was carried out based
on the methods of DeFilippi [12] and Lopez-Gallego et al.
[13]. Refinement of immobilized cells to produce active
catalyst was carried out according to the methods of
Pierce et al. [10,11]. Evaluation of anti-P. destructans ac-
tivity of fixed-cell catalyst and fermentation cell-paste was
determined in co-culture assays with P. destructans co-
nidia and mycelial plugs with various amounts of control
agent (<1.0 g), as described previously. Efficacy was deter-
mined by observation of germule formation as compared
to unexposed controls for growth from conidia, and as
percent reduction in radial growth of mycelial plugs.
Ex vivo anti-infectivity assay
The potential for induced R. rhodochrous to inhibit
fungal growth on bat skin explants was evaluated using
an ex vivo model of WNS. A 10-mm-diameter biopsy
punch was used to collect full-thickness samples of skin
(n = 40) from the patagium of bats (n = 2) immediately
after euthanasia. The explants were adhered to a mesh
support with tissue adhesive (TissueTek®) so that they
would retain their shape and could be supported at the
medium surface without allowing media to come in con-
tact with the inoculated surface of the skin. The skin
explants were then maintained on Eaglesmodifiedmi-
nimal essential medium supplemented with antibiotics
(kanamycin, 100 μg/ml: amikacin, 20 μg/ml; and vanco-
mycin 50 μg/ml). A suspension of spores was placed onto
the center of the explant and allowed to dry. The inocu-
lated explants were incubated in a shared air-space with
induced R. rhodochrous. Uninoculated control explants
were incubated alone or with uninduced R. rhodochrous.
Initial experiments were conducted at 7°C. Anti-infective
efficacy was determined by visual and microscopic eva-
luation of bat wing membrane tissue cultures exposed to
induced R. rhodochrous as compared to unexposed and
uninduced controls.
Results
Anti-P. destructans activity of induced R. rhodochrous
Initial experiments with induced cells of R. rhodochrous
demonstrated complete inhibition of growth from conidia
of P. destructans when cultured with a shared air-space at
15°C (Figure 1a-c). Uninduced cells of R. rhodochrous
showed no signs of inhibition, and were comparable to
unexposed controls. Subsequent testing at 4°C demon-
strated fungistatic activity of induced cells of R. rhodo-
chrous and resulted in slower germination and reduced
total mycelial growth as compared to uninduced cells of
R. rhodochrous and unexposed controls (Figure 1d-f).
Inclusion of activated carbon into the shared air-space
abolished the anti-P. destructans activity of induced R.
rhodochrous (Figure 1c). Mycelial plugs of P. destructans
cultured in a shared air-space with induced R. rhodochrous
Figure 1 Shared air-space co-culture of P. destructans conidia with R. rhodochrous.Uninduced cells (e), induced cells (b, c and f) and
P. destructans control (a, d) were incubated in a shared air-space at 15°C (top panel) and 4°C (bottom panel). Induced R. rhodochrous fails to
inhibit growth from conidia when activated carbon is included in the head-space (c).
Cornelison et al. BMC Microbiology 2014, 14:246 Page 3 of 7
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had a significant reduction in radial mycelial extension as
compared to control plugs cultured in the absence of in-
duced cells of R. rhodochrous (Figure 2). Radial growth of
induced R. rhodochrous-exposed P. destructans at 28 days
post inoculation indicated a 35% reduction in radial myce-
lial extension as compared to unexposed controls. This
inhibitory activity was statistically significant (p 0.05) on
days 8, 12, 16, and 20 across all replicates (Figure 2).
Induced R. rhodochrous permanently and persistently
inhibits conidia germination
Slide agar overlays inoculated with P. destructans conidia
and exposed to induced R. rhodochrous failed to produce
germules 21 days after removal of R. rhodochrous (Figure 3).
Conidia exposed to induced cells of R. rhodochrous for only
24 hours revealed no signs of germule formation, whereas
conidia exposed for 4 and 7 days exhibited early signs of
germination but no obvious germules (Figure 3).
Ex vivo anti-infectivity activity of induced R. rhodochrous
Induced R. rhodochrous completely inhibited the colo-
nization of bat wing explants by P. destructans conidia in
all replicates (n = 20) when incubated in a shared air-space
for up to 21 days at 7°C (Figure 4). Explants exposed to
uninduced R. rhodochrous and unexposed explants were
fully colonized at 14 days post inoculation. Histopatho-
logical assessments of explants were conducted. However,
in this experiment no fungal growth was detected on any
induced Rhodococcus exposed explants. Therefore the
histopathology of otherwise healthyexplants provided
no additional data to this experiment. Histopathology of
the control explants adheres to the histopathology of
WNS in bats as described by Cryan et al. [15]. Spore
germination assays, and the bat wing explant study relied
upon qualitative visual and microscopic evaluation and
Figure 3 Persistent suppression of P. destructans germination by induced R. rhodochrous.P. destructans conidia are unable to recover after
24-hour exposure to induced R. rhodochrous. P. destructans control slide (a) produced significant mycelia growth and conidiation (white arrow) after
5 days.P.destructansconidia exposed to induced Rhodococcus for 24 hours (b),72hours(c) and 7 days (d) failed to form germules 21 days after
removal of induced R. rhodochrous. Halted germination was observed in 72-hour and 7-day exposures (black arrows). All images were captured at
200X magnification.
Figure 2 Induced R. rhodochrous inhibits radial mycelial growth
of P. destructans.Growth areas of P. destructans plugs exposed to
induced R. rhodochrous compared to P. destructans control plugs. All
trials were conducted at 15°C. * indicates days post inoculation with
statistically significant (P 0.05) radial growth inhibition.
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produced definitive results (i.e. no exposed explants de-
veloped fungal growth) therefore a statistical evaluation is
unwarranted and omitted.
Evaluation of fixed-cell catalyst and fermentation
cell-paste
Fixed-cell catalyst [8,10,11] failed to inhibit or slow growth
from conidia of P. destructans when grown in a shared
air-space. Fermentation cell-paste in quantities of 1.0 g,
0.5 g, and 0.25 g completely inhibited growth from conidia
of P. destructans for greater than 80 days (Figure 5a-c).
Discussion and conclusion
Since its initial documentation in 2006, WNS has spread
to twenty-four states and four provinces and has been
implicated in the mortality of millions of North American
bats [16-18] which may have a significant impact on
North American agricultural practices [19]. WNS is char-
acterized by invasive mycelial growth on the wings, muzzle
and ears of hibernating bats that perturbs physiological
functions of the host tissues leading to mortality [15]. Cave
closures and culling of infected individuals appears to have
little to no impact on the spread and mortality associated
with this devastating disease [20]. Classic disease manage-
ment practices applied in agriculture, such as vaccination
and broad-spectrum dissemination of antibiotics, present
many challenges in the management of disease in wild,
highly disseminated, and migratory animal populations.
Consequently, the development of novel treatment options
are needed to avert the spread of WNS and reduce the
mortality associated with currently infected hibernacula.
To this end, the development of biologically-based control
tools is the preferred option for application in karst
environments.
Since the publication of the national response plan [4],
several groups have initiated investigations to identify
potential biological control agents for P. destructans
[21-23]. Several of the investigations have relied on tra-
ditional sources of biocontrol agents or probiotics such
as bacilli and lactobacilli, or competitive exclusion fungi
such as Trichoderma sp., as well as attempts to isolate
bat-skin-associated microbes with anti-P. destructans
activity [21-23]. While these approaches have proven
successful in agricultural and human health applications
[24-27], their application in the attempted remediation
Figure 4 Induced R. rhodochrous prevents fungal colonization
of bat tissue when contained in a shared air-space. Bat wing
tissue explants in a shared air-space with induced R. rhodochrous
21 days post-inoculation with P. destructans conidia (a). Magnified
image of a control explant with visible fungal colonization 21 days
post-inoculation (b).
Figure 5 Non-growth cell-paste of R. rhodochrous inhibits
growth from conidia of P. destructans.Non-growth fermentation
cell-paste of induced R. rhodochrous was incubated in a shared air-space
with P. destructans conidia inoculated plates. Quantities of 1.0 g, 0.5 g,
and 0.25 g (a,b,andc)all demonstrated complete inhibition of growth
from conidia of P. destructans as compared to unexposed controls
(d,whitearrow).Imagetaken21 days post-inoculation.
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of WNS in bats has not been demonstrated. The require-
ment for contact with P. destructans and the bat hosts is a
major hurdle for any agents reliant on competitive exclu-
sion or non-volatile antimicrobial compound production.
These potential control agents may prove to have limited
efficacy against P. destructans in situ and potentially be
harmful to the bat hosts. In contrast, the evaluation of in-
duced R. rhodochrous strain DAP 96253 for application as a
biological control agent of P. destructans aligns ideally with
the needs of wildlife management agencies tasked with
combatting WNS and is the first documented contact-
independent microbial antagonism of P. destructans.
The evolutionary lineage of R. rhodochrous lends itself
to VOC-based fungistasis due to its terrestrial ancestry
[28-30]. The global prevalence of fungistatic soils is a
measure of the natural antagonisms that exists in these
complex environments [28-32]. Due to the ubiquity of
R. rhodochrous in soils [5], it can be expected that
R. rhodochrous as well as many other soil-dwelling
bacteria have the potential to contribute to VOC-based
fungistasis observed in these environments [29,30]. How-
ever, the development of induction methodologies is re-
quired to optimize this activity for biocontrol applications
and is a decidedly advantageous quality of R. rhodochrous
strain DAP 96253 as a potential biological control agent of
WNS [33]. Leveraging this naturally evolved antagonism
for control efforts has many benefits, particularly in the
case of WNS. The complexity of soil ecology selects for
antagonisms that are effective at low concentrations in
diverse, compartmentalized environments where soluble
diffusion may be limited [29]. Therefore, the production
of antagonistic VOCs provides a viable means for soil-
dwelling bacteria to compete with soil-dwelling fungi for
resources and equates favorably with the environmental
conditions of susceptible bat hibernacula. The ability of
R. rhodochrous to detect and interfere with volatile signals
has also been demonstrated in its delayed fruit-ripening
activity [8] and is hypothesized to mediate the observed
anti-P. destructans activity.
While the efficacy of urea-induced R. rhodochrous
under growth conditions is promising for in situ man-
agement of WNS, the need for growth media supple-
mentation poses problems for field application. The long
term in vitro efficacy of non-growth-condition cell-paste
at 4°C allows for increased confidence in forecasting the
efficacy of this biocontrol agent in managing WNS in
the field as this temperature is a sound approximation of
average winter temperature of North American bat
hibernacula [34]. The lack of growth media reduces the
costs associated with application as well as reduces the
likelihood of cross-contamination of control agent media
with native cave microflora. In addition, the contact-
independent basis of the non-growth antagonism will
allow for in situ application methods that will reduce the
potential for ecological impacts associated with intro-
ducing exogenous organisms to karst environments. The
ecological impacts of any potential control agent are of
significant concern for wildlife management agencies
and the evaluation of potential ecological impacts must
be assessed in order to circumvent ecological disasters
associated with augmenting cave microflora (e.g. Lascaux
cave) [4].
The evaluation of R. rhodochrous using ex vivo bat
tissue explants as an indicator of anti-infective activity
was paramount to establishing R. rhodochrous as a viable
biocontrol agent of P. destructans. This was the first
demonstration of inhibition of fungal colonization of bat
tissue by a biological control agent. This ex vivo efficacy
justifies further in vivo studies with live bats and should
be pursued vigorously.
The ability of dormant conidia to remain viable in
host-free environments increases long-term impacts of
fungal pathogens and renders contaminated environ-
ments inhospitable to re-colonization [35]. The impact
of WNS in locations such as New York has been tre-
mendous, vastly reducing the populations of insectivor-
ous bats over a broad geographic range. The permanent
and persistent inhibition of conidia germination is a
promising result and indicates that treatment of pre-
viously decimated hibernacula to inactivate resident co-
nidia prior to re-colonization attempts may be feasible
by applying induced R. rhodochrous in these environ-
ments. However further investigations are needed to
confirm the applicability of this approach.
The evaluation of R. rhodochrous strain DAP 96253
has demonstrated the tremendous potential of this or-
ganism for application as a biological control agent of
P. destructans. This is the first and only demonstration
of contact-independent antagonism of P. destructans
and represents a significant step toward the develop-
ment of biologically-based treatment tools for WNS.
Competing interests
GEP and SAC are contributing authors on the seminal patents for the
induction and application of Rhodococcus rhodochrous DAP 96253 cited in
the text. These patents are held by Georgia State University Research
Foundation.
Authorscontributions
CTC, SAC, and GEP conceived and designed the experiments conducted at
GSU. KTG TAT and CKB developed and carried out the methodology to
assess and produce induced R. rhodochrous as well as collected and
analyzed data. MKK provided P. destructans isolates from his diagnostic work
as well as designed and conducted the experiments with bat tissue explants
at UGA. CTC, KTG, and MKK wrote the manuscript. All authors read and
approved the final version of the manuscript.
Acknowledgements
This work was funded by Bat Conservation International through a WNS
research grant as well as the Georgia State University Environmental
Research Program. The authors would like to thank Lisa Last and Page
Luttrell for assistance with the maintenance of tissue explants. The authors
Cornelison et al. BMC Microbiology 2014, 14:246 Page 6 of 7
http://www.biomedcentral.com/1471-2180/14/246
would also like to thank Ian Sarad, Blake Cherney, and Ben Poodiak for their
contributions to this effort.
Author details
1
Applied and Environmental Microbiology, Georgia State University, 161
Jesse Hill Jr. Dr, Atlanta, GA, USA.
2
Department of Pathology Microbiology &
Immunology, University of California Davis, One Shields Avenue, Davis, CA,
USA.
Received: 3 June 2014 Accepted: 11 September 2014
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Cite this article as: Cornelison et al.:A preliminary report on the contact-
independent antagonism of Pseudogymnoascus destructans by Rhodococcus
rhodochrous strain DAP96253. BMC Microbiology 2014 14:246.
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... Bacteria and fungi isolated from bats and the environment have been shown to inhibit the growth of P. destructans in vitro (Hoyt et al., 2015;Raudabaugh and Miller, 2015;Micalizzi et al., 2017;Rusman et al., 2020), and application of Pseudomonas fluorescens on bat skin over winter was recently shown to increase survival for one species of bat (Hoyt et al., 2019). P. destructans conidia have also been shown to be inhibited by the volatile organic compounds produced by R. rhodochrous (Cornelison et al., 2014) and trans-farnesol, a sesquiterpene made by the yeast Candida (Raudabaugh and Miller, 2015). In addition, Oidiodendron truncatum was shown to produce various compounds that reduced the growth of P. destructans at low concentrations (Rusman et al., 2020). ...
... However, there may be other factors influencing mycelium morphology, such as volatile organic compounds (VOCs). P. destructans mycelium growth and conidiation germination were persistently suppressed by the VOCs produced by R. rhodochrous (Cornelison et al., 2014). Therefore, the effect of P. yamanorum GZD14026 on the inhibition of P. destructans mycelium was a multiple action process. ...
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White-nose syndrome, a disease that is caused by the psychrophilic fungus Pseudogymnoascus destructans, has threatened several North America bat species with extinction. Recent studies have shown that East Asian bats are infected with P. destructans but show greatly reduced infections. While several factors have been found to contribute to these reduced infections, the role of specific microbes in limiting P. destructans growth remains unexplored. We isolated three bacterial strains with the ability to inhibit P. destructans, namely, Pseudomonas yamanorum GZD14026, Pseudomonas brenneri XRD11711 and Pseudomonas fragi GZD14479, from bats in China. Pseudomonas yamanorum, with the highest inhibition score, was selected to extract antifungal active substance. Combining mass spectrometry (MS) and nuclear magnetic resonance (NMR) spectroscopy analyses, we identified the active compound inhibiting P. destructans as phenazine-1-carboxylic acid (PCA), and the minimal inhibitory concentration (MIC) was 50.12 μg ml-1 . Whole genome sequencing also revealed the existence of PCA biosynthesis gene clusters. Gas chromatography-mass spectrometry (GC-MS) analysis identified volatile organic compounds. The results indicated that 10 ppm octanoic acid, 100 ppm 3-tert-butyl-4-hydroxyanisole (isoprenol) and 100 ppm 3-methyl-3-buten-1-ol (BHA) inhibited the growth of P. destructans. These results support that bacteria may play a role in limiting the growth of P. destructans on bats.
... Spores of Pd were isolated from Pd cultivated at 15 °C for 14 days on SDA media. As previously described by Cornelison et al. 24 , spore suspensions were created by scraping large quantities of mycelial growth off solid media and filtering the suspension through sterile glass wool. The concentration of spores in solution was quantified with a hemocytometer, and spore solutions were stored at 4 °C for no longer than 4 weeks. ...
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Pseudogymnoascus destructans (Pd) is the causative agent of white-nose syndrome, which has resulted in the death of millions of bats in North America (NA) since 2006. Based on mortalities in eastern NA, the westward spread of infections likely poses a significant threat to western NA bats. To help prevent/reduce Pd infections in bats in western NA, we isolated bacteria from the wings of wild bats and screened for inhibitory activity against Pd. In total, we obtained 1,362 bacterial isolates from 265 wild bats of 13 species in western Canada. Among the 1,362 isolates, 96 showed inhibitory activity against Pd based on a coculture assay. The inhibitory activities varied widely among these isolates, ranging from slowing fungal growth to complete inhibition. Interestingly, host bats containing isolates with anti-Pd activities were widely distributed, with no apparent geographic or species-specific pattern. However, characteristics of roosting sites and host demography showed significant associations with the isolation of anti-Pd bacteria. Specifically, anthropogenic roosts and swabs from young males had higher frequencies of anti-Pd bacteria than those from natural roosts and those from other sex and age-groups, respectively. These anti-Pd bacteria could be potentially used to help mitigate the impact of WNS. Field trials using these as well as additional microbes from future screenings are needed in order to determine their effectiveness for the prevention and treatment against WNS.
... Fig. 6). Interestingly, one of the isolates (CCB 311.5) was determined to be a species of Rhodococcus, a genus previously identified with anti-Pd activity (Cornelison et al. 2014). Taken together these results suggest that the host microbial community may influence or be influenced by fungal pathogen invasion, however the interactions between fungus and individual antifungal bacterial taxa is complex. ...
... Bacteria can also react to changes in their environment by producing volatiles that will have a specific effect on different types of target organisms. Among the most well-known effects of these volatiles are antimicrobial (Rybakova et al. 2017;Kai et al. 2018;Montes Vidal et al. 2017), antifungal (Riclea et al. 2012), and microbiostatic activities (Kanchiswamy et al. 2015;Audrain et al. 2015b;Cornelison et al. 2014;van Agtmaal et al. 2015), along with repellent or chemoattractant properties (Schulz-Bohm et al. 2017a;Effmert et al. 2012). Conversely, growth stimulation capacity toward other species of bacteria, fungi, or oomycetes has also been observed (Schulz-Bohm et al. 2017a;Audrain et al. 2015b). ...
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Emission of volatile organic compounds (VOCs) has emerged as important mean of communication between bacteria and other organisms. Most of the knowledge accumulated so far in this field has been obtained with model organisms grown in pure culture. However, in nature, bacteria are part of complex ecosystems and communities encompassing other bacteria, fungal, oomycete, protist, plant, and animal partners. In such communities, bacterial emission of volatiles will be influenced by the surrounding partners and their own volatile emission. This chapter aims at placing bacterial volatile-mediated communication in its global context and summarizing the available literature on how interactions between bacteria and other organisms shape volatile emissions as well as the outcome of biological interactions.
... Consistent with our second hypothesis, the composition of the microbiota included several taxa known to inhibit Pd growth in vitro, and which have been found on bats persisting after WNS [e.g., Rhodococcus (Cornelison et al., 2014;Hamm et al., 2017), Pseudomonas ]. Pseudonocardia was also the most abundant microbial taxa in pre-captivity samples and, although it declined in abundance by the end of the experiment, it is known to have antifungal activity (Sen et al., 2009). ...
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