Faculty of Science
INHIBITION OF PSEUDOGYMNOASCUS DESTRUCTANS, THE CAUSATIVE AGENT
OF WHITE-NOSE SYNDROME, BY ENVIRONMENTAL MICROORGANISMS
2017 | ROBYN LYNNE MCARTHUR

B.Sc. Honours thesis - Biology

Inhibition of Pseudogymnoascus destructans, the causative agent of white-nose syndrome,
by environmental microorganisms
by
Robyn Lynne McArthur

A THESIS SUBMITTED IN PARTIAL FULFILLMENT
OF THE REQUIREMENTS FOR THE DEGREE OF
BACHELOR OF SCIENCE (HONS.)
in the
DEPARTMENT OF BIOLOGICAL SCIENCES
(Cellular, Molecular, Microbial Biology)

This thesis has been accepted as conforming to the required standards by:
Naowarat (Ann) Cheeptham (Ph.D.), Thesis Supervisor, Dept. Biological Sciences
Soumya Ghosh (Ph.D.), Co-supervisor, Dept. Biological Sciences
J. Norman Reed (Ph.D.), Examining Committee member, Dept. Chemistry

Dated this 2nd day of June, 2017, in Kamloops, British Columbia, Canada

© Robyn Lynne McArthur, 2017

ABSTRACT
White-nose syndrome, a disease that affects hibernating bats, has caused mass mortality in many
bat populations since it was first detected in North America in 2006. This disease has both
ecological and economic impacts, since bats are relied upon by the agricultural sector for natural
pest-control. White-nose syndrome is caused by the psychrophilic fungus Pseudogymnoascus
destructans, which is believed to be an invasive species in North America. Although biological
and chemical agents able to inhibit the growth of P. destructans have been found, none of these
have been put into widespread use, so the search for inhibitory agents continues. Environmental
microorganisms are a diverse source of antibiotics and antifungals; therefore, the goal of this
project was to isolate bacteria and fungi from three unique environments and screen these
microorganisms for inhibitory activity towards P. destructans. Infected plant (rotten leaves, bark,
wood) and mushroom samples collected from the area around Bush Lake and Timber Lake,
British Columbia were plated on Sabouraud dextrose agar plates and incubated at 25°C to
promote microbial growth. Additionally, fungi were isolated from dwarf mistletoe plants and soil
samples collected at a mushroom farm in Summerland, British Columbia. Morphologically
distinct bacterial and fungal colonies were isolated in pure culture and screened for inhibitory
activity towards P. destructans. Ninety-three bacterial isolates were pre-screened using a direct
contact streak assay, with 30 exhibiting inhibitory activity towards P. destructans. These 30
bacterial isolates plus 77 fungal isolates were then grown in V8 broth and the broth supernatant
was collected and filtered before being used in a Kirby-Bauer style agar diffusion assay. Thirteen
bacteria and seven fungi exhibited inhibitory activity towards P. destructans in the agar diffusion
assay. Further characterization of the positive candidates could lead to the production of a novel
biological control agent that could be used for treatment and/or prevention of WNS.
Thesis Supervisor: Associate Professor Naowarat (Ann) Cheeptham

ii

ACKNOWLEDGEMENTS
I would like to thank Dr. Naowarat (Ann) Cheeptham for her kindness, support, and guidance
while acting as my supervisor. I would also like to thank Dr. Soumya Ghosh for his feedback and
enthusiasm throughout this project and Dr. Norman Reed for acting as a member of my
examining committee. I am grateful to Christine Petersen, Jennifer Petersen, Jennifer Barden,
Lucas Hampel, Keegan Koning, and Brian Callow (What the Fungus Mushroom Farm,
Summerland, BC) for their assistance with sample collection. I would like to thank Dr. Cori
Lausen for her support and Dr. JP Xu of McMaster University for his guidance and providing
Pseudogymnoascus destructans samples. I would also like to thank the TRU Department of
Biological Sciences for some of the supplies used in this project and Dr. Louis Gosselin for his
role in the Honors program. I would like to acknowledge the funding provided through Dr.
Cheeptham’s and Lausen’s USFWS grant (F16AP00028).

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TABLE OF CONTENTS
ABSTRACT.............................................................................................................................................................. II
ACKNOWLEDGEMENTS .................................................................................................................................... III
LIST OF FIGURES ...................................................................................................................................................V
LIST OF TABLES .................................................................................................................................................. VI
INTRODUCTION .................................................................................................................................................... 1
MATERIALS AND METHODS .............................................................................................................................. 7
GROWTH AND CULTURE OF PSEUDOGYMNOASCUS DESTRUCTANS..................................................................................... 7
ISOLATION OF PD CONIDIA ....................................................................................................................................................... 8
COLLECTION AND PURIFICATION OF ENVIRONMENTAL BACTERIAL AND FUNGAL ISOLATES....................................... 8
PRE-SCREENING OF BACTERIAL ISOLATES FOR ANTI-PD ACTIVITY .................................................................................. 9
PREPARATION OF MICROBIAL SUPERNATANTS FOR ANTI-PD BIOASSAY ......................................................................... 9
SCREENING OF MICROBIAL SUPERNATANTS WITH AN ANTI-PD BIOASSAY .................................................................. 10
RESULTS ............................................................................................................................................................... 11
PRE-SCREENING OF BACTERIAL ISOLATES FOR ANTI-PD ACTIVITY ............................................................................... 11
SCREENING OF MICROBIAL SUPERNATANTS WITH AN ANTI-PD BIOASSAY .................................................................. 13
DISCUSSION ......................................................................................................................................................... 20
LITERATURE CITED .......................................................................................................................................... 26
APPENDICES ........................................................................................................................................................ 30

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LIST OF FIGURES
FIGURE 1. THE SPREAD MAP OF REPORTED SUSPECT AND CONFIRMED CASES OF WHITE-NOSE SYNDROME BY
COUNTY/DISTRICT FOR EACH BAT HIBERNATION PERIOD FROM WINTER 2006 TO MAY 9, 2017 (U.S. FISH
AND WILDLIFE SERVICE, 2017). ...................................................................................................................................... 1

FIGURE 2. A LITTLE BROWN BAT WITH FUNGAL GROWTH CHARACTERISTIC OF WHITE-NOSE SYNDROME ON THE
MUZZLE AND WINGS (A) (MORIARTY, 2009) AND A SCANNING ELECTRON MICROGRAPH OF P. DESTRUCTANS
WITH CONIDIA COLORED PURPLE (B) (SPRINGER, 2013). .......................................................................................... 2

FIGURE 3. THE FOUR TYPES OF P. DESTRUCTANS INHIBITION EXHIBITED BY BACTERIAL ISOLATES DURING THE PRESCREENING EXPERIMENTS. ANY ISOLATES CREATING A ZONE OF INHIBITION WERE SUBJECTED TO FURTHER
TESTING USING THE ANTI-PD BIOASSAY........................................................................................................................ 11

FIGURE 4. ZONES OF INHIBITION CREATED IN P. DESTRUCTANS FUNGAL LAWNS BY FILTERED BROTH SUPERNATANT
OF THREE FUNGAL ISOLATES COLLECTED SEVEN DAYS AFTER BROTH INOCULATION. THE PHOTOGRAPHS
WERE TAKEN ON DAY 12 (#123) AND DAY 10 (#156 AND #158) AFTER THE PLATE WAS INOCULATED. .. 14

FIGURE 5. ANTI-PD BIOASSAY PLATE WITH A P. DESTRUCTANS FUNGAL LAWN AND INHIBITORY ZONES CREATED BY
FILTERED BACTERIAL SUPERNATANT COLLECTED SEVEN DAYS AFTER BROTH INOCULATION. THE
PHOTOGRAPH WAS TAKEN 10 DAYS AFTER THE PLATE WAS INOCULATED. BACTERIAL ISOLATES CREATING
ZONES OF INHIBITION ARE LABELED WITH THEIR ASSIGNED SCREENING NUMBER. CONTROLS ARE LABELED
WITH A LETTER TO INDICATE THEIR IDENTITY. B=10% BLEACH, P=1.5% PEROXIGARD, W=STERILE WATER,

V8=STERILE V8 BROTH. .................................................................................................................................................. 15
FIGURE 6. ANTI-PD BIOASSAY PLATE WITH A P. DESTRUCTANS FUNGAL LAWN AND INHIBITORY ZONES CREATED BY
FILTERED BACTERIAL SUPERNATANT COLLECTED 14 DAYS AFTER BROTH INOCULATION. THE PHOTOGRAPH
WAS TAKEN 10 DAYS AFTER THE PLATE WAS INOCULATED. BACTERIAL ISOLATES CREATING ZONES OF
INHIBITION ARE LABELED WITH THEIR ASSIGNED SCREENING NUMBER. CONTROLS ARE LABELED WITH A
LETTER TO INDICATE THEIR IDENTITY. B=10% BLEACH, P=1.5% PEROXIGARD, W=STERILE WATER,

V8=STERILE V8 BROTH. .................................................................................................................................................. 17
FIGURE 7. ZONES OF INHIBITION CREATED IN P. DESTRUCTANS FUNGAL LAWNS BY FILTERED BROTH SUPERNATANT
OF SEVEN FUNGAL ISOLATES COLLECTED 14 DAYS AFTER BROTH INOCULATION. THE PHOTOGRAPHS WERE
TAKEN 10 DAYS AFTER THE PLATE WAS INOCULATED. .............................................................................................. 18

FIGURE 8. COMPARISON OF AVERAGE INHIBITORY ZONE DIAMETERS (MM) CREATED IN P. DESTRUCTANS FUNGAL
LAWNS BY BACTERIAL AND FUNGAL FILTERED BROTH SUPERNATANT COLLECTED SEVEN (GREY) AND 14

(BLUE) DAYS AFTER BROTH INOCULATION. ERROR BARS REPRESENT STANDARD ERROR (N=3). .................... 20

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LIST OF TABLES
TABLE 1. INHIBITION TYPE FOR BACTERIAL ISOLATES AND CONTROLS (WATER, BLEACH, PEROXIGARD) CREATING
INHIBITORY ZONES IN P. DESTRUCTANS FUNGAL LAWNS ON DAY 10 AND DAY 15 AFTER INOCULATION. ........ 12

TABLE 2. THE NUMBER OF ISOLATES CREATING EACH TYPE (COMPLETE, SUBSURFACE, SURFACE, OR PARTIAL
SURFACE) OF INHIBITORY ZONE IN P. DESTRUCTANS FUNGAL LAWNS ON DAY 10 AND DAY 15 AFTER
INOCULATION. .................................................................................................................................................................... 13

TABLE 3. AVERAGE DIAMETER (MM), TYPE, AND LENGTH OF PRESENCE FOR INHIBITORY ZONES CREATED IN P.
DESTRUCTANS FUNGAL LAWNS BY BACTERIAL OR FUNGAL FILTERED BROTH SUPERNATANT COLLECTED SEVEN
DAYS AFTER BROTH INOCULATION. INHIBITORY ZONES WERE MEASURED 10 DAYS AFTER THE PLATE WAS
INOCULATED (EXCEPT ISOLATE 123 AND THE CORRESPONDING CONTROLS THAT WERE MEASURED ON DAY

17) AND MONITORED UNTIL DAY 21. THE HORIZONTAL LINES IN THE TABLE SEPARATE THE RESULTS FROM
DIFFERENT SCREENING PLATES. ..................................................................................................................................... 16

TABLE 4. AVERAGE DIAMETER (MM), TYPE, AND LENGTH OF PRESENCE FOR INHIBITORY ZONES CREATED IN P.
DESTRUCTANS FUNGAL LAWNS BY BACTERIAL OR FUNGAL FILTERED BROTH SUPERNATANT COLLECTED 14
DAYS AFTER BROTH INOCULATION. INHIBITORY ZONES WERE MEASURED 10 DAYS AFTER THE PLATE WAS
INOCULATED AND MONITORED UNTIL DAY 21. THE HORIZONTAL LINES IN THE TABLE SEPARATE THE
RESULTS FROM DIFFERENT SCREENING PLATES. ......................................................................................................... 18

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INTRODUCTION
White-nose syndrome (WNS) is a disease that has led to the mass mortality of hibernating
insectivorous bats in North America since it was first documented in winter 2006/2007 (U.S.
Fish and Wildlife Service, 2017). In the 10 years since it was first detected in New York, WNS
has spread across Eastern North America into 31 U.S. states and five Canadian provinces (New
Brunswick, Nova Scotia, Ontario, Prince Edward Island, Quebec) (Figure 1). Additionally, on
March 11, 2016 a bat found in Washington State was submitted to the U.S. Geological Survey
(USGS) National Wildlife Health Centre for testing and was confirmed to have WNS (Haman et
al., 2016). This is the westernmost case to date, with a distance of over 2000 km between
Washington and the previous westernmost case.

Figure 1. The spread map of reported suspect and confirmed cases of white-nose syndrome by
county/district for each bat hibernation period from winter 2006 to May 9, 2017 (U.S. Fish and
Wildlife Service, 2017).

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Although WNS is primarily transmitted from bat to bat during hibernation, the causative agent
may also be transmitted by humans on clothing or gear, which may explain the sudden
appearance of WNS in Washington State (U.S. Fish and Wildlife Service, 2017). In North
America, eight bat species (including two endangered species, and one threatened species) have
been confirmed with diagnostic symptoms of WNS and the causative agent of WNS has been
found on an additional seven bat species (including one endangered species).
One of the symptoms of WNS is fungal growth on the wings, ears, and muzzles of the bats
(Gargas et al., 2009) (Figure 2). The causative fungus was identified by Gargas et al. (2009)
based on small subunit (SSU) and internal transcribed spacer (ITS) rRNA gene sequencing and
named Geomyces destructans. G. destructans was further confirmed as the cause of WNS by
Lorch et al. (2011), who demonstrated that healthy little brown bats (Myotis lucifigus) would
develop WNS if exposed to pure cultures of G. destructans or if they had direct contact with
infected bats. However, Minnis and Lindner (2013) conducted a phylogenetic analysis of
Geomyces and relatives based on DNA sequence data from five genes/regions and concluded
that Geomyces and Pseudogymnoascus are genetically distinct, and G. destructans was renamed
Pseudogymnoascus destructans (Pd) based on its placement in their phylogenetic tree.

A

B

Figure 2. A little brown bat with fungal growth characteristic of white-nose syndrome on the
muzzle and wings (A) (Moriarty, 2009) and a scanning electron micrograph of P. destructans with
conidia colored purple (B) (Springer, 2013).

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Microscopically, Pd has septate hyphae with unique curved conidia produced on branched
conidiophores and hyphae (Chaturvedi et al., 2010; Verant et al., 2012) (Figure 2). Pd is
classified as a psychrophilic fungus since its optimum temperature for growth is 12.5°C-15.8°C,
with no growth observed at or below -10°C or above 19.0°C-19.8°C, depending on the strain.
Although Pd grows very slowly on artificial media, the ability to do so indicates that Pd may be
a facultative, rather than obligate, pathogen (Gargas et al., 2009; Reynolds and Barton, 2014).
Pd infection involves penetration and colonization of the sub-cutaneous tissue of the bat muzzle,
wings, ears, and/or tail by fungal hyphae, which is promoted by the secretion of various enzymes
(O’Donoghue et al., 2015). Although the Pd secretome includes peptidases, lipases,
glycosidases, and redox enzymes, one of the most important proteins for virulence is Destructin1, a serine endopeptidase with the ability to degrade collagen. The ability of Destructin-1 to
cleave cross-links in collagen, an important component of mammalian connective tissue, is
believed to cause structural instability and promote deep tissue penetration in the bat host.
Another virulence factor that plays a key role in WNS is the secretion of riboflavin (vitamin B2)
by Pd, which accumulates in bat skin lesions due to decreased metabolism and perfusion during
hibernation (Flieger et al., 2016). Riboflavin is cytotoxic at high concentrations since it can lead
to the production of free radicals and cause oxidative stress, apoptosis, and necrosis during
arousal from hibernation. This worsens skin lesions, especially in the bat wings, which are
required for thermoregulation, water regulation, gas exchange, and immune function (Flieger et
al., 2016; Warnecke et al., 2013). The disruption of the wing tissue therefore leads to increased
levels of carbon dioxide in the blood, which stimulates arousal from torpor to correct the CO2
levels and blood pH (Verant et al., 2014).
Although cycles of torpor and arousal are normal during bat hibernation, increased arousal
duration and/or frequency caused by WNS can lead to mortality due to a lack of food available
during hibernation coupled with the high energy demand and fat depletion associated with
arousal (Reeder et al., 2012; Thomas et al., 1990). For the majority of the hibernation period,
bats are in torpor, meaning that body temperature will drop to near ambient (2°C-8°C), metabolic
rate will be highly reduced (may be <5% of basal metabolic rate), and the innate and adaptive

3

immune systems will be less active (Flieger et al., 2016; Reeder et al., 2012). When in torpor,
bats are able to conserve their energy stored as body fat and survive through the winter without
eating, since insects are scarce (Reeder et al., 2012). Periodically during hibernation bats will
briefly arouse from torpor, body temperature will increase to approximately 37°C, metabolic rate
will increase, and bats may become active (Thomas et al., 1990). Although arousal periods may
allow bats to re-activate their immune systems, arousal is responsible for 80-90% of energy
consumption during hibernation (Reeder et al., 2012). Research indicates that WNS both
increases metabolic rate during hibernation, as well as decreases the length of torpor bouts by
approximately half due to increased arousal frequency (Reeder et al., 2012; Verant et al., 2014).
This, in addition to wing damage, leads to increased energy usage, water loss, dehydration,
electrolyte depletion, starvation due to depletion of fat reserves, and ultimately, death (Verant et
al., 2014; Warnecke et al., 2013).
Interestingly, although Pd has been found on hibernating bats throughout Europe, no mortality
due to WNS has been recorded (Puechmaille et al., 2011). The lack of WNS-associated mortality
in Europe along with an absence of closely related Pseudogymnoascus species in North America
suggests that Pd may have originated in Europe, where co-evolution with European bats may
have occurred before it became an invasive species in North America (Minnis and Lindner,
2013; Puechmaille et al., 2011). The high degree of sequence polymorphism in European Pd
isolates compared to the lack of variation detected in North American Pd isolates, as well as the
sharing of a common Pd haplotype between North American and some European Pd populations
provides further support for a European origin for WNS and introduction of Pd into North
America (Leopardi et al., 2015). Due to the distance between Europe and North America, it is
most likely that this inter-continental Pd movement was a result of anthropogenic activity.
Recent research suggests that Pd is also present beyond Europe, into the West Siberian Plain of
Russia, and although Pd is highly prevalent in Europe and Russia, bats have developed tolerance
to the pathogen in these areas (Zukal et al., 2016).
In North America, there is evidence that some bat populations are developing resistance or
tolerance to Pd that allows them to persist with the fungus; however, some species are still at risk
of extinction due to the large environmental reservoirs of Pd that exist in some hibernacula

4

(Frick et al., 2017). Hoyt et al. (2015a) demonstrated that Pd conidia can remain viable for over
five years under laboratory conditions, suggesting that conidia shed into the environment by
infected bats may stay viable in hibernacula for long periods of time and prevent recolonization
of these sites. Additionally, Reynolds and Barton (2014) found that Pd is capable of secreting
enzymes used for saprotrophic growth, indicating that it may be possible for Pd to grow in
hibernacula outside of a bat host.
Although bats in North America may be slowly developing resistance or tolerance to Pd, bats
have a very low reproductive rate so populations will take a long time to recover from the WNSassociated mass mortality that has occurred over the last decade (Bat Conservation International,
2017). This may have lasting impacts, since bats play a crucial role in ecosystems and act as a
form of natural pest control. Insectivorous bats in North America consume a large number of
forest and crop pests, which reduces crop/forest losses, pesticide application, and the likelihood
of insects becoming pesticide resistant (Boyles et al., 2011). This is estimated to save the
agricultural sector an estimated 3.7 billon dollars per year; however, this number could be as
high as 53 billion dollars per year. Bats also add to the biodiversity of ecosystems, help maintain
ecosystem stability, consume insects that are human/animal pathogens, and redistribute nutrients
through their guano (Kunz et al., 2011). Bat guano acts as a natural fertilizer rich in nitrogen and
phosphorous and is the primary source of organic matter that supports some cave ecosystems.
Since North American bats are highly susceptible to WNS and they play such a crucial role in
the ecosystem, a portion of WNS research over the past decade has focused on finding fungicides
or fungistatic agents that can be used for treatment or prevention of WNS. Despite the ability of
chemical agents such as bleach, Lysol, antifungals, fungicides, and some biocides to kill or
inhibit the growth of Pd, these agents would likely harm the cave ecosystem or the bats, so have
little application in hibernacula (Chaturvedi et al., 2011; Shelley et al., 2013). Thus, most studies
of this nature have focused on potentially non-toxic natural products, such as cold-pressed
terpeneless orange oil, 1-octen-3-ol (mushroom oil), and 1-hexanol, all of which are able to
inhibit Pd growth and have been proposed as treatments for infected hibernacula (Boire et al.,
2016; Padhi et al., 2017).

5

There have also been a number of studies testing bacteria against Pd for the purpose of finding a
biological control agent against WNS. For example, 6 bacterial isolates from bat skin swabs
belonging to the genus Pseudomonas and 36 Actinobacteria (mostly Streptomyces spp.) isolated
from WNS-free bats inhibited the growth of Pd in vitro and therefore have biological control
agent potential (Hamm et al., 2017; Hoyt et al., 2015b). However, the most promising biological
control agent to date is Rhodococcus rhodochrous strain DAP96253, a Gram-positive bacterium
that produces volatile organic compounds with fungistatic activity towards Pd (Cornelison et al.,
2014). This treatment is advantageous because of its contact-independent nature and was able to
successfully treat 75 bats suffering from WNS (Gill, 2016).
Overall, less research has been conducted to investigate the use of fungal agents against Pd;
however, one fungal species and one compound derived from Candida albicans have also shown
biological control potential. Zhang et al. (2015) found that strain WPM 39143 of Trichoderma
polysporum, collected in a cave air sample, exhibited fungicidal activity specific to Pd. Trans,
trans-farnesol, a quorum-sensing compound produced by C. albicans, was also able to inhibit the
germination of Pd conidia and inhibit hyphal growth under laboratory conditions (Raudabaugh
and Miller, 2015).
Due to the limited amount of research in this area and the need for a reliable biological control
agent against Pd, we chose to screen bacteria and fungi isolated from the area around Bush Lake
and Timber Lake, B.C., as well as fungi isolated from dwarf mistletoe plants and a mushroom
farm in Summerland, B.C. for inhibitory activity towards Pd. The choice to screen bacteria and
fungi from these locations was based on the success of previous research that involved screening
microorganisms for anti-Pd activity, as demonstrated above, as well as the knowledge that
environmental microorganisms have long been a source of novel antibiotics and antifungal
agents (Wohlleben et al., 2016). For example, Magnusson et al. screened over 1200
environmental isolates of lactic acid bacteria for antifungal activity and found that 10% of the
isolates had inhibitory activity towards Aspergillus fumigatus and some also had activity towards
other molds (Magnusson et al., 2003). There are also numerous examples of fungal biocontrol
agents active against fungal plant pathogens, nematodes, weeds, and insects (Perotto et al.,
2013). In general, fungi may be used for biocontrol because they can compete with pathogenic

6

fungi for nutrients and space or produce inhibitory substances. For example, the use of fungi to
control Fusarium head blight, a fungal disease of wheat crops, and die-back syndrome of the
cosmopolitan common reed caused by the fungal pathogen Gibberella fujikuroi have been
investigated with promising results.
In order to obtain pure bacterial and fungal cultures that could be used in the screening assays,
morphologically distinct colonies in mixed microbial cultures grown from plant and mushroom
samples (Bush Lake and Timber Lake, B.C), dwarf mistletoe plants, and mushroom compost
samples (What the Fungus Mushroom Farm, Summerland, B.C.) were streaked for pure culture.
Pd was grown until mature and a conidia isolation procedure was carried out to obtain conidia
for inoculating the screening plates. Bacteria were pre-screened for inhibitory activity towards
Pd using a direct contact streak assay and isolates producing zones of inhibition were subjected
to further testing using a Kirby-Bauer agar diffusion assay, along with the fungal isolates. To the
extent of our knowledge, this is the first study to screen bacteria and fungi from the
abovementioned environments for inhibitory activity towards Pd, as well as only the second
study to test fungi against Pd. The long-term goal of this study is the production of a novel
biological control agent for the treatment or prevention of WNS.

MATERIALS AND METHODS
Growth and culture of Pseudogymnoascus destructans
Stock plates of Pseudogymnoascus destructans were prepared by plating small agar plugs taken
from a previously-grown stock culture of Pseudogymnoascus destructans M3906-2 on
Sabouraud Dextrose Agar (SDA) (Product # M063, HiMedia, India) containing 34 µg/mL of
chloramphenicol (Product # BP904, Fisher Scientific, Fairlawn, NJ) to inhibit bacterial growth.
Plates were parafilmed, inverted, and placed inside of a plastic container in a 15°C incubator for
approximately two months. These stock plates were used to inoculate SDA plates and YM broth
(Product # 271120, Difco, Sparks, MD) for the creation of additional Pd cultures. All Pd cultures
were grown at 15°C and YM broth cultures were grown shaking at 110 rpm. YM broth cultures
of Pd were spread on SDA plates (34 µg/mL chloramphenicol) and allowed to grow for
approximately 11 days at 15°C to produce conidia.

7

Isolation of Pd conidia
Pd conidia were harvested from cultures of Pd that were approximately 11 days old (see above)
using the protocol described by Cornelison et al. (2014), with minor adjustments suggested by
Hoyt et al. (2015b). The Pd colonies on each plate were submerged in 10 mL of Conidia
Harvesting Solution (CHS) (0.05% Tween 80, 0.9% NaCl) for five minutes before the colonies
were vigorously scraped with a sterile loop to dislodge conidia. The CHS from each plate was
filtered through sterile glass wool and centrifuged at 4000 rcf for 10 minutes. The supernatant
was removed and conidia were washed in phosphate buffered saline (PBS, pH 7), resuspended in
PBS, and filtered through sterile glass wool. A 10 µl sample of the final conidia suspension was
removed and pipetted into a hemocytometer to count the conidia. The approximate concentration
of conidia in the suspension was determined using the following equation: Average # of conidia
in 1 square x dilution factor x 104 = # of conidia/mL. Conidia suspensions were stored in PBS at
4°C until needed.
Collection and purification of environmental bacterial and fungal isolates
Mushrooms from the phylloplane, tree bark, and infected plant wood and leaves were collected
from the area around Bush Lake near Lac le Jeune Road (Lat 50.531823; Lon 120.452856) and
Timber Lake Road (Lat 50.572227; Lon 120.450068), B.C. These samples were laid down on
Sabouraud Dextrose Agar (SDA) plates, which were incubated at 25°C until bacterial and fungal
growth was observed. Morphologically distinct bacterial and fungal colonies were streaked for
pure culture on SDA (34 µg/mL chloramphenicol) and plates were placed at 25°C until growth
was observed (two to six days, depending on the organism). Plates were restreaked again if
necessary until pure cultures were obtained. Pure cultures were parafilmed and stored at 4°C
until testing.
Fungi subcultured from dwarf mistletoe plants collected in the Kamloops area and isolated in
pure culture were obtained from Lucas Hampel (Ross-Friedman Lab, Thompson Rivers
University) and stored at 4°C.
Three mushroom compost samples were collected at What the Fungus Mushroom farm in
Summerland, B.C. One sample was taken from the exterior of the compost pile, while the other

8

two samples were taken from the interior of the pile. Samples were collected with a trowel,
placed in plastic zip-top bags, and transported in a Styrofoam container kept at approximately
4°C to the Thompson Rivers University microbiology laboratory, where they were stored at 4°C.
Ten grams of mushroom compost was added to 250 mL Erlenmeyer flasks containing 50 mL of
isolation solution (2% Tween20, 0.9% NaCl). The flasks were placed shaking at 120 rpm at
37°C for three hours. The flasks were then taken off of the shaker and left undisturbed for 10
minutes before 1000 µl of supernatant was collected. The supernatant was serially diluted and
plated on SDA. Plates were incubated at 25°C until fungal growth was observed.
Morphologically distinct fungal colonies were streaked for pure culture on SDA (34 µg/mL
chloramphenicol) and plates were placed at 25°C until growth was observed and stored at 4°C.
Pre-screening of bacterial isolates for anti-Pd activity
SDA medium was prepared according to the manufacturer’s instructions and 85 µl of 100
mg/mL chloramphenicol dissolved in ethanol (Product # 676829, Sigma Aldrich, USA) and 1
mL of conidia suspension at approximately 1 x 106 conidia/mL were added per 250 mL of SDA
after autoclaving for final concentrations of 34 µg /mL and 4000 conidia/mL, respectively. The
medium was mixed and poured into large bioassay plates (Nunc®, 245 mm x 245 mm x 25 mm)
or individual petri plates and allowed to cool in the laminar flow hood. For individual petri
plates, bacterial isolates were streaked in the center of each plate using a sterile pipet tip. The
large bioassay plates were divided into 36 squares and approximately 31 bacterial isolates were
streaked on each plate using the previously mentioned technique. Sterile water was used as a
negative control and 10% bleach (London Drugs, prod by RW packaging Ltd, Win, MB), and
1.5% peroxigard (Bayer, Toronto, Canada) were used as positive controls. 40 µl of each of these
substances was pipetted onto sterile 8 mm paper discs (Toyo Roshi Kaisha Ltd., Japan), which
were air dried and placed on plates with sterile forceps. All plates were then parafilmed and
inverted at 15°C. Plates were observed from day 7 to day 26, with photographs being taken when
confluent Pd growth was observed on the surface of the plate.
Preparation of microbial supernatants for anti-Pd bioassay
Modified V8 broth medium (20% low sodium V8 vegetable cocktail supernatant, 1%
Pharmamedia, 0.5% calcium carbonate, pH 6) was prepared, separated into 5 mL aliquots in
glass test tubes, and autoclaved at 121°C for 15 minutes. A sterile loop was used to pick a
9

bacterial colony or scrape fungal hyphae from each pure culture and inoculate V8 broth. Tubes
were incubated shaking at 110 rpm at 15°C for 14 days. On both day 7 and day 14 after
inoculation, 1 mL of broth was removed from each test tube and stored in a sterile microfuge
tube. Broth containing bacteria was centrifuged at 4°C at 14000 rpm for 20 minutes, and broth
containing fungi was centrifuged at 4°C at 14000 rpm for five minutes. Supernatants were then
filtered through VWR sterile syringe filters (0.2 µm cellulose acetate membrane, NA catalog
#28145-477) to ensure removal of bacteria and fungi. Filtered supernatants were used
immediately or stored at -20°C overnight. Once the filtered supernatants had been tested, they
were stored at -20°C.
Screening of microbial supernatants with an anti-Pd bioassay
SDA medium was prepared as per the manufacturer’s instructions and autoclaved for 15 minutes
at 121°C. After the medium had cooled to approximately 50°C, 85 µl of 100 mg/mL
chloramphenicol dissolved in ethanol and 50-100 µl of conidia suspension at approximately 4.9 x
107 conidia/mL were added per 250 mL of SDA. The concentration of chloramphenicol and
conidia in the medium were 34 µg /mL and 9800-19600 conidia/mL, respectively. The medium
was then gently mixed, poured into bioassay plates (Nunc®, 245 mm x 245 mm x 25 mm), and
allowed to solidify in a laminar flow hood. Before the medium was added, grids were drawn on
the bottom surface of each plate to allow 30 bacterial supernatants plus controls (6 x 6 grid) or 26
fungal supernatants plus controls (6 x 5 grid) to be tested at one time. 50 µl of filtered
supernatant or control substance was pipetted onto each 8 mm paper disc (Toyo Roshi Kaisha
Ltd., Japan) and these were allowed to dry before being placed on the bioassay plates with sterile
forceps. Sterile water and V8 broth were used as negative controls, and 10% bleach and 1.5%
peroxigard were used as positive controls. Plates were then parafilmed and placed upright at
15°C. After one to two days, plates were inverted. Plates were observed from day 7 to day 21,
with photographs being taken when confluent Pd growth was observed on the surface of the
plate. Isolates creating zones of inhibition were recorded and the zones diameters were measured
in three directions with an electronic Vernier caliper (Guangxi China, Mainland) 10 days after
plates were inoculated with Pd. The average diameters of inhibitory zones were calculated from
the three zone diameter measurements taken for each zone using the Average function in Excel.
The standard deviation of the measurements was calculated using the STDEV.S function in

10

Excel for each inhibitory zone. Average zone diameters were visualized as a bar chart in Excel,
with error bars representing the standard deviation calculated for each zone.

RESULTS
Pre-screening of bacterial isolates for anti-Pd activity
A direct contact streak assay was used to pre-screen bacterial isolates for anti-Pd activity in order
to increase the efficiency of screening with the subsequent anti-Pd bioassay. Pd growth was
visible in the agar seven days after the plate was inoculated, and some inhibitory zones were also
starting to become visible at this time. By day 10, confluent Pd lawn growth was observed on the
agar surface and zones of inhibition were clearly visible. Fifteen days after inoculation, some
zones of inhibition were no longer visible or had become reduced in size or intensity. Four types
of Pd inhibition were observed on the screening plates: complete, subsurface, surface, and partial
surface inhibition (Figure 3). Inhibition was considered to be complete when no conidial
germination leading to Pd colonies was observed within or on the agar surface in the area
surrounding the bacterial streak. Subsurface inhibition was considered to be a lack of Pd
germination in the agar, with some Pd colony growth observed on the surface of the plate.
Surface inhibition was considered to be a lack of Pd colony growth on the agar surface
surrounding the bacterial streak with Pd germination and/or growth observed within the agar.
Partial surface inhibition was considered to be a weaker form of surface inhibition, with more Pd
growth observed in the agar or some minimal Pd growth observed on the agar surface.

Figure 3. The four types of P. destructans inhibition exhibited by bacterial isolates during the prescreening experiments. Any isolates creating a zone of inhibition were subjected to further testing
using the anti-Pd bioassay.

11

The type of Pd inhibition observed ten and fifteen days after plate inoculation was recorded for
each bacterial isolate creating a zone of inhibition, as well as the negative (water) and positive
(10% bleach and 1.5% peroxigard) controls (Table 1, summarized in Table 2). Water did not
create a zone of Pd inhibition. Bleach created a zone of surface inhibition, which decreased in
size by day 15. Peroxigard created a zone of complete inhibition. One bacterial isolate (#47)
created a zone of complete inhibition that was visible on both day 10 and day 15 after
inoculation. Zones of subsurface inhibition that persisted for 15 days were created in Pd lawns
by isolate numbers 14, 37, 38, 51, and 53. All other inhibitory zones created by bacterial isolates
were zones of surface inhibition, with some decreasing in size, becoming zones of partial surface
inhibition, or disappearing altogether by day 15 after inoculation.
Table 1. Inhibition type for bacterial isolates and controls (water, bleach, peroxigard) creating
inhibitory zones in P. destructans fungal lawns on day 10 and day 15 after inoculation.

Inhibition type
Assigned screening number
3
10
12
13
14
19
20
21
32
34
35
36
37
38
41
47
51
53
54
55
56

Day 10

Day 15

Surface
Surface
Surface
Surface
Subsurface
Surface
Surface
Surface
Surface
Surface
Surface
Surface
Subsurface
Subsurface
Surface
Complete
Subsurface
Subsurface
Surface
Surface
Surface

Surface
None
Partial surface
Partial surface
Subsurface
None
Partial surface
Partial surface
Partial surface
Surface
Surface
Surface
Subsurface
Subsurface
Surface
Complete
Subsurface
Subsurface
Surface
Surface
Surface
12

70
71
77
80
81
84
86
89
90
Water
Bleach
Peroxigard

Surface
Surface
Surface
Surface
Surface
Surface
Surface
Surface
Surface
None
Surface
Complete

Partial surface
Partial surface
Partial surface
Surface
Surface
Partial surface
None
None
Partial surface
None
Surface
Complete

Table 2. The number of isolates creating each type (complete, subsurface, surface, or partial
surface) of inhibitory zone in P. destructans fungal lawns on day 10 and day 15 after inoculation.

Type of inhibition

Number of isolates creating inhibitory zone in P. destructans
fungal lawn
Day 10
Day 15

Complete

1

1

Subsurface

5

5

Surface

24

10

Partial surface

0

10

Screening of microbial supernatants with an anti-Pd bioassay
An anti-Pd bioassay was used to determine if inhibitory activity would persist in the absence of
bacterial cells and to screen fungal isolates for inhibitory activity towards Pd. Bacteria exhibiting
anti-Pd activity in the pre-screening experiment, as well as all of the fungal isolates, were grown
in V8 broth cultures for a total of 14 days. On both day 7 and day 14 after the broth was
inoculated with the microbial isolates, a sample was aliquoted into a microfuge tube, centrifuged,
and the broth supernatant was filtered and used for the anti-Pd bioassay. The plates were
photographed and the inhibitory zone diameters were measured on day 10 after plate inoculation.

13

The positive controls (10% bleach and 1.5% peroxigard) created zones of inhibition in the Pd
lawn. The negative controls (water and sterile V8 broth) did not create inhibitory zones. Fungal
isolate numbers 123, 156, and 158 created zones of inhibition in the Pd lawn when supernatant
collected seven days after broth inoculation was used (Figure 4). Isolate number 123 created a
small zone of partial surface inhibition, number 156 created a larger zone of partial surface
inhibition, and number 158 created a small zone of complete inhibition.

Figure 4. Zones of inhibition created in P. destructans fungal lawns by filtered broth supernatant of
three fungal isolates collected seven days after broth inoculation. The photographs were taken on
day 12 (#123) and day 10 (#156 and #158) after the plate was inoculated.

Bacterial isolate numbers 3, 13, 21, 32, 36, 55, 56, 81, 84, and 90 all created zones of surface
inhibition in the Pd fungal lawn when broth supernatant collected on day 7 was used (Figure 5).
The largest zones, which were all over 33 mm in diameter (Table 3), were created by isolates 3,
13, and 90; however, bacterial growth was observed around the paper discs containing the
filtered broth supernatant from these isolates. All zones of inhibition created by bacterial broth
supernatants collected on day 7 were over 18 mm in diameter (Table 3). Apart from isolate
numbers 3, 13, and 90, no zones of inhibition created by bacteria were still present on day 21
after plate inoculation. However, these three zones of inhibition persisted due to unwanted
bacterial growth on the plate surface from the supernatant. The zone diameters created by filtered
fungal broth supernatant numbers 123, 156, and 158 measured 11.7 mm, 20.6 mm, and 11.1 mm,
respectively. However, only fungal isolate number 123 still had an inhibitory zone present after
21 days of Pd growth, although the zone size was greatly reduced by day 21.

14

Figure 5. Anti-Pd bioassay plate with a P. destructans fungal lawn and inhibitory zones created by
filtered bacterial supernatant collected seven days after broth inoculation. The photograph was
taken 10 days after the plate was inoculated. Bacterial isolates creating zones of inhibition are
labeled with their assigned screening number. Controls are labeled with a letter to indicate their
identity. B=10% bleach, P=1.5% peroxigard, W=sterile water, V8=sterile V8 broth.

15

Table 3. Average diameter (mm), type, and length of presence for inhibitory zones created in P.
destructans fungal lawns by bacterial or fungal filtered broth supernatant collected seven days after
broth inoculation. Inhibitory zones were measured 10 days after the plate was inoculated (except
isolate 123 and the corresponding controls that were measured on day 17) and monitored until day
21. The horizontal lines in the table separate the results from different screening plates.

Screening
number
bleach
peroxigard
3
13
21
32
36
55
56
81
84
90
bleach
peroxigard
123
bleach
peroxigard
156
158

Average inhibitory
zone diameter (mm)
11.2
12.0
33.7
34.9
21.6
20.4
20.9
26.0
18.0
24.4
25.2
33.6
0.0
10.0
11.7
13.7
14.7
20.6
11.1

Inhibition type
surface
complete
surface
surface
surface
surface
surface
surface
surface
surface
surface
surface
none
surface
partial surface
surface
complete
partial surface
complete

Zone still present on day
21
no
no
yes
yes
no
no
no
no
no
no
no
yes
no
yes
yes
no
yes
no
no

When the broth supernatant was collected on day 14 after broth inoculation, bacterial isolate
numbers 3, 13, 14, 21, 32, 34, 36, 55, 56, 80, 81, 84, and 90 all created a zone of surface
inhibition in the Pd fungal lawn (Figure 6). All of the average bacterial inhibitory zone diameters
were greater than 16 mm, with the largest diameter being 28.8 mm (Table 4). Peroxigard (1.5%)
created a zone of complete inhibition measuring 14.0 mm in diameter and bleach (10%) created a
zone of surface inhibition measuring 13.3 mm in diameter. Sterile water and sterile V8 broth,
which were used as negative controls, did not create inhibitory zones in the Pd lawn.

16

Figure 6. Anti-Pd bioassay plate with a P. destructans fungal lawn and inhibitory zones created by
filtered bacterial supernatant collected 14 days after broth inoculation. The photograph was taken
10 days after the plate was inoculated. Bacterial isolates creating zones of inhibition are labeled
with their assigned screening number. Controls are labeled with a letter to indicate their identity.
B=10% bleach, P=1.5% peroxigard, W=sterile water, V8=sterile V8 broth.

The filtered broth supernatants from fungal isolate numbers 123, 124, 125, 127, 139, 156 and
158 also created zones of Pd inhibition when the broth was collected 14 days after inoculation
(Figure 7). Isolate numbers 124 and 158 created zones of complete inhibition measuring 13.0
mm and 14.4 mm in diameter, respectively (Table 4). Isolate number 123 created a zone of
surface inhibition with a 25.5 mm diameter. The fungal broth supernatant from fungal isolate
numbers 125, 127, 139, and 156 created zones of partial surface inhibition with zone diameters
of 12.8 mm, 13.2 mm, 13.0 mm, and 19.9 mm, respectively.

17

Figure 7. Zones of inhibition created in P. destructans fungal lawns by filtered broth supernatant of
seven fungal isolates collected 14 days after broth inoculation. The photographs were taken 10 days
after the plate was inoculated.

Of the zones of inhibition created by broth supernatants collected 14 days after inoculation, only
isolate number 36 still had a zone of inhibition by day 21 of Pd growth (Table 4). However, this
zone was reduced in size and had an irregular shape by day 21. Peroxigard had a zone of
complete Pd inhibition that persisted until day 21 but did decrease in size; however, the zone of
surface inhibition created by bleach did not persist until day 21.
Table 4. Average diameter (mm), type, and length of presence for inhibitory zones created in P.
destructans fungal lawns by bacterial or fungal filtered broth supernatant collected 14 days after
broth inoculation. Inhibitory zones were measured 10 days after the plate was inoculated and
monitored until day 21. The horizontal lines in the table separate the results from different
screening plates.

Screening number
bleach
peroxigard
3
13
14
21
32
34
36
55
56
80
81
84
90
bleach
peroxigard
123

Average inhibitory
zone diameter (mm)
13.3
14.0
25.1
28.8
16.4
24.3
20.8
22.1
19.3
27.8
27.6
24.3
23.7
27.9
28.0
23.2
17.4
25.5

Inhibition type
surface
complete
surface
surface
partial surface
surface
surface
surface
surface
surface
surface
surface
surface
surface
surface
surface
complete
surface

Zone still present on
day 21
no
yes
no
no
no
no
no
no
yes
no
no
no
no
no
no
no
yes
no

18

124
125
bleach
peroxigard
127
139
bleach
peroxigard
156
158

13.0
12.8
16.6
14.7
13.2
13.0
27.0
15.1
19.9
14.4

complete
partial surface
surface
complete
partial surface
partial surface
surface
complete
partial surface
complete

no
no
no
yes
no
no
no
yes
no
no

In order to compare the size of inhibitory zones created in Pd fungal lawns by broth collected on
day 7 versus day 14 after inoculation, a bar chart of average zone diameters was created in Excel,
with the error bars representing standard deviation (Figure 8, see also Supplementary Table 2).
For isolate numbers 14, 34, 80, 124, 125, 127, and 139, no inhibition was observed when day 7
supernatant was used; however, inhibitory zones were observed when day 14 supernatant was
applied to the plates. The day 7 supernatant created a larger zone of inhibition than the day 14
supernatant for isolate numbers 3, 13, 36, 81, 90, and 156. The day 14 supernatant created a
larger zone of inhibition than the day 7 supernatant for isolate numbers 21, 32, 55, 56, 84, 123,
and 158.

19

Figure 8. Comparison of average inhibitory zone diameters (mm) created in P. destructans fungal
lawns by bacterial and fungal filtered broth supernatant collected seven (grey) and 14 (blue) days
after broth inoculation. Error bars represent standard error (n=3).

DISCUSSION
The goal of this study was to screen bacteria and fungi for inhibitory activity towards Pd in order
to identify candidates that could have potential for use as a biological control agent. The
ecological and economic importance of bats combined with the high death tolls due to WNS and
the continued spread of the disease across North America exemplify why this is an important
area of research. Initially, 93 bacterial isolates collected in the areas near Bush Lake and Timber
Lake, B.C. were pre-screened for anti-Pd activity using a direct contact streak assay. Thirty of
the 93 isolates created a zone of inhibition in the Pd fungal lawn, which is a relatively high
percentage of the isolates (32%) compared to previous studies. Hamm and colleagues (2017)
isolated 632 Actinobacteria from bats, with only 36 isolates (6%) showing inhibitory activity
20

towards Pd and Hoyt and colleagues (2015b) isolated 133 bacterial morphotypes from bats, with
only six isolates (4.5%) observed with anti-Pd activity. This suggests that although bats may
harbor some bacteria able to inhibit Pd growth, our sample location may be a better source of
bacterial anti-Pd agents. However, it should be noted that bacterial isolates were not genetically
identified in this study, so it is possible that some of the isolates are identical, leading to a
potential overestimation of the percentage of bacterial strains from our sampling location
exhibiting anti-Pd activity.
Although the types of inhibition varied, all 30 of the bacteria creating inhibitory zones in the prescreening experiment were subjected to further testing using a Kirby-Bauer style agar diffusion
assay, which is referred to as an anti-Pd bioassay. The fungal isolates were not pre-screened,
since a direct contact assay led to the overgrowth of the fungal isolates being tested due to a
difference in the growth rates of Pd and the fungal isolates. Therefore, the fungi were only
screened for anti-Pd activity using the anti-Pd bioassay, which involved growing bacteria and
fungi in V8 broth, collecting and filtering the supernatants on day 7 and day 14 of growth, and
spotting the filtered supernatants on paper discs that were placed on the surface of agar seeded
with Pd conidia. Since the supernatant was filtered, the assay tested for inhibitory substance
production and Pd inhibition in the absence of microbial cells. This is a novel assay for anti-Pd
activity screening, since, to the extent of our knowledge, no other published studies have used
the seeded agar technique or filtered microbial broth supernatant for screening microorganisms
for anti-Pd activity.
Of the bacteria that produced anti-Pd activity in the pre-screening experiment, 10 exhibited antiPd activity when the day 7 filtered supernatant was tested against Pd, and the same 10 isolates
plus an additional three of the original 30 positive candidates exhibited anti-Pd activity when the
day 14 filtered supernatant was used. The reduction in the number of candidates exhibiting antiPd activity when progressing from the pre-screening experiment to using the filtered broth
supernatant could be a result of using different media to grow the bacteria in the two
experiments. With the exception of isolate number 14, the bacterial supernatants that produced
zones of surface inhibition in the anti-Pd bioassay corresponded to bacteria that produced large
zones of surface inhibition in the pre-screening experiment. The largest zones of inhibition
observed in our study when either filtered day 7 supernatant or filtered day 14 supernatant were

21

used were both produced by isolate number 13 and measured 34.9 mm and 28.8 mm in diameter,
respectively. The zones of inhibition produced by Actinobacteria tested by Hamm and colleagues
(2017) ranged from 1 mm to 45 mm in diameter, with most of their isolates producing zones of
inhibition less than 30 mm in diameter, although the difference in screening methods makes it
difficult to accurately compare studies. In this study, all of the zones of inhibition created by
bacterial broth supernatants were larger than the zones of inhibition produced by the positive
controls (bleach and peroxigard), however the zones created by the positive controls persisted for
longer periods of time than those produced by the bacterial supernatants, which only lasted for
one to two days after confluent Pd growth was observed. Although it is difficult to determine
why the zones of inhibition did not persist without knowing the mechanism of Pd inhibition, it is
possible that Pd degrades the inhibitory substance over time or the inhibitory substance is only
able to slow the growth or germination of Pd rather than stopping germination completely.
During the fungal isolate screening, five of the 53 fungi collected from the area around Bush
Lake and Timber Lake, B.C., none of the 16 fungi collected from the mushroom farm in
Summerland, B.C., and two of the eight fungi collected from dwarf mistletoe plants exhibited
anti-Pd activity. Of the seven positive fungal candidates, only three created an inhibitory zone
when filtered day 7 supernatant was used, but all seven displayed anti-Pd activity when filtered
day 14 supernatant was used. When testing day 7 supernatant, the largest inhibitory zone was
created by fungal isolate number 156 (20.6 mm diameter). Both isolate number 156 and number
123 created inhibitory zones larger than the zones created by the positive controls when day 7
supernatant was used. The largest inhibitory zone created by a fungal supernatant collected on
day 14 was 25.5 mm in diameter (isolate number 123). However, most of the positive fungal
candidates created inhibitory zones that were smaller than the zones created by positive controls
when the day 14 supernatant was used.
Overall, bacteria appear to be a better source of anti-Pd activity than fungi, based on this study.
A greater number of bacterial isolates produced anti-Pd activity compared to the fungal isolates
and the zones of inhibition produced by bacteria tended to be larger than those produced by
fungi. The ability to use a pre-screen and the ease of working with bacteria compared to fungi
also favors future studies investigating bacteria for anti-Pd activity over fungi. Additionally,
collecting bacterial and fungal broth supernatant on day 14 rather than day 7 should be used for

22

future studies since a greater number of isolates exhibited activity when day 14 supernatant was
used and a greater proportion of the inhibitory zones were larger when day 14 supernatant was
used, compared to day 7 supernatant. It is possible that a greater variety of metabolites with antiPd activity are produced closer to day 14 or that the amounts of these metabolites increase over
time, leading to increased efficacy of the day 14 supernatants. However, there may be a day in
between day seven and 14 or after day 14 that is optimal, which would require further research,
such as the completion of a fermentation time course experiment.
The appearance of the three types of inhibitory zones (complete, subsurface, surface) in the two
screening assays was likely dependent on whether the anti-Pd activity was fungistatic (Pd
germination and growth slowed/inhibited) or fungicidal (Pd conidia killed) (Shelley et al., 2013),
the mechanism of growth inhibition, and the rate of agar diffusion if a secreted inhibitory
substance was responsible for inhibition. However, none of these factors were examined during
this study, so the reasons for the varying types of inhibition cannot be determined. The zone of
complete inhibition could have been caused by fungistatic or fungicidal activity, but the zones of
surface inhibition were likely due to fungistatic activity since conidial germination did occur and
the zones of inhibition did not persist. Unlike the zones of inhibition created on the agar surface,
the zones of complete and subsurface inhibition did not appear to have any conidial germination.
If zones of inhibition were due to fungistatic activity, it is possible that this was caused by the
binding of an inhibitory agent to the conidia coat and the subsequent inhibition of conidial
germination by the bound agent acting on germ tube formation (Shelley et al., 2013). Little is
known about the mechanisms of Pd inhibition by other biocontrol agents; however, the
sensitivity of Pd to amphotericin B and certain azole antifungals is caused by the inhibition of
ergosterol (an important fungal cell membrane component) biosynthesis or the binding of the
antifungal to ergosterol in the cell membrane (Chaturvedi et al., 2011). Shelley et al. (2013) also
found that effective antifungal agents contained a long-chain alkyl group in their chemical
structure, indicating that chemical structure may play a key role in growth inhibition of Pd.
Another key factor affecting the type and size of inhibitory zone observed is the ability of the
inhibitory substance to diffuse through the agar. Diffusion through agar can depend on many
factors, such as concentration of the substance, amount of the substance, depth of the agar, size
of the molecules, and the incubation temperature (Koch, 1999). When bacteria are streaked on

23

the agar surface or a paper disc containing a substance is placed on the agar, diffusion occurs
both vertically and radially from the source of the substance. Koch (1999) determined that the
greater the radial distance from the source, the lower the concentration of the substance will be at
the bottom surface of the agar at that point and the more time it will take for the concentration of
the substance to equalize between the top and bottom surface of the agar. Keeping this in mind, it
may be possible that the substance(s) being produced by certain bacteria or fungi have varying
abilities to diffuse through the agar, affecting the size or type of inhibitory zone observed. For
instance, if the conidia near the agar surface were exposed to high concentrations of a substance
diffusing from the agar surface and the conidia near the bottom of the agar were exposed to a
very low concentration of this substance due to unequal diffusion through the agar, this may lead
to a zone of surface inhibition, since conidia near the agar bottom may not experience the same
effects as the conidia near the surface and may still be able to germinate. This may give
misleading results, since the agar diffusion ability of the substance may determine the level of
anti-Pd activity observed, rather than the potency of the agent itself. This is a limit of the method
used, since rather than having one layer of conidia on the agar surface, the conidia are dispersed
throughout the agar, meaning that any anti-Pd agent must diffuse from the surface of the agar to
the bottom surface of the plate at a high enough concentration to kill or inhibit conidial
germination in order to create a zone of complete inhibition. In future studies, this could be
avoided by spreading conidia on the agar surface rather than embedding them in the agar,
although fungal lawns created using this method are not as uniform.
Further experiments could also be conducted to determine if the observed anti-Pd activity was
fungicidal or fungistatic. For example, a germule suppression assay could be used to examine
conidia during germination (Cornelison et al., 2014), or a sporicidal assay could be used to
determine if spores can germinate after being soaked in an inhibitory agent (Shelley et al., 2013).
It may also be interesting to view Pd spores using a scanning electron microscope to determine if
inhibition involves any distortion of the spores, something that hasn’t been done in any other
studies involving Pd. Additionally, the isolates with strongest anti-Pd activity (13, 90, 3, 55, 84)
should undergo further testing such as whole-genome sequencing, transcriptomic analysis, and
metabolic profiling in order to determine if they could be used for the biological control of Pd.
Ideally, if the active compounds produced by these isolates could be identified, then potentially
the corresponding genes could be transformed into Escherichia coli and the compound(s) could
24

be produced on a large scale. Testing the compound(s) for safety for use on bats or in bat
hibernacula would also need to be conducted before any novel biological control agent could be
used in the field.
In summary, 93 bacterial isolates and 77 fungal isolates were screened for anti-Pd activity with
30 bacterial isolates and seven fungal isolates exhibiting anti-Pd activity. Our results indicate
that microorganisms isolated from the areas around Bush Lake and Timber Lake B.C. have a
high level of antimicrobial activity and are promising candidates in the search for a novel
biological control agent for Pd, the causative agent of WNS. To the extent of our knowledge, this
study is the first to use embedded Pd conidia and microbial broth supernatants to screen
microorganisms for anti-Pd activity. Additionally, this is the first study to screen bacteria and
fungi from Bush Lake and Timber Lake B.C., a mushroom farm, and dwarf mistletoe plants, as
well as one of the only studies to screen fungi for anti-Pd activity.

25

LITERATURE CITED
Bat Conservation International (2017). Bats Are: Threatened. Available
http://www.batcon.org/why-bats/bats-are/bats-are-threatened [accessed 15 May 2017].

from

Boire, N., Zhang, S., Khuvis, J., Lee, R., Rivers, J., Crandall, P., Keel, M.K., and Parrish, N.
(2016). Potent Inhibition of Pseudogymnoascus destructans, the Causative Agent of White-Nose
Syndrome in Bats, by Cold-Pressed, Terpeneless, Valencia Orange Oil. PLOS ONE 11,
e0148473.
Boyles, J.G., Cryan, P.M., McCracken, G.F., and Kunz, T.H. (2011). Economic Importance of
Bats in Agriculture. Science 332, 41–42.
Chaturvedi, S., Rajkumar, S.S., Li, X., Hurteau, G.J., Shtutman, M., and Chaturvedi, V. (2011).
Antifungal Testing and High-Throughput Screening of Compound Library against Geomyces
destructans, the Etiologic Agent of Geomycosis (WNS) in Bats. PLOS ONE 6, e17032.
Chaturvedi, V., Springer, D.J., Behr, M.J., Ramani, R., Li, X., Peck, M.K., Ren, P., Bopp, D.J.,
Wood, B., Samsonoff, W.A., et al. (2010). Morphological and Molecular Characterizations of
Psychrophilic Fungus Geomyces destructans from New York Bats with White Nose Syndrome
(WNS). PLOS ONE 5, e10783.
Cornelison, C.T., Keel, M.K., Gabriel, K.T., Barlament, C.K., Tucker, T.A., Pierce, G.E., and
Crow, S.A. (2014). A preliminary report on the contact-independent antagonism of
Pseudogymnoascus destructans by Rhodococcus rhodochrousstrain DAP96253. BMC Microbiol.
14, 246.
Flieger, M., Bandouchova, H., Cerny, J., Chudíčková, M., Kolarik, M., Kovacova, V.,
Martínková, N., Novák, P., Šebesta, O., Stodůlková, E., et al. (2016). Vitamin B2 as a virulence
factor in Pseudogymnoascus destructans skin infection. Sci. Rep. 6.
Frick, W.F., Cheng, T.L., Langwig, K.E., Hoyt, J.R., Janicki, A.F., Parise, K.L., Foster, J.T., and
Kilpatrick, A.M. (2017). Pathogen dynamics during invasion and establishment of white-nose
syndrome explain mechanisms of host persistence. Ecology 98, 624–631.
Gargas, A., Trest, M.T., Christensen, M., Volk, T.J., and Blehert, D.S. (2009). Geomyces
destructans sp. nov. associated with bat white-nose syndrome. Mycotaxon 108, 147–154.
Gill, J. (2016). Bat disease treatment a step forward, but not a cure, researcher says. Available
from
http://www.cbc.ca/news/canada/new-brunswick/white-nose-syndrome-nov-nin-si1.3843537 [accessed 15 May 2017].
Haman, K., Hibbard, C., and Lubeck, M. (2016). Bat with white-nose syndrome confirmed in
Washington state. Wash. Dep. Fish Wildl.
Hamm, P.S., Caimi, N.A., Northup, D.E., Valdez, E.W., Buecher, D.C., Dunlap, C.A., Labeda,
D.P., Lueschow, S., and Porras-Alfaro, A. (2017). Western Bats as a Reservoir of Novel
Streptomyces Species with Antifungal Activity. Appl. Environ. Microbiol. 83, e03057-16.

26

Hoyt, J.R., Langwig, K.E., Okoniewski, J., Frick, W.F., Stone, W.B., and Kilpatrick, A.M.
(2015a). Long-Term Persistence of Pseudogymnoascus destructans, the Causative Agent of
White-Nose Syndrome, in the Absence of Bats. EcoHealth 12, 330–333.
Hoyt, J.R., Cheng, T.L., Langwig, K.E., Hee, M.M., Frick, W.F., and Kilpatrick, A.M. (2015b).
Bacteria Isolated from Bats Inhibit the Growth of Pseudogymnoascus destructans, the Causative
Agent of White-Nose Syndrome. PLOS ONE 10, e0121329.
Koch, A.L. (1999). Diffusion through agar blocks of finite dimensions: a theoretical analysis of
three systems of practical significance in microbiology. Microbiol. Read. Engl. 145 ( Pt 3), 643–
654.
Kunz, T.H., de Torrez, E.B., Bauer, D., Lobova, T., and Fleming, T.H. (2011). Ecosystem
services provided by bats. Ann. N. Y. Acad. Sci. 1223, 1–38.
Leopardi, S., Blake, D., and Puechmaille, S.J. (2015). White-Nose Syndrome fungus introduced
from Europe to North America. Curr. Biol. 25, R217–R219.
Lorch, J.M., Meteyer, C.U., Behr, M.J., Boyles, J.G., Cryan, P.M., Hicks, A.C., Ballmann, A.E.,
Coleman, J.T.H., Redell, D.N., Reeder, D.M., et al. (2011). Experimental infection of bats with
Geomyces destructans causes white-nose syndrome. Nature 480, 376–378.
Magnusson, J., Ström, K., Roos, S., Sjögren, J., and Schnürer, J. (2003). Broad and complex
antifungal activity among environmental isolates of lactic acid bacteria. FEMS Microbiol. Lett.
219, 129–135.
Minnis, A.M., and Lindner, D.L. (2013). Phylogenetic evaluation of Geomyces and allies reveals
no close relatives of Pseudogymnoascus destructans, comb. nov., in bat hibernacula of eastern
North America. Fungal Biol. 117, 638–649.
Moriarty, M. (2009). Little brown bat with white-nose syndrome in Greeley Mine, Vermont.
O’Donoghue, A.J., Knudsen, G.M., Beekman, C., Perry, J.A., Johnson, A.D., DeRisi, J.L., Craik,
C.S., and Bennett, R.J. (2015). Destructin-1 is a collagen-degrading endopeptidase secreted by
Pseudogymnoascus destructans, the causative agent of white-nose syndrome. Proc. Natl. Acad.
Sci. 112, 7478–7483.
Padhi, S., Dias, I., and Bennett, J.W. (2017). Two volatile-phase alcohols inhibit growth of
Pseudogymnoascus destructans, causative agent of white-nose syndrome in bats. Mycology 8,
11–16.
Perotto, S., Angelini, P., Bianciotto, V., Bonfante, P., Girlanda, M., Kull, T., Mello, A.,
Pecoraro, L., Perini, C., Persiani, A.M., et al. (2013). Interactions of fungi with other organisms.
Plant Biosyst. - Int. J. Deal. Asp. Plant Biol. 147, 208–218.
Puechmaille, S.J., Wibbelt, G., Korn, V., Fuller, H., Forget, F., Mühldorfer, K., Kurth, A.,
Bogdanowicz, W., Borel, C., Bosch, T., et al. (2011). Pan-European Distribution of White-Nose

27

Syndrome Fungus (Geomyces destructans) Not Associated with Mass Mortality. PLOS ONE 6,
e19167.
Raudabaugh, D.B., and Miller, A.N. (2015). Effect of Trans, Trans-Farnesol on
Pseudogymnoascus destructans and Several Closely Related Species. Mycopathologia 180, 325–
332.
Reeder, D.M., Frank, C.L., Turner, G.G., Meteyer, C.U., Kurta, A., Britzke, E.R., Vodzak, M.E.,
Darling, S.R., Stihler, C.W., Hicks, A.C., et al. (2012). Frequent Arousal from Hibernation
Linked to Severity of Infection and Mortality in Bats with White-Nose Syndrome. PLOS ONE 7,
e38920.
Reynolds, H.T., and Barton, H.A. (2014). Comparison of the White-Nose Syndrome Agent
Pseudogymnoascus destructans to Cave-Dwelling Relatives Suggests Reduced Saprotrophic
Enzyme Activity. PLOS ONE 9, e86437.
Shelley, V., Kaiser, S., Shelley, E., Williams, T., Kramer, M., Haman, K., Keel, K., and Barton,
H.A. (2013). Evaluation of strategies for the decontamination of equipment for Geomyces
destructans, the causative agent of white-nose syndrome (WNS). J. Cave Karst Stud. 75, 1–10.
Springer, D.J. (2013). Original false-colored scanning electron micrograph of G. destructans on a
Little Brown Bat (Myotis lucifugs).
Thomas, D.W., Dorais, M., and Bergeron, J.-M. (1990). Winter Energy Budgets and Cost of
Arousals for Hibernating Little Brown Bats, Myotis lucifugus. J. Mammal. 71, 475–479.
U.S. Fish and Wildlife Service (2017). About white-nose syndrome. Available from
https://www.whitenosesyndrome.org/about-white-nose-syndrome [accessed 15 May 2017].
Verant, M.L., Boyles, J.G., Jr, W.W., Wibbelt, G., and Blehert, D.S. (2012). TemperatureDependent Growth of Geomyces destructans, the Fungus That Causes Bat White-Nose
Syndrome. PLOS ONE 7, e46280.
Verant, M.L., Meteyer, C.U., Speakman, J.R., Cryan, P.M., Lorch, J.M., and Blehert, D.S.
(2014). White-nose syndrome initiates a cascade of physiologic disturbances in the hibernating
bat host. BMC Physiol. 14, 10.
Warnecke, L., Turner, J.M., Bollinger, T.K., Misra, V., Cryan, P.M., Blehert, D.S., Wibbelt, G.,
and Willis, C.K.R. (2013). Pathophysiology of white-nose syndrome in bats: a mechanistic
model linking wing damage to mortality. Biol. Lett. 9, 20130177.
Wohlleben, W., Mast, Y., Stegmann, E., and Ziemert, N. (2016). Antibiotic drug discovery.
Microb. Biotechnol. 9, 541–548.
Zhang, T., Chaturvedi, V., and Chaturvedi, S. (2015). Novel Trichoderma polysporum Strain for
the Biocontrol of Pseudogymnoascus destructans, the Fungal Etiologic Agent of Bat White Nose
Syndrome. PLOS ONE 10, e0141316.

28

Zukal, J., Bandouchova, H., Brichta, J., Cmokova, A., Jaron, K.S., Kolarik, M., Kovacova, V.,
Kubátová, A., Nováková, A., Orlov, O., et al. (2016). White-nose syndrome without borders:
Pseudogymnoascus destructans infection tolerated in Europe and Palearctic Asia but not in North
America. Sci. Rep. 6, 19829.

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APPENDICES
Supplementary Table 1. A summary of bacterial and fungal samples collected from Bush and
Timber Lakes, What the Fungus mushroom farm, and dwarf mistletoe plants and their inhibitory
activity towards P. destructans in the bacterial pre-screening and/or the anti-Pd bioassay.

Sample
location

Isolate
numbers
& total
number
of
isolates

Bush Lake &
Timber
Lake,
B.C.

1-93
(bacteria)

Number Bacterial
Bacterial
of
positive
positive
bacteria candidate
candidate
prenumbers
numbers
screened
(pre(supernatant
screen)
screen)
3, 10, 12,
13, 14,
19, 20,
21, 32,
34, 35,
36, 37,
38, 41,
47, 51,
53, 54,
55, 56,
70, 71,
77, 80,
81, 84,
86, 89, 90

3, 13, 14, 21,
32, 34, 36,
55, 56, 80,
81, 84, 90

53

125, 127,
139, 156,
158

n/a

n/a

n/a

16

n/a

n/a

n/a

n/a

8

123, 124

125-177
(fungi)

Dwarf
mistletoe
plants

101-116
(fungi)

Fungal
positive
candidate
numbers

93

Total:
146

What the
Fungus
Mushroom
Farm,
Summerland,
B.C.

Number of
fungi
screened
(supernatant
screen)

Total: 16

117-124
(fungi)
Total: 8

30

Supplementary Table 2. Comparison of average diameter (mm) of inhibitory zones created in P.
destructans fungal lawns by bacterial or fungal filtered broth supernatants collected either seven or
14 days after broth inoculation. Inhibitory zones were measured 10 days after the plate was
inoculated with Pd spores. The bleach and peroxigard preceding the list of isolate numbers
correspond to the positive controls on the same plate as those bacterial or fungal candidates.

Screening
number
bleach
peroxigard
3
13
14
21
32
34
36
55
56
80
81
84
90
bleach
peroxigard
123
124
125
bleach
peroxigard
127
139
bleach
peroxigard
156
158

Average inhibitory zone diameter
(mm) for day 7 supernatant
11.2
12.0
33.7
34.9
n/a
21.6
20.4
n/a
20.9
26.0
18.0
n/a
24.4
25.2
33.6
0.0
10.0
11.7
n/a
n/a
n/a
n/a
n/a
n/a
13.7
14.7
20.6
11.1

Average inhibitory zone diameter
(mm) for day 14 supernatant
13.3
14.0
25.1
28.8
16.4
24.3
20.8
22.1
19.3
27.8
27.6
24.3
23.7
27.9
28.0
23.2
17.4
25.5
13.0
12.8
16.6
14.7
13.2
13.0
27.0
15.1
19.9
14.4

31