Faculty of Science
PLANTS, FUNGI, AND FREELOADERS: EXAMINING ENDOPHYTIC SPECIES
RICHNESS CHANGES OVER THE GROWING SEASON OF ARCEUTHOBIUM
AMERICANUM
2016 | LUCAS D. HAMPEL

B.Sc. Honours thesis

PLANTS, FUNGI, AND FREELOADERS:
EXAMINING ENDOPHYTIC SPECIES RICHNESS CHANGES OVER THE GROWING
SEASON OF ARCEUTHOBIUM AMERICANUM
by
Lucas D. Hampel
A THESIS SUBMITTED IN PARTIAL FULFILLMENT OF THE REQUIREMENTS FOR
THE DEGREE OF BACHELOR OF SCIENCE (HONS.)
in the
DEPARTMENT OF BIOLOGICAL SCIENCES
General Biology

This thesis has been accepted as conforming to the required standards by:
Cynthia Ross Friedman (Ph.D.), Thesis Supervisor, Dept. Biological Sciences
Naowarat Cheeptham (Ph.D.), Co-supervisor, Dept. Biological Sciences
Nancy Flood (Ph.D.), Committee Member, Dept. Biological Sciences
Dated this 29th day of April, 2016, in Kamloops, British Columbia, Canada

© Lucas D. Hampel, 2016

ABSTRACT
Arceuthobium americanum, or the lodgepole pine dwarf mistletoe, is a tree parasite in northern
Canada. The plant has negatively impacted the lumber industry, causing significant financial
losses, particularly in the west. Recently, endophytic fungi have been found in this plant, which
have been discovered to function in protecting the plant from surface pathogens. The current
study aimed to examine the endophytic fungi in more depth, looking for changes in species
richness over the plant’s growing season and determining whether these richness changes
differed between males and females. This information could yield a new management strategy,
which would involve applying a surface pathogen spray at a time when the endophytic fungal
diversity is lower and the plant is presumably more susceptible to pathogens.
Using pure culture techniques, endophytic fungi were isolated from surface sterilized A.
americanum males and females weekly from the end of April to the beginning of September.
Isolated fungi were characterized macroscopically to generate a database of morphologically
unique forms that corresponded to individual species.

Endophytic fungi that appeared

consistently throughout the sampling period were sent to be sequenced by MACROGEN™
sequencing in Korea to determine their identities based on ITS rDNA sequences.
Throughout the study period, 47 distinct morphological strains of fungi were isolated.
Endophytic fungal species abundance was found to increase throughout the growing season in
both males and female A. americanum plants. There was no significant difference in the species
richness between males and females, and the two groups shared 87.5% species similarity based
on Sørensen’s coefficient of similarity. Sequenced endophytes found in this study came from a
diverse array of genera, including Trichoderma, Serpula, Alternaria, and Tremella, which may
function as mutualistic symbionts within the plant.
Endophytic fungal species richness follows the trend shown by all other plants to date: it
increases throughout the growing season in new aerial shoots. Future work should aim to further
characterize endophytic communities using metagenomic techniques and comparing endophyte
communities of A. americanum to those of the host tree to see if there is crossover between the
two organisms.
Thesis Supervisor: Professor Cynthia Ross Friedman (Ph.D.)
ii

ACKNOWLEDGEMENTS
I would like to thank my supervisor, Dr. Cynthia Ross Friedman, co-supervisor, Dr. Naowarat
Cheeptham and all of the members of the Faculty of Science at Thompson Rivers University
who have helped me throughout this project with their input and recommendations. In particular,
I would also like to acknowledge committee member Dr. Nancy Flood for her assistance in
examining the data and assisting me with statistical analysis. I would like to recognise May Al
Fouadi for her assistance in the laboratory, where she led me through aspects of laboratory
techniques and protocols during the beginning of this project. I would also like to thank the
Natural Sciences and Engineering Research Council of Canada for their generous funding of my
project through an Undergraduate Student Research Award. Special thanks to the Department of
Biological Sciences Chairperson Dr. Mairi MacKay, who provided moral support throughout the
bureaucratic process of applying for funding.

iii

TABLE OF CONTENTS
ABSTRACT.................................................................................................................................... ii
ACKNOWLEDGEMENTS ........................................................................................................... iii
TABLE OF CONTENTS ............................................................................................................... iv
LIST OF FIGURES ........................................................................................................................ v
LIST OF TABLES .......................................................................................................................... v
INTRODUCTION .......................................................................................................................... 1
ARCEUTHOBIUM AMERICANUM: A UNIQUE PLANT-PLANT PARASITE ..................... 1
ENDOPHYTIC FUNGI: A POSSIBLE TOOL FOR BIOCONTROL? .................................... 2
PURPOSE ................................................................................................................................... 3
MATERIALS AND METHODS .................................................................................................... 4
SAMPLE COLLECTION........................................................................................................... 4
ENDOPHYTE CULTURING AND ISOLATION .................................................................... 4
ENDOPHYTE CHARACTERIZATION ................................................................................... 5
DATA ANALYSIS ..................................................................................................................... 7
RESULTS ....................................................................................................................................... 8
CHARACTERIZATION OF ENDOPHYTIC FUNGAL SPECIES IN A. AMERICANUM ..... 8
ENDOPHYTIC SPECIES RICHNESS CHANGES BETWEEN MALE AND FEMALE
PLANTS ................................................................................................................................... 13
SIMILARITY MEASURES IN THE ENDOPHYTE COMMUNITIES OF MALE AND
FEMALE A. AMERICANUM ................................................................................................... 14
DISCUSSION ............................................................................................................................... 15
CHARACTERIZATION OF ENDOPHYTES REVEALS A HIGH DIVERSITY OF FUNGI
PRESENT WITHIN A. AMERICANUM THROUGHOUT THE GROWING SEASON ........ 15
MALE AND FEMALE ENDOPHYTE COMMUNITIES EXHIBIT HIGH SIMILARITY .. 16

iv

SPECIES RICHNESS SEEMS TO INCREASE OVER THE GROWING SEASON IN BOTH
MALES AND FEMALES ........................................................................................................ 18
FUTURE WORK ...................................................................................................................... 20
CONCLUSION ......................................................................................................................... 21
LITERATURE CITED ................................................................................................................. 22
APPENDIX ................................................................................................................................... 25

LIST OF FIGURES
Figure 1. Endophytic species richness in male and female plants throughout the growing season.
Triangles denote male species richness and circles denote female species richness at each
sampling point. Regression lines for males (dashed line, R2 = 0.71) and females (solid line, R2 =
0.61) are included………………………………………………………………………..…….....13

LIST OF TABLES
Table 1. Macroscopic morphologies of fungi isolated from A. americanum. Each isolate has a
distinct two to three-letter name assigned in order to organize each morphologically distinct
isolate…………………………………………………………………………………………...…9
Table 2. Sequenced ITS region identities of endophytic fungal species isolated from A.
americanum. BLAST searches were performed with a resolution value of 97% sequence
similarity delineating a positive identification of a species. Literature searches were used to
explore the general biological niche each isolate………………………………………………..11
Table A1. Regression analysis of male species richness changes throughout the growing season
in A. americanum………………………………………………………………………………...25
Table A2. Regression analysis of male species richness changes throughout the growing season
in A. americanum……………………………………………………………………………...…25

v

INTRODUCTION

ARCEUTHOBIUM AMERICANUM: A UNIQUE PLANT-PLANT PARASITE
Plants of the Arceuthobium genus are unique in that they are plant-plant parasites of a variety
of host trees. The genus contains 42 different species, each of which infects a variety of trees
from both Pinaceae and Cupressaceae (Hawksworth and Wiens 1996). Morphologically,
Arceuthobium plants are small, evergreen herbs with haustorial tissues that permeate the host
tree and reduce timber quality of the wood. The shoots (i.e., the portion of the plant growing
out of the host) display the plant’s reproductive status. Members of the genus are dioecious,
meaning that the female (pistillate) and male (staminate) plant parts are found on separate
individuals. Of particular import in northwestern Canada is the Lodgepole Pine Dwarf
Mistletoe, Arceuthobium americanum Nutt. ex Engelm.

This species has a complex

reproductive cycle in which the female flowers develop over two years, culminating in
explosive discharge of the seed at the end of the summer, while the male flowers grow and
disperse their pollen on a yearly basis (Hawksworth and Wiens 1996). Seeds reaching a host
remain exposed on the host branches over the winter before germinating in the spring.
Typically, A. americanum infects two important lumber species present in British Columbia:
Pinus contorta Dougl. ex. Loud. (lodgepole pine) and Pinus banksiana Lamb. (jack pine).
The infection of these trees renders the plant a significant pest in western North America,
causing losses in timber volume annually throughout the region (Drummond 1982; Shamoun
et al. 2003).
Presently, there are a variety of management strategies in place to help mitigate the damage
A. americanum inflicts on forests, but most of them aim to limit the spread of the parasite to
neighbouring trees by selective logging of infected trees or by the subsequent planting of
lumber trees resistant to Arceuthobium infection (Baranyay and Smith 1972). While these
methods of control are capable of inhibiting the spread of infection in isolated stands, when
infections become more widespread, they become harder to manage. New methods of control
need to be found in order to mitigate any additional damage these parasites can cause to the
industry.

1

ENDOPHYTIC FUNGI: A POSSIBLE TOOL FOR BIOCONTROL?
Recent research on A. americanum has revolved around the exploration of endophytic fungal
symbionts within the plant (Martin et al. 2012). Endophytic fungi are organisms that live
within the host plant’s tissues. Greater than ninety percent of all plants are suspected to
harbour symbiotic fungi that interact with the host in a mutualistic or commensal fashion
(Rodriguez et al. 2009). Until recently, the phenomenon of plant endophytes in parasitic
plants was poorly documented. A paper published by Martin et al. (2012) reported the
extensive colonization of Arceuthobium americanum by endophytic fungal hyphae, a novel
observation of endophytes within the genus. The presence of fungal endophytes could reveal
a new avenue for the management of A. americanum. The study showed that the endophytes
helped inhibit the growth of the surface pathogen Cladosporium, a mold fungus. It could be
suspected that these endophytes play a role in helping the dwarf mistletoe resist pathogen
infection. Loss of endophytes in the flowering plants Quercus (Wang et al. 2006), Solanum
melangena (Narisawa et al. 2002) and Zea (Danielsen and Jensen 1999) can cause a marked
decrease in the plants’ ability to resist pathogen infections. The same could be true of A.
americanum, which is also a flowering plant.
Martin et al. (2012) did not determine whether the fungal communities within A. americanum
shoots changed over the growing season during shoot development. Previous studies have
shown that endophyte communities within an individual plant can change over its lifespan
(Faeth and Hammon 1997; Wearn et al. 2012).

Using pure-culture techinques, the

researchers were able to demonstrate the existence of an increasing trend in fungal endophyte
species richness present within the host’s tissues. If the endophytic communities in A.
americanum do change in some fashion (e.g., species loss or gain), the observation would be
the first for any parasitic plant currently known.
As mentioned, Arceuthobium species, including A. americanum, are dioecious (Hawksworth
and Wiens 1996). The study by Martin et al. (2012) did not address whether the endophyte
communities differed between genders.

Previous research in another dioecious plant,

Antenarria dioica showed that endophyte communities varied significantly between the sexes
(Vega-Frutis et al. 2013). However, there has been very little research on endophytic fungi
2

in dioecious plants. Studies in this area will broaden and deepen our knowledge on the
ecological interactions between A. americanum and its symbiotic partners, and provide more
information that could help improve our understanding of endophytic fungal communities
throughout the plant kingdom.

PURPOSE
The purpose of this project is to examine the endophytic fungi of A. americanum in more
depth, looking for changes in species richness over the plant’s growing season and
differences between genders. Specifically, this project aims to 1) examine and characterize
the endophytic fungi present within A. americanum; 2) determine if there are any significant
differences in species richness between the genders with respect to their culturable endophyte
communities; and 3) assess whether the communities of these endophytes change over the
growing season (both as a whole and between the male and female plants). The results of
such an exploration into the endophytic communities in A. americanum should reveal novel
information about the complicated fungal assemblage residing within the plant, and could
possibly yield a new management strategy based on endophytic organisms.

3

MATERIALS AND METHODS

SAMPLE COLLECTION
The methods used followed those of Martin et al. (2012). Samples of five growing A.
americanum female shoots with two fruit generations and five male shoots were collected
from five infected Pinus contorta (lodgepole pine) trees located near Stake Lake in the area
of Kamloops, British Columbia, Canada (50°31’N, 120°28’W). The specific age of the
shoots was selected based on the sheer abundance of female shoots available on trees in the
region. Said female shoots were pruned so that only the first year shoots were used for
culturing, thereby standardizing the age of the shoots between the male and female plants.
Weekly collection began April 26 of 2015 and ended September 1 of 2015. Samples were
taken from A. americanum shoots at eye level from all sides of the infected trees, and trees
sampled were marked with flagging tape so that the same mistletoe plants could be
repeatedly sampled. Shoots having damage or blemishes resulting from infection by other
pathogens were avoided, reducing the probability that non-endophytic fungi (surface
pathogens) were cultured, and increasing the likelihood that the organisms being cultured
from the sterilized samples were endophytic in nature.

ENDOPHYTE CULTURING AND ISOLATION
Culturing and endophyte isolation and characterization occurred simultaneously each week
over the collection period. The collected shoots were surface sterilized using 6% v/v sodium
hypochlorite solution for 10 minutes to remove any surface organisms, after which each
shoot was washed with sterile deionized water. Next, using a sterile razor blade, plant stems
were cut longitudinally along each shoot and placed onto 100 mm (diameter) X 20 mm
(deep) Petri plates (Fisherbrand™) with Potato Dextrose Agar, “PDA” (Sigma Aldrich)
(Atlas 2010). The plates were incubated at 28°C for three weeks, being checked every three
days for fungal growth. Fungi that grew from the sterilized tissue cultures was extracted and
subcultured onto new PDA agar plates for isolation based on differences in morphology. The
pure cultures were used to characterize the fungi based on macroscopic morphology.

4

ENDOPHYTE CHARACTERIZATION
Identification of genera present was made based on macroscopic morphology using
references to the taxonomic fungal databases as defined by J. A. Stalpers (1978) and Kirk et
al. (2008). Fungi were characterized based on their macroscopic morphology in pure culture,
and were defined by their form (general colony shape), colony diameter, elevation of the
colony, margin, surface texture, colour of the aerial and embedded colony, and architecture
of the aerial mycelium. Pure-culture studies have been used extensively in order to observe
endophytic fungi from a wide diversity of plant species (Faeth and Hammon 1997; Wearn et
al. 2012; McPherson et al. 2013; Min et al. 2014). By using similar pure-culture methods
supplemented with sequencing, a clear preliminary picture of the organisms present in A.
americanum was obtained.
Colour classes were arranged into three broad, categories to sort the fungi, with more specific
colour terms being used as defined by Ridgway (1912). There were three broad categories
used in this study: Black, Cream/White, and Orange/Yellow. Particular groups of fungi have
mycelial colours that are characteristic to a particular genus or species (Scurti et al. 1978;
Iotti et al. 2002). In identifying colour categories, and by extension determining the colour of
a particular isolate, one could be better able to elucidate the genera isolated from A.
americanum. These broad categories were also used in the general sorting of the fungi found
based on their morphology. The colours of multi-coloured colonies were recorded from the
centre to the edge of the colony, separated by forward slashes (e.g., Saccardo’s Olive/ Olive
Ochre/ Cream).
Aerial mycelial mats were described using terminology adapted from Stalpers (1978). Terms
for the mycelial mat were assigned to each pure culture:
a.

absent: mycelium only submerged. The surface of the agar may be even or chamoislike.

b.

downy: with fine, short, erect hyphae. The whole colony is usually transparent.

c.

farinaceous: mealy, powdery.

d.

granular: covered with minute grains.

5

e.

silky: with long, radiating parallel hyphae or hyphal bundles, more or less prostrate,
often glossy.

f.

cottony: rather long, single mycelial hyphae spreading in all directions.

g.

woolly: fairly long interwoven hyphae or groups of hyphae, somewhat matted,
resembling woollen cloth.

h.

floccose: small hyphal tufts, standing out from the agar or from the aerial mycelium.

i.

plumose: mycelial tufts with short or long hyphae or groups of hyphae radiating from
the central axis, often in fan-like arrangement.

j.

pellicular or subfelty: covered with thin, low, coherent mycelium.

k.

felty: cottony or woolly mycelium, which has become matted or packed; emerging
hyphae absent.

l.

velvety: a dense mat of erect, straight hyphae, usually short.

m.

crustose: hyphae forming a solid, hard crust, usually dark brown (many
Hymenochaetaceae) but sometimes cream or white (Radulomyces).

n.

lacunose: mycelial surface depressed or indented.

o.

zonate: with concentric bands or segments of different texture.

A random sampling of isolated fungi was sent to be sequenced by MACROGEN™ in Seoul,
Korea. Of the 20 samples sent, 15 could be sequenced. Sequences were compared to the
NCBI database to determine the identities of these fungi. The 97% sequence similarity in the
ITS region between two sequenced samples has been conventionally used as the threshold to
delineate two separate samples as the same species (O’Brien et al. 2005). Roles of these
fungi were elucidated by searching the literature and their potential function(s) in A.
americanum are explored in the discussion.

6

DATA ANALYSIS
Fugal species abundance was evaluated over the growing season in both males and females.
The sum total of species present in males or females at each collection time was determined
and plotted versus time. The time scale was set to ordinal dates (“day-of-year") using a
perpetual Julian date calendar (non-leap year). Regression analysis was done using
Minitab™, and a third order polynomial regression line was used to determine if there was a
trend over time in species richness of the endophytes within A. americanum male and female
flowers.

The resulting values were compared to determine whether or not there were

differences in the raw species abundance between the male and female A. americanum
plants.
Species similarity between male and female A. americanum species was also examined.
Using Sørensen’s coefficient of similarity (Sørensen 1948). Species similarity was examined
at each data point and overall to see how similar the communities inhabiting the dioecious
plants really were. The equation for the coefficient is as follows [Equation 1]:
𝐶𝐶𝐶𝐶𝐶𝐶𝐶𝐶𝐶𝐶𝐶𝐶𝐶𝐶𝐶𝐶𝐶𝐶𝐶𝐶𝐶𝐶 𝑜𝑜𝑜𝑜 𝐶𝐶𝐶𝐶𝐶𝐶𝐶𝐶𝐶𝐶𝐶𝐶𝐶𝐶𝐶𝐶𝐶𝐶 (𝑆𝑆𝑆𝑆𝑆𝑆𝑆𝑆𝑆𝑆𝑆𝑆𝑆𝑆𝑛𝑛′ 𝑠𝑠 𝑐𝑐𝑐𝑐𝑐𝑐𝑐𝑐𝑐𝑐𝑐𝑐𝑐𝑐𝑐𝑐𝑐𝑐𝑐𝑐𝑐𝑐) =

2�𝑠𝑠𝑥𝑥𝑥𝑥 �
𝑠𝑠𝑥𝑥 +𝑠𝑠𝑦𝑦

x 100%

[Equation 1]

where sxy is the number of species the two groups share, sx is the total number of species
observed in males and sy is the total number of species observed in females.

7

RESULTS

CHARACTERIZATION OF ENDOPHYTIC FUNGAL SPECIES IN A. AMERICANUM
Throughout the study period, 47 morphologically distinct fungal isolates were identified. The
endophytic fungi identified had numerous distinct morphologies, and some isolates fell
outside the three predetermined broad colour categories described in the methods. These
outlier groups were placed under a fourth grouping, labelled “Variable” in Table 1. These
fungi had a wide array of colours, including reds, greens, and blends of colours between the
other colour classes (Table 1).

8

Table 1. Macroscopic morphologies of fungi isolated from A. americanum. Each isolate had a distinct two to three-letter name assigned in order to
organize each morphologically distinct isolate.
Abbreviated name

Form

Size

Elevation

Margin

Surface

Aerial Mycelium

Colour (aerial)

Colour (Embedded)

Irregular

4.5cm

Convex

Undulate

Pitted, Wrinkled

Floccose, velvety

Black/ Brownish Olive

None/Black

(A) BLACK
BP
BFF

Filamentous

BC

Irregular

BF

Circular

FWB

Fills Plate

BS

Circular

BR

Circular/Rhizoid

0.81.1cm
1.8cm
Fills
Plate
Fills
Plate
Fills
Plate
7.9cm

Convex

Filiform

Dull

Velvety

Yellowish Olive

None

Raised

Undulate

Rough

Crustose

Black/Chrome/ white

Not Visible

Entire/ Filiform

Dull

Felty

Saccardo's Olive. Black at 3wk

None
Black/ Saccardo's Olive/ Cream.
All Black at 3wk

Not Visible

NA

NA

Woolly, clumpy

White

Black patches

Raised

Entire/Filiform

Dull

Tufted, with granular, farinose powder

White Mycelia/powder

White/Black patches

Raised

Lobate

Dull

Felty

Black/ Saccardo's Umber

Black/ Saccardo's Umber/White

Raised

Lobate, Filiform

Dull

Cottony

Cream buff/ White

Cream buff

(B) CREAM/WHITE

CF

Circular

CM
CW

Irregular
Circular

Fills
Plate
Fills
Plate
1.75cm
2.2cm

CWR

Irregular

3.6cm

BM

Irregular

Flat

Entire

Dull

Sparsley cottony to Farinaceous

Cream

None

Raised
Raised

Entire
Entire

Absent, Small tuft in centre
absent

Cream
Cream/ White

None
none

Raised

Entire

Glistening
Dull
Dull, wrinkled
in centre

Absent, Farinaceous white ring near edge

Cream

None
None

PF

Circular

4.5cm

Flat

Entire/ Filiform

Dull

Downy

Pale Lemon yellow/ Maritus Yellow.
Turned peach at 3 wk

PP

Irregular

2cm

Convex/
conical

Lobate

Pitted

Small, Felty around perimeter

Warm Buff

None

PU

Circular

5cm

Umbonate

Entire

Dull

Absent, Floccose tufts

Ochraceous Salmon/ Light Buff

None

PW

Circular

2.2cm

Raised

Entire

Wrinkled

Absent, Floccose tufts at apex

Light Ochraceous Buff/ Buff Yellow

None

SC

Circular

Entire/ Filiform

Dull

Absent w Dense Pellicular at apex

Sepia/ Cream

None

Circular

1.9cm
Fills
Plate
Fills
plate

Umbonate

WBD

Flat

Entire/ Filiform

Dull

Downy/absent at 3wk

Cream/ White

White, Mars Brown at 3wk

Flat

Curled

Dull

Cottony

White/ Cream

Cream

1.1cm

Raised

Entire

Dull

Downy

White

none

Convex

Filiform

Dull

Felty

White

None

Dull

Filiform

Dull

Densely Felty

None

Absent, floccose tuft at apex

White
Cream/ Yellow Ochre speckles/
Cream

WCC

Circular

WD

Circular/
irregular

WF

Circular

WFC

Circular

Fills
Plate
4.8cm

WR

Circular

1.6cm

Umbonate

Entire/Filiform

Dull

WS

Circular

Raised

Filamentous

Dull

Farinaceous/ downy

White

none

WU

Circular

Fills
Plate
0.7cm

Umbonate

Entire

Dull

Pellicular with floccose tuft

White

Cream

None

9

Abbreviated name

Form

Size

Elevation

Margin

Surface

Aerial Mycelium

Colour (aerial)

Colour (Embedded)

OC

Circular

3cm

Raised

Entire/ Filiform

Dull

Downy

Tawny/ Cream

None

ODF

Circular

7.2cm

Flat

Entire

Dull

Downy

Faintly Yellow Ocher

None

OF

Circular

2.9cm

Flat

Entire/ Filifrorm

Dull

Absent/ Pellicular

Xanthine Orange/ Cadmium Orange

OG
OI

Circular
Irregular

2.4cm
2.2cm

Raised
Raised

Entire
Lobate, Filiform

Dull
Dull

Pellicular
downy

Clay colour/ Deep Colonial Buff
Ochraceous Orange
Ochraceous Orange/ Pale OrangeYellow
Honey Yellow

(C) ORANGE/YELLOW

OL

Circular

7.2cm

Flat

Entire/Curled

Warty

Absent

OM

Irregular

5.5cm

Raised

Entire

Glistening

lacunose

Xanthine Orange/ Cadmium
Orange
None
Ochraceous Orange
Ochraceous Orange/ Pale
Orange-Yellow
Honey Yellow
Primuline Yellow/ Buckthorn
Brown band 1cm from centre
None

OP

Irregular

5.3cm

Raised

Irregular

Dull

Farinaceous

White

OTW

Irregular

3.3cm

Raised

Entire

Wrinkled

Absent

OW

Circular

2.1cm

Umbonate

Entire

Dull/Wrinkled

Absent

Olive Ocher / Primuline Yellow
Yellow Ocher/Antimony
Yellow/Dresden Brown

OWF

Irregular

3.9cm

Raised

Undulate, Filiform,
Curled

Dull

Floccose, In spiky tufts, arranged in Curled
pattern

White

Cadmium Yellow/ Light
Orange-Yellow

YC

Irregular

2.8cm

Flat

Irregular/ Filiform

Covered

Cottony

Lemon Chrome

None

YF

Circular

5.6cm

Flat

Entire

Dull

Floccose centre/absent

YM

Irregular

0.6cm

Raised

Filiform

Dull

Absent

YT

Circular

5.6cm

Flat

Entire

Dull

Floccose centre/absent

YW/YPR

None

Light Cadmium bands, Mustard
yellow
Buff Yellow, producing Yellow
surfactants
Light Cadmium bands, Mustard
yellow

Ochraceous Orange

Pellicular, downy

Lemon Chrome, turns Russet at wk3

Yellow, turns Russet at wk3

Yellow-Green/ White margin
White/ Warm Buff. Yellow Ocher at
2 weeks
White mycelia, Phlox Pink/ White
Mycelia Black with White surface
Cameo Brown/ Light Congo Pink
White mycelia, Amber Yellow
surface/White

Alizarine Pink at 17 days
Black/ Ferrugineous/ Cream
colour
Cream
Honey Yellow
Medal Bronze/ Buff Yellow
Black/ Deep Colonial Buff/
White

Irregular

2.3cm

Raised

Entire

Dull, Wrinkled
in centre

3.7cm
Fills
Plate
3.9cm
6.5cm
3.2cm
Fills
Plate

Raised

Undulate

Dull

Raised

Entire

Dull

Flat
Raised
Raised

Entire
Entire
Lobate

Dull
Dull
Veined

Pellicular
felty centre, cottony outside with silky
strands
downy
Felty
Granular, with reflective droplets

Raised

Entire

Dull

Woolly, clumped

None

None

(E) VARIABLE
GI

Irregular

ORS

Circular

PD
RB
RC

Circular
Circular
Irregular

TCB

Circular

10

Of the 47 fungal isolates, 34 (72%) fell into the Cream/White and Orange/Yellow categories,
while the rest fell into the other two colour classes, Black and Variable The macroscopic
morphologies of the isolated endophytic fungi present within each of the classes were highly
variable relative to the endophytes isolated previously by Martin et al. (2012), who found
fungi with six aerial mycelial morphologies. By comparison, the present study had more than
13 different aerial mycelial morphologies.
Of the 20 isolates sent to be sequenced; 15 were returned with readable sequence data. A
literature search was done to examine the general function these organisms performed in the
environment (Table 2):
Table 2. Sequenced ITS region identities of endophytic fungal species isolated from A.
americanum. BLAST searches were performed with a resolution value of 97% sequence
similarity delineating a positive identification of a species.
Abbreviated
Name

BLAST result

Ecological Characteristics

BF

Alternaria

Opportunistic pathogen endophyte in other plants,
causing leaf blight. (Fisher et al. 1992)

BS

Botryotinia/ Baudoinia/
Botrytis

Latent pathogens of woody plants
(Slippers and Wingfield 2007)

GI

Nectria

Pathogenic wood canker-causing fungus
(Hopkins et al. 2009)

OF

Sordariomycetes

ORS

Galerina/Coprinus

Highly diverse group of fungi, present in many
environments. Some species are endophytic in nature.
(Zhang et al. 2006)
Saprotrophic fungi
(Davey 2013; Noordeloos and Gulden 2012)

OTW

Sordariomycetes

Highly diverse, some endophytic
(Zhang et al. 2006)

OWF

Coniochaeta

Fungal group that is pathogenic to trees.
(Weber 2002)

PBR

Alternaria

Opportunistic leaf-blight pathogen endophyte in plants.
(Fisher et al. 1992)

PD

Lecythophora

Wood soft rot fungi present in forest environments
(Damm et al. 2010)

PP

Anthostomella

Saprotrophic fungus in a variety of plants
(Cooke 1959)

PW

Anthostomella

Saprotrophic fungus in a variety of plants
(Cooke 1959)

11

YC

Serpula

Dry rot fungi, commonly found in built timber
structures and boreal forests
(Eastwood et al. 2011)
Fungal group that is pathogenic to trees.
(Weber 2002)

YF

Coniochaeta

YM

Tremella

Wood-inhabiting fungi mycoparasites
(Vizzini 2007, Jeffries and Young 1994)

YPR

Trichoderma

Fungi with active cellulase enzymes.
(Druzhinina et al. 2010)

Sequenced isolates were identified to the genus level, as further resolution can prove
challenging and tends to require other techniques (e.g., DNA-DNA hybridization) to define
species (O’Brien et al. 2005). What was sequenced and examined with a small literature
search reveals that there is a high diversity of endophytes present within A. americanum.
These endophytes came primarily from the colour groups Orange/Yellow, Black, and
Variable. The fungi had a wide diversity of symbiotic syndromes with plants. Interestingly,
there were a number of wood-inhabiting fungi that are pathogenic to trees.

12

ENDOPHYTIC SPECIES RICHNESS CHANGES BETWEEN MALE AND FEMALE
PLANTS
Endophytic fungal species richness steadily increased over the growing season (Figure 1). A
polynomial regression line of the third order was used because it fit the data better; the
relationship between species richness and date was significant for both sexes (for males
F = 10.8, p = 0.0007 and for females, F= 6.8, p = 0.0051). It should be noted that species
richness declined in the latter part of the sampling period. Overall, richness did not differ
substantially between male and female plants.

Figure 1. Endophytic species richness in male and female plants throughout the growing
season. Open triangles denote male species richness and closed circles denote female species
richness at each sampling point. Regression lines for males (dashed line, R2 = 0.71) and
females (solid line, R2 = 0.61) were included.

13

As is evidenced in Figure 1, the change in the species richness in both the males and females
of A. americanum was found to increase at roughly the same pace throughout the growing
season. It should be noted that there is a switch between the male and female species
richness in the latter portion of the growing season through July, with the female plants
having an increased number of endophytes present through the mid-summer, but the
observation is likely nonsignificant. Full regression analyses are presented in the Appendix,
Table A1 and Table A2.

SIMILARITY MEASURES IN THE ENDOPHYTE COMMUNITIES OF MALE AND
FEMALE A. AMERICANUM
The sum total of the male and female plants’ endophyte isolates throughout the entire
sampling period was compared using the Sørensen’s coefficient. The male and female
endophyte communities were 87.5% similar.

14

DISCUSSION

CHARACTERIZATION OF ENDOPHYTES REVEALS A HIGH DIVERSITY OF FUNGI
PRESENT WITHIN A. AMERICANUM THROUGHOUT THE GROWING SEASON
The sequencing of a third of all isolated endophytes revealed that almost every
morphologically distinct strain (12 of the 15 that were returned with readable sequences) was
associated with a particular genus of fungus. In pure culture studies, a particular medium (in
this case, potato dextrose agar) is usually only capable of supporting in-vitro growth of a
fraction of the organisms present in a sample. This selection bias by a given medium, defined
here as “culture bias”, can cause a skewing of the observed richness of a community, leading
to an underrepresentation of the members present in a particular sample (reviewed in
Handelsman 2004). The high diversity of fungi observed here reinforces the idea that pureculture studies can be used for examining fungi. Unlike in bacterial samples, pure culture
techniques return a large proportion of fungal isolates without significant losses of individual
isolates due to culture bias (Fröhlich and Hyde 1999; Lacap et al. 2003).
Compared to other plants such as Plantago lanceolata, Rumex acetosa, and Cirsium
arevense, A. americanum had a reasonable number of morphologically distinct isolates (47
isolates in A. americanum, as compared to P. lanceolata’s 55, R. acetosa’s 36, and C.
arvense’s 25) (Wearn et al. 2012). The diversity of isolated fungi indicates that the plant
experiences a wide array of ecological interactions, each of which may influence the health
of the plant itself, or the health of the associated host. The fact that there are a number of
latent plant pathogens within the mistletoe’s tissues indicates that like other, more widely
studied plants (Theobroma cacao, for instance), mistletoe is constantly bombarded by fungal
pathogens, which threaten to invade its shoot tissues. These external, potentially pathogenic
fungi could be inhibited by the internal fungal endophytes that have been previously
described by Martin et al. (2012) as being able to inhibit plant pathogen growth. The fact
that the plants are not immediately damaged by the pathogens could indicate that the
mutualistic endophytes observed previously are constitutively present within the shoots of A.
americanum, and may assist in inhibiting the growth of more pathogenic endophytes within
the host tissues.
15

It is to be noted that Martin et al. (2012), revealed a number of different fungal genera living
in A. americanum, including Sydowia, Phoma, Phacidiopycnis, Davidiella, and
Cladosporium. These fungi had diverse morphologies, but most of them (Sydowia, Phoma,
and Phacidiopycnis) had white aerial myceliao f varying densities. In the present study, the
samples that were sent to be sequenced by MACROGEN™ in Korea did not include fungi
with similar macroscopic morphologies, although there were fungi with similar morphologies
observed throughout the sampling period (Table 1, isolate WFC and BM). It may be that
some of the isolates in the present study are in fact the same genera that were isolated in the
previous experiments, but were merely missed due to the sample selection procedure used
here.

MALE AND FEMALE ENDOPHYTE COMMUNITIES EXHIBIT HIGH SIMILARITY
Sørensen’s coefficient of similarity showed that that the male and female morphs of A.
americanum were very similar with respect to their endophyte communities. The manner by
which the mistletoe plant acquires its endophyte fungi might provide an explanation for the
similarity. With the plant being present on the branches of its host and having continuous
vasculature with its tree host, endophyte transmission between the pair of plants could be
readily occurring through their connected vasculatures and the transmission of fluids from
tree to parasite, which could result in a high degree of similarity between the fungal
endophytes of the host tree and the parasitic A. americanum male and female plants. The
host trees constitute a large “reservoir” of potential endophytic fungi, which could strongly
influence the endophyte communities of the male and female plants.
Alternatively, A. americanum could be acquiring its endophytes through a more conventional
mechanism. In forests, the acquisition of endophytic fungi by plants lower in the canopy can
be strongly influenced by a “drip-down effect” from the branches and leaves of other trees
nearby (Arnold and Herre 2003). A. americanum tends to be nestled on the branches of its
host tree, or within the brooming structures that occur due to the parasite’s infection of the
tree. It might be that at least some of the various endophytic fungi (parasitic, wood-rot,
saprophytic) could have ended up within A. americanum through this mechanism.

16

This is not to say that the communities of A. americanum and P. contorta are completely
identical, or even that A. americanum only obtains its endophytes horizontally, either from
external inputs (drip-down effects from the host tree’s branches) or transmission from the
vasculature of the host tree . Martin et al. (2012) observed that endophytic fungi emerged
from the seed tissue of the plant. This indicates that A. americanum has endophytes that are
vertically transmitted from parent to offspring. Vertically transmitted fungal symbionts can
play very important roles in the development of their photosynthetic partner. In the reed
species, Phragmites australis, endophytic fungi function in a unique way, helping to
stimulate growth and proliferation of the host plant (Ernst et al. 2003). The grass Festuca
arundinace has vertically-transmitted endophytes that help resist heavy metals within the soil
by increasing the plant’s ability to exude phenolic chelators into the earth (Malinowski and
Belesky 1999).
A. americanum seeds, which are deposited on the host trees in the fall, are exposed to a
number of stressors and challenges before infection occurs the following spring. As in P.
australis, or F. arundinace, endophytic fungi within A. americanum could be helping
facilitate this first challenging step in the plant life cycle, improving the ability of the plant to
survive and thrive during the six or seven months before host infection.
The endophytes that were sequenced in this study could have similar effects to those seen in
other plants. Lecythophora, Trichoderma, Serpula, Botrytinia, and many organisms from the
Sordariomycetes were all found in the samples that were sent to be sequenced. Fungi in these
groups play a diversity of roles. All are capable of breaking down cellulose, and cause a
variety of rotting diseases in woody flora (Table 2). However, these organisms could be
present in A. americanum as beneficial endophytes rather than pathogens.
During the initial stages of the A. americanum life-cycle, the seeds have to burrow through
the thick, woody tissue of their tree hosts in order to proceed through their lifecycle. If these
endophytes were present in the seed, they could break out when the seed germinates,
softening the bark where the plant would subsequently invade the tree host, thereby
improving the chance that the plant parasite will establish itself on the tree in question.

17

At maturity, A. americanum itself is a lignified plant, having a large amount of woody tissue
within its stems and also in the haustorial tissues that infiltrate the host tree. These potentially
pathogenic fungal endophytes within A. americanum could be infecting the parasitic plant
itself, and are being repressed by the aforementioned symbionts that protected the plant from
surface pathogen infection (Martin et al. 2012). A more interesting implication of the
presence of these pathogens in the mature A. americanum plant could revolve around the
maintenance of the plant-plant parasitic relationship. The parasitic plant could be hosting
wood-degrading pathogens in order to improve its ability to infect the host tree; these lignindegrading and necrotizing fungi could help maintain fluid flow from the host tree to the
parasitic A. americanum. In conifers, lignification, the production of resins, and the synthesis
of toxic phenolic compounds are all mechanisms by which a tree tries to prevent pathogens
from infecting phloem tissues (Hudgins et al. 2004). When A. americanum infects a pine, the
host’s defence mechanisms will be initiated, and lignification of pine tissues surrounding the
site of parasite invasion will occur. The wood rot endophytic fungi could help prevent the
host tree from stopping the flow of fluids through its vasculature into A. americanum. The
endophytes described here could represent a novel change of role from pathogens (as far as
the host pine is concerned) to beneficial mutualists (for the dwarf mistletoe).

SPECIES RICHNESS SEEMS TO INCREASE OVER THE GROWING SEASON IN
BOTH MALES AND FEMALES
Over the course of the growing season, the sum-total of endophytes present in male and
female A. americanum plants was found to increase. However, there was a slight shift in
species richness between male and female plants (from males having higher diversity at the
beginning of the season to females having higher diversity at the end of the season). The
shift, which was not statistically significant, could be attributed to random variation due to
relatively low sample size for each data point (n = 5). Nonetheless, the trend is interesting
and worth exploring in the future.
The results of the current study also indicate that A. americanum does follow common plant
trends in endophyte species richness over its growing season (Thompson et al. 1993; Faeth
and Hammon 1997; Mei et al. 2013). As other plants age and proceed towards senescence, a

18

number of important stressors will impact the microbial community associated with the plant
tissues. Initially, when the plant is first beginning shoot growth, the number of endophytes
present within the new shoots will be low merely due to the new tissue’s lack of exposure to
the environment (Arnold and Herre 2003). As was mentioned earlier, “drip-down effects”
can strongly influence the species richness of endophytes within plants. Rainwater drainage
from overhanging plant tissues causes young seedlings to be exposed to endophytes,
allowing for the horizontal transmission of the fungi from one plant to another.
Due to the fact that A. americanum tends to infect the branches of its host tree, the
acquisition of endophytes by the mistletoe could be facilitated through the drip down of
water onto the plant.

During the initial stages of seed dispersal and germination, A.

americanum seeds are exposed to the environment.

For up to year, the seeds will

progressively make their way to the axis between a host tree’s leaf (needle) and the bark of
the tree (Hawksworth and Wiens 1996). As water passes through the branches above the
seed and washes the seed closer to the base of the needle, the fungal spores present within the
host tree’s tissues could also fall down, coming into contact with, and potentially infecting,
the mistletoe seed, and eventually the entire plant. Once the A. americanum infection is
established, new shoots of mistletoes that are infecting the host could also acquire more
novel endophytes from further drip-down from the surrounding canopy, causing the
increased endophyte species richness depicted here.
More interesting would be the potential of an alternate mechanism of endophyte inoculation.
When A. americanum infects its host, Pinus contorta var. latifolia, the small parasite’s
vasculature becomes continuous with that of its host. This means that there is continuous
flow of nutrients and fluids between the two plants. It would therefore be feasible for
endophytic organisms native to the tree to invade A. americanum plants “from below” so to
speak rather than raining down “from above” from canopy drainage.

In either case,

endophytes will be accumulating over time as the new shoot tissue develops from the A.
americanum mass located within the tree.

19

FUTURE WORK
Endophytic fungi in plants provide a variety of potentially beneficial roles to their plant
hosts. In the present study, the endophytic species richness was examined in the male and
female forms of A. americanum to determine whether species richness changed over time in
this species, and if the species richness differed between the sexes. The results presented here
regarding the diversity of endophytes present within mistletoe and the similarity between the
endophyte communities of male and female plants opens a new avenue for further research
regarding the functioning of the fungal symbionts within A. americanum. Future work could
begin to explore the 12.5% difference in the Sørensen coefficient of community between
males and females, and explore the small shift in species richness observed by the end of the
growing season. These fungi could have biological roles that are specific to the particular sex
of their associated plant host. In the dioecious plant Antennaria. dimorpha, it was observed
that symbiotic fungi associated with the roots of the female plants were more abundant
during the flowering season, and that the increase in mutualistic fungal symbionts in the roots
allowed for increased nutrient uptake during the production of inflorescences (Vega-Frutis et
al. 2013). Based on these observations in other plants, it could be reasoned that the slight
differences between the sexes of A. americanum, with respect to their endophyte
communities could also have implications for the health or virility of the male or female
morphs. Examining fungal isolates specific to male or female plants would be useful in
understanding these sex-specific interactions in the plant. An exploration into the presence of
endophytes within the seeds of A. americanum would also be conducive to understanding
aspects of the plant’s relationship with fungi and their potential ability to assist in
germination and proliferation of the mistletoe seeds.
More broadly, the project here was performed in pure culture, which, while reliable for fungi,
still has culturing bias. Some species will be more abundant, and have a chance to crowd out
slower-growing species on a plate. The species richness trends reflected here could be
reinforced by using a more robust technique, like ITS sequencing of fungal symbionts. The
usage of molecular techniques could also allow for comparisons of the endophyte
communities in the host tree and the parasitic mistletoe plant. If there are similarities between
the two groups, it could indicate horizontal transfer of endophytes between the plant and its
host: a novel observation for parasitic plants worldwide.
20

CONCLUSION
Through this study, it was revealed that there was a high diversity of endophytic fungal
symbionts housed within the tissues of A. americanum. Based on a literature search, the
endophytes in question had a wide diversity of ecological roles, ranging from mutualists to
pathogens to saprophytes. Species richness was found to be highly similar between both male
and female plants, exhibiting 87.5% similarity in populations of endophytes grown from
internal plant tissues. It was determined that, as in other plants, the endophytic species
richness increased over the growing season in both male and female plants. These results do
not shed much light on biocontrol implications of endophytic communities within A.
americanum, but they do open up windows into aspects of the symbiotic fungal partners that
have not been previously observed in a plant-plant parasitic interaction. Unveiling the fungal
partner identities of the A. americanum plant could reveal novel metabolites that modulate
pathogen growth within the plant or its associated tree host. These findings shed light on the
biology of A. americanum, and provide researchers with a new picture of the world of
endophytes within parasitic plants.

21

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24

APPENDIX
Table A1. Regression analysis of male species richness changes throughout the growing
season in A. americanum.
SUMMARY OUTPUT: MALES
Regression Statistics
Multiple R
0.845088116
R Square
0.714173923
Adjusted R Square
0.64821406
Standard Error
1.86937943
Observations
17
ANOVA
df
Regression
Residual
Total

3
13
16

SS
113.5116436
45.42953287
158.9411765

MS
37.83721453
3.494579452

F
10.82740142

P-Value
0.000768249

Table A2. Regression analysis of male species richness changes throughout the growing
season in A. americanum.
SUMMARY OUTPUT: FEMALES
Regression Statistics
Multiple R
0.783229836
R Square
0.613448976
Adjusted R Square
0.524244894
Standard Error
2.201137444
Observations
17
ANOVA
df
Regression
Residual
Total

3
13
16

SS
MS
F
99.95609786 33.31869929 6.876915935
62.98507861 4.845006047
162.9411765

P-Value
0.005137652

25