Microb Ecol
DOI 10.1007/s00248-011-9851-6
PLANT MICROBE INTERACTIONS
Host Associations Between Fungal Root Endophytes
and Boreal Trees
Gavin Kernaghan & Glenn Patriquin
Received: 1 October 2010 / Accepted: 23 March 2011
# Springer Science+Business Media, LLC 2011
Abstract Fungal root endophytes colonize root tissue
concomitantly with mycorrhizal fungi, but their identities
and host preferences are largely unknown. We cultured
fungal endophytes from surface-sterilized Cenococcum geophilum ectomycorrhizae of Betula papyrifera, Abies balsamea, and Picea glauca from two boreal sites in eastern
Canada. Isolates were initially grouped on the basis of
cultural morphology and then identified by internal transcribed spacer ribosomal DNA sequencing or by PCR
restriction fragment length polymorphism. Phylogenetic
analysis of the sequence data revealed 31 distinct phylotypes
among the isolates, comprising mainly members of the
ascomycete families Helotiaceae, Dermateaceae, Myxotrichaceae, and Hyaloscyphaceae, although other fungi were
also isolated. Multivariate analyses indicate a clear separation among the endophyte communities colonizing each host
tree species. Some phylotypes were evenly distributed across
the roots of all three host species, some were found
preferentially on particular hosts, and others were isolated
from single hosts only. The results indicate that fungal root
endophytes of boreal trees are not randomly distributed, but
instead form relatively distinct assemblages on different host
tree species.
Introduction
Fungal endophytes colonize plant tissue internally and
asymptomatically for at least some of their lifecycle [75]
G. Kernaghan (*) : G. Patriquin
Biology Department, Mount St. Vincent University,
166 Bedford Hwy.,
Halifax, NS B3M 2J6, Canada
e-mail: [email protected]
and appear to be ubiquitous within stems, leaves, bark,
and roots [54]. Plant roots harbor characteristic assemblages of fungal endophytes that are distinct from those of
above-ground plant tissue [2, 62]. Although the ecological
roles of root endophytes are largely unknown, they
represent a significant component of the below-ground
microbial community and are thought to be at least as
common as mycorrhizal fungi [41, 73]. In boreal trees,
commonly occurring fungal root endophytes include
species of Cryptosporiopsis [69, 72], Oidiodendron [56],
Umbelopsis [29], and members of the “Rhizoscyphus
ericae aggregate” (Helotiaceae) [25, 70]. The best known,
however, are commonly referred to as “dark septate
endophtes” or DSE, which includes members of the
Phialocephala fortinii complex and other fungi with
melanized hyphae. Much of our understanding of fungal
root endophytes is based on studies of DSE, as they are
common (especially in boreal soils), easily observed and
easily cultured (but not easily identified) [1, 20, 24, 33,
44, 51, 77].
There have been several recent studies of tree root
endophyte species composition and diversity [3, 16, 19, 35,
44, 45], but few have documented the differences in
naturally occurring endophyte communities across host
plant species. Those that have shown differences among
host plants generally detect evidence of host preference [20,
38, 64], although the influence of variation in abiotic
factors among sampling sites is usually not considered.
The phenomenon of host specificity (or host preference
if the relationship is not strictly exclusive) has been
demonstrated in leaf endophytes [31, 50], mycorrhizal
fungi [36, 46], and fungal root pathogens [17], and is
assumed to play an important role in plant community
ecology [55]. For example, any positive [57, 68] or
negative [77] effects of colonization would not be shared
G. Kernaghan, G. Patriquin
equally throughout the plant community. Levels of host
specificity also factor heavily into estimates of global
fungal biodiversity in that all plant species are assumed to
support a certain number of host-specific fungi [28, 78].
Despite these considerations, information regarding levels
of host specificity among root endophytes is still sparse,
and their ubiquity has led earlier authors to consider them
to be non-host specific [16, 20]. This impression was based
largely on studies of DSE, which when considered as a
group, do colonize a wide range of plant hosts [33].
However, the concept of DSE encompasses a wide variety
of ascomycetous fungi, and even the best known root
endophyte, P. fortinii, is now considered to be a species
complex comprised of several cryptic species [22, 23], each
of which may potentially exhibit its own level of host
preference or specificity.
In order to compare fungal root endophyte communities
across boreal tree hosts and to investigate levels of host
preference, we isolated endophytes from surface sterilized
Cenococcum geophilum ectomycorrhizae of three cooccurring tree species. We focused on ectomycorrhizal
tissue, as it is relatively unexplored with respect to fungal
endophytes and may harbor species localized in the
metabolically active root tips. We also chose to isolate
fungi from surface sterilized tissue to ensure that all isolates
were endophytic.
Methods
Collection of Roots and Isolation of Fungi
Sampling was conducted at two boreal sites in eastern
Canada. One was located on Mount Mackenzie, Cape
Breton Highlands National Park, Nova Scotia (46o45′ N,
60o50′ W) at 380 m elevation. The second was in the
southern boreal mixed wood forest, located in the Lac
Duparquet Teaching and Research Forest, northwestern
Québec (48o29′ N, 79o25′ W) at 300 m elevation. The two
sites were approximately 1,400 km apart.
Both sites support mature (over 70 years) mixtures of
Betula papyrifera, Abies balsamea, and Picea glauca.
Daily average temperatures near Mount McKenzie range
from −6.3°C in February to 18.3°C in August, with a total
annual rainfall of 1,391 mm. The Lac Duparquet site is
colder and dryer with daily average temperatures from
−18.2°C in January to 16.9°C in July and an annual average
precipitation of 889 mm (30-year normals, Environment
Canada). Soil surveys have not been conducted in northern
Cape Breton, but Mount McKenzie soils are likely Gleyed
Humo-Ferric or Gleyed Ferro-Humic Podzols (Keys 2007,
K. Keys, personal communication). Soils at Lac Duparquet
are Gray Luvisols [7].
Four 2-m2 plots were established at each site. Plots
were spaced approximately 50 m apart and supported at
least one mature A. balsamea, one mature B. papyrifera,
and one mature P. glauca. For each tree species on each
plot, one major root was traced from the base of the tree to
the fine roots (12 major roots per site), and all the fine
roots were collected. Root tips were examined under a
dissecting microscope and 20 fine root tips colonized by
C. geophilum (ectomycorrhizae) were identified on the
basis of morphology and removed from each sample for
isolation of endophytes. Only root tips colonized by C.
geophilum were used for endophye isolation in order to
avoid any possible differences in endophyte assemblages
between root tips colonized by different species of
ectomycorrhizal fungi. Ectomycorrhizae were surface
sterilized [27] by rinsing for 1 h in cold tap water,
sonicating for 6 min, dipping in 95% ethanol for 1 min,
then 15% hydrogen peroxide (6 min for Abies and Picea
and 4 min for Betula—optimal surface sterilization times
for each host species were determined previously).
Sterilized ectomycorrhizae were plated (one tip per plate)
onto malt-yeast media (15 g Bacto malt extract, 1 g Bacto
yeast extract, and 15 g agar) supplemented with 100 ppm
oxytetracycline, 50 ppm streptomycin sulfate, and 50 ppm
penicillin G. Plates were incubated at 20°C in the dark.
Emergent hyphae were transferred to water agar for
subsequent hyphal tip transfers onto oatmeal–salts and
Czapek’s media [12]. Sucrose in the Czapek’s medium
was reduced to 15 g/L.
For each of the two sampling sites, cultures were
sorted into morphological groups on the basis of color,
texture, growth habit, growth rate, and sporulation [5, 39]
when growing on malt–yeast, oatmeal–salts, and Czapek’s
media; dark septate isolates, preliminarily identified as P.
fortinii sensu lato, were also grown on malt extract agar
with or without 100 mg/L cycloheximide [22], as well as
on pectin based media in order to better distinguish among
species.
DNA Extraction
Fungal tissue was removed from agar plates, frozen at −20 oC,
then placed in 600 μl 2× cetyl trimethylammonium bromide
extraction buffer, ground in a ceramic mortar and incubated
at 65 oC for 1 h in a micro-centrifuge tube with 100 μg/ml
proteinase K. Six hundred microliters chloroform/isoamyl
alcohol (24:1) was then added followed by a 15-min
centrifugation at 20,000 g. DNA was then precipitated by
removing the upper aqueous layer, adding 600 μl cold
isopropanol, cooling to −20°C for 30 min and centrifuging at
20,000×g for 15 min. The resulting pellet was washed twice
with 70% ethanol, air dried, and re-suspended in 100 μl
sterile distilled water.
Host Associations Between Root Endophytes and Boreal Trees
Identification of Isolates
Between 33% and 100% of the isolates in each morphological group were selected for internal transcribed spacer
(ITS) sequencing (58% of isolates overall). The percentage
of isolates sequenced was dependent on the number of
isolates in the group, with a smaller proportion of the most
abundant types being sequenced. Sequenced isolates
included representatives of either end of any subtle
morphological gradients within the group.
PCR amplification and sequencing were as follows:
50 μl reactions included 25 μl GoTaq® master mix
(Promega Corp., Madison, WI, USA), 1 μl DNA template,
2.5 μmol of the primers ITS1-F [18] and ITS4 [74] and
14 μL H2O. Unsuccessful PCR reactions were repeated
using DNA template diluted to 1:25 or 1:250 in H2O. The
thermal parameters were as described in DeBellis et al.
[14]. The resulting PCR products were sequenced at the
McGill University and Genome Québec Innovation Centre
with an ABI PRISM 3730XL DNA analyzer system with
ITS1 (forward) and ITS4 (reverse) primers [74].
Sequence contigs were assembled for each isolate, edited
using Sequencher 4.9 (Gene Codes, Ann Arbor, MI, USA)
and compared to GenBank sequences using nucleotide–
nucleotide BLAST (blastn). As the majority of sequences
grouped among either the Helotiaceae, Dermateaceae,
Hyaloscyphaceae, or Myxotrichaceae (Leotiomycetes, Ascomycota), separate maximum parsimony analyses were
conducted for each of these families. Sequences from our
isolates and closely matching GenBank sequences (reference
sequences) were aligned automatically in MUSCLE [15]
using the default settings, then manually adjusted in Bioedit
(Ver. 7.0.9.0) [26]. Alignments were between 460 and
590 bp in length (including gaps), with between 54 and
155 parsimony informative characters. Whenever possible,
the GenBank sequences used as references were those
derived from ex-type or identified cultures, rather than from
environmental samples not supported by cultures. Maximum
parsimony analyses were performed using PAUP* 4.0b10
[63] with midpoint rooting, heuristic search, TBR branch
swapping, 100 trees maximum, and 1,000 bootstrap replications. The small number of isolates not belonging to the
four dominant families was identified by BLAST searches
only (Table 1). Isolate groupings were then adjusted on the
basis of the sequence data. In most cases, this simply
amounted to pooling smaller morphological groups into
larger, sequence based groups (phylotypes).
For the remaining (non-sequenced) isolates, within clade
homogeneity was confirmed by restriction fragment length
polymorphism (RFLP) analysis. DNA extractions and amplifications were performed as above and the resulting amplicons
digested with the restriction enzymes TspR I and Tsp509 I
(New England Biolabs, Ipswich, MA, USA) and run on 2%
agarose gels stained with ethidium bromide. TspR I and
Tsp509 I were selected for their ability to differentiate among
the previously sequenced phylotypes, determined using
NEBcutter V2.0 (http://tools.neb.com/NEBcutter2).
All cultures are stored on malt agar slants and in sterile
water at 4 oC [53] at Mount Saint Vincent University. At least
one sequenced isolate representing each phylotype has also
been deposited in the University of Alberta Microfungus
Collection and Herbarium, Edmonton, AB, Canada, under
the accession numbers UAMH 11124–11133, 11165–11175,
11194–11205, 11207, and 11220–11224. All ITS sequences,
including those representing the UAMH accessions, have
been deposited in GenBank as HQ157833 to HQ157959.
Statistical Analysis
Species accumulation curves were produced for each host
using Estimates 8.2.0 (http://viceroy.eeb.uconn.edu/esti
mates) and the number of potentially undetected phylotypes
estimated by subtracting the observed species richness from
the estimated species richness calculated with the bootstrap
estimator of species richness [61].
Differences in isolation frequency among the three host
trees were calculated for the 19 non-singleton phylotypes
using a randomization test of goodness of fit [43] with
10,000 randomizations. Standardized niche breadth [32, 37]
was also calculated for the 19 non-singletons (using data
from both sites). Shannon diversity indices were calculated
for root endophytes cultured from each host–site combination using PC-ORD version 4 [42]. Analysis of similarity
(ANOSIM) among endophyte assemblages on each tree
species on each site was calculated using the Morisita index
with PAST version 2.04 (http://folk.uio.no/ohammer/past/).
Relationships among root endophytes, host tree species,
and sites were assessed by detrended correspondence
analysis (DCA) using CANOCO [66]. The input for the
ordination was a matrix of non-transformed counts of
phylotypes from each host tree. ITS sequences with 97%
similarity or greater [48] were treated as discrete phylotypes.
The ordination was detrended by 26 segments and rare
species were down-weighted.
Results
Isolation and Identification of Root Endophytes
Two hundred thirty isolates of fungal root endophytes were
obtained from the 480 surface sterilized root tips (20 root
tips×3 tree species×4 plots×2 sites), giving an overall
endophyte isolation frequency of 48%. A further 5% of the
isolations yielded the ectomycorrhizal symbiont Cenoccocum geophilum (the ectomycorrhizal fungus on all root tips
(93%)
(99%)
(99%)
(98%)
(93%)
(97%)
(96%)
(91%)
(97%)
677/722
558/559
556/561
579/587
473/507
392/401
434/448
589/646
587/60
94
97
90
97
88
99
93
99
86
1064
1075
1007
1035
736
688
754
852
1011
EU846251
AF527058
AF527058
Z48815
DQ494677
FJ872076
EU816388
AJ876493
DQ888724
Mycena tenax voucher OSC 11374
Penicillium montanense
Penicillium montanense
Trichoderma polysporum CBS 820.68
Mycena plumbea isolate AFTOL-ID 1631
Umbelopsis isabellina
Umbelopsis isabellina isolate ODHO4
Umbelopsis isabellina
Umbelopsis ramanniana
190907.23II/UAMH 11174
180507.8/UAMH 11198
230507.17/UAMH 11199
190907.26/UAMH 11133
170507.37/UAMH 11130
230507.15/UAMH 11125
170507.25II
180507.11/UAMH 11205
230507.20 (c)/UAMH 11194
ARSL
ARSL
ARSL
ARSL
ARSL
ARSL
ARSL
ARSL
ARSL
Umbelopsis sp. II
Hypocrea pachybasioides
Tricholomataceae sp. I
Umbelopsis sp. I
Cape Breton
Quebec
Quebec
Cape Breton
Quebec
Quebec
Quebec
Quebec
Quebec
Mycena sp.
Penicillium montanense
Abies
Picea
Betula
Abies
Abies
Betula
Abies
Picea
Betula
485/486 (99%)
503/533 (94%)
79
82
893
808
DQ093680
AF178542
Lecythophora mutabilis isolate aurim 1180
Chaetosphaeria chloroconia
ARSL 060907.9/UAMH 11173
ARSL 060907.80/UAMH 11124
Cape Breton
Cape Breton
Lecythophora mutabilis
Chaetosphaeria sp.
Betula
Betula
Isolate no.
Host
Site
Phylotype
Table 1 BLAST results for 11 phylotypes not included in phylogenetic trees
Best GenBank match
GenBank
accession
Total
score
Query
coverage (%)
Identities
G. Kernaghan, G. Patriquin
sampled) and were excluded from further analyses. Most
isolations yielded a single fungus, but in 7% of the
isolations, two different fungi grew from a single root tip.
The initial grouping of isolates based on cultural morphology on different media resulted in 17 groups and 54
singleton isolates from the Cape Breton site and 24 groups
and 50 singleton isolates from the Quebec site.
Initial BLAST searches revealed that the majority (96%) of
the endophyte isolates were members of the ascomycete
families Helotiaceae, Dermateaceae, Myxotrichaceae, and
Hyaloscyphaceae. The remaining 4% were other ascomycetes,
basidomycetes (Tricholomataceae), or Mucorales (Umbelopsis
spp.), with low isolation frequencies (Table 1). Separate
maximum parsimony analyses of each of the four dominant
families revealed 11 phylotypes in the Helotiaceae, six in the
Dermateaceae, two in the Myxotrichaceae, and four in the
Hyaloscyphaceae (Figs. 1, 2, 3, and 4). The helotialian
phylotypes fell into two main groups; one comprised of
species of Meliniomyces, a genus belonging to the R. ericae
aggregate [25, 70] and the other comprised of seven
phylotypes of an unidentified complex, close to, but not part
of, the R. ericae aggregate (Fig. 1). For this latter group, no
close matches to any named isolates were found in GenBank.
In the Dermateaceae, three phylotypes were referable to
Phialocephala, two to Cryptosporiopsis and one phylotype
not assignable to a known species is designated “Dermateaceae sp. I” (Fig. 2). The Phialocephala isolates include
P. sphaeroides, an unidentified Phialocephala species, and
members of the P. fortinii complex, which may include
cryptic species not distinguishable by ITS sequencing [22].
The Cryptosporiopsis isolates include C. ericae and an
unidentified Cryptosporiopsis species.
The Myxotrichaceae (Fig. 3) are represented by Oidiodendron maius and a second Oidiodendron species, for
which there are no matching culture-derived sequences in
GenBank. The Hyaloscyphaceae (Fig. 4) are represented by
four unidentified isolates, all phylogentically close to
Hyphodiscus hymeniophilus.
RFLP analysis of the unsequenced isolates confirmed
that they had been correctly grouped on the basis of
morphology, with the exception of three isolates: one
Phialocephala sp., one Dermateaceae sp. I, and one
Helotiaceae sp. VI. These isolates were easily re-assigned
to their correct groups on the basis of the RFLP data.
Testing of P. fortinii s.l. isolates on MEA medium with
cycloheximide revealed a gradient of inhibition from strong
to weak and did not demonstrate distinctive morphological
groupings among the isolates.
Endophyte Communities
The number of isolate groupings originally distinguished on
the basis of morphology was reduced after sequencing, to
Host Associations Between Root Endophytes and Boreal Trees
Ascocalyx abietina (FJ746661)
Godronia sp. DAOM 233257 (EF672237)
Gremmeniella laricina (GLU72262)
ARSL 230507.35/UAMH 11201 Q B
Uncultured Pezizomycotina clone (FJ554013)
100
ARSL190907.38/UAMH 11169 F
Uncultured Helotiales clone (FJ475664)
ARSL 060907.20 CB B
ARSL 180907.11 CB S
94 ARSL 180907.23 CB S
ARSL 180907.34 CB S
ARSL 180907.19 CBS
91
ARSL 190907.24 CB F
ARSL 170507.37II Q F
ARSL 70907.34 CB S
ARSL 170507.43I Q F
Uncultured fungus clone (EF433994)
ARSL 220507.53 Q S
ARSL 220507.16I Q S
ARSL 220507.58 Q S
ARSL 180507.12/UAMH 11170 Q S
ARSL 70907.35/UAMH 11202 CB S
Uncultured Leotiomycetes clone (FJ152529)
99 ARSL 190907.75 CB F
ARSL 190907.55/UAMH 11171 CB F
ARSL 190907.41 CB F
ARSL 190907.54 CB F
Uncultured Leotiomycetes (AY394893)
ARSL 170507.50 Q F
100 ARSL 170507.46 Q F
ARSL 190907.66 CB F
ARSL 190907.2 CB F
ARSL 070907.9/UAMH 11172 CB S
ARSL190907.35 CB F
ARSL 190907.71 CB F
ARSL 190907.19I CB F
ARSL 190907.15/UAMH 11168 CB F
Rhizoscyphus ericae (AY762622)
ARSL 180907.22 CB S
99 ARSL 190907.74/UAMH 11175 CB F
Meliniomyces bicolor (AY394885)
ARSL 170507.36 Q F
93
72 ARSL 070907.13 CB S
90 ARSL 070907.12/UAMH 11204 CB S
ARSL 250507.3 Q B
ARSL 230507.30II Q B
Meliniomyces vraolstadiae strain T G1 (AJ292199)
93
ARSL 170507.42I Q F
ARSL 230507.6 Q B
Meliniomyces vraolstadiae strain G2 (AJ292200)
ARSL 170507.42II Q F
96 ARSL 230507.46 Q B
ARSL 060907.18II CB B
ARSL 60907.26/UAMH 11128 CB B
ARSL60907.27 CB B
ARSL 170507.25I Q F
ARSL 060907.1 CB B
ARSL 70907.4 CB S
ARSL 230507.7 Q B
ARSL 70907.19 CB S
ARSL 180907.39 CB S
100
ARSL 230507.30I Q B
Meliniomyces variabilis (EF093173)
90 ARSL 220507.11 Q S
ARSL 220507.2 Q S
ARSL 220507.4I Q S
ARSL 70907.15/UAMH 11129 CB S
ARSL 190907.72 CB F
ARSL 60907.24 CB B
10
ARSL 190907.5 CB F
ARSL 190907.17 CB F
ARSL 190907.8 CB F
100
Helotiaceae sp. I
Helotiaceae sp. II
Helotiaceae sp. III
Helotiaceae sp. IV
84
100
78
Helotiaceae sp. VI
Helotiaceae sp. VII
Meliniomyces bicolor
Meliniomyces vraolstadiae
Meliniomyces sp.
Rhizoscyphus ericae aggregate
Figure 1 One of 100 most parsimonious midpoint rooted trees
comparing ITS sequences of cultured root endophytes within the
Helotiaceae with GenBank sequences (in bold). Consistency index=
0.720, retention index=0.956, and tree length=408. Clades, which
Helotiaceae sp. V
Meliniomyces variabilis
contain reference sequences from uncultured environmental samples
only, were given operational names (Helotiaceae sp. I–VII). Bootstrap
values>70% are shown. Scale bar=10 substitutions
G. Kernaghan, G. Patriquin
Phialocephala sphaeroides (AY524844)
ARSL 070907.7/UAMH11132 CB S
ARSL 220507.6II Q S
ARSL 60907.65 CB B
ARSL 230507.33 Q B
ARSL 230507.36 Q B
86
ARSL 170507.56 Q F
ARSL 230507.57 Q B
ARSL 190907.49I/UAMH 11207 CB F
ARSL 190907.50 CB F
Acephala applanata (AY078147)
Phialocephala helvetica (AY347408)
Phialocephala turiciensis (AY347389)
ARSL 220507.12 Q S
ARSL 190907.7 CB F
ARSL 190907.9 CB F
ARSL 070907.28 CB S
ARSL 220507.18 Q S
ARSL 180907.1 CB S
ARSL 190907.6 CB F
ARSL 070907.31 CB S
ARSL 190907.20 CB F
ARSL 070907.21 CB S
ARSL 180907.2 CB S
Phialocephala fortinii (AY664502)
ARSL 250507.1 Q B
ARSL 220507.49 Q S
ARSL 070907.20 CB S
ARSL 070907.39 CB S
ARSL 190907.33/UAMH 11197 CB F
ARSL 070907.26 CB S
ARSL 220507.35 Q S
ARSL 220507.22 Q S
Phialocephala letzii (AY347396)
Phialocephala europaea (AY347403)
ARSL 230507.43 Q B
ARSL 220507.55 Q S
ARSL 180507.5 Q S
ARSL 220507.43 Q S
ARSL 180507.2 CB S
ARSL 180507.18 Q S
ARSL 060907.60 CB B
100 ARSL 060907.18I CB B
ARSL 170507.11 Q F
ARSL 190907.53/UAMH 11131 CB F
Neofabraea alba (AF141190)
Pezicula sporulosa (AF141172)
98
Cryptosporiopsis ericae (AY853167)
95
ARSL 190907.12/UAMH 11126 CB F
93
ARSL 190907.56 CB F
Dermea hamamelidis (AF141157)
ARSL 170507.22/UAMH 11127 Q F
93
ARSL 170507.49 Q F
ARSL 190907.51 CB F
84
100
89
100
100
Phialocephala sphaeroides
Phialocephala sp.
Phialocephala fortinii
complex
Dermataceae sp. I
Cryptosporiopsis ericae
Cryptosporiopsis sp.
1
Figure 2 One of 100 most parsimonious midpoint rooted trees
comparing ITS sequences of cultured root endophytes within the
Dermateaceae with GenBank sequences (in bold). Consistency
index=0.797, retention index=0.962, and tree length=276. Bootstrap values >70% are shown. Scale bar=1 substitution
give an overall total of 31 distinct phylotypes across both
sites (Figs. 1, 2, 3, and 4, Table 1). For the Cape Breton
site, the 71 morphological groups (including 54 singleton
isolates) were reduced to 23 phylotypes (including seven
singletons). For the Québec site, the original 74 groups
(including 50 singletons) were reduced to 19 phylotypes
(with five singletons). For individual host trees, phylotype
richness was highest on Abies with S=26, followed by
Picea and Betula, both with S=15.
Although species accumulation curves (not shown)
indicated that further sampling would have detected more
endophyte phylotypes, comparisons of our observed richness values with bootstrap estimated richness values
indicates that our 160 isolations per host captured 82.7%,
82.2%, and 84.3% of the endophyte richness of Abies,
Betula, and Picea, respectively.
The undetected phylotypes are most probably rare,
however, and are unlikely to have had a significant impact
on our conclusions regarding the host associations of the
more common phylotypes.
The overall Shannon diversity index for root endophytes was somewhat higher for the Cape Breton site
(H′ = 2.58) than for the Québec site (H′ = 2.21). For
individual host trees, the highest endophyte diversity was
on Abies (H′=2.66), followed by Betula (H′=2.22) and
then Picea (H′ = 1.74), but there were no significant
differences (P>0.05) in diversity among hosts or among
the 15 host–site combinations.
Host trees also varied in the degree of overlap in endophyte
phylotypes, with Picea and Abies sharing 14, Betula and
Abies sharing 11, and Betula and Picea sharing nine. Twelve
phylotypes were isolated only from the Cape Breton site,
Host Associations Between Root Endophytes and Boreal Trees
Figure 3 One of 54 most parsimonious midpoint rooted trees comparing
ITS sequences of cultured root endophytes within the Myxotricaceae
with GenBank sequences (in bold). Consistency index=0.697, retention
index=0.823, and tree length=155. Bootstrap values >70% are shown.
Scale bar=1 substitution
eight phylotypes were isolated only from the Québec site,
and 11 phylotypes were isolated from both sites, giving a
35% overlap between sites. Overlap between sites increases to
58% if singletons are disregarded. Results from the ANOSIM,
which takes the relative proportions of phylotypes on each
host–site combination into account (Table 2), indicate that
there are no significant differences (α=.05) between the root
endophyte communities of host trees of the same species
across sites. Furthermore, within the Cape Breton site, the
root endophyte communities are significantly different among
all three tree species. However, on the Québec site, endophyte
communities are not significantly different between Abies and
Picea and between Betula and Picea. Differences between
different host tree species across sites are mainly significant,
with the exception of Cape Breton Picea vs Québec Abies
and Cape Breton Picea vs Québec Betula.
The DCA also indicates differences in endophyte
assemblages colonizing the three host tree species
(Fig. 6a, b), as the hosts fall into relatively distinctive
groupings regardless of site. The first and second axes of
the ordination explain a total of 23.6% of the variation in
the data (14.5% and 9.1%, respectively; γ1 =0.612, γ2 =
0.382, total inertia=4.205). In Fig. 6a, site scores (host
trees) are mainly separated along the first axis, with most of
G. Kernaghan, G. Patriquin
Figure 4 Midpoint rooted parsimony tree comparing ITS
sequences of cultured root
endophytes within the Hyaloscyphaceae with GenBank
sequences (in bold). Consistency index=0.769, retention index
=0.629, and tree length=283.
Bootstrap values >70% are
shown. Scale bar=1 substitution
Hyaloscypha daedaleae (AY789416)
Axenic ericoid root isolate (AJ430215)
97
Lachnellula calyciformis (U59145)
100
Lachnum bicolor (AJ430394)
Cistella acuum (U57492)
ARSL 230507.52/UAMH 11166 Q B
Hyaloscyphaceae sp. I
85
ARSL 170507.13/UAMH 11200 Q F
Hyaloscyphaceae sp. II
ARSL 190907.62/UAMH 11165 CB F Hyaloscyphaceae sp. III
87
ARSL 180907.20/UAMH 11167 CB S
1
Hyaloscyphaceae sp. IV
Uncultured fungus isolate RFLP67 (AF461628)
72
Hyphodiscus hymeniophilus (DQ227264)
the Abies toward the left of the diagram, most of the Betula
toward the right (although some are toward the bottom left),
and all of the Picea occupying a central position.
Abies was colonized by Cryptosporiopsis ericae, Cryptosporiopsis sp., Helotiaceae sp. V, and Helotiaceae sp. VI
to a greater extent than Betula and Picea (Fig. 5). These
fungi fall mainly on the left side of the ordination (Fig. 6b).
Picea forms a distinct group, delineated from the other
hosts by the frequency of Helotiaceace III and P. fortinii s.l.
(Figs. 5 and 6b). The endophyte assemblages of Betula are
more variable, with the Québec Betula characterized by O.
maius and Phialocephala sphaeroides. The two Cape
Breton Betula trees toward the bottom left of the diagram
are separated from the others mainly by Meliniomyces sp.
(Figs. 5 and 6b).
Host Associations
The fungal root endophytes detected fell into four general
categories with respect to host associations. The first
category included infrequent phylotypes (<1% overall
isolation frequency) for which there was not enough data
to make inferences as to their distributional patterns. These
included the 12 singletons, Chaetospheria sp., Helotiaceae
sp. I, II, IV, and VII, Hyaloscyphaceae sp. I–IV, Hypocrea
pachybasioides, Tricholomataceae sp. I, and Umbelopsis sp.
II, as well as other low frequency phylotypes such as
Mycena sp., Meliniomyces bicolor, Lecythophora mutabilis,
Umbelopsis sp. I, Phialocephala sp., Penicillium montenese,
and Oidiodendron sp., many of which were detected from
only one of the two sites (Fig. 5).
The second category included those relatively common
phylotypes, which appear to lack host preference, i.e.
Meliniomyces variabilis, Meliniomyces vraolstadiae, Meliniomyces sp., and Dermateaceae sp. I (Fig. 5). These
phylotypes have relatively large niche breadth indices (BA
from 0.488 to 1) and host distributions not significantly
different from expected based on goodness of fit (Table 3).
The third group included phylotypes which appeared to
exhibit host preference on one site, but were absent or at
low frequency on the other site. These included C. ericae,
Cryptosporiopsis sp., and Helotiaceae sp. V that all
occurred only on Abies where detected, as well as P.
sphaeroides and O. maius, that both occurred mainly on
Betula on the Québec site (Fig. 5). Phylotypes in this
second group had relatively small niche breadth indices (BA
from 0 to 0.372), and their host distributions were
significantly different from expected (Table 3).
The final group consisted of the phylotypes that were
common (at least eight isolates per site) and that appeared to
exhibit preference for a particular host. These included
Helotiaceae sp. III, Helotiaceae sp. VI, and P. fortinii s.l.
(Fig. 5). These three phylotypes had significant goodness of
fit test results and were widely distributed across individuals
Host Associations Between Root Endophytes and Boreal Trees
Table 2 Results of ANOSIM test (p values) comparing endophyte
assemblages on each tree species at each site
Betula
Picea
Abies
Betula
Picea
0.032
0.02
0.033
0.085
0.037
0.365
0.031
0.087
0.09
0.044
0.021
0.027
0.305
0.198
0.124
Values in bold are significant (p<0.05). Results of comparisons
between trees of the same species on different sites are in italics
of their preferred host (Table 3, Fig. 5). Helotiaceae sp. III
and VI each had small niche breadth indices (0.161 and
0.155, respectively), while P. fortinii s.l. was broader at
0.635 (Table 3).
Discussion
Our results demonstrate that species assemblages of fungal
root endophytes of boreal trees differ from host to host. We
have also shown that the differences in root endophytes
across hosts are not due to edaphic or micro-climactic
conditions, as these were controlled for by sampling from
small plots containing intertwined roots of the different host
50
30
25
20
15
10
Mycena sp. nd
Meliniomyces bicolor nd
Lecythophora mutabilis nd
Umbelopsis sp. I nd
Phialocephala sp.
Dermateaceae sp. I
Penicillium montanense nd
Cryptosporiopsis sp. nd
Oidiodendron sp.
Cryptosporiopsis ericae nd
Meliniomyces sp.
Meliniomyces vraolstadiae
Phialocephala sphaeroides
Helotiaceae sp. III
Helotiaceae sp. VI
0
Helotiaceae sp. V nd
5
Phialocephala fortinii s.l.
Root tips colonized
Figure 5 Number of root tips
colonized by fungal root endophytes on each tree species. The
first and second bars for each
phylotype represent the number
of isolations from the Cape
Breton and Québec sites, respectively. Black, Abies; white,
Betula; grey, Picea, nd, not
detected. Twelve singletons not
shown
Oidiodendron maius
Quebec
Abies
Betula
Picea
Abies
Betula
Quebec
Meliniomyces variabilis
Cape Breton
Cape Breton
species. Although many of the phylotypes detected occurred
at frequencies too low to allow for inferences about their
distributional patterns, several were relatively common, and
a proportion of these appear to exhibit distinct associations
with particular hosts. Of course, these distributional patterns
pertain only to the three host species sampled and only to
Cenococcum ectomycorrhizae on those hosts. We cannot
extrapolate to other host species.
The most commonly encountered root endophyte was P.
fortinii s.l. It was most commonly isolated from Picea, least
common on Betula at the Quebec site, and absent from
Betula on the Cape Breton site. However, P. fortinii s.l. is
somewhat problematic in the context of host associations,
as recent genetic studies divide European isolates into
several cryptic species, some of which are not distinguishable on the basis of ITS sequence analysis [22]. As it is
very likely that cryptic species of P. fortinii also exist in
North America, and each may display its own host
preference, the distribution of P. fortinii seen in the current
study likely represents an overall pattern of a group of
closely related fungal endophytes.
Conversely, P. sphaeroides was most common on Betula
on one of our sites. P. sphaeroides was originally isolated
from a range of herbaceous and woody host plants
(including B. papyrifera) in a sphagnum-dominated wetland
[76]. Again, as with all of the phylotypes detected in the
current study, more sites and more host species would
undoubtedly reveal broader host ranges.
G. Kernaghan, G. Patriquin
a
4
Figure 6 a, b Detrended correspondence analysis (DCA)
depicting relationships among
host trees and sites on the basis
of fungal root endophyte colonization. Site scores (a) are
separated from species scores
(b) for clarity. Twelve singletons
not shown (b). In Fig 6a, CB,
Cape Breton; Q, Québec; black
triangles, Abies; white triangles,
Betula; grey triangles, Picea
CB
CB
CB
CB
Q
CB
Q
Q
CB
Q
Q
Q
CB
Q
CB CB
Q
Q
Q
Q
Q
CB
CB
CB
6
-1
-1
7
b
Meliniomyces bicolor
Helotiaceae sp. V
Cryptosporiopsis ericae
Mycena sp.
Helotiaceae sp. VI
Meliomyces variabilis
Phialocephala sp.
Phiacephala fortinii s.l.
Phialocephala sphaeroides
Oidiodendron sp.
Cryptosporiopsis sp.
Helotiaceae sp. III
Meliniomyces vraolstadiae
Oidiodendron maius
Penicillium montenese
Umbelopsis sp. I
Dermataceae sp. I
-1
7
-1
Lecythophora mutabilis
Meliniomyces sp.
The most obvious host–endophyte associations were
seen within the Helotiaceae, specifically among Helotiaceae
sp. III, V, and VI, for either Picea or Abies. However, these
fungi remain unidentified, other than that they appear to be
a group of species close to, but not part of the Rhizoscypus
ericae aggregate [25, 70].
Both C. ericae and Cryptosporiopsis sp. were isolated
solely from the roots of Abies, although each was found on
Host Associations Between Root Endophytes and Boreal Trees
Table 3 Results of randomization tests for goodness of fit, standardized niche breadth indices (BA), percentage of individual trees of each species
colonized, and the preferred host for 19 fungal root endophytes
Root endophyte
Difference among hosts (goodness of fit p values)
Individual trees colonized (%)
Preferred host
Both sites
Cape Breton
Quebec
BA
Phialocephala fortinii s.l.
Helotiaceae sp. VI
Meliniomyces variabilis
Helotiaceae sp. III
Oidiodendron maius
Phialocephala sphaeroides
Meliniomyces sp.
Meliniomyces vraolstadiae
Helotiaceae sp. V
Oidiodendron sp.
<0.0001
<0.0001
0.4700
<0.0001
0.0117
0.0129
0.0476
0.7420
0.0035
0.1282
<0.0001
0.0003
0.1776
0.0016
1
0.3354
0.0530
0.1132
0.0037
0.1148
0.0209
0.0003
0.1597
0.0600
0.0001
0.0083
0.7736
0.1407
nd
0.7750
0.635
0.155
0.899
0.161
0.372
0.337
0.488
0.954
0
0.461
50
0
37.5
12.5
62.5
37.5
37.5
37.5
0
0
75
75
37.5
37.5
12.5
12.5
25
25
25
37.5
87.5
25
37.5
87.5
25
25
12.5
12.5
0
25
Picea
Abies
nd
Picea
Betula
Betula
Betula
nd
Abies
nd
Cryptosporiopsis ericae
Cryptosporiopsis sp.
Dermateaceae sp. I
Penicillium montanense
Phialocephala sp.
Umbelopsis sp. I
Lecythophora mutabilis
Meliniomyces bicolor
Mycena sp.
0.0035
0.0361
0.5640
1
0.7805
1
0.3312
0.3295
0.1135
0.0031
nd
0.7810
nd
0.1148
nd
0.3428
0.3355
0.1148
nd
0.0367
1
1
1
1
nd
nd
nd
0
0
0.5
1
0
0.4
0.5
0.5
0
0
0
25
12.5
0
12.5
12.5
0
0
37.5
25
25
12.5
12.5
12.5
12.5
12.5
12.5
0
0
0
12.5
12.5
12.5
0
12.5
0
Abies
Abies
nd
nd
nd
nd
nd
nd
nd
Betula
Abies
Picea
p values in bold are significant (p<0.05). Phylotypes are listed from most to least commonly isolated. Twelve singletons not analyzed. A preferred
host was assigned only when the result of the goodness of fit test was significant
nd not detected
only one site. C. ericae was originally described from
ericaceous roots [60] and has since also been isolated from
Populus roots [72].
Similarly, O. maius was predominantly isolated from
Betula on one of our sites, although other authors have
found it to be relatively common on the roots of Picea [56]
and Pinus [45]. The ecological niche of O. maius appears
very wide, as it also forms mycorrhizae on ericaceous
plants [13] and grows saprophytically on sphagnum [52].
The other, as of yet unidentified, species of Oidiodendron
did not exhibit host specificity.
All four phylotypes referable to Meliniomyces (M.
bicolor, M. varabilis, M. vraolstadiae, and Meliniomyces
sp.) were fairly evenly distributed across hosts. The genus
Meliniomyces is composed of sterile, root-associated species with the potential to colonize a wide range of plants
[25]. For example, M. vraolstadiae forms ectomycorrhize
on Betula, Picea, and Pinus [71], Meliniomyces varabilis is
capable of forming ericoid mycorrhizae on ericaceous hosts
[49], and M. bicolor is reported to form either type of
mycorrhizae, depending on the host plant colonized [21].
Therefore, in the case of M. bicolor, it is possible that our
isolates may have been acting as endophytes within C.
geophilum (the mycorrhizal fungus colonizing all roots
sampled), or they may have themselves been involved in
concomitant ectomycorrhizal associations with Cenococcum (dual ectomycorrhizal colonization).
We used a culture based approach (from surfacesterilized root tips) followed by PCR, rather than direct
amplification of fungal DNA from root tips. Although we
recognize that our method does not detect unculturable
endophytes, we felt it preferable to use direct PCR for our
objective, in that we can be certain that our isolates
represent fungi colonizing the root tips internally, as
opposed to those residing on the root surface [27]. Direct
PCR does not discern between these two groups of fungi
and may detect non-host specific soil fungi on the root
surface, perhaps confounding our data on host associations.
Even with surface sterilization by peroxide or hypochlorite
(bleach), the DNA of these superficial fungi may still be
detected by PCR; the fungi may be killed, but amplifyable
DNA may remain [34]. Our cultural approach also avoids
the problem of concurrent colonization of root tips by
ectomycorrhizal (ECM) fungi, the DNA of which would
likely swamp the endophyte DNA. Amplification of ECM
fungal DNA can be avoided by using ascomycete-
G. Kernaghan, G. Patriquin
specific PCR primers to amplify endophytes within
basidiomycetous ECM [64], but this approach does not
detect basidiomycete endophytes and cannot be used for
ascomycetous ECM such as those sampled here. Although
isolation of pure cultures from surface sterilized roots
tends to detect fewer fungal species than direct PCR [4, 9],
each approach appears to have its own biases. When both
methods were used to detect root associated fungi of
conifer seedlings [45], P. fortinii and Oidiodendron were
frequently isolated from surface sterilized mycorrhizae,
but were rarely (or never) detected by direct PCR.
The patterns of host preference displayed by some of the
endophytes in the current study are characteristic of
biotrophic [33] or mutualistic [57], rather than necrotrophic,
relationships. Saprophytes tend to exhibit substrate specificity rather than host specificity [10], and biotrophic fungi
(including biotrophic mutualists such as the mycorrhizal
fungi) exhibit greater host specificity than necrotrophs [8,
40]. Patterns of host specificity in the ectomycorrhizal
(ECM) fungi are fairly well understood; although some
hosts such as Alnus and Larix support very specific ECM
mycobionts [46, 65], studies of mixed conifer stands [11,
30] found that commonly occurring ECM fungi lacked
specificity, and only a few uncommon species were host
specific. In a study of boreal forest ECM (conducted in the
same research forest as our current Québec site), the most
common ECM fungi were generalists, less common fungi
often preferred particular hosts, and some uncommon
species exhibited apparent host specificity [36]. Although
we recognize that rarity and specificity are interrelated,
due to uncommon species occurring on fewer hosts by
chance alone, the general pattern of host preference
described for ECM fungi is also evident in our root
endophyte data.
Root endophytic fungi and ECM fungi differ, however,
in that endophyte species likely vary greatly in their
relationship with the plant host, making it difficult to
predict the ramifications of root endophyte host preference.
Some root endophytes may be latent pathogens, causing
disease symptoms in weakened or damaged roots [59],
while many others are beneficial, improving plant growth
[58], defending from disease [47], improving drought
tolerance [6], or mineralizing organic nutrient sources
[67]. Therefore, the asymmetric distributions of the fungal
root endophytes detected on our sites may potentially
influence interspecific plant competition.
Acknowledgements This work was made possible by a grant
from the Natural Sciences and Engineering Research Council of
Canada (341671-2007). We thank Emily Cormier and Erica Fraser
for technical assistance, Cape Breton Highlands National Park and
the Lac Duparquet Teaching and Research Forest for field
Logistics, and Lynne Sigler for comments on an earlier version
of the manuscript.
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