PLANT MICROBE INTERACTIONS

Host Associations Between Fungal Root Endophytesand 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 tissueconcomitantly with mycorrhizal fungi, but their identitiesand host preferences are largely unknown. We culturedfungal endophytes from surface-sterilized Cenococcum geo-philum ectomycorrhizae of Betula papyrifera, Abies balsa-mea, and Picea glauca from two boreal sites in easternCanada. Isolates were initially grouped on the basis ofcultural morphology and then identified by internal tran-scribed spacer ribosomal DNA sequencing or by PCRrestriction fragment length polymorphism. Phylogeneticanalysis of the sequence data revealed 31 distinct phylotypesamong the isolates, comprising mainly members of theascomycete families Helotiaceae, Dermateaceae, Myxotri-chaceae, and Hyaloscyphaceae, although other fungi werealso isolated. Multivariate analyses indicate a clear separa-tion among the endophyte communities colonizing each hosttree species. Some phylotypes were evenly distributed acrossthe roots of all three host species, some were foundpreferentially on particular hosts, and others were isolatedfrom single hosts only. The results indicate that fungal rootendophytes of boreal trees are not randomly distributed, butinstead form relatively distinct assemblages on different hosttree species.

Introduction

Fungal endophytes colonize plant tissue internally andasymptomatically for at least some of their lifecycle [75]

and appear to be ubiquitous within stems, leaves, bark,and roots [54]. Plant roots harbor characteristic assemb-lages of fungal endophytes that are distinct from those ofabove-ground plant tissue [2, 62]. Although the ecologicalroles of root endophytes are largely unknown, theyrepresent a significant component of the below-groundmicrobial community and are thought to be at least ascommon as mycorrhizal fungi [41, 73]. In boreal trees,commonly occurring fungal root endophytes includespecies of Cryptosporiopsis [69, 72], Oidiodendron [56],Umbelopsis [29], and members of the “Rhizoscyphusericae aggregate” (Helotiaceae) [25, 70]. The best known,however, are commonly referred to as “dark septateendophtes” or DSE, which includes members of thePhialocephala fortinii complex and other fungi withmelanized hyphae. Much of our understanding of fungalroot endophytes is based on studies of DSE, as they arecommon (especially in boreal soils), easily observed andeasily cultured (but not easily identified) [1, 20, 24, 33,44, 51, 77].

There have been several recent studies of tree rootendophyte species composition and diversity [3, 16, 19, 35,44, 45], but few have documented the differences innaturally occurring endophyte communities across hostplant species. Those that have shown differences amonghost plants generally detect evidence of host preference [20,38, 64], although the influence of variation in abioticfactors among sampling sites is usually not considered.

The phenomenon of host specificity (or host preferenceif the relationship is not strictly exclusive) has beendemonstrated in leaf endophytes [31, 50], mycorrhizalfungi [36, 46], and fungal root pathogens [17], and isassumed to play an important role in plant communityecology [55]. For example, any positive [57, 68] ornegative [77] effects of colonization would not be shared

G. Kernaghan (*) :G. PatriquinBiology Department, Mount St. Vincent University,166 Bedford Hwy.,Halifax, NS B3M 2J6, Canadae-mail: gavin.kernaghan@msvu.ca

Microb EcolDOI 10.1007/s00248-011-9851-6

equally throughout the plant community. Levels of hostspecificity also factor heavily into estimates of globalfungal biodiversity in that all plant species are assumed tosupport a certain number of host-specific fungi [28, 78].Despite these considerations, information regarding levelsof host specificity among root endophytes is still sparse,and their ubiquity has led earlier authors to consider themto be non-host specific [16, 20]. This impression was basedlargely on studies of DSE, which when considered as agroup, do colonize a wide range of plant hosts [33].However, the concept of DSE encompasses a wide varietyof ascomycetous fungi, and even the best known rootendophyte, P. fortinii, is now considered to be a speciescomplex comprised of several cryptic species [22, 23], eachof which may potentially exhibit its own level of hostpreference or specificity.

In order to compare fungal root endophyte communitiesacross boreal tree hosts and to investigate levels of hostpreference, we isolated endophytes from surface sterilizedCenococcum geophilum ectomycorrhizae of three co-occurring tree species. We focused on ectomycorrhizaltissue, as it is relatively unexplored with respect to fungalendophytes and may harbor species localized in themetabolically active root tips. We also chose to isolatefungi from surface sterilized tissue to ensure that all isolateswere endophytic.

Methods

Collection of Roots and Isolation of Fungi

Sampling was conducted at two boreal sites in easternCanada. One was located on Mount Mackenzie, CapeBreton Highlands National Park, Nova Scotia (46o45′ N,60o50′ W) at 380 m elevation. The second was in thesouthern boreal mixed wood forest, located in the LacDuparquet Teaching and Research Forest, northwesternQuébec (48o29′ N, 79o25′ W) at 300 m elevation. The twosites were approximately 1,400 km apart.

Both sites support mature (over 70 years) mixtures ofBetula papyrifera, Abies balsamea, and Picea glauca.Daily average temperatures near Mount McKenzie rangefrom −6.3°C in February to 18.3°C in August, with a totalannual rainfall of 1,391 mm. The Lac Duparquet site iscolder and dryer with daily average temperatures from−18.2°C in January to 16.9°C in July and an annual averageprecipitation of 889 mm (30-year normals, EnvironmentCanada). Soil surveys have not been conducted in northernCape Breton, but Mount McKenzie soils are likely GleyedHumo-Ferric or Gleyed Ferro-Humic Podzols (Keys 2007,K. Keys, personal communication). Soils at Lac Duparquetare Gray Luvisols [7].

Four 2-m2 plots were established at each site. Plotswere spaced approximately 50 m apart and supported atleast one mature A. balsamea, one mature B. papyrifera,and one mature P. glauca. For each tree species on eachplot, one major root was traced from the base of the tree tothe fine roots (12 major roots per site), and all the fineroots were collected. Root tips were examined under adissecting microscope and 20 fine root tips colonized byC. geophilum (ectomycorrhizae) were identified on thebasis of morphology and removed from each sample forisolation of endophytes. Only root tips colonized by C.geophilum were used for endophye isolation in order toavoid any possible differences in endophyte assemblagesbetween root tips colonized by different species ofectomycorrhizal fungi. Ectomycorrhizae were surfacesterilized [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 Piceaand 4 min for Betula—optimal surface sterilization timesfor 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 Bactoyeast extract, and 15 g agar) supplemented with 100 ppmoxytetracycline, 50 ppm streptomycin sulfate, and 50 ppmpenicillin G. Plates were incubated at 20°C in the dark.Emergent hyphae were transferred to water agar forsubsequent hyphal tip transfers onto oatmeal–salts andCzapek’s media [12]. Sucrose in the Czapek’s mediumwas reduced to 15 g/L.

For each of the two sampling sites, cultures weresorted 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’smedia; dark septate isolates, preliminarily identified as P.fortinii sensu lato, were also grown on malt extract agarwith or without 100 mg/L cycloheximide [22], as well ason pectin based media in order to better distinguish amongspecies.

DNA Extraction

Fungal tissue was removed from agar plates, frozen at −20oC,then placed in 600 μl 2× cetyl trimethylammonium bromideextraction buffer, ground in a ceramic mortar and incubatedat 65oC for 1 h in a micro-centrifuge tube with 100 μg/mlproteinase K. Six hundred microliters chloroform/isoamylalcohol (24:1) was then added followed by a 15-mincentrifugation at 20,000 g. DNA was then precipitated byremoving the upper aqueous layer, adding 600 μl coldisopropanol, cooling to −20°C for 30 min and centrifuging at20,000×g for 15 min. The resulting pellet was washed twicewith 70% ethanol, air dried, and re-suspended in 100 μlsterile distilled water.

G. Kernaghan, G. Patriquin

Identification of Isolates

Between 33% and 100% of the isolates in each morpho-logical group were selected for internal transcribed spacer(ITS) sequencing (58% of isolates overall). The percentageof isolates sequenced was dependent on the number ofisolates in the group, with a smaller proportion of the mostabundant types being sequenced. Sequenced isolatesincluded representatives of either end of any subtlemorphological 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] and14 μL H2O. Unsuccessful PCR reactions were repeatedusing DNA template diluted to 1:25 or 1:250 in H2O. Thethermal parameters were as described in DeBellis et al.[14]. The resulting PCR products were sequenced at theMcGill University and Genome Québec Innovation Centrewith an ABI PRISM 3730XL DNA analyzer system withITS1 (forward) and ITS4 (reverse) primers [74].

Sequence contigs were assembled for each isolate, editedusing Sequencher 4.9 (Gene Codes, Ann Arbor, MI, USA)and compared to GenBank sequences using nucleotide–nucleotide BLAST (blastn). As the majority of sequencesgrouped among either the Helotiaceae, Dermateaceae,Hyaloscyphaceae, or Myxotrichaceae (Leotiomycetes, Asco-mycota), separate maximum parsimony analyses wereconducted for each of these families. Sequences from ourisolates and closely matching GenBank sequences (referencesequences) 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 and590 bp in length (including gaps), with between 54 and155 parsimony informative characters. Whenever possible,the GenBank sequences used as references were thosederived from ex-type or identified cultures, rather than fromenvironmental samples not supported by cultures. Maximumparsimony analyses were performed using PAUP* 4.0b10[63] with midpoint rooting, heuristic search, TBR branchswapping, 100 trees maximum, and 1,000 bootstrap repli-cations. The small number of isolates not belonging to thefour dominant families was identified by BLAST searchesonly (Table 1). Isolate groupings were then adjusted on thebasis of the sequence data. In most cases, this simplyamounted to pooling smaller morphological groups intolarger, sequence based groups (phylotypes).

For the remaining (non-sequenced) isolates, within cladehomogeneity was confirmed by restriction fragment lengthpolymorphism (RFLP) analysis. DNA extractions and ampli-fications were performed as above and the resulting ampliconsdigested 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 andTsp509 I were selected for their ability to differentiate amongthe previously sequenced phylotypes, determined usingNEBcutter V2.0 (http://tools.neb.com/NEBcutter2).

All cultures are stored on malt agar slants and in sterilewater at 4oC [53] at Mount Saint Vincent University. At leastone sequenced isolate representing each phylotype has alsobeen deposited in the University of Alberta MicrofungusCollection and Herbarium, Edmonton, AB, Canada, underthe accession numbers UAMH 11124–11133, 11165–11175,11194–11205, 11207, and 11220–11224. All ITS sequences,including those representing the UAMH accessions, havebeen deposited in GenBank as HQ157833 to HQ157959.

Statistical Analysis

Species accumulation curves were produced for each hostusing Estimates 8.2.0 (http://viceroy.eeb.uconn.edu/estimates) and the number of potentially undetected phylotypesestimated by subtracting the observed species richness fromthe estimated species richness calculated with the bootstrapestimator of species richness [61].

Differences in isolation frequency among the three hosttrees were calculated for the 19 non-singleton phylotypesusing a randomization test of goodness of fit [43] with10,000 randomizations. Standardized niche breadth [32, 37]was also calculated for the 19 non-singletons (using datafrom both sites). Shannon diversity indices were calculatedfor root endophytes cultured from each host–site combina-tion using PC-ORD version 4 [42]. Analysis of similarity(ANOSIM) among endophyte assemblages on each treespecies on each site was calculated using the Morisita indexwith PAST version 2.04 (http://folk.uio.no/ohammer/past/).

Relationships among root endophytes, host tree species,and sites were assessed by detrended correspondenceanalysis (DCA) using CANOCO [66]. The input for theordination was a matrix of non-transformed counts ofphylotypes 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 rarespecies were down-weighted.

Results

Isolation and Identification of Root Endophytes

Two hundred thirty isolates of fungal root endophytes wereobtained from the 480 surface sterilized root tips (20 roottips×3 tree species×4 plots×2 sites), giving an overallendophyte isolation frequency of 48%. A further 5% of theisolations yielded the ectomycorrhizal symbiont Cenocco-cum geophilum (the ectomycorrhizal fungus on all root tips

Host Associations Between Root Endophytes and Boreal Trees

sampled) and were excluded from further analyses. Mostisolations yielded a single fungus, but in 7% of theisolations, two different fungi grew from a single root tip.The initial grouping of isolates based on cultural morphol-ogy on different media resulted in 17 groups and 54singleton isolates from the Cape Breton site and 24 groupsand 50 singleton isolates from the Quebec site.

Initial BLAST searches revealed that the majority (96%) ofthe endophyte isolates were members of the ascomycetefamilies Helotiaceae, Dermateaceae, Myxotrichaceae, andHyaloscyphaceae. The remaining 4% were other ascomycetes,basidomycetes (Tricholomataceae), or Mucorales (Umbelopsisspp.), with low isolation frequencies (Table 1). Separatemaximum parsimony analyses of each of the four dominantfamilies revealed 11 phylotypes in the Helotiaceae, six in theDermateaceae, two in the Myxotrichaceae, and four in theHyaloscyphaceae (Figs. 1, 2, 3, and 4). The helotialianphylotypes fell into two main groups; one comprised ofspecies of Meliniomyces, a genus belonging to the R. ericaeaggregate [25, 70] and the other comprised of sevenphylotypes of an unidentified complex, close to, but not partof, the R. ericae aggregate (Fig. 1). For this latter group, noclose matches to any named isolates were found in GenBank.

In the Dermateaceae, three phylotypes were referable toPhialocephala, two to Cryptosporiopsis and one phylotypenot assignable to a known species is designated “Derma-teaceae sp. I” (Fig. 2). The Phialocephala isolates includeP. sphaeroides, an unidentified Phialocephala species, andmembers of the P. fortinii complex, which may includecryptic species not distinguishable by ITS sequencing [22].The Cryptosporiopsis isolates include C. ericae and anunidentified Cryptosporiopsis species.

The Myxotrichaceae (Fig. 3) are represented by Oidio-dendron maius and a second Oidiodendron species, forwhich there are no matching culture-derived sequences inGenBank. The Hyaloscyphaceae (Fig. 4) are represented byfour unidentified isolates, all phylogentically close toHyphodiscus hymeniophilus.

RFLP analysis of the unsequenced isolates confirmedthat they had been correctly grouped on the basis ofmorphology, with the exception of three isolates: onePhialocephala sp., one Dermateaceae sp. I, and oneHelotiaceae sp. VI. These isolates were easily re-assignedto their correct groups on the basis of the RFLP data.

Testing of P. fortinii s.l. isolates on MEA medium withcycloheximide revealed a gradient of inhibition from strongto weak and did not demonstrate distinctive morphologicalgroupings among the isolates.

Endophyte Communities

The number of isolate groupings originally distinguished onthe basis of morphology was reduced after sequencing, toT

able

1BLASTresults

for11

phylotyp

esno

tinclud

edin

phylog

enetic

trees

Phy

lotype

Site

Host

Isolateno

.BestGenBankmatch

GenBank

accession

Total

score

Query

coverage

(%)

Identities

Lecytho

phoramutab

ilis

CapeBreton

Betula

ARSL06

0907

.9/UAMH

1117

3Lecytho

phoramutab

ilisisolateaurim

1180

DQ09

3680

893

7948

5/48

6(99%

)

Cha

etosph

aeriasp.

CapeBreton

Betula

ARSL06

0907

.80/UAMH

1112

4Cha

etosph

aeriachloroconia

AF17

8542

808

8250

3/53

3(94%

)

Mycenasp.

CapeBreton

Abies

ARSL19

0907

.23II/UAMH

1117

4Mycenatena

xvo

ucherOSC1137

4EU84

6251

1064

9467

7/72

2(93%

)

Penicillium

mon

tanense

Quebec

Picea

ARSL18

0507

.8/UAMH

1119

8Penicillium

mon

tanense

AF52

7058

1075

9755

8/55

9(99%

)

Quebec

Betula

ARSL23

0507

.17/UAMH

1119

9Penicillium

mon

tanense

AF52

7058

1007

9055

6/56

1(99%

)

Hypocreapa

chybasioides

CapeBreton

Abies

ARSL19

0907

.26/UAMH

1113

3Tricho

derm

apo

lysporum

CBS82

0.68

Z48

815

1035

9757

9/58

7(98%

)

Tricho

lomataceaesp.I

Quebec

Abies

ARSL17

0507

.37/UAMH

1113

0Mycenaplum

beaisolateAFTOL-ID

1631

DQ49

4677

736

8847

3/50

7(93%

)

Umbelopsissp.I

Quebec

Betula

ARSL23

0507

.15/UAMH

1112

5Umbelopsisisab

ellin

aFJ872

076

688

9939

2/40

1(97%

)

Quebec

Abies

ARSL17

0507

.25II

Umbelopsisisab

ellin

aisolateODHO4

EU81

6388

754

9343

4/44

8(96%

)

Quebec

Picea

ARSL18

0507

.11/UAMH

1120

5Umbelopsisisab

ellin

aAJ876

493

852

9958

9/64

6(91%

)

Umbelopsissp.II

Quebec

Betula

ARSL23

0507

.20(c)/UAMH

1119

4Umbelopsisraman

nian

aDQ88

8724

1011

8658

7/60

(97%

)

G. Kernaghan, G. Patriquin

10

Ascocalyx abietina (FJ746661) Godronia sp. DAOM 233257 (EF672237)

Gremmeniella laricina (GLU72262) ARSL 230507.35/UAMH 11201 Q B

Uncultured Pezizomycotina clone (FJ554013)ARSL190907.38/UAMH 11169 F

Uncultured Helotiales clone (FJ475664)ARSL 060907.20 CB BARSL 180907.11 CB SARSL 180907.23 CB SARSL 180907.34 CB SARSL 180907.19 CBS

ARSL 190907.24 CB FARSL 170507.37II Q FARSL 70907.34 CB SARSL 170507.43I Q FUncultured fungus clone (EF433994) ARSL 220507.53 Q SARSL 220507.16I Q SARSL 220507.58 Q SARSL 180507.12/UAMH 11170 Q S

ARSL 70907.35/UAMH 11202 CB SUncultured Leotiomycetes clone (FJ152529)ARSL 190907.75 CB FARSL 190907.55/UAMH 11171 CB FARSL 190907.41 CB FARSL 190907.54 CB F

Uncultured Leotiomycetes (AY394893)ARSL 170507.50 Q FARSL 170507.46 Q FARSL 190907.66 CB FARSL 190907.2 CB FARSL 070907.9/UAMH 11172 CB SARSL190907.35 CB FARSL 190907.71 CB FARSL 190907.19I CB F

ARSL 190907.15/UAMH 11168 CB F Rhizoscyphus ericae (AY762622)ARSL 180907.22 CB SARSL 190907.74/UAMH 11175 CB FMeliniomyces bicolor (AY394885)

ARSL 170507.36 Q FARSL 070907.13 CB SARSL 070907.12/UAMH 11204 CB SARSL 250507.3 Q B

ARSL 230507.30II Q BMeliniomyces vraolstadiae strain T G1 (AJ292199)ARSL 170507.42I Q FARSL 230507.6 Q B

Meliniomyces vraolstadiae strain G2 (AJ292200) ARSL 170507.42II Q FARSL 230507.46 Q BARSL 060907.18II CB BARSL 60907.26/UAMH 11128 CB BARSL60907.27 CB BARSL 170507.25I Q FARSL 060907.1 CB BARSL 70907.4 CB SARSL 230507.7 Q BARSL 70907.19 CB S

ARSL 180907.39 CB SARSL 230507.30I Q BMeliniomyces variabilis (EF093173) ARSL 220507.11 Q SARSL 220507.2 Q SARSL 220507.4I Q SARSL 70907.15/UAMH 11129 CB SARSL 190907.72 CB FARSL 60907.24 CB BARSL 190907.5 CB FARSL 190907.17 CB FARSL 190907.8 CB F

Helotiaceae sp. III

Helotiaceae sp. IV

Helotiaceae sp. II

Helotiaceae sp. I

Helotiaceae sp. V

Meliniomyces bicolor

Meliniomyces vraolstadiae

Meliniomyces sp.

Meliniomyces variabilis

Helotiaceae sp. VI100

100

78

93

99

90

72

93

96

100

100

99

84

91

94

100

90

Helotiaceae sp. VII

Rhizoscyphus ericae aggregate

Figure 1 One of 100 most parsimonious midpoint rooted treescomparing ITS sequences of cultured root endophytes within theHelotiaceae with GenBank sequences (in bold). Consistency index=0.720, retention index=0.956, and tree length=408. Clades, which

contain reference sequences from uncultured environmental samplesonly, were given operational names (Helotiaceae sp. I–VII). Bootstrapvalues>70% are shown. Scale bar=10 substitutions

Host Associations Between Root Endophytes and Boreal Trees

give an overall total of 31 distinct phylotypes across bothsites (Figs. 1, 2, 3, and 4, Table 1). For the Cape Bretonsite, the 71 morphological groups (including 54 singletonisolates) were reduced to 23 phylotypes (including sevensingletons). For the Québec site, the original 74 groups(including 50 singletons) were reduced to 19 phylotypes(with five singletons). For individual host trees, phylotyperichness was highest on Abies with S=26, followed byPicea and Betula, both with S=15.

Although species accumulation curves (not shown)indicated that further sampling would have detected moreendophyte phylotypes, comparisons of our observed rich-ness values with bootstrap estimated richness valuesindicates 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 impacton our conclusions regarding the host associations of themore common phylotypes.

The overall Shannon diversity index for root endo-phytes was somewhat higher for the Cape Breton site(H′=2.58) than for the Québec site (H′=2.21). Forindividual host trees, the highest endophyte diversity wason Abies (H′=2.66), followed by Betula (H′=2.22) andthen Picea (H′=1.74), but there were no significantdifferences (P>0.05) in diversity among hosts or amongthe 15 host–site combinations.

Host trees also varied in the degree of overlap in endophytephylotypes, with Picea and Abies sharing 14, Betula andAbies sharing 11, and Betula and Picea sharing nine. Twelvephylotypes were isolated only from the Cape Breton site,

1

Phialocephala sphaeroides (AY524844)ARSL 070907.7/UAMH11132 CB SARSL 220507.6II Q SARSL 60907.65 CB BARSL 230507.33 Q BARSL 230507.36 Q B

ARSL 170507.56 Q FARSL 230507.57 Q B

ARSL 190907.49I/UAMH 11207 CB FARSL 190907.50 CB F

Acephala applanata (AY078147)Phialocephala helvetica (AY347408)

Phialocephala turiciensis (AY347389)ARSL 220507.12 Q SARSL 190907.7 CB FARSL 190907.9 CB FARSL 070907.28 CB SARSL 220507.18 Q SARSL 180907.1 CB SARSL 190907.6 CB FARSL 070907.31 CB SARSL 190907.20 CB FARSL 070907.21 CB SARSL 180907.2 CB S

Phialocephala fortinii (AY664502)ARSL 250507.1 Q BARSL 220507.49 Q SARSL 070907.20 CB SARSL 070907.39 CB SARSL 190907.33/UAMH 11197 CB FARSL 070907.26 CB S

ARSL 220507.35 Q SARSL 220507.22 Q SPhialocephala letzii (AY347396)

Phialocephala europaea (AY347403)ARSL 230507.43 Q BARSL 220507.55 Q S

ARSL 180507.5 Q SARSL 220507.43 Q SARSL 180507.2 CB SARSL 180507.18 Q S

ARSL 060907.60 CB BARSL 060907.18I CB BARSL 170507.11 Q FARSL 190907.53/UAMH 11131 CB F

Neofabraea alba (AF141190)Pezicula sporulosa (AF141172)

Cryptosporiopsis ericae (AY853167)ARSL 190907.12/UAMH 11126 CB FARSL 190907.56 CB F

Dermea hamamelidis (AF141157)ARSL 170507.22/UAMH 11127 Q FARSL 170507.49 Q FARSL 190907.51 CB F

100

100

89

84

86

100

100

93

9895

93

Cryptosporiopsis ericae

Cryptosporiopsis sp.

Dermataceae sp. I

Phialocephala fortinii complex

Phialocephala sp.

Phialocephala sphaeroides

Figure 2 One of 100 most parsimonious midpoint rooted treescomparing ITS sequences of cultured root endophytes within theDermateaceae with GenBank sequences (in bold). Consistency

index=0.797, retention index=0.962, and tree length=276. Boot-strap values >70% are shown. Scale bar=1 substitution

G. Kernaghan, G. Patriquin

eight phylotypes were isolated only from the Québec site,and 11 phylotypes were isolated from both sites, giving a35% overlap between sites. Overlap between sites increases to58% if singletons are disregarded. Results from the ANOSIM,which takes the relative proportions of phylotypes on eachhost–site combination into account (Table 2), indicate thatthere are no significant differences (α=.05) between the rootendophyte communities of host trees of the same speciesacross sites. Furthermore, within the Cape Breton site, theroot endophyte communities are significantly different amongall three tree species. However, on the Québec site, endophytecommunities are not significantly different between Abies and

Picea and between Betula and Picea. Differences betweendifferent host tree species across sites are mainly significant,with the exception of Cape Breton Picea vs Québec Abiesand Cape Breton Picea vs Québec Betula.

The DCA also indicates differences in endophyteassemblages colonizing the three host tree species(Fig. 6a, b), as the hosts fall into relatively distinctivegroupings regardless of site. The first and second axes ofthe ordination explain a total of 23.6% of the variation inthe data (14.5% and 9.1%, respectively; γ1=0.612, γ2=0.382, total inertia=4.205). In Fig. 6a, site scores (hosttrees) are mainly separated along the first axis, with most of

Figure 3 One of 54most parsimoniousmidpoint rooted trees comparingITS sequences of cultured root endophytes within the Myxotricaceaewith 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

Host Associations Between Root Endophytes and Boreal Trees

the Abies toward the left of the diagram, most of the Betulatoward the right (although some are toward the bottom left),and all of the Picea occupying a central position.

Abies was colonized by Cryptosporiopsis ericae, Cryp-tosporiopsis sp., Helotiaceae sp. V, and Helotiaceae sp. VIto a greater extent than Betula and Picea (Fig. 5). Thesefungi fall mainly on the left side of the ordination (Fig. 6b).Picea forms a distinct group, delineated from the otherhosts by the frequency of Helotiaceace III and P. fortinii s.l.(Figs. 5 and 6b). The endophyte assemblages of Betula aremore variable, with the Québec Betula characterized by O.maius and Phialocephala sphaeroides. The two CapeBreton Betula trees toward the bottom left of the diagramare separated from the others mainly by Meliniomyces sp.(Figs. 5 and 6b).

Host Associations

The fungal root endophytes detected fell into four generalcategories with respect to host associations. The firstcategory included infrequent phylotypes (<1% overallisolation frequency) for which there was not enough datato make inferences as to their distributional patterns. Theseincluded the 12 singletons, Chaetospheria sp., Helotiaceaesp. I, II, IV, and VII, Hyaloscyphaceae sp. I–IV, Hypocreapachybasioides, 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 fromonly one of the two sites (Fig. 5).

The second category included those relatively commonphylotypes, which appear to lack host preference, i.e.Meliniomyces variabilis, Meliniomyces vraolstadiae, Meli-niomyces sp., and Dermateaceae sp. I (Fig. 5). Thesephylotypes have relatively large niche breadth indices (BA

from 0.488 to 1) and host distributions not significantlydifferent from expected based on goodness of fit (Table 3).

The third group included phylotypes which appeared toexhibit host preference on one site, but were absent or atlow frequency on the other site. These included C. ericae,Cryptosporiopsis sp., and Helotiaceae sp. V that alloccurred only on Abies where detected, as well as P.sphaeroides and O. maius, that both occurred mainly onBetula on the Québec site (Fig. 5). Phylotypes in thissecond group had relatively small niche breadth indices (BA

from 0 to 0.372), and their host distributions weresignificantly different from expected (Table 3).

The final group consisted of the phylotypes that werecommon (at least eight isolates per site) and that appeared toexhibit preference for a particular host. These includedHelotiaceae sp. III, Helotiaceae sp. VI, and P. fortinii s.l.(Fig. 5). These three phylotypes had significant goodness offit test results and were widely distributed across individuals

1

Hyaloscypha daedaleae (AY789416)

Axenic ericoid root isolate (AJ430215)

Lachnellula calyciformis (U59145)

Lachnum bicolor (AJ430394)

Cistella acuum (U57492)

ARSL 230507.52/UAMH 11166 Q B

ARSL 170507.13/UAMH 11200 Q F

ARSL 190907.62/UAMH 11165 CB F

ARSL 180907.20/UAMH 11167 CB S

Uncultured fungus isolate RFLP67 (AF461628)

Hyphodiscus hymeniophilus (DQ227264)

Hyaloscyphaceae sp. I

Hyaloscyphaceae sp. II

Hyaloscyphaceae sp. III

Hyaloscyphaceae sp. IV

100

97

87

72

85

Figure 4 Midpoint rooted par-simony tree comparing ITSsequences of cultured rootendophytes within the Hyalo-scyphaceae with GenBanksequences (in bold). Consisten-cy index=0.769, retention index=0.629, and tree length=283.Bootstrap values >70% areshown. Scale bar=1 substitution

G. Kernaghan, G. Patriquin

of their preferred host (Table 3, Fig. 5). Helotiaceae sp. IIIand VI each had small niche breadth indices (0.161 and0.155, respectively), while P. fortinii s.l. was broader at0.635 (Table 3).

Discussion

Our results demonstrate that species assemblages of fungalroot endophytes of boreal trees differ from host to host. Wehave also shown that the differences in root endophytesacross hosts are not due to edaphic or micro-climacticconditions, as these were controlled for by sampling fromsmall plots containing intertwined roots of the different host

species. Although many of the phylotypes detected occurredat frequencies too low to allow for inferences about theirdistributional patterns, several were relatively common, anda proportion of these appear to exhibit distinct associationswith particular hosts. Of course, these distributional patternspertain only to the three host species sampled and only toCenococcum ectomycorrhizae on those hosts. We cannotextrapolate to other host species.

The most commonly encountered root endophyte was P.fortinii s.l. It was most commonly isolated from Picea, leastcommon on Betula at the Quebec site, and absent fromBetula on the Cape Breton site. However, P. fortinii s.l. issomewhat problematic in the context of host associations,as recent genetic studies divide European isolates intoseveral cryptic species, some of which are not distinguish-able on the basis of ITS sequence analysis [22]. As it isvery likely that cryptic species of P. fortinii also exist inNorth America, and each may display its own hostpreference, the distribution of P. fortinii seen in the currentstudy likely represents an overall pattern of a group ofclosely related fungal endophytes.

Conversely, P. sphaeroides was most common on Betulaon one of our sites. P. sphaeroides was originally isolatedfrom 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 thecurrent study, more sites and more host species wouldundoubtedly reveal broader host ranges.

Hel

otia

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VI

Hel

otia

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25

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50

Phi

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ena

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nd nd nd nd nd nd nd nd

Figure 5 Number of root tipscolonized by fungal root endo-phytes on each tree species. Thefirst and second bars for eachphylotype represent the numberof isolations from the CapeBreton and Québec sites, re-spectively. Black, Abies; white,Betula; grey, Picea, nd, notdetected. Twelve singletons notshown

Table 2 Results of ANOSIM test (p values) comparing endophyteassemblages on each tree species at each site

Cape Breton Quebec

Betula Picea Abies Betula Picea

Cape Breton Abies 0.032 0.02 0.085 0.031 0.021

Betula 0.033 0.037 0.087 0.027

Picea 0.365 0.09 0.305

Quebec Abies 0.044 0.198

Betula 0.124

Values in bold are significant (p<0.05). Results of comparisonsbetween trees of the same species on different sites are in italics

Host Associations Between Root Endophytes and Boreal Trees

The most obvious host–endophyte associations wereseen within the Helotiaceae, specifically among Helotiaceaesp. III, V, and VI, for either Picea or Abies. However, thesefungi remain unidentified, other than that they appear to be

a group of species close to, but not part of the Rhizoscypusericae aggregate [25, 70].

Both C. ericae and Cryptosporiopsis sp. were isolatedsolely from the roots of Abies, although each was found on

-1 6

-14

CB

CB

CB

CB

CB

CB

CB CB

CB

CB

CB

CB

Q

QQ

Q

Q

Q

Q

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-1 7

-17

Dermataceae sp. I

Lecythophora mutabilis

Penicillium montenese Umbelopsis sp. I

Oidiodendron sp.

Oidiodendron maius

Meliniomyces sp.

Phialocephala sp.

Helotiaceae sp. III

Helotiaceae sp. V

Meliniomyces bicolor

Cryptosporiopsis ericae

Phiacephala fortinii s.l.

Phialocephala sphaeroides

Meliniomyces vraolstadiae

Mycena sp.

Helotiaceae sp. VI

Cryptosporiopsis sp.

Meliomyces variabilis

a

b

Figure 6 a, b Detrended corre-spondence analysis (DCA)depicting relationships amonghost trees and sites on the basisof fungal root endophyte colo-nization. Site scores (a) areseparated from species scores(b) for clarity. Twelve singletonsnot shown (b). In Fig 6a, CB,Cape Breton; Q, Québec; blacktriangles, Abies; white triangles,Betula; grey triangles, Picea

G. Kernaghan, G. Patriquin

only one site. C. ericae was originally described fromericaceous roots [60] and has since also been isolated fromPopulus roots [72].

Similarly, O. maius was predominantly isolated fromBetula on one of our sites, although other authors havefound it to be relatively common on the roots of Picea [56]and Pinus [45]. The ecological niche of O. maius appearsvery wide, as it also forms mycorrhizae on ericaceousplants [13] and grows saprophytically on sphagnum [52].The other, as of yet unidentified, species of Oidiodendrondid not exhibit host specificity.

All four phylotypes referable to Meliniomyces (M.bicolor, M. varabilis, M. vraolstadiae, and Meliniomycessp.) were fairly evenly distributed across hosts. The genusMeliniomyces is composed of sterile, root-associated spe-cies with the potential to colonize a wide range of plants[25]. For example, M. vraolstadiae forms ectomycorrhizeon Betula, Picea, and Pinus [71], Meliniomyces varabilis iscapable of forming ericoid mycorrhizae on ericaceous hosts[49], and M. bicolor is reported to form either type ofmycorrhizae, depending on the host plant colonized [21].Therefore, in the case of M. bicolor, it is possible that ourisolates may have been acting as endophytes within C.

geophilum (the mycorrhizal fungus colonizing all rootssampled), or they may have themselves been involved inconcomitant ectomycorrhizal associations with Cenococ-cum (dual ectomycorrhizal colonization).

We used a culture based approach (from surface-sterilized root tips) followed by PCR, rather than directamplification of fungal DNA from root tips. Although werecognize that our method does not detect unculturableendophytes, we felt it preferable to use direct PCR for ourobjective, in that we can be certain that our isolatesrepresent fungi colonizing the root tips internally, asopposed to those residing on the root surface [27]. DirectPCR does not discern between these two groups of fungiand may detect non-host specific soil fungi on the rootsurface, perhaps confounding our data on host associations.Even with surface sterilization by peroxide or hypochlorite(bleach), the DNA of these superficial fungi may still bedetected by PCR; the fungi may be killed, but amplifyableDNA may remain [34]. Our cultural approach also avoidsthe problem of concurrent colonization of root tips byectomycorrhizal (ECM) fungi, the DNA of which wouldlikely swamp the endophyte DNA. Amplification of ECMfungal DNA can be avoided by using ascomycete-

Table 3 Results of randomization tests for goodness of fit, standardized niche breadth indices (BA), percentage of individual trees of each speciescolonized, 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 Betula Abies Picea

Phialocephala fortinii s.l. <0.0001 <0.0001 0.0209 0.635 50 75 87.5 Picea

Helotiaceae sp. VI <0.0001 0.0003 0.0003 0.155 0 75 25 Abies

Meliniomyces variabilis 0.4700 0.1776 0.1597 0.899 37.5 37.5 37.5 nd

Helotiaceae sp. III <0.0001 0.0016 0.0600 0.161 12.5 37.5 87.5 Picea

Oidiodendron maius 0.0117 1 0.0001 0.372 62.5 12.5 25 Betula

Phialocephala sphaeroides 0.0129 0.3354 0.0083 0.337 37.5 12.5 25 Betula

Meliniomyces sp. 0.0476 0.0530 0.7736 0.488 37.5 25 12.5 Betula

Meliniomyces vraolstadiae 0.7420 0.1132 0.1407 0.954 37.5 25 12.5 nd

Helotiaceae sp. V 0.0035 0.0037 nd 0 0 25 0 Abies

Oidiodendron sp. 0.1282 0.1148 0.7750 0.461 0 37.5 25 nd

Cryptosporiopsis ericae 0.0035 0.0031 nd 0 0 37.5 0 Abies

Cryptosporiopsis sp. 0.0361 nd 0.0367 0 0 25 0 Abies

Dermateaceae sp. I 0.5640 0.7810 1 0.5 25 25 0 nd

Penicillium montanense 1 nd 1 1 12.5 12.5 12.5 nd

Phialocephala sp. 0.7805 0.1148 1 0 0 12.5 12.5 nd

Umbelopsis sp. I 1 nd 1 0.4 12.5 12.5 12.5 nd

Lecythophora mutabilis 0.3312 0.3428 nd 0.5 12.5 12.5 0 nd

Meliniomyces bicolor 0.3295 0.3355 nd 0.5 0 12.5 12.5 nd

Mycena sp. 0.1135 0.1148 nd 0 0 12.5 0 nd

p values in bold are significant (p<0.05). Phylotypes are listed from most to least commonly isolated. Twelve singletons not analyzed. A preferredhost was assigned only when the result of the goodness of fit test was significant

nd not detected

Host Associations Between Root Endophytes and Boreal Trees

specific PCR primers to amplify endophytes withinbasidiomycetous ECM [64], but this approach does notdetect basidiomycete endophytes and cannot be used forascomycetous ECM such as those sampled here. Althoughisolation of pure cultures from surface sterilized rootstends to detect fewer fungal species than direct PCR [4, 9],each approach appears to have its own biases. When bothmethods were used to detect root associated fungi ofconifer seedlings [45], P. fortinii and Oidiodendron werefrequently isolated from surface sterilized mycorrhizae,but were rarely (or never) detected by direct PCR.

The patterns of host preference displayed by some of theendophytes in the current study are characteristic ofbiotrophic [33] or mutualistic [57], rather than necrotrophic,relationships. Saprophytes tend to exhibit substrate speci-ficity rather than host specificity [10], and biotrophic fungi(including biotrophic mutualists such as the mycorrhizalfungi) exhibit greater host specificity than necrotrophs [8,40]. Patterns of host specificity in the ectomycorrhizal(ECM) fungi are fairly well understood; although somehosts such as Alnus and Larix support very specific ECMmycobionts [46, 65], studies of mixed conifer stands [11,30] found that commonly occurring ECM fungi lackedspecificity, and only a few uncommon species were hostspecific. In a study of boreal forest ECM (conducted in thesame research forest as our current Québec site), the mostcommon ECM fungi were generalists, less common fungioften preferred particular hosts, and some uncommonspecies exhibited apparent host specificity [36]. Althoughwe recognize that rarity and specificity are interrelated,due to uncommon species occurring on fewer hosts bychance alone, the general pattern of host preferencedescribed for ECM fungi is also evident in our rootendophyte data.

Root endophytic fungi and ECM fungi differ, however,in that endophyte species likely vary greatly in theirrelationship with the plant host, making it difficult topredict the ramifications of root endophyte host preference.Some root endophytes may be latent pathogens, causingdisease symptoms in weakened or damaged roots [59],while many others are beneficial, improving plant growth[58], defending from disease [47], improving droughttolerance [6], or mineralizing organic nutrient sources[67]. Therefore, the asymmetric distributions of the fungalroot endophytes detected on our sites may potentiallyinfluence interspecific plant competition.

Acknowledgements This work was made possible by a grantfrom the Natural Sciences and Engineering Research Council ofCanada (341671-2007). We thank Emily Cormier and Erica Fraserfor technical assistance, Cape Breton Highlands National Park andthe Lac Duparquet Teaching and Research Forest for fieldLogistics, and Lynne Sigler for comments on an earlier versionof the manuscript.

References

1. Addy HD, Hambleton S, Currah RS (2000) Distribution andmolecular characterization of the root endophyte Phialocephalafortinii along an environmental gradient in the boreal forest ofAlberta. Mycol Res 104:1213–1221

2. Addy HD, Piercey MM, Currah RS (2005) Microfungal endo-phytes in roots. Can J Bot 83:1–13

3. Ahlich K, Sieber TN (1996) The profusion of dark septateendophytic fungi in non-ectomycorrhizal fine roots of forest treesand shrubs. New Phytol 132:259–270

4. Allen T, Millar T, Berch S, Berbee M (2003) Culturing and directDNA extraction find different fungi from the same ericoidmycorrhizal roots. New Phytol 160:255–272

5. Arnold AE, Henk DA, Eells RL, Lutzoni F, Vilgalys R (2007)Diversity and phylogenetic affinities of foliar fungal endophytesin loblolly pine inferred by culturing and environmental PCR.Mycologia 99:185–206

6. Barrow J, Osuna-Avila P, Reyes-Vera I (2004) Fungal endophytesintrinsically associated with micropropagated plants regeneratedfrom native Bouteloua eriopoda torr. and Atriplex canescens(pursh). Nutt In Vitro Cell Dev-Pl 40:608–612

7. Belleau A, Brais S, Pare D (2006) Soil nutrient dynamics afterharvesting and slash treatments in boreal aspen stands. Soil SciSoc Am J 70:1189–1199

8. Borowicz VA, Juliano SA (1991) Specificity in host–fungusassociations: do mutualists differ from antagonists? Evol Ecol5:385–392

9. Bougoure DS, Cairney JWG (2005) Assemblages of ericoidmycorrhizal and other root-associated fungi from Epacris pul-chella (Ericaceae) as determined by culturing and direct DNAextraction from roots. Environ Microbiol 7:819–827

10. Cooke RC, Whipps JM (1980) The evolution of modes ofnutrition in fungi parasitic on terrestrial plants. Biol Rev55:341–362

11. Cullings KW, Vogler D, Parker V, Finley S (2000) Ectomycor-rhizal specificity patterns in a mixed Pinus contorta and Piceaengelmannii forest in Yellowstone National Park. Appl EnvironMicrobiol 66:4988–4991

12. Davet P, Rouxel F (2000) Detection and isolation of soil fungi.Science, New Hampshire, p 188

13. Douglas GC, Heslin MC, Reid C (1989) Isolation of Oidioden-dron maius from Rhododendron and ultrastructural characteriza-tion of synthesized mycorrhizas. Can J Bot 67:2206–2212

14. DeBellis T, Kernaghan G, Widden P (2007) Plant communityinfluences on soil microfungal assemblages in boreal-mixed woodforests. Mycologia 99:356–367

15. Edgar RC (2004) MUSCLE: multiple sequence alignment withhigh accuracy and high throughput. Nucleic Acids Res 32:1792–1797

16. Fisher PJ, Petrini O, Petrini LE (1991) Endophytic ascomycetesand deuteromycetes in roots of Pinus sylvestris. Nova Hedwig52:11–15

17. Garbelotto M, Ratcliff A, Bruns TD, Cobb FW, Otrosina WJ(1995) Use of taxon specific competitive priming PCR to studyhost specificity, hybridization, and intergroup gene flow. Phyto-pathology 86:543–551

18. Gardes M, Bruns TD (1993) ITS primers with enhancedspecificity for basidiomycetes: application to the identification ofmycorrhizae and rusts. Mol Ecol 2:113–118

19. Girlanda M, Luppi-Mosca AM (1995) Microfungi associated withectomycorrhizae of Pinus halepensis Mill. Allionia 33:93–98

20. Girlanda M, Ghignone S, Luppi AM (2002) Diversity of sterileroot-associated fungi of two Mediterranean plants. New Phytol155:481–498

G. Kernaghan, G. Patriquin

21. Grelet GA, Johnson D, Vrålstad T, Alexander IJ, Anderson IC(2010) New insights into the mycorrhizal Rhizoscyphus ericaeaggregate: spatial structure and co-colonization of ectomycorrhizaland ericoid roots. New Phytol 188:210–222

22. Grünig CR, Duo A, Sieber TN, Holdenrieder O (2008) Assign-ment of species rank to six reproductively isolated cryptic speciesof the Phialocephala fortinii s.l.-Acephala applanata speciescomplex. Mycologia 100:47–67

23. Grünig CR, McDonald BA, Sieber TN, Rogers SO, HoldenriederO (2004) Evidence for subdivision of the root-endophytePhialocephala fortinii into cryptic species and recombinationwithin species. Fungal Genet Biol 41:676–687

24. Grünig CR, Queloz V, Sieber TN, Holdenrieder O (2008) Darkseptate endophytes (DSE) of the Phialocephala fortinii s.l.–Acephala applanata species complex in tree roots: classification,population biology, and ecology. Botany 86:1355–1369

25. Hambleton S, Sigler L (2005) Meliniomyces, a new anamorphgenus for root-associated fungi with phylogenetic affinities toRhizoscyphus ericae (≡ Hymenoscyphus ericae), Leotiomycetes.Stud Mycol 53:1–27

26. Hall TA (1999) BioEdit: a user-friendly biological sequencealignment editor and analysis program for Windows 95/98/NT.Nucl Acid S Ser 41:95–98

27. Johannes H, Berg G, Schulz B (2006) Isolation procedures forendophytic microorganisms. In: Schulz B, Boyle C, Sieber TN(eds) Microbial root endophytes. Springer, Berlin, pp 299–319

28. Hawksworth DL (2001) The magnitude of fungal diversity: the1.5 million species estimate revisited. Mycol Res 105:1422–1432

29. Hoff JA, Klopfenstein NB, McDonald GI, Tonn JR, Kim MS,Zambino PJ, Hessburg PF, Rogers JD, Peever TL, Carris LM(2004) Fungal endophytes in woody roots of Douglas-fir(Pseudotsuga menziesii) and ponderosa pine (Pinus ponderosa).Forest Pathol 34:255–271

30. Horton T, Bruns T (1998) Multiple-host fungi are the mostfrequent and abundant ectomycorrhizal types in a mixed stand ofDouglas fir (Pseudotsuga menziesii) and bishop pine (Pinusmuricata). New Phytol 139:331–339

31. Higgins KL, Arnold AE, Miadlikowska J, Sarvate SD, Lutzoni F(2006) Phylogenetic relationships, host affinity, and geographicstructure of boreal and arctic endophytes from three major plantlineages. Mol Phylogenet Evol 42:543–555

32. Hurlbert SH (1978) The measurement of niche overlap and somerelatives. Ecology 59:67–77

33. Jumpponen A, Trappe JM (1998) Dark septate endophytes: areview of facultative biotrophic root-colonizing fungi. New Phytol140:295–310

34. Kemp BM, Smith DG (2005) Use of bleach to eliminatecontaminating DNA from the surfaces of bones and teeth.Forensic Sci Int 154:53–61

35. Kernaghan G, Sigler L, Khasa D (2003) Mycorrhizal and rootendophytic fungi of containerized Picea glauca seedlings assessedby rDNA sequence analysis. Microb Ecol 45:128–136

36. Kernaghan G, Widden P, Bergeron Y, Légaré S, Paré D (2003)Biotic and abiotic factors affecting ectomycorrhizal diversity inboreal mixed-woods. Oikos 102:497–505

37. Krebbs CJ (1999) Ecological methodology. Benjamin/Cummings,Menlo Park, p 620

38. Kwaśna H, Bateman GL, Ward E (2008) Determining speciesdiversity of microfungal communities in forest tree roots by pure-culture isolation and DNA sequencing. Appl Soil Ecol 40:44–56

39. Lacap DC, Hyde KD, Liew ECY (2003) An evaluation of thefungal ‘morphotype’ concept based on ribosomal DNA sequences.Fungal Divers 12:53–66

40. Lucas JA (1998) Plant pathology and plant pathogens. Blackwell,Oxford, p 274

41. Mandyam K, Jumpponen A (2005) Seeking the elusive functionof the root-colonising dark septate endophytic fungi. Stud Mycol53:173–189

42. McCune B, Mefford MJ (1999) Multivariate analysis of ecolog-ical data, version 4.26. MjM Software Design, Oregon

43. McDonald JH (2009) Handbook of biological statistics, 2nd edn.Sparky House, Baltimore, 293 pp

44. Menkis A, Allmer J, Vasiliauskas R, Lygis V, Stenlid J, Finlay R(2004) Ecology and molecular characterization of dark septatefungi from roots, living stems, coarse and fine woody debris.Mycol Res 108:965–973

45. Menkis A, Vasiliauskas R, Taylor A, Stenlid J, Finlay R (2005)Fungal communities in mycorrhizal roots of conifer seedlings inforest nurseries under different cultivation systems, assessed bymorphotyping, direct sequencing and mycelial isolation. Mycor-rhiza 16:33–41

46. Molina R, Massicotte H, Trappe J (1992) Specificity phenomenain mycorrhizal symbioses: community ecological consequencesand practical implications. In: Allen MF (ed) Mycorrhizalfunctioning: an integrated plant-fungal process. Chapman & Hall,New York, pp 357–423

47. Narisawa K, Usuki F, Hashiba T (2004) Control of Verticilliumyellows in Chinese cabbage by the dark septate endophytic fungusLtVB3. Phytopathology 94:412–418

48. Nilsson RH, Kristiansson E, Ryberg M, Hallenberg N, Larsson K(2008) Intraspecific ITS variability in the kingdom fungi asexpressed in the international sequence databases and its implica-tions for molecular species identification. Evol Bioinform 4:193–201

49. Ohtaka N, Narisawa K (2008) Molecular characterization andendophytic nature of the root-associated fungus Meliniomycesvariabilis (LtVB3). J Gen Plant Pathol 74:24–31

50. Petrini O (1996) Ecological and physiological aspects of host-specificity in endophytic fungi. In: Redlin SC, Carris LM (eds)Endophytic fungi in grasses and woody plants: systematics,ecology, and evolution. APS, Minnisota, pp 87–100

51. Queloz V, Grünig CR, Sieber TN, Holdenrieder O (2005)Monitoring the spatial and temporal dynamics of a communityof the tree-root endophyte Phialocephala fortinii sl. New Phytol168:651–660

52. Rice AV, Currah RS (2006) Oidiodendron maius: Saprobe insphagnum peat, mutualist in ericaceous roots. In: Schulz B, BoyleC, Sieber T (eds) Microbial root endophytes. Springer, Berlin, pp227–246

53. Richter DL (2008) Revival of saprotrophic and mycorrhizalbasidiomycete cultures after twenty years in cold storage in sterilewater. C J Microbiol 54:595–599

54. Rodriguez RJ, White JF, Arnold AE, Redman RS (2009) Fungalendophytes: diversity and functional roles. New Phytol 182:314–330

55. Sanders IR (2003) Preference, specificity and cheating in thearbuscular mycorrhizal symbiosis. Trends Plant Sci 8:143–145

56. Schild DE, Kennedy A, Stuart MR (1988) Isolation of symbiontand associated fungi from ectomycorrhizas of Sitka spruce. Eur JForest Pathol 18:51–61

57. Schulz B, Boyle C (2006) Mutualistic interactions with fungalroot endophytes. In: Schulz B, Boyle C, Sieber TN (eds)Microbial root endophytes. Springer, Berlin, pp 261–279

58. Schulz B, Boyle C, Draeger S, Römmert AK, Krohn K (2002)Endophytic fungi: a source of novel biologically active secondarymetabolites. Mycol Res 106:996–1004

59. Schulz B, Rommert AK, Dammann U, Aust HJ, Strack D (1999)The endophyte–host interaction: a balanced antagonism? MycolRes 103:1275–1283

60. Sigler L, Allan T, Sea Ra L, Berbee M, Berch S (2005) Two newCryptosporiopsis species from roots of ericaceous hosts in westernNorth America. Stud Mycol 53:53–62

Host Associations Between Root Endophytes and Boreal Trees

61. Smith EP, Van Belle G (1984) Nonparametric estimation ofspecies richness. Biometrics 40:119–129

62. Summerbell RC (2005) Root endophyte and mycorrhizospherefungi of black spruce, Picea mariana, in a boreal forest habitat:influence of site factors on fungal distributions. Stud Mycol53:121–145

63. Swofford DL (2002) PAUP*. Phylogenetic analysis using parsi-mony (*and related methods). Version 4.10b. Sinauer, Sunderland

64. Tedersoo L, Pärtel K, Jairus T, Gates G, Põldmaa K, Tamm H(2009) Ascomycetes associated with ectomycorrhizas: moleculardiversity and ecology with particular reference to the Helotiales.Environ Microbiol 11:3166–3178

65. Tedersoo L, Suvi T, Jairus T, Ostonen I, Põlme S (2009)Revisiting ectomycorrhizal fungi of the genus Alnus: differentialhost specificity, diversity and determinants of the fungal commu-nity. New Phytol 182:727–735

66. ter Braak CJF, Smilauer P (2002) CANOCO 4.5 reference manualand CanoDraw for Windows. User’s guide to Canoco forWindows: software for canonical community ordination. Micro-computer Power, New York, p 499

67. Upson R, Newsham KK, Bridge PD, Pearce DA, Read DJ (2009)Taxonomic affinities of dark septate root endophytes of Coloban-thus quitensis and Deschampsia antarctica, the two nativeAntarctic vascular plant species. Fungal Ecol 2:184–196

68. Varma A, Verma S, Sudha, Sahay N, Bütehorn B, Franken P(1999) Piriformospora indica, a cultivable plant-growth-promoting root endophyte. Appl Environ Microb 65:2741–2744

69. Verkley GJM, Zijlstra JD, Summerbell RC, Berendse F (2003)Phylogeny and taxonomy of root-inhabiting Cryptosporiopsisspecies, and C. rhizophila sp. nov., a fungus inhabiting roots ofseveral Ericaceae. Mycol Res 107:689–698

70. Vrålstad T, Myhre E, Schumacher T (2002) Molecular diversityand phylogenetic affinities of symbiotic root-associated ascomy-cetes of the Helotiales in burnt and metal polluted habitats. NewPhytol 155:131–148

71. Vrålstad T, Schumacher T, Taylor AFS (2002) Mycorrhizalsynthesis between fungal strains of the Hymenoscyphus aggregateand potential ectomycorrhizal and ericoid hosts. New Phytol153:143–152

72. Wang W, Tsuneda A, Gibas C, Currah RS (2007) Cryptospor-iopsis species isolated from the roots of aspen in central Alberta:identification, morphology and interactions with the host, in vitro.Can J Bot 85:1214–1226

73. Weishampel P, Bedford B (2006) Wetland dicots and monocotsdiffer in colonization by arbuscular mycorrhizal fungi and darkseptate endophytes. Mycorrhiza 16:495–502

74. White TJ, Bruns T, Lee S, Taylor J (1990) Amplification anddirect sequencing of fungal ribosomal RNA genes for phyloge-netics. In: Innis MA, Gelfand DH, Sninsky JJ, White TJ (eds)PCR protocols: a guide to methods and applications. Academic,New York, pp 315–322

75. Wilson D (1995) Endophyte—the evolution of a term, andclarification of its use and definition. Oikos 73:274–276

76. Wilson BJ, Addy HD, Tsuneda A, Hambleton S, Currah RS(2004) Phialocephala sphaeroides sp nov., a new species amongthe dark septate endophytes from a boreal wetland in Canada. CanJ Bot 82:607–617

77. Wilcox HE, Wang CJK (1987) Mycorrhizal and pathologicalassociations of dematiaceous fungi in roots of 7-month-old treeseedlings. Can J Forest Res 17:884–889

78. Zhou D, Hyde KD (2001) Host-specificity, host-exclusivity, andhost-recurrence in saprobic fungi. Mycol Res 105:1449–1457

G. Kernaghan, G. Patriquin