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The cultivable endophytic community of Norway spruceectomycorrhizas from microhabitats lacking ericaceous hostsis dominated by ericoid mycorrhizal Meliniomyces variabilis

Martin VOHN�IKa,b,*, Libor MRNKAa, Tereza LUKE�SOV�Aa,b, Mar�ıa Clara BRUZONEc,Petr KOHOUTa,b,1, Judith FEHRERd

aDepartment of Mycorrhizal Symbioses, Institute of Botany ASCR, Pr�uhonice 252 43, Czech RepublicbDepartment of Plant Experimental Biology, Faculty of Science, Charles University in Prague, Prague 128 44, Czech RepubliccLaboratorio de Microbiolog�ıa Aplicada y Biotecnolog�ıa, Centro Regional Universitario Bariloche, INIBIOMA (Universidad Nacional del

Comahue e CONICET), San Carlos de Bariloche, R�ıo Negro, ArgentinadDNA Laboratory, Institute of Botany ASCR, Pr�uhonice 252 43, Czech Republic

a r t i c l e i n f o

Article history:

Received 21 August 2012

Revision received 7 February 2013

Accepted 22 February 2013

Available online 14 May 2013

Corresponding editor:

Kevin K. Newsham

Keywords:

Cadophora finlandica

Dark septate endophytes

Ectomycorrhiza

Endophytic fungi

Ericaceae

Ericoid mycorrhiza

Meliniomyces bicolor

Rhizoscyphus ericae

Root associated fungi

* Corresponding author. Department of Myco271015331.

E-mail addresses: martin.vohnik@ibot.ca1 Present address: Group of Mycology, Inst

1754-5048/$ e see front matter ª 2013 Elsevhttp://dx.doi.org/10.1016/j.funeco.2013.03.006

a b s t r a c t

Most of the temperate conifers associate with ectomycorrhizal fungi, but their roots also

harbour a wide range of endophytes. We focused on ascomycetes associating with basidio-

mycetous ectomycorrhizasofNorway spruce ina temperatemontane forest in central Europe

and found that the majority of the co-associated fungi belonged to the Rhizoscyphus ericae

aggregate (REA), being dominated by Meliniomyces variabilis. We further tested the ability of

representative isolates to colonize spruce root tips and European blueberry (Vaccinium myr-

tillus) hair roots in an agar system as well as their effect on blueberry growth in a peat-agar

system. M. variabilis intracellularly colonized spruce (Picea abies) root tip cortex, formed eri-

coidmycorrhizas in blueberry and enhanced blueberry shoot and root growth in comparison

with non-inoculatedplants. Our findings suggest that spruce ectomycorrhizasmay represent

selective niches for ericoid mycorrhizal fungi in habitats lacking suitable ericaceous hosts.

ª 2013 Elsevier Ltd and The British Mycological Society. All rights reserved.

Introduction range from facultative saprobes and parasites to neutral

Any healthy or dead plant tissue contains fungal endobionts

(hereafter referred to as mycobionts), both above- and below-

ground (Sieber 2002; Summerbell 2005). These mycobionts

rrhizal Symbioses, Instit

s.cz, vohnik@ibot.cas.cz (itute of Ecology and Earthier Ltd and The British M

endophytes to mutualistic symbionts positively affecting host

plant fitness (Rodriguez et al. 2009; Newsham 2011). While

there seems to be only a small overlap between the fungi

inhabiting plant stems and roots (Sieber 2002, 2007), some

ute of Botany ASCR, Pr�uhonice 252 43, Czech Republic. Tel.: þ420

M. Vohn�ık).Sciences, University of Tartu, 14a Ravila, Tartu 50411, Estonia.

ycological Society. All rights reserved.

282 M. Vohn�ık et al.

widespread mycobionts apparently colonize both aerial and

underground plant tissues (Menkis et al. 2004; Rodriguez et al.

2009). Aerial mycobionts can influence longevity of plant tis-

sues by protection against water stress and herbivores and

may accelerate their senescence (Carroll 1988; Arnold et al.

2003; Rodriguez et al. 2009), thus directly affecting plant bio-

mass available for carbon capture through photosynthesis. In

contrast, root mycobionts directly influence plant mineral

nutrition through scavenging nutrients from recalcitrant

substrates unavailable to host plants (mycorrhizal fungi),

protecting roots from biotic and abiotic stresses (mycorrhizal

and endophytic fungi) and/or influencing their longevity

(mycorrhizal fungi, fungal root endophytes, parasites and

casual saprobes) (Addy et al. 2005; Smith & Read 2008).

Roots of the large majority of terrestrial plants form

mutualistic symbioses with mycorrhizal fungi which pro-

foundly affect their anatomy, morphology, distribution and

metabolism (Brundrett 2002). In temperate and boreal forests,

most dominant trees form ectomycorrhizas (EcM). EcM fungi

mostly belong to the Basidiomycota with a relatively smaller

part belonging to the Ascomycota (LoBuglio et al. 1996;

Tedersoo et al. 2006). Ectomycorrhizas additionally harbour

diverse spectra of co-associated, mostly ascomycetous fungi

(Kernaghan et al. 2003; Urban et al. 2008; Tedersoo et al. 2009;

Kernaghan & Patriquin 2011). Due to their fast growth on

nutrient media, these may be preferentially isolated from the

respective ectomycorrhizas and mistaken for true EcM fungi

(Summerbell 2005 and references therein). Likewise, DNA of

the co-associated fungi may interfere with DNA of true EcM

mycobionts during PCR and/or may be preferentially ampli-

fied by the commonly used fungal primers (Gr€unig et al. 2008a;

Tedersoo et al. 2009). Significance of the EcM co-associated

fungi is largely unknown, but was hypothesized to include

specific inter-fungal interactions helping to maintain EcM

fungi diversity (Urban et al. 2008) or alteration of nutrient

uptake by the respective ectomycorrhizas (Jones et al. 2009).

Intriguingly, EcM co-associated fungi often display affinities to

Helotiales which commonly inhabit roots of Ericaceae

(Kernaghan et al. 2003; Addy et al. 2005; Tedersoo et al. 2009)

and at least some of them seem to be capable of forming eri-

coid mycorrhizas under controlled conditions (Vohn�ık et al.

2007a; Grelet et al. 2009).

Although EcM co-associated fungi comprise wide spectra

of fungal symbionts, two assemblages seem to be more fre-

quent in temperate and boreal forests of the Northern Hemi-

sphere: members of the Rhizoscyphus ericae aggregate (REA)

(Vr�alstad et al. 2000) and the dark septate endophytes (DSE)

displaying affinities or belonging to the Phialocephala fortinii

s.l.eAcephala applanata species complex (PAC) (Gr€unig et al.

2008a). Both groups contain species able to form ectomycor-

rhizas with developed Hartig net; Meliniomyces bicolor

(belonging to REA) produces the Piceirhiza bicolorata EcM mor-

photype (Vr�alstad et al. 2000; Villarreal-Ruiz et al. 2004) and

Acephala macrosclerotiorum (related to PAC) forms the Pinirhiza

sclerotia EcM morphotype (M€unzenberger et al. 2009). In addi-

tion, REA comprises the well-known ericoidmycorrhizal (ErM)

fungus R. ericae, and there is evidence supporting ericoid

mycorrhizal status of M. bicolor and M. variabilis (Grelet et al.

2009). On the other hand, despite DSE being frequently

observed, isolated and molecularly detected in ericaceous

roots, and that some of them seem to form intracellular

hyphal coils resembling ericoid mycorrhizas (Usuki &

Narisawa 2005; Vohn�ık & Albrechtov�a 2011), it is so far

unclear whether they form functional mycorrhizal symbioses

with Ericaceae.

In contrast to arbuscular mycorrhizal and EcM fungi, we

know very little about the extent, distribution and fate of the

mycelium of ErM fungi in the soil environment. It is believed

that the ErM extraradical mycelium reaches only a few milli-

metre from the root surface into the surrounding substratum

(Smith & Read 2008), and it has been suggested that genets of

M. variabilis are relatively small (<13 cm; Grelet et al. 2010).

While Oidiodendron maius, a typical ErM fungus, is an efficient

saprobe which can persist in the soil without contact with

Ericaceae (Rice & Currah 2006), the REAmembers are typically

detected in plant roots. Congruently, Bergero et al. (2003) found

that a consortium of ErM fungi persisted and maintained

mycorrhizal ability in the soil in a Mediterranean habitat

lacking ericaceous hosts but the authors failed to detect any

REA members. On the other hand, there is conflicting infor-

mation about the effect of Ericaceae on the REA members

interacting with EcM roots. For example, Kohout et al. (2011)

reported that the presence of Vaccinium spp. inhibited the

ability of M. bicolor to form ectomycorrhizas with Pinus spp.

while genetically identical isolates of Meliniomyces variabilis

were commonly detected in neighbouring Pinus sylvestris

ectomycorrhizas and Vaccinium vitis-idaea hair roots (Grelet

et al. 2010). Although several authors have hypothesized that

Ericaceae might be interconnected with neighbouring EcM

conifers through shared mycelium of some REA mycobionts,

especially Cadophora finlandica and M. bicolor (e.g., Villarreal-

Ruiz et al. 2004; Vr�alstad 2004), annotations of ITS nrDNA

sequences deposited in GenBank suggest that these two spe-

cies are only rarely detected in Ericaceae roots, in contrast to

M. variabilis and R. ericae (cf. Suppl. Fig 1). These findings

suggest possible differences within the R. ericae aggregate in

preferences for different host plants as well as in the ability to

develop and persist in the soil environment outside host roots

(cf. Baldrian et al. 2012).

The Bohemian Forest NP lies in a mountain range

extending fromSouth Bohemia, the Czech Republic, to Austria

and Germany. It comprises forested hills/mountains at an

elevation between 800 and 1 400 m a.s.l. and covers an area of

approx. 700 km2. Most of its montane ecosystems are formed

by secondary Norway spruce (Picea abies) forests with erica-

ceous, grass and hygrophilous understorey, such vegetation

being characteristic for many European temperate and boreal

regions. Detailed information on fungi associating with ecto-

mycorrhizas in this ecosystem type is lacking; to our knowl-

edge, the only local report is from serpentinite sites in Austria

(Urban et al. 2008). We, therefore, investigated the diversity of

ascomycetous fungi living in endorhizal symbiosis with Nor-

way spruce basidiomycetous ectomycorrhizas at two sites in

the Bohemian Forest NP. Basidiomycetous EcMs were chosen

as they form the great majority of spruce ectomycorrhizas at

the two sites (M. Vohn�ık, personal observation) and basidio-

mycetous EcM mycelia seem to dominate the fungal soil

community detected at a nearby experimental site (Baldrian

et al. 2012). We specifically focused on microsites lacking eri-

caceous understory which led us to the expectation that the

Fig 1 e Symbiotic potential of selected isolates obtained in this study in roots of Picea abies and Vaccinium myrtillus

seedlings. (A) Acephala applanata EF093158 D P. abies: intercellular hyphae in the cortex (arrows) accompanied by a circle of

microsclerotia (asterisks) around the endodermis. Hand-sectioned root tip, bar [ 50 mm. (B) Meliniomyces variabilis

EF093169 D P. abies: intercellular hyphae in the cortex cells (arrows). Hand-sectioned root tip stained with trypan blue,

bar [ 50 mm. (C) Typical morphology of P. abies roots colonized by Cadophora finlandica EF093179. Bar [ 500 mm. (D)

Cadophora finlandica EF093179 D P. abies: intercellular hyphae forming Hartig net (arrows) accompanied by intracellular

hyphae (arrowheads) in the cortex. Hand-sectioned root tip stained with aniline blue, bar [ 12.5 mm. (E) Meliniomyces

variabilis EF093169 D V. myrtillus: an intracellular hyphal coil in a rhizodermal cell, morphologically identical to ericoid

mycorrhiza. Stained with trypan blue, bar [ 25 mm. (F) Cadophora finlandica EF093179 D V. myrtillus: the rhizodermal cells

are filled with dense hyphal loops identical to ericoid mycorrhizas. Stained with trypan blue, bar [ 25 mm. (G) Cadophora

finlandica EF093155 D V. myrtillus: melanized dark septate hyphae forming loose intracellular loops while spreading from

cell to cell in the rhizodermis. An arrow points at a thin hypha penetrating the host cell wall. Stained with trypan blue,

bar [ 20 mm. (H) Meliniomyces bicolor EF093180 D V. myrtillus: rhizodermal cells filled with dense hyphal coils

morphologically identical to ericoid mycorrhiza. Stained with trypan blue, bar [ 25 mm. (I) Acephala applanata

EF093158 D V. myrtillus: dark septate hyphae occasionally forming intracellular microsclerotia (asterisks). Bar [ 30 mm.

Meliniomyces variabilis in spruce ectomycorrhizas 283

284 M. Vohn�ık et al.

endophytic fungal community would be dominated by non-

ericaceous mycobionts. As we intended to screen endo-

phytes living inside the screened ectomycorrhizas, rather

than their superficial associates which are often detected by

culture-independent approaches, we employed a culture-

dependent approach which additionally favoured growth of

ascomycetes over basidiomycetes. Isolates of EcM co-

associated fungi were also needed to test their symbiotic

interactions with Norway spruce and European blueberry

(Vaccinium myrtillus). In the symbiotic tests, we wanted to find

out whether: (1) the isolates have endophytic ability, i.e., they

can intracellularly colonize spruce root tips without apparent

negative effects e this would mean that they are likely true

endophytes of the respective ectomycorrhizas from which

they were isolated; (2) at least some isolates have ectomy-

corrhizal ability, i.e., they form the Hartig net e this would

mean that they could participate in formation of the respec-

tive ectomycorrhizas; and (3) at least some isolates have the

ericoid mycorrhizal ability, i.e., they form the characteristic

morphological structures in Ericaceae hair roots and pos-

itively affect the colonized host plants e this wouldmean that

ectomycorrhizas could serve as micro-reservoirs of functional

ericoid mycorrhizal inoculum in habitats lacking suitable

ericaceous hosts.

Materials and methods

Field site and root sampling

The two investigated sites are located in the central part

of the Bohemian Forest NP at an elevation of approx.

1 075 m a.s.l (N49�00, E13�290 and N48�590, E13�290). The valley

is dominated by a secondary Norway spruce (P. abies) forest

(>80 yr old) with understory consisting of ericaceous plants

(V. myrtillus and V. vitis-idaea), grass (mostly Avenella flexuosa

and Calamagrostis villosa), and bryophytes (Sphagnum spp.);

the prevailing soil type is podzol. In total, 19 3e5 yr old spruce

seedlings inhabiting different niches within the two sites

were sampled on two occasions during the vegetation sea-

son. The different niches included dead Norway spruce

stumps, mosses, fallen decaying spruce trunks, the forest

floor covered by spruce needles and waterlogged Sphagnum

undergrowth. These microhabitats were chosen to capture

the heterogeneous ecological amplitude of spruce seedlings

regenerating within the National Park. However, we omitted

spruce seedlings regenerating in the vicinity of Ericaceae or

directly within ericaceous shrubs as these situations are a

subject of another ongoing study. Seedlings were carefully

dug up not to destroy their fine ectomycorrhizal roots,

packed in plastic bags and kept refridgerated until the iso-

lation of the ectomycorrhiza co-associated fungi on the fol-

lowing day.

Isolation of fungal cultures

The roots of the seedlings were thoroughly washed with tap

water, transferred to250mlflaskswith100mlofdistilledwater

with two drops of Tween 20 and shaken for 10 min. Basidio-

mycetous EcM root tips were recognized by the presence of

clamped hyphal mantles and/or emanating clamped hyphae

and typical EcM root tip morphology. According to their mor-

phology (Agerer 1987e2002, A. Lep�sov�a pers. com.) the collected

ectomycorrhizas belonged to Amphinema byssoides, Hebeloma

velutipes, Lactarius spp., Piceirhiza guttata (¼Tylospora fibrillosa),

Russula ochroleuca and Thelephora terrestris. In total, 360 basi-

diomycetous EcM tips of healthy appearance were randomly

selected from the collected seedlings, cut from the main roots

(length approx. 3 mm), surface-sterilized (0.5 % NaClO for

10 min), serially washed in sterile deionized water, aseptically

placed onto modified Melin Norkrans medium (MMN), 15 EcM

tips per 9 cm diam. Petri dish, and incubated for 2 months at

20 �C in the dark. The dishes were periodically checked for

mycelial growth and possible occurrence of contaminants.

According to Schild et al. (1988) and our previous testing, this

medium favours the growth of ascomycetes over basidiomy-

cetes, while addition of benomyl leads to a higher yield of

putatively EcM basidiomycetes. Fast growing and intensively

sporulating fungi were discarded and the remaining isolates

were transferred onto new MMN media for subculture. Their

radial growth onmalt extract agar (SigmaeAldrich, Germany),

MMN, corn meal agar, oatmeal agar and potato dextrose agar

(all HiMedia Laboratories, India) was measured each second

day for 3 weeks, and mycelial characteristics were recorded

after 2 weeks of growth. The isolates were then conservatively

grouped intodistinct culturemorphotypesandrepresentatives

of eachmorphotype (2e3 isolates if available)were subjected to

molecular identification.

DNA extraction and amplification

Fungal DNA was extracted from fresh mycelia using the sor-

bitol method (�Storchov�a et al. 2000). DNA was dissolved in

10 mM Tris/HCl (pH 8.0) and kept in a freezer (�20 �C). DNA

amplifications of the ITS nrDNA regionswere performed using

an Eppendorf Master Cycler (Eppendorf AG, Germany) using

the primers ITS1F and ITS4. The PCR mix consisted of 5 ml of

10� PCR buffer without MgCl2 (Fermentas International Inc,

Canada), 200 nM concentration of dNTPmixture, 2 mMMgCl2,

0.8 mM of each primer, 1 U of Taq DNA polymerase, 32 ml of

sterile ddH2O and 4 ml of 1:4 diluted template DNA. The fol-

lowing cycling parameters were used: initial denaturation

step for 4 min at 94 �C, 35 cycles consisting of a denaturation

step at 94 �C for 30 s, annealing at 55 �C for 30 s, extension at

72 �C for 70 s, with a final extension at 72 �C for 10 min. PCR

products were checked for length and quality/quantity by gel

electrophoresis (1.5 % agarose) and purified using the High

Pure PCR product purification kit (Roche Holding Ltd, Swit-

zerland). Sequencing was done by GATC Biotech AG (Ger-

many) using the PCR primers, sequence electropherograms

were edited manually. The obtained sequences were sub-

mitted to the GenBank database (accession numbers

EF093147eEF093185, Table 1).

Species identification

As a first step towards species identification, the obtained

ITS sequences were subjected to BLAST searches (GenBank,

NCBI) to retrieve the most similar sequences. Most of them

could be assigned to particular species (sequence identity

Table 1 e The identity of isolates obtained in this study and their symbiotic status based on results of a monoxenic re-synthesis experiment

GenBank Cluster Identitya Closest GenBankmatchb

Coverage/Similarity (%)

Re-synthesiswith Picea abiesc

Re-synthesiswith Vaccinium

myrtillusc

EF093155 1 Cadophora finlandica AB190393 Cadophora

finlandica

98/99 Hyaline hyphal mantle

� Hartig net, IH

IH resembling

DSE-association

AF011327 C. finlandica 100/98

EF093179 1 Cadophora finlandica AF486119 C. finlandica 100/97 Ectendomycorrhiza-like

with Hartig net

IH resembling

ericoid mycorrhiza

EU938675 Chloridium

paucisporum

96/97

EF093180 1 Meliniomyces bicolor AY394885 Meliniomyces

bicolor

100/100 Few IH in cortex IH resembling

ericoid mycorrhiza

AY394907 Rhizoscyphus

ericae

100/95

EF093181 1 Meliniomyces bicolor As EF093180

EF093182 1 Meliniomyces bicolor As EF093180

EF093183 1 Meliniomyces bicolor As EF093180

EF093163 1 Meliniomyces variabilis HM190128 Meliniomyces

variabilis

100/100

HM190126 M. variabilis 100/100

EF093165 1 Meliniomyces variabilis As EF093163

EF093170 1 Meliniomyces variabilis As EF093163 IH in cortex IH resembling

ericoid mycorrhiza

EF093171 1 Meliniomyces variabilis As EF093163

EF093172 1 Meliniomyces variabilis HM190128 M. variabilis 100/99

HM190126 M. variabilis 100/99

EF093173 1 Meliniomyces variabilis As EF093172 No colonization Few loose IH

EF093164 1 Meliniomyces variabilis AY838789 M. variabilis 96/99 No colonization No colonization

AY838785 M. variabilis 96/99

EF093166 1 Meliniomyces variabilis As EF093164

EF093177 1 Meliniomyces variabilis As EF093164

EF093167 1 Meliniomyces variabilis AY838789 M. variabilis 96/99

AY838785 M. variabilis 96/98

EF093168 1 Meliniomyces variabilis As EF093167 96/98

EF093169 1 Meliniomyces variabilis AY838789 M. variabilis 99/99 IH in cortex IH resembling

ericoid mycorrhiza

AY838785 M. variabilis 99/99

EF093176 1 Meliniomyces variabilis AY838789 M. variabilis 100/98

AY838785 M. variabilis 100/98

EF093178 1 Meliniomyces cf.

variabilis

AY394885 M. bicolor 99/93 No colonization IH resembling

ericoid mycorrhiza

AY394907 Rhizoscyphus

ericae

99/93

EF093174 1 Meliniomyces sp. AY838789 M. variabilis 100/92 No colonization Few loose IH

AY838785 M. variabilis 100/92

EF093175 1 Meliniomyces sp. AY394885 M. bicolor 100/92 No colonization Few loose IH

AY838789 M. variabilis 96/92

EF093158 2 Acephala applanata d IH in cortex, MS

around endodermis

IH, MS

EF093161 2 Phialocephala subalpina d IH in cortex,

without MS

IH, MS

EF093156 2 Phialocephala turiciensis d Few IH in cortex MS

EF093157 2 P. turiciensis d

EF093162 2 P. turiciensis d No colonization IH

EF093159 2 P. fortinii CSP 17 d No colonization MS

EF093160 2 P. fortinii CSP 17 d

EF093148 3 Helotiales sp. EU040232 Hyalodendriella

betulae

100/95

FR717230 Chalara longipes 95/94

EF093150 3 Articulospora tetracladia 100/97

(continued on next page)

Meliniomyces variabilis in spruce ectomycorrhizas 285

Table 1 e (continued )

GenBank Cluster Identitya Closest GenBankmatchb

Coverage/Similarity (%)

Re-synthesiswith Picea abiesc

Re-synthesiswith Vaccinium

myrtillusc

EU998918 Articulospora

tetracladia

EU998917 A. tetracladia 100/97

EF093184 3 Varicosporium elodeae GQ152148 V. elodeae 95/100 No colonization No colonization

GQ411275 V. elodeae 93/100

EF093185 3 V. elodeae As EF093184 No colonization No colonization

EF093147 3 cf. Neobulgaria HM051080 N. pura 89/97

HM116745 Neobulgaria sp. 99/93

EF093149 3 cf. Neobulgaria As EF093147

EF093153 3 Tolypocladium inflatum AB255606 Tolypocladium

inflatum

100/100

AB103381 T. inflatum 100/100

EF093154 3 Thysanophora

penicillioides

AB213268 Thysanophora

penicillioides

99/99

AB213265 T. penicillioides 99/99

EF093151 4 Hebeloma sp. AF430291 Hebeloma

incarnatulum

98/99

(Basidiomycota) AY309960 Hebeloma bryogenes 96/100

EF093152 4 Mycena galopus HM240534 M. galopus 96/99

(Basidiomycota) JF908484 M. galopus 94/99

a According to BLAST and/or phylogenetic analyses; the strains in bold were used in monoxenic cultures with Picea abies and Vaccinium

myrtillus.

b Preference was given for sequences derived from cultures (with deposited vouchers if available) with scientific names.

c IH ¼ intracellular hyphae; MS ¼ microsclerotia; DSE ¼ dark septate endophytes.

d These sequences fell within the Phialocephala fortiniieAcephala applanata species complex. As ITS nrDNA sequences are not suitable for

classification of Phialocephala-like strains to cryptic species, they were identified according to Gr€unig et al. (2008b).

286 M. Vohn�ık et al.

�97 %). For a more reliable identification, the isolates

belonging to the R. ericae aggregate were subjected to phy-

logenetic analyses. Further sequences, derived preferentially

from cultured isolates deposited in international culture

collections, as well as suitable outgroup taxa were retrieved

from GenBank to represent the genetic variability of the

isolates as comprehensively as possible (Suppl. Table 1).

Sequences were aligned manually using BioEdit V7.0.0 (Hall

1999). Maximum parsimony (MP) and minimum evolution

(ME) analyses were done with PAUP* V4.0b10 (Swofford

2002). MP analysis was performed as a heuristic search

with 100 random addition sequence replicates, saving no

more than 100 trees of length greater than or equal to 1 per

replicate, and TBR branch swapping. Bootstrapping was

done with the same settings and 1 000 replicates, but with-

out branch swapping. ME analysis was run as a heuristic

search using HKY85 distances with TBR branch swapping,

bootstrapping was done as for MP analysis. Bayesian analy-

sis was done using MrBayes V3.1 (Ronquist & Huelsenbeck

2003) with six substitution rates, gamma distribution and a

proportion of invariable sites as priors. MCMC analyses were

performed in two parallel runs with four chains each for 4

million generations, sampling every 1 000th tree. All stat-

istical parameters indicated that convergence was reached.

The first 25 % of the trees were discarded as burn-in, and the

remaining 75 % trees were summarized. Fungal isolates

belonging to the P. fortinii s.l.eA. applanata species complex

were kindly identified to cryptic species by C. Gr€unig

according to Gr€unig et al. (2008b).

Symbiotic potential of selected isolates in Picea andVaccinium

P. abies and V. myrtillus seedswere surface-sterilizedwith 30 %

hydrogen peroxide for 10 and 3 min, respectively, and placed

on MMN agar to check for possible contamination. Non-

contaminated seeds were left to germinate for 3 weeks.

Lower compartments of split Petri dishes (9 cm in diam.) with

perforated central septa were filled with MMN (10� diluted

sugars), overlaid with a sterile cellophane membrane to pre-

vent growth of roots into the medium and inoculated with

agar plugs cut from margins of colonies of 17 selected fungal

isolates actively growing on MMN (Table 1). The dishes with

the plugs were left for 3 weeks at room temperature in the

dark to produce vegetative mycelium. Then, 3-week-old

seedlings were transferred to the dishes (five seedlings per

each isolate/host combination), the empty upper compart-

ments accommodating their shoots and the lower compart-

ments their roots. The roots of the seedlings were covered

with a piece of sterile moistened filter paper. The dishes were

sealed with air-permeable film, lower compartments covered

with aluminium foil, and placed in a vertical position in a

growth chamber (16/8 hr and 21/15 �C day/night cycle, irra-

diation 250 m mol m�2 s�1). After 5 months, the plants were

extracted and their roots were separated and processed as

follows: V. myrtillus roots were cleared with 10 % KOH (20 min

at 121 �C), washed with tap water, acidified (1 min in 3 % HCl),

washed with tap water, stained with trypan blue (1 hr at

121 �C) and de-stained overnight in lactoglycerol; P. abies roots

Meliniomyces variabilis in spruce ectomycorrhizas 287

were hand-sectioned and thin sections were stained with

aniline or trypan blue. Both dyes were prepared in lactogly-

cerol (lactic acid:glycerol:deionized water ¼ 1:1:3) as 0.05 %

solutions. The stained roots and thin sections were observed

using an Olympus BX60microscope equippedwith DIC at high

magnifications (400� and 1 000�). Pictures were taken with an

Olympus DP70 camera, graphic documentation was modified

for clarity in Paint.NET (Brewster, Jackson and contributors þMicrosoft Corporation) when needed.

Effect of selected isolates on V. myrtillus growth

The potential ericoid mycorrhizal status of selected REA iso-

lateswas also tested in a peat-agar system. Briefly, 10ml of 1 %

(w/v) autoclaved water agar was poured into sterile 50 ml

plastic vials and a small piece of mycelium of each of the

selected isolates was subsequently transferred onto its sol-

idified surface. Sealed vials were kept at room temperature in

the dark and periodically checked for new hyphal growth.

After 28 d new mycelium covered the agar surface in all ino-

culated vials; at this point approx. 10 ml of twice-autoclaved

white peat (Br�at-Velkoobchod s.r.o., Czech Republic; pH 4.0)

were added into each vial, which were then treated as above.

After 29 d, one axenic 48-d-old V. myrtillus seedling was

aseptically inserted into peat, the vials were covered and

sealed with an air-permeable film and incubated in a growth

chamber (modified from Vohn�ık et al. 2012). Due to space

limitations it was not possible to use all REA isolates; there-

fore, we tested one representative of each REA clade (two in

the case of the new Meliniomyces sp. clade, see Suppl. Fig 1)

randomly selected from the isolates used in monoxenic cul-

tures with P. abies and V. myrtillus (Table 2). An isolate of

R. ericae, obtained in an unpublished study from surface-

sterilized ErM roots of Calluna vulgaris collected in the same

area, and an O. maius isolate JF304942 were used as positive

controls. Negative control vials were treated as above but

without inoculation with fungal mycelium (we assumed that

possible nutrient input to the experimental systems through

the inoculation was minimal). There were five vials with

healthy V. myrtillus seedlings for each treatment, including

the positive and negative control treatments.

Table 2 e The effect of selected isolates on the growth of Vacci

Isolatea Fresh root weight (mg)H7,37 ¼ 15.83; p ¼ 0.027

Cadophora finlandica EF093179 [n ¼ 4] 6.43 � 1.26 ab

Meliniomyces bicolor EF093180 [3] 6.70 � 1.46 ab

Meliniomyces variabilis EF093169 [5] 9.06 ± 1.13 b

Meliniomyces sp. EF093174 [5] 7.44 � 1.13 ab

Meliniomyces sp. EF093175 [5] 6.50 � 1.13 ab

Oidiodendron maius JF304942 [5] 3.62 � 1.13 ab

Rhizoscyphus ericaeb [5] 7.44 � 1.13 ab

Control (not inoculated) [5] 2.72 ± 1.13 a

The values are means � SE, different letters indicate significantly differen

comparisons of means ( p ¼ 0.05).

a Identity according to Table 1.

b This isolate was obtained from an ericoid mycorrhizal Calluna vulgaris

c Not included in the statistical analysis.

After 132 d, seedlings were carefully pulled from peat,

washed with tap water and their roots were separated from

shoots, dried with paper towels and immediately weighed.

Roots were then cleared in 10 % KOH, acidified in 3 % HCl,

stained with trypan blue at room temperature and de-stained

in lactoglycerol. ErM colonization was assessed by a single

person (C.M.B.), using the samemicroscope as above at 1 000�magnification, in 25 consecutive rhizodermal cells observed in

20 random fields of view per each root system (i.e., 500 cells

per each seedling), including negative control plants. Root

colonization was documented as above. Shoots were dried for

2 hr at 60 �C, and weighed. The collected data (fresh root

weight, dry shoot weight and ericoid mycorrhizal colo-

nization) were checked for homogeneity of variances and

normal distribution; fresh root weight and dry shoot weight

were not normally distributed while ericoid mycorrhizal col-

onization had neither homogeneity of variances nor normal

distribution, even after log and sqrt transformations. We,

therefore, used non-parametric KruskaleWallis ANOVA fol-

lowed by post-hoc multiple comparisons of means. STATIS-

TICA 10 (StatSoft, USA) was used for the statistical analyses. A

piece of peat was aseptically picked from several randomly

chosen vials and placed in Petri dishes with MMN to verify

viability of the inoculated strains at the end of the experiment.

Results

Isolation and identification of fungal cultures

The 360 surface-sterilized basidiomycetous ectomycorrhizas

yielded 128 relatively slow-growing non-sporulating isolates

which were sorted into 39 morphotypes differing in growth

and mycelial characteristics. The rest of the ectomycorrhizas

(ca. 64 %) either yielded fast-growing sporulating con-

taminants or produced no mycelium within the cultivation

period. By comparing their ITS nrDNA sequences with public

databases and with the help of phylogenetic analyses, we

were able to assign at least putative genus names to 38 mor-

photypes (36 ascomycetous, two basidiomycetous) belonging

to four distinct clusters (Table 1). The first cluster comprised

nium myrtillus in a peat-agar system

Dry shoot weight (mg)H7,37 ¼ 27.57; p ¼ 0.0003

Ericoid mycorrhizalcolonization (%) H5,28 ¼ 21.46;

p ¼ 0.0007

11.13 � 1.17 ab 0c

11.37 � 1.36 ab 30.27 � 7.79 ab

15.20 ± 1.05 b 69.20 ± 6.04 b

11.82 � 1.05 ab 22.76 � 6.04 ab

10.06 � 1.05 ab 8.80 ± 6.04 a

2.70 ± 1.05 a 26.08 � 5.91 ab

8.78 � 1.05 ab 69.16 ± 6.04 b

2.64 ± 1.05 a 0c

t groups according to KruskaleWallis ANOVA followed bymultiple

hair root and served as positive control.

288 M. Vohn�ık et al.

the R. ericae aggregate (Suppl. Fig 1 and Suppl. Table 1)

encompassing C. finlandica, M. bicolor, M. variabilis and two

unidentified ascomycetous isolates (EF093174 and

EF093175 ¼ a putatively new Meliniomyces species, referred to

as “Meliniomyces sp. Bohemian Forest”). The second cluster

comprised the P. fortinii s.l.eA. applanata species complex

encompassing A. applanata, Phialocephala subalpina, Phialoce-

phala turiciensis and two unidentified Phialocephala-related

isolates (EF093159 and EF093160¼ putative P. fortinii s.l. cryptic

species CSP 17, C. Gr€unig, pers. com.). The third cluster com-

prised various ascomycetes from the Eurotiales, Helotiales

and Hypocreales, including those with affinities to known

aquatic hyphomycetes which sometimes inhabit terrestrial

roots. The fourth cluster comprised the only two basidiomy-

cetes isolated, the ectomycorrhizal Hebeloma cf. bryogenes/

incarnatum and the saprotrophic Mycena galopus, suggesting

that our approach indeed favoured ascomycetes over

basidiomycetes.

All methods of phylogenetic analyses resulted in generally

the same trees with similar branch support (given that pos-

terior probabilities of �0.94 in Bayesian analyses and boot-

strap support of �70 % in all other analyses are significant). As

expected, phylogenetic relationships were largely independ-

ent of the isolates’ geographic origin or host species. The REA

isolates (Suppl. Fig 1) consisted of several more or less well-

defined (putative) Meliniomyces species in which C. finlandica

and R. ericae were embedded as monophyletic clades.

According to their placement in the tree and significant stat-

istical support, the two Meliniomyces-related morphotypes

indeed represent a new species within REA; no other

sequences represented in GenBank to date aremore similar to

these isolates than those included in the tree (maximal iden-

tity 93 %). In contrast, the rest of our REA morphotypes clus-

tered with well-defined species in this clade which were,

again, independent of host species or geographic origin. Our

analyses, thus, show that central European REA samples,

whichwere previously underrepresented in GenBank, fall into

the range of intraspecific variation for most REA species

(provided that they were correctly identified by their authors).

Symbiotic potential of selected isolates in Picea andVaccinium

In the monoxenic cultures with P. abies seedlings, none of the

five tested PAC isolates formed ectomycorrhizas with Hartig

net and hyphal mantle. However, some isolates (A. applanata

EF093158, P. turiciensis EF093156 and P. subalpina EF093161)

produced intracellular hyphae in the cortex cells of P. abies

roots. Additionally, A. applanata formed a circle of dense

microsclerotia around the endodermis, which was accom-

panied by dense intra- and intercellular colonization (Fig 1A).

Generally, roots of the plants inoculated with DSE isolates

lacked hair roots and resembled “pseudomycorrhizal” roots as

described by Melin (1922).

Several REA isolates (M. bicolor EF093180, M. variabilis

EF093169 and EF093170) produced intracellular colonization in

the P. abies cortex cells which was usually accompanied with

apparent darkening and thickening of their cell walls (cf.

Fig 1B and D). C. finlandica EF093155 and EF093179-inoculated

roots lacked root hairs and superficially resembled

ectomycorrhizas, but not the EcM morphotype P. bicolorata

(Fig 1C). Microscopic observation of these roots showed that

both strains formed an intraradical association resembling

ectendomycorrhiza, i.e., intercellular cortical Hartig net

accompanied by a few intracellular hyphae (Fig 1D). Spruce

roots with such ectendomycorrhizal association were

embedded in loose wefts of hyphae, but without dense hyphal

mantles. None of theM. bicolor norM. variabilis isolates formed

ectomycorrhiza or ectendomycorrhiza with spruce seedlings.

About one-half of the tested REA isolates, including the

putatively new Meliniomyces species, produced no inter- or

intracellular colonization. Similarly, neither Varicosporium

elodeae isolates (EF093184 and EF093185) colonized spruce

roots (Table 1).

Except for one M. variabilis isolate (EF093164) and both

V. elodeae isolates, all the remaining isolates colonized roots of

V. myrtillus intracellularly. Most of the REA isolates formed

dense intracellular hyphal coils resembling ericoid mycor-

rhiza, including C. finlandica EF093179 and M. bicolor EF093180

(Fig 1E, F and H). However, loose intracellular loops formed by

melanized and relatively thick hyphae of C. finlandica EF093155

resembled colonization typical for DSE-association rather

than ericoid mycorrhiza (Fig 1G). The putative new Melinio-

myces species infrequently formed loose to dense intracellular

hyphal loops and coils. Further in vitro experiments are nee-

ded to decide whether this colonization pattern represents

ericoid mycorrhiza. On the other hand, the typical DSE colo-

nization pattern was produced by all PAC isolates; most of

them formed vigorous intraradical colonization and often

produced intracellular microsclerotia which usually filled the

whole lumen of the colonized rhizodermal or cortical cells

(Fig 1I). None of the tested PAC strains formed intracellular

coils typical of ericoid mycorrhiza (Table 1).

Effect of selected isolates on V. myrtillus growth

All blueberry seedlings were green and healthy-looking at the

time of harvest except two inoculated withM. bicolor EF093180

and one inoculated with C. finlandica EF093179, which died

during the course of the experiment. Ericoid mycorrhizas

were formed in the roots inoculated with M. bicolor EF093180,

M. variabilis EF093169,Meliniomyces sp. EF093174 and EF093175,

O. maius JF304942 and R. ericae, though at different levels (see

below); non-inoculated control plants as well as those inocu-

lated with C. finlandica EF093179 were free of ericoid mycor-

rhizal colonization and were, therefore, excluded from the

statistical analysis of ErM colonization. All peat pieces trans-

ferred onto MMN producedmycelial colonies morphologically

identical to the respective inoculated isolates, including

C. finlandica EF093155; we did not detect any fast-growing

sporulating contaminants. This test, therefore, showed that at

the end of the experiment the screened vials contained viable

non-contaminated inoculum.

Inoculation had significant effects on all measured

parameters, i.e., fresh root weight (H7,37 ¼ 15.83, p¼ 0.027), dry

shoot weight (H7,37 ¼ 27.57, p ¼ 0.0003) and ErM colonization

(H5,28 ¼ 21.46, p ¼ 0.0007). Post-hoc multiple comparisons of

means indicated that plants forming ericoid mycorrhiza with

M. variabilis EF093169 produced significantly more fresh root

biomass than the non-inoculated plants, and their dry shoot

Meliniomyces variabilis in spruce ectomycorrhizas 289

weightwas significantly higher than in the case of theO.maius

JF304942-inoculated and non-inoculated plants. Additionally,

roots of the plants inoculated with M. variabilis EF093169 and

R. ericae had significantly higher ericoid mycorrhizal colo-

nization than those inoculated withMeliniomyces sp. EF093175

(Table 2).

Discussion

The EcM co-associated fungi only recently gained systematic

attention and their ecophysiological function remains largely

unclear. We still do not have answers for some basic ques-

tions, e.g., whether each single ectomycorrhiza possesses

endophytes. In our study from central Europe, approx. 36 % of

the screened ectomycorrhizal root tips yielded an endophyte.

This is comparable to 33.5 % obtained by Tedersoo et al. (2009),

who screened ectomycorrhizas from two Tasmanian wet

sclerophyll forests and a bit less than 48 % obtained by

Kernaghan & Patriquin (2011), who focused on ectomycor-

rhizas formed by the ascomycete Cenococcum geophilum at two

boreal sites in east Canada. It remains to be established where

is the actual living space of the co-associates in the respective

ectomycorrhizas, as they may dynamically inhabit hyphal

mantles and/or root intercellular and intracellular spaces;

intracellular colonization by apparently non-EcM mycelium

may be high in senescing ectomycorrhizas but is usually

absent in young turgescent EcM root tips (cf. Fig 2A and B). It is

plausible that intraradical competition for space and/or

resources ultimately leads to antagonism between EcM fungi

and their co-associates with yet unknown consequences to

Fig 2 e Typical anatomy of basidiomycetous ectomycorrhizas c

ectomycorrhiza with frequent intracellular hyphal colonization

(arrowheads) and a hyphal mantle (hm). Bar [ 50 mm. (B) Healt

(arrowheads) and a hyphal mantle (hm), with no intracellular c

permission of A. Lep�sov�a.

the host plant. Furthermore, ectomycorrhiza ageing ulti-

mately leads to termination of the active carbon flow from the

host plant to the EcM fungus which might give a competitive

advantage to its co-associate. However, these hypotheses

need to be further investigated.

In this study the frequency of mycobionts from the R. ericae

aggregate exceeded that of DSE and four different REA species

were obtained: C. finlandica, M. bicolor, the putatively new

Meliniomyces sp. and the dominant M. variabilis. Congruently,

Kernaghan & Patriquin (2011) detected M. bicolor and M. vari-

abilis in C. geophilum ectomycorrhizas in eastern Canada, and

Grelet et al. (2010) detected M. variabilis as the most frequent

co-associate of P. bicolorata-like ectomycorrhizas in two

P. sylvestris forests in northeeast Scotland. However, none of

our isolates belonged to the prominent ErM fungus R. ericae,

similarly to Kernaghan & Patriquin (2011) and Grelet et al.

(2010). This lack cannot be simply explained by absence in

the studied ecosystem because R. ericae, as well asM. variabilis,

were regularly isolated from hair roots of Ericaceae growing in

the investigated area in the Bohemian Forest NP (M. Vohn�ık,

unpublished). On the other hand, M. variabilis (similarly to

R. ericae and in contrast to Meliniomyces vraolstadiae) did not

belong among the major soil fungal OTUs found in the same

area by Baldrian et al. (2012). These observations suggest that

M. variabilis is an obligate biotroph rather than a free-living

soil dweller. Additionally, this species seems to have the

broadest ecological niche among the R. ericae aggregate

members e it has been detected as an endophyte in the roots

of a taxonomically diverse spectrum of plants including

Fagaceae, Orchidaceae and Salicaceae (Hambleton & Sigler

2005), Betulaceae and Pinaceae (Kernaghan & Patriquin

ollected in the vicinity of the sampling sites. (A) Senescing

in the cortex cells (arrows), a senescing Hartig net

hy turgescent ectomycorrhiza with a developed Hartig net

olonization in the cortex. Bar [ 50 mm. Photos used with

290 M. Vohn�ık et al.

2011), and Poaceae and Primulaceae (Tejesvi et al. 2013), but

seems to prefer Ericaceae, the only group which forms true

mycorrhizas with M. variabilis (Othaka & Narisawa 2008).

Thus, given its ability to intracellularly colonize conifer root

tips (Piercey et al. 2002; Vohn�ık et al. 2007b; the present study),

these most likely represent suitable refuges for M. variabilis in

microhabitats lacking ericaceous hosts. The outcome of col-

onization by M. variabilis for the EcM plant remains unclear

but the colonized roots often show signs of defence against

penetrating hyphae (Othaka & Narisawa 2008; the present

study).

C. finlandica and M. bicolor have been detected as EcM co-

associated fungi but they also form characteristic ecto- and

ectendomycorrhizas (Wang&Wilcox 1985; Vr�alstad et al. 2000,

2002; Villarreal-Ruiz et al. 2004). Additionally, both species

have been shown to form structures resembling ericoid

mycorrhizas in vitro (Vr�alstad et al. 2002; Villarreal-Ruiz et al.

2004; the present study), but they are only rarely detected in

Ericaceae roots under natural conditions (cf. Suppl. Fig 1).

These findings indicate an intriguing difference in life strat-

egies of the closely related REA mycobionts where some spe-

cies seem to prefer roots of Ericaceae (ericoid mycorrhizal

R. ericae), some roots of conifers (endophytic/ectomycorrhizal

C. finlandica and M. bicolor), some seem to be endophytic gen-

eralists as well as mycorrhizal specialists (M. variabilis, pos-

sibly also the newly discovered Meliniomyces sp.) while others

possibly form ectomycorrhizas with conifers and at the same

time represent a significant part of the soil fungal mycelium

(M. vraolstadiae, cf. Baldrian et al. 2012).

The in vitro potential of some REA members to form ecto-

mycorrhizas as well as ericoid mycorrhizas might be of great

ecological significance because Ericaceae often form a domi-

nant part of understory of EcM trees. In this study, both DSE

and REA isolates colonized spruce and blueberry seedlings;

however, DSE failed to form ectomycorrhizas or ericoid

mycorrhizas and M. bicolor, M. variabilis and Meliniomyces sp.

formed only ericoid mycorrhizas. Congruently, Vr�alstad et al.

(2002) reported a delimitation of EcM- and ErM-forming iso-

lates within REA. Intriguingly, in the present study one

C. finlandica strain formed ectendomycorrhizawith spruce and

associated with blueberry roots in a manner morphologically

resembling ericoidmycorrhiza in the agar-based system. Prior

to our report, there was only one re-synthesis study showing a

REA isolate with dual mycorrhizal ability (Villarreal-Ruiz et al.

2004); this isolate (M. bicolor LVR4069) formed characteristic P.

bicolorata ectomycorrhizas with pine, and the authors noted

that while interacting with blueberry, its “hyphae entered the

cells of the hair roots and formed coils characteristic of ericoid

mycorrhizas”. However, Tedersoo et al. (2009) noted that soil

saprobes belonging to Helotiales frequently display root

endophytic lifestyle and suggested that Ericaceae evolved

capacities to host them and to stimulate the formation of

intracellular hyphal coils for improved nutrient exchange.

This most probably led to the evolution of ErM symbiosis, yet

Ericaceae apparently kept the ability to host a wide range of

helotialean endophytes (Walker et al. 2011). Although C. fin-

landica EF093179 and M. bicolor LVR4069 formed structures

morphologically corresponding to ericoid mycorrhizas in vitro

on agar media, bi-directional nutrient transfer similar to that

observed by Grelet et al. (2009) needs to be demonstrated to

prove their ErM capabilities. Additionally, the incidence of C.

finlandica and M. bicolor in ericaceous roots under natural

conditions should be investigated to decide on ecophysio-

logical significance of their realized ericoid mycorrhizal

potential.

In the present study, Phialocephala-related DSE fungi repre-

sented the second most frequent group of EcM co-associated

fungi. These mycobionts generally inhabit living, decaying or

dead, mycorrhizal or non-mycorrhizal roots as well as stumps,

living stems, needles and fine or coarse woody debris (Menkis

et al. 2004, 2006; Gr€unig et al. 2009) although they seem to pre-

fer the cortex of primary roots and the periderm of lignified

roots of conifers (Gr€unig et al. 2008a). In the early period of

ectomycorrhiza research, DSE were often isolated from

healthy-looking roots or ectomycorrhizas (Mycelium radicis

atrovirens) and considered as true EcM fungi. However, inocu-

lation experiments showed that despite intraradical colo-

nization, they were not capable of forming EcM symbiosis and

often suppressed growth of the inoculated plants; hence their

early collective name “pseudomycorrhizal” fungi. Congruently,

none of the DSE isolates tested in the present study formed

ectomycorrhizaswith spruce seedlings. Instead, they colonized

spruce root cortex intracellularly, often formed typical micro-

sclerotia and the colonized roots morphology corresponded to

pseudomycorrhiza (Wang & Wilcox 1985). These results are in

agreement with a large body of literature showing neutral/

negative effects of P. fortinii-related DSE when inoculated in

non-mycorrhizal (but otherwise mycotrophic) coniferous

plants (e.g., Tellenbach et al. 2011). On the other hand, com-

parably less is known about physiological consequences of

their interactions with hosts possessing mycorrhizas. Based on

the few published experimental reports available, it appears

that they are neutral or rather negative (e.g., Hashimoto &

Hyakumachi 2001; Vohn�ık et al. 2005; Jones et al. 2009). While

in conifer plants from temperate regions most of the so far

investigated DSE seem to form harmful pseudomycorrhiza, the

effects of DSE may shift to positive in other plant guilds and/or

other climatic regions (e.g., Newsham 1999; Newsham et al.

2009). Additionally, the apparent ectomycorrhizal ability of

some DSE (Hashimoto & Hyakumachi 2001; Kaldorf et al. 2004)

deserves further attention. However, to our knowledge, A.

macrosclerotiorum is so far the only DSE observed to form ecto-

mycorrhizas in nature and at the same time confirmed to be an

EcM fungus in vitro (M€unzenberger et al. 2009).

In addition to the Phialocephala- andR. ericae-related isolates,

we obtained several other Helotiales with affinities to known

aquatic hyphomycetes (Articulospora tetracladia, V. elodeae, Neo-

bulgaria sp.) reported as common endophytes in submerged

aquatic plants (e.g., Kohout et al. 2012). Here, we obtained

aquatic species as endophytes in ectomycorrhizas but these

subsequently failed to colonize spruce and blueberry roots.

Further experiments, possibly in conditionsmore reflecting the

aquatic environment, are therefore needed to experimentally

confirm their endophytic lifestyle.

To conclude, our study showed that at least 36 % of appa-

rently healthy basidiomycetous ectomycorrhizas harboured

mostly ascomycetous endophytes. In contrast to our hypoth-

esis, the endophytic community was dominated by ericoid

mycorrhizal M. variabilis. The ericoid mycorrhizal potential of

this mycobiont was demonstrated in two independent trials

Meliniomyces variabilis in spruce ectomycorrhizas 291

where it formed characteristic intracellular structures in hair

roots and enhanced growth of an ericaceous host. The total

lack of the typical ErM fungus R. ericae in the screened ecto-

mycorrhizas suggests that these may represent selective

niches for certain ErM fungi andmay serve asmicro-reservoirs

of ErM inoculum in habitats lacking suitable ericaceous hosts.

It remains to be demonstrated where is the actual living space

ofM. variabilis inside the colonized ectomycorrhizas, andwhat

is the outcome of such colonization for the respective ecto-

mycorrhizas and consequently for the host plant.

Acknowledgements

This study was financially supported by the Czech Science

Foundation (GACR 206/09/P340) and the Czech Ministry of

Education, Youth and Sports (OC10058) and is a part of the

long-term research projects of the Institute of Botany ASCR

(RVO 67985939) and Charles University in Prague (SVV265203/

2012). Authors thank C. Gr€unig for identification of the Phia-

locephala-related isolates, O. Koukol for help with collection of

the field samples and A. Lep�sov�a for help with morphotyping

and permission to use her photos. Valuable comments of

anonymous reviewers and Kevin K. Newsham (editor) are

greatly appreciated. M.C.B. is grateful for a travel stipendium

from the Czech Ministry of Education, Youth and Sports

(AMB12AR014).

Supplementary material

Supplementary data related to this article can be found online

at http://dx.doi.org/10.1016/j.funeco.2013.03.006.

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