Mycorrhiza DOI 10.1007/s00572-014-0584-5

ORIGINAL PAPER

Culturable fungal endophytes in roots of Enkianthus campanulatus (Ericaceae) Keisuke Obase & Yosuke Matsuda

Received: 15 February 2014 / Accepted: 21 April 2014 # Springer-Verlag Berlin Heidelberg 2014

Abstract Roots of plants in the genus Enkianthus, which belongs to the earliest diverging lineage in the Ericaceae, are commonly colonized by arbuscular mycorrhizal (AM) fungi. We documented the community of fungal root endophytes associated with Enkianthus species using a culture-based method for better understanding the members of rootcolonizing fungi, except for AM fungi. Fungal isolates were successfully obtained from 610 out of 3,599 (16.9 %) root segments. Molecular analysis of fungal cultures based on ribosomal internal transcribed spacer (ITS) sequences yielded 63 operational taxonomical units (OTUs: 97 % sequence similarity cutoff) from 315 representative isolates. Further phylogenetic analysis showed that most (296 isolates) belonged to Ascomycota and were either members of Helotiales (Dermataceae, Hyaloscyphaceae, Phialocephala and Rhizoscyphus ericae aggregate), Oidiodendron, or other Pezizomycotina. Twenty-three out of 63 OTUs, which mainly consisted of Leotiomycetes, showed high similarities with reference sequences derived from roots of other ericaceous plants such as Rhododendron. The results indicated that Enkianthus houses variable root mycobionts including

Electronic supplementary material The online version of this article (doi:10.1007/s00572-014-0584-5) contains supplementary material, which is available to authorized users. K. Obase (*) Department of Plant Pathology, University of Florida, 2523 Fifield Hall, Gainesville, FL 32611-0680, USA e-mail: [email protected] K. Obase e-mail: [email protected] Y. Matsuda Laboratory of Forest Pathology and Mycology, Graduate School of Bioresources, Mie University, Kurimamachiya 1577, Tsu, Mie 514-8507, Japan

putative endophytic and mycorrhizal fungi in addition to AM fungi. Keywords Endophyte . Ericaceous plant . Evolution . Molecular identification . Mycorrhiza

Introduction Plants harbor a wide variety of fungi in their healthy roots (Vandenkoornhuyse et al. 2002; Schulz et al. 2006). The vast majority of terrestrial plants are associated with mycorrhizal fungi (Wang and Qiu 2006), but simultaneously, their roots are colonized by other diverse fungi such as dark septate endophytes (DSE) (Jumpponen and Trappe 1998). Relationships of plants with root fungal endophytes range from facultatively pathogenic to neutral to mutualistic (Schulz et al. 2006). The ecological and physiological interactions between hosts, fungal root endophytes, and other sympatric root mycobionts (including mycorrhizal fungi) are still poorly understood. Nonetheless, endophytic fungal diversity in roots is likely a significant factor that influences the establishment of host plants by affecting mineral nutrient uptake from the soil by hosts (Jumpponen et al. 1998; Mandyam and Jumpponen 2005) and protecting plants from biotic and abiotic stresses (Redman et al. 2002; Schulz et al. 2002). The Ericaceae, which comprises 8 subfamilies, 126 genera, and 3,995 species (Stevens 2014), includes plants that form several distinct types of mycorrhizal associations with fungi (Fig. 1). Recent studies revealed that each lineage within the Ericaceae hosts their own unique yet diverse assemblages of fungal root endophytes. In arbutoid or monotropoid mycorrhizal roots of plants in the subfamily Monotropoideae, Basidiomycota such as Russulaceae, which form ectomycorrhizas (EcMs) in roots of adjacent woody plants, are the dominant fungal symbionts (e.g., Hynson and Bruns

Mycorrhiza Fig. 1 The occurrence of mycorrhizal types (AM arbuscular mycorrhizal, AtM arbutoid mycorrhizal, MtM monotropoid mycorrhizal, ErM ericoid mycorrhizal) (Katenin 1964; Wang and Qiu 2006) in the phylogeny of the Ericaceae and other closely related Ericalean families Clethraceae and Cyrillaceae (Steven 2014)

2009; Matsuda et al. 2011, 2012). Members of the subfamily Arbutoideae form arbutoid mycorrhizas and associate with a wide variety of EcM fungi (Krpata et al. 2007; Kennedy et al. 2012). On the other hand, members of the subfamilies Cassiopoideae, Ericoideae, Harrimanelloideae, Styphelioideae, and Vaccinioideae have fine hair roots and form ericoid mycorrhizas (ErMs) (Katenin 1964; Wang and Qiu 2006). These plants harbor the ascomyceteous members of Helotiales (e.g., Berch et al. 2002; Vrålstad et al. 2002; Zhang et al. 2009) and Oidiodendron (e.g., Couture et al. 1983; Zhang et al. 2009) as well as basiodiomycetes members of the Sebacinales in their ErM roots (Allen et al. 2003; Bougoure et al. 2007; Selosse et al. 2007). Recent studies demonstrated that several species in the genus Vaccinium and/ or Calluna form ErM-like associations with basidiomycetes that are otherwise considered to be EcM fungi (Villarreal-Ruiz et al. 2012) and non-EcM basidiomycete with affinities to Trechisporales (Vohník et al. 2012). By contrast, plants in the genus Enkianthus, which belongs to the earliest diverging lineage (subfamily Enkianthoideae) that represents the sister group of all other ericaceous taxa (Kron et al. 2002) (Fig. 1), have relatively thick fine roots (approximately 0.20–0.25 mm in diameter) and form relationships with soil fungi that form hyphal coil-like structures (Zhuang and Chan 1997; Abe 2005; Fukuchi et al. 2011; Obase et al. 2013) as well as vesicular-like hyphal structures in roots (Fukuchi et al. 2011; Obase et al. 2013). A recent molecular phylogenetic analysis of root-associated fungi from Enkianthus campanulatus showed ubiquitous colonization by members of Glomus spp. (Obase et al. 2013), which belong to the Glomus group A lineage in the Glomeromycota (Schüßler et al. 2001). Thus, hyphal structures that have been observed in the previous reports (Zhuang and Chan 1997; Abe 2005;

Fukuchi et al. 2011; Obase et al. 2013) appeared to include arbuscular mycorrhizas (AMs) of the Paris type (Gallaud 1905; Smith and Smith 1997). However, the previous study used AM-specific PCR primers for the detection of root endophytes (Obase et al. 2013), and thus, it is possible that other fungal root endophytes besides AM fungi may colonize the roots of Enkianthus. In this study, we examined culturable fungi colonizing the roots of plants in the genus Enkianthus to improve our understanding of the root-associating fungal biota of this group. We isolated the fungi colonizing surface sterilized roots of several Enkianthus species obtained from four sites located in different regions of Japan. We identified the fungal isolates based on barcoding of the internal transcribed spacer (ITS) region of the nuclear ribosomal DNA (Schoch et al. 2012). We hypothesized that, like other ericaceous plants, Enkianthus would harbor a wide variety of root endophytes, including putatively EcM or ErM fungi.

Materials and methods Site descriptions and sampling procedures Between August 2011 and July 2012, roots of four Enkianthus species (E. campanulatus, E. sikokianus and E. perulatus, and E. cernuus f. rubens) were collected from 1–7 tree individuals in four study sites in central Japan (Kyoto, Mie, Shiga, and Tochigi) (Table 1). Root samples collected in 2011 were the same set of roots collected in the previous study (Obase et al. 2013). All trees were mature (>2 m tall) and planted or transplanted 10–40 years ago. Study sites in Kyoto and Tochigi are in botanical gardens. Shiga is a forest park in a

Mycorrhiza Table 1 Numbers of isolates included in each OTU (n), frequencies of trees colonized by each OTU, and host plant species that harbored rootcolonizing fungi, showing high similarity (more than 98 %) and coverage (more than 90 %) with each OTU in BLAST search OTU

Accession no. of representative isolates

N

Frequencies of seedlings colonized by each OTUa Ec K (/1)

M (/1)

T (/6)

S11' (/7)

S12' (/6)

Ascomycota Chaetosphaeria Chloridium Cladophialophora Conlarium Cryptosporiopsis

AB846972 AB846979 AB847068 AB847000 AB847002

1 4 1 1 10

Cylindrocarpon

AB846995

2

Dermataceae Dothideomycetes 1 Dothideomycetes 2 Dothideomycetes 3 Dothideomycetes 4 Dothideomycetes 5 Dothideomycetes 6 Dothideomycetes 7 Eurotiomycetes 1 Eurotiomycetes 2 Eurotiomycetes 3 Eurotiomycetes 4 Eurotiomycetes 5 Helotiales 1

AB847060 AB847071 AB847078 AB846998 AB847012 AB847026 AB847028 AB846962 AB847013 AB847061 AB847017 AB847052 AB847065 AB846988

6 11 1 2 1 1 1 3 25 1 4 1 1 47

Helotiales 2 Helotiales 3

AB847064 AB847010

5 10

Helotiales 4

AB847075

3

Helotiales 5 Helotiales 6 Helotiales 7

AB847058 AB847076 AB847035

1 5 1

Helotiales 8 Herpotrichiellaceae 1 Herpotrichiellaceae 2 Ilyonectria

AB847059 AB847033 AB847008 AB847001

1 1 4 1

Lachnum 1 Lachnum 2

AB846994 AB847038

15 2

Lachnum 3

AB847020

1

Leohumicola Leptodontidium

AB847009 AB846993

5 3

1 2

Lulwoana Oidiodendron 1

AB847044 AB847062

1 16

3

Oidiodendron 2 Phialocephala

AB846983 AB846999

6 48

Host plants speciesb,c

Es

Ecr

Ep

M (/3)

M (/1)

M (/3)

S12' (/1)

1 2 1 1

1 2

1

1

1

2 1 1 1 1

1 1

2 1

1 1

1

1 1

2

3

2 1

1 1 1 3

1

1

1

1 1 1

1 1

1 6

1

3 3

1

1

1

1 1 2

1

1 1 1 1

2 1

1

1

1

4

1 1 1 3 1

1

1

2

4

2 5

1

3

1

2

1 1

Sc Acb, Rhl Pia, Polsp – Abb, Casp, Dro, Epm, Epp, Fre, Fov, Gas, Lep, Lis, Pia, Pit, Polsp, Potre, Psa, Rhf, Rhsp, Sam, Vam, Vao Acr, Ansp, Ap, Cysp, Fisp, Non, Org, Pis, Pit, Piss, Pod, Pot, Prp Acb, Bas, Cav, Polsp, Rhf, Vav – Dea, Lid Acr – – – – Piss, Rhf, Rhsp – Dea, Org – – Fas, Lis, Pia, Pima, Pyj, Qua, Qup, Rhok, Scsm Dsp, Rhf, Rhl, Rhsp, Piss Cab, Gofl, Gop, Orsp, Pinsp, Psa, Rhf, Rhl, Rhsp Dro, Epm, Ors, Pia, Pic, Pim, Psa, Polsp, Posp, Pyr, Pysp, Rhf, Scsm, Tsh, Vam Caf Cab, Rhl Caf, Cil, Epm, Epp, Hoc, Lep, Lol, Pitab, Rhl, Scsc, Wop – – Rhsp Cel, Epm, Eph, Epa, Gam, Gasi, Kosp, Lid, Noc, Orsp, Pag, Paq, Pia, Pis, Poa, Pod, Pot, Pop, Pysp, Rhl Dea, Rhl, Rhsp Bee, Casp, Dea, Def, Dro, Epm, Kosp, Lid, Nys, Pip, Pot, Psa, Pysp, Tre, Vam Casp, Kosp, Polsp, Posp, Rhf, Pysp, Vav, Vasp Bar, Epm, Era, Gas, Ise, Psa, Pysp, Tsh Ap, Casp, Cel, Epip, Epm, Eph, Epa, Kosp, Liu, Pea, Pia, Pis, Plh, Ptn, Pod, Pot, Psa, Raa, Rhd, Sac, Sai, Pia, Pig, Pis, Pot, Qu, Sua Anp, Cad, Cav, Cya, Pima, Pip, Pit, Psa, Qup, Rhf, Rhl, Rhok, Rhsp, Scsm, Vac, Vam Epp, Gap, Lol, Poa, Rhd, Wop Abb, Ben, Bep, Cya, Emn, Fas, Kosp, Lid, Pia, Pig, Pisp, Picl, Pip, Pis, Pitab, Polsp, Posp, Pyp, Rhf, Vav

Mycorrhiza Table 1 (continued) OTU

Accession no. of representative isolates

N

Frequencies of seedlings colonized by each OTUa Ec K (/1)

Rhizoscyphus ericae aggregate 1 Rhizoscyphus ericae aggregate 2 Sordariomycetes 1 Sordariomycetes 2 Sordariomycetes 3 Sordariomycetes 4 Sordariomycetes 5 Sordariomycetes 6 Sordariomycetes 7 Sordariomycetes 8 Basidiomycota Agrocybe Atractiellales Auriculariales Galerina Mycena 1 Mycena 2 Rectipilus Russulales Stephanosporaceae Mucoromycotina Mucoromycotina 1 Mucoromycotina 2 Mucoromycotina 3 Mucoromycotina 4 Mucoromycotina 5

AB847066

8

AB847027

21

AB847072 AB847081 AB847015 AB847034 AB847039 AB846980 AB847036 AB846990

5 1 2 1 1 1 2 1

AB846974 AB847006 AB847046 AB846961 AB846991 AB847043 AB847016 AB846966 AB846964

1 1 1 1 1 1 1 3

AB846969 AB846975 AB846960 AB846970 AB846971

2 1 1 1 3

M (/1)

T (/6)

1

S11' (/7)

S12' (/6)

1

4

2

4

Host plants speciesb,c

Es

Ecr

Ep

M (/3)

M (/1)

M (/3)

S12' (/1) Cya, Pia, Pima

2

Cav, Cya, Posp, Psa, Pym, Rhd – Rhl – – Pod – – –

4 1 1 1 1 1 1 1 1

1 1

Org, Piss, Pit, Pod, Pop Glm – Dro, Pip, Ror Cad – – –

1 1 1 1 1 1 1

1 1

1 1 1 1

1

1

– – – – –

a

Ec, Enkianthus campanulatus; Es, E. sikokianus; Ecr, E. cernuus f. rubens; Ep, E. perulatus; K, Kyoto; M, Mie; T, Tochigi; S11' and S12', Shiga in 2011 and 2012, respectively

b

Abb, Abies balsamifera; Acb, Acer barbatum; Acr, Acer rubrum; Anp, Andromeda polifolia; Ansp, Anthurium sp.; Ap, Apple; Bar, Bauera rubioides; Bas, Banksia spinulosa; Bee, Betula ermanii; Ben, Betula nana; Bep, Betula papyrifera; Cab, Cavendishia bracteata; Cad, Castanea dentata; Caf, Castanopsis fargesii; Casp, Carex sp.; Cav, Calluna vulgaris; Cel, Cephalanthera longifolia; Cil, Cistus laurifolius; Cya, Cypripedium acaule; Cysp, Cymbidium sp.; Dea, Decalepis arayalpathra; Def, Deschampsia flexuosa; Dro, Dryas octopetala; Dsp, Dendrobium sp.; Emn, Empetrum nigrum; Epa, Epipogium aphyllum; Eph, Epipactis helleborine; Epip, Epipactis palustris; Epm, Epacris microphylla; Epp, Epacris pulchella; Era, Erica arborea; Fas, Fagus sylvatica; Fisp, Ficus sp.; Fov, Forsythia viridissima; Fre, Fraxinus excelsior; Gam, Gaultheria mucronata; Gap, Gaultheria poeppigii; Gas, Gaultheria shallon; Gasi, Gastrodia similis; Glm, Glycine max; Gofl, Goodyera foliosa var. laevis; Gop, Goodyera procera; Hoc, Hoffmannseggella cinnabarina; Kosp, Kobresia sp.; Ise, Isoetes echinospora; Lep, Leptospermum polygalifolium; Lid, Lithocarpus densiflorus; Lis, Liquidambar styraciflua; Liu, Littorella uniflora; Lol, Lomandra longifolia; Noc, Nothofagus cunninghamii; Non, Nothofagus nervosa; Nys, Nyssa sylvatica; Org, Oryza granulate; Ors, Orthilia secunda; Orsp, Orchidaceae sp.; Pag, Panax ginseng; Paq, Panax quinquefolius; Pea, Persicaria amphibia; Pia, Picea abies; Pic, Pinus contorta; Picl, Pinus contorta var. latifolia; Pig, Picea glauca; Pim, Pinus muricata; Pima, Pinus massoniana; Pip, Pinus pinaster; Pis, Pinus sylvestris; Pinsp, Pinus sp.; Pisp, Picea sp.; Piss, Pisonia sechellarum; Pitab, Pinus tabulaeformis; Pit, Pinus taeda; Plh, Platanthera hyperborea; Poa, Pomaderris apetala; Polsp, Polygonum sp.; Pop, Poa pratensis; Posp, Potentilla sp.; Pod, Populus deltoides; Pot, Populus trichocarpa; Potre, Populus tremuloides; Pop, Populus deltoides x Populus nigra; Prp, Prunus persica; Psa, Pseudorchis albida; Ptn, Pterostylis nutans; Pyj, Pyrola japonica; Pym, Pyrola media; Pyp, Pyrola picta; Pyr, Pyrola rotundifolia; Pysp, Pyrola sp.; Qu, Quercus bicolor x Q. robur; Qua, Quercus alba; Qup, Quercus petraea; Raa, Ranunculus adones; Rha, Rhododendron argyrophyllum; Rhd, Rhododendron decorum; Rhf, Rhododendron fortunei; Rhl, Rhododendron lochiae; Rhok, Rhododendron obtusum var. kaempferi; Rhsp, Rhododendron sp.; Ror, Rosa rugosa; Sac, Salix caprea; Sai, Saussurea involucrata; Sam, Salvia miltiorrhiza; Sc, Sugarcane cultivar; Scsc, Schizachyrium scoparium; Scsm, Schizocodon soldanelloides var. magnus; Sua, Subularia aquatica; Tra, Triticum aestivum; Tre, Trientalis europaea; Tsh, Tsuga heterophylla; Vac, Vaccinium corymbosum; Vam, Vaccinium membranaceum; Vao, Vaccinium ovalifolium; Vav, Vaccinium vitis-idaea; Wop, Woollsia pungens c

Abbreviations were described in italics when their root-colonizing fungi were are derived from ectomycorrhizal root tips. Abbreviations of ericaceous plants and Schizocodon were underlined. Abbreviations were described in bolds when their root-colonizing fungi have been proved proven to form ericoid mycorrhizae with their hosts in previous studies.

Mycorrhiza

mountainous region dominated by tree species of Quercus, Fraxinus, and Picea. Mie is an arboretum planted with various Rhododendron spp. (Ericaceae). At each site, we traced the roots from the stem of each tree until fresh, white roots were visible. One root segment of 5– 10 cm in length was collected from three different points and then pooled together in each tree. Each root sample was composed of white, unsuberized roots. The distances between adjacent trees ranged from 2 m to several tens of meters. All samples were stored in plastic bags at 4 °C, transported to the laboratory within 12 h, and processed within 24 h of collection. Isolation of fungi from roots Roots were washed vigorously in running tap water over a 0.5-mm mesh sieve to remove adhering soil. Roots were then surface-sterilized in 30 % H2O2 for 5–7 min, soaked in two changes of sterilized distilled water, cut into 0.5-mm pieces, and then plated on full-strength potato dextrose agar (PDA) in 90-mm Petri dishes. Surface sterilization was effective at removing epiphytic fungi (Obase et al. 2010). Fungal isolates were incubated at 25 °C in the dark. Plates with bacteria or with rapidly growing fungi detected within 2 days were considered to be contaminants and discarded. Fungal cultures derived from a single species were subcultured whereas any cultures putatively included more than two fungal species were discarded. Isolates were grouped on the basis of gross morphology under the dissecting microscope (e.g., color, texture, and growth habit). One to three representatives from each morphotype from each tree were selected for molecular identification. Identification of fungal isolates DNA was extracted from fungal isolates (approximately 0.5× 0.5-cm-thin mycelia mats) with a DNeasy Plant Mini kit (Qiagen) according to the manufacturer’s instructions. We amplified the ITS region (ITS1-5.8 S-ITS2) using Ex Taq DNA polymerase (Takara) with primers ITS1 and ITS4 or with ITS1F and ITS4 (White et al. 1990; Gardes and Bruns 1993). The following PCR amplification condition with ITS1 and ITS4 was used: 95 °C for 3 min, followed by 30 cycles of 95 °C for 30 s, 52 °C for 30 s, and 72 °C for 2 min and then a final extension at 72 °C for 10 min. PCR with ITS1F and ITS4 was performed with the same regime except denaturing at 94 °C and annealing at 50 °C. Successful amplicons were cleaned with an Illustra GFX PCR DNA and Gel Band Purification Kit (GE Healthcare) and then sequenced bidirectionally with the relevant primer pairs using a BigDye Terminator v. 3.1 Cycle Sequencing Kit on an ABI3700 DNA sequencer (Applied Biosystems). The fungal DNA sequence data were submitted

to DDBJ under accession numbers AB846959 to AB847083 (Table S1). All unique sequences were subjected to BLAST searches (Altschul et al. 1997) against GenBank (http://blast.ncbi.nlm. nih.gov). Neighbor-joining (NJ) analyses were performed using datasets aligned by the CLUSTALX algorithm, with bootstrap analysis of 1,000 replications (Felsenstein 1985) in the MEGA 5 software (Tamura et al. 2011), in order to infer similarity among the isolates obtained from this study as well as known fungal taxa. In the NJ analysis, the evolutionary distances were computed by the maximum composite likelihood method (Tamura et al. 2004) and are presented as the number of base substitutions per site. All ambiguous positions within each sequence pair were removed with the pairwise deletion option. Percentage of sequence similarity was calculated between all pairs of DNA sequences obtained from this study using the MEGA 5 software package (Tamura et al. 2011). Fungal DNA sequences within 3 % nucleotide difference were categorized into a single operational taxonomic unit (OTU) (Smith et al. 2007). The OTUs with affinities to only one genus with high bootstrap values (>90 %) in the NJ trees were designated by the genus name. The OTUs with affinities to several genera within a family were designated by the family name. Similarity of fungal biota among tree species We conducted an analysis of similarity (ANOSIM) based on presence/absence data, to compare the biota of root endophytes among tree species. The ANOSIM was computed with 9,999 permutations and based on the Raup-Crick similarity index (Raup and Crick 1979). Tree individuals with low sampling efforts (less than 100 root segments used for fungal isolation) and of E. cernuus f. rubens (only one individual was examined) were removed from the analysis. Next, we conducted nonmetric multidimensional scaling (NMDS) based on presence/absence data and using the Raup-Crick similarity index, to understand the variations in the biota of root endophytes among tree individuals. Both analyses were performed using PAST (Hammer et al. 2001).

Results Molecular identification of fungal isolates From 3,599 root segments from all Enkianthus trees, 610 fungal isolates were successfully obtained (433 isolates from 1,999 root segments in E. campanulatus, 64 isolates from 600 root segments in E. sikokianus, 25 isolates from 200 root segments in E. cernuus f. rubens, and 88 isolates from 800 root segments in E. perulatus) (Table S2). For molecular analysis, 340 out of 610 isolates were analyzed, but PCR

Mycorrhiza

products could not be obtained from 23 isolates, and sequencing failed for two additional isolates. Of the 315 successful sequences, 125 representative sequences were used for the final analysis. BLAST and phylogenetic analysis identified all of the 315 ITS sequences to the subphylum or phylum level: Basidiomycota (n = 11), Ascomycota (n = 296), and Mucoromycotina (n = 8) (Figs. S1–S20). Among the Ascomycota, 117 isolates could be assigned to the genus level (e.g., Chaetosphaeria, Chloridium, Cladophialophora, Conlarium, Cryptosporiopsis, Ilyonectria, Lachnum, Leohumicola, Leptodontidium, Lulwoana, Oidiodendron, and Phialocephala). Twenty-nine isolates belonged to two different taxa in the R. ericae aggregate (Fig. 2). Eleven isolates could be assigned to the families Dermataceae and Herpotrichiellaceae, 73 could be assigned to the order Helotiales, and 66 isolates could be assigned to the class level (Dothideomycetes, Eurotiomycetes, or Sordariomycetes). Among the Basidiomycota, five isolates could be assigned to the genus level (e.g., Agrocybe, Galerina, Mycena, and Rectipilus). Three isolates could be assigned to the family Stephanosporaceae whereas the remaining three taxa could only be assigned to order level (Atractiellales, Auriculariales, and Russulales). Eight isolates within Mucoromycotina could not be assigned to the genus level. Overall, fungal isolates could be classified into 63 OTUs (Table 1). Frequency of isolation in each OTU OTUs of Phialocephala (48 isolates) and Helotiales 1 (47 isolates) were frequently isolated from Enkianthus (Table 1). Other frequently detected fungi outside of Phialocephala and Helotiales 1 include Eurotiomycetes 1 (n=25), R. ericae aggregate 2 (n=21), Oidiodendron 1 (n=16), Lachnum 1 (n= 15), Dothideomycetes 1 (n=11), Helotiales 3 (n=10), and Cryptosporiopsis (n=10). Thirty-three OTUs were represented by only a single isolate. Seven commonly occurring OTUs were isolated from both E. campanulatus and other Enkianthus taxa (Table 1). Phialocephala and Eurotiomycetes 1 were obtained from all four Enkianthus taxa and were commonly isolated at all of the E. campanulatus study sites except for Shiga despite relatively low sampling effort at some sites. Oidiodendron 1 was obtained only from Shiga whereas several other OTUs were obtained from more than two study sites.

L a c h n u m 2 a n d 3 ; O i d i o d e n d ro n 1 a n d 2 ; a n d Phialocephala) appear closely related to root-colonizing fungi derived not only from EcM root tips of herbaceous (Carex, Polygonum, and Potentilla) or woody plants (Abies, Betula, Castanopsis, Fagus, Nothofagus, Picea, Pinus, Pisonia, Pomaderris, Quercus, Tsuga) but also those from roots of Ericaceae (Calluna, Epacris, Gaultheria, Pyrola, Rhododendron, Vaccinium, and Woollsia). Four OTUs (Cylindrocarpon, Helotiales 5, Lulwoana, and R. ericae aggregate 1) were closely related to root-colonizing fungi derived from EcM root tips. Nine OTUs (Chloridium, Helotiales 3 and 6, Herpotrichiellaceae 2, Lachnum 1, Leohumicola, Leptodontidium, R. ericae aggregate 2, Sordariomycetes 2) were closely related to root-colonizing fungi derived from roots of ericaceous plants. Twenty-seven OTUs that were distantly related to any root-colonizing fungi were most closely related to fungi derived from soils, plant parts besides roots, or from fungal sporocarps. Seven OTUs (Cryptosporiopsis, Helotiales 1–4, Leohumicola, and Oidiodendron 1) were closely related to root-colonizing fungi that have been experimentally demonstrated to form ErMs in roots of Rhododendron species (Usuki et al. 2003; Zhang et al. 2009) (except for Leohumicola which forms ectoendomycorrhizal-like structures—see Bergero et al. (2000)). A root-colonizing fungus that was phylogenetically closely related to OTU Eurotiomycetes 1 did not form ErMs in roots of Rhododendron fortunei in inoculation study (Zhang et al. 2009). Similarity of fungal biota among tree species The ANOSIM detected significant but relatively weak dissimilarity in the taxa occurrence among three Enkianthus species E. campanulatus, E. sikokianus, and E. perulatus (R=0.26, p=0.018). However, the ANOSIM did not detect significant differences when data of rare OTUs that include only one isolate were removed from the analysis (R=0.094, p=0.23). When looking at the variability of the fungal biota among tree individuals using NMDS analysis, the plots of different species were intermingled and did not clearly separate each other in the ordination. The results were almost unchanged when data of rare OTUs were removed from the analysis (Fig. S21).

Discussion Similarity of each OTU with root-colonizing fungi Thirty-six OTUs showed high (≥97 %) similarity with reference sequences derived from root-colonizing fungi in taxonomically diverse herbaceous and woody plants (Table 1). Of these, 13 OTUs (Cryptosporiopsis; Dermataceae; Eurotiomycetes 1; Helotiales 1, 2, 4, and 7; Ilyonectria;

We identified representative fungal isolates obtained from inside surface-sterilized root segments of several Enkianthus species. The results indicated that Enkianthus not only hosts AM fungi (Obase et al. 2013) but also acts as the host for other diverse fungi, including DSE (e.g., Leptodontidium and Phialocephala) and putatively pathogenic fungi (e.g.,

Mycorrhiza

Fig. 2 Neighbor-joining phylogenetic tree based on ITS sequences of root-colonizing fungi (OTU Helotiales 8 and Rhizoscyphus ericae aggregates 1 and 2) obtained from Enkianthus collected in Japan. The tree is rooted with Mollisia cinerea (AJ430222) and Neocudoniella radicella

(AY524843). The matrix of the ITS regions of 43 fungal taxa contained a total of 549 positions in the final dataset. The optimal tree had a total branch length of 0.45. Bootstrap values higher than 80 % are indicated at the nodes (1,000 replications)

Cylindrocarpon and Ilyonectria). Although ecological, functional, and physiological relationships between Enkianthus species and the root mycobionts were not explicitly documented in this study, it is likely that the commonly detected fungi in the genus Oidiodendron and putative ErM fungi in Helotiales do form ErM with Enkianthus. However, a previous study found that E. campanulatus did not form ErM when inoculated with a putative member of the R. ericae aggregate (since DNA sequence data are not available for the fungal isolate, the identity of this fungus is uncertain) (Gorman and Starrett 2003). Therefore, even though we frequently detected

putative ErM fungi in the roots of Enkianthus species, inoculation tests are essential to determine whether or not these fungi form specific ErM hyphal structures in root cortical cells and enhance the growth of Enkianthus species. Indeed, recent studies have shown that several ErM fungi can colonize roots of non-ErM host plants as endophytes without forming mycorrhiza-like structures (Chambers et al. 2008; Vohník et al. 2013). In our previous study, Obase et al. (2013) found high colonization ratio of AM-like hyphal coils in Enkianthus species and also common and dominant colonization by a

Mycorrhiza

specific OTU of Glomus sp. along E. campanulatus individuals. In contrast, this study showed relatively low isolation ratio of root endophytes. Biota of root endophyte was likely similar among Enkianthus species, but relatively commonly observed taxa were Phialocephala and Eurotiomycetes 1, which are supposed to be endophytic and not mycorrhizal fungi. These findings indicated that the genus Enkianthus forms an intimate association with specific taxa of AM fungi, while association with other putative mycorrhizal fungi is likely rather uncommon. Low isolation ratio of root endophyte is also possibly and partly due to competence among sympatric root mycobionts (e.g., AM fungi vs other root endophytes) (Rillig et al. 2014), even though intense surface sterilization performed in this study would also negatively affect the isolation ratio of root endophytes. ITS barcoding of representative isolates (n=315) revealed that most (n=296) belonged to Ascomycota, which comprised 49 out of the 63 total OTUs. Of these, 239 isolates (22 OTUs) were closely related with root-colonizing fungi which have been detected in roots of other ericaceous plant species such as Rhododendron and Vaccinium. Most of the remaining OTUs (37 out of 41 OTUs) included only 1–3 isolates, indicating that they are rare fungi in Enkianthus roots. These findings inferred that biota of root fungal endophyte in Enkianthus is relatively similar with those of other ericaceous plants. On the other hand, the present study has shown R. ericae, which is one of the most common ErM fungi and colonizes a wide variety of ericaceous plants (e.g., Upson et al. 2007; Walker et al. 2011), to be absent in Enkianthus species. Two different taxa belonged to a R. ericae aggregate were clearly different from R. ericae (Fig. 2): the identity of R. ericae aggregate 1 was uncertain, while R. ericae aggregate 2 closely related to Hyaloscypha leuconica var. bulbopilosa (JN033451). It is likely that association with R. ericae is absent or very rare in Enkianthus species. Thus, there would be subtle but clear difference in fungal biota of root endophyte between Enkianthus and other ericaceous plants. However, it should be noted that methodological bias resulting from culturebased community analysis would cause low or lack of occurrence of slow-growing fungi such as R. ericae (Walker et al. 2011). Because Enkianthus is the earliest diverging lineage within Ericaceae, it is a key to understanding evolution of fungal root symbioses in this group of plants. Two hundred fourteen isolates belonged to Leotiomycetes (20 OTUs), and most were members of Helotiales (n=186, 17 OTUs). Frequent and dominant colonization by the members of Leotiomycetes, especially Helotiales and/or Oidiodendron, has often been documented in other ericaceous plants such as Calluna (Sharples et al. 2000; Bougoure et al. 2007), Cassiope (Walker et al. 2011), Empetrum (Walker et al. 2011), Epacris (Bougoure and Cairney 2005a; Curlevski et al. 2009), Gaultheria (Berch et al. 2002; Allen et al. 2003),

Rhododendron (Usuki et al. 2003; Bougoure and Cairney 2005b; Zhang et al. 2009; Tian et al. 2011; Sun et al. 2012), and Vaccinium (Bougoure et al. 2007; Ishida and Nordin 2010; Gorzelak et al. 2012; Walker et al. 2011). These plants belong to the subfamilies Cassiopoideae, Ericoideae, Styphelioideae, and Vaccinioideae (Fig. 1). Similar communities of root-colonizing fungi among many of the ericaceous plants that have been surveyed (including in species of Enkianthus of subfamily Enkianthoideae) suggest that the common ancestor of all ericaceous plants may have associated with similar communities of diverse ascomycete fungi. In contrast, plants in the Monotropoideae, such as Pyrola (Hynson and Bruns 2009; Toftegaard et al. 2010; Matsuda et al. 2012) and Monotropa (Young et al. 2002; Yang and Pfister 2006; Shen et al. 2012), and plants in the Arbutoideae, such as Arbutus (Richard et al. 2005) and Arctostaphylos (Krpata et al. 2007), are rarely colonized by ErM fungi and are instead typically colonized by diverse EcM fungi. This discrepancy between the fungi found in Monotropoideae and Arbutoideae and the fungi found with the remaining subfamilies in Ericaceae indicates specialization toward EcM fungi in these plant lineages after diversification from the common ancestor of sister groups of Enkianthoideae. This study examined the community of fungal root endophytes within species of Enkianthus using culture-based methods, which might represent a restricted subset derived from potential root fungal communities. Using direct methods such as cloning, recent studies revealed that there were several unculturable fungi such as Sebacinales (Allen et al. 2003) that also regularly colonize the roots of ericaceous plants (e.g., Bougoure and Cairney 2005a, 2005b; Oberwinkler et al. 2013). Indeed, our previous work indicated the prevalence of AM fungi in the Enkianthus species (Obase et al. 2013). Therefore, it is indispensable to further examine the comprehensive fungal assemblages for better understanding the rootfungal associations in this genus Enkianthus. Knowing more about the mycorrhizal status in this group should give further insights into the evolutionary trajectories of the mycorrhizal symbiosis (Moyersoen 2006; Yukawa et al. 2009), such as origin of EcM and ErM symbioses in the phylogeny of Ericaceae (Selosse et al. 2007). In this context, understanding the morphological and functional interactions between root endophytes and ericaceous plants would provide further clues for changes of fungal partners in the evolution of Ericaceae. Acknowledgments This study was carried out partly with the financial support of Grants-in-Aid for Scientific Research from the Ministry of Education, Science, Sports and Culture of Japan to YM (22688011, 23658124, and 25304026). We are grateful for sampling supports of Kyoto Botanical Gardens (Kyoto City, Kyoto Prefecture), Mie Prefecture Forestry Research Institute (Tsu City, Mie Prefecture), Mr. Keizo Kanazawa (Tsu City, Mie Prefecture), Dr. Hirosuke Oba (Nikko Botanical Gardens, Graduate School of Science, The University of Tokyo), and Ohmi-Fuji Karyoku Park (Yasu City, Shiga Prefecture), and for sequence

Mycorrhiza analyses of Ms. Tomiko Chikada (Life Science Research Center, Center for Molecular Biology and Genetics, Mie University). We specially thank Dr. Shin-ichiro Ito (Graduate School of Bioresources, Mie University) and the members of the Laboratory of Forest Pathology and Mycology, Mie University, for their assistance and Dr. Matthew E Smith for critical comments and editing the manuscript.

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