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Synthesis of Japanese Boletus edulisectomycorrhizae with Japanese red pine

Naoki Endo a,*, Fuminori Kawamura b, Ryoko Kitahara d,Daisuke Sakuma e, Masaki Fukuda a,b, Akiyoshi Yamada a,b,c

aDepartment of Bioscience and Food Production Science, Interdisciplinary Graduate School of Science and Technology,

Shinshu University, 8304, Minami-minowa, Nagano 399-4598, JapanbDepartment of Bioscience and Biotechnology, Faculty of Agriculture, Shinshu University, 8304, Minami-minowa,

Nagano 399-4598, JapancDivision of Rural Environmental and Symbiotic Science, Institute of Mountain Science, Shinshu University, 8304,

Minami-minowa, Nagano 399-4598, JapandGraduate School of Agriculture, Hokkaido University, Sapporo, Hokkaido 060-8589, JapaneOsaka Museum of Natural History, 1-23, Nagai-koen, Higashisumiyoshi-ku, Osaka, Osaka 546-0034, Japan

a r t i c l e i n f o

Article history:

Received 9 August 2013

Received in revised form

18 November 2013

Accepted 19 November 2013

Available online

Keywords:

Ectomycorrhizal morphology

In vitro mycorrhization

ITS phylogeny

Pinus densiflora

Porcini mushroom cultivation

* Corresponding author. Tel.: þ81 265 77 163E-mail address: caesareae@yahoo.co.jp (N

1340-3540/$ e see front matter ª 2014 The Mhttp://dx.doi.org/10.1016/j.myc.2013.11.008

a b s t r a c t

Boletus edulis is a well-known ectomycorrhizal mushroom. Although cultivation has been

widely attempted, no artificial fruiting has been achieved owing to difficulties associated

with mycorrhizal synthesis and acclimatization in fields. We collected fifteen B. edulis

basidiomata samples from locations in Japan and identified them microscopically and by

phylogenetic analysis of their nuclear ribosomal internal transcribed spacer (ITS) regions.

Pure culture isolates of B. edulis were established efficiently on malt extract agar medium,

and one isolate, EN-63, was inoculated to axenic Pinus densiflora seedlings in vitro. Brownish

ectomycorrhizal tips were observed on the pine lateral roots within four months of inoc-

ulation. Ten pine seedlings that formed ectomycorrhizae were acclimatized under labo-

ratory and greenhouse conditions. At four months after transplant, mycorrhizal

colonization by B. edulis was observed on newly grown root tips under laboratory condi-

tions, but no B. edulis ectomycorrhiza survived under greenhouse conditions. These results

suggest that B. edulis ectomycorrhizae synthesized in vitro with P. densiflora requires

additional steps for acclimatization to greenhouse conditions.

ª 2014 The Mycological Society of Japan. Published by Elsevier B.V. All rights reserved.

1. Introduction

Boletus edulis Bull. is a delicious edible mushroom known as

“king bolete”, “cep”, or “porcini” (Hall et al. 1998; Boa 2004; de

Roman and Boa 2004; Wang and Hall 2004; Arora 2008; Agueda

1; fax: þ81 265 77 1629.. Endo).ycological Society of Jap

et al. 2008a; Dentinger et al. 2010; de la Varga et al. 2012; Feng

et al. 2012). Boletus edulis belongs to the family Boletaceae in

the order Boletales and phylum Basidiomycota. It is naturally

andwidely distributed in the Northern Hemisphere, is present

in New Zealand as an exotic (Wang et al. 1995; Hall et al. 1998;

Dentinger et al. 2010; Feng et al. 2012), and forms

an. Published by Elsevier B.V. All rights reserved.

my c o s c i e n c e 5 5 ( 2 0 1 4 ) 4 0 5e4 1 6406

ectomycorrhizal associations with various tree taxa (Agerer

and Gronbach 1990; Hall et al. 1998; Meotto et al. 1999; Smith

and Read 2008; Agueda et al. 2008ab; de la Varga et al. 2012).

Related species such as B. reticulatus Schaeff. (¼B. aestivalis

(Paulet) Fr.), B. pinophilus Pilat & Dermek, and B. aereus Bull.,

also named porcini, are mushrooms with high commercial

value in Europe, North America, and China (Singer 1986; Hall

et al. 1998; Sitta and Floriani 2008; Agueda et al. 2008a;

Dentinger et al. 2010; Feng et al. 2012). The wholesale price

of fresh porcinimushrooms in the USwas aroundUS $60/kg in

2009 and reached a price of US $200/kg (Dentinger et al. 2010).

Over the past 40 years, many attempts to synthesize

porcini ectomycorrhizae have been made in the course of

mushroom cultivation trials (Hall et al. 1998; Agueda et al.

2008a). Froidevaux and Amiet (1975) first reported the syn-

thesis of B. edulis ectomycorrhizae with Pinus mugo Turra,

Tozzi et al. (1980) reported the synthesis of mycorrhizae with

Quercus pubescens Willd. Molina and Trappe (1982a,b) synthe-

sized B. edulis ectomycorrhizae with eight plant species

including Arbutus menziesii Pursh, Arctostaphylos uva-ursi (L.)

Spreng., Larix occidentalis Nutt., Picea sitchensis (Bong.) Carr.,

Pinus contorta Dougl. ex Loud., P. ponderosa Dougl. ex Laws., P.

monticola Dougl. ex D. Don, and Tsuga heterophylla (Raf.) Sarg.,

demonstrating its wide host range. Poitou et al. (1982) syn-

thesized ectomycorrhizae of B. edulis and B. aereus with Pinus

radiata D. Don and compared morphological characteristics

between the two species. Dunabeitia et al. (1996) established B.

pinophilus ectomycorrhizae with P. radiata seedlings by inoc-

ulation with spore suspensions at concentrations of 106e107

spores per plant. Meotto et al. (1999) succeeded in the ecto-

mycorrhization of B. edulis with European chestnut (Castanea

sativa Mill.) using mycelial cultures and maintained its

mycorrhizal status under field conditions for 6 years. Agueda

et al. (2008a) succeeded in themycorrhization of three porcini,

B. edulis, B. reticulatus, and B. aereus, in vitro with rockrose

hosts (Cistus ladanifer L., and C. albidus L.). However, almost no

successful instances of porcini fruiting after the outplanting of

established mycorrhizal seedlings have been reported, with

the exception of the accidental introduction of B. edulis fruits

under oak stands in New Zealand (Hall et al. 1998). The only

know experimental fruiting of porcini was the formation of B.

reticulatus primordium on nutrient agar medium without a

host (Yamanaka et al. 2000).

Morphological and anatomical characteristics have been

described for naturally established B. edulis ectomycorrhiza on

Picea abies (K.) Karst., C. ladanifer, and C. albidus hosts

(Gronbach 1988; Agerer and Gronbach 1990; Franz and Acker

1995; Agueda et al. 2006, 2008a,b). Mycorrhizal tips were

white to yellowish, becomingmore yellowwith age. The outer

and middle mantle layers were plectenchymatous ring-like

arrangements of hyphal bundles (Type A; Agerer 1991), the

inner mantles were arranged with broad streaks of parallel

hyphae, the rhizomorphs were highly differentiated, thick

hyphae formed most of the core, and the septa were often

partially or completely dissolved (Type F; Agerer 1991).

Although other common hosts of B. edulis such as Pinus spp.

are known, limited morphological data on mycorrhizae for-

mation on pine are available (Ortega-Martınez et al. 2011;

Martınez-Pena et al. 2012; de la Varga et al. 2012). Accumu-

lated morphological and anatomical data may be valuable for

testing mycorrhizal synthesis with candidate hosts for

mushroom cultivation trials (Yamada et al. 2001ab, 2014;

Fangfuk et al. 2010; Murata et al. 2013ab). Japanese red pine,

Pinus densiflora Sieb. et Zucc., is a well-known host for in vitro

ectomycorrhization (Kawai 1997; Yamada et al. 2001a, b, 2007,

2010; Fangfuk et al. 2010; Endo et al. 2013). However, the

combination of this species and porcini mushrooms has not

been tested in vitro.

In this study, we focused on the taxonomy of Japanese

porcini, especially B. edulis, because of previous confusion in

distinguishing between B. edulis sensu lato and B. reticulatus

(Kawamura 1908, 1929, 1954; Imazeki and Hongo 1957; Ito

1959; Imazeki et al. 1970), although both of which (in the

strict sense) has been reported to be phylogenetically distant

in other geographic samples (Leonardi et al. 2005; Dentinger

et al. 2010). In fact, B. reticulatus occurs commonly in Japan,

and B. edulis sensu stricto was recently reported in northern

and subalpine areas, but comprehensive comparative data for

the two species have yet to be reported. Therefore, we aimed

to identify Japanese porcini specimens based on morphology

and phylogenetic analysis of ribosomal DNA sequences. We

also aimed to establish B. edulis ectomycorrhiza in vitro using

cultures of Japanese origin, and then sustain its mycorrhizal

status under non-sterile conditions. To date, only European

and North American populations of this fungal species have

been tested for mycorrhization despite the probable circum-

boreal distribution of this fungus in nature. Comparing

mycorrhizal characteristics of B. edulis obtained from Japan

and other geographic regionswill lead to better understanding

of the biology of this fungus.

2. Materials and methods

2.1. Basidiomata collection and morphologicalidentification

Basidiomata of B. edulis and B. reticulatus were collected from

Nagano, Yamanashi, Osaka, and Hokkaido, Japan, during

2003e2012 (Table 1). Fifteen samples of B. eduliswere collected

from Abies, Quercus, Betula, and Fagus forests, and four sam-

ples of B. reticulatus were collected from Quercus and Pinus

forests. All samples were tested using the fungal isolation

procedure described below then freeze-dried, heated at 70 �Covernight, and stored in the laboratory as specimens. All

specimens were then deposited to the herbarium of National

Museumof Nature and Science, Japan (TNS) or OsakaMuseum

of Natural History, Japan (OSA). Morphological identification

of specimens was according to the descriptions of Nagasawa

(1989), Munoz (2005), and Korhonen et al. (2009). For mea-

surement and observation of basidiospores, a small portion of

hymenial tubes from each specimen was immersed in a drop

of 70% ethanol for 1 min at room temperature, transferred to

distilledwater to remove the ethanol, andmountedwith lactic

acid on a glass slide. For the observation of pileipellis hyphal

structure, a small portion of dried pileipellis from each spec-

imen was hand-sectioned with a razor and mounted on slides

by the same method used for hymenial tubes. Each slide was

observed under a differential interference contrast (DIC) mi-

croscope (AXIO Imager A1, Carl Zeiss, Inc., Gottingen,

Table 1 e Boletus specimens collected from Japan.

Specimenname

Sampling data DDBJ accessionnumber of ITSsequences

Specimen numberin the herbariumc

Date Site Forest canopyvegetationb

B. edulis KS209 21 Sep 2006 Mt. Norikuradake, Matsumoto, Nagano Qc, Bp AB821446 TNS-F-55572

B. edulis S-40a 20 Aug 2009 Mt. Yatsugatake, Chino, Nagano Av AB821447 TNS-F-55573

B. edulis S-41a 20 Aug 2009 Mt. Yatsugatake, Chino, Nagano Av TNS-F-55574

B. edulis S-42a 20 Aug 2009 Mt. Yatsugatake, Chino, Nagano Av TNS-F-55575

B. edulis S-50a 23 Aug 2009 Mt. Yatsugatake, Hara, Nagano Av, Ah AB821448 TNS-F-55576

B. edulis S-115a 8 Aug 2010 Mt. Yatsugatake, Chino, Nagano Av AB821449 TNS-F-55577

B. edulis S-229a 18. Jul 2011 Mt. Norikuradake, Matsumoto, Nagano Av, Qc, Bp AB821450 TNS-F-55578

B. edulis S-251a 15 Aug 2011 Mt. Yatsugatake, Chino, Nagano Av, Be AB821451 TNS-F-55579

B. edulis S-258a 9 Sep 2011 Makkari, Hokkaido As AB821452 TNS-F-55580

B. edulis S-293a 23 Jun 2012 Togakushi, Nagano Ah AB821453 TNS-F-55581

B. edulis S-297 22 Jul 2012 Takayama, Nagano Ah, Be, Fc AB821454 TNS-F-55582

B. edulis S-303-1 29 Aug 2012 Mt. Yatsugatake, Chino, Nagano Av AB821455 TNS-F-55583

B. edulis S-303-2 2 Sep 2012 Mt. Yatsugatake, Chino, Nagano Av AB821456 TNS-F-55584

B. edulis S-304a 30 Aug 2012 Mt. Fujisan, Fuji-yoshida, Yamanashi Av, Td AB821457 TNS-F-55585

B. edulis S-315a 15 Sep 2012 Mt. Norikuradake, Matsumoto, Nagano Bp, Av AB821458 TNS-F-55586

B. reticulatus S-4a 23 Jul 2009 Ooshika, Nagano Qs, Pd AB821459 TNS-F-55587

B. reticulatus S-17a 25 Jul 2009 Matsukawa, Nagano Qs, Pd AB821460 TNS-F-55588

B. reticulatus

Sakuma2003

27 Jun 2003 Izumi, Osaka Qs, Pd AB821462 OSA-MY3698

B. reticulatus

Shimono2005

17 Jul 2005 Minoo, Osaka Cc, Qg, Qs, Pd AB827926 OSA-MY4415

a Specimens tested for fungal isolation.b Ah: Abies homolepis, As: A. sachalinensis, Av: A. veitchii, Be: Betula ermanii, Bp: B. platyphylla var. japonica, Cc: Castanopsis cuspidata, Fc: Fagus crenata,

Pd: Pinus densiflora, Qc: Quercus crispula, Qg: Q. glauca, Qs: Q. serrata, Td: Tsuga diversifolia.c TNS: National Museum of Nature and Science, Japan, OSA: Osaka Museum of Natural History, Japan.

myc o s c i e n c e 5 5 ( 2 0 1 4 ) 4 0 5e4 1 6 407

Germany) with a �100 objective immersion lens. Because

Boletus species are generally difficult to identify (Munoz 2005;

Korhonen et al. 2009; Dentinger et al. 2010), we measured

100 basidiospores for length, width, and length/width (¼Q)

ratio in the following selected specimens all of which had fully

sporulated: B. edulis S-115, S-251, S-297, S-303-01, S-303-02, S-

304.

2.2. Fungal isolation

Eleven samples of B. edulis and two samples of B. reticulatus

were tested for fungal isolation (Table 1). The isolation pro-

cedure of Endo et al. (2013) was followed, with minor modifi-

cations. Fresh basidiomata surfaces were cleaned using

cotton moistened with 70% ethanol. The inner tissues were

removed in 5� 5-mmpieces with a blade andwere inoculated

ontomodified Norkrans’s C (MNC; Yamada and Katsuya 1995),

modified MelineNorkrans (MMN; Marx 1969), or malt agar

[MA; 20 gmalt extract (Difco, BD, Franklin Lakes, NJ, USA), 15 g

agar, 1000 ml distilled water] media. Fungus-inoculated agar

media plates were incubated at 20 � 2 �C for 2 mo. When

mycelial growth of the desired funguswas observed on a given

agar plate, themyceliumwas subcultured to a fresh plate with

MA or MNC to establish an isolate by cutting out a 5 � 5-mm

mycelial plug. Established pure culture isolates were sub-

cultured once onto malt yeast agar [MYA; 10 g malt extract

(Difco), 1 g yeast extract (Difco), 15 g agar, 1000 ml distilled

water] plates, stored as MYA slant cultures at 4 �C in a

refrigerator, and used for the following experiments.

2.3. Molecular identification of fungal species in thespecimens and established isolates

To identify the species in the specimens and established iso-

lates, sequences of internal transcribed spacer (ITS) region of

genomic ribosomal RNA gene (rDNA) were analyzed phylo-

genetically. The DNA extraction and polymerase chain reac-

tion (PCR) procedures of Fangfuk et al. (2010) and Endo et al.

(2013) were followed with minor modification. A 5 � 5-mm

plug of cultured mycelium from each agar plate or a portion

of dried parent basidiomata (ca. 50 mg) was tested. To amplify

the fungal ITS region by PCR, NS7 or ITS-1F was used as the

forward primer, and LB-W or Tw14 was used as the reverse

one, respectively (White et al. 1990; Gardes and Bruns 1993;

Taylor et al. 2003; Tedersoo et al. 2008). The PCR products

were purified using the QIAquick PCR Purification Kit (Qiagen,

Inc., Hilden, Germany). Cycle sequencing reactions were per-

formed on both forward and reverse strands using a BigDye

Terminator v. 3.1 Cycle Sequencing Kit (Life Technologies,

Inc., Carlsbad, CA, USA). Reactions were performed in 10 ml

with 4 ml purified PCR product, 1 ml of 1.6 mM primers (ITS-1,

ITS-2, ITS-3, ITS-4, or LB-W), 2 ml of 5� sequencing buffer, 1 ml

Dye Terminator Ready Reaction Mix, and 2 ml ultrapure water.

The reactions proceeded under the following conditions: 96 �Cfor 1 min, 25 cycles at 96 �C for 10 s, 50 �C for 5 s, and 60 �C for

4min. The reaction products (10 ml) were purifiedwith ethanol

and then sequenced using the ABI Prism 3100 Genetic

Analyzer (Life Technologies). The nucleotide sequences ob-

tained for each strand were assembled and complementarity

my c o s c i e n c e 5 5 ( 2 0 1 4 ) 4 0 5e4 1 6408

between the strands was confirmed. Full ITS sequences were

deposited in the DNA Data Bank of Japan (DDBJ; http://www.

ddbj.nig.ac.jp/). The DNA sequence were initially aligned

with MISHIMA v2.0.6 server (http://esper.lab.nig.ac.jp/study/

genome/?page¼mishima_server_stable) and manually

refined with Seaview v4.4.0 software (http://pbil.univ-lyon1.

fr/software/seaview.html). The refined alignment (725 bp)

was submitted to TreeBase (http://www.treebase.org/; acces-

sion no. S14457). Phylogenetic analyses were constructed by

Maximum Likelihood (ML) or Maximum Parsimony (MP) with

MEGA v5.1 software (Tamura et al. 2011) using the default

setting. Statistical support values were obtained using

nonparametric bootstrapping with 1000 replicates. The best

substitution model, Kimura 2-parameter (K2) þ Has Invariant

sites (I), was applied to the ML analysis. The trees generated

were rooted to Boletus variipes Peck. as outgroup. Sequences of

Japanese B. edulis specimens were compared with known se-

quences of European and North American B. edulis that were

used for taxonomic studies (Leonardi et al. 2005; Korhonen

et al. 2009; Dentinger et al. 2010; Feng et al. 2012).

2.4. In vitro mycorrhization

Boletus edulis isolate EN-63 (NBRC 109674; DDBJ accession no.

AB821449) obtained from specimen S-115 (Tables 1, 2) was

tested for mycorrhizal synthesis in vitro. Colony diameters of

B. edulis EN-63 on MYA plates grew ca. 17.0 mm/mo. A 10-ml

volume of MY liquid medium was poured into a 70-ml wide-

mouth glass bottle (no. 0323-05-83-01; TGK, Inc., Tokyo,

Japan) and autoclaved at 121 �C for 20 min. Mycelia of B. edulis

EN-63 growing on a MYA plate were axenically cut into 1 � 1-

cm plugs, four of which were inoculated into the liquid me-

dium tomake an inoculum formycorrhizal synthesis. Fungus-

inoculated bottles were incubated at 20 �C in the dark for

1e2 mo.

Seeds of P. densiflora were donated by the Ibaraki Prefec-

tural Forestry Institute and stored at 4 �C. Although P. densi-

florawas not the natural host of B. edulis EN-63, it was adopted

for mycorrhizal synthesis based on the following two reasons:

(1) pine species has been known to be susceptible with Euro-

pean B. edulis, and (2) P. densiflora is known to be associated

with wide range of basidiomycetous ectomycorrhizal fungi

in vitro (Yamada and Katsuya 1995; Yamada et al. 2001a, b,

2010; Endo et al. 2013). Seeds were transferred to a test tube

Table 2 e Established Boletus pure culture isolates.

Species and isolatea Medium

Specimen

B. edulis EN-47 MNC S-40

B. edulis EN-63 MA S-115

B. edulis EN-100 MA S-229

B. edulis EN-101 MA S-258

B. edulis EN-110 MA S-293

B. edulis EN-111 MA S-304

B. reticulatus EN-83 MNC S-4

B. reticulatus EN-84 MNC S-17

a In the ITS sequence of each isolate, please refer to the parent basidiomb NBRC: NITE Biological Resource Center, Japan.

containing autoclaved 0.01% polyoxyethylene sorbitan mon-

ooleate (Tween 80) solution and vortexed for several minutes

to wash the seed surfaces.Washed seedswere transferred to a

100-ml glass bottle containing 50 ml of 5% calcium hypo-

chlorite solution and stirred on a magnetic stirrer for 5 min to

sterilize seed surfaces. Sterilized seeds were rinsed three

times with autoclaved distilled water and then placed on

sterilized filter paper to remove excess water. Seeds were

transferred in groups of 10 to individual MNC agar plates. The

plates were incubated at 20 �C and illuminated continuously

at 140 mmol m�2 s�1 in a growth chamber (Eyela FLI-2000A,

Tokyo Rikakikai Co. Ltd., Tokyo, Japan) for 2 wk to germinate.

Sphagnum moss, previously minced with scissors, and

vermiculite were mixed at a 1:40 (v/v) ratio as a soil substrate,

and 1000 ml soil substrate was soaked with 600 ml intact or

dilutedMY liquidmedia containing 0.2%malt extract. A 75-ml

volume of prepared substrate was placed in a wide-mouth

glass bottle (no. 0323-05-83-02, 140-ml volume; TGK, Inc.)

with an attached transparent polycarbonate cap. An aeration

hole was punched in the cap and then sealed with a fluoro-

carbonmembrane filter (pore size 0.45 mm;Milliseal, Millipore,

Yonezawa, Japan). The glass bottles with substrate were

autoclaved at 121 �C for 20 min and cooled overnight before

fungal inoculation. Liquid-cultured mycelia (ca. 0.2 g fresh

weight) were washed with sterilized distilled water, dissected

into four to six segments, and dispersed throughout the sub-

strate in the glass bottle. A P. densiflora seedling axenically

germinated on an agar plate was transplanted into each glass

bottle. Five seedlings were placed in each nutrient condition

(total of ten seedlings). The bottleswere incubated at 20 �C and

illuminated continuously at 140 mmol m�2 s�1 in a growth

chamber (Eyela FLI-2000A) for 4 mo. A total of 5 ml sterilized

distilled water (autoclaved at 121 �C for 20min) was axenically

supplied to each bottle monthly to compensate for evapora-

tive water loss. Four months after inoculation, pine seedlings

were removed from the substrate in the glass bottles. The root

systems were gently washed with flowing tap water on a 0.56-

mm-mesh standard sieve. Substrate particles attached to the

pine roots were removed with a fine brush and fine forceps,

and the roots were washed with distilled water. Several root

tips colonized with mycelia were inspected using a micro-

scope as described below. The number of mycorrhizal tips in

each mycorrhized seedling was counted to estimate mycor-

rhizal colonization levels. Numerical data were statistically

Parent basidiomata NBRC numberb

Developmental stage

Immature 109785

Immature 109674

Immature 109786

Immature 109787

Immature 109788

Mature 109789

Mature 109790

Mature 109791

ata (Table 1).

myc o s c i e n c e 5 5 ( 2 0 1 4 ) 4 0 5e4 1 6 409

compared using Student’s t-test at P < 0.05 (KaleidaGraph ver.

3.6J, Hulinks, Inc., Tokyo, Japan) to evaluate differences be-

tween nutrient conditions for mycorrhizal synthesis.

2.5. Acclimatization of mycorrhizal seedlings under non-sterile conditions

Eight seedlings colonized with B. edulis EN-63 were trans-

planted to a straight-sided, polycarbonate-based, wide-mouth

jar (no. 2116-0250, 250-ml volume; Thermo Scientific, Inc.,

Rochester, NY, USA) filled with autoclaved soil. Another jar

was inverted and placed on top of the planted jar so that the

mouth of both the top and bottom jars were connected, then

sealed with vinyl tape. Four aeration holes were punched in

the top jar, each of which was sealed with a fluorocarbon

membrane filter (pore size 0.45 mm; Milliseal). Another two

pine seedlings colonized with B. edulis EN-63 were trans-

planted individually to clay pots (each 500 ml in volume) filled

with autoclaved soil. Granite-based soil was used for both

acclimatizations. The soil was sampled from the A and B

layers of the soil horizon in a P. densiflora forest in Ina, Nagano,

Japan and then sieved with a 5-mm-mesh standard sieve,

homogenized, and autoclaved at 121 �C for 60 min. The

mycorrhizal seedlings transplanted to the polycarbonate jars

were incubated at 23 �C with continuous light at

100 mmol m�2 s�1 in the laboratory. Distilled water was added

when the soil surface appeared to be dry. Water loss was

estimated by measuring the total weight of the polycarbonate

jar at watering. Clay pots containing transplantedmycorrhizal

seedlings were buried in the ground of the greenhouse in Dec

2011. Soil temperature in the greenhouse was controlled by

water flow (water temperature was 10e25 �C, depending on

the season) in a metal pipe that was set in the ground at a 5-

cm depth. Soil temperature ranged from 11.0 to 16.6 �C dur-

ing the incubation period. Light was not controlled in the

greenhouse. About 100 ml of tap water was added to the clay

pots almost daily. Four months after transplant (Apr 2012, in

the case of clay pot acclimatization), seedlings were removed

from bottles or pots, and the root systems were washed with

tap water. Several root tips colonized with mycelium were

inspected using a microscope as described below. Another

four or five acclimatized mycorrhizal tips were transferred to

a 1.5-ml microtube and stored at �65 �C for DNA analysis as

described below. The number of mycorrhizal tips on each

tested seedling was counted to estimate mycorrhizal coloni-

zation levels.

2.6. Molecular identification of mycorrhizal tips

To confirm whether ectomycorrhizae sampled from poly-

carbonate jars were colonized with the inoculated fungal

species, PCR-restriction fragment length polymorphism (PCR-

RFLP) analysis of the ITS region was conducted. The analysis

followed the procedure of Endo et al. (2013) with minor mod-

ifications. DNA was extracted from a 5 � 5-mm plug of

cultured EN-63 mycelium removed from an agar plate or four

ectomycorrhizal tips from each seedling. To amplify the

fungal ITS region specifically by PCR, ITS-1F and ITS-4 oligos

(Gardes and Bruns 1993) were used as forward and reverse

primers, respectively. A total of 4 ml of PCR product was

digested separately withHaeIII,HinfI, or EcoRI, according to the

manufacturer’s recommendations (TaKaRa Biochemicals,

Inc., Otsu, Japan). The choice of endonucleases used was

based on sequence data for the EN-63 isolate. Digested sam-

pleswere electrophoresed on 3% agarose gels (Nacalai Tesque,

Inc., Kyoto, Japan) for 1 h at 100 V (Mupid-2plus, Advance Co.

Ltd., Tokyo, Japan) and visualized by ethidium bromide

staining. The RFLP pattern of the mycorrhizal samples was

compared to that of cultured EN-63 mycelium.

2.7. Microscopic observations and descriptions ofectomycorrhizae

Mycorrhizal tips sampled and washed as described above

were submerged in a Petri dish filled with tap water, brushed

again to remove vermiculite/sphagnummoss substrate or soil

particles, and observed under a dissecting microscope (Stemi

2000C, Carl Zeiss). A fewmycorrhizal tips per in vitro seedling

or at least four tips per seedling in the acclimatized mycor-

rhizae were used for further microscopy as described below.

Additional microscopic observations were conducted under a

DICmicroscope (AXIO Imager A1) with�40 and�100 objective

lenses. Mycorrhizal tips were hand-sectioned with a razor

blade both transversally and longitudinally andmountedwith

lactic acid on a glass slide for microscopy. Ectomycorrhizal

structures, i.e., Hartig net and fungal mantle formations, were

observed in the sectioned mycorrhizal tips. The latter struc-

ture was further observed in “plan view” (Agerer 1987e2012,

1991) to measure the diameter of extraradical mycelium,

and the frequency of clamp connections in the acclimatized

mycorrhizae.

3. Results

3.1. Basidiomata collection and morphologicalidentification

Most B. edulis specimens in this study were collected from

boreal fir forests. External morphologies and colors of B. edulis

specimens (Fig. 1A, B) corresponded to the description of B.

edulis Bull. (Munoz 2005; Korhonen et al. 2009). Basidiospores

were observed in all specimens, but some had limited

numbers and were pale yellow in color due to immature

basidiomata. In mature basidiomata, basidiospores were

fusiform in shape, smooth, yellowish-brown in color, and

thick walled (Fig. 1C). Basidiospore sizes in mature specimens

S-297, S-303-01, S-303-02, and S-304 (mean length

15.7e18.3 mm, mean width 5.3e6.6 mm, mean Q ratio 2.5e3.1;

Supplement Table 1) corresponded to the descriptions of B.

edulis Bull. (Munoz 2005; Korhonen et al. 2009). Specimens S-

115 and S-251 were relatively young basidiomata, and the

immature basidiospores were pale yellow in color and smaller

in size (mean length 14.2e14.3 mm, mean width 4.9e5.5 mm,

mean Q ratio 2.6e2.9; Supplement Table 1). Pileipellis hyphae

of all B. edulis specimens examined were short, cylindrical or

slightly clavate, and very round at the tips, and the inter-

hyphal space was filled with gelatinous matrix (Fig. 1D). All B.

edulis specimens examinedweremorphologically identified as

B. edulis.

Fig. 1 e Morphological characteristics of Japanese B. edulis basidiomata. A: Basidioma of B. edulis S-297. B: Basidioma of B.

edulis S-303-02. C: Basidiospores of B. edulis S-303-02. D: pileipellis hyphae of B. edulis S-303-02. E: Basidiospores of B.

reticulatus S-4. F: Pileipellis hyphae of B. reticulatus S-4. Bars: 20 mm.

my c o s c i e n c e 5 5 ( 2 0 1 4 ) 4 0 5e4 1 6410

External morphological characteristics of the B. reticulatus

specimens examined corresponded to the descriptions of B.

reticulatus Schaeff. (Nagasawa 1989; Korhonen et al. 2009) and

B. aestivalis (Paulet) Fr. (Munoz 2005). Basidiospores were

fusiform in shape, smooth, yellowish-brown, and thick walled

(Fig. 1E). Basidiospore sizes of B. reticulatus specimens exam-

ined (Supplement Table 1) corresponded to the descriptions of

B. reticulatus Schaeff. (Nagasawa 1989; Korhonen et al. 2009)

and B. aestivalis (Paulet) Fr. (Munoz 2005). Pileipellis hyphae of

all B. reticulatus specimens examinedwere short, cylindrical or

slightly clavate, very round at the tip, and slightly pointed; no

gelatinous matrix was observed in the interhyphal space

(Fig. 1F). All B. reticulatus specimens examined were morpho-

logically identified as B. reticulatus (¼B. aestivalis).

3.2. Molecular phylogenetic analysis and speciesidentification

All Japanese B. edulis specimens examined grouped with Eu-

ropean and North American B. edulis specimens in a single

clade, which was distant from both B. pinophilus and B. retic-

ulatus clades (Fig. 2). The B. edulis clade was supported with

high ML and MP bootstrap values (100/99). All the B. edulis

sequences showed 99e100% homology when compared to the

full ITS sequence.

Boletus reticulatus specimens S-4 and S-17 in our study

grouped in a clade that was independent from the European B.

reticulatus clade (Fig. 2).

3.3. Establishment of pure culture for two Boletusspecies

Six B. edulis and two B. reticulatus isolates were established

from 11 and two tested specimens, respectively (Tables 1, 2).

Each of these isolates had ITS sequences identical to the

parent basidioma specimen, which was described in the note

of each deposited sequences in DDBJ. Most isolates of B. edulis

were obtained from young and immature basidiomata (Table

2). In contrast, two B. reticulatus isolates were obtained from

mature basidiomata, although immature samples were not

Fig. 2 e Phylogenetic tree of Boletus species based on ITS sequences. Topology was inferred from ML analysis. The two

values at each node represent the percentage of ML bootstrap support/MP bootstrap support. Each sample name following

species epithet is given as the specimen number in this study or the DDBJ or UNITE (http://unite.ut.ee/) accession number

with its locality: JP: Japan, SW: Sweden, SP: Spain, IT: Italy, DM: Denmark, FN: Finland, FR: France.

myc o s c i e n c e 5 5 ( 2 0 1 4 ) 4 0 5e4 1 6 411

tested for isolation. Boletus edulis isolates were established

efficiently on MA medium, but no mycelial growth from

inocula occurred on MMN medium (Table 3). The color of B.

edulis colonies on MYA plates ranged from white to pale yel-

low, and B. reticulatus colonies on MYA were white to pale

yellow or brownish-yellow.

3.4. In vitro mycorrhizal synthesis

Inoculated B. edulis EN-63 mycelia grew on both vermiculite

particles and sphagnum moss within a week, and growth on

pine lateral roots was observed within 2 mo of inoculation.

The growth was observable from outside the glass bottles.

Mycelia grew better on the soil substrate containing MY me-

dium than on that containing diluted MY. Although distinct

fungal mantle formation was not observed on lateral root tips

from outside the glass bottle within 4 mo after inoculation,

mantle formation was confirmedwhen the whole root system

was observed under a dissectingmicroscope. Ectomycorrhizal

formation was confirmed on all 10 pine seedlings tested; the

mean number of mycorrhizal tips per seedling was 63.4 with

diluted MY medium and 37.0 in MY medium. These mean

values were not significantly different (P ¼ 0.19).

Hartig net structures were confirmed in all mycorrhizal

samples under a DIC microscope (Fig. 3B). Mycorrhizal tips

were dichotomous and straight or slightly curved (Fig. 3A). The

mantle surface was smooth or loosely ramified and yellowish-

brown to reddish-brown (Fig. 3A). The mantle showed a

plectenchymatous ring-like pattern (Type A) without distinct

layer differentiation (Fig. 3C, D). The hyphal diameter of

extraradical mycelia was 2.5e5.5 mm. No clamp connections

were observed on hyphal septa. Rhizomorphs were not

observed.

3.5. Acclimatization of mycorrhizal seedlings

Five of eight mycorrhizal seedlings tested sustained B. edulis

ectomycorrhizae for 4 mo following transplant to

Table 3 e The isolation ratio of two Boletus species on three medium conditions.

Species Number of specimens produced a pure culture isolate/tested on the following media:

MNC MMN MA

Maturebasidiomata

Immaturebasidiomata

Maturebasidiomata

Immaturebasidiomata

Maturebasidiomata

Immaturebasidiomata

B. edulis NTa 1/9 NT 0/7 1/1 4/6

B. reticulatus 2/2 NT NT NT NT NT

a NT: Not tested.

my c o s c i e n c e 5 5 ( 2 0 1 4 ) 4 0 5e4 1 6412

polycarbonate jars. Colonization by the inoculated fungal

species was confirmed by PCR-RFLP analysis of ITS regions

(Fig. 4). Themean number of ectomycorrhizal tips per seedling

(N ¼ 8) was 36.0, which was less than but not significantly

different from that for in vitro seedlings (P ¼ 0.10). In the clay

pots, the mycorrhizal seedlings tested failed to sustain robust

ectomycorrhizal tips, and most were darkly discolored. The

newly developed lateral root tips were non-mycorrhizal.

Acclimatized mycorrhizal tips in polycarbonate jars were

dichotomous and straight or slightly curved. The mantle

surface was smooth or loosely ramified and white or silvery to

yellowish-brown in color (Fig. 5A) with a plectenchymatous

ring-like pattern (Type A) and without distinct layer differ-

entiation. It sometimes had a gelatinous matrix between the

hyphae (Type C) (Fig. 5BeD). Mantle mycelium sometimes

contained intracellular oily droplets. Hyphal surfaces of the

mantle surface mycelium and the extraradical mycelium

sometimes appeared granular (Fig. 5E). The hyphal diameters

of the extraradical mycelium were 3.0e5.7 mm. No clamp

connections were observed on any hyphal septa.

Fig. 3 e Morphological characteristics of B. eduliseP. densiflora e

morphology. B: Transverse section showing Hartig net. C: Outer

hyphae (hn), fungal mantle (fm), cortical cell (co), epidermal cel

Rhizomorphs were present, though not abundant, and were

whitish in color and 16.0e38.5 mm in diameter. The inner

structure was boletoid (Type F) with nodes, inflated cells, and

vessel-like hyphae (Fig. 5F). Hyphal surfaces of rhizomorphs

also sometimes appeared granular. No cystidia were observed

on either fungal mantles or rhizomorphs.

4. Discussion

This is the first report of the isolation of B. edulis Bull. in Japan.

Identification was supported by both microscopic character-

istics and phylogenetic analysis. Distribution of B. edulis sensu

stricto in East Asia was reported from China (Feng et al. 2012),

and identification was based on phylogenetic analysis. These

results suggest that B. edulis has circumboreal distribution in

the Northern Hemisphere. On the other hand, Japanese B.

reticulatus specimens collected in this study and identified by

microscopic observation as a single morphological species

consisted of two sister clades, both of which were different

ctomycorrhizae synthesized in vitro. A: External

mantle layer. D: Inner mantle layer. Abbreviation: Hartig net

l (ep). Bars: A 1 mm; BeD 20 mm.

Fig. 4 e RFLP patterns of the ITS region of B. edulis

ectomycorrhizae acclimatized in polycarbonate jars for

four months. Undigested PCR products (lanes 1, 2) and

digested samples with each of the three endonucleases

(lanes 3e8). My: mycorrhiza sample, Cu: cultured

mycelium, M: molecular ladder markers between 100 and

1000 bps.

Fig. 5 e Morphological characteristics of B. eduliseP. densiflora e

External morphology. B: Outer mantle layer. C: Inner mantle lay

Extraradical hyphae. F: A rhizomorph showing the inner vascu

myc o s c i e n c e 5 5 ( 2 0 1 4 ) 4 0 5e4 1 6 413

from the European B. reticulatus clade. Therefore, Japanese B.

reticulatus clades need further taxonomic study. Because we

analyzed only a limited number of specimens of Japanese B.

reticulatus, further analysis with additional specimens is

necessary to resolve this issue. In this study, Japanese B. edulis

was found in the subalpine mountain areas of the central and

eastern regions of Honshu and Hokkaido Islands. Above 2000-

m elevation on Mt. Yatsugatake in central Honshu Island, B.

edulis fruit bodies appeared inmidsummer to early autumn. In

Hokkaido, fruiting occurred in summer to late autumn. In

contrast, Japanese B. reticulatus occurs in the lowland

temperate areas of Honshu and Hokkaido Islands and fruits in

early summer to early autumn (Nagasawa 1989). Feng et al.

(2012) suggested that B. edulis clade fungi, i.e., B. edulis and B.

pinophilus, exhibit a geographic distribution associated with

low temperature that resulted from climate change after the

Eocene andOligocene periods. This hypothesis is applicable to

the Japanese B. edulis population but may not apply to Japa-

nese B. reticulatus.

ctomycorrhizae acclimatized in polycarbonate jars. A:

er. D: Inner mantle layer with intercellular matrix. E:

lar hyphae. Bars: A 1 mm; BeF 20 mm.

my c o s c i e n c e 5 5 ( 2 0 1 4 ) 4 0 5e4 1 6414

Pure culture of B. edulis is generally known to be difficult in

both its establishment and maintenance because mycelial

growth is slow on nutrient agar media and can be rapidly lost

(Wang et al. 1995; Hall et al. 1998). In this study, 11 Japanese B.

edulis specimens were tested for fungal isolation, and five

isolates were established on MA medium, all of which grew

when subcultured on MA and MYA media. Brundrett et al.

(1997) reported that Boletales mushrooms prefer malt-based

media such as MA and MMN. However, no B. edulis isolate

was established on MMN medium in this study. These results

suggest that some elements inMMNor its composition are not

suitable for isolation of Japanese B. edulis. However, Agueda

et al. (2008a) established isolates of B. edulis and three rela-

tive species, B. aereus, B. reticulatus, and B. pinophilus, on Baf

mediumwithoutmalt extract (Oort 1981). These differences in

the isolation success on a same medium condition suggest

that there are significant physiological differences between

Japanese and European B. edulis.

Host infections of B. edulis often ceasewhen infected plants

are transferred to non-sterile substrates (Hall et al. 1998). In

this study, synthesized ectomycorrhizae of B. edulis survived

on five of eight pine seedlings tested in polycarbonate jars for

4 mo. However, B. edulis ectomycorrhizae acclimatized under

greenhouse conditions disappeared during the same incuba-

tion period. These results suggest that B. edulis ectomycor-

rhizae synthesized in vitro with P. densiflora require additional

steps for successful acclimatization under greenhouse con-

ditions. Fluctuations in abiotic factors such as light and tem-

perature under greenhouse conditions may negatively affect

the ability of small pine seedlings to sustain B. edulis ecto-

mycorrhizae. Veselkov (1975) and Hall et al. (1998) reported

that B. edulis mycorrhizae synthesized by the inoculation of

non-sterile basidiospores were stable under non-sterile con-

ditions, unlike mycorrhizae established by pure culture inoc-

ulation, in spite of heavy contamination with other

ectomycorrhizal fungi and rhizospheric organisms. Specific

soil microflora may be required for stable B. edulis infection

(Hall et al. 1998), or some B. edulis individuals that are more

compatible to host or are tolerant under a given biotic and

abiotic stress may survive. Wu et al. (2012) reported that

inoculation with a mycorrhizal helper bacterium, Bacillus ce-

reus Frankland & Frankland, significantly increased ectomy-

corrhizal colonization by B. edulis. Therefore, helper bacteria

would be valuable for the acclimatization of in vitro B. edul-

iseP. densiflora system. Another factor in stable mycorrhizal

development of B. edulis on a host is the carbon balance in the

symbiosis. Boletus is generally considered a late-stage fungus

that produces fruit bodies in mature stands and is thought to

require a higher level of carbon supply to sustain the mycor-

rhizal system (Dighton andMason 1985; Ortega-Martınez et al.

2011; Martınez-Pena et al. 2012). Therefore, exposure of

mycorrhizal seedlings to high light intensity and/or high CO2

levelsmay support stablemycorrhizal development through a

higher level of photosynthesis and increased translocation of

sugars to roots (Danell 1994; Yamada et al. 2001a).

Boletus edulis ectomycorrhizae produced in vitro in this

study showed similarmantle structures to those on Cistus spp.

(Agueda et al. 2006, 2008ab) and Picea abies (Agerer and

Gronbach 1990; Franz and Acker 1995). Furthermore, the

acclimatized ectomycorrhizae in this study developed

rhizomorphs as in naturally occurring mycorrhizae (Agerer

and Gronbach 1990; Franz and Acker 1995). The synthesis of

B. edulis ectomycorrhizae on Japanese red pines reported here

conserved the morphological characteristics that occur under

natural conditions. In addition, the acclimatized mycorrhizae

sometimes showed a fully developed Type C mantle structure

with differentiated boletus-type rhizomorphs. This suggests

that Japanese red pine, P. densiflora, which is allopatric with B.

edulis in nature, is fully compatible with B. edulis in the

mycorrhizal morphogenesis. However, because Japanese B.

edulis was only found in boreal forests containing tree species

such as Abies spp. Q. crispula, and Betula spp., sympatric nat-

ural hosts may provide more stable and robust B. edulis ecto-

mycorrhizae even under experimental acclimatization

conditions.

In conclusion, we isolated Boletus edulis basidiomata from

various boreal forests in Japan and identified them morpho-

logically and phylogenetically. We established pure culture of

B. edulis and conducted ectomycorrhization with Japanese red

pine seedlings in vitro. The synthesized mycorrhizae were

acclimatized successfully under laboratory condition for four

months, and the mycorrhizal tips developed morphological

characteristics similar to naturally established ones. These

results suggest that Japanese B. edulis isolates can be used for

mushroom cultivation studies with native host plants as well

as Japanese red pine.

Acknowledgment

We thank Yuichi Taneyama, Mika Taneyama, Kohei Yama-

moto, and Muneyuki Ohmae for donation of basidiomata

samples.

Appendix A. Supplementary data

Supplementary data related to this article can be found at

http://dx.doi.org/10.1016/j.myc.2013.11.008.

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