「森林総合研究所研究報告」(Bulletin of FFPRI) Vol. 22-No. 1 (No. 465) 13–16 March 2023

13

ノート (Note)
A semisolid plate method to isolate mycelia maintaining chlamydospore
formation in Tricholoma bakamatsutake
Hitoshi MURATA 1)*, Noritaka NAKAMURA 1), Akira OHTA 2) and Hiroyuki ICHIDA 3)
Key words: mutants, mushroom, matsutake
Introductionchange their mycelial morphology in the course of mycelial

Tr i c h o l o m a b a k a m a t s u t a k e i s a n e c t o m y c o r r h i z a ltransplantation to agar media, generally from “slow-growing

agaricomycete with fruiting bodies resembling those ofbrown” to “fast-growing white” mycelia, the latter being

Tricholoma matsutake, the prized mushroom known as

phenotypically stable on agar media, and do not become thematsutake, in Fagaceae forests (Ogawa 1978, Yamada et al.

brown ones in general. Brown mycelia display numerous

2014, Herrera et al. 2022). Certain T. bakamatsutake isolates

chlamydospores generated asexually from hyphae (Fig. 1; Kües

Fig. 1. Trichloma bakamatsutake hyphae and chlamydospores observed under a light microscope. A.
Brown mycelia of SF-Tf08 with numerous chlamydospores. These brown mycelia were regenerated
from the white mycelia using MMN+V8+0.3% SeaKem® GTG agarose™. B. White mycelia of SFTf08 without chlamydospores. C. Brown mycelia of SF-Tf09. D. Chlamydospores present in fast
growing hyphal sector area of SF-Tf09.
バカマツタケ菌糸体から厚壁胞子形成能を維持した菌株を選抜する半流動培地
村田 仁 1)*、中村 慎崇 1)、太田 明 2)、市田 裕之 3)
Received 1 November 2022, Accepted 6 December 2022
1) Department of Mushroom Sciences and Forest Microbiology, Forestry and Forest Products Research Institute (FFPRI)
2) Kansai Research Center, FFPRI
3) RIKEN Nishina Center for Accelerator-Based Science* Department of Mushroom Sciences and Forest Microbiology, FFPRI,
1 Matsunosato, Tsukuba, Ibaraki, 305-8687 JAPAN; E-mail: murmur@ffpri.affrc.go.jp
原稿受付：令和 4 年 11 月 1 日 原稿受理：令和 4 年 12 月 6 日
1) 森林総合研究所 きのこ・森林微生物研究領域
2) 森林総合研究所 関西支所
3) 理化学研究所 仁科加速器研究センター* 森林総合研究所 きのこ・森林微生物研究領域 〒 305-8687 茨城県つくば市松の里 1、E-mail: murmur@ffpri.affrc.go.jp

Hitoshi MURATA et al.

142000). In this study, we developed an agar culture method thatcomponents: MMN+V8 medium or the half-strength version ofallows for distinguishing brown and white mycelial colonies inthe medium, and 0.3% SeaKem® GTG agarose™.

a single white mycelium of T. bakamatsutake. This approach

T. bakamatsutake variants were isolated as follows. The

allowed for reisolating strains with the original phenotype, i.e.,

mycelia were cultured in MMN+V8+0.3% SeaKem ® GTG

slow-growing brown mycelia, from spontaneously occurring

agarose™ medium at 23°C until reaching a decent size, i.e.,variants, the fast-growing white mycelia, of T. bakamatsutake.

a diameter of 15 mm. The mycelia were then cut into pieces
with a sterile surgical scalpel (Feather No. 21) and observable

Materials and methods

mycelial pieces were removed from the agar medium. The

We used T. bakamatsutake strains SF-Tf05, SF-Tf08, andmedium was then supplemented with fresh MMN+V8+0.3%

SF-Tf09 as described previously (Murata et al. 2013a, b).

SeaKem® GTG agarose™ to fill the agar plate and incubated

Unless stated otherwise, these fungal strains were cultured

further at 23°C until mycelial colonies appeared.

in the MMN+V8 liquid medium or on MMN+V8 containing

The DNA analysis-based taxon identification was performed

1.5% agar at 23°C (Murata et al. 1999). Semisolid agar

using an rRNA gene internal transcribed spacer region (ITS1-plates to differentiate the original strains from phenotypic

5.8S-ITS2) as described previously (Gardes and Bruns 1993,

T. bakamatsutake variants were made of the following

Murata et al. 2013a, Aoki et al. 2022).
Results and discussion
T. bakamatsutake SF-Tf05 grew in the form of fully brown
mycelia, exhibiting the same apparent colony morphology asthat of the first fruiting body isolate in the MMN+V8 agar. Incontrast, SF-TF08 and SF-Tf09 grew as white mycelia andbrown mycelia with white aerial hyphae, which had originallygrown in the form of totally brown mycelia (Fig. 2, 3A,4A). T. bakamatsutake SF-Tf08 formed white mycelia withFig. 2. Trichloma bakamatsutake mycelial
morphology grown in theMMN+V8+1.5% agar slants.

uniform appearance and has never formed brown myceliaeither on MMN+V8+1.5% agar slants or plates (Fig. 2, 3A).
Therefore, the original strain SF-Tf08 exhibiting browncolony morphology isolated from the fruiting body of SF-Fig. 3. Trichloma bakamatsutake SF-Tf08
mycelial morphology. A. SF-Tf08 strain
grown in the MMN+V8+1.5% agar
plate. B. SF-Tf08 strain grown in theMMN+V8+0.3% SeaKem® GTG agarose™
plate. Note the appearance of brown(yellow arrows) and white (white arrows)
colonies. C-D. Strain SF-Tf08 reisolated
from the MMN+V8+0.3% SeaKem® GTGagarose™ plate; stable growth of brown(C) and white (D) mycelia.
森林総合研究所研究報告 第 22 巻 1 号, 2023

Fig. 4. Trichloma bakamatsutake SF-Tf09
mycelial morphology. A. SF-Tf09
strain grown in the MMN+V8+1.5%
agar plate. B. SF-Tf09 strain grown inthe MMN+V8+0.3% SeaKem® GTG
agarose™ plate. Note the appearanceof brown (yellow arrows) and white(white arrows) colonies. C-D. Growth ofstrain SF-Tf09 in the MMN+V8+0.3%
SeaKem® GTG agarose™ plate.

Isolating chlamydospore-forming T. bakamatsutake

Tf08 has no longer exists (Fig. 2). However, when culturing®

™

15(Fig. 5).

MMN+V8+0.3% SeaKem GTG agarose as described in theWhen T. bakamatsutake SF-Tf05 was cultured on thematerials and methods section, SF-Tf08 generated various

MMN+V8+0.3% SeaKem® GTG agarose™ plate, the brown

mycelial colonies that appeared to grow from chlamydospores

mycelial colonies predominantly appeared with a few white(Fig. 3B). Both brown and white mycelia isolated fromcolonies (Fig. 6). T. bakamatsutake SF-Tf09 exhibited the®

™

MMN+V8+0.3% SeaKem GTG agarose stably grew on thesame trend as SF-Tf08 by culturing MMN+V8+0.3% SeaKem®

standard MMN+V8 with 1.5% agar with seldom generating

GTG agarose™ (Fig. 4B–D). DNA analysis confirmed that allvariants (Fig. 3C, D).

representing isolates with the above-described traits were T.

Microscopic observations did not allow for detecting

bakamatsutake.

any chlamydospores in the white mycelia unlike the brown

When isolating mycelial colonies in 0.3% SeaKem ®

ones (Fig. 1). However, based on variant isolation from T.

GTG agarose ™ , the various observed T. bakamatsutake

bakamatsutake SF-Tf08 mycelia using MMN+V8+0.3%

morphological phenotypes could be attributed to an agar plateSeaKem® GTG agarose™, a small number of chlamydosporeswith pure agar that loosely solidified the medium, allowing thecould have potentially scattered in the white mycelia, giving

fungus to better form chlamydospores that spread that medium.

rise to both brown and white mycelial colonies. Notably, theThe described protocol is thus useful in isolating variants fromhyphae of T. bakamatsutake SF-Tf08 with brown mycelial

T. bakamatsutake strains and reisolating the original isolatecolony are quite thicker than that with white mycelial colony

from a subculture that changed its phenotypic trait. Henceforth,
the semisolid agar culture method may be useful in reisolating
T. bakamatsutake strains from the single mycelial colony thatmight behave differently among the population in association
with host plants and substrate cultivation for fruiting. In
fact, we are currently examining the difference between T.
bakamatsutake strains exhibiting brown mycelial colonies
and those exhibiting white ones on agar plate culture in termsof mycorrhiza synthesis and spawn cultivation for fruiting.
Furthermore, we isolated and characterized Tricholoma

Fig. 5. Trichloma bakamatsutake SF-Tf08 hyphae observed
under a light microscope showing that hyphae ofwhite mycelia are thinner than those of brown ones.
A. Hyphae of white mycelia. B. Hyphae of brownmycelia.

matsutake mutants generated by irradiation mutagenesis(Murata et al. 2019, 2021). The agar plate screening system
could also be useful in such analyses.
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