Ⅲ-4. Radiation Chemistry & Biology

RIKEN Accel. Prog. Rep. 52 (2019)

Pleiotropic mutant of plant-symbiotic edible mushroom Tricholoma
matsutake induced by argon-ion beam†
H. Murata,∗1 T. Abe,∗2 H. Ichida,∗2 Y. Hayashi,∗2 T. Yamanaka,∗1 T. Shimokawa,∗1 and K. Tahara∗1Tricholoma matsutake is an ectomycorrhizal fungus
that produces prized mushrooms “matsutake” in association with conifers.1) Currently, there are no fungal strains as useful in cultivating fruits as commercial
saprophytic edible mushrooms. Developing the strainsthat are suitable for spawn cultivation will greatly
contribute to the artiﬁcial cultivation of mycorrhizal
mushrooms. Previously, we reported that an argonion beam (40 Ar17+ , 95 MeV/nucleon) eﬃciently killed
T. matsutake strains on agar plates at a dose of over
100 Gy and generated some mutants whose mycelial
morphology was diﬀerent from that of the wild-type.2)
In this study, we document that the irradiation ofargon-ion beam on T. matsutake induces a mutant thathas pleiotropically altered phenotypes in mycelial morphology and degrading enzymatic activities.
The mutant Ar 59 was obtained by irradiating themycelia of T. matsutake NBRC 33136 on modiﬁed
Melin-Norkrans agar containing 1.5% V8 juice withan argon-ion beam at a dose of 500 Gy. Next, themycelial colony was separated into pieces and transferred onto fresh agars.2) The Ar 59 strain formed
a hedgehog-like colony on the potato dextrose agar(PDA) containing 0.1% azurin-crosslinked (AZCL)amylose and 0.1% AZCL-hydroxyethyl (HE)-cellulose
substrates, unlike the wild-type strain that had a ﬂatmycelial mat with ﬂower-like areal hyphae on the inoculation plug (Figs. 1 (a–d)). The Ar 59 strain exhibited a signiﬁcantly higher amylose- and cellulosedegrading activities than the wild-type strain. In addition, clear halo zones, resulting from the conversionof water-insoluble dye-linked substrates into water soluble substances, were formed around the colonies during the incubation period of 21 days. These clear halozones extended for seven additional days (Figs. 1 (e–h)).
The phenotype of strain Ar 59 diﬀered substantiallyfrom those of T. matsutake NBRC 33136, which raises
a concern whether Ar 59 is a contaminant. Therefore,
the relationship between the NBRC33136 and Ar 59
strains was clariﬁed by analyzing the sequences of theirrRNA gene ITS (867 bp) and IGS1 (361 bp) regions,and the phylum-speciﬁc mobile DNA megB1 (479 bp).
The results showed that the strain sequences were identical. Therefore, strain Ar 59 was proven to be a mutant of T. matsutake NBRC33136.
We have isolated a pleiotropic mutant whose colonymorphology diﬀers from that of the wild-type T. mat†∗1
∗2Fig. 1. Traits of T. matsutake mutant Ar 59 on PDA containing AZCL-amylose and AZCL-HE-cellulose. (a–d)
Colony morphology. (a, c) NBRC 33136 on PDA containing AZCL-amylose and AZCL-HE-cellulose, respectively. (b, d) Strain Ar 59 on PDA containing AZCLamylose and AZCL-HE-cellulose, respectively. (e–h)
Depolymerizing activities. (e, g) NBRC 33136 on PDA
containing AZCL-amylose and AZCL-HE-cellulose. (f,h) Strain Ar 59 on PDA containing AZCL-amylose
and AZCL-HE-cellulose, respectively. Clear halo zones
around the colonies were scored as enzymatic activities(e–h).

sutake strain NBRC33136 and has acquired high levels of amylose- and cellulose-degrading activities. Its
pleiotropic feature indicates that argon-ion beams may
have deleted a part of negative regulatory region that
controls various traits. Whether heavy-ion beams thatbreak double-stranded regions of DNA ranging in size
from a few bp to over 1 kilobase positively inﬂuence
the life cycle of fungus, rendering fruiting in spawn
cultivation, remains to be clariﬁed. However, irradiation with heavy-ion beams may be useful to create newtraits in T. matsutake.
References
1) A. Yamada et al., Mycoscience 55, 27 (2014).
2) H. Murata et al., RIKEN Accel. Prog. Rep. 51, 244(2018).

Condensed from the article in Mycorrhiza 28, 171 (2018)
Forestry and Forest Products Research Institute
RIKEN Nishina Center- 219 -