III-4. Radiation Chemistry & Biology

RIKEN Accel. Prog. Rep. 54 (2021)

RIKEN Accel. Prog. Rep. 54 (2021)

Behaviors of the Saprophytic Tricholoma matsutake Mutants G1 andAr 59 In Vitro Substrate Cultivation: the former exhibited
Morphological Changes while the latter did not†H. Murata,∗1 T. Yamanaka,∗1 T. Shimokawa,∗1 T. Abe,∗2 H. Ichida,∗2 Y. Hayashi,∗2 and A. Ohta∗1Tricholoma matsutake produces a specialty mushroom“matsutake” only in a symbiotic association with live
trees in the wild, unlike cultivated mushrooms based onwood-decaying fungi. Another ectomycorrhizal mushroom Lyophyllum shimeji is commercially cultivated toproduce the gourmet mushroom “shimeji” using spawn
substrates comprising barley and saw dust for a fewdecades.1) The key to successful L. shimeji fruiting isthat the isolates that can grow as spawn without hostplants and easily produce fruiting bodies using a protocol similar to that used for cultivated mushrooms.1)
We previously reported T. matsutake mutants G1 andAr 59; the former was isolated after irradiating the wildtype NBRC 33136 (a.k.a., Y1) with γ-rays, and the latter was isolated after irradiating the wild-type with anargon-ion beam.2) Both mutants exhibited signiﬁcantly
higher amylase and cellulase activities compared withthe wild-type; however, G1 gained more saprophytic
traits, and it became lethal rather than symbiotic to Pinus densiflora seedlings, whereas Ar 59 remained symbiotic. Because of such phenotypic conversions, we examined if these mutants could exhibit some morphologicalchanges relevant to fruiting body production.

Fig. 1. Morphological changes in T. matsutake G1 after substrate cultivation at 23◦ C for 3 months followed by 16◦ Cfor 6 months. (A–C) Lumps arising from the spawn are
indicated by arrows. (D) Cross-section of lump indicatestissue diﬀerentiation. Scale bars 5 mm.
†∗1∗2

Condensed partly from the article in Bulletin of FFPRI 19, 2153 (2020)
Forest Research and Management Organization
RIKEN Nishina Center

Fig. 2. Mycelial growth of T. matsutake NBRC 33136 (=Y1), Ar 59, and G1 after the substrate cultivation forca. 1 month. Scale bars 10 mm.

The T. matsutake mutants G1 and Ar 59 and theirwild-type NBRC 33136 were separately cultivated witha modiﬁed barley-based substrate. G1 developed several tiny (∼5–7 mmϕ) lumps arising from the spawn(Fig. 1A–C). Cross-sections of the lumps indicated thatthey were not simple aggregates of aerial hyphae but
tissue-like, although some uncertainty remains as towhether the lumps are related to fruiting bodies suchas a remnant of the inner veil around the pileal margin.
The lumps occurred in the G1 spawn in three consecutive independent experiments did not grow into fruiting
bodies. Like G1, Ar 59 grew better in the substrate than
the wild-type (Fig. 2); however unlike G1, it did not exhibit any morphological changes as observed in NBRC
33136.
The lumps developed in the G1 spawn consecutively inthe three independent experiments, and they developed
sporadically for nearly 2 years, while other traits of G1
that characterize this mutant, including colony morphology on agar plates, increased degrading enzymatic activities, and harmful eﬀects on plants, were maintained.2)
Morphological changes associated with sexual reproduction requires an environment desirable for such vital
life cycle events including nutrient availability, temperature, and moisture ﬂuctuations. In the habitat of T.
matsutake, such moisture and temperature ﬂuctuations
occur in the soil and in the atmosphere, even within asingle day. To resolve this problem, we are currently
inducing mutations in T. matsutake G1 by irradiatingwith heavy-ion beams so that we can obtain mutants
that can easily enter the sexual reproduction stage andfruit in artiﬁcial cultivation only with substrates. Thisline of irradiation breeding can eventually yield cultivarsof T. matsutake.
References

1) A. Ohta, Mycoscience 35, 147 (1994).
2) H. Murata et al., Botany 97, 463 (2019).

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