Ⅲ-4. Radiation Chemistry & Biology

RIKEN Accel. Prog. Rep. 49 (2016)

Particle beam radiation of the ectomycorrhizal basidiomycete Tricholoma
matsutake that produces the prized, but uncultivable, mushroom “matsutake”
H. Murata,∗1 T. Abe,∗2 H. Ichida,∗2 Y. Hayashi,∗2 T. Shimokawa,∗1 H. Neda,∗1 T. Yamanaka,∗1M. Sunagawa,∗1 and A. Ohta∗3Tricholoma matsutake is an ectomycorrhizal basidiomycete that produces the economically important edible mushroom “matsutake” in association withPinaceae plants. The annual, worldwide yield of matsutake is estimated to be 2,000 t, which represents a
total retail value of over US$500 million. In Japan,
the annual yield of matsutake in the past decade
ranged from 50 to 150 t per year, which is much lower
than previous annual yields, which reached 12,000 t in
1941. Such a concerning downward trend in the matsutake yield has also occurred elsewhere, including inSouth Korea, the northeastern provinces of China, andBhutan.
Mushrooms are fruiting bodies (i.e., the sexual reproductive stage) that contain a tremendous amount ofbasidiospores. In general, homokaryotic basidiosporesare produced after meiosis in dikaryotic hyphae, which
result from mating between two monokaryotic hyphae, or between monokaryotic and dikaryotic ones1,2) .
While some ectomycorrhizal mushrooms that are regarded as early colonizers frequently propagate andexpand their colonies through spore dispersal, thereby
heavily relying on sexual reproduction, and spread
elsewhere, those regarded as late colonizers, such as
T. matsutake, expand their colonies through vegetative hyphal growth, thereby persisting in a preexisting area. However, it has been proven recently thatlate colonizers also require spore dispersal, thus making them genetically mosaic3) .
Currently, no method is available to cultivate T.
matsutake to produce fruiting bodies, although methods for artiﬁcial mycorrhization and shiro formation
have been established in vitro. In fact, in axenic invitro culture systems, T. matsutake associates with abroad range of tree species, including those that naturally harbor arbuscular mycorrhizal fungi, albeit via
a root endophytic association, as well as with Betula platyphylla, a deciduous broad-leaved tree thatshares the same habitat as Pinus densiflora and Tsuga
sieboldii 4) . The most important issue in matsutake
cultivation may be the lack of any suitable cultivars.
There is a precedent for developing cultivars of fungithat are diﬃcult to cultivate, as the ectomycorrhizal
mushroom Lyophyllum shimeji is now commerciallycultivated to produce the prizedstrains from naturethat grow well in a barley-based spawn. Such L.
shimeji strains were further bred to generate cultivars
∗1
∗2
∗3

Forestry and Forest Products Research Institute
RIKEN Nishina Center
Shiga Forest Research Center- 266 -that were suitable for large-scale mushroom production5) . Therefore, the creation of strains that are suitable for fruiting, or even spawning like L. shimeji, is a
key factor for the artiﬁcial cultivation of matsutake.
In the present study, we determined whether heavy
particle beams could be used to isolate T. matsutake
mutants. Because we previously obtained some mutants whose traits may be desirable for spawn cultivation and which exhibited a survival rate of ca. 30%
following gamma-ray irradiation (500 Gy), we used Arand Fe-ions at radiation doses ranging from 0 to 500
Gy. Of these heavy ion beams, Ar irradiation (400–500Gy) resulted in a survival rate of 12%, while Fe irradiation (large particles, 100–300 Gy) resulted in a survival
rate of ca. 65%; following irradiation, some mutants
exhibited abnormal mycelial morphologies on an agar
plate at the ﬁrst screening (Fig. 1). However, suchmutant phenotypes reverted to the wild-type during
the second screening. This phenomenon may be attributed to the fact that we picked a piece of mycelia,rather than a single hypha, wherein the survivor withthe wild-type phenotype was mixed with, and eventually overtook, the mutant mycelia. Another issue isthat the T. matsutake hypha is composed of multinucleated cells, and even a monokaryon isolated from asingle spore becomes multinucleated during vegetative
growth6) . Therefore, we conclude that irradiation withheavy particle beams is useful for developing cultivars
for matsutake fruiting, although some issues regarding
how to obtain stable mutants need to be addressed.

Fig. 1. A putative mutant generated by Fe-ion radiation(300 Gy: left) that generates a large amount of aerial
hypae unlike the wild-type (right)

References
1) K. Babasaki et al., Biosci. Biotechnol. Biochem. 76, 100(2003).
2) J. Heitman et al., Sex in fungi, ASM Press (2007).
3) H. Murata et al., Mycorrhiza 15, 505 (2005).
4) H. Murata et al., Mycorrhiza 25, 237 (2015).
5) A. Ohta, Mycoscience 35, 147 (1994).
6) H. Murata et al., Mycoscience 56, 287 (2015).