Environ. Control in Biol. 33 (1), 59-64, 1995Original Paper

An Instance of the Fruiting-Body Formation of

Tricholoma matsutake

Kazunari INABA, Toyokazu YOSHIDA, Yoshinori TAKANO,*

Yoshikazu MAYUZUMI,** Toshio MITSUNAGA and Tetsuo KOSHIJIMA

Faculty of Agriculture, Kinki University, Nara 631, Japan*Norin Kinrui , Koshiji-machi, Niigata 949-54, Japan

**Myougi Kinokoen , Matsuida-machi, Gunma 379-01, Japan

(Received June 21, 1994)

In 1985-1987, the ecology of Shiro which had produced fruiting-body of Tricholoma

matsutake was studied in the forest of Pinus densiflora, Sadogashima, Niigata Prefecture.

There are three types of Shiro showing circular and crescent with pine root and independent

circular with pine root. The latter one developed in the soil which the root of pine tree

did not grow well. When the mycelia from the latter Shiro are mixed with the sawdust

medium containing several nutrients and LVD (separated sulphite pulp waste from soft-

wood) and incubated in the plastic case at 24•Ž, the fruiting-body of T. matsutake was

formed 90 days after the mixing. The growth of mycelia (Z-1 strain) isolated from the

fruiting-body formed in the plastic case was better than those produced in nature. More-

over, Z-1 strain formed some small fruiting-bodies in the sawdust nutrient medium con-

taining LVD and Sphagnum sp. (commercial plant). Other strains, however, did not form

any fruiting-body in the same culture conditions. Therefore, the mycological and physio-

logical characters of Z-1 strain isolated will be examined in further investigation.

INTRODUCTION

Tricholoma matsutake (Ito et Imai) Singer, the most famous edible mushroom in

Japan, is an ectomycorrhizic fungus. Since it forms mycorhiza on the rhizoplane of

Pinus densiflora, the growth depends facultatively on living plants. Recently, the pro-

duction of this mushroom has reduced drastically year after year, because the ecological

environments in soil, especially rhizosphere has altered to reduce mycorrhiza formation.

On the artificial cultivation of this mushroom, several reports have published on

the environmental improvements of Shiro and maintenance and artificial formation of

Shiro (fungal colony) by planting the pine seedling infected with T. matsutake (Kawai

and Abe, 1976; Kawai and Terada, 1976; Oyama et al., 1974). On the other hand,

Ogawa and Hamada (1975) reported that the aerial hyphae of T. matsutake were

formed abundantly on the nutrient agar medium contained the sterilized decoction of

soil, then the primordia developed at 17-19•Ž. This primordia formed in vitro was

confirmed morphologically to be identical with those in nature, but any mature

fruiting-body did not formed by this method.

Afterwards, Kawai and Ogawa (1976) reported the formation of primordia on a

vermiculite medium containing the homogenized mycelia of T. matsutake for 3 months

culture at 17•Ž and the development of fruiting-body from some primordia. The

Vol. 33, No. 1 (1995) (59) 59

further mycological information, however, on the strain formed primordium has not

yet been reported.

In this paper, the Shiro type which the mycelium of T. matsutake developed in nature and the formation of fruiting-body in the case which the Shiro's mycelia were cultured in the sawdust medium containing LVD (separated sulphite pulp waste from softwood) was studied and some characters of the strain isolated from the fruiting-body which was formed in the medium containing Sphagnum sp. (commercial plant) in vitro culture were described.

MATERIALS AND METHODS

1. Used Shiro which formed fruiting-body o f T. matsutake in the forest o f Pinus densi f lora

Since 1985 the Shiro, fungal colony of T. matsutake, was studied for 3 years in Kanai-cho, Sado-gun, Niigata Prefecture. On the soil grown Shiro moisture and pH value were analyzed by using moisture meter (Kett) and pH meter (HORIBA F-8E type), respectively.

We found a novel small independent Shiro which has not been influenced with P. densiflora. This Shiro showed a circular and independent forming fairy ring of diameter about 50 cm.

2. Cultivation of T. matsutake in artificial medium

Isolation of the mycelia of T. matsutake from Shiro: Aluminum sampler shown in

Fig. 1 was used to isolate the mycelium from Shiro in 20 cm depth of soil.

Culture implements and ingredient of medium : Plastic case (20 •~ 20 •~ 90 cm) was

employed for the culture. As shown in Fig. 2, the tube (13.5 mm in diam.) with many

holes (1 mm in diam.) was installed to flow

water, nutrients and air during the culti-

vation. The basal medium was the sawdust

one which consists of P. densiflora sawdust

(40%) and PDYL (60%) reported previ-

ously (Table 1) (Inaba et al., 1993), and

sterilized at 120•Ž for 2 h. The water

content of the medium was regulated to

keep 50% during the cultivation.

3. Inoculation and fruiting-body induction

culture

The preincubated inocula of T. matsu-

take were inoculated by injection with

aluminum sampler into the sawdust

medium. Then the inoculated plastic case

was covered with acryl board having 8

holes (30 mm in diam.) which were sealed

with cotton plugs, and subsequently

cultured 24•Ž for 90 days. During 46-90

days after inoculation, fresh air was sup-

plied every 7 days, and the nutrients and

water were flowed every 10 days through

the tube.

Fig. 1 Sampler of the mycelia of T.

matsutake from soil.

Environ. Control in Biol.60 (60)

Fig. 2 Plastic case of artificial culture.

Table 1 Basal medium composition for the mycelial growth of Tricholoma spp.

The values in the table indicate percentages of contents in the medium.

* Separated sulphite pulp waste from softwood .

To induce primordium and form fruiting-body the case was transferred to 18•Ž

from 90 days after the inoculation.

4. Isolation of the mycelium from fruiting-body of T. matsutake and its culture as an

inoculum

The mycelium was isolated from fruiting-body of T. matsutake which was formed

in the sawdust medium inoculated with the novel Shiro and grown on the preincu-

bation medium (Table 1) at 24•Ž for 40 days. The preincubated mycelia were

grown in large quantity of the same medium to employ as an inoculum for artificial

cultivation in the glass bottle.

5. Bottle cultivation of T. matsutake

Medium for artificial cultivation was prepared by the Sphagnum sp. absorbing

PDYL containing LVD (Table 1). Water content was regulated to 50%. The medium

was packed into culture bottle (140 •~ 140 •~ 180 mm) before sterilization at 120•Ž for

2 h. The culture was grown at 24•Ž for 120 days. The fresh air was supplied to the

surface of medium for 15 min, every one h for 35 days, then the induction of fruiting-

body was carried out by transfer to 18•Ž.

Vol. 33, No. 1 (1995) (61) 61

RESULT AND DISCUSSION

1. Ecological environment for Shiro development

The pine forest used in ecological investigation of Shiro situated on the mountain

of 150 m above the sea, Sadogashima, Niigata Prefecture. Soil type was brown forest

soil (BA type) and P. densiflora were

60-70 year-olds. In the forest,

Clevera ochnacea DC. grew up spon-

taneously except pine tree and the

surface of Ao layer was covered by

fresh fallen leaves. The form of

Shiro in the forest was circular and

crescent and the growth of active

mycelia in Shiro was recognized

outward Shiro along the passage of

tunnel for animals. This aspect

means that supply of fresh air may

be essential for the mvcelial growth.

On the contrary, the novel small

Shiro which developed independent-

ly with the roots of P. densiflora

was found in the forest. This Shiro

showed a circular and formed fairy

ring, about 50 cm in diam., produc-

ing only one fruiting-body on the

Shiro in both 1986 and 1987. These

Shiro developed usually on the half-

way slope from moutain ridge where

the soil condition showed good

draining, 35-50% moisture and pH

5.1-5.4. The Shiro of like this type

had been observed also in Yoshino

district, Nara Prefecture.

2. Culture of Shiro's mycelia in

Plastic case

The mvcelial masses collected

from 3 types of Shiro were respec-

tively cultured in the plastic cases.

One fruiting-body (Fig. 3) of T.

mnatsutake was appeared when the

mycelia from the novel Shiro was

cultured. However, all mycelia from

usual Shiro being depend on the

former case, the primordium (Fig.

3(a)) was formed 20 days after the in-

duction treatment of fruiting-body,

that is, transfer to 18•Ž from 24•Ž,

Fig. 3 Fruiting-body formation of T. naatsulake

in the sawdust medium.

Arrows show (a) early of fruiting-body,

(b) fruiting-body and (c) primordium.

Environ. Control in Biol62 (62)

and the mature fruiting-body was formed 30 days after the treatment (Fig. 3(b)). The fresh weight of fruiting-body was 76g showing 80 mm pileus in diam. and 100 mm stipe in length and having a strong characteristic aroma of T. matsutake.

In Fig. 3(c), three primordia, about 15 nmm in diam., were appeared, but these did not continue further development.

3. Property of the strain from the tissue of fruiting-body formed in plastic case

To compare the property of the mycelia (Z-1 strain) isolated from the fruiting-body formed in plastic case (Fig. 3(b)) with that in nature , both strains were cultured in the sawdust medium. The mvcelial growth of Z-1 strain was the highest among all strains tested. It is suggested that the mvcelial growth of Z-l strain in nature differs from other ones in the character of facultative saprophytism.

4. Culture of mycelia from Z-1 strain in glass bottle

The mvcelial growth of Z-1 strain was the highest in the medium containing 40% Sphagnum sp. 60% PDYL added 0.5% LVD.

In this cultivation, the pri-mordia of fruiting-body were formed 4 months after inoculation and the fruiting-body was formed 7 weeks after adjusting the moisture at 45% RH in the medium. The sizes of pri-mordium, pileus and stipe are 10 mm in diam., 18 mm in diam. and 30 mm in length, respectively (Fig. 4(a) and (b)). Although the culti-vation was further continued, the fruiting-body no longer developed in size.

In the previous paper (Inaba et al., 1993), the mycelial growth of T. matsutake is promoted remarkably by addition of LVD and in this experiment LVD also showed a inducible effect on the fruiting-body formation.

In conclusion, it must be needed to study the multiplication of mvcelial mass for producing mature fruiting-body of T. matsutake in the different kinds of artificial media in glass bottle. Moreover, the corre-lation between the fruiting-body formation and physiological and mycological properties of mycelial mass will be elucidated on the basis of present data using Z-1 strain.

Fig. 4 Fruiting-body formation of T. matsutake

in the bottle containing Sphagnum sp.

medium.

Arrows show (a) primordium and (b) fruit-

ing-body.

Vol. 33, No. 1 (1995) (63) 63

REFERENCES

Inaba, K., Yoshida, T., Mitsunaga, T., Koshijima, T. 1993. Acceleration of the growth of

Tyicholoma matsutake mycelium by a fraction of sulphite pulping waste. Mokuzai Gakkaishi

39: 710-715.

Kawai, M., Abe, S. 1976. Studies on the artificial reproduction of Tricholoma matsutake (Ito et

Imai) Sing. I. Effect of carbon and nitrogen sources in media on the vegetative growth of T.

matsutake. Trans. Mycol. Soc. Jpn. 17: 159-167.

Kawai, M., Terada, O. 1976. Studies on the artificial reproduction of Tyicholoma matsutake (Ito

et Imai) Sing. II. Effects of vitamines, nucleic acid and relating substances, phytohormones

and metal ions in media on the vegetative growth of T. matsutake. Trans. Mycol. Soc. Jpn.

17: 168-174.

Kawai, M., Ogawa, M. 1976. Studies on the artificial reproduction of Tricholoma matsutake (Ito

et Imai) Sing. IV. Studies on a seed culture and trial for the cultivation on solid media.

Trans. Mycol. Soc. Jpn. 17: 499-505.

Ogawa, M., Hamada, M. 1975. Primodia formation of Tricholoma matutake (Ito et Imai) Sing. in

pure culture. Trans. Mycol. Soc. Jpn. 16: 406-415.

Oyama, Y., Yoshida, T., Taguchi, H. 1974. The artificial cultivation of mycorrhiza-forming

basidiomycetes. In •gProc. 9th Internatl. Sci. Congress on the Cultivation of Edible Fungi•h

(ed. by Mori, K.), Mushroom Sci. IX (Part 1), The Mushroom Research Institute, Tokyo.

p 719-731.

<和文抄録>

人工培地におけるマツタケの子実体形成の一例

稲 葉 和 功 ・吉 田 豊 和 ・高 野 吉 則*・ 黛 栄 長**・ 光 永 俊 郎 ・越 島 哲 夫

近畿大学農学部,*農 林菌類,**妙 義 きのこ園

新潟県佐渡島で3年 間(1985～1987)に わ た りマツタケの シロを調査 した.そ の結果,松 を中心に菌

糸の生息が円形,三 日月形に観察 され,ま た,ア カマツの根の少ない ところに直径約50cmの 菌糸の

生息 した新 しい単独型 の計3種 類 の シロが観察 され た.シ ロの一部を採取 し,栄 養源を吸収 させた鋸屑

培地で栽培 した ところ単独型 の シロか ら採取 した菌株のみに マツタケの子実体形成が認め られた.こ の

人工栽培で形成 した子 実体 か ら分離 した菌株(Z-1株)の 菌糸生育は良好であ り,さ らに ミズ ゴケを培

地に用いたびんによる栽培 では,小 さい子実体が形成 した.

Environ. Control in Biol.64 (64)