Commercial cultivation of Lyophyllum shimeji.

ABSTRACT: Lyophyllum shimeji ("Hon-shimeji" in Japanese) is an ectomycorrhizal fungus that grows in association with Japanese red pine and/or oak trees. The fungus is prized as the most delicious and the next most expensive mushroom to Matsutake (Tricholoma matsutake) in Japan. L. shimeji has been successfully cultivated experimentally in pure culture using the selected strains capable of growing saprobically.

However, productivity was not good for commercial cultivation because of using limited wild strains and inadequate substrate. Large amounts of costly materials such as barley grains have been used in experimental cultivation. Yamasa Corporation has cultivated the fungus commercially using high-quality and high-yield strains developed by selective breeding. Moreover, an optimum and inexpensive substrate for commercial cultivation of L. shimeji was recently developed. L. shimeji is cultivated in facilities with automatic cultivation machines using polypropylene bottles but the scale of production currently is small.

Key words: Lyophyllum shimeji; Ectomycorrhizal fungus; Commercial cultivation; Bottle culture; Liquid spawn

INTRODUCTION

Lyophyllum shimeji (Kawam.) Hongo is a member of the family Tricholomataceae. This species is considered an ectomycorrhizal fungus in Japan although the species of genus Lyophylum Karsten have been considered saprophytic fungi in Europe. The general name "Shimeji" has been widely used to describe some of the best Japanese gourmet mushrooms and has been assigned to about 20 mushroom species. However L. shimeji has been called "Hon-shimeji" (Hon means true in Japanese, true-Shimeji) in Japan because the fungus is the richest flavored mushroom among mushrooms with the general name Shimeji. A Japanese proverb states "for fragrance, Matsutake (Tricholoma matsutake); for flavor, Shimeji (Hon-shimeji)" or "Matsutake is noted for its aroma and Shimeji is noted for its taste." Nevertheless, wild fruiting of L. shimeji has become more rare year after year, and provisions for marketing of the fruit bodies collected from the wild have decreased sharply.

This mycorrhizal fungus has been regarded as a species that is difficult to cultivate artificially in pure culture. However, some selected wild strains of the fungus have the ability to form primordia in pure culture using a rye medium without the host plant (Ohta, 1994a). Moreover, Ohta (1994b) and Yoshida & Fujimoto (1994) succeeded in experimental artificial cultivation of L. shimeji in bottles and bags, respectively. Thereafter, Ohta (1998a) has improved the substrate materials, bottle types and casing materials to cultivate it commercially. However, the cost of barley grain for substrate was too expensive and the yield was relatively low. In 2004, Takara Bio Inc., and Yamasa Corporation (a soy sauce manufacturing company) developed new commercial cultivation methods for L. shimeji. Only 40 tons of L. shimeji are sold in the market as the most exclusive cultivated mushroom.

The purpose of this paper is to review the progress from basic research and experimental cultivation to the commercial production of L. shimeji by the two companies.

Biological Characteristics of L. shimeji

Description

Pileus 2-8 cm across, hemispherical when young, later convex with an incurved margin, eventually plane, surface smooth, slightly lubricous, dark gray when young, later gray-brown to light gray. Flesh white, thick. Lamellae white to slightly cream, small depressed or slightly decurrent. Stipe 3-8 cm, white, usually ventricose when young, later cylindric. Spores globose, smooth, 4-6 um.

Distribution

Widely in Japan and China.

Natural Habitats

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Mainly in Quercus serrata and Pinus densiflora forests, ectomycorrhized with these tree species, usually clustered, crowded, or in fairy rings, more rare solitary.

Ecological Characteristics

This fungus forms a mat of fungal mycelium (Shiro in Japanese) in the soil at the fruiting location. Fairy rings of the fungus enlarge outwards 30 to 50 cm a year (Fujita et al. 1982). Yamanaka (1999) used electron microscopy to examine the Hartig net between cortical cells and a mantle layer formed on the surface of ectomycorrhizas of P. densiflora colonized by L. shimeji.

Mycelial Characteristics

Vegetative hyphae have clamp connections. The fungus has gelatinous, fine hyphae, i.e. 0.7-2.0 [micro]m in diameter, 0.20-0.26 [micro] m in cell wall thickness (Yamanaka & Ohta, 1998). The average optimum temperature for mycelial growth of L. shimeji on a medium consisting of rye grains was 24.9[degrees]C (Ohta, 1994a).

Chemical Components of the Flavor

The mushroom flavor is predominantly a result of the presence of monosodium glutamate, further improved by the addition of minor amounts of nucleotides, such as 5'-guanidine monophosphate (5'-GMP) and adenosine monophosphate (AMP). The content of 5'-GMP in broth after boiling of L. shimeji fruit bodies is the highest in major edible mushrooms, and the content is about 1.4 times the levels in Lentinula edodes and Pleurotus ostreatus. Such a high content of 5'-GMP in the broth of L. shimeji is the reason why this fungus has been prized as the most delicious mushroom.

Fruit Body Formation Capability

Ohta (1994a) reported that three strains among 45 wild strains of L. shimeji had the ability to form primordia on the rye grain medium. Yoshida & Fujimoto (1994) reported fruit body formation in all 26 experimental wild strains using a medium composed of a mixture of peat moss and soluble starch. We recognized that all of the wild strains collected from the whole of Japan formed fruit bodies using a newly developed substrate although the yield and the morphology of the fruit bodies were different among the strains tested. Moreover, we could induce fruit bodies in Chinese strains of L. shimeji.

Starch Utilization

Some ectomycorrhizal fungi have the ability to utilize polysaccharides such as starch. Ohta (1997) reported that L. shimeji grew rapidly on both starch and amylose medium. He mentioned that the quantity of starch used as a carbon source supplied the factor that allowed successful fruit body formation without raising the osmotic pressure of the medium. The ability of starch utilization of L. shimeji is effective for screening strains that form fruit bodies under artificial cultivation. Amylase and glucoamylase in fruit body-forming strains of L. shimeji show a higher activity level than those of strains that do not form fruit bodies (Terashita et al. 2000). Kusuda et al. (2004) found that extracellular glucoamylase was a dominant hydrolytic enzyme produced in the barley medium during the vegetative mycelial growth of L. shimeji and they purified the extracellular glucoamylase from the fungal medium. It is considered that two types of extracellular amylases, glucoamylase and the endo-type amylase (alpha-amylase), are associated with nutritional decomposition of barley starch composing 25 percent amylose and 75 percent amylopectin (Terashita et al. 2000; Kusuda et al. 2004).

Cultivation Methods

Available Strains

Ohta (1994a, 1998a) recommended the wild strain SF-Ls6 as having high productivity and high quality. However, this strain readily forms warty structures on the surface of the cap. Warts on the cap surface notably decrease the quality and market value of the mushroom. Yoshida & Fujimoto (1994) have used only wild strains to form fruit bodies. These wild strains in experimental cultivation were low yield and low quality for cultivation of L. shimeji commercially. Wild strains vary in their morphology and in the color of the mushrooms at maturity. It is supposed that Takara Bio Inc., also uses the wild strains for the commercial cultivation in sophisticated and automated facilities. Fruit bodies produced by Takara have a darker colored cap and the gill compared with general wild strains of this fungus. Yamasa Corporation has produced fruit bodies commercially using new high quality and high yielding strains developed by mating between excellent wild strains. The fruit bodies are extremely similar morphologically to those of wild strains.

Substrate

Ohta (1994b) found that a mixture of barley grain and beech sawdust supplemented with synthetic nutrients was the best substrate to form fruit bodies in bottle culture of L. shimeji. Table 1 shows the culture conditions for the production of this fungus devised by Ohta (1998b). Yoshida & Fujimoto (1994) used a solid medium for fruit body formation, adding 750 ml of liquid medium (consisting of: soluble starch 100 g, D-glucose 25 g, pectin 1 g, yeast extract 3g, KH2PO4 0.5 g, MgS04 0.5 g, thiamine-MCl 1 mg, CaCO3 5 g, charcoal powder 5 g, water 860 ml) to 120 g of peat moss.

Barley grains are suitable material as a starch source for the substrate to produce L. shimeji fruit bodies. However, barley grain medium is immensely expensive for commercial cultivation. In addition, barley grain medium results in a non-porous condition in the substrate and retards mycelial colonization because of swelling and viscosity of barley grains after autoclaving. Therefore, Takara uses the substrate based on hardwood sawdust supplemented with corn grits and/or corn meal. At Yamasa, the basal medium for L. shimeji production is composed mainly of a hardwood/softwood sawdust mixture, corn meal and barley grain.

Table 1. Substrate for L. shimeji cultivation (from Dhta 1998b)

Barley grain 875g (dry wt), hardwood sawdust 542g (dry wt). Moisture content of the Substrate: 70 % on a wet weight basis

Synthetic medium: Citric acid, 0.5 g; [KH.sub.2][PO.sub.4], 0.1g; Mg[SO.sub.4], 0.2g; acetylacetone, 0.05 ml; Fe[Cl.sub.3], 50 mg; Zn[SO.sub.4], 1.0 mg; Mn[SO.sub.4], 0.03 mg; Cu[SO.sub.4], 1.5 mg; Co[SO.sub.4], 0.3 mg; Ni[SO.sub.4], 0.1 mg; HEPES, 7 g; distilled water, 1,000 ml; pH; 5.4

Synthetic medium (1,000 ml) is added to the barley grain/hardwood sawdust and, after mixing, pH was adjusted to 5.4 with 1 M HCI 

Cultivation Bottles & Filling

Ohta (1998b) produced the fruit bodies using 400 ml of barley/hardwood sawdust substrate contained in 800 ml polypropylene bottles with a large opening. However, such half-filling of the substrate into the bottles is inefficient and uneconomical for commercial cultivation because bottles cannot be half-filled with substrate using automatic filling machines. Moreover, the cropping of the fruit bodies produced inside the bottle is very difficult. Takara fills the substrate into 1,100 ml bottles with the opening of 82 mm in diameter, and then mechanically drills five holes into the substrate to inoculate with liquid spawn. Yamasa uses 800 ml polypropylene bottles with an opening of 75 mm in diameter, that are filled mechanically with substrate, and that contains approximately 640 g of substrate per bottle.

Utilization of Liquid Spawn

Solid spawn produced on a sawdust substrate has been used in experimental cultivation. Takara has used liquid spawn for commercial production of L. shimeji. We also confirmed that liquid spawn inoculation resulted in faster colonization of the substrate by fungal mycelia and gave high fruit body yields of good quality in the commercial cultivation of L. shimeji.

Spawn Run

In the cultivation of L. shimej using 800 ml bottles filled with 400 ml of substrate (Ohta 1998b), the inoculated bottles are placed in an incubation room at 20-23[degrees]C and 60-70 percent relative humidity (RH) for spawn running. Forty to fifty days after inoculation, mycelia have fully colonized the substrate. According to Yoshida & Fujimoto (1994), 870 g of a substrate composed of peat moss/liquid medium contained in a polypropylene bag was incubated at 23[degrees]C and 70-80 percent RH for 90 days after inoculation. In the commercial production of L. shimei in Takara, 1,100 ml polypropylene bottles containing the substrate are initially incubated at 21[degrees]C for 40 days and then for 70 days additionally after the inoculation. In commercial cultivation at Yamasa, inoculated bottles of 800 ml size are placed in an incubation room at 23[degrees]C, 65-70 percent RH for 80-85 days.

Casing & Casing Materials

According to Ohta's cultivation manual (1998b), the surface of the substrate was covered with autoclaved peat moss as a casing material and the substrate was incubated for an additional 5-7 days after casing. The casing material, consisting of 20 liters of peat moss, 100 g of CaCO3 and 10 liters of water, was adjusted to pH 5.0-5.4 before autoclaving. However, peat moss is totally inadequate as a casing material for the commercial cultivation of L. shimeji because peat moss falls between the stipes of fruit body clusters and soils white stipes. Consumers must then wash the mushroom with water. Takara initiates the primordial formation by Kinkaki (in Japanese, raking off the surface of substrate to stimulate fruiting) without the need for casing. Yamasa uses Kanuma-soil (porous, lightweight granular soil) as the casing medium. The substrate covered with casing layer is incubated at 20-23[degrees]C for 10-14 days.

Primordium formation (Medashi in Japanese)

After Kinkaki, and additional incubation for approximately 10 to 14 days after casing, the bottles are placed in a pinning room at 15-16[degrees]C, 80-90 percent RH and 600-1,000 ppm C[O.sub.2] concentration to stimulate primordium formation (Ohta, 1998b). Illumination (500-600 lux) in the daytime is required for the induction of primordia. Primordia are formed on the surface of the substrate (Takara), or the surface of the casing layer (Yamasa), 10 to 14 days after transfer of the bottles to the pinning room controlled at 15-16[degrees]C, 95 percent RH.

Growing

After 25 to 35 days, when the primordia appear on the surface of either the substrate or the casing layer, the primordia develop into mature fruit bodies ready for harvest. The cultivation cycles from inoculation to harvest in the commercial production of L. shimeji are approximately 130 days at Takara and 90-100 days at Yamasa.

Harvesting

Crop yields using the technique of Ohta (1998b) are in the range of 53 to 69 g per 800 ml bottle containing 400 ml of barley/sawdust substrate. Fruit body yields are lower under commercial cultivation. On the other hand, it is estimated that fruit body yields of 120 to 150 g per 1,100 ml bottle with the large opening are harvested at the Takara commercial cultivation facility. At Yamasa, yields are in the range of 110 to 160 g per 800 ml bottle with the large opening. Commercial production yields of L. shimeji are considerably lower compared with yields obtained through the commercial cultivation of Flammulina velutipes and Hypsizygus marmoreus.

In the near future, new strains of L. shimeji having improved cultivation characteristics, e.g. high productivity, high quality and short cultivation cycle, should be developed by breeding for desired fruit body features.

The prices of wild L. shimeji fruit bodies in season can range from US $110 to $200 per kilogram in the retail market. The retail price of cultivated L. shimeji fruit bodies ranges from US $30 to $80 per kilogram.

OUTLOOK

As previously mentioned, growers and companies producing L. shimeji should improve the substrate, casing materials and cultivation methods for the commercial production of this mushroom. Utilization of cheap materials for the growth substrate, effective substrate formulations, and shorter term cultivation periods are important to reduce production costs and to provide for stable cultivation of this fungus. Overly expensive prices for this cultivated edible mushroom are not acceptable to consumers although its taste and texture are immensely agreeable. The breeding of excellent hybrid strains with desirable features is strongly expected and will lead to a more solid consumption of L. shimeji.

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REFERENCES

Fujita H, Itoh T, Kobayashi F and Ogawa M. 1982. Ecological studies of Lyophyllum shimeji in Pinus densiflora forest. Trans. Mycol. Soc. Japan, 23:391-403 (in Japanese).

Kusuda M, Ueda M, Konishi Y, Matsuzawa K, Shirasaka N, Nakazawa M, Miyatake K and Terashita T. 2004. Characterization of extracellular glucoamylase from the ectomycorrhizai mushroom Lyophylium shimemi. Mycoscience, 45:383-389.

Ohta, A. 1994a. Some cultural characteristics of mycelia of a mycorrhizal fungus, Lyophyllum shimeji. Mycoscience, 35: 83-87.

Ohta A. 1994b. Production of fruit-bodies of a mycorrhizal fungus, Lyophyllum shimeji, in pure culture. Mycoscience, 35: 147-151.

Ohta A. 1997. Ability of ectomycorrhizal fungi to utilize starch and related substrates. Mycoscience. 38:403-408.

Ohta A. 1998a. Culture conditions for commercial production of Lyophyllum shimeji Trans. Mycol. Soc. Japan, 39: 13-20 (in Japanese).

Ohta A 1998b. (Shiga Prefectural Forest Center) Cultivation Manual for Lyophyllum shimemi. Pp 6 (in Japanese).

Terashita T, Kitao T, Nagai M, Yoshikawa K and Sakai T. 2000. Amylase production during the vegetative mycelial growth of Lyophyllum shimeji. Mush. Sci. Biotechnol. 8:61-69 (in Japanese).

Yamanaka, K. 1990. Ecological characteristics of Lyophyllum shimeji. The Bull. Nara For. Exp. Stn. 20:1-11 (in Japanese).

Yamanaka K, Ohta C. 1998. Nutritional Requirement for mycelial growth in Lyophyllum shimeji and Lyophyllum fumosum. Mush. Sci. Biotechnol. 1998, 6:159-165 (in Japanese).

Yoshida H, Fujimoto S. 1994. A trial cultivation of Lyophyllum shimeji on solid media. Trans. Mycol. Soc. Japan, 35:192-195 (in Japanese).

K. Yamanaka

Kyoto Mycological Inst.

Misasagi, Yamashina,

Kyoto, 607-8406

Japan

[e-mail]

Presented at the 6th International Conference on Mushroom Biology & Mushroom Products Bonn, Germany Sept. 29 - Oct. 3, 2008