KOBORI Hajime, TADA Naoto, MORIZONO Toshihiro
Iwade Research Institute of Mycology Co. Ltd., 1-9 Suehiro, Tsu, Mie, 514-0012, Japan

At the same time, there are currently concerns about the impacts of damage from bamboo species through abandoned thickets, centred around the moso bamboo (Phyllostachys heterocycla). As one part of examinations on the valid use of bamboo material derived from these abandoned thickets, mushroom cultivation on bamboo sawdust has been carried out with species such as shiitake, maitake (Grifola frondosa), nameko (Pholiota microspore), enoki, bunashimeji (Hypsizygus marmoreus), oyster mushrooms (Pleurotus ostreatus), eryngii (P. eryngii), abalone mushrooms (P. abalonus), and pioppini/yanagi matsutake (Agrocybe cylindracea). The cultivation of mushrooms on substrate using bamboo as a base up to now has not, however, been incorporated into industry, and there is much anticipation for further research in this area.

It has also been reported that breeding conditions such as substrate composition have an effect on the pharmacological activity of the mushroom species reishi (Ganoderma lucidum). From this fact, we can see possibilities in suggesting new insights regarding methods for cultivating Ganoderma species and their uses, through examining the use of bamboo as a new substrate material for its cultivation. The research presented here uses two species of Ganoderma from the many that belong to the genus - Ganoderma lucidum (reishi) and Ganoderma sinense - and attempts substrate cultivation making use of bamboo chips taken from an abandoned bamboo thicket. In this cultivation trial we examine the number of days required for mycelial colonisation of the substrate, as well as the weight of the fruiting body, with the aim of getting insights into the effective use of felled bamboo from abandoned thickets in the cultivation of Ganoderma species.

The bamboo chips used were produced from felled bamboo (predominately moso bamboo) that was ground into pieces with particle size 5mm using a wood chipper (Ohashi Inc. GS400D). The raw material for the sawdust was broadleaf trees from Mie prefecture, ground to a particle size below 1mm and purchased from Matsusaka Iinan Forestry Association Mushroom Cultivation Substrate Mushroom Cultivation Centre. The Ganoderma test strains used in this research were Ganoderma lucidum and Ganoderma sinense. Both were (non-patented) strains kept at the Iwase Research Institute of Mycology Group, with the inoculum being test strains each inoculated into bottles (Toyo Glass Co. milk bottles 900K-N) filled with substrate (sawdust : rice bran = 9:1 [volume ratio], water content = 65% [wet basis]; after adjustment, high pressure sterilisation for 2 hours at 121°C), and incubated for around one month.

A sawdust substrate (sawdust : rice bran = 9:1 [volume ratio], water content = 65% [wet basis]) was used for the control group. The test group used bamboo chips in the sawdust, changing the volume ratios to 10%, 20%, 30%, 40%, 50%, and 100%. The formulated substrates were, for both control and test groups, filled 6L each into cultivation bags (`Sunbag` SS37, Suntomi Industries) with two holes opened (left and right) and then high-pressure sterilised for 2 hours at 121°C. After cooling overnight, the substrate was inoculated with the test strains by dropping 10g of the inoculum onto the surface and the two opened holes using an inoculation needle.

The inoculated substrate was incubated for around 60 days at 25°C. Once incubation was complete, the substrate was taken out of the cultivation bag and 8 of each were moved into cultivation containers (internal dimensions: length 580mm, width 420mm, height 180mm), before instigating fruiting through covering the surface of the substrates with a peatmoss pH adjusted to around 7.0 through the addition of oyster shell (Seishin Industries, by-product oyster shell lime). The number of cultivation containers prepared was 8-15 for each test group and also for the control group (64-120 total).

These cultivation containers were moved to a fruiting room set with continuous illumination using fluorescent lights. Fruiting was instigated using the following conditions: temperature and humidity inside the fruiting room was the same as natural environmental conditions for the time of year (May-August 2019, April-July 2020); and ventilation and watering of the containers was carried out when required. Fruiting bodies were developed for between 45-60 days, and harvested when the cap had become brown to the edges, and spores had formed. For a further 2-4 weeks a second cap formation was stimulated, with these newly formed caps also harvested. For each container the wet weight of the harvested fruiting bodies (water content 60-65%) was measured immediately, before the dry weight was measured after drying for 14 hours at 50°C and 2 hours at 70°C (water content 5-7%).

The values (excluding Figure 2) for fruiting weight taken from each test, as well as the number of days required for colonisation, are shown as average values ± standard error, produced from a Dunnett`s multiple comparison using BellCurve for Excel (Social Survey Research Information Co.), a statistical processing program.

The mycelial growth regulated activity test making use of paper discs was carried out as follows: first, extracts were produced from sawdust and bamboo chips with, separately, hot water (99°C, 2 hours), ethanol (25°C, 24 hours), and ethyl acetate (25°C, 24 hours), with each extract dried under reduced pressure; after weighing the extracts, each type was dissolved in a solvent, and then soaked into paper discs (Toyo Filter Paper Co., Advantec antibiotic testing paper discs, thickness φ 8mm). For the control paper discs, only the solvent was soaked into them, and, after air-drying, were sterilised for 20 minutes at 121°C. In the middle of a Petri dish was placed a potato dextrose agar medium inoculated with mycelium from either G. lucidum or G. sinense, with the following paper discs each placed an equal distance from the mycelium: a paper disc containing the sawdust extract was placed at the 12 o`clock position; a paper disc containing the bamboo chip extract was placed at the 6 o`clock position; and the control paper discs were placed at the 3 and 9 o`clock positions. The Petri dishes were incubated at 25°C in a dark place for 18 days, with the area surrounding the paper discs containing each of the extracts observed continuously.

The harvest quantity of fruiting bodies per single container for both control and test groups, and for G. lucidum and G. sinense, are shown in Figure 2. In comparison to the harvest quantity (wet weight, dry weight) for the sawdust substrate, it was observed that there was a fall in quantity for both G. lucidum
and G. sinense as the ratio of bamboo in the substrate was increased. In particular, there was a notable decrease in harvest quantity for G. sinense, in the 100% bamboo chip substrate.

Building on these results we then conducted a further test, to examine a more optimal sawdust/bamboo chip ratio to reduce the number of days for mycelial colonisation. The required number of days for full mycelial colonisation by G. lucidum and G. sinense for the control group (sawdust substrate) and test groups (10%, 20%, 30%, 40%, 50% bamboo chip substrate) are shown in Figure 3. For both G. lucidum and G. sinense, there was a statistically significant shortening of the number of days required for the completion of mycelial colonisation of the substrate for all test groups in comparison to the control group.

The harvest quantity of the fruiting bodies for the two strains per single container, and for each of the control (sawdust substrate) and test groups (10%, 20%, 30%, 40%, 50% bamboo chip substrate) is shown in Figure 4. In contrast to the results shown in Figure 2, there was no statistically significant increase or reduction in harvest quantity observed in any of the test groups in comparison to the control group. This suggests that, taken together with the results from Figure 2, substituting the base substrate with bamboo chips even up to 50% for both G. lucidum and G. sinense does not have a major impact. From Figures 1, 3, and 4, we can see the possibility, for substrate that replaces sawdust with 10-50% of bamboo chips, of reducing the incubation period in substrate cultivation without impacting the harvest quantity of fruiting bodies.

Regarding these results, we observed the possibility that the cause may be in differences in the composition of the substrates, and so took extracts from the sawdust and bamboo chips by, in order, hot water extraction, ethanol extraction, and ethyl acetate extraction. We then used each of these as part of a paper disc mycelial growth regulated activity test on G. lucidum and G. sinense. In the results, no stimulatory or inhibitory activity on mycelial growth was observed (Figure 5).

As such, we can surmise that the cause of the increase in speed of mycelial growth in the substrate containing 10-50% bamboo chips was not in differences in composition between the sawdust and bamboo chips, but rather in its material properties. Bamboo fibre has high tensile strength with straight threads not seen in other plant fibres, has an aspect ratio (ratio between fibre length and diameter) of over 100 even while the fibre length is short, and for young bamboo a cavity (lumen) can be seen in the centre of the fibre. It is, generally, easier for mushroom mycelium to grow directionally along fibres from, for example, timber. As such, for G. lucidum and G. sinense there is a possibility of an increase in the speed of mycelial growth and a reduction in the number of days required for completion of mycelial colonisation of substrate containing a certain proportion of bamboo chips, as the material properties of the bamboo such as particle size, increased strength, and contained lumens come to have an impact. However, the fact that the 100% bamboo chip substrate not only decreased incubation time but also reduced harvest quantity suggests the importance of including a certain proportion of sawdust in the base substrate.

Ikeda, Y. (2009). Take seni no tokuchō to sono yōto kaihatsu (Characteristics of bamboo fiber and development of its uses). Sen`i Gakkaishi, 65:45-48.

Iwade, I. (1929). Kinshi no junsui baiyō (Pure cultivation of mycelium). In: Shokuyō kinjinrui to sono baiyō (Edible mushrooms and their cultivation). Tokyo: Nishigahara Kankōkai, 90-106.

Iwade, I. (1958). Nokokuzu saibai (Sawdust cultivation). In: Kinokorui no baiyōhō (Cultivation methods for mushrooms). Tokyo: Chikyūsha, 336-338.

Kobori, H., Sekiya, A., Suzuki, T., Choi, J-H., Hirai, H., and Kawagishi, H. (2014). Bioactive Sesquiterpene Aryl Esters from the Culture Broth of Armillaria sp.. Journal of Natural Products, 78:163-167.

Konuma, R. (1995). Kinoko saibai no chichi: Morimoto Hikosaburō (The father of mushroom cultivation: Morimoto Hikosaburō). Chiba Mycological Club Bulletin, 10:9.

Kumakura, K., Igarashi, K., Eguchi, F., and Sameshima, M. (2009). Mannentake kinkabu to baichi kizai ga shijitsutai Keisei to angiotenshin henkan kōso sogaisei ni ataeru eikyō (Influence of Ganoderma lucidum strains and culture medium on fruit body formation and angiotensin I-converting enzyme inhibition). Mushroom Science and Biotechnology, 17(4):161-165.

Kumakura, K., Maehara, K., Yoshidome, T., Kikukawa, T., and Eguchi, F. (2013). Anti-inflammatory effect of extracts from Ganoderma lucidum cultured in medium including Prunus mume flesh. Mushroom Science and Biotechnology, 20: 205-208.

Nogami, T. and Kitamoto, Y. (1995). Kinoko no kiso jikken gijutsu jikken kōza (Practical course on fundamental experimental techniques for mushrooms). Mushroom Science, 2, 115-124.

Ohga, S. (1999). Suitability of bamboo powder for the sawdust-based cultivation of edible mushrooms. Mushroom Science and Biotechnology, 7:19-22.

Shida, K. (1967). Shokuyōkin saibai ni okeru nokokuzu no riyō (The use of sawdust in the cultivation of edible fungi). Journal of the Hokkaido Forest Products Research Institute, August 1967, 22-25.

Shizuoka Prefecture Department for Environment and Forestry (2004). Chikurin seibi tokuhon (Manual for managing bamboo thickets). Shizuka Prefecture, 2-3, 9-11.

Takabatake, K. (2012). Chikuzai o riyō shita nameko kinshō saibai (Utility of bamboo materials for sawdust based cultivation of Pholiota nameko). Bulletin of the Toyama Forestry Research Institute, 4:25-30.

Takabatake, K., Nakada, Y., and Yoshida, M. (2016). Chikuzai ogako ni yoru hiratake kinshō saibai (Utilization of bamboo sawdust for sawdust based cultivation of Pleurotus ostreatus). Mushroom Science and Biotechnology, 24(2); 71-76.

Takai, Y., Kikuzaki, H., Akiyama, K., Kōno, B., Fujita, T., Nakazawa, M., Ueda, M., Nakatani, N. and Miyatake, M. (2004). Effects of culture substrates on antioxidant activities of extracts from Ganoderma lucidum (Rei-shi). Mushroom Science and Biotechnology, 12: 113-118.