For group B and group BS we put approximately 2,600g and 2,200g respectively fresh weight of the substrate with the amount of added fossilised shell that showed the best mycelial growth in the cultivation test and the substrate of the same group with ±0.5% shell added, as well as the negative control groups, into heat-resistant bags. These were then sterilised with high-pressure steam at 120℃ for 1.5 hours, cooled and inoculated with approximately 20g of sawdust spawn and incubated at 23±2℃ in the dark. After 30 days when the mycelium had spread throughout the substrate, we gave them maturation periods of 10, 20 and 30 days, so they incubated for a total of 40, 50 and 60 days. Following that, we used a cutter to create slits about 5mm deep in three places on the wide surfaces of the substrate block (130mm long vertically) and one place on the narrow surface (150mm long diagonally), totaling eight places, to promote growth. They were grown at 23±2℃ at a humidity level of 96±2% under 200 lux lighting for eight hours (then 16 hours of darkness), and ventilated as appropriate to prevent deformation of the fruiting bodies from CO2 exposure. Seven days after the start of the growing phase, they were watered daily with a sprinkler for 15 minutes until the end of the harvest phase. The optimal time for harvesting was determined to be when the periphery of the fruiting body caps started to show undulations, and harvesting continued for two months after the growth phase. Each group consisted of 6-7 substrate blocks per subgroup and the test was repeated three times. In the negative control groups, however, the test was repeated twice.  We calculated the yield for each group by dividing the cumulative dried yield by the number of substrate blocks. We used the Dunnett test to compare the yields between the group B and BS subgroups with added fossilised shell and the negative control subgroups in each incubation period, and the Tukey test to compare the yields between all group B and BS subgroups with varying amounts of added fossilised shell, excluding the negative control groups.

The results of the cultivation tests based on added fossilised shell amounts established in the mycelial growth tests are shown in Table 5 (group B) and Table 6 (group BS). These tests were conducted with the amounts of added fossilised shell that had the best mycelial growth, which for group B were 3.0±0.5% and for group BS were 1.0±0.5%, as well as 0% for both groups. As a result, a significant increase in yield with the addition of fossilised shell was observed in all incubation periods for both the B and BS groups compared to the 0% negative control groups. From this, it is clear the yields in both group B and group BS increased greatly with the addition of fossilised shell. However, based on the difference in yields between group B and group BS, it is apparent that substituting cedar sawdust for beech sawdust, which has increased in price recently, was not effective under the conditions of this study. Further, there was no significant difference in yield between the amounts of fossilised shell added and the length of incubation of each group. This suggests that for this strain it is possible to reduce the incubation period for A. polytricha, which generally has an incubation period of over 60 days, to 40-50 days by adding fossilised shell (See Tables 5 and 6). We expect that in future reducing the incubation period for other practical strains with the addition of fossilised shell will be investigated. With the recent revitalisation of domestic A. polytricha production, there has been a steady increase in the release of cultivation manuals by public testing and research institutes across Japan as well as in the number of registration applications for cultivation varieties. In this context, the results from this study will contribute to strengthening the foundation of Japan’s A. polytricha production by increasing yield and reducing the cultivation periods.