Comparative Trial of Six Strains in Substitute-Material Cultivation of Lentinula edodes Using Liquid Spawn

In recent years, China's shiitake (Lentinula edodes) production has developed rapidly, with substitute-material cultivation of shiitake being particularly prominent. The traditional solid spawn production technology for shiitake requires a rather cumbersome three-stage process of mother culture, stock culture and cultivation spawn before it can finally be expanded to the production of cultivation bags. Its high labor intensity, cumbersome process, high rate of contaminant mold infection and long growth cycle all greatly restrict the development of shiitake. In this experiment, liquid spawn of 6 shiitake strains was cultured and compared in cultivation, so as to provide a certain theoretical basis for the promotion and application of liquid spawn for shiitake.

1 Materials and Methods

1.1 Test strains L135 (Huazhong Agricultural University), L939 (Huazhong Agricultural University), L241-4 (Edible Fungi Laboratory of Xinyang Agricultural College), L103 (Xixia County Nongfeng Mushroom Industry Company), L9608 (Henan Academy of Agricultural Sciences), L9015 (Institute of Biology, Henan Academy of Sciences).

1.2 Culture medium formulas ① Liquid medium (CYM): peptone 2 g, yeast extract 2 g, magnesium sulfate 0.5 g, potassium dihydrogen phosphate 0.46 g, dipotassium hydrogen phosphate 1.0 g, glucose 20 g, agar 20 g, distilled water 1000 mL. ② Cultivation medium: sawdust 77%, wheat bran 10%, rice bran 10%, sucrose 0.7%, potassium dihydrogen phosphate 0.3%, gypsum 1%, quicklime 1%. Water content 55%-60%.

1.3 Liquid spawn culture method Different shiitake strains were respectively inoculated onto slant medium and cultured at (25±1)°C for 9 d. After the mycelium had covered the slant, they were respectively transferred into liquid medium, with an inoculation amount of 3 blocks of 0.5 sq cm mycelium per bottle, and 3 bottles per treatment (formula). They were cultured statically at 25°C for 48 h, then cultured with shaking at a rotation speed of 120 r/min and a temperature of (25±1)°C for 6 d; culture was terminated when many fine and dense mycelial pellets had formed in the bottles.

1.4 Determination of nutritional components of liquid spawn mycelium Six shiitake strains were inoculated into liquid medium, with 0.5 sq cm mycelial blocks inoculated into 100 mL culture liquid per bottle, 5 bottles per replicate. After culture was terminated, the mature fermentation liquid was filtered through a 60-mesh sieve to collect the mycelium and filtrate of each strain. The mycelium was rinsed twice with distilled water and dried at 60°C to constant weight, and the dry weight of the mycelium was weighed with an electronic balance to calculate biomass. pH was measured with precise pH test paper.

1.5 Comparison of liquid spawn culture characteristics This trial used cultivation bags with specifications of 55 cm × 15 cm × 0.045 cm, with 1.5 kg dry material per bag. After inoculation, they were placed under low-temperature conditions of 20-25°C for culture, with 20 bags per variety. When the mycelium began to germinate, a straight ruler was used to measure mycelial growth rate. After the mycelium had fully colonized the bags, they were moved into the mushroom house, removed from the bags and induced to fruit, and the fruiting characteristics were compared.

2 Results and Analysis

2.1 Determination of mycelial biomass and culture liquid pH of liquid spawn of different shiitake strains

Six shiitake strains were inoculated into liquid medium of the same formula, with 100 mL culture liquid per bottle and 5 replicates per treatment. After culture was completed, the dry weight of mycelium and the initial and final pH among different shiitake strains were measured, and the results are shown in Table 1.

Table 1 Biomass (mg/mL) and pH among different shiitake strains

Variety Mycelial dry weight Initial pH Final pH pH change value

　　L960816.764.21.8

　　L241-416.464.21.8

　　L9399.763.92.1

　　L1359.963.92.1

　　L90158.964.21.8

　　L1037.863.92.1

As can be seen from Table 1, among the 6 shiitake strains, the mycelial dry weights of L9608 and L241-4 were relatively high. The results indicate that these two strains decomposed and utilized various nutrients relatively quickly in liquid medium, which was conducive to mycelial absorption, growth and increased vigor, and they were suitable for liquid culture. The mycelial dry weights of the other 4 strains were relatively low, indicating that these strains were not suitable for liquid culture. The pH change ranges of the 6 shiitake strains were not large.

2.2 Mycelial growth of different strains in substrate bags The liquid spawn of 6 shiitake strains was inoculated onto cultivation substrate of the same formula, using cultivation bags of the same size and specifications, with the same amount of substrate per bag. After inoculation, they were cultured at 20-25°C, and with 10 d as an interval, a straight ruler was used to measure the mycelial growth rate of each bag and calculate the average value. The mycelial growth results are shown in Table 2.

Table 2 Mycelial growth of different strains in mushroom bags (mm)

Mycelial growth L9608 L9015 L103 L939 L241-4 L135

Mycelial growth rate 32.19 28.72 27.14 29.32 32.30 31.28

　　36.8727.6328.3228.2637.4329.33

　　31.6435.2829.2434.3430.3027.29

Average value 33.57 30.55 28.23 30.64 33.34 29.30

Note: The above are values measured over 10 days.

As can be seen from Table 2, when the 6 shiitake strains were used in cultivation, the four strains L103, L135, L939 and L9015 had relatively weak resistance and slow mycelial growth. L9608 and L241-4 had relatively fast mycelial growth and relatively low contamination. L9608 had the fastest mycelial growth, with a 10-day average of 33.57 mm, while L103 had the slowest mycelial growth, indicating that there were certain differences in mycelial vitality among the strains.

2.3 Fruiting situation of different strains The cultivation test results of each strain are shown in Table 3.

Table 3 Fruiting characteristics of different strains (units: g, %)

Strain Fruiting time Fruiting characteristics Cap size Stipe length Fruiting body color Total yield Biological efficiency Rank

L241-4 Medium Single Medium-large Medium Yellow-brown 23952 79.42 2

L9608 Medium Single Medium Short Light brown 23955 79.83 1

L939 Medium Single Medium Short Brown 20601 68.67 1

L135 Early Single/cluster Medium Medium Tea brown 20958 69.86 4

L9015 Medium-early Single/cluster Small Short Light brown 21426 71.42 3

L103 Early Single Large Small Short Brown 19725 65.76 6

Average value 21769 72.33

As can be seen from Table 3, among the 6 shiitake strains, L241-4, L939 and L9608 fruited late and belonged to late-maturing strains. The highest yield was L9608, followed by L241-4; biological efficiency was L9608 > L241-4 > L9015 > L135 > L939 > L103.

3 Discussion

Through analysis of the liquid spawn cultivation characteristics of 6 shiitake strains, this trial shows that among the 6 strains, L9608 and L241-4 had certain advantages in mycelial vitality, growth vigor and contamination resistance, and are suitable for substitute-material cultivation with liquid spawn. Further trials are still needed to compare the culture characteristics, fruiting characteristics and yield of the same strain under different ecological factors such as temperature.

Note: During the experiment, we received strong support from Professor Lu Zuozhou of Huazhong Agricultural University, for which we express our deep gratitude.