Screening out the non-sterilization cultivation method of oyster mushroom with fresh Giant Juncao grass
The Giant Juncao grass that we chose are shown in Figure 1, and the grass powder are shown in Figure 2. The results showed that the mycelia of oyster mushroom (Pleurotus sapidus P969 strain) in each group recovered normally in the first three days. The mycelial growth speed of the oyster mushroom in the no saturated lime water treatment group (LW×0) and the low-weight lime water treatment groups were even slightly faster than that in the high-weight lime water treatment groups. However, as shown in Figure 3, from the third day onwards, in the LW×0 group, the mycelia of a white mold began to spread near the oyster mushroom mycelia and across some places of the mushroom cultivation bags. At subsequent stages, the mycelia of this white mold became more preponderant, the growth of the oyster mushroom mycelia was hampered and the oyster mushroom mycelia were covered by the white mold mycelia. After 21 days, the oyster mushroom mycelia were almost entirely disappeared, as shown in Table 3. In the same-weight saturated lime water treatment group (LW×1), the mycelia growth of white mold was less than that of the LW×0 group, and the inhibition on the growth of the oyster mushroom mycelia was also alleviated. In the inoculated part, some oyster mushroom mycelia would not be covered by the white mold mycelia, and a small part of the oyster mushroom mycelia remained even after 21 days, as shown in Table 3. The mycelial growth of the white mold was further diminished in the treatment group subjected to twice the weight of lime water (LW×2), which was associated with enhanced growth of the mycelial growth of the oyster mushroom. After 21 days, about 1/3 of the oyster mushroom mycelia did not subside, as shown in Table 3. Starting with four times the weight of the saturated lime water treatment group (LW×4), the white mold only appeared sporadically in the form of small mycelial plaque or weak gauze, which did not affect the growth of the oyster mushroom mycelia. In this group, after approximately 21 days, the oyster mushroom mycelia could grow to full extent in the mushroom cultivation bags and did not subside, as shown in Table 3. There were no significant differences observed between the eight times the weight of saturated lime water treatment group (LW×8) and LW×4 group, and each repetition grew normally, with high stability, as shown in Table 3. Therefore, these groups can facilitate normal growth of the oyster mushroom mycelia.
As shown in Table 3, In each treatment group, addition of corn flour was associated with a slight increase in the white mold abundance and moderately stronger oyster mushroom mycelia, which was not significant. Based on these observations and previous studies, we deduced that addition of appropriate amount of corn flour is beneficial to improve the mycelial growth of oyster mushroom in this non-sterilization cultivation method. As such, the formula consisting of 0.5% corn flour in the middle of LW×4 group was selected for mushroom production experiments. As shown in Figure 4, the mushroom production experiment revealed that after the oyster mushroom mycelia grew to full extent by the average growth speed of 0.43 cm/d in bags, and was incubated for approximately 14 days, the mushroom bags would turn predominantly white and began to exhibit mushroom primordia. Mushrooms grew three or four times in total, the conversion rate was 95% in average (Ratio of total weight of fresh mushroom to dry weight of culture material), and the water content was 91.5%.
Isolation, purification and identification of white mold
Morphological identification of the white mold
Sequencing and comparison of ITS sequence of the white mold
As shown in Figure 5, when the white mold grew to approximately 2/3 of the plate on PDA medium, black sporangia became preponderant in most of the area, with the aerial mycelia exhibiting a curly morphology and the peripheral mycelia appearing white. Microscopy was used to discern the morphology of sporangium, spore and mycelia as well as the interval between mycelia, as shown in Figure 6. The genomic DNA was extracted from the white mold as shown in Figure 7, the ITS fragment was obtained through PCR as shown in Figure 8, and the positive clones of TA cloning were identified through PCR as shown in Figure 9. Compared the ITS sequence to the nucleotide data base in GenBank via NCBI BLAST, the comparison results corresponded to Mucor circinelloides. The ITS sequence is available in the Genbank, MN744376.1, and the length is 662 bp. This sequence-based data corroborated the results obtained via morphological identification. Full-length gels are presented in Supplementary Fig. 7, Fig. 8 and Fig. 9 of the Supplementary Information.
Measuring the concentration of soluble substances in each group of culture materials with a refractometer
Compared the concentration of soluble substances in each group of culture materials with a refractometer (The result is presented in Supplementary Chart 1 of the Supplementary Information) with the result of screening out the non-sterilization cultivation method of oyster mushroom with fresh Giant Juncao grass, we found that, with the increase of saturated lime water in treatments, the concentration of soluble substances and the contamination caused by M. circinelloides decreased, and the growth of oyster mushroom mycelia increased accordingly. For all groups with the concentration of soluble substances measured by refractometer equal to or less than 2%Brix, the M. circinelloides grew very week, which did not affect the growth of the oyster mushroom mycelia, and after approximately 21 days, the oyster mushroom mycelia could grow to full extent and did not subside.
The performance of reducing the concentration of soluble substances in culture materials to 2%Brix with Giant Juncao grass in other growth periods
According to the results as shown in Table 4, in the control groups, the group with Giant Juncao grass growing for 3 months and the group with Giant Juncao grass growing for 1 year still had high soluble substances, reached 11%Brix, the M. circinelloides grew vigorously, and the oyster mushroom mycelia could not grow effectively. The group with branches and leaves of dead Giant Juncao grass, had relatively low soluble substances, only 5%Brix, the M. circinelloides grew like gauze, the oyster mushroom mycelia could grow to full but week and had big black patches. In all the experimental groups with the treatment of saturated lime water, the M. circinelloides only appeared sporadically and the oyster mushroom mycelia could grow vigorously to full extent and did not subside in each group.