The original strain (T0), a commercially cultivated strain V844, was conserved in the College of Food Science and Engineering, Gansu Agricultural University, China.

Succession strains were obtained by tissue isolation as follows: T0 was cultivated, egg-shaped fruiting body were obtained, the fruiting body were cut open, and a small piece was cut at the stalk-cap junction to generate the first generation tissue culture, named T1. The same method was followed 19 times to obtain total of 19 generations named T1–T19 (Supplementary Figure S1), with three replicates of each strain. All strains were stored in liquid paraffin at 20°C.

Media for strain cultivation were as follows. Potato dextrose broth (PDB) medium: 200 g fresh potato, 20 g glucose, 1.0 g KH₂PO₄, 1.0 g MgSO₄-7H₂O, in 1000 mL distilled water. Potato dextrose agar (PDA) medium: 20% agar in PDB. Spawn substrate/cultivation medium (w/w): 88% cotton seed hulls, 10% bran, 1% gypsum, 1% lime, and 65% water content (pH 8–9). Cultivation substrate (w/w): 97% waste cotton, 3% lime, and 70% water content (pH 9–10).

The colony and mycelium were examined as described by Fu et al. (2015). All strains were inoculated onto PDA medium and the colony morphology of the succeeding strains was observed and photographed after 3 days of incubation at 30°C.

The mycelial growth rate was determined according to Chung et al. (2020). A mycelium block of 1 cm diameter was taken with a hole punch and inoculated in the center of a PDA plate. After incubation for 72 h at 30°C, the colony diameter was marked on the plate by the cross-marking method and the mycelial growth rate was calculated as follows:

Mycelium biomass was determined by referring to the method of Li et al. (2020). The cellophane was cut into a size slightly smaller than the petri dish, and after sterilization, the sterile cellophane was covered on the surface of the solidified PDA plate. The respective strains were inoculated with 1 piece of mycelium (6 mm diameter punch) on PDA plate at 30°C for 3 days. The mycelium was then gently scraped off, weighed, and the data were recorded.

Mycelial branching was determined as described by Guo et al. (2020). V. volvacea strains T0, T4, T8, T12, T16, and T19 that were preserved in liquid paraffin were taken out for uniform activation in triplicates. The mycelial blocks (1 cm diameter) of respective strains were inoculated in the center of a PDA plate, a clean sterile slide was inserted into the plate at an angle of 45°, and the plate was incubated at 30°C for 3 days. Afterward, the plate was opened, the slide was gently removed. The slide was placed under a light microscope for observation and photography, and the number of mycelial branches of each strain was counted in the same field of view.

The cell nuclei number was estimated according to Gao et al. (2019). The mycelia of T0, T4, T8, T12, T16, and T19 strains were grown on PDA plates at 30°C for 3 days as described in Section 2.5. When the mycelium grew to occupy about 1/3rd of the inserted slide, 10 μL of DAPI (2-(4-amidinophenyl)-6-indolecarbamidine dihydrochloride) staining solution was poured onto the slide, the excess dye was removed with the help of a tissue paper. The DAPI-stained slide was observed under an inverted fluorescence microscope and the number of nuclei per 100 micron length of mycelia was counted.

Cultivation characteristics were determined following the method of Liu et al. (2011) with slight modifications. T0–T19 strains were cultured uniformly on PDA plates for 5 days. Then, three mycelium blocks (10 mm diameter) were cut off and transferred to 500 mL seed medium in a culture flask for incubation at 30°C for 10 days. Later the culture was inoculated in plastic frames (40 cm × 20 cm × 10 cm) with 1 kg of cultivation medium. Three parallels were set for each strain. During the cultivation period, parameters such as the primordium formation time, fruiting body diameter, fruiting body number, production cycle, and biological efficiency of the respective strain were recorded.

Primordium formation time denoted the number of days required for the first primordium to sprout from inoculation to cultivation medium. Production cycle denoted the number of days required for the fruiting body to grow into the harvest period, i.e., the egg-shaped period, after inoculation to the cultivation medium. The number of fruiting bodies denoted the number of sub-entities in the egg-shaped period in the respective plastic frame during the harvest period. The diameter of the fruiting body was calculated as the average value from 5 randomly selected egg-shaped fruiting bodies using a vernier caliper. Biological efficiency was calculated as follows:

Nitroblue tetrazolium chloride (NBT) staining was performed as described by Parkhey et al. (2012). T0, T4, T12, and T19 strains were uniformly activated. A mycelial block (1 cm in diameter) from the respective strain was attached to the PDA medium with a punch and a slide was inserted into the medium. The strain was incubated at 30°C for 3 days. When the V. volvacea mycelium grew to occupy 1/3rd of the slide, staining was performed with 0.3 mmol/L NBT working solution at room temperature for 20 min. 30% glycerol was added onto the coverslip before observation under a light microscope.

T0-T19 strains were inoculated in the PDB medium for 3 days and the mycelium was collected. The intracellular levels of hydrogen peroxide (H₂O₂), superoxide anion (O₂⁻) and antioxidant enzymes (SOD, CAT, GPX, and GR) were estimated using commercial kits (Beijing Solaibao Technology Co., Ltd., Beijing, China) following the manufacturer’s instructions.

Quantitative real-time polymerase chain reaction (qRT-PCR) analysis of four antioxidant enzyme genes (sod, cat, gpx, and gr) was performed as described by Livak and Schmittgen (2001). The V. volvacea housekeeping gene, glyceraldehyde phosphate dehydrogenase (gapdh), was used as the internal reference gene. The respective primers were designed using Primer Premier 7.0 software (sequences were listed in Supplementary Table S1). V. volvacea V23 was used as a reference.¹

T0–T19 strains were incubated in the PDB medium at 30°C for 4 days. Mycelium was collected and washed 2–3 times with sterile distilled water. The harvested mycelium was ground to a fine powder in liquid nitrogen. Total RNA from the mycelium powder was extracted using RNA simple Total RNA Kit [Tiangen Biochemical Technology (Beijing) Co., Ltd., Beijing, China]. PrimeScript^™ RT Master Mix (TaKaRa, Shiga, Japan) was used to synthesize the first strand complementary DNA (cDNA), followed by RT-PCR amplification using a Real-Time PCR instrument (LightCycler Model 480, Roche, Germany). The relative gene expressions were calculated using the 2^−ΔΔCT method (Livak and Schmittgen, 2001).

Mitochondrial staining was performed as described by Xu et al. (2019). The activated T0, T4, T12, and T19 strains were removed on the ultra-clean bench with a sterilized 1 cm diameter punch. The corresponding mycelium block was inoculated in the center of a PDA plate, and a sterile slide (one per plate) was inserted obliquely into the medium at an angle of 45°. The plate was incubated at 30°C for 4 days. Afterward, the slide was rinsed 2–3 times with phosphate buffer saline (PBS) and then stained with Mito-Tracker Green for 20 min at room temperature. The excess stain was removed by washing with PBS 2–3 times and finally, the slide was observed under an inverted fluorescence microscope.

Mitochondrial membrane potential was measured according to Moghadasi et al. (2019). The activated T0, T4, T12, and T19 strains were grown on a slide using the PDA plate as described in Section 2.5. When mycelium grew about 1/3rd of the slide, it was fixed in 3.7% paraformaldehyde, rinsed 2–3 times with PBS for 20 min, and then added with Mito-Tracker Red CMXRos dropwise for 20 min. The excess stain was washed 2–3 times with PBS (pH 7.5) and the slide was observed under an inverted fluorescence microscope.

All experiments had three replicates and the measurement data were expressed as mean ± standard deviation (SD). Data were statistically processed using Microsoft Excel 2010, and one-way ANOVA significance of data was performed using SPSS19.0 (SPSS Inc., United States); Origin 9.0 was used for data plotting.

Total of 19 succession strains (T1–T19) of V. volvacea were obtained by tissue isolation. The strain T19 was obtained after 18 successions. We found that T19 failed to produce fruiting body. T0–T19 were incubated uniformly on corresponding PDA plates, and the colony morphology was observed and photographed after 3 days. The results were shown in Figure 1A. The change in aerial mycelial density was not obvious for T0 and T4. However, beginning from the T8 succession, with the increase in succession generations, the colony diameter gradually decreased, the aerial mycelium density became sparse.

With the increase in succession, both the mycelial growth rate and mycelial biomass first increased (up to T4) and then decreased (Figure 1B). There was an obvious positive correlation between mycelial growth rate and mycelial biomass. The mycelial growth rate began to decrease after peaking at T4 and further accelerated after T12. The mycelial growth rate of T19 was 14.7% lower than that of T0. Similarly, the mycelial biomass peaked at T4 and then decreased; the mycelial biomass of T19 was 8.9% lower than that of T0.

Microscopic observations of the mycelium from succeeding strains were shown in Figure 2A. At the same magnification, the mycelium of T0 and T4 strains showed the least branches, however, the mycelium branching began to increase from the T12 generation. The T19 strain exhibited the highest branching of mycelium and the mycelium intertwined with each other into a network (Figure 2B).

The number of nuclei in mycelium was estimated by fluorescence microscopy. In general, the number of nuclei first increased and then decreased with the increase in succession generations (Figure 2C). The number of nuclei was the highest at T4 and the lowest at T19. The number of nuclei was smaller in T8, T12, T16, and T19 than in T0 (Figure 2D).

We conducted uniform cultivation of V. volvacea succeeding strains and recorded their production characteristics. T0–T18 could produce fruiting bodys, while T19 failed to do so (Figure 3A). The production cycle gradually increased with the increasing number of passaged cultures. Compared with T0, the production cycle of T18 was prolonged by 6 days. Also, the increasing passage gradually decreased the biological efficiency of strains; T18 was at 7.87%, which was 64.50% lower than that of T0 (Figure 3B).

The number and diameter of the fruiting body first increased and then decreased with the increase in succeeding generations (Table 1). Meanwhile, the primordium formation time first decreased and then increased. The number and diameter of the fruiting body at the T4 generation were 40.78 ± 2.13 and 33.45 ± 1.15 mm respectively, which were 21.2% and 29.3% higher than those of T0; the number and diameter of the fruiting body at the T18 generation were 8.14 ± 1.65 and 19.03 ± 1.23 mm respectively, which were 75.8 and 26.5% lower than those of T0. The primordium formation time for T4 and T18 was 5.43 ± 0.83 (19.4% less than that of T0) and 9.63 ± 0.66 (43.3% more than that of T0) days, respectively (Table 1).

We performed NBT staining to examine the change in ROS content in the mycelium of V. volvacea strains. The stained mycelia were shown in Figure 4A. Compared with later generations, the color of T0 and T4 mycelia was lighter but not so different from each other. As the number of generations increased, the mycelium color gradually deepened; the color of T12 mycelia was dark blue and the T19 mycelium-stained indigo. It indicated that the ROS content in the T12 and T19 strains was significantly higher than that in T0 and T4 strains.

The contents of O₂⁻ and H₂O₂, the two main components of ROS, were measured (Figure 4B). The O₂⁻ content showed slow increase in fluctuation until the T12 generation, and then rapid increase from T12 to T19; the O₂⁻ content increased by 50.1% in the T19 strain compared with the T0 strain. The H₂O₂ content increased rapidly with the increasing generation; the H₂O₂ content was 8.6 times higher in T19 than in T0.

The enzyme activities of ROS-scavenging antioxidant enzymes were measured in V. volvacea succeeding strains (Figures 5A–D). Both GPX and GR activities showed first increasing trend and then decreasing, while the SOD activity decreased rapidly with the increase in succession number until T8 and then became stable. The CAT activity remained relatively stable until T13. The differences were not significant for GPX activity until T4 and for GR activity until T10, which then decreased rapidly with the increasing generations. Compared with T0, at T19, SOD, CAT, GPX, and GR activities decreased by 59.0%, 77.1%, 88.6%, and 40.3%, respectively.

The relative gene expression levels of sod, cat, gpx, and gr were analyzed by qRT-PCR using T0 as the control group strain and T4, T8, T12, T16, and T19 as the experimental group strains (Figures 6A–D). With the increasing generations, the expression of gr and cat first increased and then decreased (Figures 6B,D), meanwhile, the expression sod and gpx showed a decreasing trend (Figures 6A,C). All four genes were expressed at the lowest level in T19. Compared with T0, the expression levels of sod, cat, gpx and gr decreased by 91.78%, 97.53%, 96.94% and 82.76%, respectively, in T19. These results were not completely consistent with the results of enzyme activity assays, which could be attributed to specific translational regulation of the respective genes.

Staining tests for estimation of mitochondrial membrane potential and mitochondrial morphology were performed on T0, T4, T12, and T19 mycelia. After staining with mitochondrial membrane potential-dependent fluorescent dye probes, both T0 and T4 mycelia showed bright red fluorescence, the red fluorescence of T12 mycelium was significantly weaker, while T19 mycelium showed no obvious red fluorescence (Figure 7A). These results demonstrated that the increase in oxidative stress reduced the mitochondrial membrane potential in V. volvacea succeeding strains.

Accordingly, for mitochondrial morphology observation, we used the specific fluorescent dye Mito-Tracker Green to label the mitochondria. The staining results showed obvious bright spots in T0 and T4 mycelia, while the numbers of such spots were reduced in T12 mycelium and were almost absent in T19 mycelium (Figure 7B). These results indicated that mycelium mitochondria shape changed significantly with the increase in successions of V. volvacea strains.