The experiment was established as a two-factor randomized block trial design, with two factors of fertilizer type and concentration. Fertilizers included biochar, bacterial fertilizer, and vermicompost; the concentrations were: undiluted, diluted 10 times, diluted 50 times, and distilled water treatment as the control ( Table 1 ).

A two-factor randomized group experimental design was used with two factors: cultivation method and fertilizer application. The cultivation method included rotational stubble (R) and continuous stubble (C), and fertilizer application included Mumeituli bio-bacterial fertilizer (J), biochar-based fertilizer (T), vermicompost (Q), and control (CK), with 8 treatments in total and each treatment replicated three times. All treatments were CCK, CJ, CT, CQ, RCK, RJ, RT, RQ. The experimental site was stubble cultivated in the previous year, with continuous stubble being land planted with C. pilosula for 2 consecutive years, and rotational stubble with Astragali radix, as the previous crop. The plot area was 5 m×3.5 m=17.5 m², with a plot spacing of 40 cm and a non-treated buffer around the experimental area, and the experimental plot was cultivated with stubble in the previous year.

C. pilosula was transplanted on April 12. Bio-bacterial fertilizer is used as Mumei Tuli compound microbial fertilizer (application rate is 364.2 kg/acre); biochar-based fertilizer (application rate is 607 kg/acre); vermicompost is used as “Taiping No. 2 Aichi”, which is sieved from cow dung after feeding for 1 month (application rate is 607 kg/acre). All treatments were applied to the soil as a one-time base fertilizer at the beginning of the planting period, and no additional fertilizer was applied during the trial period. When the soil in the field was dry, we needed to water in time and removed weeds diligently to prevent grass shortage, reflecting regular field management.

Preparation of the rhizosphere soil extract (Lin et al., 2022): air-dried rhizosphere soil was sieved through a 20-mesh sieve, and 100 g was weighed into a 500 mL beaker, distilled water was added to the total volume of 400 mL, and solution was sonicated for 1 h and left overnight; then, the extract was filtered through a filter paper.

Preparation of biochar extract (Guo et al., 2020): 10 g of biochar sample was weighed and mixed with distilled water in a 100 mL conical flask with a charcoal-to-water ratio of 1:5, shaken at 150 r/min for 2 h at room temperature, vacuum filtered with a double layer of medium speed filter paper; the charcoal residue was rinsed with 20 mL of deionized water, and the filtrate was stock solution (T0). The filtrate was diluted 10 (T10) and 50 times (T50) when used.

Preparation of the Mumeituli bio-bacterial fertilizer extract (Li et al., 2018): 10 g of bacterial fertilizer compound was weighed in a 250 mL triangular flask, 100 mL of sterile water was added, and the flask was shaken for 30 min at room temperature at 200 r/min; the solution was filtered through a filter paper to obtain the required stock solution (J0). The extract was diluted 10 (J10) and 50 times (J50) for use.

Preparation of vermicompost extract (Liu et al., 2017): the vermicompost was fully air-dried and crushed, passed through a 20-mesh sieve, and a 20 g sample was weighed into a 250 mL conical flask with 100 mL of deionized water, shaken for 30 min, and then placed at room temperature for 24 h. The larger sized residue was first filtered through gauze, and then the filtrate was filtered through a double layer of medium-speed filter paper until it was free of particles to obtain the desired vermicompost extract (Q0). The extract was diluted 10 (Q10) and 50 times (Q50) for use.

Seed germination test (Mackenzie and Naeth, 2019): full, unbroken seeds of C. pilosula were selected. Seeds were soaked in distilled water for 6 h before the test, then surface-sterilized with 5% sodium hypochlorite solution for 15 min, and then rinsed several times with sterile water. Petri dishes were lined with a double layer of filter paper (both petri dishes and filter paper were autoclaved) and the treated seeds were evenly arranged in 90 mm size petri dishes with 50 seeds per dish. Three different concentrations of extracts (no dilution, 10-fold dilution and 50-fold dilution) were used in germination experiments, with three replications of each treatment; distilled water was used as the control (CK). In each petri dish, 2 mL of root soil extract and 2 mL of different dilutions of the treatment solution were added to each dish, and the seeds were incubated at 25°C for 15 d. During the incubation period, the petri dishes were supplemented with 2 mL of distilled water every 2 d. The seeds were observed and any changes recorded daily. The germination rate, germination potential, and germination index of the seeds were calculated based on total number of germinated seeds and the number of seeds sprouted on the day of maximum sprouting. The formula is as follows (Equations 1–3):

1) Seed germination indicators:

After 15 days of seed germination, SOD enzyme activity, POD enzyme activity, MDA content, and total chlorophyll content of whole seeding of C. pilosula were measured in random sampling. Superoxide dismutase (SOD) activity was determined with the nitrogen blue tetrazolium method, peroxidase (POD) activity with the guaiacol method, malondialdehyde (MDA) content with the thiobarbituric acid TBA colorimetric method, and total chlorophyll content was determined using the acetone method (Zhang et al., 2019).

2) Physiological and biochemical indices:

Inside the experimental field, 20 C. pilosula whole seedlings were randomly selected using the five-point sampling method and their physiological parameters were measured on July 10, August 10, September 10, and October 10. The whole seedlings were weighed on a balance, placed in a mortar, a small amount of calcium carbonate and quartz sand were added, and an appropriate amount of 80% acetone solution was added and ground thoroughly to form a homogenous paste, which was then subjected to subsequent operations. Superoxide dismutase (SOD) activity was determined with the nitrogen blue tetrazolium method, peroxidase (POD) activity with the guaiacol method, free proline content with the acid ninhydrin colorimetric method, soluble protein content with the Komas Brilliant Blue G-250 reagent staining method, and malondialdehyde (MDA) content with the thiobarbituric acid TBA colorimetric method (Zhang et al., 2019).

All three fertilizer extracts significantly promoted the germination of C. pilosula seeds. Among them, both bacterial fertilizer and vermicompost at the 10-dilution concentration had the most significant effect on promoting germination. Germination potential increased by 30.6 and 47.9%, respectively, for bacterial fertilizer and vermicompost compared to the control group. Biochar at a 50-fold dilution also had a significant effect on germination; increasing germination rate by 46.5% compared to the control group. All treatments, except for the undiluted biochar, showed significant differences in germination rate and potential compared to the control group. The germination rate and potential of C. pilosula seeds treated with T0 (control) were not significantly different from the control group. However, the increase in germination rate and potential in T10 was 14.3 and 12.7%, respectively, and in T50, it was 23.5, and 31.0% compared to the control group. The germination indices of C. pilosula seeds in all three treatments were higher than those of the control group. The most significant increase in germination rate and germination index of C. pilosula seeds was observed in J10 and Q10, with increases of 24.5 and 26.5% for J10, and 31.0 and 47.9% for Q10, respectively over those in CK. Thus, all three fertilizer extracts with different concentrations can alleviate the effects of continuous cropping stress on the germination of C. pilosula seeds ( Table 2 ).

SOD enzyme activity in C. pilosula seedlings generally showed an increasing trend in different treatments between July to August, with a slight decrease in the CCK (continuous cropping CK) and RT (rotational cropping CK) treatments. Subsequently, there was a moderate increase in the CCK, CJ (continuous cropping J), and RT treatments between August to September, while the other treatments exhibited a decrease. During the harvest period from September to October, the SOD enzyme activity in C. pilosula seedlings showed a decreasing trend in all treatments. However, the SOD enzyme activity in each treatment of rotational cropping was still higher than the corresponding treatments in continuous cropping. The RT, RJ, and RQ treatments exhibited significant differences not only compared to RCK (rotational cropping CK), but also compared to CCK (continuous cropping CK). All three treatments, including bacterial fertilizer, biochar, and vermicompost, improved the SOD enzyme activity in field-grown C. pilosula plants. Because an enhancement in SOD enzyme activity helps to remove and reduce the damage from the reactive oxygen species in plants, these treatments contributed to improving the growth potential of continuously cropped C. pilosula plants ( Table 3 ).

The overall trend in POD enzyme activity in C. pilosula leaves showed an initial increase followed by a decrease. The most significant changes in enzyme activities were observed in August and September, with enzyme activities in September being significantly higher than those in August for most treatments, except for the RT (rotational cropping CK) treatment. Additionally, in August, the POD enzyme activities were significantly higher compared to July for all treatments, except for the RQ (rotational cropping Q) treatment. The POD enzyme activities under rotational cropping treatments were higher than those under continuous cropping for all stages. In October, when the above-ground parts of the plants started to wilt, the POD enzyme activities in C. pilosula leaves for all treatments slightly decreased compared to September. However, POD enzyme activities in the rotational cropping treatments remained higher than those in the continuous cropping treatments. There was no significant difference in enzyme activities between the same fertilizer treatments applied to different cultivation methods ( Table 4 ).

The effects of T, J, and Q treatments on the MDA content in C. pilosula leaves varied and changed over time. In July, the MDA content of C. pilosula leaves in all treatments, except for RCK (continuous cropping CK) and RJ (rotational cropping J), was higher in continuous cropping compared to rotational cropping. In August, the MDA content showed a slight decrease in the RCK and RJ treatments compared to July, while it increased in the remaining treatments. The MDA content in plants in all continuous cropping treatments was higher than that in rotational cropping. RCK, RT, RJ, and RQ treatments, MDA was significantly reduced by 31.8, 28.5, 41.6, and 31.8% respectively, compared to the CCK, CT, CJ, and CQ treatments. The MDA content in C. pilosula reached its peak in September, with the highest value of 4.45 μmol/g in the CCK treatment, which was significantly different from the other treatments in both cultivation methods, and it was 2.28 times higher than in the RT treatment. In October, the MDA content decreased for all treatments, except for the RT treatment, which showed a slight increase ( Table 5 ). Considering four months of realizations, all three fertilizers effectively prevented the excessive accumulation of MDA content in C. pilosula. Specifically, the RT and RJ treatments appeared more effective and consistent in their potential capacity to reduce lipid peroxidation in plant cell membranes and mitigate damage to the membranes.

The free proline content in C. pilosula plants showed an increasing trend in July to October, with significant changes mostly observed in September and October. The free proline content in rotational cropping treatments was higher than that in continuous cropping. In July, proline contents in continuous cropping CJ were greater than in CCK, while those in CT and CQ treatments were significantly lower than in CCK, with reductions of 25.3 and 36.2%, respectively. The RJ (rotational cropping J) and RQ (rotational cropping Q) treatments exhibited significant increases of 21.2 and 46.7%, respectively, in free proline content compared to the CCK treatment. In October, the free proline content in C. pilosula was significantly greater in T under both cultivation methods than in CCK but not RCK (rotational cropping CK). The continuous cropping CQ treatment also showed a significant difference in proline from CCK. The rotational cropping treatments of RT, RJ, and RQ exhibited significantly higher free proline contents than RCK, with increases of 39.0, 23.4, and 32.1%, respectively. All three fertilizers enhanced free proline content in C. pilosula to different extents, with J (vermicompost) and Q (biochar) showing more pronounced and stable enhancements under both cultivation methods, while T (bacterial fertilizer) performed better in later stages of growth ( Table 6 ).

The soluble protein content in C. pilosula was consistently lower in the continuous cropping treatment compared to the rotational cropping treatment over the four-month period of study. In the continuous cropping treatment, the soluble protein content increased until September, after which it started to decrease. In contrast, the rotational cropping treatment showed an increasing trend in soluble protein content until August, followed by a slow decrease, although the decrease was still greater than that observed in the continuous cropping treatment. In October, the soluble protein content in both cultivation methods decreased compared to September. There were no significant differences among the three fertilizers in either of the cropping methods. However, all three fertilizers showed a significant difference compared to CCK and RCK ( Table 7 ). It is evident that all three fertilizers were capable of enhancing the soluble protein content in C. pilosula to varying extents, thereby mitigating the damage caused by osmotic and nutrient dysregulation in C. pilosula under continuous cropping stress.