Abatement effects of different soil amendments on continuous cropping of Codonopsis pilosula

Introduction Codonopsis pilosula is widely sought-after in China as a substitute for the more expensive ginseng. Continuous cropping of C. pilosula supports a vibrant health-supplement industry but requires significant inputs of fertilizers which increase production costs and degrade the environment. Methods Here, three environmentally-friendly natural fertilizers, including biochar, bacterial fertilizer, and vermicompost, were used at different concentrations (undiluted, diluted 10 times, diluted 50 times) to determine their efficacy in seed germination and growth physiology of C. pilosula in continuous cropping. Results The results showed that biochar, bacterial fertilizer, and vermicompost with different concentrations of leachate could all increase the germination rate, germination potential and germination index of C. pilosula seeds treated with inter-root soil leachate of continuous C. pilosula; increase the activity of antioxidant enzymes (superoxide dismutase and peroxidase) in C. pilosula seedlings under the stress of inter-root soil leachate of continuous C. pilosula, reduce the over-accumulation of malondialdehyde (MDA) content, and increase the resistance of C. pilosula seedlings. After transplanting, superoxide dismutase (SOD) activity increased by an average of 16.1%. Peroxidase (POD) levels showed an average increase of 16.4%. Additionally, there was a significant reduction in the MDA content, with an average decrease of 50%, and the content of osmotic-regulating substances (free proline content and soluble protein content) exhibited a significant increase. Discussion In conclusion, biochar, bacterial manure, and vermicompost have the potential to overcome the challenges of extensive fertilizer use in continuous cropping of C. pilosula.


Introduction
The Chinese herbal medicine Codonopsis pilosula has been recognized for its ability to strengthen the spleen and lungs, nourish the blood, generate fluids, and supplement human diet.It has been listed in the catalog of medicinal food, and its market demand has shown a rising trend in 2018 (Huang et al., 2021).Gansu Province in China is the main production area of C. pilosula, and the main cultivated species is Codonopsis pilosula Nannf.var.modesta (Nannf.)L.T. Shen (National Pharmacopoeia Committee, 2020).
Obstacles to continuous cropping are defined as the abnormal growth and development of crops caused by continuous cultivation of the same crop or related crops in the same field.Currently, China's grain crops, oilseed crops, vegetables, melons, fruits, and other cash crops, along with a majority of medicinal plants, are grown in a continuous cropping system which often fails various degrees of continuous cropping obstacles due to poor quality of soil, soil degradation, and nutrient depletion (Liu et al., 2010;Durre et al., 2023).As a result, there has been a significant decline in both yield and quality of crops.Continuous cropping obstacle can significantly reduce the yield of Asparagus officinalis and a 15% reduction in yield and deterioration in the quality of rice (Shi et al., 2018;Yin et al., 2019).Grapes grown in a continuous cropping system exhibit decreased plant growth and higher incidence of disease (Shi et al., 2018;Yin et al., 2019).
Medicinal plants may be more affected than vegetable and fruit crops.Studies indicate that affects more than 70% of plant roots, and this limits the growth of Panax quinquefolius, Panax notoginseng, and Radix ginseng as succession plants.After nearly a year of continuous Radix ginseng and Panax quinquefolius growth, seedling survival plummeted to 30%.Panax notoginseng perished in severe circumstances of continuous cropping, due to its tractability and specificity of growth.In general, Panax notoginseng can only be replanted every 10 years (Guo et al., 2006;Jian et al., 2008;Zhao et al., 2014).
Chemosensitizers, which cause the continuous cropping obstacle to develop, are typically distributed in plant reproductive and some nutrient organs.They can be released into the environment via a number of different pathways, including exudation with root secretions (Deng et al., 2017;Jouni et al., 2023;Nwe et al., 2023).The germination and seedling growth may be hindered by autotoxic chemicals found in soil extracts (He et al., 2018).In a study by Zhao et al. (Zhao et al., 2023), the root secretions of Salvia miltiorrhiza under continuous cropping exhibited inhibitory and toxic effects on the germination and growth of Salvia miltiorrhiza seeds.
Similar to other crops, continuous cropping of C. pilosula is linked to growth slowness, yield decrease, quality degradation, and pest and disease severity (Hosseinzadeh et al., 2018).The primary field manifestations of continuous cropping impediment in C. pilosula are dead seedlings, reduced seedling growth, occurrence of various pests and diseases, and incidence of root rot disease that can reach more than 30% (Wu et al., 2013;Yan et al., 2019).Because the root rot pathogen can spread through soil and seedlings, the prevalence of root rot in C. pilosula is on the rise (Zhang and Wang, 2019).Although there is a preliminary understanding of the causes of C. pilosula continuous cropping obstacle, its abatement is still in the exploratory stage (Guo et al., 2012;Ren et al., 2022) due to the complexity of the causes and mechanisms.
Numerous preliminary studies addressed the effectiveness of organic fertilizers in mitigating the challenges posed by continuous cropping in various crops.For example, Zhou et al. (2021) andLi F. G. et al. (2019) demonstrated that both organic fertilizer and silicacalcium-potassium-magnesium fertilizer can mitigate the continuous cropping obstacle of C. tangshen by enhancing photosynthetic metabolism and regulating the soil biochemical environment promoting higher yields.Biochar is a carbon-rich material produced through the thermal decomposition of biomass under conditions of limited oxygen availability.When mixed into agricultural soils, biochar can be used as a fertilizer to promote soil fertility and plant growth by providing and retaining nutrients in agroecosystems (Lin et al., 2023;Liu et al., 2024).Jin Wook Kim (Kim et al., 2022) applied bacterial fertilizer treatment to cucumber and the results showed that cucumber applied with bacterial fertilizer was better than the control in plant height, stem thickness, number of leaves and yield, indicating that bacterial fertilizer was able to stimulate plant growth.Vermicompost also contains many bacteria, actinomycetes, as well as a large number of amino acids, enzymes and bio-hormones and other biologically active substances, which can accelerate the transformation of nutrients and facilitate plant growth (Zhao et al., 2017).Additionally, the use of microbial fertilizer has been shown to enhance the quality of C. tangshen.However, limited research has been conducted on the issue of continuous cropping in C. pilosula.We aimed to investigate the effects of environmentally friendly biochar, bacterial fertilizer, and vermicompost as soil amendments on seed germination and seedling physiology of C. pilosula under C. pilosula continuous cropping obstacles.Additionally, we examined the impact of these three fertilizers on the growth of continuously cropped C. pilosula in the field.Assuming that these three fertilizers have some degree of mitigating effect on continuous C. pilosula, the study will provide new insights into pollution-free production of C. pilosula and sustainable soil utilization (Hosseinzadeh et al., 2016;Zhao et al., 2017;Liu et al., 2020).

Test materials
The seeds and seedlings of C. pilosula were obtained from Lichuan Town Liuhe Herbal Cooperative in Tanchang County, Gansu Province, China.The seeds of C. pilosula had an average thousand grain weight of (9.70 ± 0.35) g, a germination rate of more than 96%, seedlings were annual, uniform in size, free from diseases, pests and mechanical damage, with an average root length of (14.53 ± 0.56) cm, and a root thickness of (3.62 ± 0.84) mm.

Study area
The study area is located in Lasha Village, Lichuan Town, Tanchang County.It is an alpine and humid area with an altitude of 2390 m, annual rainfall of 450 mm, annual average temperature of 10°C, annual solar radiation of 124.9 kcal/cm, frost-free period of 180 d, and annual sunshine hours of 2081.1 h.The climate is alpine and arid, with plenty of sunshine, and plenty of rain in the spring and autumn.Soil has a pH of 8.3, organic matter content of 12.6 g/ kg, fast-acting phosphorus of 23.7 mg/kg, nitrate nitrogen of 16.2 mg/kg, ammonium nitrogen of 12.8 mg/kg.

Effects of three fertilizer extracts on seed germination and seedling resistance physiology of C. pilosula in continuous cropping
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).

Effects of three fertilizers on the resistance physiology of transplanted C. pilosula
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 m 2 , 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.

Soil extract preparation
Preparation of the rhizosphere soil extract (Lin et al., 2022): airdried 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.

Fertilizer extract preparation
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
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:
Germination rate = (total number of germinated seeds(15d) ÷ total number of seeds for testing) Â 100 % (1) Germination potential = (the number of seeds sprouted on the day of maximum sprouting ÷ total number of seeds for testing) Â 100% (2) Germination index (GI) = o (Gt = Dt), Gt is the number of germinated seeds on day t and Dt is the corresponding number of days to germination) 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 F. 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 F. et al., 2019).

Statistical analysis
Data analysis of the trial was performed using Office 2016 and IBM SPSS Statistics version 26.0 (IBM SPSS Inc., Chicago, USA) statistical analysis software.One-way analysis of variance (ANOVA) using SPSS 27.0 with LSD method of significance test (p<0.05).

Results
3.1 Effects of three fertilizer extracts on seed germination and seedling resistance physiology in C. pilosula under continuous cropping

Seed germination
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
The differences in SOD enzyme activities among the T0, Q0, and Q50 treatments, and CK were not statistically significant; however, SOD enzyme activities of the remaining treatments were significantly higher than those of the CK seedlings (Figure 1).Among them, the T10, T50, and J0 treatments exhibited the most significant increases of 24.6, 23.6, and 21.0%, respectively.The SOD enzyme activity among the three concentrations in the J treatment were significantly higher than in the CK.The SOD enzyme activity in seedlings decreased with increasing dilution concentration of the fertilizer extracts.The T and J treatments were more effective than the Q treatment in enhancing the SOD enzyme activity in C. pilosula seedlings.Additionally, the SOD enzyme activity in all treatments was higher than that in the CK group.These findings indicate that all three fertilizer extracts, at different concentrations, have the potential to enhance the antioxidant capacity in C. pilosula seedlings.

POD enzyme activity
All treatments showed significant differences in POD compared to the CK group, except for the T10 and T50 treatments, which were not significantly different from the CK group.Specifically, the T0, J10, J50, and Q10 treatments exhibited significant increases in POD enzyme activity of 15.5, 27.5, 23.3, and 47.7%, respectively.On the other hand, the J0, Q0, and Q50 treatments led to significant decreases in POD enzyme activity of 49.2, 25.2, and 14.7%, respectively.Notably, the effect of J treatment at concentrations J10 and J50 showed the highest efficacy in enhancing POD enzyme activity in C. pilosula seedlings.Among the Q-treated seedlings, the Q10 treatment demonstrated the most significant increase in POD enzyme activity compared to Q0 and Q50, surpassing the effectiveness of other treatments (Figure 2).These results indicate that all treatments, except for J0, Q0, and Q50, increased the POD enzyme activity, thus enhancing the antioxidant capacity of C. pilosula seedlings.

MDA content
There was no significant difference observed between the J10 and J50 treatments compared to the CK group.Among all the treatments, the J10 treatment had the highest MDA content.Further, the MDA content in C. pilosula seedlings increased in T and Q treatments with increasing dilution of the solution.However, it was significantly lower than the MDA content of the CK group.Specifically, the reductions in MDA content in the T0, T10, T50, Q0, Q10, and Q50 treatments were 69.5, 60.0, 42.0, 82.5, 78.1, and 73.6%, respectively.Overall, all treatments, except for J10 and J50, effectively reduced the excessive accumulation of MDA in C. pilosula seedlings under continuous cropping.Among them, the three treatments corresponding to vermicompost exhibited the most significant effects (Figure 3).

Total chlorophyll content
All treatments exhibited a significant difference in chlorophyll content, except for the J0 treatment.The total chlorophyll content in C. pilosula seedlings in both T and Q treatments was significantly higher than in the CK group.The J0 treatment showed a significantly higher chlorophyll content compared to the CK group, while the J10 and J50 treatments exhibited significant reductions of 14.6 and 10.6%, respectively, compared to the CK group.Total chlorophyll content in the Q treatment was significantly higher than that in the T, J, and CK treatments.Both T and Q treatments exhibited higher chlorophyll content at a 10fold dilution.Among all the treatments, Q enhanced the total chlorophyll content most, followed by T. Total chlorophyll content in C. pilosula seedlings in J treatment was consistently lower than in the CK group (Figure 4).

SOD enzyme activity
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).

POD enzyme activity
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).

MDA content
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 mmol/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.

Free proline content
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).

Soluble protein content
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.

Discussion and conclusions
Allelochemical, which are one of the causes of cropping obstacle, are usually distributed in the nutrient organs and some reproductive organs of plants and can be released into the environment through a variety of pathways, including emission through root secretions (Sun and He, 2019;Miao et al., 2021).Autotoxins contained in the interroot soil leachate of plants with a high incidence of cropping obstacle can indeed impede the germination of seeds and the growth of seedlings of their own or other plants (Deng et al., 2017).The aqueous phase extract of biochar has a fraction of highly active substances that are thermally stable and able to remain stable in aqueous solution for a long period of time, and this fraction is the main component that promotes seed germination (Gao et al., 2023).Mumeituli biobacterial fertilizer and vermicompost contains a large number of beneficial active flora, microbial activity secretion of certain substances can also play a role in promoting the effect of C. pilosula seed germination and seedling growth, in addition to vermicompost contains a variety of amino acids and growth hormones, which are able to alleviate the successive cropping of the soil extracts of liquid stress on the inhibition of the germination of C. pilosula seeds and seedling growth.
The germination of C. pilosula seeds under continuous cropping was improved by biochar, bacterial fertilizer, and vermicompost at different concentrations.Among these, the 10 times diluted extract of biofertilizer stimulated seed germination most of the extracts tested and the other two concentrations.Additionally, because biochar is only weakly alkaline, adding too much would result in a high pH of the environment for seed germination, which is harmful to seed germination (Liao et al., 2014;Kolton et al., 2017;Tian et al., 2017).Except for individual treatments, all three fertilizers had a positive effect on the increase in antioxidant enzyme activity, osmoregulatory compounds, and total chlorophyll content in continuous cropping of C. pilosula seedlings under simulated stress in root soil extract.In comparison to the other treatments, bacterial fertilizer diluted 10 and 50 times, and vermicompost diluted 10 times significantly boosted POD enzyme activity in C. pilosula seedlings.Biochar and vermicompost treatments were found to be more effective than biofertilizer treatments in reducing excessive accumulation of MDA and increasing the chlorophyll content in C. pilosula seedlings.The chlorophyll content in all C. pilosula seedlings treated with biofertilizer was lower compared to the control group.This suggests that the biofertilizer treatment negatively affected the photosynthetic capacity of C. pilosula.Reduced chlorophyll levels  are often linked to inhibited plant growth and decreased grain yield (Han et al., 2013;Du et al., 2021).
Continuous cropping obstacles will lead to a decline in plant growth, the plant is subjected to adversity stress, the level of antioxidant enzyme activity in the plant, the accumulation of osmoregulatory substances and the good or bad resistance of the plant have a direct impact.The three fertilizers had a better regulating effect on antioxidant enzyme activity, the accumulation of malondialdehyde content and the content of osmoregulatory substances in different stubble C. pilosula compared with the control, because the improvement of soil physicochemical properties by the three fertilizers facilitated the nutrient uptake by C. pilosula plants, stimulated the growth of C. pilosula, improved C. pilosula resistance, and helped C. pilosula to resist the stress of the continuous cropping obstacles, which is in line with the studies by Zhu Yihao (Zhu et al., 2017) and Li Zhen et al. (Li Z. et al., 2019).The results of Zhu Yihao's study showed that the application of biochar could improve the antioxidant enzyme activity of lily, increase the accumulation of osmoregulatory substances in lily, and improve the resistance of lily.Li Zhen et al. showed that the addition of biochar was able to improve the physiological indices of continuously cropped Andrographis paniculata, bringing them close to those of the positive crop and shifting them in the direction of favoring plant growth.Mumeituli bio-bacterial fertilizer has been applied more in mitigating apple replanting diseases, and the application of Mumeituli bio-bacterial fertilizer has resulted in robust apple plants, developed root systems, and obvious promotion and recovery effects on disease-affected and weakened plants (Yang et al., 2020).Vermicompost (Feng et al., 2018) was found to be effective in improving the growth and physiology of continuous cucumber and continuous ginger (Yin et al., 2017), and was helpful in reducing the persecution of plant growth due to continuous cropping obstacles.Based on our findings, it remains a challenge to uncover the mechanism of abatement effects of different soil conditioner on continuous cropping of C. pilosula.A comprehensive investigation on the effects of continuous cropping on medicinal plants would be appropriate for a future study to fully understand the underlying intricacies.
In addition, other important indicators of C. pilosula succession, such as yield, aboveground biomass, disease index, and nutritional quality.Our group has completed the determination and the results are as follows: The extracts of three fertilizers with different concentrations had a certain inhibitory effect on two root rot pathogens (Fusarium oxysporum and  Lower case letters within columns indicate significant differences among treatments at the P < 0.05 level. Fusarium acuminatum) of C. pilosula.All of the three treatments can promote the growth of the embryonic roots of the C. pilosula seedlings inoculated with Fusarium acuminatum and the growth of hypocotyls of the C. pilosula seedlings inoculated with Fusarium oxysporum, and obviously improve the seed viability of the C.
pilosula inoculated with two root rot pathogenic bacteria.All that three fertilizer can improve the activities of sucrase, catalase, urease and alkaline phosphatase in the rhizosphere soil of C. pilosula in different crop rotation; The contents of soil organic matter, available phosphorus, nitrate nitrogen and ammonium nitrogen were   increased, and the diversity of soil microbial environment was improved.All the three fertilizers were beneficial to the yield increase and quality improvement of C. pilosula (Ma et al., 2024).

FIGURE 1
FIGURE 1Effects of three extracts on SOD enzyme activity in C. pilosula seedlings.

FIGURE 2
FIGURE 2Effects of three extracts on POD enzyme activity in C. pilosula seedlings.Lower case letters indicate significant differences among treatments at the P<0.05 level.

FIGURE 3
FIGURE 3Effects of three extracts on MDA content in C. pilosula seedlings.

FIGURE 4
FIGURE 4Effects of three extracts on total chlorophyll content in C. pilosula seedlings.

TABLE 2
Effects of three extracts on seed germination in C. pilosula.
different lowercase letters within columns indicate significant differences among treatments at the 5% level (P < 0.05).

TABLE 3
Effects on SOD activity in C. pilosula.

TABLE 4
Effects of three kinds of fertilizers on POD activity in C. pilosula.

TABLE 7
Effects of three fertilizers on soluble protein content in C. pilosula.Lower case letters within columns indicate significant differences among treatments at the P < 0.05 lever.

TABLE 5
Effects of three fertilizers on MDA content of C. pilosula.Lower case letters within columns indicate significant differences among treatments at the P < 0.05 level.

TABLE 6
Effects of three fertilizers on proline content in C. pilosula..
Lower case letters within columns indicate significant differences among treatments at the P < 0.05 lever.