Method for identifying and screening autotoxic substances in continuous cropping soil of pilose asiabell root, application and autotoxic substances

Abstract

The invention relates to a method for identifying and screening autotoxic substances in continuous cropping soil of codonopsis punctata, which comprises the following steps: collecting rhizosphere soil of continuous cropping codonopsis punctata, performing water extraction, performing gradient extraction by using an organic solvent in sequence, and performing seed germination and seedling growth activity test on each extraction part to obtain an n-butyl alcohol phase and a residual water phase; analyzing the obtained n-butyl alcohol phase and the residual water phase by adopting a liquid chromatography-mass spectrometry technology to realize baseline separation of compounds in the n-butyl alcohol extraction phase and the residual water phase; and carrying out discriminant analysis on the obtained data through partial least squares, and screening out seven autotoxic substances. According to the method, the LC-MS is combined with the PLS-DA model, the specific autotoxic substances of the ardisia punctata are systematically identified and screened for the first time, the accuracy is high, the efficiency is high, and blindness of a traditional bioassay method is avoided.

Description

Identification and screening methods, uses, and autotoxic substances in soils continuously cropped with Codonopsis pilosula Technical Field

[0001] This invention relates to the fields of agricultural ecology and plant cultivation technology, specifically to a method for identifying and screening autotoxic substances in soils where Codonopsis pilosula is continuously cropped, and also to autotoxic substances identified by the above method. Background Technology

[0002] Codonopsis pilosula (also known as Chinese ginseng) is valued for its invigorating and qi-boosting properties, and market demand is steadily increasing. However, due to limited arable land resources, monoculture has become widespread, leading to continuous cropping obstacles, which have become a bottleneck hindering the sustainable development of the industry. The causes of continuous cropping obstacles are complex, with autotoxicity considered a key factor. For example, Qiu Yajuan et al., in their study "The Effects of Continuous Cropping on the Growth, Physiological Characteristics, and Autotoxic Substances of Codonopsis pilosula," pointed out that autotoxicity under continuous cropping conditions is the main cause of continuous cropping obstacles in Codonopsis pilosula. Accurately identifying the autotoxic substances in the growth process of Codonopsis pilosula will provide a theoretical basis for developing scientific strategies to control continuous cropping obstacles.

[0003] Currently, the screening of autotoxic substances faces the following problems:

[0004] (1) The lack of scientific and reasonable statistical and analytical methods leads to low accuracy;

[0005] The commonly used method is to assume that the compound with the highest or highest relative content during the qualitative analysis is an autotoxic substance, without considering its biological activity in the screening process. The biggest drawback of this approach is low accuracy and significant deviation from actual results. This is because an autotoxic substance is not necessarily the most abundant substance, and using the most abundant substance as the autotoxic substance will lead to substantial biases in the screening results, making them inconsistent with reality.

[0006] (2) Low goal orientation and high degree of blindness

[0007] Traditional bioassays for measuring substances with high concentrations do not have a minimum concentration threshold. Therefore, they can only blindly screen from massive amounts of data, which lacks scientific rigor.

[0008] (3) It consumes a lot of time and energy.

[0009] Blindly filtering from massive amounts of data using the method in (2) will consume a lot of time and energy, and will not yield the most accurate results.

[0010] Therefore, how to accurately screen and identify the autotoxic substances in the continuous cropping process of Codonopsis pilosula is the key problem that this invention aims to solve. Summary of the Invention

[0011] To address the aforementioned technical problems, this invention provides a method for identifying and screening autotoxic substances in soils continuously cropped with Codonopsis pilosula. By combining LC-MS with the PLS-DA model, this method systematically identifies and screens Codonopsis pilosula-specific autotoxic substances for the first time. It is highly accurate and efficient, avoiding the blindness of traditional bioassay methods and saving a significant amount of time and effort.

[0012] This invention provides a method for identifying and screening autotoxic substances in soils continuously cropped with Codonopsis pilosula, comprising the following steps:

[0013] (1) Collect rhizosphere soil of continuously cropped Codonopsis pilosula, extract it with water and then perform gradient extraction with petroleum ether, chloroform, ethyl acetate and n-butanol in sequence. Perform seed germination and seedling growth activity tests on each extracted part to obtain the n-butanol phase with the strongest activity and the remaining aqueous phase.

[0014] (2) The n-butanol phase and the remaining aqueous phase obtained in step (1) were analyzed by liquid chromatography-mass spectrometry. The chromatographic conditions were: HILIC column, mobile phase A was an aqueous solution containing 25 mM ammonium acetate and 25 mM ammonia, and B was acetonitrile. A gradient elution program was used to achieve baseline separation of the compounds in the n-butanol extract phase and the remaining aqueous phase within 12 min.

[0015] (3) By processing the data obtained in steps (1) and (2) through partial least squares discriminant analysis, compounds with a projection value greater than 1 on the germination index variable of Codonopsis pilosula seeds and a positive regression coefficient were screened out. Then, the autotoxic substances in the soil of Codonopsis pilosula continuous cropping were identified as 2,4-di-tert-butylphenol, diphenylamine, dibutyl phthalate, cis-hexadienedia acid, n-butyric acid, palmitic acid, and sulfoacetic acid.

[0016] The core principle of this invention lies in transforming complex agricultural ecological problems into quantifiable chemical and biological problems, and establishing a quantitative relationship model between chemical components and biological inhibitory effects through the combined use of multidisciplinary technologies.

[0017] The beneficial effects of this invention are:

[0018] (1) The types of autotoxic substances have been identified.

[0019] This invention, through LC-MS combined with PLS-DA analysis, systematically screened and identified seven key autotoxic substances (2,4-di-tert-butylphenol, diphenylamine, dibutyl phthalate, cis-hexadienediaic acid, n-butyric acid, palmitic acid, and sulfoacetic acid) in soils continuously cropped with Codonopsis pilosula for the first time. This discovery fills the gap in the study of autotoxic substances in Codonopsis pilosula continuous cropping obstacles and provides a clear target for solving this problem at its root.

[0020] (2) The identification method has high accuracy.

[0021] Compared to the commonly used method of defaulting to the compound with the highest or highest relative content during the qualitative analysis as an autotoxic substance, this invention uses partial least squares discriminant analysis to process the data and screen out compounds with a projection value greater than 1 on the importance of the seed germination index variable of Codonopsis pilosula and a positive regression coefficient, thereby identifying autotoxic substances in the soil of Codonopsis pilosula in continuous cropping. Obviously, the method of this invention overcomes the shortcomings of traditional methods, such as cumbersome identification process and inability to accurately locate key substances. In particular, it avoids the problem that traditional methods are prone to large deviations in results. The accuracy of this invention is high. The correlation analysis of QC samples shows a correlation coefficient >0.9, which proves the stability and reliability of the method.

[0022] (3) The identification method is highly efficient.

[0023] This invention establishes an LC-MS analysis method and optimizes the gradient elution procedure to achieve baseline separation of compounds within 12 minutes, resulting in high analytical efficiency.

[0024] This invention enables accurate identification and rapid detection of self-toxic substances. Traditional methods rely on bioactivity assays, which are cumbersome and cannot accurately locate key substances.

[0025] (4) It overcomes the blindness and subjectivity of traditional methods.

[0026] Traditional methods blindly sift through massive amounts of data, which not only consumes a lot of time and energy, but also yields inaccurate results. This invention applies chemometrics (PLS-DA) to screen for agricultural autotoxic substances, using variable importance projection values ​​(VIP>1) and regression coefficients (positive values) as objective quantitative standards to quickly identify key harmful substances from compounds, avoiding the blindness and subjectivity of traditional bioassay methods.

[0027] This invention clarifies the effective inhibitory concentrations of two key autotoxic substances (2,4-di-tert-butylphenol and n-butyric acid) in soil; and demonstrates through extensive physiological experimental data the significant inhibitory effects of these two autotoxic substances on seed germination and seedling growth of Codonopsis pilosula at the aforementioned effective inhibitory concentrations.

[0028] (5) It provides a reliable tool for early diagnosis and risk warning of soil in continuous cropping of Codonopsis pilosula.

[0029] This invention establishes a highly sensitive and accurate quantitative detection method for key substances (such as 2,4-di-tert-butylphenol and n-butyric acid), providing a reliable tool for early diagnosis and risk warning of continuously cropped soils. This invention goes beyond the methodological level, extending to the substances themselves and their applications, demonstrating extremely high application value.

[0030] This invention, through the combined use of multiple technologies, not only successfully solved the key problem of unclear autotoxic material basis in the continuous cropping obstacle of Codonopsis pilosula, but also provided a replicable and scalable research paradigm, which has important reference value for the study of continuous cropping obstacles of other medicinal plants or crops. Attached Figure Description

[0031] Figure 1 shows the PCA analysis of the overall sample in positive (left) and negative (right) ion modes in Example 2;

[0032] Figure 2 shows the correlation spectrum of QC samples under positive (left) and negative (right) ion modes in Example 2;

[0033] Figure 3 shows the percentage of metabolites identified in Example 2 in each chemical category;

[0034] Figure 4 shows the chromatograms of n-butanol in positive (left) and negative (right) ion modes for detection in Example 2;

[0035] Figure 5 shows the chromatograms of the remaining aqueous phase in positive (left) and negative (right) ion modes for detection in Example 2;

[0036] Figure 6 shows the PLS-DA analysis of the positive ion mode compound in Example 2;

[0037] Figure 7 shows the analysis of PLS-DA, the negative ion mode compound in Example 2;

[0038] Figure 8 shows the germination of seeds under the treatment of two autotoxic substances in Example 2;

[0039] Figure 9 shows the effects of two autotoxic substances on seed germination rate, germination potential, germination index and radicle length in Example 2.

[0040] Figure 10 shows the seedling growth under the treatment of two autotoxic substances in Example 2;

[0041] Figure 11 shows the effects of two autotoxic substances on seedling fresh weight, seedling length and root length in Example 2.

[0042] Figure 12 shows the effects of two autotoxic substances on seedling antioxidant enzymes in Example 2;

[0043] Figure 13 shows the effect of two autotoxic substances on the MDA content of seedlings in Example 2;

[0044] Figure 14 shows the effect of two autotoxic substances on the Pro content of seedlings in Example 2;

[0045] Figure 15 shows the effect of the two autotoxic substances on the soluble sugar content of seedlings in Example 2;

[0046] Figure 16 shows the effect of two autotoxic substances on the chlorophyll content of seedlings in Example 2. Detailed Implementation

[0047] To clearly illustrate the technical features of this solution, the following detailed implementation method will be used to describe the solution.

[0048] Example 1: Material Testing Indicators and Methods

[0049] 1.1 Materials

[0050] In October 2022, rhizosphere soils from Codonopsis pilosula planted for one year and three years were collected from Wenxian County, Gansu Province, and Codonopsis pilosula seeds were purchased locally.

[0051] 1.2 Methods

[0052] 1.2.1 Study on the autotoxic activity of different extract fractions of rhizosphere soil aqueous extract

[0053] 1.2.1.1 Extraction and separation of rhizosphere soil aqueous extract

[0054] 1500 g of rhizosphere soil from *Codonopsis pilosula* plants that had been continuously cropped for 1 year and 3 years, respectively, were weighed and added to 6000 mL of distilled water. The soil was ultrasonically extracted for 2 h and then soaked for 24 h, with frequent stirring during this period at 4000 r·min. -1 After centrifugation for 20 min, the supernatant was filtered. The resulting filtrate was then concentrated to approximately 100 mL by rotary evaporation and extracted three times each with equal volumes of petroleum ether, chloroform, ethyl acetate, and n-butanol. The same extracts were combined and rotary evaporated to dryness, yielding four organic extracts and the remaining aqueous phase from the rhizosphere soil of *Codonopsis pilosula* plants grown for one and three years. Two samples were prepared using the same method; one was used for seed germination testing, and the other for LC-MS identification.

[0055] 1.2.1.2 Effects of different extraction fractions of continuously cropped soil aqueous extract on the germination of Codonopsis pilosula seeds

[0056] The samples obtained in 1.2.1.1 were dissolved in 120 mL of the corresponding organic solvent to obtain stock solutions X1. These stock solutions were then diluted 1.25 times and 2.5 times to obtain solutions of concentrations X2 and X3, respectively. 15 mL of each solution was measured into a sterile petri dish. After the solution evaporated, two layers of sterile filter paper were placed on top, and 4 mL of distilled water was added. The seeds of *Codonopsis pilosula* were disinfected by immersion in 70% alcohol (20 s) and 2% sodium hypochlorite solution (20 min) sequentially. After rinsing with sterile water, 100 plump seeds were neatly arranged in a petri dish and incubated at 25 ℃ for 12 h·d. -1Alternating light and dark culture was used. A reagent blank control was provided using the corresponding solvent, and a sample blank control was provided using distilled water. During culture, 1 mL of distilled water was added every other day to keep the filter paper moist. Each concentration treatment was performed in triplicate. The number of germinated seeds was recorded every 24 hours (germination was defined as the radicle breaking through the seed coat). Statistics were terminated when no new seeds germinated within 3 consecutive days. After germination, the germination rate, germination potential, germination index, and radicle length were calculated.

[0057] 1.2.2 Identification of Autotoxic Substances

[0058] 1.2.2.1 Preparation of the test solution

[0059] Based on the results of seed germination tests, the part with the strongest autotoxic activity was selected, and each sample was tested in triplicate.

[0060] 1.2.2.2 Preparation of QC Samples

[0061] Take equal amounts of 10 μL of each of the prepared test sample solutions, mix them, and use them as QC samples. Cross-inject these samples with the test samples under the same conditions.

[0062] 1.2.2.3 Chromatographic conditions

[0063] Samples were separated using an Agilent 1290 Infinity LC ultra-high performance liquid chromatography (UHPLC) system with a HILIC column; column temperature 25 ℃; flow rate 0.5 mL·min. -1 The injection volume was 2 μL; the mobile phase composition was A: water + 25 mM ammonium acetate + 25 mM ammonia, and B: acetonitrile; the gradient elution program is shown in Table 1. Throughout the analysis, the sample was placed in the autosampler at 4 ℃. To avoid the influence of instrument signal fluctuations, the samples were analyzed continuously in a random order. QC samples were inserted into the sample queue to monitor and evaluate the system's stability and the reliability of the experimental data.

[0064] Table 1. LC-MS Gradient Elution Program Time (min) A (%) B (%) 0.0 -0.5 595 0.5 -7.0 3565 7.0 -8.0 4040 8.0 -9.0 6040 9.0 -9.1 595 9.1 -12.0595 surface

[0065] 1.2.2.4 Mass Spectrometry Conditions

[0066] After separation by a Vanquish LC ultra-high performance liquid chromatography (UHPLC) system, the sample was analyzed using an Orbitrap Exploris system. TMMass spectrometry analysis was performed using a Thermo 480 mass spectrometer, employing electrospray ionization (ESI) in both positive and negative ion modes. The ESI source and mass spectrometry settings were as follows: Auxiliary heating gas 1 (Gas1): 50, Auxiliary heating gas 2 (Gas2): 2, Ion source temperature: 350 ℃, Spray voltage (ISVF): 3500 V for positive ion mode, 2800 V for negative ion mode; Primary mass-to-charge ratio detection range: 70–1200 Da, resolution: 60000, cumulative scan time: 100 ms; Secondary mass-to-charge ratio was acquired using a segmented acquisition method, with a scan range of 70–1200 Da, secondary resolution: 60000, cumulative scan time: 100 ms, and dynamic exclusion time: 4 s.

[0067] 1.2.3 Analysis of the content of typical autotoxic substances in soils continuously cropped with Codonopsis pilosula

[0068] 1.2.3.1 Preparation of the test solution

[0069] Weigh 15 g of rhizosphere soil from three consecutive years of cropping, extract it with 100 mL of methanol using ultrasound for 1 h, then soak it for 12 h, centrifuge and filter the supernatant, recover the solvent under reduced pressure until dry, dissolve and bring to a final volume using a mixed solvent of 25 mL methanol:acetonitrile:water = 2:2:1, and then use it for content determination.

[0070] 1.2.3.2 Preparation of reference solution

[0071] Accurately weigh 1 mg of 2,4-di-tert-butylphenol reference standard and accurately pipette 1.04 mL of n-butyric acid reference standard. Dissolve each in a mixed solvent of methanol:acetonitrile:water = 2:2:1 and bring the volume to 10 mL to obtain a concentration of 100 μg·mL⁻¹. -1 Two reference standard mother liquors.

[0072] 1.2.3.3 Chromatographic conditions

[0073] Instrument: UHPLC-QQQ-MS / MS (1290-6460, Agilent); Chromatographic conditions: mobile phase: water (A) and acetonitrile (B); flow rate: 0.3 mL / min -1 Column temperature 30 °C, injection volume: 5 μL, gradient elution method is shown in Table 2.

[0074] Table 2 LC-MS gradient elution program

[0075] 1.2.3.4 Mass Spectrometry Conditions

[0076] The ion source was an Agilent 6460 QQQ jet focusing electrospray source (ESI), with ion monitoring mode (SIM) selected; the drying gas was N2, the temperature was 300 ℃, and the flow rate was 6 L·min. -1 The atomizing gas pressure is 15 psi; the capillary voltage is 4000 V (+) or 3500 V (-).

[0077] 1.2.3.5 Examination of Linear Relationships

[0078] Using a mixed solvent of methanol:acetonitrile:water = 2:2:1, the two reference standard stock solutions were serially diluted to concentrations ranging from 1 to 10 μg / mL. -1 The solution was injected for analysis. A standard curve was plotted with concentration on the x-axis and peak area on the y-axis, as shown in Table 3.

[0079] Table 3 Linear equations for reference standards. Linear equation R for reference standards. 2 2,4-Di-tert-butylphenol y = 12871x + 3559.3R 2 = 0.9982 n-butyric acid y = 22302x + 146585R 2 = 0.9983 surface

[0080] 1.2.3.6 Precision Test

[0081] Accurately pipette 1 μL of each of the two reference solutions and repeat the injection 6 times under the same conditions. The results were obtained by parallel determination and the RSD value of 2,4-di-tert-butylphenol was 2.25%, and the RSD value of butyric acid was 1.09%.

[0082] 1.2.3.7 Repeatability Test

[0083] Six samples of the same rhizosphere soil were taken, and test solutions were prepared according to the method in 1.2.4.1. The samples were then injected and tested under the same conditions. The RSD values ​​of 2,4-di-tert-butylphenol and butyric acid were 2.78% and 1.38%, respectively.

[0084] 1.2.4 Study on the autotoxic activity of typical autotoxic substances

[0085] 1.2.4.1 Preparation of solutions with different concentrations

[0086] Based on the above content determination results, appropriate amounts of the compounds were weighed and dissolved in methanol to prepare solutions. The concentration of n-butyric acid was recorded as D2, D2 reduced by 10 times was recorded as D1, D2 increased by 10 times was recorded as D3, and D2 increased by 100 times was recorded as D4. 2,4-di-tert-butylphenol solutions were prepared in the same manner according to the content determination results, and their concentrations were recorded as E1~E4, respectively, for use in seed germination tests and seedling growth tests.

[0087] 1.2.4.2 Effects of typical autotoxic substances on the germination of Codonopsis pilosula seeds

[0088] Accurately pipette 10 mL of each of the above-prepared autotoxic substance solutions of different concentrations into petri dishes. After the methanol has evaporated, place two layers of sterile filter paper in each dish and conduct seed germination tests using the same method as described in 1.2.1.2, with a blank methanol reagent as a control. Each treatment was performed in triplicate. After the experiment, the seed germination rate, germination potential, germination index, and radicle length were measured.

[0089] 1.2.4.3 Effects of typical autotoxic substances on the growth of Codonopsis pilosula seedlings

[0090] Place 250 g of sterilized nutrient soil in each germination box. Pour 150 mL of a solution of a different concentration of autotoxin into each germination box. Use methanol as a blank control. Perform three replicates for each treatment. Place the boxes in a ventilated area until the nutrient soil is completely dry and the alcohol odor has dissipated. Then, thoroughly water the nutrient soil with distilled water. Select plump, disease-free seeds of Codonopsis pilosula and disinfect them (using the same method as in 1.2.1.2). Sow them evenly into the germination boxes. Incubate the boxes at 25 ℃ for 12 h·d in a constant temperature and light incubator. -1 The seedlings were cultured under alternating light and dark conditions. During the culture period, approximately 100 mL of distilled water was added every other day to keep the substrate moist. After 30 days of culture, the growth indicators and physiological and biochemical indicators of the seedlings were measured.

[0091] 1.2.4.4 Measurement Indicators

[0092] After the germination test, 10 germinated seeds were randomly selected from each petri dish, and the radicle length (RL) was measured with a ruler. Germination rate (%) = number of germinated seeds / number of tested seeds × 100%; Germination potential (%) = total number of germinated seeds on the day with the highest number of newly germinated seeds / number of tested seeds × 100%; Germination index (GI) = Σ (Gt / Dt), where Gt represents the number of germinated seeds on day t, and Dt represents the number of days the corresponding seed has germinated.

[0093] After 30 days of seedling culture, 10 seedlings were randomly selected from each germination box, and their root length (RL) and seedling length (SL) were measured with a ruler. The total fresh weight (FW) of the 10 seedlings was weighed using a 0.01% balance. The chlorophyll content was determined by the ethanol-acetone colorimetric method. The peroxidase (POD) activity of the seedlings was determined by the guaiacol method. The catalase (CAT) activity was determined by ultraviolet spectrophotometry. The superoxide dismutase (SOD) activity was determined by the nitroblue tetrazolium photoreduction method. The malondialdehyde (MDA) content was determined by the 2-thiobarbituric acid method. The proline (Pro) content was determined by the ninhydrin colorimetric method. The soluble sugar content was determined by the anthrone colorimetric method.

[0094] The allelopathic index was calculated using the method of Bruce et al., and the formula is as follows:

[0095] Where C represents the average value of the index in the control group, and T represents the average value of the index in the treatment group. When RI > 0, it indicates that the treatment has an allelopathic promoting effect on the growth of the tested plant; when RI < 0, it indicates that the treatment has an allelopathic inhibiting effect on the growth of the tested plant. The magnitude of |RI| can be used to determine the intensity of the allelopathic effect.

[0096] The formula for calculating the overall perceptual effect index (SE) is as follows:

[0097]

[0098] SE is the average value of the allelopathic effect index (RI) of each measured index. When SE > 0, it indicates allelopathic promotion; when SE < 0, it indicates allelopathic inhibition. The magnitude of |SE| indicates the strength of the allelopathic effect.

[0099] 1.3 Data Processing

[0100] The experimental data were statistically analyzed and plotted using Microsoft Office Excel 2010, and one-way ANOVA and significance tests (Duncan's method, α=0.05) were performed using IBM SPSS Statistics 26.0.

[0101] Example 2: Results and Analysis of the Detection Indicators in Example 1

[0102] 2.1 Study on the autotoxicity of different extract fractions of water extract from continuously cropped soil

[0103] 2.1.1 Effect of blank reagent on germination index of Codonopsis pilosula seeds

[0104] Table 4. Effects of Blank Reagent on Seed Germination Indicators Reagent GR (%) GP (%) GIRL (cm) Petroleum Ether 91.7±0.01 a 40.0±0.03 a 36.3±2.91 a 3.81±0.02 a Ethyl Acetate 93.3±0.02 a 44.7±0.11 a 36.1±3.84 a 3.70±0.06 a Chloroform 92.7±0.01 a 32.3±0.07 a 33.0±1.31 a 3.74±0.09 a n-Butanol Blank Reagent 94.7±0.02 a 40.7±0.09 a 36.5±2.02 a 3.71±0.06 a Water (CK) 92.0±0.04 a 36.0±0.05 a 32.4±0.90 a 3.83±0.28 a surface

[0105] As shown in Table 4, there were no significant differences between the blank reagent treatment and the control results for the four measured indicators: germination rate, germination index, germination potential, and radicle length, indicating that the solvents in each phase had no significant effect on the germination test results (P<0.05). Gradient extraction of soil aqueous extracts was performed using solvents of different polarities. Combined with seed germination tests, the allelopathic effect index (|SE|) ​​was used to quantify the biological activity of different extraction fractions, precisely identifying the n-butanol phase and the remaining aqueous phase, which exhibited the strongest autotoxic activity.

[0106] 2.2.2 Effects of different extraction fractions of continuously cropped soil aqueous extract on the germination rate of Codonopsis pilosula seeds

[0107] Table 5. Effects of different extraction fractions of soil aqueous extract from continuously cropped soil on seed germination rate.

[0108] As shown in Table 5, the germination rate of Codonopsis pilosula seeds generally decreased with increasing concentration of each extract phase. The effects of different extract phases on seed germination rate varied in continuously cropped soils. Specifically, under the petroleum ether extract phase, except for the X1 concentration which was lower than the control after 3 years of continuous cropping, the germination rates of all other treatments were higher than the control. Only the X3 concentration treatment in the soil after 1 year of continuous cropping showed a significant difference from the control (P<0.05). Under the ethyl acetate extract phase, the germination rates of the X2 and X3 concentration treatments in the soil after 1 year of continuous cropping were higher than the control, while the X1 concentration treatment was lower, but the differences were not significant (P<0.05). Under all concentration treatments after 3 years of continuous cropping, the seed germination rate was lower than the control under all concentrations, with the X1 concentration treatment being significantly lower than the control by 17.0% (P<0.05). Under the chloroform extract phase, the seed germination rate of the X3 concentration in the soil after 1 year of continuous cropping was higher than the control, while the X1 and X2 concentrations were lower, but these effects were not significant. After three years of continuous cropping, all concentrations were lower than the control. Specifically, the seed germination rates at concentrations X1 and X2 were significantly lower than the control by 30.8% and 6.2%, respectively (P<0.05). Under both the n-butanol and residual aqueous phases, the seed germination rates of *Codonopsis pilosula* were lower than the control. While there was no significant difference in the X1 concentration of the soil n-butanol extract between the one-year and three-year continuous cropping treatments compared to the control, the X1 and X2 concentrations were significantly lower than the control by 42.8% and 34.8%, and 98.9% and 60.5%, respectively. The X1–X3 concentrations in the residual aqueous phase of the soil after one year and three years of continuous cropping were significantly lower than the control by 82.2%–8.7% and 100%–58.3%, respectively (P<0.05). This indicates that the seed germination rate decreased most significantly under the residual aqueous phase treatment compared to the control, suggesting that the autotoxic substances in the treatment have a strong inhibitory effect on seed germination.

[0109] 2.2.3 Effects of different extraction fractions of continuously cropped soil aqueous extract on the germination potential of Codonopsis pilosula seeds

[0110] Table 6. Effects of different extraction fractions of soil aqueous extract from continuously cropped soil on seed germination potential.

[0111] As shown in Table 6, in the petroleum ether phase, the germination potential of soil at concentration X3 after one year of continuous cropping was higher than that of the control. When the solution concentration was increased to X1, the seed germination potential was lower than that of the control, but the difference was not significant. After three years of continuous cropping, the germination potential of soil at concentration X3 was higher than that of the control, but not significantly, while X1 and X2 were both lower than that of the control, with X1 being significantly lower than that of the control by 35.2% (P<0.05). In the ethyl acetate phase, the germination potential of soil at concentrations X2 and X3 after one year of continuous cropping was higher than that of the control, while the germination potential of soil at concentrations X1 was lower than that of the control, but the above effects were not significant. However, after three years of continuous cropping, the germination potential of soil at concentrations of X1 to X3 was significantly lower than that of the control by 74.1% to 19.4% (P<0.05). Under the treatments of chloroform, n-butanol, and residual aqueous phase at various concentrations after one year and three years of continuous cropping, the seed germination potential was lower than that of the control. After one year of continuous chloroform cropping, the soil X3 concentration was not significantly different from the control, while the X1 and X2 concentrations were significantly lower than the control by 52.8% and 21.3%, respectively. After three years of continuous cropping, the X1-X3 concentrations were significantly lower than the control by 96.3%-20.4% (P<0.05). Under the n-butanol phase and residual aqueous phase, after one year of continuous cropping, the soil X1-X3 concentrations were significantly lower than the control by 88.0%-30.6% and 100%-87%, respectively. After three years of continuous cropping, the X1-X3 concentrations were significantly lower than the control by 100%-92.6% and 100%-93.5%, respectively (P<0.05).

[0112] 2.2.4 Effects of different extraction fractions of continuously cropped soil aqueous extract on the germination index of Codonopsis pilosula seeds

[0113] Table 7. Effects of different extraction fractions of soil aqueous extract from continuously cropped soil on seed germination index.

[0114] As shown in Table 7, in the petroleum ether phase, the germination index of soil treated with concentrations X2 and X3 after one year of continuous cropping was significantly higher than the control by 13.3% and 14.6%, respectively, while the X1 concentration treatment showed no significant difference from the control. In soil treated after three years of continuous cropping, the X3 concentration treatment was significantly higher than the control by 10.4%, while the X1 and X2 concentrations were significantly lower than the control by 7.9% and 6.9%, respectively (P<0.05). In the ethyl acetate phase, the germination index of soil treated with concentration X3 after one year of continuous cropping was significantly higher than the control by 12.9%, while the X2 concentration showed no significant change. When the concentration was increased to X1, the seed germination index was slightly lower than the control. After three years of continuous cropping, the X1 and X2 concentration treatments were significantly lower than the control by 36.7% and 18.3%, respectively, while the X3 concentration was higher than the control but not significantly. Under the chloroform phase X3 concentration treatment, the germination index was slightly higher than the control after one year of continuous cropping, and slightly lower than the control after three years of continuous cropping, but there was no significant difference. As the concentration increased, the X2 concentration after one year of continuous cropping began to be lower than the control, and was significantly lower than the control by 9.0% at the X1 concentration. After three years of continuous cropping, the X2 and X1 concentrations were significantly lower than the control by 11.4% and 65.4%, respectively (P<0.05). Under the n-butanol and residual aqueous phase treatments, the germination index was lower than the control after both one and three years of continuous cropping. After one year of continuous cropping, the n-butanol phase X3 concentration showed no significant difference from the control, while the X2 and X1 concentrations were significantly lower than the control by 57.9% and 64.4%, respectively. After three years of continuous cropping, the germination index at the n-butanol phase X1~X3 concentrations was significantly lower than the control by 36.4%~99.4% (P<0.05). Under the residual aqueous phase treatment, the number of crops continuously cropped for 1 year (X1~X3) was significantly reduced by 31.8%~91.9% compared with the control, and the number of crops continuously cropped for 3 years (X1~X3) was significantly reduced by 72.9%~100% compared with the control.

[0115] 2.2.5 Effects of different extraction fractions of continuously cropped soil aqueous extract on the radicle length of Codonopsis pilosula seeds

[0116] Table 8. Effects of different extraction fractions of soil aqueous extract from continuously cropped soil on seed radicle length.

[0117] As shown in Table 8, in the petroleum ether phase, the seed radicle length of seeds treated with concentrations X1 to X3 after one year of continuous cropping was significantly higher than that of the control by 6.0% to 55.0%, and after three years of continuous cropping, the X3 concentration was significantly higher than that of the control by 14.6%, while the X2 and X1 concentrations were significantly lower than those of the control by 0.7% and 14.0%, respectively. In the ethyl acetate phase, the X3 concentration after one year of continuous cropping and after three years of continuous cropping was significantly higher than that of the control by 22.9% and 9.6%, respectively, while the X2 to X1 concentrations were significantly lower than those of the control by 11.2% to 17.4% and 24.7% to 27.1%, respectively. Seed radicle lengths treated with chloroform, ethyl acetate, and residual aqueous phase were all significantly lower than those treated with the control. The concentrations of chloroform in consecutive cropping for 1 and 3 years (X1-X3) were significantly lower than those of the control by 22.4%-14.5% and 65.0%-34.8%, respectively. The concentrations of n-butanol in consecutive cropping for 1 and 3 years (X1-X3) were significantly lower than those of the control by 84.1%-24.3% and 92.8%-34.9%, respectively. The concentrations of residual aqueous phase in consecutive cropping for 1 and 3 years (X1-X3) were significantly lower than those of the control by 91.0%-38.7% and 100%-47.8%, respectively.

[0118] 2.2.6 Analysis of the allelopathic effect index of different extracts from continuously cropped soil aqueous extracts on Codonopsis pilosula seeds

[0119] The allelopathic effect index can reflect the strength of the allelopathic effect, as shown in Table 9:

[0120] Table 9. Allelopathic effect index of different extracts from continuously cropped soil aqueous extracts on Codonopsis pilosula seeds.

[0121] The effects of different extraction fractions of soil aqueous extracts from consecutive cropping for 1 year and 3 years on the germination rate, germination potential, germination index, and radicle length of Codonopsis pilosula seeds showed a significant concentration gradient effect. At lower concentrations, the petroleum ether and ethyl acetate extracts showed a certain promoting effect on all germination indicators, but as the concentration increased to a certain range, they began to show an inhibitory effect, which intensified with increasing concentration. Except for the chloroform extract, which promoted the germination rate and germination index of Codonopsis pilosula seeds at a concentration of X3 after 1 year of consecutive cropping, all concentrations of chloroform, n-butanol, and the remaining aqueous phase showed an inhibitory effect on all germination indicators, which gradually intensified with increasing concentration. At the same concentration and for the same number of years, the relative germination rate (RI) of the remaining aqueous phase was highest at |RI| of the n-butanol phase, followed by the chloroform phase, then the ethyl acetate phase, and finally the petroleum ether phase, indicating that the remaining aqueous phase had the strongest inhibitory effect on the germination ability of Codonopsis pilosula seeds, while the petroleum ether extract had the weakest inhibitory effect. Furthermore, at the same concentration in the same extraction fraction, the inhibitory effect of soil extracts from three consecutive years of cropping was stronger than that from one year of cropping. Combined with the comprehensive allelopathic effect index, the |SE| values ​​of the n-butanol phase and the remaining aqueous phase after three years of cropping were 0.978 and 1.000, respectively, which were higher than the |SE| values ​​of other fractions. In conclusion, this study selected the n-butanol phase and the remaining aqueous phase from three years of cropping to identify and analyze the types and contents of autotoxic substances contained therein.

[0122] 2.2 Identification of components from the site with the strongest autotoxic activity

[0123] 2.2.1 Evaluation of Experimental Data Quality

[0124] 2.2.1.1 Principal Component Analysis (PCA) of the Population Sample

[0125] All peaks extracted from the experimental and QC samples were subjected to PCA analysis, as shown in Figure 1. Green dots represent QC samples, blue dots represent n-butanol extract phase samples, and red dots represent the remaining aqueous phase samples. The results show that QC samples are tightly aggregated in both positive and negative ion modes, indicating good reproducibility of the experiment.

[0126] 2.2.1.2 Correlation Analysis of QC Samples

[0127] Pearson correlation analysis was performed on the QC samples, as shown in Figure 2. The horizontal and vertical axes represent each QC sample, and the points in each small grid represent the ion peaks (metabolites) extracted from the QC sample. The horizontal and vertical axes represent the logarithmic values ​​of the ion peak signal intensity values. Generally, the correlation coefficient is greater than 0.9. The results show that the correlation coefficients among the QC samples are all above 0.9, indicating good experimental repeatability.

[0128] 2.2.2 LC-MS Analysis Results

[0129] The compounds identified by LC-MS are shown in Figure 3. According to the chemical classification information, the compounds with a matching score greater than 90 were selected, as shown in Tables 10 and 11. Among them, 59 effective compounds were detected in positive ion mode and 37 effective compounds were detected in negative ion mode. Figures 4 and 5 are the chromatograms of detection in positive and negative ion modes.

[0130] Table 10. Detection Results of Positive Ion Mode LC-MS | No. | English Name | Chinese Name | No. | English Name | Chinese Name | M1. gamma-aminobutyric acid | γ-aminobutyric acid | M31. Deoxyadenosine | M21. Aminocyclopropanecarboxylic acid | 1-aminocyclopropanecarboxylic acid | M32. Dioctyl phthalate | M31. O-octadecyl-2-O-methyl-sn-glycerol | 1-O-octadecyl-2-O-methyl-sn-glycerol | M33. Diphenylamine | M41. 1,2-diamino-2-methylpropane | 1,2-diamino-2-methylpropane | M34. Gabapentin | M51. 2-hydroxydodecanoic acid | 1,2-hydroxydodecanoic acid | M35. Gentiopiroside | M62-aminophenol | 2-aminophenol | M36. Hippuric acid | M72,2,6,6-tetramethyl-4 piperidone 2,2,6,6-Tetramethylpiperidinone M37 Hirsuteine ​​M82'-deoxycytidine M38 Hypoxanthine M92'-deoxyinosine M39 Indole-3-acetonitrile 3-indoleacetonitrile M102(1h)-pyridinone 2-hydroxypyridine M40 Isopropylcocaine M112s-amino-4e-pentadecene-1,3r-diol 2S-amino-4E-pentadecene-1,3R-diol M41L-leucyl-l-leucine methyl ester L-leucine-L-leucine methyl ester M123-pyridylacetic acid 3-pyridylacetic acid M42 Lauramidopropylbetaine Lauramidopropylbetaine M134-benzofuranethanamine 2,3-dihydro-.alpha.-methyl-2,3-dihydro-α-methyl-4-benzofuran ethylamine M43 Melamine M144-hydroxyquinoline M44-methylguanidine M154-methyl-1h-pyrazole M45 Myristamine oxide N-oxide-n-tetradecyl-N,N-dimethylamine M164-methylcinnamicacid M46 Myristic Myristic acid (M174-quinolinecarboxylate, M47N-acetyl-beta-d-mannosamine, M185-aminolevulinica, M48N-methyl-gamma-oxo-3-pyridinebutanamide, M19-acetaminophen, M49N-palmitoyl-d-sphingosine, M20-adenine, M50N.epsilon.-acetyl-l-lysine, M21-adenosine, M51-norharmane, M22-arachidic acid) Acids: arachidic acid (M52), ouabain (M23), 2,4-di-tert-butylphenol (M53), palmitamide (M24), benzotriazole (M54), Phe-Ser-Lys (M25), Benzyl alcohol (M55), Prednisolone 21-acetate (M26), Betaine (M56), Dibutyl phthalate (M27), Ciprofloxacin (M57), Serricornin (M28), Clozapine (M58), Tetrandrine (M29), Cytidine (M59), Trigonelline (M30), D-glucosaminic acid (D-glucosinolate). surface

[0131] Table 11 LC-MS Detection Results in Negative Ion Mode | No. | English Name | Chinese Name | No. | English Name | Chinese Name | M60 | 1,11-undecanedicarboxylic acid | Tegdecanediic acid | M79 | Cis,cis-muconic acid | cis-hexadienedicarboxylic acid | M61 | 1,2-benzenedicarboxylic acid | Phthalic acid | M80 | D-myo-inositol-3,4,5,6-tetraphosphate | D-inositol-3,4,5,6-tetraphosphate | M62 | 13-hydroxy-9z,11e-octadecadienoic acid | 13(s)-hydroxy-9z,11e-octadecadienoic acid | M81 | DL-valine | DL-valine | M63 | 16-hydroxyhexadecanoic acid | 16-hydroxypalmitic acid | M82 | Ethyl laurate | M64 | 2-benzothiazolsulfonic acid | 2-methoxybenzenesulfonic acid | M83 | Ethyl Diethyl sulfate (MSB) M65 2-chloro-6-fluorophenol M84 Glycolate M66 3-hydroxybenzaldehyde M85 Indole-3-carboxylicacid M67 3-hydroxyoctanoic acid M86 N-lauroylsarcosine M684-aminosalicylicacid M87 Nonanoic acid M69 4-hydroxy-5e,7z,10z,13z,16z,19z-docosahexaenoic acid 4-hydroxy-5e,7z,10z,13z,16z19z-Docosahexaenoic acid (M88) Butanoic acid (M704) hydroxybenzaldehyde (M89) Oleic acid (M714) pyridoxic acid (M90) p-coumaric acid (M726) hydroxyhexanoate (M91) Palmitic acid (M737) hydroxycoumarin (M92) Phenol (M74) Azelaic acid (M93) Propionic acid (M75) Benzenesulfonic acid (M94) Pyruvaldehyde (M76) Octadecanoicacid (M95) Sulfoacetic acid (M77) Cholesterylsulfate (M96) Uracil (M78) Cis-9-palmitoleic acid surface

[0132] 2.2.3 Screening of typical autotoxic substances

[0133] Chemical composition identification and key substance screening: LC-MS non-targeted metabolomics analysis was performed on the active sites to obtain a comprehensive chemical composition profile. Furthermore, partial least squares-discriminant analysis (PLS-DA) was innovatively applied to establish a mathematical model using chemical composition (X variable) and seed germination indicators (Y variable). By analyzing the variable importance projection values ​​(VIP>1), the compounds that contributed most to germination inhibition were identified. Positive regression coefficients further confirmed a positive correlation between the concentration of these compounds and the inhibitory intensity, thus accurately locating seven key autotoxic substances from massive amounts of data.

[0134] Specifically, the following was performed: The dimensionless peak area of ​​the compounds under both positive and negative ion modes was used as the independent variable (X), and the dimensionless values ​​of the germination indices (germination rate, germination index, and radicle length) of seeds treated with n-butanol extract and residual aqueous phase for three consecutive years (as described in section 2.1) were used as the dependent variable (Y). Partial least squares discriminant analysis (PLS-DA) was conducted to obtain the variable importance projection (VIP) values ​​and standardized regression coefficients. The results are shown in Figures 6 and 7. In Figure 6, uppercase letters A, B, C, and D represent germination rate, germination potential, germination index, and radicle length, respectively; the same applies to Figure 7. Under positive ion mode, the VIP values ​​of all indicators M43, M31, M20, M29, M16, M56, M14, M37, M38, M15, M44, M13, M33, and M23 are greater than 1. Among them, the regression coefficients of M23, M33, and M56 are positive, showing a positive correlation with each germination indicator. Under negative ion mode, the VIP values ​​of all indicators M91, M88, M79, M96, M64, M95, M67, and M71 are greater than 1. Among them, the regression coefficients of M79, M88, M91, and M95 are positive, showing a positive correlation with each germination indicator.

[0135] In summary, based on the PLS-DA analysis results, compounds with a VIP value greater than 1 and a positive regression coefficient in the seed germination index under positive and negative ion modes were selected as potential autotoxic substances (7 types), namely 2,4-di-tert-butylphenol, diphenylamine, dibutyl phthalate, cis-hexadienoic acid, n-butyric acid, palmitic acid, and sulfoacetic acid.

[0136] 2.3 Analysis of the content of typical autotoxic substances in continuously cropped soils

[0137] Functional verification and mechanism analysis: Exogenous addition experiments were conducted on the screened key substances to verify their autotoxic effects under controlled conditions. The mechanism of action was analyzed in depth from two levels: growth and development indicators and physiological and biochemical indicators, to complete the scientific verification from correlation to causation.

[0138] Based on the screening results above, this paper selected two compounds, 2,4-di-tert-butylphenol and n-butyric acid, from the compounds detected in positive and negative ion modes to analyze their content in the soil of continuous cropping of Codonopsis pilosula. The detection results are shown in Table 12.

[0139] Table 12. Analysis results of the content of two autotoxic substances in continuously cropped soil. Compound concentration (μg·mL) -1 ) content (μg·g -1 2,4-Di-tert-butylphenol 0.38g 0.13g butyric acid 0.42g 0.15g surface

[0140] 2.4 Effects of two autotoxic substances on the germination of Codonopsis pilosula seeds

[0141] Changes in seed plumules and radicles can directly reflect the effects of autotoxic substances on seed germination. As shown in Figure 8, the growth of plumules and radicles is inhibited as the concentration of the two autotoxic substances in the solution increases.

[0142] 2.4.1 Effects of two autotoxic substances on the germination index of Codonopsis pilosula seeds

[0143] As shown in Figure 9, within the experimental concentration range, the effects of the two autotoxic substances on the germination indices of Codonopsis pilosula seeds showed consistent trends: germination rate, germination potential, germination index, and radicle length all gradually decreased with increasing solution concentration. At low concentrations, the germination rates of 2,4-di-tert-butylphenol and n-butyric acid solutions were slightly lower than the control, but not significantly different. With increasing concentrations of both autotoxic substances, the germination rate decreased by 18.2%–29.6% in the E2–E4 range and significantly decreased by 8.0%–35.2% in the D2–D4 range (P<0.05). The germination potential of seeds treated with 2,4-di-tert-butylphenol was significantly lower than the control at all concentrations, decreasing by 19.6%–57.0% in the E1–E4 range. The lowest concentration of n-butyric acid showed no significant difference from the control, but was significantly lower in the D2–D4 range, decreasing by 22.4%–55.1% (P<0.05). Seed germination index showed low sensitivity to the two autotoxic substances. The radicle, as the structure directly in contact with the culture medium, showed the most significant inhibition in length. Treatment with 2,4-di-tert-butylphenol solution at concentrations E1–E4 resulted in a significant reduction in radicle length of 12.1%–42.8% compared to the control (P<0.05), while treatment with butyric acid solution at concentrations D1–D4 resulted in a significant reduction in radicle length of 10.9%–39.9% (P<0.05). In conclusion, both autotoxic substances inhibited various germination indices of Codonopsis pilosula seeds to varying degrees. This indicates that the two autotoxic substances comprehensively hinder the germination and early growth of Codonopsis pilosula seeds by interfering with germination rate, delaying germination process, and inhibiting radicle elongation, with the effect intensifying with increasing concentration.

[0144] 2.5 Effects of two autotoxic substances on the growth of Codonopsis pilosula seedlings

[0145] As shown in Figure 10, the degree of inhibition on the growth of Codonopsis pilosula seedlings gradually increased with the increase of the concentration of the two autotoxic substances. When the concentration was the highest, the seedling length and root length of Codonopsis pilosula seedlings were lower than those of the control and low concentration treatments. It can be seen that the treatment with the two autotoxic substances can affect the growth of Codonopsis pilosula seedlings.

[0146] 2.5.1 Effects of two autotoxic substances on growth indicators of Codonopsis pilosula seedlings

[0147] As shown in Figure 11, the two autotoxic substances had the same effect on the fresh weight and seedling length of Codonopsis pilosula seedlings. When the 2,4-di-tert-butylphenol and n-butyric acid solutions were treated at lower concentrations (i.e., E1 and D1), the fresh weight and seedling length of the seedlings were not significantly different from the control. When the concentration was increased to the range of E2 to E4, the fresh weight and seedling length of the seedlings were significantly reduced by 21.8% to 49.4% and 17.3% to 34.6% respectively compared with the control (P<0.05). When the n-butyric acid concentration was at D2 to D4, the fresh weight and seedling length of the seedlings were significantly reduced by 11.8% to 46.9% and 12.4% to 20.2% respectively compared with the control (P<0.05). Within the experimental concentration range (E1~E4), 2,4-di-tert-butylphenol significantly reduced seedling root length compared to the control, with a decrease of 8.5%~43.1%. However, the butyric acid (D1) treatment showed no significant difference from the control, but within the D2~D4 range, it significantly reduced root length by 20.1%~40.8% compared to the control (P<0.05). These results indicate that both autotoxic substances significantly inhibited the fresh weight, root length, and seedling length of *Codonopsis pilosula* seedlings, severely impacting their growth and development. This may further affect subsequent plant development and stress resistance. Therefore, the inhibitory effect of autotoxic substances on the growth and development of *Codonopsis pilosula* seedlings is one of the important manifestations of their influence on plant growth.

[0148] 2.5.2 Effects of two autotoxic substances on the physiological and biochemical indicators of Codonopsis pilosula seedlings

[0149] As shown in Figure 12, both 2,4-di-tert-butylphenol and n-butyric acid treatments exhibited concentration-dependent regulatory effects on the activities of antioxidant enzymes (POD, SOD, CAT) in seedlings, demonstrating a "low concentration promotes, high concentration inhibits" phenomenon. In the 2,4-di-tert-butylphenol treatment, the activities of the three enzymes at concentration E1 were higher than the control but not significantly different. At concentration E2, CAT activity significantly increased by 23.1% compared to the control, while POD activity significantly decreased, and SOD activity showed no significant change (P<0.05). The inhibitory effect was significantly enhanced at concentrations E3-E4, with POD and SOD activities decreasing by 24.3%–67.4% and 26.0%–35.3%, respectively, and CAT activity significantly decreased by 24.8% compared to the control at the highest concentration E4 (P<0.05). Butyric acid solution enhanced the activities of all three enzymes at concentrations D1-D2, with SOD activity significantly increasing by 22.1% compared to the control at concentration D2, while POD and CAT activities showed no significant change (P<0.05). However, at concentrations D3-D4, an inhibitory effect was observed, with the highest concentration, D4, showing significant decreases in the activities of the three enzymes by 66.5%, 59.7%, and 85.5%, respectively, compared to the control (P<0.05). In summary, the four autotoxic substances exhibit a biphasic regulatory effect on the seedling antioxidant enzyme system, with the intensity of the effect being concentration-dependent; low concentrations show a promoting effect, while high concentrations produce a significant inhibitory effect.

[0150] As shown in Figures 13 and 14, the MDA and Pro contents of seedlings treated with 2,4-di-tert-butylphenol and n-butyric acid were both higher than those of the control, and gradually increased with increasing solution concentration. At 2,4-di-tert-butylphenol concentrations E1–E4, the MDA and Pro contents of seedlings were significantly higher than the control by 13.2%–56.1% and 19.5%–88.3%, respectively (P<0.05). At n-butyric acid concentration D1, the MDA content of seedlings was higher than the control but not significantly higher; however, at concentrations D2–D3, it was significantly higher than the control by 28.1%–54.4% (P<0.05). The Pro content of seedlings was significantly higher than the control at concentrations D1–D4, with an increase of 14.9%–76.6% (P<0.05).

[0151] As shown in Figure 15, the soluble sugar content of *Codonopsis pilosula* seedlings showed a trend of first increasing and then decreasing under treatment with both autotoxic substances. At low concentrations (E1 and D1), the soluble sugar content of seedlings was higher than that of the control, but the difference was not significant. The soluble sugar content of seedlings treated with 2,4-di-tert-butylphenol at concentrations E2 to E4 was lower than that of the control. The soluble sugar content of seedlings treated with butyric acid at concentration D2 was significantly higher than that of the control, while at concentrations D3 and D4 it was significantly lower than that of the control by 5.9% and 30.8%, respectively (P<0.05).

[0152] As shown in Figure 16, the chlorophyll content in the leaves of *Codonopsis pilosula* seedlings generally decreased with increasing solution concentration under both compound treatments. The concentrations of 2,4-di-tert-butylphenol (E1 and E2) were lower than the control, while E3 and E4 were significantly lower than the control by 19.0% and 26.0%, respectively (P<0.05). The concentration of butyric acid (D1) was higher than the control, while the concentration of butyric acid (D2) was lower. The concentrations of butyric acid (D3 and D4) were significantly lower than the control by 16.2% and 12.8%, respectively (P<0.05). These results collectively indicate that the two autotoxic substances exerted a significant stress effect on the physiological metabolism of *Codonopsis pilosula* seedlings, and the inhibitory effect of 2,4-di-tert-butylphenol was more significant than that of butyric acid.

[0153] The above experiments show that:

[0154] (1) This invention uses four organic solvents of different polarities—petroleum ether, chloroform, ethyl acetate, and n-butanol—to extract and separate the aqueous extract of Codonopsis pilosula from continuously cropped soil, and conducts autotoxicity tests. The results show that the inhibitory effect on the germination of Codonopsis pilosula seeds gradually increases with the increase of the extract concentration. In addition, at the same concentration, the autotoxicity intensity of each extract fraction is as follows: residual aqueous phase > n-butanol phase > chloroform phase > ethyl acetate phase > petroleum ether phase. This result may be closely related to the polarity and solubility of allelochemicals. Fractions with stronger polarity may contain more water-soluble allelochemicals, which are more likely to interact with the cell membranes of seeds and seedlings, thereby producing a stronger inhibitory effect.

[0155] (2) In order to further identify the types of autotoxic substances in Codonopsis pilosula, this invention uses LC-MS technology and combines it with PLS-DA analysis to screen typical autotoxic substances in Codonopsis pilosula, including 2,4-di-tert-butylphenol, diphenylamine, dibutyl phthalate, cis-hexadienoic acid, n-butyric acid, palmitic acid and sulfoacetic acid.

[0156] Two typical autotoxic substances, 2,4-di-tert-butylphenol and n-butyric acid, were selected for autotoxicity studies. Seed germination experiments showed that both autotoxic substances, at certain concentration thresholds, significantly inhibited seed germination indicators of Codonopsis pilosula, including germination rate, germination potential, germination index, and radicle length. This indicates that autotoxic substances inhibit both seed germination and early seedling growth in Codonopsis pilosula. The significance of these conclusions obtained through the method of this invention lies in laying the foundation for developing strategies to alleviate the inhibition of seed germination or seedling growth in Codonopsis pilosula. Targeted reduction of the aforementioned autotoxic substances can alleviate the inhibition of seed germination or seedling growth, thereby achieving the goal of controlling continuous cropping obstacles and increasing the yield of Codonopsis pilosula.

[0157] (3) In this invention, the MDA and Pro contents of *Codonopsis pilosula* seedlings increased with increasing autotoxic substance concentration, while the soluble sugar content first increased and then decreased. At low concentrations, plants may enhance osmotic regulation and energy supply by accumulating soluble sugars and Pro, while simultaneously activating the antioxidant system to cope with ROS accumulation. However, as the concentration of autotoxic substances increases, stress intensifies, and the damage of autotoxic substances to cell membranes and cell metabolism worsens, leading to a further increase in MDA content and a decrease in soluble sugar content, ultimately affecting the normal growth and development of plants. In addition, autotoxic substance treatment inhibited the chlorophyll content in *Codonopsis pilosula* seedling leaves, indicating that autotoxicity inhibits photosynthesis or alters the overall metabolic state. This may be related to factors such as autotoxic substances inhibiting chlorophyll synthesis, accelerating its degradation, or damaging the structure and function of photosynthetic organs such as chloroplasts and thylakoids.

Claims

1. A method for identifying and screening autotoxic substances in soils continuously cropped with Codonopsis pilosula, characterized in that: Includes the following steps: (1) Collect rhizosphere soil from continuously cropped Codonopsis pilosula, extract with water, and then perform gradient extraction with petroleum ether, chloroform, ethyl acetate, and n-butanol. Seed germination and seedling growth activity tests were conducted on each extracted fraction to obtain the most active n-butanol phase and the remaining aqueous phase. (2) Analyze the n-butanol phase and the remaining aqueous phase obtained in step (1) using liquid chromatography-mass spectrometry (LC-MS). The chromatographic conditions were: HILIC column, mobile phase A being an aqueous solution containing 25 mM ammonium acetate and 25 mM ammonia, and mobile phase B being acetonitrile. A gradient elution program was used to ensure that the compounds in the n-butanol extract and the remaining aqueous phase were separated at 12... Baseline separation was achieved within min; (3) The data obtained in steps (1) and (2) were processed by partial least squares discriminant analysis to screen out compounds whose importance projection value on the seed germination index variable of Codonopsis pilosula was greater than 1 and whose regression coefficient was positive. Then, the autotoxic substances in the soil of Codonopsis pilosula continuous cropping were identified as 2,4-di-tert-butylphenol, diphenylamine, dibutyl phthalate, cis-hexadienedia acid, butyric acid, palmitic acid, and sulfoacetic acid.

2. The method for identifying and screening autotoxic substances in soils continuously cropped with Codonopsis pilosula according to claim 1, characterized in that: In step (2), a gradient elution program is used to achieve baseline separation of 2,4-di-tert-butylphenol and n-butyric acid in the chromatogram.

3. The method for identifying and screening autotoxic substances in soils continuously cropped with Codonopsis pilosula according to claim 1, characterized in that: In step (2), the mass spectrometry conditions are electrospray ionization positive and negative ion mode, nebulizer gas pressure 50 psi, ion source temperature 350℃, scanning range 70~1200 Da, and resolution 60000.

4. The method for identifying and screening autotoxic substances in soils continuously cropped with Codonopsis pilosula according to claim 1, characterized in that: In step (1), the n-butanol phase and the remaining aqueous phase are based on the fact that their comprehensive allergic reaction index |SE| value on Codonopsis pilosula seeds is greater than 0.

9.

5. The application of autotoxic substances screened by the identification and screening method of claim 1 in the prevention and control of continuous cropping obstacles, characterized in that, The aforementioned prevention and control refers to using screened autotoxic substances as targeted substances to reduce their levels.

6. The application of the identification and screening method of claim 1 in alleviating the inhibition of Codonopsis pilosula seed germination or seedling growth, characterized in that, The inhibition of seed germination or seedling growth of Codonopsis pilosula can be alleviated by targeting and reducing the autotoxic substances of claim 1.

7. The autotoxic substance that inhibits seed germination or seedling growth of Codonopsis pilosula, obtained by the identification and screening method of claim 1, is characterized in that: It includes at least one of the seven autotoxic substances identified in claim 1.

8. The autotoxic substance that inhibits seed germination or seedling growth of Codonopsis pilosula, obtained by the identification and screening method of claim 1, is characterized in that: Based on soil dry weight, the concentration range of 2,4-di-tert-butylphenol and / or n-butyric acid in the autotoxic substances is 0.13–0.15 μg·g⁻¹. -1 .

Images