Study site
This experiment was conducted in Tanchang County, Longnan City, Gansu Province (E 104°01', N33°46', altitude 2390 m). The area is in the Longnan temperate humid zone within temperate continental climate, with an average annual temperature of 9.3℃, an average annual precipitation of 583.9 mm, an average annual sunshine duration of 1986.5 h, and an average annual frost-free period of 181d. The soil of the study site had a pH of 8.2, an organic matter content of 12.5 g/kg, nitrate nitrogen of 15.7 mg/kg, available phosphorus of 22.6 mg/kg, and available potassium of 178.1 mg/kg. Previously, the site was cultivated with Astragalus memeranaceus (Fisch.) Bge. Var. mongholicus (Bge.) Hsiao.
The seeds and seedlings of C. pilosula Nannf. var. modesta (Nannf.) L. T. Shen, a plant of Codonopsis platycodon were purchased from Tanchang Traditional Chinese Medicinal Materials Trade City.
Measurement indicators and methods
Collection of aqueous leachate from root secretions of C. pilosula.
The intact root systems of 100 harvested from well-established, disease- and pest-free C. pilosula seedlings from the test site. The root system was washed thoroughly with deionised water to remove soil, soaked in 1% KMnO4 aqueous solution for 15 minutes for root disinfection, and then cleaned 2–3 times with deionized water. Root samples were taken to the laboratory as soon as possible and continued to culture the cleaned C. pilosula seedlings.
Hydroponics (Aoi et al. 2022) was used to cultivate C. pilosula seedlings. Specifically, cleaned and sterilised C. pilosula seedlings were planted on evenly perforated foam boards and moved into hydroponic tanks, with a total of five hydroponic tanks and twenty C. pilosula seedlings planted in each tank. Tanks were replenished with 1/2 Hoagland nutrient solution (Delden et al. 2020) {Pure water: Concentrate A masterbatch (contains mainly calcium nitrate and potassium nitrate), Concentrate B masterbatch (contains mainly ammonium phosphate and magnesium sulphate), Concentrate C masterbatch (contains mainly ferrous sulphate heptahydrate, disodium ethylene diamine tetraacetic acid) = 17:1:1:1}. Aeration and light were provided for 12 h per day, and the nutrient solution was changed once in 5 d to ensure normal growth of the plants.
The root secretions of C. pilosula were collected at the early growth stage (30 May), growth middle stage (29 July), and harvesting stage (28 September) as follows: fifteen C. pilosula plants were taken and the root surface was cleaned to adequately remove residual components of the nutrient solution; subsequently, they were placed in a beaker containing 1 L of sterile deionised water, the beaker was wrapped in a black cloth to protect roots from light, while leaves were exposed to light, and after 4 hours, the root system was removed. The 1 L aqueous solution of root secretion was concentrated to 50, 100, and 150 mL, and placed in a 4 ℃ refrigerator to use as a treatment solution for seed germination experiments (Li et al. 2023). The aqueous solution of root secretion collected at 3-day intervals using the same method as above, for a total of 3 collections; the aqueous solutions of root secretions collected during the same growth period of C. pilosula were combined.
Detection of Allelopathic autotoxicity
Seed germination experiment
Distilled water was used as the control and three concentrations of root secretion aqueous leachate were used as treatment groups for a total of 12 treatments, and each treatment replicated three times. Fifty uniformly sized, intact, and undamaged C. pilosula seeds (sterilised by soaking in 15% NaClO3 solution for 10 min) were placed on a petri dish with 5 mL of each treatment solution; petri dishes with seeds were incubated in a thermostatic incubator at 25°C with a light intensity of 4,000 µmol·m− 2·s− 1 and 12 h of light. Water was sprayed periodically to keep the filter paper moist. The number of germinated seeds in each petri dish was counted every day (the germination criterion was that the seed radicle broke through the seed coat and reached half of the length of the seed), and germinated seeds were removed (1995).
Seedling growth experiments
The “small cup method” was used (Zhe et al. 2023) to grow seedlings. Specifically, seedlings grown for 7 d were transferred to histoculture flasks for further growth with the appropriate concentration of immersion solution and returned to the incubator. Growth indicators (stem length, root length, and total biomass) and physiological indicators of C. pilosula seedlings were measured on the 20th day of growth.
Stem length was measured on straightened rhizomes of C. pilosula seedlings with vernier calipers at the obvious demarcation line between the above-ground and the underground part to the tip of the leaf blade, expressed in cm.
Root length was measured on straightened roots of C. pilosula seedlings with vernier calipers at the obvious demarcation line between the above-ground and the underground part to the tip of the root, expressed in cm.
Total fresh weight was determined as mass of seedlings in three petri dishes per treatment.
Superoxide dismutase (SOD) activity was determined using the nitrogen blue tetrazolium (NBT) photoreduction method. Peroxidase (POD) activity was determined using the guaiacol method, and the malondialdehyde (MDA) content with the two-component spectrophotometric method (Günes et al. 2019).
Identification of allelopathic and autotoxic substances
Gas chromatography-mass spectrometry (GC-MS) analysis was used to qualitatively identify the chemical constituents in an aqueous solution of root secretion of C. pilosula at a concentration of 125 mg/mL (Dalluge et al. 2002). Detection instrument: Agilent 7890B-7000D triple quadrupole gas chromatography-mass spectrometry (Agilent, USA) with HP-5MS (30 m×0.25 mm×0.25 µm) capillary column. The detection conditions were as follows: GC: injection volume of 3 µL, injection temperature of 250℃, programmed temperature increase: the initial column temperature was 50℃ or 2 min, and then the temperature was increased to 210℃ at 5℃/min for 10 min. Mass spectrometry conditions included anion source 70 ev, scanning range 35–600 m/z, ion source temperature of 230℃, quadrupole temperature of 150℃, with He as the carrier gas at flow rate of 1 mL/min. The mass spectra were analysed by applying the NIST mass spectrometry database computer retrieval system (https://chemdata.nist.gov/) for identification of unknowns, and the peak area normalisation method was used to calculate the relative content of each component.
Verification of allelopathic effects of monomer compounds
The most abundant monomer compounds were screened and configured into solutions with concentrations of 1, 0.1, and 0.01 g/L. There were three concentration treatments with three replications per treatment, and a control group. In each replication, 50 uniformly sized C. pilosula seeds were randomly selected for seed germination and seedling growth tests (the same method as in 1.3.2.1 and 1.3.2.2) to verify the autotoxicity effects of the monomer compounds.
The Williamson allelopathic response index (Ir) was used to measure the type and intensity of allelopathic effects, \({I}_{r}=1-CK÷T\) (when T ≥ CK) or \({I}_{r}=T÷CK-1\) (when T < CK) (Ghimire et al. 2019), where CK is the mean of the control value indicators and T is the mean of the treatment group indicators; Ir < 0 indicates inhibition of seed germination and seedling growth and Ir > 0 indicates promotion of seed germination and seedling growth, and the magnitude of the absolute value is consistent with the strength of promotion or inhibition.
The allelopathic comprehensive effect index (Mr.) is the arithmetic mean of the percentage of inhibition or promotion of multiple measured indicators(Germination rate, germination strength, stem length,root length, fresh weight) of C. pilosula seedlings in the same treatment. The calculation formula is:
$${M}_{r}=\left({\sum }_{j=1}^{n}{I}_{j}\right)÷n$$
where Mr is the chemosensory composite effect index, I is the Ir value, and n is the total number of indicators measured. Mr >0 indicates a facilitating effect and Mr <0 indicates an inhibitory effect.
Data processing
IBM SPSS Statistics version 26 (IBM SPSS Inc., Chicago, USA) software was used for ANOVA
We used SPSS 26.0 (IBM SPSS Inc., Chicago, USA) software for one-way analysis of variance (ANOVA) with a significance level of α = 0.05. Correlation analyses and graphing were performed using Origin 2021 (www.OriginLab. com) software.