An inducing agent and a method for inducing paeoniae suffruticosae to accumulate benzopyranone derivatives

Abstract

The present application relates to a kind of induction agent and the method for inducing paeonia obovata accumulate benzopyranone derivative.The preparation method includes the following steps: 1) obtain Fusarium oxysporum Strain, after scale-up culture, safety treatment is carried out, and the safety treatment includes: 1-1) the strain obtained by scale-up culture is collected, and inactivated bacteria powder is prepared by high temperature inactivation or ultraviolet irradiation treatment;And / or 1-2) after filtering the culture solution produced by scale-up culture, the obtained extracellular polypeptide and low molecular organic acid are separated by organic solvent separation, silica gel column chromatography or chromatography separation treatment, which are the purified inducer;2) the inactivated bacteria powder or the purified inducer is mixed with carrier adjuvant, and a water-dispersible preparation is prepared.The preparation method is safety treated, so that the prepared plant inducer does not cause plant diseases or environmental pollution.

Description

Technical Field

[0001] This invention relates to the field of plant secondary metabolism engineering technology, and in particular to an inducer and a method for inducing the accumulation of benzopyranone derivatives in Paeonia suffruticosa. Background Technology

[0002] Mangrove plant peony ( Acanthus ilicifolius *Acanthaceae* is an evergreen shrub belonging to the Acanthaceae family, widely distributed in the intertidal zone of subtropical and tropical coastal areas. Its stems, leaves, and roots are rich in secondary metabolites such as flavonoids, terpenoids, and benzopyranones. 2-(2,4-dimethoxyphenyl)-3,5,6,7,8-pentamethoxychromen-4-one is a typical benzopyranone derivative with significant antioxidant, anti-inflammatory, and hepatoprotective activities, possessing important application value in the pharmaceutical and functional food fields. However, extraction from natural sources faces bottlenecks such as low yield, large seasonal fluctuations, and unsustainable resources, making it difficult to meet industrialization needs; traditional chemical synthesis processes are cumbersome, costly, and prone to environmental pollution.

[0003] The phenylpropanoid pathway in plants is a key hub in the synthesis of secondary metabolites. This pathway involves the deamination of phenylalanine to cinnamic acid, which is then converted to p-coumaroyl-CoA via cinnamic acid 4-hydroxylase (C4H) and 4-coumaryl-CoA ligase (4CL). Subsequently, under the action of branched enzymes such as chalcone synthase (CHS), various flavonoids, anthocyanins, and benzopyranones are produced. As a benzopyranone derivative, it is also a potential core strategy to enhance the accumulation of 2-(2,4-dimethoxyphenyl)-3,5,6,7,8-pentamethoxybenzopyran-4-one by regulating the phenylpropanoid pathway in plants.

[0004] Existing research indicates that using microorganisms or their metabolites as inducers to activate plant secondary metabolism is a research hotspot for improving the yield of natural products. For example: Fusarium Extracts from fungi can enhance the synthesis of flavonoids, saponins, and phenolic acids in various medicinal plant cell culture systems; Sclerotinia sclerotiorum Mycelial proteins can induce the accumulation of ginseng cell saponins; Aspergillus This strain significantly increased the lycopene and capsaicin content in tomatoes and peppers. (Partial) FusariumWhen endophytic fungi interact with host plants, they can induce the production of signaling molecules such as ethylene, jasmonic acid, and salicylic acid, thereby activating the expression of key enzymes (such as PAL, C4H, 4CL, and CHS) in the phenylpropanoid metabolic pathway. However, excessive endophytic fungi can harm plants. Specifically, an overabundance of endophytic fungi can lead to physiological imbalances, inhibit plant growth, and even cause plant diseases. Excessive infection can cause carbon loss, inhibit photosynthesis and nutrient absorption, and reduce biomass. Furthermore, under certain extreme conditions, an excessive number of fungi can exceed the plant's tolerance, leading to metabolic disorders and physiological diseases, affecting plant health and yield. Summary of the Invention

[0005] The present invention aims to disclose an inducer and a method for inducing the accumulation of benzopyranone derivatives in Paeonia suffruticosa, in order to solve one or more technical problems existing in the prior art and provide at least one beneficial alternative or create conditions.

[0006] To achieve the above objectives, the present invention provides the following technical solution:

[0007] The first aspect of this invention is to provide a method for preparing a plant inducer. The preparation method includes the following steps: 1) Obtain Fusarium oxysporum The strain, after being cultured on a large scale, undergoes a safety treatment, which includes: 1-1) Collect the strains obtained from the expansion culture, and prepare inactivated bacterial powder by high-temperature inactivation or ultraviolet irradiation treatment; and / or 1-2) After filtering the culture medium produced by the expanded culture, the extracellular polypeptides and low molecular weight organic acids obtained are purified inducers by separation with organic solvents, silica gel column chromatography or chromatographic separation. 2) The inactivated bacterial powder or the purified inducer is mixed with the carrier excipient to prepare a water-dispersible formulation.

[0008] The preparation method employs a safety treatment process to ensure that the resulting plant inducer will not cause plant diseases or environmental pollution.

[0009] In a further embodiment of the first aspect of the present invention, the high-temperature inactivation treatment conditions in step 1-1) are 121°C for 15 minutes.

[0010] In a further embodiment of the first aspect of the present invention, the ultraviolet irradiation treatment conditions in step 1-1) are ultraviolet light irradiation at a wavelength of 254 nm for 30 minutes.

[0011] In a further embodiment of the first aspect of the present invention, the filtration in steps 1-2) is aseptic filtration through a filter membrane with a pore size of 0.22 μm.

[0012] In a further embodiment of the first aspect of the present invention, the final concentration of the inactivated bacterial powder is 0.1~2.0 g / L.

[0013] In a further embodiment of the first aspect of the present invention, the final concentration of the purification inducer is 50-500 mg / L.

[0014] In a further embodiment of the first aspect of the present invention, the carrier excipient is selected from at least one of pectin, silk fibroin, and sodium alginate.

[0015] In a further embodiment of the first aspect of the present invention, the water-dispersible formulation is a spray or a root soaking suspension.

[0016] A second aspect of the present invention is to provide a plant inducer prepared by the preparation method described in the first aspect of the present invention.

[0017] A third aspect of this invention lies in providing the application direction of the plant inducer shown in the second aspect of this invention. Specifically, it is applied to Paeonia suffruticosa (Paeonia lactiflora). Acanthus ilicifolius Using the plant inducer, a large amount of 2-(2,4-dimethoxyphenyl)-3,5,6,7,8-pentamethoxybenzopyran-4-one is accumulated in the plant.

[0018] In a further embodiment of the third aspect of the present invention, the plant inducer is applied by foliar spraying, rhizosphere irrigation, or a combination of both.

[0019] In a further embodiment of the third aspect of the present invention, the plant inducer is applied once every 7 to 10 days, for a total of 3 to 4 times.

[0020] In a further embodiment of the third aspect of the present invention, the environmental conditions for the induction process are: an average temperature of about 28 to 32 °C and a relative humidity of 70 to 90%.

[0021] In a further embodiment of the third aspect of the present invention, the method further includes collecting cartilage peony root samples 3 to 5 days after the induction treatment, extracting the samples by reflux with 70% ethanol, concentrating under reduced pressure, and then quantitatively analyzing the content of 2-(2,4-dimethoxyphenyl)-3,5,6,7,8-pentamethoxybenzopyran-4-one by high performance liquid chromatography.

[0022] Verification showed that after applying the plant inducer, the experimental group of *Paeonia suffruticosa* exhibited a 2-5 fold increase in the cumulative content of 2-(2,4-dimethoxyphenyl)-3,5,6,7,8-pentamethoxybenzopyran-4-one compared to the control group. This demonstrates that the plant inducer is simple, cost-effective, environmentally friendly, and easily industrialized in achieving efficient enrichment of 2-(2,4-dimethoxyphenyl)-3,5,6,7,8-pentamethoxybenzopyran-4-one. Detailed Implementation

[0023] The following embodiments further illustrate the content of the present invention, but should not be construed as limiting the present invention. Any modifications and substitutions made to the methods, steps, or conditions of the present invention without departing from the spirit and essence of the present invention are within the scope of the present invention.

[0024] Unless otherwise specified, the technical means used in the embodiments are conventional means well known to those skilled in the art.

[0025] Example 1: Preparation and application method of the plant inducer Preparation process: 1) Take Fusarium oxysporum The strain was inoculated onto PDA plates or potato dextrose liquid medium and cultured at 37°C for 7-10 days. It was harvested when the colonies grew vigorously and the mycelia were dense.

[0026] 2-1) Mix and suspend the mycelia and spores obtained from the culture, filter to remove the culture medium, autoclave at 121℃ for 15 min, cool and dry at 60℃ for 24 h, and grind into inactivated bacterial powder.

[0027] 2-2) Filter the culture supernatant through filter paper with a pore size of 0.22 μm to remove bacterial cells; separate the organic phase with ethyl acetate or n-butanol and recover the organic phase; after concentrating the organic phase, separate it by silica gel column chromatography or countercurrent chromatography, and the extracellular polypeptides and low molecular weight organic acids obtained are the purification inducers; after vacuum drying the purification inducers, store them for later use.

[0028] 3) The inactivated bacterial powder and / or purified inducer are used as active ingredients. The active ingredients are mixed with the carrier excipients at a mass ratio of 1: (1~5), and an appropriate amount of deionized water is added to prepare a water-dispersible spray or root soaking suspension. After homogenization, emulsification, ultrasonic sterilization, and packaging, the suspension is stored at 4°C. The carrier excipients are selected from at least one of pectin, silk fibroin and sodium alginate.

[0029] Induction treatment conditions: Select robust Paeonia lactiflora plants with 5-10 leaves, retain a taproot of more than 30 cm, and cultivate them in mangrove soil with a salinity of about 20.8‰, an average temperature of about 28-32 ℃, and a relative humidity of 70-90%, for more than one week.

[0030] The prepared spray is applied evenly to the underside and surface of the leaves using a foliar spray method; the prepared root soaking suspension is applied as a root drenching solution at a concentration of 50 mL per plant; it can be applied alone or in combination. The final concentration of the inactivated bacterial powder in the plant inducer is 0.1–2.0 g / L; the final concentration of the purified inducer is 50–500 mg / L. Apply once every 7–10 days, for a total of 3–4 times. After treatment, maintain the original growth environment and avoid rain erosion.

[0031] Example 2: Gradient screening of inactivated bacterial powder concentration The effects of different concentrations of inactivated bacterial powder on the accumulation of 2-(2,4-dimethoxyphenyl)-3,5,6,7,8-pentamethoxybenzopyran-4-one in Paeonia suffruticosa, a mangrove plant.

[0032] 1) Bacterial cell treatment: Following the preparation method provided in Example 1, only the inactivated bacterial cell powder provided in step 2-1) is used as the active ingredient.

[0033] 2) Plant inducer formulation: Add inactivated bacterial powder to deionized water to prepare a water-dispersible spray, and adjust the final concentration to 0.1, 0.5, 1.0, 1.5 and 2.0 g / L respectively.

[0034] 3) Treatment method: Select 5-leaf stage Paeonia lactiflora plants with uniform growth status, 10 plants per group, for a total of 6 groups (control group and 5 groups with different concentrations). Use foliar spraying to evenly spray until the underside and surface of the leaves are moist, once every 7 days, for a total of 3 treatments.

[0035] 4) Detection method: Leaves were collected 5 days after the last treatment. After extraction by reflux with 70% ethanol, the content of 2-(2,4-dimethoxyphenyl)-3,5,6,7,8-pentamethoxybenzopyran-4-one was analyzed by HPLC.

[0036] Table 1 - Content Analysis

[0037] The results are shown in Table 1. The inactivated bacterial powder significantly promoted the accumulation of 2-(2,4-dimethoxyphenyl)-3,5,6,7,8-pentamethoxybenzopyran-4-one in Paeonia suffruticosa, exhibiting a concentration-dependent effect. At a concentration of 1.0 g / L, the content of 2-(2,4-dimethoxyphenyl)-3,5,6,7,8-pentamethoxybenzopyran-4-one reached its highest level, 4.80 times that of the control group. Further increases in concentration to 1.5 g / L and 2.0 g / L slightly decreased the content of 2-(2,4-dimethoxyphenyl)-3,5,6,7,8-pentamethoxybenzopyran-4-one, but it remained at a relatively high level. This indicates that 1.0 g / L is the optimal induction concentration, which can maximize the accumulation of 2-(2,4-dimethoxyphenyl)-3,5,6,7,8-pentamethoxybenzopyran-4-one while saving on the amount of inducing agent used.

[0038] Further observation of plant morphology revealed that the leaves of the induced treatment group showed a slight dark green change, but no obvious growth inhibition or toxicity symptoms were observed, indicating that the inactivated bacterial powder inducer has good safety.

[0039] Example 3, Comparison of Induction Methods Based on the optimal concentration of inactivated bacterial powder (1.0 g / L) determined in Example 2, this example compares different induction methods.

[0040] 1) Preparation of plant inducers: Following the preparation method provided in Example 1, only the inactivated bacterial powder provided in step 2-1) was used as the active ingredient; spray and root soaking suspension were prepared at a final concentration of 1.0 g / L of inactivated bacterial powder.

[0041] 2) Treatment methods: Select 5-leaf stage Paeonia suffruticosa plants with uniform growth, 10 plants per group, for a total of 4 groups: A. Control group (no treatment); B. Foliar spray group (spray the agent evenly until the underside and surface of the leaves are moist); C. Root zone irrigation group (each plant is irrigated with 50 mL of root soaking suspension); D. Combined treatment group (foliar spray and root zone irrigation are performed simultaneously). Treatments are given every 7 days, for a total of 3 treatments.

[0042] 3) The detection method is the same as in Example 2.

[0043] Table 2 - Content Analysis

[0044] The results are shown in Table 2. All induction treatments significantly increased the accumulation of 2-(2,4-dimethoxyphenyl)-3,5,6,7,8-pentamethoxybenzopyran-4-one, with the combined treatment showing the best effect, reaching 5.21 times that of the control group; foliar spray alone was the second most effective, reaching 4.90 times that of the control group; and root irrigation had a weaker effect, but still reached 3.56 times that of the control group. This indicates that... Fusarium oxysporum The induction effect of inactivated bacterial powder is mainly achieved through absorption by plant leaves, and combined with rhizosphere irrigation, the induction effect can be further enhanced.

[0045] This example demonstrates that using a combination of foliar spraying and rhizosphere irrigation with inactivated bacterial powder at a concentration of 1.0 g / L is the optimal method to increase the accumulation of 2-(2,4-dimethoxyphenyl)-3,5,6,7,8-pentamethoxybenzopyran-4-one in Paeonia suffruticosa.

[0046] Example 4: Purification inducer concentration gradient screening The effects of different concentrations of purified inducers on the accumulation of 2-(2,4-dimethoxyphenyl)-3,5,6,7,8-pentamethoxybenzopyran-4-one in Paeonia suffruticosa were evaluated.

[0047] 1) Purification of the inducer: Following the preparation method provided in Example 1, only the purified inducer provided in steps 2-2) was used as the active ingredient. Specifically, the culture medium obtained from the expanded culture was filtered through a 0.22 μm filter membrane, and then sequentially extracted with ethyl acetate, followed by silica gel column chromatography (chloroform:methanol = 9:1~7:3 gradient elution) and semi-preparative HPLC purification to obtain a brown powdery purified inducer.

[0048] 2) Formulation of plant inducers: The purified inducer was added to deionized water to prepare water-dispersible spray and root soaking suspension, and the final concentration was adjusted to 50, 100, 200, 350 and 500 mg / L, respectively.

[0049] 3) Treatment method: Select 5-leaf stage Paeonia lactiflora plants with uniform growth status, 10 plants per group, for a total of 6 groups (control group and 5 groups with different concentrations). Combined treatment of foliar spray and root drenching was adopted. 50 mL of root soaking suspension was drenched into the roots of each plant, and the spray was evenly sprayed on the back and front of the leaves until moist. Treatment was carried out once every 7 days, for a total of 3 treatments.

[0050] 4) The detection method is the same as in Example 2.

[0051] Table 3 - Content Analysis

[0052] The results are shown in Table 3. The purified inducer significantly promoted the accumulation of 2-(2,4-dimethoxyphenyl)-3,5,6,7,8-pentamethoxybenzopyran-4-one in Paeonia suffruticosa compared to the inactivated bacterial powder, and this effect also showed a concentration-dependent characteristic. At a concentration of 350 mg / L, the content of 2-(2,4-dimethoxyphenyl)-3,5,6,7,8-pentamethoxybenzopyran-4-one reached its highest level, 6.59 times that of the control group. Further increasing the concentration to 500 mg / L resulted in a content of 2-(2,4-dimethoxyphenyl)-3,5,6,7,8-pentamethoxybenzopyran-4-one comparable to that of the 350 mg / L group, with no significant difference. This indicates that 350 mg / L is the optimal induction concentration, maximizing the accumulation efficiency of 2-(2,4-dimethoxyphenyl)-3,5,6,7,8-pentamethoxybenzopyran-4-one.

[0053] Further LC-MS analysis revealed that the main active ingredient in the purified inducer was a glycoprotein with a molecular weight of approximately 2.8 kDa. It is speculated that this glycoprotein may specifically induce the expression of key enzymes in the phenylpropanoid metabolic pathway by activating plant defense responses and signal transduction pathways, thereby promoting the synthesis and accumulation of 2-(2,4-dimethoxyphenyl)-3,5,6,7,8-pentamethoxybenzopyran-4-one.

[0054] Compared to inactivated bacterial powder, purified inducers have the advantages of higher activity, lower dosage, and more significant effects, but the purification process is more complex. Considering both induction effect and production cost, purified inducers are more suitable for large-scale production applications of high-value medicinal plants.

[0055] Example 5: Screening of different ratios of inactivated bacterial powder and sodium alginate 1) Obtain inactivated bacterial powder according to the method provided in Example 1, and adjust the final concentration to 1.0 g / L.

[0056] 2) Sodium alginate was selected as the carrier excipient.

[0057] 3) Mix inactivated bacterial powder with sodium alginate at different mass ratios (1:1, 1:2, 1:3, 1:5), and add deionized water to prepare water-dispersible sprays and root soaking suspensions.

[0058] 4) Select 5-leaf stage Paeonia lactiflora plants with uniform growth status, with 10 plants in each group. Use a combination of foliar spraying and root irrigation treatment. Irrigate the roots of each plant with 50 mL of root soaking suspension, and spray the spray evenly on the underside and surface of the leaves until moist. Treat once every 7 days, for a total of 3 treatments.

[0059] 5) The detection method is the same as in Example 2.

[0060] Table 4 - Content Analysis

[0061] The results are shown in Table 4. The combination of inactivated bacterial powder and sodium alginate significantly promoted the accumulation of 2-(2,4-dimethoxyphenyl)-3,5,6,7,8-pentamethoxybenzopyran-4-one in Paeonia suffruticosa, and this effect showed a certain ratio-dependent characteristic. When the mass ratio of bacterial powder to sodium alginate was 1:3, the content of 2-(2,4-dimethoxyphenyl)-3,5,6,7,8-pentamethoxybenzopyran-4-one reached its highest level, which was 4.89 times that of the control group. When the ratio of sodium alginate was further increased to 1:5, the induction effect decreased slightly, possibly because excessive carrier excipients affected the release of the active ingredient.

[0062] Compared to the 5.21 increase in induction efficiency achieved by the combined treatment with inactivated bacterial powder in Example 3, the decrease in induction efficiency due to the combined use with sodium alginate may be related to the lower content of active ingredients in the inactivated bacterial powder. However, considering the simple and low-cost preparation process of inactivated bacterial powder, and the fact that it can still achieve a nearly 5-fold increase in the content of 2-(2,4-dimethoxyphenyl)-3,5,6,7,8-pentamethoxybenzopyran-4-one after being combined with sodium alginate at a 1:3 ratio, it has good economic efficiency and feasibility in large-scale production applications and can be used as an alternative to purifying the inducer.

[0063] Example 6: Screening of different ratios of low-concentration purification inducer and sodium alginate 1) Obtain the purified inducer according to the method provided in Example 1, and adjust the final concentration to 200 mg / L.

[0064] 2) Sodium alginate was selected as the carrier excipient.

[0065] 3) The purified inducer was mixed with sodium alginate at different mass ratios (1:1, 1:2, 1:3, 1:5), and deionized water was added to prepare water-dispersible sprays and root soaking suspensions.

[0066] 4) Select 5-leaf stage Paeonia lactiflora plants with uniform growth status, with 10 plants in each group. Use a combination of foliar spraying and root irrigation treatment. Irrigate the roots of each plant with 50 mL of root soaking suspension, and spray the spray evenly on the underside and surface of the leaves until moist. Treat once every 7 days, for a total of 3 treatments.

[0067] 5) The detection method is the same as in Example 2.

[0068] Table 5 - Content Analysis

[0069] The results are shown in Table 5. The combination of low-concentration purified inducer (200 mg / L) and sodium alginate significantly promoted the accumulation of 2-(2,4-dimethoxyphenyl)-3,5,6,7,8-pentamethoxybenzopyran-4-one in *Paeonia lactiflora*, exhibiting a ratio-dependent effect. When the mass ratio of purified inducer to sodium alginate was 1:3, the content of 2-(2,4-dimethoxyphenyl)-3,5,6,7,8-pentamethoxybenzopyran-4-one reached its highest level, 5.25 times that of the control group. Further increasing the sodium alginate ratio to 1:5 slightly decreased the induction effect.

[0070] Compared to the higher concentration of purification inducer in Example 4 (6.86 times higher than the control group), the induction effect of the lower concentration of purification inducer was slightly weaker, but still significantly better than the inactivated bacterial powder in Example 5 (4.89 times higher than the control group). This indicates that the induction effect is positively correlated with the concentration of purification inducer, but a good induction effect can still be obtained at a lower concentration by combining it with an appropriate proportion of sodium alginate carrier.

[0071] Considering the cost factors in practical applications, the low-concentration purification inducer (200 mg / L) combined with sodium alginate in a 1:3 ratio can be used as an economical and efficient induction scheme. This not only significantly reduces the amount of purification inducer used, but also achieves a significant effect of increasing the content of 2-(2,4-dimethoxyphenyl)-3,5,6,7,8-pentamethoxybenzopyran-4-one by 5.25 times. It has good economic and practical value in large-scale production applications.

[0072] Example 7: Screening of different carrier excipient ratios 1) Preparation of inducer: Inactivated bacterial powder (1.0 g / L) and purified inducer (200 mg / L) were obtained according to the method provided in Example 1.

[0073] 2) Carrier excipient selection: Pectin, silk fibroin and sodium alginate are selected as three natural polymers as carrier excipients.

[0074] 3) Inducer formulation: Inactivated bacterial powder and purified inducer are mixed at a 1:1 ratio to prepare the active ingredient. The active ingredient is then mixed with the three carrier excipients mentioned above at different mass ratios (1:1, 1:2, 1:3, 1:5), and deionized water is added to prepare water-dispersible sprays and root soaking suspensions. The final concentration of the active ingredient is adjusted to 350 mg / L.

[0075] 4) Treatment method: Select 5-leaf stage Paeonia lactiflora plants with uniform growth status, with 10 plants in each group. Use a combination of foliar spraying and root irrigation. Irrigate the roots of each plant with 50 mL of root soaking suspension, and spray the spray evenly on the underside and surface of the leaves until moist. Treat once every 7 days, for a total of 3 treatments.

[0076] 5) The detection method is the same as in Example 2.

[0077] Table 6 - Content Analysis

[0078] Table 7 - Content Analysis

[0079] The results are shown in Tables 6 and 7. Among the three natural polymeric carrier excipients, sodium alginate exhibited the best induction and synergistic effect, followed by silk fibroin and pectin. Further studies on different ratios revealed that when the mass ratio of purified inducer to sodium alginate was 1:3, the content of 2-(2,4-dimethoxyphenyl)-3,5,6,7,8-pentamethoxybenzopyran-4-one reached the highest value (5.56 mg / g dry weight), which was 6.86 times that of the control group. When the ratio was increased to 1:5, the induction effect decreased slightly, possibly because excessive carrier excipients affected the release and absorption of the active ingredient.

[0080] Sodium alginate, as a natural polysaccharide, possesses excellent biocompatibility and sustained-release properties. It can effectively protect active ingredients from environmental factors (such as ultraviolet radiation and high temperatures), prolong their residence time on plant surfaces, and improve their efficiency in penetrating the plant epidermis. Simultaneously, the alginate contained in sodium alginate may also act as a synergistic factor for plant growth regulators, further enhancing their induction effects.

[0081] In conclusion, mixing the purification inducer with sodium alginate at a mass ratio of 1:3 achieves the best induction effect while ensuring good dispersibility and stability of the formulation, making it the optimal choice of carrier excipient.

[0082] Example 8, Effect of sodium alginate on the stability of purification inducer 1) The purified inducer was obtained according to the method in Example 1, and the final concentration was adjusted to 500 mg / L.

[0083] 2) Experimental grouping: The purified inducer solution was divided into two groups. Group A contained only the purified inducer, and Group B contained the purified inducer and sodium alginate at a mass ratio of 1:3.

[0084] 3) Storage conditions: Dispense samples A and B into sterile reagent bottles and store them in a constant temperature environment of 25℃ away from light.

[0085] 4) Stability test: Samples were taken on days 0, 3, 7, 14 and 30 after preparation to determine the content of active polysaccharides and induction activity in each group of samples.

[0086] 5) Evaluation of induction activity: Ten plants of Paeonia suffruticosa at the 5-leaf stage with uniform growth were selected in each group. Each plant was injected with 50 mL of inducing agent solution stored for different durations via rhizosphere irrigation. Leaves were collected one week later, and the content of 2-(2,4-dimethoxyphenyl)-3,5,6,7,8-pentamethoxybenzopyran-4-one was analyzed by HPLC.

[0087] Table 8 - Stability Comparison

[0088] The results are shown in Table 8. Under 25℃ conditions, the purified inducer without sodium alginate (Group A) had poor stability. After 3 days of storage, the retention rate of active polysaccharides dropped to 68.5%, and the induction effect decreased significantly. The content of 2-(2,4-dimethoxyphenyl)-3,5,6,7,8-pentamethoxybenzopyran-4-one was only 2.85 times that of the control group. After 7 days of storage, the retention rate of active polysaccharides dropped to 42.3%, and the induction effect decreased further. After 14 days of storage, it almost lost its inducing activity.

[0089] In contrast, the purified inducer with added sodium alginate (Group B) showed excellent stability. After 30 days of storage, the retention rate of active polysaccharides still reached 86.4%, and the content of 2-(2,4-dimethoxyphenyl)-3,5,6,7,8-pentamethoxybenzopyran-4-one induced was 6.07 times that of the control group, maintaining good inducing activity.

[0090] Sodium alginate, as a natural polysaccharide, can form a protective complex with the active polysaccharides in the purification inducer through non-covalent interactions such as hydrogen bonding, effectively slowing down enzymatic and oxidative degradation processes. Furthermore, sodium alginate has strong hygroscopic and film-forming properties, which can reduce the contact between the active ingredients and air, lower water activity, inhibit microbial contamination and free radical oxidation reactions, thereby significantly extending the shelf life of the purification inducer.

[0091] It will be apparent to those skilled in the art that the present invention is not limited to the details of the exemplary embodiments described above, and that the invention can be implemented in other specific forms without departing from the spirit or essential characteristics of the invention. Therefore, the embodiments should be considered in all respects as exemplary and non-limiting, and the scope of the invention is defined by the appended claims rather than the foregoing description. Thus, it is intended that all variations falling within the meaning and scope of equivalents of the claims be included within the present invention.

Claims

1. A method for preparing a plant inducer, characterized in that, include: 1) Obtain Fusarium oxysporum The strain, after being cultured on a large scale, undergoes a safety treatment, which includes: 1-1) Collect the strains obtained from the expansion culture, and prepare inactivated bacterial powder by high temperature inactivation or ultraviolet irradiation treatment; and / or 1-2) After filtering the culture medium produced by the expanded culture, the extracellular polypeptides and low molecular weight organic acids obtained are purified inducers by separation with organic solvents, silica gel column chromatography or chromatographic separation. 2) The inactivated bacterial powder or purified inducer is mixed with the carrier excipient to prepare a water-dispersible formulation.

2. The preparation method according to claim 1, characterized in that, Step 1-1) The high-temperature inactivation treatment conditions are 121 °C for 15 minutes; the ultraviolet irradiation treatment conditions are ultraviolet light irradiation with a wavelength of 254 nm for 30 minutes.

3. The preparation method according to claim 1, characterized in that, The carrier excipient is selected from at least one of pectin, silk fibroin and sodium alginate.

4. The preparation method according to claim 1, characterized in that, The water-dispersible formulation is a spray or a root soaking suspension.

5. A plant inducer, characterized in that, It is prepared by any one of the preparation methods of claims 1 to 4.

6. The use of the plant inducer of claim 5 in inducing the accumulation of 2-(2,4-dimethoxyphenyl)-3,5,6,7,8-pentamethoxybenzopyran-4-one in Paeonia suffruticosa.

7. The application according to claim 6, characterized in that, The plant inducer can be applied by foliar spraying, root irrigation, or a combination of both.

8. The application according to claim 6, characterized in that, The plant inducer is applied once every 7 to 10 days, for a total of 3 to 4 times.

9. The application according to claim 6, characterized in that, The environmental conditions for the induction process are: an average temperature of approximately 28-32 ℃ and a relative humidity of 70-90%.

10. The application according to claim 6, characterized in that, The procedure also included collecting cartilage peony root samples 3-5 days after the induction treatment, extracting the samples by reflux with 70% ethanol, concentrating under reduced pressure, and then quantitatively analyzing the content of 2-(2,4-dimethoxyphenyl)-3,5,6,7,8-pentamethoxybenzopyran-4-one using high performance liquid chromatography.

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