Cultivation method for promoting growth of rhizoma bletillae and accumulation of active ingredients

Abstract

The invention discloses a cultivation method for promoting the growth of rhizoma bletillae and the accumulation of active ingredients, which comprises the following steps: carrying out foliage spraying on planted annual rhizoma bletillae seedlings by adopting a mesoporous silica loaded jasmonic acid methyl ester solution as a growth promoting agent, and meanwhile, applying trichoderma asperellum microcapsules to the roots of the annual rhizoma bletillae seedlings, the purposes of promoting growth of rhizoma bletillae and accumulation of active ingredients are achieved; by adopting the method, the growth of the rhizoma bletillae can be remarkably promoted, the content of active ingredients such as total polysaccharides, total phenols and total flavonoids in the rhizoma bletillae is effectively increased, meanwhile, the content of antioxidant ingredients is greatly increased, the yield and quality are both improved, and the method has important application value in rhizoma bletillae quality improvement cultivation.

Description

Technical Field

[0001] The present invention belongs to the technical field of Chinese medicinal material cultivation, and specifically relates to a cultivation method for promoting the growth of Bletilla striata and enhancing the accumulation of its active ingredients through the synergistic effect of a mesoporous silica composite growth promoter and Trichoderma aspergillus microcapsules. Background Art

[0002] Bletilla striata ( Bletilla striata ) is a perennial herbaceous medicinal plant of the Orchidaceae family and one of the traditional precious Chinese medicinal materials (Gou K, Li Y, Qu Y, et al. Advances and prospects of Bletilla striata polysaccharide as promising multifunctional biomedical materials[J].Materials & Design, 2022, 223: 111198). Its dried tubers are rich in active ingredients such as polysaccharides, total phenols and total flavonoids (Jiang L, Lin M, Wang H, et al. Haplotype-resolved genome assembly of Bletilla striata (Thunb.) Reichb.f. to elucidate medicinal value[J]. ThePlant Journal, 2022, 111(5): 1340-1353). It has pharmacological effects such as antioxidant, wound healing, immune regulation, and anti-tumor (Bai L, Wang T, Deng Q, et al. Dual properties ofpharmacological activities and preparation excipient: Bletilla striatapolysaccharides[J]. International Journal of Biological Macromolecules, 2024,254: 127643). The demand for Bletilla striata in the fields of medicine, daily chemicals and food continues to grow, while wild resources have been listed as national second-class protected plants due to over-exploitation. At present, artificial cultivation has become the only way to ensure resource supply. However, existing cultivation technologies generally have problems such as long growth cycle, frequent pests and diseases, and insufficient accumulation of active ingredients, resulting in low planting efficiency. In traditional planting processes, the use of a single growth promoter or a single plant probiotic agent is difficult to achieve a dual increase in yield and quality (Wang Q, Xu S, Wen Z, et al. Combined Plant Growth-Promoting Bacteria Inoculants Were More Beneficial than Single Agents for Plant Growth and CdPhytoextraction of Brassica juncea L. during Field Application[J]. Toxics, 2022, 10(7): 396). Therefore, developing a green and efficient cultivation technology system to achieve a dual increase in the yield and quality of Bletilla striata has become an urgent need for the sustainable development of the industry.

[0003] To enhance the growth and accumulation of active ingredients in traditional Chinese medicines (TCMs), existing technologies mostly rely on single exogenous hormones or microbial agents. However, these conventional single agents have low survival rates in soil and weak competitiveness with native microorganisms, leading to technical bottlenecks such as inconsistent effectiveness in practical applications. To overcome these bottlenecks, a coordinated regulatory system combining sustained release of foliar signals and optimized root microbiome is urgently needed.

[0004] Currently, the cross-integration of nanomaterials and microbial engineering technologies has opened up new paths for the green cultivation of medicinal plants. Nanocarrier technologies, represented by mesoporous silica, can increase the loading capacity of exogenous hormones by virtue of their high specific surface area, adjustable pore size, and surface functionalization, significantly alleviating the problems of rapid decomposition and poor absorption of traditional foliar sprays (Feng Haibo, Du Xihui, Huang Ling, et al. Research Progress on the Application of Mesoporous Silica Nanoparticles in Biomaterials [J]. Journal of Southwest University for Nationalities (Natural Science Edition), 2025, 51(01): 26-33). The concurrently developed microencapsulation technology using sodium alginate-chitosan as an embedding material has improved the survival rate of beneficial microorganisms by constructing a three-dimensional network structure through ionic crosslinking, and has also achieved root microenvironment-triggered release (Yuan Xiaolu, Li Baoxia, Huang Yayan, et al. Progress in the preparation and application of sodium alginate microcapsules [J]. Chemical Industry and Engineering Progress, 2022, 41(06): 3103-3112). However, the current research on the “nanocarrier-plant hormone-beneficial microorganism” synergistic effect targeting the unique growth habits and physiological needs of Bletilla striata is still in its infancy. Summary of the Invention

[0005] The present invention provides a cultivation method for promoting the growth of Bletilla striata and the accumulation of active ingredients. The method establishes a "nano slow-release induction-microcapsule colonization and growth promotion" synergistic system, and achieves dual regulation of Bletilla striata growth and active ingredient accumulation through the combined application of foliar spraying of a mesoporous silica-loaded methyl jasmonate solution growth promoter and root application of Trichoderma spinulosa microcapsules.

[0006] The cultivation method for promoting the growth of Bletilla striata and the accumulation of active ingredients of the present invention comprises the following steps: during the planting process of one-year Bletilla striata, using a mesoporous silica-loaded methyl jasmonate solution as a growth promoter, spraying the solution on the leaves of the planted one-year Bletilla striata seedlings, and simultaneously applying Trichoderma aspergillus microcapsules to the roots of the one-year Bletilla striata seedlings, combining the two treatments, treating the two treatments once every seven days, spraying the growth promoter and applying the microcapsules in the evening each time, and continuously treating the two treatments for six months, wherein the amount of the mesoporous silica-loaded methyl jasmonate solution used is 1.0-1.5 mL per plant, and the amount of the Trichoderma aspergillus microcapsules used is 10-15 g per plant.

[0007] The mesoporous silica-loaded methyl jasmonate is prepared by mixing a methyl jasmonate-anhydrous ethanol solution with a mesoporous silica-anhydrous ethanol solution, stirring overnight at room temperature, centrifuging, washing the solid with pure water, and drying. The mass ratio of methyl jasmonate to mesoporous silica is 1:2-3; and the mass volume ratio (g:mL) of the mesoporous silica-loaded methyl jasmonate to water is 1:4000-5000.

[0008] The Trichoderma aculeatus microcapsules are prepared by inoculating Trichoderma aculeatus into a PDA culture medium and culturing the microcapsules at 28°C. After the Trichoderma aculeatus has grown all over the culture medium, sterile water is added to scrape the mycelia, and the microcapsules are filtered through gauze to obtain a spore suspension. The spore suspension is mixed with a sodium alginate solution having a mass volume concentration (g:mL) of 3%, and the mixture is then dropped into a calcium chloride solution having a mass volume concentration of 2% for cross-linking and solidification. The mixture is shaken at 200 r / min for 15-20 minutes, and the solid-liquid separation is performed. The solid is washed with sterile water, and the excess water is removed by filtration to obtain a spore content of ≥1×10 8 / g of microcapsules; the volume ratio of spore suspension to sodium alginate solution is 1:2-3.

[0009] The Trichoderma spinulosa ( Trichoderma asperellum ) has been disclosed in non-patent literature before the application date of this invention, namely, in "Study on the mechanism of the effect of Trichoderma aspergillus on the growth promotion and disease prevention of Panax notoginseng. Master's thesis of Kunming University of Science and Technology, 2024". The applicant guarantees that the biological material will be released to the public within twenty years from the application date.

[0010] The beneficial effects of the present invention are: By loading methyl jasmonate on mesoporous silica, the stability of the growth promoter was improved; by embedding Trichoderma aspergillus with sodium alginate, the survival rate of the bacteria was improved. Compared with the traditional method, the present invention can not only give full play to the growth-promoting effect of the growth promoter, but also improve the soil microenvironment, providing a suitable environment for the growth of Bletilla striata. Practical application shows that after the implementation of the method of the present invention, compared with the control group, the treatment group can increase the yield of Bletilla striata by 20.91%, the total polysaccharide content by 15.63%, the total phenol content by 11.44%, the total flavonoid content by 7.53%, and the antioxidant activity by 3.66%-6.71%, thereby achieving the simultaneous optimization of yield and quality. This synergistic system is easy to operate and has stable effects. It provides an innovative solution for the standardized cultivation of Chinese medicinal materials and is of great value in ensuring the quality of medicinal materials and promoting the sustainable development of the industry. BRIEF DESCRIPTION OF THE DRAWINGS

[0011] Figure 1 1. It is the tuber size diagram of Bletilla striata of the treatment group of the present invention and the control group; Figure 2 The statistical results of the agronomic trait parameters of the Bletilla striata treated group and the control group of the present invention are as follows; Figure 3 The results of chlorophyll content determination of Bletilla striata leaves in the treatment group and the control group of the present invention; Figure 4 The results of total polysaccharides, total phenols and total flavonoids determination in the tubers of Bletilla striata in the treatment group and the control group of the present invention; Figure 5 These are the results of measuring the antioxidant capacity of the Bletilla striata tubers in the treatment group and the control group of the present invention. DETAILED DESCRIPTION

[0012] The present invention will be further described below with reference to the accompanying drawings and specific examples, but the examples do not limit the present invention in any way. Unless otherwise specified, the reagents, culture media, methods and equipment used in the present invention are all conventional reagents, culture media, methods and equipment in the art.

[0013] Example 1: Cultivation and processing of Bletilla striata For cultivation of Bletilla striata, warm, shady, humid, deep, fertile and well-drained sandy loam or humus loam should be selected. Weed and till the soil by 20-25 cm 1-2 months before planting. Apply 1000-1200 kg of decomposed organic fertilizer or 30-40 kg of potassium sulfate compound fertilizer per mu, and make ridges 1.3-1.5 meters wide and 20-30 cm high. Use tuber propagation, select tubers with tender buds and no damage of the current year, plant in autumn from September to October, cut the pieces and disinfect them with wood ash, dig holes with a row spacing of 25-30 cm and a hole spacing of 20-25 cm, plant 3 tubers in each hole, with the buds facing outwards, cover with soil and water to establish roots. Cultivate and weed 4 times a year, pay attention to shallow hoeing, water in the morning and evening during droughts, and drain to prevent waterlogging in the rainy season.

[0014] Methyl jasmonate and anhydrous ethanol were ultrasonically mixed at a ratio of 1:20 (g:mL), mesoporous silica and anhydrous ethanol were mixed at a ratio of 1:20 (g:mL), and ultrasonic treatment was performed. The methyl jasmonate solution and the ultrasonically treated mesoporous silica solution were mixed at a volume ratio of 1:2, stirred overnight at room temperature, centrifuged at 10,000 rpm for 20 minutes, and the precipitate was taken and washed with pure water multiple times. After washing, the precipitate was dried to obtain mesoporous silica-loaded methyl jasmonate. The mesoporous silica-loaded methyl jasmonate was mixed with water at a ratio of 1:4500 (g:mL) and sprayed on the leaves of one-year-old Bletilla striata seedlings at a dosage of 1.2 mL / plant.

[0015] Trichoderma aspergillus was inoculated into PDA medium and cultured in a 28°C incubator. After the culture medium was filled with Trichoderma aspergillus, sterile water was added and the mycelium was scraped and filtered through gauze to collect the spore suspension. Sodium alginate was weighed and added to distilled water. The mixture was stirred and dissolved in a 45°C water bath to prepare a 3% (g:mL) sodium alginate solution. The spore suspension and sodium alginate solution were mixed at a volume ratio of 1:2 and the mixture was dropwise added to a 3% (g:mL) calcium chloride solution for cross-linking and curing. The microcapsules obtained after cross-linking and curing were transferred to a shaker and shaken at a constant speed (200 r / min) for 20 minutes. After solid-liquid separation, the microcapsules were washed multiple times with sterile water to remove unreacted cross-linking agent, solvent, and other soluble impurities. After washing, excess water was removed by filtration to obtain microcapsules with a spore content of 1×10 912 g / plant of Trichoderma acanthoides microcapsules were applied to the roots of one-year-old Bletilla striata seedlings. The treatment was carried out every 7 days, with each spraying of the growth promoter and application of the microcapsules carried out in the evening for six consecutive months. The cultivation method of the control group of Bletilla striata was as follows: a 0.0001 g / mL aqueous solution of methyl jasmonate was sprayed on the leaves of one-year-old Bletilla striata seedlings as a growth promoter, and a spore content of 1×10 9 A spore suspension of Trichoderma aspergillus at a concentration of 100 / mL was placed on the roots of one-year-old Bletilla striata seedlings, and the other cultivation measures were the same as those of the above treatment group.

[0016] The cultivation method of Bletilla striata in the blank group was as follows: the same amount of sterile water as that of the treatment group was sprayed on the leaves, and the same amount of sterile water as that of the treatment group was applied to the roots. The other cultivation measures were the same as those of the above treatment group.

[0017] After 6 months of treatment according to the above method, 20 Bletilla striata plants were randomly selected from the treatment group and the control group, and the growth and agronomic trait-related parameters and active ingredient content were measured. The antioxidant capacity was also measured.

[0018] Example 2: Determination of agronomic trait parameters of Bletilla striata The method for measuring the agronomic traits of Bletilla striata was referred to the method in Yang Pingfei et al. (2017) (Yang Pingfei, Song Zhiqin, Liu Hai, et al. Agronomic traits and quality of Bletilla striata in different growth years during the vigorous growth period [J]. Northern Horticulture, 2017, (10): 140-144), and statistical analysis of the relevant agronomic traits was performed. The results are shown in Figure 1 、 2 , it can be seen from the figure that the average plant height of Bletilla striata in the treatment group is 33.62cm, and that of the control group is 31.05cm; the average tuber fresh weight of the treatment group is 8.73g, and that of the control group is 7.22g. The yield of the treatment group is increased by about 20.91% compared with that of the control group; other agronomic traits such as stem thickness, fresh weight of the aboveground part, and leaf shape are all higher in the treatment group than in the control group. In summary, the cultivation method involved in the present invention has a significant promoting effect on the growth of Bletilla striata, and can significantly improve a number of key agronomic traits such as plant height, fresh weight of the aboveground part, and fresh weight of tubers of Bletilla striata, reflecting the superiority and application value of the cultivation method in Bletilla striata cultivation.

[0019] Example 3: Determination of chloroplast pigment content in Bletilla striata leaves Chlorophyll is an important pigment for plant photosynthesis, and its content affects plant photosynthetic performance. The total chlorophyll content in leaves was determined according to the method described in the literature (Zhou Yong, Fan Xiaolei, Lin Yongjun, et al. Determination of Chlorophyll Content in Rice [J]. Biological Experimental Protocol, 2018).

[0020] See the results Figure 3The results showed that the chlorophyll a content of the control group was 1.14 mg / g, and that of the treated group was 1.24 mg / g, with the latter being significantly higher than the former; as for the chlorophyll b content, the control group was 0.50 mg / g, and the treated group was 0.51 mg / g, with the two being similar and having no significant difference; in terms of total chlorophyll content, the control group was 1.61 mg / g, and the treated group was 1.76 mg / g, with the total chlorophyll content of the treated group increasing by approximately 9.32% compared to that of the control group. In summary, compared with the control group, the treated group was able to significantly increase the chlorophyll a and total chlorophyll contents. Therefore, the cultivation method of the invention can increase the chlorophyll content in the leaves of Bletilla striata, help improve photosynthesis, and thus promote the accumulation of organic matter in Bletilla striata.

[0021] Example 4: Determination of the active ingredient contents of total polysaccharides, total phenols and total flavonoids in Bletilla striata 1. Determination of the content of total polysaccharides, total phenols and total flavonoids in Bletilla striata was carried out according to the method in the reference (Liu Jiawei, Cao Guofan, Wu Mingkai, et al. Determination of the content of total phenols, total flavonoids and total polysaccharides in different parts of "Guiji No. 1" and study on their biological activities [J]. Chinese Traditional Patent Medicine, 2024, 46(06): 1968-1972).

[0022] See the results Figure 4 The results showed that the total polysaccharide content of the control group was 10.24 mg / g, and the total polysaccharide content of the treatment group was 11.84 mg / g, which was 15.63% higher than that of the control group; the total phenol content of the control group was 5.42 mg / g, and the total phenol content of the treatment group was 6.04 mg / g, which was 11.44% higher than that of the control group; the total flavonoid content of the control group was 2.92 mg / g, and the total flavonoid content of the treatment group was 3.14 mg / g, which was about 7.53% higher than that of the control group.

[0023] In summary, total polysaccharides, total phenolics, and total flavonoids are important indicators of Bletilla striata's quality; increased levels of these polysaccharides and flavonoids indicate an increase in its medicinal value. Therefore, combined foliar application of a mesoporous silica-based growth promoter containing methyl jasmonate and root application of Trichoderma aspergillus microcapsules significantly improved Bletilla striata's quality.

[0024] Example 5: Determination of Antioxidant Capacity of Water Extract of Bletilla striata Tuber The antioxidant capacity of the water extract of Bletilla striata tubers was analyzed by detecting two important antioxidant indicators: DPPH free radical and ABTS free radical scavenging rates. The detection method is described in Chi Xuejie and Wang Juan (2025). (Chi Xuejie, Wang Juan. Determination of total flavonoids content and comparative antioxidant activity of Scutellaria barbata from different origins [J]. Journal of Shanxi University of Traditional Chinese Medicine, 2025, 26(01): 35-40).

[0025] The tubers of Bletilla striata were dried at 60°C, crushed, and passed through a 60-mesh sieve. 2 g of sample powder was weighed, added with 10 mL of distilled water, mixed well, and heated in an 85°C water bath for extraction for 2 h. The mixture was centrifuged at 8000 rpm for 10 min, and the supernatant was collected for later use.

[0026] See the results Figure 5 The results showed that the DPPH free radical scavenging rate of the control group was about 45.87%, and the DPPH free radical scavenging rate of the treatment group was about 49.53%, which was about 3.66% higher than that in the control group; the ABTS free radical scavenging rate of the control group was about 41.07%, and the ABTS free radical scavenging rate of the treatment group was about 47.78%, which was about 6.71% higher than that in the control group.

[0027] In summary, the Bletilla striata cultivation method of the present invention significantly increases the chlorophyll content in Bletilla striata leaves, enhances photosynthesis, and provides sufficient energy and substances for plant growth and metabolite production. It also increases the accumulation of active ingredients such as Bletilla striata polysaccharides, total phenols, and total flavonoids. At the same time, it significantly improves the antioxidant capacity of Bletilla striata, achieving the dual effects of promoting growth and improving quality.

Claims

1. A cultivation method for promoting the growth of Bletilla striata and the accumulation of active ingredients, characterized in that: Mesoporous silica-loaded methyl jasmonate solution was used as a growth promoter to spray the leaves of one-year-old Bletilla striata seedlings. At the same time, Trichoderma aspergillus microcapsules were applied to the roots of the one-year-old Bletilla striata seedlings to achieve the purpose of promoting the growth of Bletilla striata and the accumulation of active ingredients.

2. The cultivation method for promoting the growth of Bletilla striata and the accumulation of active ingredients according to claim 1, characterized in that: The mesoporous silica-loaded methyl jasmonate is prepared by mixing a methyl jasmonate-anhydrous ethanol solution with a mesoporous silica-anhydrous ethanol solution, stirring the mixture overnight at room temperature, centrifuging the mixture, washing the solid with purified water, and drying the mixture.

3. The cultivation method for promoting the growth of Bletilla striata and the accumulation of active ingredients according to claim 2, characterized in that: The mass ratio of methyl jasmonate to mesoporous silica is 1:2-3.

4. The cultivation method for promoting the growth of Bletilla striata and the accumulation of active ingredients according to claim 1, characterized in that: The mass volume ratio of mesoporous silica-loaded methyl jasmonate to water (g:mL) is 1:4000-5000.

5. The cultivation method for promoting the growth of Bletilla striata and the accumulation of active ingredients according to claim 1, characterized in that: The microcapsules of Trichoderma aspergillus are prepared by inoculating Trichoderma aspergillus into a PDA culture medium and culturing at 28°C. After the Trichoderma aspergillus has grown all over the culture medium, sterile water is added to scrape the mycelium, and the spore suspension is filtered through gauze to obtain a spore suspension. The spore suspension is mixed with a sodium alginate solution having a mass volume concentration of 3%, and then the mixture is dropped into a calcium chloride solution having a mass volume concentration of 2% for cross-linking and solidification. The mixture is shaken at 200 r / min for 15-20 minutes, solid-liquid separation is performed, the solid is washed with sterile water, and excess water is removed by filtration to obtain a spore content of ≥1×10 8 Microcapsules per gram.

6. The cultivation method for promoting the growth of Bletilla striata and the accumulation of active ingredients according to claim 5, characterized in that: The volume ratio of spore suspension to sodium alginate solution is 1:2-3.

7. The cultivation method for promoting the growth of Bletilla striata and the accumulation of active ingredients according to claim 1, characterized in that: The usage amount of the mesoporous silica-loaded methyl jasmonate solution is 1.0-1.5 mL / plant, and the usage amount of the Trichoderma aspergillus microcapsules is 10-15 g / plant.

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