A composite microbial fertilizer for promoting the growth of polygala tenuifolia root and application thereof

Abstract

The application provides a composite microbial fertilizer for promoting the growth of underground parts of polygalae and application thereof, and the composite microbial fertilizer for promoting the growth of underground parts of polygalae comprises rice straw biochar, grass charcoal soil, potassium dihydrogen phosphate, organic fertilizer and mucor circinelloides. The composite microbial fertilizer is simple in preparation process, clear in components, can significantly promote the growth of underground parts of polygalae, and has high application value in the ecological cultivation for promoting the yield and quality of polygalae.

Description

Technical Field

[0001] This invention belongs to the field of plant fertilizer technology, and relates to a compound microbial fertilizer that promotes the underground growth of Polygala tenuifolia and its application. Background Technology

[0002] As a traditional Chinese medicine from Shaanxi and Shanxi provinces, Polygala tenuifolia has a high market demand due to its effects of calming the mind and improving intelligence, harmonizing the heart and kidneys, and its potential in treating diseases such as insomnia, memory disorders, and neurosis.

[0003] Currently, the use of growth regulators, such as swelling agents, and other chemical products is quite common in the cultivation of Polygala tenuifolia. However, long-term excessive application of chemical fertilizers and other agricultural chemicals can lead to problems such as soil compaction, acidification, salinization, and environmental pollution, resulting in reduced soil fertility, decreased fertilizer efficiency, and increased pests and diseases, thereby affecting the yield and quality of medicinal plants. Therefore, the development of new green and efficient fertilizers is crucial for promoting the sustainable development of Polygala tenuifolia production.

[0004] Mucor truncatula is a filamentous fungus, and its application as a microbial fertilizer in agriculture has begun to attract attention. As an environmentally friendly fertilizer, microbial fertilizers can improve crop yield, enhance crop disease resistance, increase soil fertility, and improve the soil environment by introducing beneficial microorganisms.

[0005] For example, CN112574892B discloses the use of Mucor DF20 (CGMCC NO.20742), which can effectively promote the root development of Salvia miltiorrhiza by co-culturing with Salvia miltiorrhiza plants, and can also effectively promote the biosynthesis and accumulation of tanshinone components.

[0006] For example, CN110922974B discloses the application of Mucor circinelloides in the degradation of lambda-cyhalothrin. This strain has simple culture conditions, rapid reproduction, and high degradation characteristics after fermentation. Utilizing this degradation ability to decompose pesticide residues has broad application value in the field of microbial strain application technology.

[0007] While microbial fertilizers are widely used in crop production, they have not yet been widely applied in the production of medicinal plants. Medicinal plants are the origin of traditional Chinese medicine, and their quality is fundamental to TCM diagnosis and treatment. Therefore, developing microbial fertilizers specifically for medicinal plants is of great significance for increasing yield and improving quality to meet usage demands while ensuring their efficacy. Summary of the Invention

[0008] In view of the shortcomings of the existing technology, the purpose of this invention is to provide a compound microbial fertilizer that promotes the underground growth of Polygala tenuifolia and its application.

[0009] To achieve this objective, the present invention adopts the following technical solution:

[0010] In a first aspect, the present invention provides a compound microbial fertilizer for promoting the growth of the underground parts of Polygala tenuifolia, the compound microbial fertilizer for promoting the growth of the underground parts of Polygala tenuifolia includes: rice straw biochar, peat soil, potassium dihydrogen phosphate, organic fertilizer and Mucor.

[0011] This invention creatively utilizes *Mucor* as a microbial fertilizer, and combines it with rice straw biochar, peat moss, potassium dihydrogen phosphate, and organic fertilizer to synergistically promote the excellent growth of the underground parts of *Polygala tenuifolia*. Specifically, this is achieved by ensuring the preservation of the medicinal components of *Polygala tenuifolia* in the root phloem. Based on the content of ketone III and 3,6'-disinyl sucrose, it significantly increased the underground biomass, root length and root diameter of Polygala tenuifolia, thereby improving the quality of Polygala tenuifolia while increasing its yield.

[0012] *Mucor* exhibits broad application potential across multiple fields. Firstly, in the biofuel sector, its accumulated lipids can be converted into biodiesel, becoming an important raw material for replacing fossil fuels. Secondly, *Mucor* can synthesize carotenoids (such as lycopene, β-carotene, and astaxanthin) and γ-linolenic acid, among other natural antioxidants and nutritional supplements, demonstrating significant antioxidant activity and medicinal value. Furthermore, in environmental remediation, *Mucor* can effectively absorb heavy metals, accumulate polyphosphates, and improve the nutrient ratio of wastewater, making it suitable for wastewater treatment and soil remediation. Studies have shown that its immobilization efficiency for heavy metals such as zinc, lead, and manganese is over 74%, and it can shorten the remediation cycle. In agriculture, *Mucor* colonizes plant roots, promoting plant growth and the accumulation of secondary metabolites. Simultaneously, it can enhance plant tolerance to heavy metals, improve antioxidant levels, and degrade pesticide residues, such as lambda-cyhalothrin, achieving a degradation rate of up to 79.7%. In addition, chitosan derived from *Mucor* can inhibit the growth of pathogenic fungi such as *Aspergillus flavus*. In conclusion, Mucor has significant value in bioenergy, nutrition and health care, environmental remediation, and agricultural production and quality improvement, and is a multifunctional microbial resource.

[0013] The aforementioned Mucor may significantly promote the growth of the underground parts of Polygala tenuifolia by improving the absorption and utilization of nutrients in the soil and fertilizer utilization rate by plants through the secretion of secondary metabolites and the optimization and improvement of the soil environment.

[0014] In the aforementioned microbial fertilizer, organic fertilizer, rice straw charcoal, and potassium dihydrogen phosphate have a synergistic effect in promoting the underground growth of Polygala tenuifolia. Organic fertilizer provides abundant nutrients for both Polygala tenuifolia and microorganisms, while rice straw charcoal, with its high specific surface area and porous structure, not only improves soil aeration and water retention but also provides an ideal habitat for microorganisms. Potassium dihydrogen phosphate directly supplements phosphorus and potassium, promoting root development and nutrient absorption. The synergistic effect of these three components significantly enhances soil fertility, optimizes the growth environment of Polygala tenuifolia, and effectively increases the biomass, root length, and root diameter of the underground parts, achieving the goal of increased yield and improved quality.

[0015] Preferably, the amount of *Mucor* in the compound microbial fertilizer is (2-10) × 10⁻⁶ per kg of biochar. 6 CFU.

[0016] Among them, the specific point values ​​in (2-10) can be independently selected as 2, 4, 6, 8, 10, etc. Other specific point values ​​within this range can also be selected, which will not be elaborated here.

[0017] Preferably, the biochar is rice straw charcoal produced by burning at 500-700℃ (e.g., 500℃, 550℃, 600℃, 630℃, 650℃, 670℃, 680℃, 700℃, etc.). Other specific values ​​within this range are also acceptable and will not be elaborated upon here.

[0018] Preferably, the organic fertilizer contains ≥30% organic matter, ≥1.3% N, ≥1.1% P2O5, and ≥1.6% K2O.

[0019] Preferably, in the microbial fertilizer, the mass ratio of rice straw biochar, peat moss, potassium dihydrogen phosphate and organic fertilizer is (150-200):(650-700):(0.5-1.0):(400-500).

[0020] Based on the potential synergistic relationship between organic fertilizer and potassium dihydrogen phosphate, and the potential synergistic relationship between peat soil and rice straw biochar, when the above-mentioned specific ratios are met, they have a better effect on promoting the underground growth of Polygala tenuifolia.

[0021] The specific point values ​​in (150-200) can be 150, 160, 170, 180, 190, 200, etc.; the specific point values ​​in (650-700) can be 650, 660, 670, 680, 690, 700, etc.; the specific point values ​​in (0.5-1.0) can be 0.5, 0.6, 0.7, 0.8, 0.9, 1.0, etc.; the specific point values ​​in (400-500) can be 400, 420, 440, 460, 480, 500, etc.; other specific point values ​​within the above ranges can also be selected, and will not be elaborated on here.

[0022] Preferably, the *Mucor circinelloides* strain has the preservation number CGMCC No. 40842 and is classified as *Mucor circinelloides*.

[0023] Preferably, the suspension of *Mucor* is prepared by a method comprising the following steps:

[0024] Mucor was activated in PDA medium, and a single colony was picked and inoculated into PDB medium for culture to obtain Mucor seed culture. The seed culture was then inoculated into PDB medium for culture to obtain the desired result.

[0025] The formula for PDA medium is: 4-6g potato extract powder, 20-25g glucose, 15-20g agar, and 1000mL distilled water; the formula for PDB medium is: 4-6g potato extract powder, 20-25g glucose, and 1000mL distilled water.

[0026] The specific values ​​selected for the 4-6g range can be 4g, 4.5g, 5g, 5.5g, 6g, etc.; the specific values ​​selected for the 20-25g range can be 20g, 22g, 24g, 25g, etc.; the specific values ​​selected for the 15-20g range can be 15g, 16g, 17g, 18g, 20g, etc.; other specific values ​​within the above ranges can also be selected, and will not be elaborated on here.

[0027] In a second aspect, the present invention provides the application of the compound microbial fertilizer for promoting the underground growth of Polygala tenuifolia according to the first aspect in the cultivation of the Chinese medicinal herb Polygala tenuifolia.

[0028] Compared with the prior art, the present invention has the following beneficial effects:

[0029] This invention creatively uses Mucor as a microbial fertilizer, and combines it with rice straw biochar, peat moss, potassium dihydrogen phosphate, and organic fertilizer to exert an excellent effect on promoting the underground growth of Polygala tenuifolia.

[0030] The compound microbial fertilizer involved in this invention has a simple preparation process, clear components, and a significant effect on promoting the growth of the underground parts of the Chinese medicinal herb Polygala tenuifolia. It has high application value in promoting the ecological cultivation of Polygala tenuifolia to increase yield and improve quality. Attached Figure Description

[0031] Figure 1 This describes the growth status of each group of Polygala tenuifolia at harvest time;

[0032] Figure 2 This is a graph showing the statistical results of root length in each group of Polygala tenuifolia.

[0033] Figure 3 This is a graph showing the statistical results of the root diameters of each group of Polygala tenuifolia.

[0034] Figure 4 This is a graph showing the statistical results of the fresh weight of the underground parts of each group of Polygala tenuifolia.

[0035] Figure 5 It is the Polygala tenuifolia phloem in each group. Statistical results of ketone III content;

[0036] Figure 6 This is a graph showing the statistical results of the 3,6'-disinic sucrose content in the phloem of Polygala tenuifolia in each group.

[0037] The strain of *Mucor circinelloides* involved in this invention is classified and named *Mucor circinelloides*. It is deposited at the China General Microbiological Culture Collection Center (CGMCC) on October 9, 2023, with accession number CGMCC No. 40842, located at No. 3, Courtyard 1, Beichen West Road, Chaoyang District, Beijing. Detailed Implementation

[0038] The technical solution of the present invention will be further illustrated below through specific embodiments. Those skilled in the art should understand that the embodiments described are merely illustrative of the present invention and should not be construed as limiting the invention in any way.

[0039] Example 1

[0040] This embodiment provides a compound microbial fertilizer, which is composed of *Mucor* sp. and the following components in parts by weight: 178 parts rice straw biochar, 667 parts peat moss, 1 part potassium dihydrogen phosphate, and 444 parts organic fertilizer; wherein the biochar is rice straw biochar produced by burning at 500℃; wherein the organic fertilizer is a product purchased from Beijing Dongxiang Environmental Technology Co., Ltd. (organic matter ≥30%, N ≥1.3%, P2O5 ≥1.1%, K2O ≥1.6%, N+P2O5+K2O ≥4.0%); wherein the potassium dihydrogen phosphate is a product purchased from Xilong Scientific Co., Ltd. (KH2PO4 ≥99.5%); and wherein the peat moss is a product purchased from PINDSTRUP Group of Denmark.

[0041] Mucor was prepared by the following method:

[0042] (1) *Mucor zh* was activated in PDA medium, and a single colony was picked and inoculated into PDB medium. The culture was carried out in the dark at 30°C and 230 rpm to obtain *Mucor zh* seed culture. The seed culture was then inoculated into fresh PDB medium and cultured under the same conditions to obtain *Mucor zh* mycelial culture. The formula for PDA medium was: 4.0 g potato starch, 20.0 g glucose, 16.0 g agar, and 1000 mL distilled water; the formula for PDB medium was: 6.0 g potato starch, 20.0 g glucose, and 1000 mL distilled water.

[0043] (2) The dosage of *Mucor truncatula* in the compound microbial fertilizer was calculated as 2.5 × 10⁻⁶ kg of biochar. 6 CFU.

[0044] Comparative Example 1

[0045] This embodiment provides a compound fertilizer composed of the following components in parts by weight: 178 parts rice straw biochar, 667 parts peat moss, 1 part potassium dihydrogen phosphate, and 444 parts organic fertilizer; wherein the biochar is rice straw biochar produced by burning at 500℃; wherein the organic fertilizer is a product purchased from Beijing Dongxiang Environmental Technology Co., Ltd. (organic matter ≥30%, N ≥1.3%, P2O5 ≥1.1%, K2O ≥1.6%, N+P2O5+K2O ≥4.0%); wherein the potassium dihydrogen phosphate is a product purchased from Xilong Scientific Co., Ltd. (KH2PO4 ≥99.5%); and wherein the peat moss is a product purchased from PINDSTRUP Group of Denmark.

[0046] Comparative Example 2

[0047] This comparative example provides a compound microbial fertilizer, which is composed of a compound inoculant and the following components in parts by weight: 178 parts rice straw biochar, 667 parts peat moss, 1 part potassium dihydrogen phosphate, and 444 parts organic fertilizer; wherein the biochar is rice straw biochar produced by burning at 500℃; wherein the organic fertilizer is a product purchased from Beijing Dongxiang Environmental Technology Co., Ltd. (organic matter ≥30%, N ≥1.3%, P2O5 ≥1.1%, K2O ≥1.6%, N+P2O5+K2O ≥4.0%); wherein the potassium dihydrogen phosphate is a product purchased from Xilong Scientific Co., Ltd. (KH2PO4 ≥99.5%); and wherein the peat moss is a product purchased from PINDSTRUP Group of Denmark.

[0048] The compound microbial agent is prepared by the following method:

[0049] (1) Weigh out water-soluble Bacillus licheniformis, water-soluble Bacillus amyloliquefaciens and water-soluble Bacillus subtilis powder (all purchased from Shandong Dehe Mingxing Biotechnology Co., Ltd.), dissolve them in sterile water, mix thoroughly to obtain bacterial solutions of Bacillus licheniformis, Bacillus amyloliquefaciens and Bacillus subtilis.

[0050] (2) Mix the bacterial solutions of Bacillus licheniformis, Bacillus amyloliquefaciens, and Bacillus subtilis to obtain a compound microbial agent, such that the ratio of viable bacteria of Bacillus licheniformis, Bacillus amyloliquefaciens, and Bacillus subtilis in the compound microbial agent is 1:1:1, and the dosage of Bacillus licheniformis in the compound microbial fertilizer is 8.33 × 10⁻⁶ per kg of biochar. 5 The dosage of CFU (Cyclopyralid) and Bacillus amyloliquefaciens in compound microbial fertilizer is 8.33 × 10⁻⁶ per kg of biochar. 5 The dosage of CFU (Bacillus subtilis) in compound microbial fertilizer is 8.33 × 10⁻⁶ per kg of biochar. 5 CFU.

[0051] Comparative Example 3

[0052] This comparative example provides a compound microbial fertilizer, which is composed of a compound inoculant, *Mucor* sp., and the following components in parts by weight: 178 parts rice straw biochar, 667 parts peat moss, 1 part potassium dihydrogen phosphate, and 444 parts organic fertilizer; wherein the biochar is rice straw biochar produced by burning at 500℃; wherein the organic fertilizer is a product purchased from Beijing Dongxiang Environmental Technology Co., Ltd. (organic matter ≥30%, N ≥1.3%, P2O5 ≥1.1%, K2O ≥1.6%, N+P2O5+K2O ≥4.0%); wherein the potassium dihydrogen phosphate is a product purchased from Xilong Scientific Co., Ltd. (KH2PO4 ≥99.5%); and wherein the peat moss is a product purchased from PINDSTRUP Group of Denmark.

[0053] The compound microbial agent and Mucor rotundifolia were prepared by the following method:

[0054] (1) Weigh out water-soluble Bacillus licheniformis, water-soluble Bacillus amyloliquefaciens and water-soluble Bacillus subtilis powder (all purchased from Shandong Dehe Mingxing Biotechnology Co., Ltd.), dissolve them in sterile water, mix thoroughly to obtain bacterial solutions of Bacillus licheniformis, Bacillus amyloliquefaciens and Bacillus subtilis.

[0055] (2) *Mucor zh* was activated in PDA medium, and a single colony was picked and inoculated into PDB medium. The culture was carried out in the dark at 30°C and 230 rpm to obtain *Mucor zh* seed culture. The seed culture was then inoculated into fresh PDB medium and cultured under the same conditions to obtain *Mucor zh* mycelial culture. The formula for PDA medium was: 4.0 g potato starch, 20.0 g glucose, 16.0 g agar, and 1000 mL distilled water; the formula for PDB medium was: 6.0 g potato starch, 20.0 g glucose, and 1000 mL distilled water.

[0056] (3) Mix Bacillus licheniformis, Bacillus amyloliquefaciens, Bacillus subtilis, and Mucor truncatula to obtain a compound microbial agent. The ratio of viable bacteria of Bacillus licheniformis, Bacillus amyloliquefaciens, Bacillus subtilis, and Mucor truncatula in the compound microbial agent is 1:1:1:3, and the dosage of Bacillus licheniformis in the compound microbial fertilizer is 4.17 × 10⁻⁶ per kg of biochar. 5 The dosage of CFU (Cyclopyralid) and Bacillus amyloliquefaciens in compound microbial fertilizer is 4.17 × 10⁻⁶ per kg of biochar. 5 The dosage of CFU (Chemical Fumarate) and Bacillus subtilis in compound microbial fertilizer is 4.17 × 10⁻⁶ per kg of biochar. 5 The dosage of CFU (Cyclocarya spp.) and Mucor zh. in compound microbial fertilizer is 1.25 × 10⁻⁶ kg of biochar. 6 CFU.

[0057] Test case

[0058] The effects of the compound fertilizer and compound microbial fertilizer prepared in Example 1 and Comparative Examples 1-3 on promoting the underground growth of Polygala tenuifolia were verified:

[0059] From October 2023 to October 2024, a small-plot experiment was conducted in the experimental greenhouse of the Institute of Medicinal Plant Development, Chinese Academy of Medical Sciences, Haidian District, Beijing. The experimental material was Polygala tenuifolia. The experiment included four treatments: compound fertilizer prepared in Example 1 and Comparative Examples 1-3, and compound microbial fertilizer, respectively.

[0060] In October 2023, rice straw biochar, peat moss, potassium dihydrogen phosphate, and organic fertilizer from each fertilizer group were mixed with the soil substrate. 5 kg of this solid substrate was applied per square meter. Plump, uniformly sized *Polygala tenuifolia* seeds were directly sown in the experimental soil. Simultaneously, *Mucor* and the compound microbial agent from each fertilizer group were applied with irrigation water. During the experiment, *Polygala tenuifolia* was treated three times with *Mucor* and the compound microbial agent on days 60, 120, and 180 after planting. The amount of *Mucor* and the compound microbial agent used in each treatment was the same. *Polygala tenuifolia* was harvested one year after planting. The fresh weight of the underground parts, root length, root diameter, and content of medicinal components (*Polygala tenuifolia*) were measured and recorded. Keto-III, 3,6'-disinyl sucrose.

[0061] The root length statistics of each group of Polygala tenuifolia are as follows: Figure 2 As shown, compared with fertilizers that did not use Mucor and fertilizers that used both Mucor and compound microbial agents, the compound microbial fertilizer involved in this invention can significantly increase the root length of Polygala tenuifolia, with the root length of Polygala tenuifolia increasing by 60.39% compared with the treatment without Mucor (P<0.05).

[0062] The statistical results of root diameter of each group of Polygala tenuifolia are as follows: Figure 3 As shown, compared with fertilizers that did not use Mucor and fertilizers that used both Mucor and compound microbial agents, the compound microbial fertilizer involved in this invention can significantly increase the root diameter of Polygala tenuifolia, with the root diameter of Polygala tenuifolia increasing by 55.23% compared with the treatment without Mucor (P<0.05).

[0063] The statistical results of the fresh weight of the underground parts of each group of Polygala tenuifolia are as follows: Figure 4 As shown, compared with fertilizers that do not use Mucor and fertilizers that use both Mucor and compound microbial agents, the compound microbial fertilizer involved in this invention can significantly increase the underground biomass of Polygala tenuifolia, increasing it by 162.14% compared with the treatment without Mucor (P < 0.05).

[0064] Polygala tenuifolia in each group of phloem The statistical results of ketone III content are as follows: Figure 5 As shown, the compound microbial fertilizer involved in this invention can ensure the quality of Polygala tenuifolia in the phloem. The ketone III content was the same as that of no microbial fertilizer applied.

[0065] The statistical results of the 3,6'-disinic sucrose content in the phloem of Polygala tenuifolia in each group are as follows: Figure 6 As shown, using the compound microbial fertilizer involved in this invention can ensure that the 3,6'-disinic sucrose content in the phloem of Polygala tenuifolia is the same as that of not applying any microbial fertilizer and applying Mucor rotundifolia and compound microbial fertilizer at the same time.

[0066] The applicant declares that the technical solution of this invention is illustrated by the above embodiments, but this invention is not limited to the above embodiments, that is, it does not mean that this invention must rely on the above embodiments to be implemented. Those skilled in the art should understand that any improvements to this invention, equivalent substitutions of raw materials for the products of this invention, addition of auxiliary components, selection of specific methods, etc., all fall within the protection scope and disclosure scope of this invention.

[0067] The preferred embodiments of the present invention have been described in detail above. However, the present invention is not limited to the specific details in the above embodiments. Within the scope of the technical concept of the present invention, various simple modifications can be made to the technical solution of the present invention, and these simple modifications all fall within the protection scope of the present invention.

[0068] It should also be noted that the various specific technical features described in the above specific embodiments can be combined in any suitable manner without contradiction. In order to avoid unnecessary repetition, the present invention will not describe the various possible combinations separately.

Claims

1. A compound microbial fertilizer for promoting the underground growth of Polygala tenuifolia, characterized in that, The compound microbial fertilizer that promotes the underground growth of Polygala tenuifolia includes: rice straw biochar, peat moss, potassium dihydrogen phosphate, organic fertilizer, and Mucor. The *Mucorcircinelloides* strain mentioned is the one with the preservation number CGMCC No. 40842 and the classification name *Mucorcircinelloides*.

2. The compound microbial fertilizer for promoting the underground growth of Polygala tenuifolia according to claim 1, characterized in that, The content of the *Mucor* strain in the microbial fertilizer is (2-10)×10⁻¹⁰ per kg of biochar. 6 CFU.

3. The compound microbial fertilizer for promoting the underground growth of Polygala tenuifolia according to claim 1, characterized in that, The biochar is rice straw charcoal produced by burning at 500-700℃.

4. The compound microbial fertilizer for promoting the underground growth of Polygala tenuifolia according to claim 1, characterized in that, The organic fertilizer contains ≥30% organic matter, ≥1.3% N, ≥1.1% P2O5, and ≥1.6% K2O.

5. The compound microbial fertilizer for promoting the underground growth of Polygala tenuifolia according to claim 1, characterized in that, In the microbial fertilizer, the mass ratio of rice straw biochar, peat moss, potassium dihydrogen phosphate and organic fertilizer is (150-200):(650-700):(0.5-1.0):(400-500).

6. The compound microbial fertilizer for promoting the underground growth of Polygala tenuifolia according to claim 1, characterized in that, The suspension of *Mucor* was prepared by a method comprising the following steps: Mucor was activated in PDA medium, and a single colony was picked and inoculated into PDB medium for culture to obtain Mucor seed culture. The seed culture was then inoculated into PDB medium for culture to obtain the desired result. The formula for PDA medium is: 4-6g potato extract powder, 20-25g glucose, 15-20g agar, and 1000mL distilled water; the formula for PDB medium is: 4-6g potato extract powder, 20-25g glucose, and 1000mL distilled water.

7. The compound microbial fertilizer for promoting the underground growth of Polygala tenuifolia according to any one of claims 1-6 is used in the cultivation of the medicinal plant Polygala tenuifolia.

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