Angelica sinensis special-purpose compound microbial agent, and preparation method and application thereof

Abstract

The present application relates to a kind of angelica dedicated composite microbial inoculant, the composite microbial inoculant is the bacterial culture fluid containing Bacillus subtilis DG-R-B-1-16 ( Bacillus subtilis ), Bacillus megaterium BHL3-15 ( Bacillus megaterium ), Bacillus amyloliquefaciens BHL1-16 ( Bacillus amyloliquefaciens ), Bacillus mucilaginosus DG-R-B-3-1 ( Bacillus mucilaginosus ), Bacillus licheniformis DG-R-B-4-17 ( Bacillus licheniformis ) and Paenibacillus peoriae DG-R-B-3-3 ( Paenibacillus peoriae ).Meanwhile, the preparation method and application of the angelica dedicated composite microbial inoculant are also disclosed.The angelica dedicated composite microbial inoculant can promote the growth of angelica, prevent and control diseases and pests, and improve the yield and quality of angelica.

Description

Technical Field

[0001] This invention relates to the field of microbial resources technology, and in particular to a special compound microbial agent for Angelica sinensis, its preparation method and application. Background Technology

[0002] Angelica sinensis ( Angelica sinensis Angelica sinensis is a perennial herb belonging to the Apiaceae family. Its root is cylindrical with branch roots at the base and numerous fleshy fibrous roots. The root is used medicinally; it is sweet, pungent, and warm in nature, and has the effects of nourishing blood, promoting blood circulation, regulating menstruation, relieving pain, and moistening the intestines. It is one of the most frequently used traditional Chinese medicines in clinical practice. Gansu is the authentic producing area of ​​Angelica sinensis; it has been cultivated in Minxian, Dangchang, Zhangxian, and Weiyuan counties for over 1800 years.

[0003] Currently, angelica cultivation is mainly carried out by small-scale farmers with limited technical expertise. Production techniques are similar to those used for ordinary crops. Under continuous cropping conditions in the farmland soil ecosystem, allelopathic and autotoxic allelopathic substances from angelica are continuously released into the soil environment. This inhibits the growth and development of angelica itself and leads to an imbalance and dysfunction of the soil microbial community structure. Pathogens proliferate and break out in large numbers, inducing continuous cropping obstacles, resulting in a continuous decline in the yield and quality of angelica, and increasingly serious pest and disease outbreaks. Furthermore, the excessive application of chemical fertilizers during production, cultivation, and field management causes soil compaction, reduces available nutrients, and severely impacts the beneficial microbial community. Farmers also use chemical pesticides far exceeding the permitted frequency and dosage for pest and disease control, leading to excessive pesticide residues in angelica, reduced quality, and lower value. This has become a significant factor affecting the efficacy and safety of angelica medicines and a major source of agricultural environmental pollution, seriously impacting regional economic benefits and the sustainable development of the angelica cultivation industry. Therefore, it is urgent to carry out research on replacing traditional chemical fertilizers with new bio-fertilizers for Angelica sinensis and to promote the green and standardized planting of Angelica sinensis.

[0004] Currently, most domestic microbial inoculants for Chinese medicinal herbs consist of only 1-2 types of functional bacteria, resulting in limited functionality and effects with poor sustainability. They cannot fundamentally solve bottleneck problems such as continuous cropping obstacles, outbreaks of pests and diseases, and decline in yield and quality of Angelica sinensis. Summary of the Invention

[0005] The technical problem to be solved by the present invention is to provide a special compound microbial agent for Angelica sinensis that promotes growth, increases yield and quality.

[0006] Another technical problem to be solved by the present invention is to provide a method for preparing the Angelica sinensis-specific compound microbial agent.

[0007] The third technical problem to be solved by the present invention is to provide the application of the Angelica sinensis-specific compound microbial agent.

[0008] To solve the above problems, the present invention provides a special compound microbial agent for Angelica sinensis, characterized in that: the compound microbial agent contains Bacillus subtilis DG-RB-1-16 (… Bacillus subtilis ), Bacillus megaterium BHL3-15 ( Bacillus megaterium ), Bacillus amyloliquefaciens BHL1-16 ( Bacillus starch liquefier ), Bacillus mucilaginosus DG-RB-3-1 ( Mucilaginous Bacillus Bacillus licheniformis DG-RB-4-17 Bacillus licheniformis ) and Bacillus dermatitidis DG-RB-3-3 ( Paenibacillus peoriae Bacterial culture medium of Bacillus subtilis DG-RB-1-16 ( Bacillus subtle The accession number for Bacillus megaterium BHL3-15 is CGMCC No. 27080; Bacillus megalith The accession number for Bacillus amyloliquefaciens BHL1-16 is CGMCC No. 27094; Bacillus starch liquefier The accession number for the Bacillus subtilis DG-RB-3-1 is CGMCC No. 27071; Mucilaginous Bacillus The accession number for Bacillus licheniformis DG-RB-4-17 is CGMCC No. 27078; Bacillus licheniformis The accession number for the *Bacillus pilaris* DG-RB-3-3 is CGMCC No. 27074; Paenibacillus peoriae The accession number of the object is CGMCC No. 27092.

[0009] Bacillus subtilis DG-RB-1-16 ( Bacillus subtilis ), Bacillus megaterium BHL3-15 ( Bacillus megaterium ), Bacillus amyloliquefaciens BHL1-16 ( Bacillus amyloliquefaciens ), Bacillus mucilaginosus DG-RB-3-1 ( Mucilaginous Bacillus Bacillus licheniformis DG-RB-4-17 Bacillus lichen-like ) and Bacillus dermatitidis DG-RB-3-3 ( Paenibacillus peoriae All samples are deposited at the China General Microbiological Culture Collection Center; Address of depository: Institute of Microbiology, Chinese Academy of Sciences, No. 3, No. 1 Beichen West Road, Chaoyang District, Beijing, China; Date of deposit: April 12, 2023.

[0010] The preparation method of the Angelica sinensis-specific compound microbial agent as described above includes the following steps: (1) Bacillus subtilis DG-RB-1-16 ( Bacillus subtilis ), Bacillus megaterium BHL3-15 ( Bacillus megaterium ), Bacillus amyloliquefaciens BHL1-16 ( Bacillus amyloliquefaciens ), Bacillus mucilaginosus DG-RB-3-1 ( Mucilaginous Bacillus Bacillus licheniformis DG-RB-4-17 ( Bacillus lichen-like ), Bacillus dermatitis DG-RB-3-3 ( Paenibacillus peoriae Six functional bacteria were inoculated onto TSA solid agar plates using the streak method and incubated in the dark at 28°C for 4-5 days, resulting in the growth of fresh colonies visible to the naked eye. (2) Preparation of single-strain fermentation broth: Six single colonies were picked and inoculated into 500 mL Erlenmeyer flasks containing 250 mL of TSA liquid medium. The flasks were then incubated in the dark at 28°C and 180 rpm with shaking for 72 h to ensure a viable bacterial concentration greater than 10⁻⁶ in the liquid fermentation broth. 9 The concentration of cfu / mL was adjusted to 0.75-0.85 using sterile water to obtain the fermentation broth of a single strain. (3) Bacillus subtilis DG-RB-1-16 ( Bacillus subtilis ), Bacillus megaterium BHL3-15 ( Bacillus megaterium ), Bacillus amyloliquefaciens BHL1-16 ( Bacillus amyloliquefaciens ), Bacillus mucilaginosus DG-RB-3-1 ( Mucilaginous Bacillus Bacillus licheniformis DG-RB-4-17 ( Bacillus lichen-like ), Bacillus dermatitis DG-RB-3-3 ( Paenibacillus peoriae The fermentation broth of a single strain of Angelica sinensis is mixed evenly in a volume ratio of 2:2:2:2:1:1 to obtain the special compound microbial agent for Angelica sinensis.

[0011] In step (1), the TSA solid plate culture medium is prepared by adding 15 g tryptone, 5 g soybean peptone, 5 g sodium chloride, and 18 g agar to 1000 mL of distilled water, mixing thoroughly, adjusting the pH to 7.0-7.2, and sterilizing at 121℃ for 20 min.

[0012] In step (2), the TSA liquid culture medium is prepared by adding 15 g of tryptone, 5 g of soybean peptone, and 5 g of sodium chloride to 1000 mL of distilled water, mixing them thoroughly, adjusting the pH to 7.0-7.2, and then sterilizing by steaming at 121℃ for 30 min.

[0013] The application of the Angelica sinensis-specific compound microbial agent as described above is characterized in that: the Angelica sinensis-specific compound microbial agent is applied to Angelica sinensis planting sites that are continuously cropped by root irrigation method.

[0014] First, dip the roots of the angelica root in water. After planting the angelica root, dilute the special compound microbial agent for angelica root with water at a volume ratio of 1:100 and put it into a spray bottle. Then, evenly irrigate the soil around the roots of the angelica root. Set four concentration gradients for the amount used: 0 kg / mu, 10 kg / mu, 20 kg / mu, and 30 kg / mu.

[0015] Compared with the prior art, the present invention has the following advantages: 1. The six functional bacteria used in the compound microbial agent of this invention are all derived from the rhizosphere and internal tissues of medicinal herbs and specialty plants in Northwest China. They can quickly adapt to the climate and soil environment of Angelica sinensis planting areas and have good ecological adaptability. Among them, four functional bacteria are Bacillus subtilis DG-RB-1-16 (… Bacillus subtilis ), Bacillus mucilaginosus DG-RB-3-1 ( Mucilaginous Bacillus Bacillus licheniformis DG-RB-4-17 ( Bacillus lichen-like ) and Bacillus dermatitidis DG-RB-3-3 ( Paenibacillus peoriae Derived from the roots of Angelica sinensis, it can quickly establish itself in the rhizosphere soil and roots of Angelica sinensis, promote the growth of Angelica sinensis, increase soil nutrient content and soil enzyme activity, inhibit the growth of pathogens, and achieve the purpose of increasing the yield and quality of Angelica sinensis and improving the soil environment.

[0016] 2. The six functional bacteria in the compound microbial agent of this invention will not cause diseases of Angelica sinensis. They can not only degrade the autotoxic allelochemicals of Angelica sinensis, but also promote the growth of Angelica sinensis, control pests and diseases, improve the soil environment, and are safe to use, have high economic value, and will not pollute the ecological environment.

[0017] 3. The ratio and dosage of different functional bacteria in this invention can be appropriately adjusted according to different climatic conditions, soil factors and nutritional needs of Angelica sinensis at different growth stages. Attached Figure Description

[0018] The specific embodiments of the present invention will be described in further detail below with reference to the accompanying drawings.

[0019] Figure 1 This study focuses on field experiments and data collection for the 2021 Angelica sinensis-specific compound microbial inoculant.

[0020] Figure 2 The present invention describes the promoting effect of Angelica sinensis-specific compound microbial inoculant on plant height (A. Dangchang County; B. Minxian County).

[0021] Figure 3 The effect of the special compound microbial agent for Angelica sinensis of this invention on the appearance (top image: Dangchang County; bottom image: Minxian County).

[0022] Figure 4 The effect of the Angelica sinensis-specific compound microbial agent of the present invention on yield (top figure: Dangchang County; bottom figure: Minxian County).

[0023] Figure 5 This is the chemical structural formula of ferulic acid and ligustilide, the active medicinal components of Angelica sinensis in this invention.

[0024] Figure 6 The effect of the Angelica sinensis-specific compound microbial agent of the present invention on the active medicinal components (top figure: ferulic acid; bottom figure: ligustilide). Detailed Implementation

[0025] To make the purpose, technical solution, and advantages of the invention clearer, the invention will be further described in detail below with reference to embodiments. The specific embodiments described herein are for illustrative purposes only and are not intended to limit the invention.

[0026] Plant rhizosphere growth-promoting bacteria (PGPR) are a large class of beneficial bacteria that colonize the rhizosphere of plants, promoting plant growth and improving the soil environment. These strains have functions such as nitrogen fixation, phosphorus solubilization, secretion of plant hormones, antagonism of pathogens, and degradation of toxic substances. Moreover, these strains are derived from natural plant soil and internal tissues, and the microbial agents prepared from them through fermentation are environmentally friendly, pollution-free, and residue-free.

[0027] Compound microbial agents refer to live bacterial preparations made from two or more non-antagonistic microbial strains. Compared with single microbial agents, they have more comprehensive functions, stronger growth-promoting effects, and wider ecological adaptability.

[0028] This invention screens plant growth-promoting bacteria obtained from crops cultivated in western China. Through degrading angelica autotoxic allelochemicals, promoting angelica growth, controlling pests and diseases, and improving the soil environment, six functional bacteria strains of angelica were screened out.

[0029] A compound microbial agent specifically for Angelica sinensis, wherein the compound microbial agent contains Bacillus subtilis DG-RB-1-16 ( Bacillus subtilis ), Bacillus megaterium BHL3-15 ( Bacillus megaterium ), Bacillus amyloliquefaciens BHL1-16 ( Bacillus amyloliquefaciens ), Bacillus mucilaginosus DG-RB-3-1 ( Bacillus mucilaginous Bacillus licheniformis DG-RB-4-17 Bacillus licheniformis ) and Bacillus dermatitidis DG-RB-3-3 ( Paenibacillus peoriaeBacterial culture medium of Bacillus subtilis DG-RB-1-16 ( Bacillus subtilis The accession number for Bacillus megaterium (BHL3-15) is CGMCC No. 27080; Bacillus megalith The accession number for *Bacillus amyloliquefaciens* is CGMCC No. 27094; *Bacillus amyloliquefaciens* BHL1-16 ( Bacillus starch liquefier The accession number for *Bacillus mucilaginosus* DG-RB-3-1 is CGMCC No. 27071. Mucilaginous Bacillus The accession number for Bacillus licheniformis DG-RB-4-17 is CGMCC No. 27078. Bacillus licheniformis The accession number for *Bacillus pilaris* is CGMCC No. 27074; *Bacillus pilaris* DG-RB-3-3 ( Paenibacillus peoriae The accession number of the object is CGMCC No. 27092.

[0030] Bacillus subtilis DG-RB-1-16 ( Bacillus subtilis ), Bacillus megaterium BHL3-15 ( Bacillus megaterium ), Bacillus amyloliquefaciens BHL1-16 ( Bacillus amyloliquefaciens ), Bacillus mucilaginosus DG-RB-3-1 ( Mucilaginous Bacillus Bacillus licheniformis DG-RB-4-17 Bacillus lichen-like ) and Bacillus dermatitidis DG-RB-3-3 ( Paenibacillus peoriae All samples are deposited at the China General Microbiological Culture Collection Center; Address of depository: Institute of Microbiology, Chinese Academy of Sciences, No. 3, No. 1 Beichen West Road, Chaoyang District, Beijing, China; Date of deposit: April 12, 2023.

[0031] A method for preparing a compound microbial agent specifically for Angelica sinensis includes the following steps: (1) Bacillus subtilis DG-RB-1-16 ( Bacillus subtilis ), Bacillus megaterium BHL3-15 ( Bacillus megaterium ), Bacillus amyloliquefaciens BHL1-16 ( Bacillus amyloliquefaciens ), Bacillus mucilaginosus DG-RB-3-1 ( Mucilaginous Bacillus Bacillus licheniformis DG-RB-4-17 ( Bacillus lichen-like ), Bacillus dermatitis DG-RB-3-3 ( Paenibacillus peoriaeSix functional bacteria were inoculated into TSA solid agar plates (90 mm in diameter, 15-20 mL of culture medium) using the streak method and incubated in the dark at 28°C for 4-5 days, at which point fresh colonies visible to the naked eye would grow.

[0032] The TSA solid plate culture medium is prepared by adding 15 g tryptone, 5 g soybean peptone, 5 g sodium chloride, and 18 g agar to 1000 mL of distilled water, mixing thoroughly, adjusting the pH to 7.0-7.2, and sterilizing at 121℃ for 20 min.

[0033] (2) Preparation of single-strain fermentation broth: Six single colonies were picked and inoculated into 500 mL Erlenmeyer flasks containing 250 mL of TSA liquid medium. The flasks were then incubated in the dark at 28°C and 180 rpm with shaking for 72 h to ensure a viable bacterial concentration greater than 10⁻⁶ in the liquid fermentation broth. 9 The concentration of cfu / mL was adjusted to 0.75-0.85 using sterile water to obtain the fermentation broth of a single strain.

[0034] The TSA liquid culture medium is prepared by adding 15 g of tryptone, 5 g of soybean peptone, and 5 g of sodium chloride to 1000 mL of distilled water, mixing thoroughly, adjusting the pH to 7.0-7.2, and then sterilizing by steaming at 121℃ for 30 min.

[0035] (3) Bacillus subtilis DG-RB-1-16 ( Bacillus subtilis ), Bacillus megaterium BHL3-15 ( Bacillus megaterium ), Bacillus amyloliquefaciens BHL1-16 ( Bacillus amyloliquefaciens ), Bacillus mucilaginosus DG-RB-3-1 ( Mucilaginous Bacillus Bacillus licheniformis DG-RB-4-17 ( Bacillus lichen-like ), Bacillus dermatitis DG-RB-3-3 ( Paenibacillus peoriae The fermentation broth of a single strain of Angelica sinensis is mixed evenly at a volume ratio of 2:2:2:2:1:1 (ml / ml) to obtain the special compound microbial agent for Angelica sinensis.

[0036] Angelica sinensis-specific compound microbial inoculant is an environmentally friendly microbial preparation that can be used as both a pesticide and fertilizer. The functional microbial strains are derived from plant tissues and rhizosphere soil, enabling them to degrade autotoxic allelochemicals in Angelica sinensis, reduce continuous cropping obstacles, promote growth, increase yield and quality, control pests and diseases, improve the rhizosphere microbial community, and enhance the soil ecological environment. This ensures the healthy, stable, and sustainable development of the Angelica sinensis planting industry, giving it greater application value and market competitiveness compared to similar products.

[0037] Angelica sinensis-specific compound microbial inoculant is suitable for use during Angelica sinensis cultivation.

[0038] Application of a compound microbial agent specifically for Angelica sinensis: This compound microbial agent for Angelica sinensis is applied to continuously cropped Angelica sinensis fields via root irrigation. It degrades autotoxic allelopathic substances in Angelica sinensis, reducing obstacles caused by continuous cropping; simultaneously, it promotes Angelica sinensis growth, reduces disease incidence, increases yield and quality, and improves the soil ecological environment. The specific process is as follows: First, dip the roots of the angelica root in water. After planting the angelica root, dilute the special compound microbial agent for angelica root with water at a volume ratio of 1:100 and put it into a spray bottle. Then, evenly irrigate the soil around the roots of the angelica root. Set four concentration gradients for the amount used: 0 kg / mu, 10 kg / mu, 20 kg / mu, and 30 kg / mu.

[0039] Unless otherwise specified, the experimental methods used in the following examples are conventional methods.

[0040] Example 1 A method for preparing a compound microbial agent specifically for Angelica sinensis includes the following steps: (1) Bacillus subtilis DG-RB-1-16 ( Bacillus subtilis ), Bacillus megaterium BHL3-15 ( Bacillus megaterium ), Bacillus amyloliquefaciens BHL1-16 ( Bacillus amyloliquefaciens ), Bacillus mucilaginosus DG-RB-3-1 ( Mucilaginous Bacillus Bacillus licheniformis DG-RB-4-17 ( Bacillus lichen-like ), Bacillus dermatitis DG-RB-3-3 ( Paenibacillus peoriae Six functional bacteria were inoculated into TSA solid agar plates (90 mm in diameter, 15-20 mL of culture medium) using the streak method and incubated in the dark at 28°C for 4-5 days, at which point fresh colonies visible to the naked eye would grow.

[0041] It should be noted that the streak plate method can gradually dilute and disperse the aggregated bacterial strain into different areas of the culture medium by repeatedly streaking the surface of the culture medium with an inoculation loop. This can result in a large number of independently distributed single cells, which will form single colonies after culturing.

[0042] (2) Preparation of single-strain fermentation broth: Six single colonies were picked and inoculated into 500 mL Erlenmeyer flasks containing 250 mL of TSA liquid medium. The flasks were incubated in the dark at 28°C and 180 rpm with shaking for 72 h to obtain fermentation broths of single bacteria. The viable cell concentration in the liquid fermentation broth was ensured to be greater than 10⁻⁶. 9The cfu / mL concentration was adjusted to 0.75-0.85 using sterile water for the fermentation broths of the six single strains. This was done to ensure that the number of viable bacteria in each fermentation broth was the same, thus making the broth practical and reproducible.

[0043] (3) Bacillus subtilis DG-RB-1-16 ( Bacillus subtilis ), Bacillus megaterium BHL3-15 ( Bacillus megaterium ), Bacillus amyloliquefaciens BHL1-16 ( Bacillus amyloliquefaciens ), Bacillus mucilaginosus DG-RB-3-1 ( Mucilaginous Bacillus Bacillus licheniformis DG-RB-4-17 ( Bacillus lichen-like ), Bacillus dermatitis DG-RB-3-3 ( Paenibacillus peoriae The fermentation broth of a single strain of Angelica sinensis is mixed evenly at a volume ratio of 2:2:2:2:1:1 (ml / ml) to obtain the special compound microbial agent for Angelica sinensis.

[0044] It should be noted that this invention verifies the activity of Bacillus subtilis DG-RB-1-16 (Bacillus subtilis DG-RB-1-16) using the cross-streaking method. Bacillus subtilis ), Bacillus megaterium BHL3-15 ( Bacillus megaterium ), Bacillus amyloliquefaciens BHL1-16 ( Bacillus amyloliquefaciens ), Bacillus mucilaginosus DG-RB-3-1 ( Bacillus mucilaginous Bacillus licheniformis DG-RB-4-17 ( Bacillus licheniformis ), Bacillus dermatitis DG-RB-3-3 ( Paenibacillus peoriae The six functional bacteria do not have antagonistic effects on each other and can be mixed as a compound microbial agent.

[0045] It should be further noted that, in this invention, different concentration ratios were set for six functional bacteria strains, and the optimal functional bacteria compound ratio was screened out through indoor plate experiments and pot experiments: Bacillus subtilis DG-RB-1-16 ( Bacillus subtle Bacillus megaterium BHL3-15 ( Bacillus megaterium Bacillus amyloliquefaciens BHL1-16 ( Bacillus amyloliquefaciens Bacillus subtilis DG-RB-3-1 ( Mucilaginous Bacillus ): Bacillus licheniformis DG-RB-4-17 ( Bacillus licheniformis ): Bacillus pilaris DG-RB-3-3 ( Paenibacillus peoriae = 2:2:2:2:1:1 (v / v).

[0046] It should be further explained that this compound microbial agent combines the advantages of six plant growth-promoting bacteria, and at the same time has the functions of degrading the autotoxic allelopathic substances of Angelica sinensis, reducing continuous cropping obstacles, promoting the growth of Angelica sinensis, controlling pests and diseases, and improving the soil environment. It is easy to operate, has good effects, and can be applied directly to the roots.

[0047] Example 2 This embodiment will verify the effectiveness of the compound microbial agent of the present invention through a field plot experiment using Angelica sinensis grown in Minxian County, the main producing area of ​​Angelica sinensis in Gansu Province.

[0048] Experimental Example: The compound microbial agent prepared in Example 1 was used in three treatments: T1: 10 kg / mu of compound microbial agent; T2: 20 kg / mu of compound microbial agent; T3: 30 kg / mu of compound microbial agent. Before use, the compound microbial agent was diluted 50-100 times with water.

[0049] Control group: Sterile TSA culture medium was used as the control, i.e., 0 kg / mu of compound microbial agent.

[0050] Application time: April 12, 2021, during the planting period of Angelica sinensis (equivalent to base fertilizer); May 20, 2021, during the seedling stage of Angelica sinensis (equivalent to topdressing).

[0051] Application methods: root dipping and watering.

[0052] Experimental location: Mazichuan Village, Mazichuan Town, Minxian County, Dingxi City, Gansu Province Figure 1 ).

[0053] Tested variety: Mingui No. 1.

[0054] Implementation Methods and Results: A field plot experiment was conducted using a compound microbial agent specifically for Angelica sinensis as described in Example 1. During the Angelica sinensis harvesting season, six small plots (1 x 1.5 m each) were randomly selected in the planting area, comprising a control group and three treatment groups. All Angelica sinensis plants within each plot were harvested. The root length, rhizome diameter, disease incidence, and yield of the compound microbial agent in the control and treatment groups of Example 1 were measured to verify the application effect of the compound microbial agent specifically for Angelica sinensis as described in Example 1 of this invention.

[0055] After application of Angelica sinensis-specific compound microbial inoculant, the plant height of Angelica sinensis during its growth period was significantly higher than that of the control. Figure 2 The diameter of the rhizome was highest in the T2 treatment group (average 27.70 mm) and lowest in the control group (average 25.16 mm). The root length was highest in the T3 treatment group (23.37 cm) and lowest in the control group (19.95 cm). The incidence rate of angelica root was highest in the control group (50.10%) and lowest in the T2 treatment group (11.67%), with a highly significant difference. Figure 3 ).

[0056] After applying the Angelica sinensis-specific compound microbial agent, the T3 treatment group had the highest yield, reaching 364.45 kg, while the control group had the lowest yield, at 262.22 kg. Specifically, the T1 treatment group showed a 10.17% increase in yield compared to the control group; the T2 treatment group showed a 22.03% increase; and the T3 treatment group showed a 38.99% increase. A dose-response relationship was observed between yield and the amount of compound microbial agent used (Table 1). Figure 4 ).

[0057] Table 1. Statistics on the Yield of Angelica sinensis from Field Plots in Minxian County Example 3 This embodiment will verify the effectiveness of the compound microbial agent of the present invention through a field plot experiment using Angelica sinensis grown in Dangchang, a major production area of ​​Angelica sinensis in Gansu Province.

[0058] Experimental Example: The compound microbial agent prepared in Example 1 was used in three treatments: T1: 10 kg / mu of compound microbial agent; T2: 20 kg / mu of compound microbial agent; T3: 30 kg / mu of compound microbial agent. Before use, the compound microbial agent was diluted 50-100 times with water.

[0059] Control group: Sterile TSA culture medium was used as the control, i.e., 0 kg / mu of compound microbial agent.

[0060] Application time: April 14, 2021, during the planting period of Angelica sinensis (equivalent to base fertilizer); May 22, 2021, during the seedling stage of Angelica sinensis (equivalent to topdressing).

[0061] Application methods: root dipping and watering.

[0062] Experiment location: Muer Township, Lichuan Town, Dangchang County, Longnan City, Gansu Province.

[0063] Tested variety: Mingui No. 1.

[0064] Implementation Methods and Results: A field plot experiment was conducted using a compound microbial agent specifically for Angelica sinensis as described in Example 1. During the Angelica sinensis harvesting season, six small plots (1 x 1.5 m each) were randomly selected in the planting area, comprising a control group and three treatment groups. All Angelica sinensis plants within each plot were harvested. The root length, rhizome diameter, disease incidence, and yield of the compound microbial agent in the control and treatment groups of Example 1 were measured to verify the application effect of the compound microbial agent specifically for Angelica sinensis as described in Example 1 of this invention.

[0065] After application of Angelica sinensis-specific compound microbial inoculant, the plant height of Angelica sinensis during its growth period was significantly higher than that of the control. Figure 2The T3 treatment group had the highest diameter of Angelica sinensis rhizome, at 31.49 mm, compared to 23.29 mm in the control group. The T3 treatment group also had the longest root, at 38.93 cm, 12.29 cm longer than the control. In terms of disease incidence, the control group had the highest incidence at 19.44%, while the T3 treatment group had the lowest at only 7.85%, a highly significant difference. Figure 3 ).

[0066] After applying the Angelica sinensis-specific compound microbial agent, the T3 treatment group had the highest yield, reaching 484.45 kg, while the control group had the lowest yield, at 360.00 kg. Specifically, the T1 treatment group showed a 13.58% increase in yield compared to the control group; the T2 treatment group showed a 17.28% increase; and the T3 treatment group showed a 34.57% increase. A dose-response relationship was observed between yield and the amount of compound microbial agent used (Table 2). Figure 4 ).

[0067] Table 2. Statistics on the Yield of Angelica sinensis in Field Plots in Dangchang County Example 4 In this embodiment, the compound microbial agent of the present invention will be used to detect the medicinal components of Angelica sinensis grown in Minxian County, Gansu Province, a major producing area of ​​Angelica sinensis, to evaluate the effect of the Angelica sinensis-specific compound microbial agent on the medicinal components.

[0068] Experimental Example: In Example 2, Angelica sinensis roots were treated with different concentrations of compound microbial agent. T1: 10 kg / mu of compound microbial agent; T2: 20 kg / mu of compound microbial agent; T3: 30 kg / mu of compound microbial agent; Control group: sterilized TSA medium was used as the control, i.e., 0 kg / mu of compound microbial agent.

[0069] Sampling time: October 22, 2021, during the Angelica sinensis harvesting season.

[0070] Testing instrument: High performance liquid chromatograph.

[0071] Detection of active pharmaceutical ingredients: ferulic acid, ligustilide ( Figure 5 ).

[0072] Tested variety: Mingui No. 1.

[0073] Implementation Methods and Results: During the harvesting period, six small plots (1x1.5m in area) of Angelica sinensis from Example 2 were randomly selected in the planting area for both the control group and three treatment groups. All Angelica sinensis roots within each plot were harvested. The roots were cleaned, dried in a cool, dry place indoors, and then pulverized. The contents of ferulic acid and ligustilide were detected using high-performance liquid chromatography (HPLC) to evaluate the effect of the Angelica sinensis-specific compound microbial agent of Example 1 on the active components of Angelica sinensis.

[0074] Quantitative analysis of the active components of traditional Chinese medicine is the most direct and important means of quality control. High-performance liquid chromatography (HPLC) was used to quantitatively detect ferulic acid and ligustilide, the medicinal active components of Angelica sinensis, in the treatment groups and the control group. The results showed that the application of the Angelica sinensis-specific compound microbial agent significantly improved the efficacy of Angelica sinensis. The increase rate of ferulic acid in the treatment groups ranged from 17.70% to 99.73% in Minxian Angelica sinensis, with the largest increase observed in treatment group T2 (applied at 20 kg / mu). The increase rate of ferulic acid in Dangchang Angelica sinensis ranged from 133.88% to 279.49%, with the largest increase observed in treatment group T3 (applied at 30 kg / mu). P <0.01 The increase in the efficacy of angelica sinensis lactone in Minxian County ranged from 18.97% to 193.19%, with the largest increase observed in treatment group T3 when 30 kg / mu was applied. The increase in the efficacy of angelica sinensis lactone in Dangchang County ranged from 16.73% to 462.78%, with the largest increase observed in treatment group T3 when 30 kg / mu was applied. P<0.001 () Figure 6 Therefore, applying a special compound microbial agent for Angelica sinensis can significantly improve the quality and content of medicinal components of Angelica sinensis, thereby enhancing its competitiveness in the market.

[0075] Field trials have shown that the application of the Angelica sinensis-specific compound microbial agent described in this invention can significantly reduce continuous cropping obstacles, improve the yield and quality of Angelica sinensis, and also significantly reduce the disease incidence of Angelica sinensis, improve its appearance, and greatly increase the content of medicinal components. Furthermore, it fully utilizes the functional microorganisms' ability to promote nutrient absorption, enhance stress resistance, regulate disease resistance and immunity, activate soil nutrients, and improve the soil ecological environment. This helps improve arable land quality and reduce the excessive use of chemical fertilizers and pesticides, resulting in significant social and ecological benefits and broad market prospects.

[0076] The above description is merely an embodiment of the present invention and does not limit the scope of the invention. Any equivalent structural or procedural transformations made based on the content of this specification, or direct or indirect applications in other related technical fields, are similarly included within the scope of protection of the present invention.

Claims

1. A compound microbial agent specifically for Angelica sinensis, characterized in that: This compound microbial agent contains Bacillus subtilis (… Bacillus subtilis DG-RB-1-16, Bacillus megaterium ( Bacillus megaterium BHL3-15, Bacillus amyloliquefaciens ( Bacillus amyloliquefaciens BHL1-16, Bacillus mucilaginosus ( Bacillus mucilaginosus DG-RB-3-1, Bacillus licheniformis ( Bacillus licheniformis DG-RB-4-17 and Bacillus terrestris ( Paenibacillus peoriae The bacterial culture medium of DG-RB-3-3; the preservation number of Bacillus subtilis DG-RB-1-16 is CGMCC No. 27080; the preservation number of Bacillus megaterium BHL3-15 is CGMCC No. 27094; the preservation number of Bacillus amyloliquefaciens BHL1-16 is CGMCC No. 27071; the preservation number of Bacillus mucilaginosus DG-RB-3-1 is CGMCC No. 27078; the preservation number of Bacillus licheniformis DG-RB-4-17 is CGMCC No. 27074; the preservation number of Bacillus pilaris DG-RB-3-3 is CGMCC No. 27092; the preparation method of this compound microbial agent includes the following steps: (1) Six functional bacteria, namely Bacillus subtilis DG-RB-1-16, Bacillus megaterium BHL3-15, Bacillus amyloliquefaciens BHL1-16, Bacillus mucilaginosus DG-RB-3-1, Bacillus licheniformis DG-RB-4-17, and Bacillus pilaris DG-RB-3-3, were inoculated into TSA solid agar plates by streak plating and then incubated in the dark at 28°C for 4-5 days. Fresh colonies visible to the naked eye will then grow. (2) Preparation of single-strain fermentation broth: Six single colonies were picked and inoculated into 500 mL Erlenmeyer flasks containing 250 mL of TSA liquid medium. The flasks were then incubated in the dark at 28°C and 180 rpm with shaking for 72 h to ensure a viable bacterial concentration greater than 10⁻⁶ in the liquid fermentation broth. 9 The concentration of cfu / mL was adjusted to 0.75-0.85 using sterile water to obtain the fermentation broth of a single strain. (3) Mix the fermentation broths of Bacillus subtilis DG-RB-1-16, Bacillus megaterium BHL3-15, Bacillus amyloliquefaciens BHL1-16, Bacillus mucilaginosus DG-RB-3-1, Bacillus licheniformis DG-RB-4-17, and Bacillus pilaris DG-RB-3-3 at a volume ratio of 2:2:2:2:1:1 to obtain the Angelica sinensis-specific compound microbial agent.

2. The Angelica sinensis-specific compound microbial agent as described in claim 1, characterized in that: In step (1), the TSA solid plate culture medium is prepared by adding 15 g tryptone, 5 g soybean peptone, 5 g sodium chloride, and 18 g agar to 1000 mL of distilled water, mixing thoroughly, adjusting the pH to 7.0-7.2, and sterilizing at 121℃ for 20 min.

3. The Angelica sinensis-specific compound microbial agent as described in claim 1, characterized in that: In step (2), the TSA liquid culture medium is prepared by adding 15 g of tryptone, 5 g of soybean peptone, and 5 g of sodium chloride to 1000 mL of distilled water, mixing them thoroughly, adjusting the pH to 7.0-7.2, and then sterilizing by steaming at 121℃ for 30 min.

4. A method of using the Angelica sinensis-specific compound microbial agent as described in claim 1, characterized in that: The Angelica sinensis-specific compound microbial agent was applied to Angelica sinensis plantations that were continuously cropped using the root irrigation method.

5. A method of using the Angelica sinensis-specific compound microbial agent as described in claim 1, characterized in that: First, dip the roots of the angelica root in water. After planting the angelica root, dilute the special compound microbial agent for angelica root with water at a volume ratio of 1:100 and put it into a spray bottle; then evenly irrigate it into the soil around the roots of the angelica root.

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