A synthetic bacterial community for improving the quality and yield of simulated wild astragalus and its application

Abstract

The present invention discloses a synthetic bacterial community for improving the quality and yield of simulated wild astragalus and its application, which belongs to the field of ecological planting of Chinese medicinal materials. The synthetic bacterial community is composed of 3 nitrogen-fixing bacteria, 2 phosphate-dissolving bacteria and 1 potassium-dissolving bacteria; the nitrogen-fixing bacteria are composed of Bacillus subtilis, Sinorhizobium and Rhizobium; the phosphate-dissolving bacteria are composed of Pseudomonas meningitidis and Pseudomonas putida; the potassium-dissolving bacteria is bidirectional Burkholderia. The synthetic bacterial community provided by the present invention is prepared into a bacterial agent, which is inoculated during the planting of astragalus, which significantly improves the yield and quality of astragalus, and the microbial community structure, soil nutrition and physical and chemical properties of the soil for planting astragalus are also significantly improved. The present invention provides a technical reference for standardizing the planting technology of Hunyuan astragalus, improving the level of planting technology, and improving the yield and quality of astragalus medicinal materials from the source.

Description

Technical Field

[0001] The invention relates to the field of ecological planting of Chinese medicinal materials, and in particular to a synthetic bacterial community for improving the quality and yield of simulated wild astragalus and an application thereof. Background Art

[0002] Astragalus is the main authentic medicinal material in Shanxi Province, and the main production area is located in Hunyuan County. The specific soil type and unique ecological environment, as well as the planting and processing methods that combine tradition and modernity, provide unique conditions for the growth of the traditional Chinese medicine Astragalus and the formation of excellent quality, and also create the "authentic" characteristics of Astragalus. However, due to the long-term "man-made and natural" planting method of Hunyuan Astragalus, the land is only planted but not raised, coupled with backward cultivation technology and lack of controllable planting standards, resulting in a decrease in soil nutrients year by year, an imbalance in microbial structure, and frequent diseases and insect pests, which in turn causes Astragalus to grow slowly, have low yields, and unstable quality. Therefore, it is urgent to improve soil fertility, standardize planting techniques, and improve the level of planting technology, so as to increase the yield and quality of Astragalus medicinal materials from the source and enhance the market competitiveness of Shanxi Astragalus. Summary of the invention

[0003] The purpose of the present invention is to provide a synthetic bacterial community and its application for improving the quality and yield of simulated wild astragalus, so as to solve the problems existing in the above-mentioned prior art. The synthetic bacterial community provided by the present invention is prepared into a bacterial agent, which is inoculated when astragalus is planted, which significantly improves the yield and quality of astragalus, and the microbial community structure, soil nutrition and physical and chemical properties of the soil planted with astragalus are also significantly improved.

[0004] To achieve the above object, the present invention provides the following solutions:

[0005] The invention provides a synthetic bacterial community specially used for simulating wild astragalus cultivation, wherein the synthetic bacterial community is composed of 3 nitrogen-fixing bacteria, 2 phosphate-dissolving bacteria and 1 potassium-dissolving bacteria; the nitrogen-fixing bacteria are composed of Bacillus subtilis, Sinorhizobium sp. and Rhizobium sp.; the phosphate-dissolving bacteria are composed of Pseudomonas mandelii and Pseudomonas putida; and the potassium-dissolving bacteria is Burkholderia ambifaria;

[0006] The deposit number of the Sinorhizobium is CGMCC No.9677;

[0007] The deposit number of the rhizobium is CGMCC No.9676.

[0008] The present invention also provides a synthetic bacterial agent, which is prepared from the synthetic bacterial community.

[0009] Furthermore, in the synthetic bacterial agent, the ratio of the number of live bacteria of the Bacillus subtilis, the Sinorhizobium, the Rhizobium, the Pseudomonas mentonii, the Pseudomonas putida and the Burkholderia bifida is 1:1:1:1:1:1:1.

[0010] The present invention also provides the use of the synthetic bacterial flora or the synthetic bacterial agent in improving the quality and yield of simulated wild astragalus.

[0011] The present invention also provides a planting method for improving the quality and yield of simulated wild astragalus, comprising the step of inoculating the synthetic bacterial community on the surface of astragalus seeds or in the soil for planting astragalus.

[0012] Furthermore, when the synthetic bacterial flora is inoculated on the surface of the astragalus seeds, the seeds are soaked or mixed with a bacterial agent containing the synthetic bacterial flora.

[0013] Furthermore, the number of viable bacteria in the bacterial agent is 1×10 8 ~1×10 10 CFU / mL.

[0014] Furthermore, the seed soaking time is 0.5 to 1 hour.

[0015] The present invention discloses the following technical effects:

[0016] The present invention constructs a synthetic bacterial community based on the types of beneficial bacterial strains separated from the root system and rhizosphere soil of different distribution areas of Astragalus in the early stage, through the complementarity of strain functions, the increase of diversity and the superposition of growth-promoting effects, with strains with similar functions in existing strains, and prepares a synthetic bacterial agent, which is inoculated when Astragalus is planted, significantly improving the yield and quality of Astragalus, and the microbial community structure, soil nutrition and physical and chemical properties of the soil for planting Astragalus are also significantly improved.

[0017] The invention provides a technical reference for standardizing the planting technology of Hunyuan Astragalus, improving the planting technology level, and improving the yield and quality of Astragalus medicinal materials from the source. BRIEF DESCRIPTION OF THE DRAWINGS

[0018] In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings required for use in the embodiments will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present invention. For ordinary technicians in this field, other drawings can be obtained based on these drawings without paying creative work.

[0019] Figure 1 This is a graph showing the effect of synthetic bacterial flora on the growth of potted seedlings;

[0020] Figure 2This is the effect of the synthetic bacterial community on the growth of Astragalus membranaceus seedlings in the field, where A is one-year-old Astragalus membranaceus; B is two-year-old Astragalus membranaceus; C is three-year-old Astragalus membranaceus;

[0021] Figure 3 This is a graph showing the effect of the synthetic bacterial flora on the content of the active ingredients of Astragalus;

[0022] Figure 4 Relative abundance of rhizosphere soil bacterial communities at the phylum (A), family (B), and genus (C) levels. DETAILED DESCRIPTION

[0023] Various exemplary embodiments of the present invention will now be described in detail. This detailed description should not be considered as limiting the present invention, but should be understood as a more detailed description of certain aspects, features, and embodiments of the present invention.

[0024] It should be understood that the terms described in the present invention are only for describing a particular embodiment and are not intended to limit the present invention. In addition, for the numerical range in the present invention, it should be understood that each intermediate value between the upper and lower limits of the scope is also specifically disclosed. The intermediate value in any stated value or stated range, and each smaller range between any other stated value or intermediate value in the described range is also included in the present invention. The upper and lower limits of these smaller ranges can be independently included or excluded in the scope.

[0025] Unless otherwise indicated, all technical and scientific terms used herein have the same meanings as those generally understood by those skilled in the art. Although the present invention describes only preferred methods and materials, any methods and materials similar or equivalent to those described herein may also be used in the implementation or testing of the present invention. All documents mentioned in this specification are incorporated by reference to disclose and describe the methods and / or materials associated with the documents. In the event of a conflict with any incorporated document, the content of this specification shall prevail.

[0026] It will be apparent to those skilled in the art that various modifications and variations may be made to the specific embodiments of the present invention description without departing from the scope or spirit of the present invention. Other embodiments derived from the present invention description will be apparent to those skilled in the art. The present invention description and examples are exemplary only.

[0027] The words “include,” “including,” “have,” “contain,” etc. used in this document are open-ended terms, meaning including but not limited to.

[0028] In the early stage of the present invention, a variety of different microorganisms were isolated from the root system and rhizosphere soil of Hunyuan Astragalus, including strains of nitrogen-fixing bacteria, phosphate-dissolving bacteria and potassium-dissolving bacteria, which have the functions of symbiotic nitrogen fixation, phosphorus dissolving, potassium dissolving, secretion of plant hormones and growth promotion. According to the principles of strain functional complementarity, diversity increase and superposition of growth-promoting effects, and referring to the analysis results, a synthetic bacterial community simulating the growth environment of wild Astragalus was finally artificially constructed in the existing strains, and the synthetic bacterial community included 3 strains of nitrogen-fixing bacteria, 2 strains of phosphate-dissolving bacteria and 1 strain of potassium-dissolving bacteria. The nitrogen-fixing bacteria include strains N1, N2 and N3; N1 is Bacillus subtilis, with a strain number of CGMCC 1.9083; N2 is Sinorhizobium sp., with a deposit number of CGMCC No.9677; N3 is Rhizobium sp., with a deposit number of CGMCC No.9676; N2 and N3 were both deposited in the General Microbiological Center of the China Microbiological Culture Collection Administration on September 18, 2014, and the deposit address is No. 3, Yard No. 1, Beichen West Road, Chaoyang District, Beijing, Institute of Microbiology, Chinese Academy of Sciences. The phosphate-solubilizing bacteria include strains P1 and P2; P1 is Pseudomonas mandelii, with a strain number of CGMCC 1.6426; P2 is Pseudomonas putida, with a strain number of CGMCC 1.8092. Potassium-dissolving bacteria K1 is Burkholderia ambifaria, with strain number CGMCC 1.10506. Strains N1, P1, P2 and K1 were purchased from the General Microbiological Center of China National Microbiological Culture Collection Administration.

[0029] Example

[0030] 1. Methods

[0031] 1. Preparation of synthetic bacterial agents

[0032] The strains N1, N2, N3, P1, P2 and K1 were inoculated into LB liquid medium and incubated at 28°C for 180 r·min. -1 Culture for 48 h. The viable bacterial count was determined to be 1×10 10 cfu·mL -1 The bacterial cultures with determined viable counts were mixed in equal proportions at a 1% inoculation rate to prepare a synthetic bacterial agent.

[0033] 2. Potted plant inoculation test with synthetic flora

[0034] Select dark brown astragalus seeds with full and uniform grains, soak them in boiling water for 1 min, then soak them in 40°C warm water for 8 h, wash them with sterile water 2-3 times, plant them with the hilum facing down in a pot filled with nutrient soil, and after the seeds germinate, dilute the prepared synthetic bacterial agent 100 times with purified water (the number of viable bacteria after dilution is 1×10 8 cfu·mL -1 ) and then evenly sprinkle it in the pot, set up 10 replicates, take the uninoculated bacteria as the control, and culture in a light room with a culture temperature of 25℃, humidity of 35%, and light for 16h. After 60d of culture, record the plant growth, measure the seedling height, root length and root diameter, and measure the root dry weight after drying in an oven at 65℃ to constant weight.

[0035] 3. Field trial of synthetic bacterial flora inoculation

[0036] ① Seed requirements: Astragalus seeds are collected from plants aged 3 to 6 years. Select seeds with full grains, no diseases or insect pests, and no mechanical damage. The seed coat is dark brown or black brown, the germination rate is above 65%, the purity is not less than 95%, the thousand-grain weight is 5.8 to 7.5g, and the water content is not higher than 10%.

[0037] ②Inoculation: dilute the synthetic bacterial agent with a volume ratio of pure water: bacterial agent = 1:100, so that the number of viable bacteria in the diluted synthetic bacterial agent is 1×10 8 CFU / mL, soak the seeds for 0.5-1h, or spray the diluted fungus agent evenly on the surface of the seeds, turning them while spraying to ensure that the fungus agent evenly covers the surface of the seeds, and they can be sown after a little drying, with the uninoculated seeds as the control.

[0038] ③Sowing: In early May, artificial row sowing is adopted. A shallow furrow with a depth of 5 cm is opened with a row spacing of 50 cm. The seeds are evenly scattered into the furrow, and the soil thickness is 1 cm. The seeds are slightly shaken and pressed. Mechanical sowing is adopted for large-scale planting. The sowing amount per mu is 1.0-1.5 kg.

[0039] 2. Results

[0040] 1. Effects of synthetic flora on the growth of potted seedlings

[0041] After inoculation with the synthetic agent, the growth indexes and the contents of the main active ingredients of the potted seedlings were measured for 60 days as shown in Tables 1 and Figure 1As shown. Although the height of the seedlings increased by 17.32% after inoculation compared with the control, there was no significant difference; however, the promoting effect on root length, root diameter and root dry weight was obvious, increasing by 43.62%, 69.42% and 69.57% respectively, all reaching a significant level. After inoculation, the contents of flavonoids and saponins in the root of Astragalus membranaceus increased significantly, among which the flavonoid content increased by 48.81% and the saponin content increased by 11.54% compared with the control. Astragalus membranaceus is a traditional Chinese medicinal material with roots as medicine. The dry weight of the root is the main indicator to measure the yield, and the content of flavonoids and saponins in the root represents the quality. It can be seen that the yield and quality of Astragalus membranaceus were significantly promoted after inoculation with synthetic bacterial agents.

[0042] Table 1 Effects of synthetic bacterial flora on the biomass of potted Astragalus membranaceus

[0043]

[0044] 2. Effects of synthetic flora on the growth and quality of Astragalus membranaceus in the field

[0045] In order to further study the inoculation application of synthetic flora in production, the present invention conducted a field inoculation test.

[0046] like Figure 2 As shown in Table 2, after the synthetic flora was mixed with seeds, the root growth of Astragalus membranaceus at different ages was significantly improved. Compared with the control group, in addition to the root length, the root diameter and root dry weight of the one-year-old Astragalus membranaceus were significantly increased, increasing by 50.91% and 116%, respectively. The root length, root diameter and root dry weight of the two-year-old Astragalus membranaceus increased by 29.38%, 45.80% and 58.05%, respectively. The root length, root diameter and root dry weight of the three-year-old Astragalus membranaceus increased by 20.52%, 58.99% and 41.90%, respectively.

[0047] Table 2 Effects of synthetic bacterial flora on Astragalus membranaceus biomass

[0048]

[0049]

[0050] The contents of three active ingredients in the three-year-old Astragalus root were further analyzed and determined, such as Figure 3 As shown. After inoculation with synthetic bacteria, the contents of total polysaccharides, total flavonoids and saponins in the root of Astragalus membranaceus were significantly increased compared with the control, among which the total polysaccharides increased by 19.93%, the total saponins increased by 31.57%, and the total flavonoids increased by 54.97. In summary, the synthetic flora not only promoted the growth of Astragalus membranaceus and increased the yield of medicinal materials, but also promoted the biosynthetic pathway of secondary metabolism, increased the content of active ingredients, and improved the quality of medicinal materials.

[0051] 3. Effects of inoculation with synthetic microbiota on the metabolome of Astragalus root

[0052] In order to further explore the mechanism of increased content of Astragalus medicinal ingredients, the present invention measured the metabolome of field Astragalus. As shown in Table 3, there were significant differences in metabolites between the treatment group and the control group. Using the non-targeted metabolome method with t-test (p<0.05) and fold change (FC>1.2 or <0.8) as the standard, a total of 26 compounds with significant differences were identified, including 10 flavonoids (NO.1-10), 3 triterpenoid saponin compounds (NO.14-16), 5 amino acid compounds (NO.17-21), and 8 other compounds. The content of these differential metabolites is reflected in the fact that the content of compounds in the root of Astragalus after inoculation with synthetic flora is significantly higher than that in the uninoculated Astragalus. Flavonoids and saponin compounds are the main medicinal ingredients of Astragalus and are the main indicators for measuring the quality of Astragalus.

[0053] Table 3 Different compounds in the non-targeted metabolomics results

[0054]

[0055]

[0056] 4. Effects of synthetic microbiota on the microbial community structure of rhizosphere soil of Astragalus membranaceus

[0057] To investigate the changes in the rhizosphere soil and root endophytic bacterial communities of Astragalus membranaceus in Hunyuan after treatment with synthetic microbial agents, the relative abundance of microbial taxa in the field Astragalus membranaceus treatment and control groups was evaluated at the phylum, family, and genus levels ( Figure 4 At the phylum level, Actinobacteriota, Proteobacteria, Acidobacteriota, and Chloroflexi were identified as the dominant phyla in the rhizosphere soil samples in both the treatment and control groups ( Figure 4 A). Among them, Actinobacteria maintained a high abundance throughout the growth period, with an average relative abundance of 32.7%. The relative abundance of Proteobacteria increased continuously during plant growth, reaching the highest proportion (23.8%) at 120 days in the treatment group. At the family level, Vicinamibacterales, Vicinamibacterace, Micrococcaceae, and Gemmatimonadaceae were the dominant bacteria in each group ( Figure 4 B); at the genus level, Arthrobacter, Gaiella and Solirubrobacter were the dominant bacteria in each group ( Figure 4C). These dominant bacteria are involved in the biosynthesis of plant terpenoids, phenylpropanoids and polyketides. It is speculated that the inoculation of synthetic microbial agents regulates the types and structure of dominant soil bacteria, which has an important impact on the regulation of Astragalus secondary metabolism, thereby promoting the accumulation of flavonoids and saponins in the roots.

[0058] 5. Effects of synthetic flora on soil nutrition of Astragalus

[0059] Soil nitrogen, phosphorus and potassium are the main sources of plant nutrients and the main indicators for evaluating soil quality, which directly affect plant growth. After inoculation with synthetic microbial agents, the N, P and K contents of field Astragalus soil increased to varying degrees (Table 4), among which the soil nitrogen and potassium contents increased significantly, with total nitrogen and effective nitrogen increasing by 27.78% and 68.66% respectively compared with the control, and total potassium and effective potassium increasing by 1.94% and 8.21% respectively compared with the control, and phosphorus content had no significant difference from the control group.

[0060] Table 4 Effects of inoculation with synthetic bacteria on soil N, P and K nutrition

[0061]

[0062] The embodiments described above are only descriptions of the preferred modes of the present invention, and are not intended to limit the scope of the present invention. Without departing from the design spirit of the present invention, various modifications and improvements made to the technical solutions of the present invention by ordinary technicians in this field should all fall within the protection scope determined by the claims of the present invention.

Claims

1. A synthetic bacterial agent, characterized in that: Prepared from synthetic bacterial flora; The synthetic bacterial community is composed of 3 nitrogen-fixing bacteria, 2 phosphate-dissolving bacteria and 1 potassium-dissolving bacteria; the nitrogen-fixing bacteria is composed of Bacillus subtilis ( Bacillus subtilis )、Sinorhizobium rhizobium( Sinorhizobium sp. ) and Rhizobium ( Rhizobium sp. ); The phosphate-dissolving bacteria are composed of Pseudomonas meningitidis ( Pseudomonas mandelii ) and Pseudomonas putida ( Pseudomonas putida ); The potassium-dissolving bacteria is biphasic Burkholderia ( Burkholderia ambifaria ); The strain number of the Bacillus subtilis is CGMCC 1.9083; The deposit number of the Sinorhizobium is CGMCC No.9677; The deposit number of the rhizobium is CGMCC No.9676; The strain number of the Pseudomonas meningitidis is CGMCC 1.6426; The strain number of the Pseudomonas putida is CGMCC 1.8092; The species number of the biphasic Burkholderia is CGMCC 1.10506; In the synthetic bacterial agent, the ratio of the number of live bacteria of the Bacillus subtilis, the Sinorhizobium, the Rhizobium, the Pseudomonas mentonii, the Pseudomonas putida and the Burkholderia bifida is 1:1:1:1:1:1:

1.

2. Use of the synthetic bacterial agent as claimed in claim 1 in improving the quality and yield of simulated wild astragalus.

3. A planting method for improving the quality and yield of simulated wild astragalus, characterized in that: The method comprises the steps of inoculating the synthetic bacterial agent according to claim 1 on the surface of astragalus seeds or in the soil in which astragalus is planted.

4. The planting method according to claim 3, characterized in that: When the synthetic bacterial flora is inoculated on the surface of the astragalus seeds, the seeds are soaked or mixed with a bacterial agent containing the synthetic bacterial flora.

5. The planting method according to claim 4, characterized in that: The number of viable bacteria in the bacterial agent is 1×10 8 ~1×10 10 CFU / mL.

6. The planting method according to claim 4, characterized in that: The seed soaking time is 0.5 to 1 hour.

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