Aspergillus tubigensis, microbial combination and application thereof in improving production and quality of epimedium

Abstract

The present application relates to the field of microbial technology, and more particularly to a Tuberibacterium, a microbial combination and application thereof in improving the yield and quality of Epimedium. Colletotrichum truncatum S5 and Clonostachys rosea RS5. The present application provides a Tuberibacterium EKYY3 with the ability to promote the growth and accumulation of medicinal ingredients of Epimedium, and simultaneously provides a complex microbial agent comprising the strain. The application of the complex microbial agent during the growth of Epimedium can effectively promote the growth and accumulation of medicinal ingredients of Epimedium, and can increase the expression of key enzymes such as PAL, FNS and F3H, improve the yield and quality of medicinal materials, and increase the clinical efficacy. This has important significance in the field of high-yield, high-efficiency and green and safe production of traditional Chinese medicinal materials.

Description

TECHNICAL FIELD

[0001] The present application relates to the technical field of microorganisms, and particularly relates to a Tuberibacterium tabinum, a microbial combination and application of the Tuberibacterium tabinum and the microbial combination in improving production and quality of Epimedium. BACKGROUND

[0002] Epimedium koreanum is a perennial herb, is an important raw material of medicine and health food, has great research and development potential, and is widely concerned at home and abroad, so the industrial development demand is huge. However, most of the medicinal materials are derived from wild, and because of the weak sexual reproduction ability, it belongs to endangered species, combined with various improper and unreasonable development and utilization, etc., the wild resources of Epimedium koreanum are very scarce, the market price is rising, and the contradiction between supply and demand is becoming increasingly serious. In addition, it is found that the quality of Epimedium koreanum medicinal materials is unstable, the content of active ingredients is low, and most of them do not meet the requirements. The total flavonoid glycoside content in the existing quality control standard of Epimedium koreanum should not be less than 0.5%, which is lower than the standard limit of 1.5% of Epimedium, Epimedium pubescens and Epimedium sagittatum. Therefore, improving the yield and quality of Epimedium koreanum is an urgent problem to be solved for the orderly development of the industry.

[0003] The vigorous growth and development of medicinal plants and the efficient accumulation of metabolic products are important prerequisites for increasing production and quality of Chinese herbal medicines and improving efficiency, but the large-scale application of pesticides and fertilizers in the production of Chinese herbal medicines at present has stressed the growth and development of plants, reduced the quality of medicinal materials, polluted the ecological environment and increased the clinical risk. Therefore, how to improve the yield and quality of medicinal materials, repair the ecological environment and ensure the safety, effectiveness, stability and controllability of traditional Chinese medicine have become scientific problems to be solved in the traditional Chinese medicine industry. SUMMARY

[0004] In order to solve the problems in the prior art, the present application provides a Tuberibacterium tabinum, a microbial combination and application of the Tuberibacterium tabinum and the microbial combination in improving production and quality of Epimedium.

[0005] In a first aspect, the present application provides a Tuberibacterium tabinum (Aspergillus tabinum) EKYY3, and biological preservation is carried out thereon, and the preservation information is as follows: Aspergillus tubingensis Preservation number: CGMCC No.41722; classification name:

[0006] ; preservation unit: China General Microbiological Culture Collection Center; preservation address: No.3, Yikhuan, Beichen West Road, Chaoyang District, Beijing, China, Institute of Microbiology, Chinese Academy of Sciences; preservation date: November 27, 2024. Aspergillus tubingensis

[0007] ​Based on the above Aspergillus tubigensis EKYY3, the application innovatively develops a compound microbial agent for improving the yield and quality of Epimedium herb, which can form a good mutualistic symbiotic system with the host plant, improve the yield and quality of the herb by promoting the growth and development of Epimedium and increasing the expression amount of key genes in the synthesis pathway of medicinal ingredients. The microbial agent can replace inorganic fertilizers, root strengthening agents, etc., to produce high-yield, high-quality and efficient green and safe Chinese herbal medicines by improving soil structure, increasing soil fertility and repairing ecological environment.

[0008] In a second aspect, the application provides a microbial combination comprising the aforementioned Aspergillus tubigensis EKYY3, and further comprising Colletotrichum truncatum S5 and Clonostachys rosea RS5.

[0009] wherein, Colletotrichum truncatum S5 and Clonostachys rosea RS5 are disclosed in Research on Endophytic and Rhizosphere Microbial Diversity and Function of Epimedium koreanum.

[0010] Further, the Aspergillus tubigensis EKYY3, Colletotrichum truncatum S5 and Clonostachys rosea The total viable bacterial number ratio of S5, RS5 and S5 is (4-6):(2-4):(3-5).

[0011] In a second aspect, the application provides a microbial agent comprising the aforementioned Aspergillus tubigensis EKYY3 or the aforementioned microbial combination.

[0012] Further, the microbial agent preferably further comprises, by weight: carrier peat 10-20 parts, sodium hydroxymethyl cellulose 0.5-1.5 parts, and sodium alginate 0.5-1.5 parts.

[0013] In a third aspect, the application provides a preparation method of the microbial agent, comprising: after the fermentation culture of the microbial combination, inoculating in a nutrient solution.

[0014] Further, the culture medium used in the fermentation culture process comprises, by weight: potato 30-70 parts, glucose 3-7 parts, agar 3-7 parts, and water 200-300 parts.

[0015] Further, the initial microbial inoculation amount of the fermentation culture is 3-12% (mass percentage content).

[0016] Further, the fermentation culture conditions include: rotation speed 150-220 r / min, 250-500 mL, temperature 22-26℃, pH 7-9.

[0017] After the fermentation culture, the total viable bacterial number of the microbial combination is 1×10 1~9cfu / mL, the inoculation amount is 3-12% (in mass percentage, inoculated into the nutrient solution); and / or,

[0018] The nutrient solution comprises, in parts by weight: beef extract 5-10 parts, sodium chloride 5-10 parts, brown sugar 5-10 parts, and soluble starch 2-5 parts.

[0019] Further, after inoculation into the nutrient solution, the culture is carried out in a shaking bed for more than 6 days, and the culture condition is 150-250 r / min and 22-25 DEG C.

[0020] Further, the method further comprises: adding dextrin and vitamin C, and irradiating with an ultraviolet lamp for 25-40 minutes.

[0021] The present application uses vitamin C as an ultraviolet protection agent, can collect most of the ultraviolet radiation, maintains the activity and stability of the viable cells, and vitamin C has the function of promoting iron absorption compared with traditional dextrin.

[0022] The microbial agent provided by the present application does not add nitrogen, phosphorus and potassium elements, and uses the properties of nitrogen fixation and phosphorus solubilization of the plant itself to promote the development of root system and the flourishing of leaves.

[0023] In a fourth aspect, the present application provides the use of the foregoing Aspergillus tubingensis EKYY3, or the foregoing microbial combination, or the foregoing microbial agent in improving the yield or quality of Epimedium.

[0024] Further, the use comprises:

[0025] Improving one or more of the dry weight, fresh weight, leaf area, PAL level, FNS level, F3H level, epimedoside A, epimedoside B, epimedoside C or icariin of Epimedium.

[0026] Further, the Epimedium is Epimedium koreanum Nakai.

[0027] The present application has the following beneficial effects:

[0028] 1. The strain used in the present application is different from the strains used in the existing microbial fertilizer products on the market, and is isolated from the plant body of Epimedium koreanum Nakai, which is an endophytic fungus of the host plant and can symbiotically coexist with the host plant without harming the plant itself. The Aspergillus tubingensis EKYY3 provided by the present application has the ability to promote the growth and medicinal ingredient accumulation of Epimedium.

[0029] 2. The present application further provides a microbial combination comprising three kinds of microorganisms, which can form a good symbiotic system with Epimedium, promote the growth and development of Epimedium, and increase the expression amount of key genes in the synthesis pathway of medicinal ingredients, thereby improving the yield and quality of medicinal materials.

[0030] 3. In addition to improving the yield indicators of Epimedium plants such as dry weight, fresh weight, and leaf area, the compound microbial agent provided by this invention can also increase the expression levels of key enzymes for the synthesis of medicinal components such as PAL, FNS, and F3H in the plant, promote the accumulation of medicinal components such as icariin, improve the quality of medicinal materials, and increase clinical efficacy. This is a function that existing microbial fertilizers on the market do not have.

[0031] 4. The process of this invention is simple and low in cost. This formula can also be prepared into a solid bacterial agent. Without changing the formula composition, adding an appropriate amount of carrier and vacuum drying at -80℃ will result in a bacterial survival rate of over 80%. Attached Figure Description

[0032] To more clearly illustrate the technical solutions in this invention or the prior art, the drawings used in the description of the embodiments or the prior art will be briefly introduced below. Obviously, the drawings described below are some embodiments of this invention. For those skilled in the art, other drawings can be obtained from these drawings without creative effort.

[0033] Figure 1 This is a diagram of the flavonol glycosides of EKYY3 and S5 provided in Example 1 of the present invention.

[0034] Figure 2 The image shown is a blank group of Epimedium photos provided in Embodiment 2 of the present invention.

[0035] Figure 3 These are photos of Epimedium from the EKYY3 experimental group provided in Embodiment 2 of the present invention.

[0036] Figure 4 These are photos of Epimedium from the RS5 experimental group provided in Embodiment 2 of the present invention.

[0037] Figure 5 This is a photograph of Epimedium in the S5 experimental group provided in Embodiment 2 of the present invention.

[0038] Figure 6 This is a photograph of Epimedium from the Epimedium growth-promoting agent group provided in Embodiment 2 of the present invention.

[0039] Figure 7 This is a stability test chart of the compound microbial agent provided in Example 3 of the present invention.

[0040] Figure 8 This is a picture of the finished product of the Epimedium growth promoter provided in Example 3 of the present invention. Detailed Implementation

[0041] To make the objectives, technical solutions, and advantages of this invention clearer, the technical solutions of this invention will be clearly and completely described below with reference to the accompanying drawings. Obviously, the described embodiments are only some, not all, of the embodiments of this invention. All other embodiments obtained by those skilled in the art based on the embodiments of this invention without creative effort are within the scope of protection of this invention.

[0042] Unless otherwise specified, the experimental methods involved in the following embodiments are conventional methods in the art. For example, you can refer to the experimental manual in the art or follow the conditions recommended in the manufacturer's instructions.

[0043] Unless otherwise specified, all experimental materials and reagents used in the following examples are commercially available.

[0044] The Epimedium used in the following examples is Korean Epimedium.

[0045] Example 1

[0046] This invention provides a compound microbial agent and its preparation method, comprising the following steps:

[0047] 1. Preparation of mother liquor: Aspergillus tabineus (… Aspergillus tubingensis EKYY3、 Colletotrichum truncatum S5 and Clonostachys rosea After activation, the three RS5 strains were fermented in a fermentation medium until the spore count reached 80% of the total volume. Fermentation was then stopped, and the cultures were centrifuged at 5000 rpm for 10 minutes at room temperature to obtain viable cell counts of 2.5 × 10⁻⁶ cells / mL. 7 CFU / mL, 1.5×10 7 CFU / mL, 2×10 7 CFU / mL;

[0048] Both the inoculum activation and fermentation were performed using PDB liquid medium: 50g potato, 5g glucose, 5g agar, and 250ml distilled water, sterilized at 121℃ for 20min. The fermentation conditions were: 50 mL / 250mL liquid volume, 6% inoculum, 180 r / min rotation speed, 30℃, and pH=9.

[0049] 2. Preparation of nutrient solution: Accurately weigh 5g beef extract, 5g sodium chloride, 7g brown sugar, and 2.5g soluble starch and mix them in a beaker. Add 250mL of distilled water to adjust the pH to 9. Sterilize at 121℃ and 101kPa for 20 minutes and let cool. Add 7g of ferrous sulfate and magnesium sulfate solution while stirring with a glass rod until fully mixed and free of lumps. Place in a 4℃ refrigerator for later use.

[0050] 3. Prepare the mixed bacterial inoculum by inoculating the stock solution from step 1 into the nutrient solution from step 2 at a 6% inoculation rate. Place the mixture in a shaker at 180 rpm and 24°C for 7 days.

[0051] 4. Add 5-10g of UV protectant dextrin and vitamin C to the above mixture, and irradiate it at a distance of 30 minutes from the UV lamp of the ultra-clean workbench to obtain the Epimedium growth promoter.

[0052] 5. Application method of Epimedium growth promoter: The total viable count of the Epimedium growth promoter obtained in step 4 is 2 × 10⁻⁶. 8 cfu / mL, dilute with water to a total viable count of 1×10⁻⁶. 6 Apply 6L of cfu / mL per acre using a root irrigation system.

[0053] The culture medium formula for bacterial counting in this invention includes: 10g peptone, 3g beef extract, 5g sodium chloride, 14g agar, 1000 mL distilled water, pH=7.0±0.1, sterilized at 121℃ for 20 min.

[0054] Example 2

[0055] 1. The present invention further relates to the embodiments described in Example 1. Aspergillus tubingensis EKYY3 Colletotrichum truncatum S5 and Clonostachys rosea The three RS5 strains were evaluated using a plate growth promotion experiment, with nitrogen fixation, phosphorus solubilization, siderophore production, and indoleacetic acid production as observation indicators. The methods included the following:

[0056] Five purified bacterial strains were inoculated into Pikovaskaia's, CAS, and Ashby media, respectively, and incubated at 28℃ for 3 days. Colony growth was observed. 20.0 mg of IAA standard was weighed and diluted with methanol to prepare standard solutions containing 2 mg / L, 10 mg / L, 20 mg / L, 40 mg / L, and 50 mg / L of IAA. 1 mL of each solution was mixed with the colorimetric solution at a 1:1 ratio, incubated in the dark for 30 min, and the OD530 value was measured to plot a standard curve.

[0057] The results are shown in the table below:

[0058] Table 1. Determination of growth-promoting ability of three bacterial strains

[0059]

[0060] This invention uses Aspergillus tabineus ( Aspergillus tubingensisUsing the genomic DNA of EKYY3 as a template, PCR amplification was performed using universal fungal primers ITS1 and ITS4, and the amplified sequences were sent to a bioengineering company. The obtained sequences were compared with the NCBI database, and a phylogenetic tree of EKYY3 and closely related bacteria was constructed using MEGA 7.0.21 software. The results showed that the sequence of EKYY3 had the highest homology (100%) with the sequence of *Aspergillus tubingensis*. Based on the *Handbook of Common Fungal Identification*, the physiological and biochemical characteristics of strain EKYY3 were identical to those of *Aspergillus tubingensis*. Combined with the above molecular identification, it was identified as *Aspergillus tubingensis*. EKYY3 was named *Aspergillus tubingensis* EKYY3.

[0061] This invention has been biologically deposited for EKYY3, and the deposit information is as follows: Deposit number: CGMCC No. 41722; Classification and nomenclature: Aspergillus tubingensis Depository Institution: China General Microbiological Culture Collection Center; Depository Address: No. 3, Courtyard 1, Beichen West Road, Chaoyang District, Beijing, China, Institute of Microbiology, Chinese Academy of Sciences; Deposit Date: November 27, 2024.

[0062] Aspergillus tubingensis EKYY3 Basic biological characteristics:

[0063] (1) Colony characteristics: Aspergillus tubingensis colonies on isolation media are usually concentric circles, with the outer ring composed of white vegetative hyphae, and the central part appearing dark brown due to the color of the conidia. The texture is velvety, and the back is pale yellow. As the incubation time increases, the colonies can cover the entire plate and produce conidia, making the entire plate dark brown. In addition, there will be a light brown exudate on the surface of the colonies.

[0064] (2) Microstructure: The peduncle is approximately 1000-1600 μm long and 15-20 μm in diameter. The conidiophores are spherical, with a diameter of 60-150 μm. The conidia are nearly spherical, with a diameter of 3 μm, and concave at both ends, forming conidial chains by connecting the concave ends. The surface of the conidia is slightly rough, with longitudinal striations parallel to the direction of the conidial chains. These morphological characteristics are consistent with the relevant descriptions of Aspergillus tabineus. Under an optical microscope, hyphae (or some kind of filamentous structure) can be seen connecting the spherical conidiophores.

[0065] (3) Physiological and biochemical characteristics: Aspergillus tubingensis can hydrolyze starch, oils and gelatin, showing its diversity in the utilization of carbon and nitrogen sources. Aspergillus tubingensis has the ability to produce citric acid, can decompose urea to release ammonia, and can decompose hydrogen peroxide.

[0066] (4) Effect of pH on optimal growth conditions: Aspergillus tubingensis showed a certain tolerance to Pb2+, and could still grow well under the condition of Pb2+ concentration of 4 mmol / L, and had a strong ability to fix Pb2+.

[0067] (5) Fermentation and secondary metabolites

[0068] During solid-state and liquid-state fermentation, *Aspergillus tubingensis* produces kaempferol (3.791 mg / mL), a secondary metabolite of *Epimedium kansui*. Other metabolites include 1-aspergillus A, 2-aspergillus B, 3-aspergillus C, 4-icariin, and 5-kaempferol. Figure 1 As shown.

[0069] 2. The present invention further conducts field trials on the Epimedium growth-promoting agent prepared in Example 1 and various strains, including the following procedures:

[0070] Prepare equal volumes of inoculant and distilled water. In spring, once the Epimedium plants have grown two true leaves, select plants with uniform growth and dilute the inoculant to a concentration of 1×10⁻⁶. 6 CFU / mL Epimedium growth promoter (compound agent), three strains ( Aspergillus tubingensis EKYY3 Colletotrichum truncatum S5 and Clonostachys rosea RS5) was prepared using the same method as in Example 1, with a single-strain inoculant and sterile water (CK) applied as a root drenching once each. Six groups of Epimedium plants were selected for the experiment (Experimental groups 1-3: compound inoculant group, Experimental group 4: EKYY3 group, Experimental group 5: S5, Experimental group 6: RS6 group), with each group replicated three times. The growth of the Epimedium plants was observed every 20 days (e.g., Figures 2-6 The growth rate and effects of each treatment were statistically analyzed. The results are shown in Tables 2 and 3.

[0071] Table 2. Changes in the yield and quality indicators of Epimedium in the 6 experimental groups - Part 1

[0072]

[0073] Table 3. Changes in the yield and quality indicators of Epimedium in the six experimental groups - Part Two

[0074]

[0075] The results above show that all three strains have a certain ability to promote the growth of Epimedium, but the Epimedium growth-promoting agent (a compound agent of the three strains) has the best effect.

[0076] Example 3

[0077] In this invention, the Epimedium growth-promoting bacterial agent prepared in Example 1 was added to a carrier of a solid bacterial agent (15g carrier peat moss, 1.5g sodium hydroxymethyl cellulose, 1.5g sodium alginate), thoroughly mixed and stirred, and then freeze-dried in a vacuum freeze dryer to obtain the Epimedium growth-promoting solid bacterial agent. The stability of the bacterial strain was tested after freeze-drying, and the results are shown in […]. Figure 7 All three strains were able to grow stably under the culture of this inoculant.

[0078] This invention further investigated the method for developing microbial agents in the Key Laboratory of Changchun University of Traditional Chinese Medicine, Jilin Province, and conducted comparative experiments on the addition of microbial agents in the Changchun Agricultural Expo Park, Jilin Province. The process included the following steps: After the Epimedium plant had grown two true leaves, plants with uniform growth were selected, and 1×10⁻⁶ microbial agents were added to each plant. 6 The roots were drenched once with a CFU / g solid inoculum agent for promoting the growth of Epimedium (prepared in Example 2) and sterile water (CK), with each group being replicated three times. The growth of Epimedium plants was observed every 20 days, and the growth rate and effect of each treatment were statistically analyzed. The results are as follows.

[0079] Table 4. Changes in the yield and quality indicators of Epimedium in the six experimental groups - Part Two

[0080]

[0081] Table 5. Changes in the yield and quality indicators of Epimedium in the six experimental groups - Part Two

[0082]

[0083] As can be seen from the above results, the composite microbial agent provided in Example 1 of this application, after being prepared into a solid agent, can also achieve superior technical effects in promoting the growth of Epimedium and the accumulation of medicinal components. A picture of the finished Epimedium growth-promoting agent is shown below. Figure 8 As shown.

[0084] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention, and not to limit them; although the present invention has been described in detail with reference to the foregoing embodiments, those skilled in the art should understand that modifications can still be made to the technical solutions described in the foregoing embodiments, or equivalent substitutions can be made to some of the technical features; and these modifications or substitutions do not cause the essence of the corresponding technical solutions to deviate from the spirit and scope of the technical solutions of the embodiments of the present invention.

Claims

1. A type of Aspergillus tubingensis EKYY3, characterized in that, The preservation number of Aspergillus tabingensis EKYY3 is CGMCC No.41722.

2. A microbial agent, characterized in that, Includes Aspergillus tabineus EKYY3 as described in claim 1.

3. The microbial agent according to claim 2, characterized in that, Also includes: The carriers are peat moss, sodium hydroxymethyl cellulose, and sodium alginate.

4. The application of Aspergillus tabineum EKYY3 as described in claim 1 in improving the yield or quality of Epimedium.

5. The application according to claim 4, characterized in that, The applications include: Increase the dry weight, fresh weight, leaf area, PAL level, FNS level, F3H level, and one or more of icariin A, icariin B, icariin C, or icariin in Epimedium.

6. The application according to claim 4 or 5, characterized in that, The epimedium mentioned is the Korean epimedium.

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