Aspergillus tubingensis, microbial composition and application of aspergillus tubingensis in improvement of yield and quality of herba epimedii

Abstract

The invention relates to the technical field of microorganisms, in particular to aspergillus tubingensis, a microorganism composition and application of the aspergillus tubingensis to improvement of the yield and quality of herba epimedii. The aspergillus tubingensis is aspergillus tubingensis EKYY3. The microbial composition is prepared from the aspergillus tubingensis, Colletotrichum truncatum S5 (5) and Clonostachys rosea RS5 (5), and the microbial composition is prepared from the aspergillus tubingensis, Colletotrichum truncatum S5 and Clonostachys rosea RS5. The invention provides Aspergillus tubingensis EKYY3 capable of promoting the growth of epimedium and the accumulation of medicinal components, and also provides a complex microbial inoculant comprising the strain. When the compound microbial agent is applied in the growth process of the epimedium koreanum, the growth of the epimedium koreanum and the accumulation of medicinal components can be effectively promoted, the expression quantity of key enzymes for synthesizing the medicinal components such as PAL, FNS and F3H can be improved, the yield and quality of medicinal materials are improved, and the clinical curative effect is improved. The method has important significance in the field of high-yield, high-efficiency, green and safe production of traditional Chinese medicinal materials.

Description

Technical Field

[0001] The invention relates to the technical field of microorganisms, and in particular to Aspergillus tubingensis, a microbial combination and application thereof in improving the yield and quality of epimedium. Background Art

[0002] Korean Epimedium is a perennial herb and an important raw material for medicine and health food. It has great research and development potential and has attracted wide attention at home and abroad, so the demand for industrial development is huge. However, most of the medicinal materials come from wild plants. Due to its weak sexual reproduction ability, it is an endangered species. In addition, due to various improper and unreasonable development and utilization, the wild resources of Korean Epimedium are very scarce, the market price continues to rise, and the contradiction between supply and demand is becoming increasingly serious. In addition, studies have found that the quality of Korean Epimedium medicinal materials is unstable, the content of effective ingredients is low, and most of them do not meet the requirements. The existing quality control standards for Korean Epimedium require that the total flavonol glycoside content must not be less than 0.5%, which is lower than the standard limit of 1.5% for Epimedium, Wollose Epimedium, and Sagittaria. Therefore, improving the yield and quality of Korean Epimedium is a key issue that needs to be solved for the orderly development of the industry.

[0003] The vigorous growth and development of medicinal plants and the efficient accumulation of metabolites are important prerequisites for increasing the production and income of Chinese herbal medicines and improving their quality and efficiency. However, the large-scale application of pesticides and fertilizers in the production of Chinese herbal medicines today has threatened the growth and development of plants, reduced the quality of medicinal materials, polluted the ecological environment, and increased clinical risks. Therefore, how to improve the yield and quality of medicinal materials, repair the ecological environment, and ensure the safety, effectiveness, stability, and controllability of Chinese medicines has become a scientific problem that needs to be solved urgently in the Chinese medicine industry. Summary of the invention

[0004] In order to solve the problems existing in the prior art, the present invention provides an Aspergillus tubingensis, a microbial combination and application thereof in improving the yield and quality of Epimedium.

[0005] In a first aspect, the present invention provides a method of treating Aspergillus tubingensis ( Aspergillus tubingensis )EKYY3, and made a biological deposit with the following information: Deposit number: CGMCC No.41722; Classification name: Aspergillus tubingensis ; Depository: General Microbiology Center, China Culture Collection Administration; Deposit address: Institute of Microbiology, Chinese Academy of Sciences, No. 3, Yard 1, Beichen West Road, Chaoyang District, Beijing, China; Deposit date: November 27, 2024.

[0006] Based on the above Aspergillus tubingensis EKYY3, the present invention innovatively develops a composite bacterial agent for improving the yield and quality of Epimedium medicinal materials, which can form a good mutualistic symbiotic system with the host plant, and improve the yield and quality of the medicinal materials by promoting the growth and development of Epimedium and increasing the expression of key genes in the synthesis pathway of the active ingredients. This bacterial agent can replace inorganic fertilizers, Zhuanggenling, etc., and produce high-yield, high-quality, and efficient green and safe Chinese medicinal materials by improving soil structure, increasing soil fertility, and repairing the ecological environment.

[0007] In a second aspect, the present invention provides a microbial combination, comprising the aforementioned Aspergillus tubingensis EKYY3, and further comprising Colletotrichum truncatum S5 and Clonostachys rosea RS5.

[0008] in, Colletotrichum truncatum S5 and Clonostachys rosea RS5 was published in "Study on the Diversity and Function of Endophytic and Rhizosphere Microorganisms of Epimedium koreana".

[0009] Furthermore, Aspergillus tubingensis EKYY3, Colletotrichum truncatum S5 and Clonostachys pink The total live bacterial count ratio of RS5 is (4~6):(2~4):(3~5).

[0010] In a second aspect, the present invention provides a bacterial agent, comprising the aforementioned Aspergillus tubingensis EKYY3, or the aforementioned microbial combination.

[0011] Furthermore, in parts by weight, the bacterial agent preferably also includes: 10-20 parts of carrier peat, 0.5-1.5 parts of sodium hydroxymethyl cellulose and 0.5-1.5 parts of sodium alginate.

[0012] In a third aspect, the present invention provides a method for preparing the bacterial agent, comprising: fermenting and culturing the microbial combination and then inoculating it into a nutrient solution.

[0013] Furthermore, the culture medium used in the fermentation culture process includes, by weight, 30-70 parts of potatoes, 3-7 parts of glucose, 3-7 parts of agar, and 200-300 parts of water.

[0014] Furthermore, the initial microbial inoculation amount of the fermentation culture is 3-12% (mass percentage).

[0015] Furthermore, the fermentation culture conditions include: rotation speed 150-220 r / min, 250-500 mL, temperature 22-26° C., and pH 7-9.

[0016] After the fermentation culture, the total viable count of the microbial combination is 1×10 1~9cfu / mL, the inoculation amount is 3-12% by mass (inoculated into the nutrient solution); and / or, Calculated by weight, the nutrient solution includes: 5-10 parts of beef extract, 5-10 parts of sodium chloride, 5-10 parts of brown sugar, and 2-5 parts of soluble starch.

[0017] Furthermore, after being inoculated in the nutrient solution, the culture was carried out in a shaking incubator for more than 6 days, and the culture conditions were 150-250 r / min and 22-25°C.

[0018] Furthermore, it also includes: adding dextrin and vitamin C, and irradiating with ultraviolet light for 25 to 40 minutes.

[0019] The present invention utilizes vitamin C as an ultraviolet protective agent, which can absorb most of the ultraviolet radiation and maintain the activity and stability of living bacteria. Compared with traditional dextrin, vitamin C has the function of promoting iron absorption.

[0020] The microelement of the microbial agent provided by the present invention does not contain nitrogen, phosphorus and potassium elements, but utilizes the nitrogen-fixing and phosphorus-dissolving properties of the plant itself to promote the development of the root system and the luxuriance of the leaves.

[0021] In a fourth aspect, the present invention provides the use of the aforementioned Aspergillus tubingensis EKYY3, or the aforementioned microbial combination, or the aforementioned bacterial agent in improving the yield or quality of Epimedium.

[0022] Furthermore, the application includes: The dry weight, fresh weight, leaf area, PAL level, FNS level, F3H level, epimedin A, epimedin B, epimedin C or icariin of Epimedium are increased.

[0023] Furthermore, the epimedium is epimedium koreana.

[0024] The present invention has the following beneficial effects: 1. The strains used in the present invention are different from the strains used in existing commercial bacterial fertilizers on the market. They are all isolated from the Korean Epimedium plant and are endophytes of the host plant. They can coexist with the host plant and are harmless to the plant itself. The provided Aspergillus tubingensis EKYY3 has the ability to promote the growth of Epimedium and the accumulation of medicinal ingredients.

[0025] 2. The present invention further provides a microbial combination comprising three microorganisms, which can form a good symbiotic system with Epimedium, thereby improving the yield and quality of the medicinal material by promoting the growth and development of Epimedium and increasing the expression of key genes in the synthesis pathway of medicinal ingredients.

[0026] 3. The composite bacterial agent provided by the present invention can not only improve the yield indicators of Epimedium plants, such as dry weight, fresh weight, and leaf area, but also increase the expression of key enzymes for the synthesis of effective ingredients such as PAL, FNS, and F3H in plants, promote the accumulation of effective ingredients such as icariin, improve the quality of medicinal materials, and increase clinical efficacy, which are functions that existing bacterial fertilizers on the market do not have.

[0027] 4. The process of the present invention is simple and low in cost. The formula can also be prepared into a solid bacterial agent. Without changing the formula composition, the survival rate of the strain can reach more than 80% after adding an appropriate amount of carrier and vacuum drying at -80°C. BRIEF DESCRIPTION OF THE DRAWINGS

[0028] In order to more clearly illustrate the technical solutions in the present invention or the prior art, the following briefly introduces the drawings required for use in the embodiments or the description of the prior art. Obviously, the drawings described below are 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.

[0029] Figure 1 This is a flavonol glycoside composition diagram of EKYY3 and S5 provided in Example 1 of the present invention.

[0030] Figure 2 This is a photo of the blank group of Epimedium provided in Example 2 of the present invention.

[0031] Figure 3 This is a photo of Epimedium in the EKYY3 experimental group provided in Example 2 of the present invention.

[0032] Figure 4 This is a photo of the Epimedium of the RS5 experimental group provided in Example 2 of the present invention.

[0033] Figure 5 This is a photo of Epimedium in the S5 experimental group provided in Example 2 of the present invention.

[0034] Figure 6 This is a photo of the epimedium in the epimedium growth-promoting bacteria agent group provided in Example 2 of the present invention.

[0035] Figure 7 This is a stability test chart of the composite bacterial agent provided in Example 3 of the present invention.

[0036] Figure 8 This is a picture of the finished product of the epimedium growth-promoting bacteria agent provided in Example 3 of the present invention. DETAILED DESCRIPTION

[0037] In order to make the purpose, technical solution and advantages of the present invention clearer, the technical solution of the present invention will be clearly and completely described below in conjunction with the drawings of the present invention. Obviously, the described embodiments are part of the embodiments of the present invention, not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without creative work are within the scope of protection of the present invention.

[0038] The experimental methods involved in the following examples, unless otherwise mentioned, are all conventional methods in the art, for example, reference may be made to experimental manuals in the art, or the conditions recommended in the manufacturer's instructions.

[0039] Unless otherwise specified, the experimental materials and reagents involved in the following examples can be obtained from commercial sources.

[0040] The epimedium involved in the following embodiments is Epimedium koreana.

[0041] Example 1 The present invention provides a composite microbial agent and a preparation method thereof, comprising the following process: 1. Preparation of mother liquor: Aspergillus tubingensis ( Aspergillus tubingensis )EKYY3, Colletotrichum truncated S5 and Clonostachys rosea After activation, the three strains RS5 were fermented in the fermentation medium until the spore count accounted for 80% of the total volume, and then the fermentation was stopped. The centrifugation was carried out at room temperature at 5000 r / min for 10 min, and the viable bacteria counts reached 2.5×10 7 CFU / mL, 1.5×10 7 CFU / mL, 2×10 7 CFU / mL; PDB liquid medium was used for strain activation and fermentation: 50g potato, 5g glucose, 5g agar, 250ml distilled water, sterilized at 121℃ for 20min. Fermentation conditions were: liquid volume 50mL / 250mL, inoculation volume 6%, rotation speed 180 r / min, temperature 30℃, pH=9.

[0042] 2. Prepare nutrient solution: accurately weigh 5g beef extract, 5g sodium chloride, 7g brown sugar, and 2.5g soluble starch, mix in a beaker, add 250mL distilled water, adjust pH=9, sterilize at 121℃101kpa for 20min, and let cool. Add 7g trace element ferrous sulfate and magnesium sulfate solution, stir with a glass rod while pouring, until fully mixed without lumps, and put in a 4℃ refrigerator for later use.

[0043] 3. Prepare a mixed bacterial agent, add the mother solution of step 1 to the nutrient solution of step 2 at a 6% inoculum amount. Place in a shaker, 180 r / min, 24°C, and culture for 7 days.

[0044] 4. Add 5-10g of UV protective agent dextrin and vitamin C to the above mixed preparation, place it near the ultraviolet lamp of the clean bench for 30 minutes, and obtain the epimedium growth-promoting agent.

[0045] 5. Application method of epimedium growth-promoting agent, the total viable count of the epimedium growth-promoting agent obtained in step 4 is 2×10 8 cfu / mL, dilute with water to a total viable count of 1×10 6 cfu / mL, apply 6L per mu, and use root irrigation system for root irrigation.

[0046] The culture medium for counting bacterial species of the present invention comprises: 10 g of peptone, 3 g of beef extract, 5 g of sodium chloride, 14 g of agar, 1000 mL of distilled water, pH=7.0±0.1, and sterilization at 121°C for 20 min.

[0047] Example 2 1. The present invention further relates to Aspergillus tubingensis EKYY3, Colletotrichum truncatum S5 and Clonostachys rosea The three strains, RS5, were tested by plate growth promotion experiment, with nitrogen fixation, phosphate solubilization, siderophore production, and indoleacetic acid production as observation indicators, including the following methods: The five purified strains were inoculated into Pikovaskaia's, CAS, and Ashby medium, respectively, and cultured at 28°C for 3 days to observe whether the colonies grew. Weigh 20.0 mg of IAA standard and dilute it with methanol to make standard solutions containing 2 mg / L, 10 mg / L, 20 mg / L, 40 mg / L, and 50 mg / L of IAA. Take 1 mL of each solution and mix it with the colorimetric solution in a 1:1 ratio, keep it away from light for 30 minutes, measure the OD530 value, and draw a standard curve.

[0048] The results are shown in the following table: Table 1 Determination of growth-promoting ability of three strains

[0049] The present invention is based on Aspergillus tubingensis ( Aspergillus tubingensis)The genomic DNA of EKYY3 was used as a template, and the fungal universal primers ITS1 and ITS4 were used for PCR amplification and sent to Sangon Sequencing Company. The obtained sequences were compared in the NCBI database, and the phylogenetic tree of EKYY3 and closely related bacteria was constructed using MEGA 7.0.21 software. Results The sequence of EKYY3 had the highest homology with that of Aspergillus tubingensis, reaching 100%. According to the Manual of Identification of Common Fungi, the above physiological and biochemical characteristics of the strain EKYY3 are the same as those of the genus Aspergillus tubingensis. Combined with the above molecular identification, it was identified as Aspergillus tubingensis. EKYY3 was named Aspergillus tubingensis EKYY3.

[0050] The present invention has made a biological deposit for EKYY3, and the deposit information is as follows: deposit number: CGMCC No.41722; classification name: Aspergillus tubingensis ; Depository: General Microbiology Center, China Culture Collection Administration; Deposit address: Institute of Microbiology, Chinese Academy of Sciences, No. 3, Yard 1, Beichen West Road, Chaoyang District, Beijing, China; Deposit date: November 27, 2024.

[0051] Aspergillus tubingensis EKYY3 Basic biological characteristics: (1) Colony characteristics: Aspergillus tubingensis colonies usually appear in concentric circles on the isolation medium. The outer ring is composed of white vegetative hyphae, and the central part appears dark brown due to the color of the conidia. The texture is velvety and the back is light yellow. As the culture time increases, the colonies can cover the entire plate and all produce conidia, making the entire plate appear dark brown. In addition, there will be light brown exudate on the surface of the colonies.

[0052] (2) Microstructure: The spore stalk is about 1000-1600 μm long and 15-20 μm in diameter. The conidial head is spherical and 60-150 μm in diameter. The conidia are nearly spherical, 3 μm in diameter, with concave ends at both ends, connected to each other at the concave parts to form a conidial chain. The conidial surface is slightly rough, with longitudinal stripes parallel to the direction of the conidial chain. These morphological characteristics are consistent with the relevant description of Aspergillus tubingensis. Observation under an optical microscope shows that the spherical conidial heads are connected by hyphae (or some kind of filamentous structure).

[0053] (3) Physiological and biochemical characteristics: Aspergillus tubingensis can hydrolyze starch, oil, and gelatin, showing its diversity in carbon and nitrogen source utilization. Aspergillus tubingensis has the ability to produce citric acid, decompose urea, release ammonia, and decompose hydrogen peroxide.

[0054] (4) Effect of pH on optimal growth conditions: Aspergillus tubingensis showed a certain tolerance to Pb2+. It could still grow well under the condition of Pb2+ concentration of 4 mmol / L and had a strong ability to fix Pb2+.

[0055] (5) Fermentation and secondary metabolites Aspergillus tubingensis produced 3.791 mg / mL of kaempferol, a secondary metabolite of Epimedium koreanum, during solid and liquid fermentation. 1-Icariin A, 2-Icariin B, 3-Icariin C, 4-icariin, 5-kaempferol, such as Figure 1 shown.

[0056] 2. The present invention further conducts field tests on the epimedium growth-promoting agent prepared in Example 1 and various strains, including the following process: Prepare the same volume of bacterial agent and distilled water. In spring, when the epimedium grows two true leaves, select plants with the same growth trend and dilute them to 1×10 6 cfu / mL of Epimedium growth-promoting agent (compound agent), three strains ( Aspergillus from Tubingen EKYY3, Colletotrichum truncatum S5 and Clonostachys rosea RS5) The bacterial agent (single bacterial agent) prepared in the same manner as in Example 1 and sterile water (CK) were respectively drenched once on the roots, and 6 groups of Epimedium plants were selected for the experiment (experimental groups 1-3: composite bacterial agent group, experimental group 4: EKYY3 group, experimental group 5: S5, experimental group 6: RS6 group), with three replicates for each group. The growth of the Epimedium plants was observed every 20 days (such as Figure 2-Figure 6 ), the growth rate and effect of each treatment were counted. The results are shown in Tables 2 and 3.

[0057] Table 2 Changes in yield and quality indicators of Epimedium in 6 experimental groups - Part I

[0058] Table 3 Changes in yield and quality indicators of Epimedium in 6 experimental groups - Part II

[0059] It can be seen from the above results that all three strains have a certain ability to promote the growth of Epimedium, but the Epimedium growth-promoting agent (a composite agent of three strains) has the best effect.

[0060] Example 3 The present invention further adds the epimedium growth-promoting bacterial agent prepared in Example 1 to the carrier of the solid bacterial agent (15g of carrier peat, 1.5g of sodium hydroxymethyl cellulose, and 1.5g of sodium alginate), fully mixes and stirs evenly, and freeze-dries in a vacuum freeze dryer to obtain an epimedium growth-promoting solid bacterial agent. After freeze-drying, the stability of the strain is tested, and the results are shown in FIG. Figure 7 All three strains can grow stably under the culture of this bacterial agent.

[0061] The present invention further conducted a research on the preparation method of the microbial agent in the key laboratory of Changchun University of Traditional Chinese Medicine in Jilin Province, and conducted a comparative test of adding microbial agents in the Agricultural Expo Park of Changchun City, Jilin Province, including the following process: wait for the epimedium to grow two true leaves, select plants with the same growth trend, and use 1×10 6 The roots were sprinkled with a solid bacterial agent for promoting growth of Epimedium (prepared in Example 2) with cfu / g and sterile water (CK) once, and each group was repeated three times. The growth of the Epimedium plants was observed after every 20 days, and the growth rate and effect of each treatment were statistically analyzed. The results are as follows.

[0062] Table 4 Changes in yield and quality indicators of Epimedium in the six experimental groups - Part II

[0063] Table 5 Changes in yield and quality indicators of Epimedium in the six experimental groups - Part II

[0064] As can be seen from the above results, the composite microbial agent provided in Example 1 of the present application can also achieve a better technical effect of promoting the growth of epimedium and the accumulation of medicinal ingredients after being prepared into a solid agent. Figure 8 shown.

[0065] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention, rather than to limit it. Although the present invention has been described in detail with reference to the aforementioned embodiments, those skilled in the art should understand that they can still modify the technical solutions described in the aforementioned embodiments, or make equivalent replacements for some of the technical features therein. However, these modifications or replacements do not deviate the essence of the corresponding technical solutions from the spirit and scope of the technical solutions of the embodiments of the present invention.

Claims

1. A tubingensis Aspergillus tubingensis ) EKYY3, characterized in that The deposit number of the Aspergillus tubingensis EKYY3 is CGMCC No.41722.

2. A microbial combination, characterized in that: The method comprises the Aspergillus tubingensis EKYY3 according to claim 1, and further comprises Colletotrichum truncatum S5 and Clonostachys rosea RS5.

3. The microbial combination according to claim 2, characterized in that: Aspergillus tubingensis EKYY3, Colletotrichum truncatum S5 and Clonostachys rosea The total live bacterial count ratio of RS5 is (4~6):(2~4):(3~5).

4. A bacterial agent, characterized in that It comprises the Aspergillus tubingensis EKYY3 described in claim 1, or the microbial combination described in claim 2 or 3.

5. The bacterial agent according to claim 4, characterized in that Also includes: Carriers: peat, sodium hydroxymethyl cellulose and sodium alginate.

6. The method for preparing the bacterial agent according to claim 4 or 5, characterized in that: include: The microbial combination according to claim 2 or 3 is fermented and then inoculated into a nutrient solution.

7. The preparation method according to claim 6, characterized in that: The culture medium used in the fermentation culture process comprises, by weight, 30-70 parts of potato, 3-7 parts of glucose, 3-7 parts of agar and 200-300 parts of water; and / or, After the fermentation culture, the total viable count of the microbial combination is 1×10 7~9 cfu / mL, the inoculum concentration is 3-12% by mass; and / or, Calculated by weight, the nutrient solution includes: 5-10 parts of beef extract, 5-10 parts of sodium chloride, 5-10 parts of brown sugar, and 2-5 parts of soluble starch.

8. Use of the Aspergillus tubingensis EKYY3 according to claim 1, or the microbial combination according to claim 2 or 3, or the bacterial agent according to claim 4 or 5 in improving the yield or quality of Epimedium.

9. The use according to claim 8, characterized in that: The applications include: The dry weight, fresh weight, leaf area, PAL level, FNS level, F3H level, epimedin A, epimedin B, epimedin C or icariin of Epimedium are increased.

10. The use according to claim 8 or 9, characterized in that: The epimedium is epimedium koreana.

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