Endophytic streptomyces coelicolor from gynostemma pentaphyllum and application thereof

Abstract

The application discloses a gentiana rigescens endophytic Streptomyces coelicoflavus and application thereof, and belongs to the technical field of microorganisms. The application relates to the gentiana rigescens endophytic Streptomyces coelicoflavus, which is preserved in the China General Microbiological Culture Collection Center on September 23, 2022, and the strain preservation name is MEP0301, and the preservation number is CGMCC No. 25778. The strain can significantly promote the increase of the total flavone content and the total triterpene content in the gentiana rigescens, and can be artificially cultured, is genetically stable, and is easy to be produced on a large scale. The strain can be used for artificial planting of the gentiana rigescens, can effectively improve the quality of medicinal materials, and has a good application prospect.

Description

Technical Field

[0001] This invention belongs to the field of microbial technology, specifically relating to an endophytic Streptomyces cerevisiae of Sedum aizoon and its applications. Background Technology

[0002] Sedum aizoon L., a plant belonging to the genus Sedum in the family Crassulaceae, is also known as "Heart-Nourishing Grass" or "Earthly Ginseng." It is a traditional Chinese medicinal and edible plant, harvested year-round, with the whole plant or root used for both medicinal and culinary purposes. Sedum aizoon is neutral in nature, sweet and slightly sour in taste, and enters the heart and liver meridians. It possesses effects such as dispersing blood stasis and relieving pain, cooling the blood and stopping bleeding, reducing inflammation, and calming the mind. It contains various effective active ingredients, including flavonoids, triterpenoids, and polysaccharides. Flavonoids are an important and diverse natural active ingredient in Sedum aizoon, often used as a quality control indicator for medicinal materials. Flavonoids have clear and broad pharmacological effects, such as antioxidant, anti-gastric ulcer, neuroprotective, anti-myocardial ischemia, antihypertensive, anti-inflammatory, antibacterial, antiviral, and antitumor activities. In recent years, due to its ease of cultivation and strong adaptability, Sedum aizoon has been widely planted and distributed in Northeast, Northwest, North my country, and the Yangtze River basin. Due to its high nutritional value, Sedum aizoon has been developed into a new type of health vegetable. With the improvement of people's living standards and the increasing awareness of health preservation, the demand for Sedum aizoon in pharmaceuticals, health foods, and other fields is constantly expanding. Artificial cultivation is an effective way to meet this increasing demand. However, current artificial cultivation of Sedum aizoon suffers from the overuse of chemical fertilizers and pesticides, leading to a shortened growth period and deteriorated quality. Therefore, finding ways to rationally reduce the use of chemical fertilizers and discover green and ecological methods to promote the growth of Sedum aizoon and improve its effective active ingredients is of great significance for ensuring the quality of Sedum aizoon medicinal materials.

[0003] Plant endophytes are a group of microorganisms that live within healthy plant tissues or organs at some stage or throughout their life cycle, without causing obvious disease symptoms in the host plant. As an important microbial resource, plant endophytes play a significant role in promoting plant growth and development, the accumulation of active ingredients, and enhancing stress resistance. The rhizosphere of Sedum aizoon harbors a large number of microorganisms closely related to its growth and development, influencing its nutrient absorption, immune regulation, and disease resistance. Therefore, applying beneficial endophytes with quality-enhancing effects to improve the rhizosphere microecological environment of Sedum aizoon and enhance its medicinal quality has enormous application potential in ensuring the efficacy and safety of its medicinal materials.

[0004] Currently, the artificial cultivation of Sedum aizoon mainly relies on traditional chemical fertilizers to promote its growth. On the one hand, the continuous use of chemical fertilizers will have a lasting impact on the soil condition, and on the surrounding environment and ecology. There is currently no record of using microbial strains to increase yield and shorten the growth cycle in the artificial cultivation of Sedum aizoon. Summary of the Invention

[0005] The purpose of this invention is to address the shortcomings of existing technologies by providing an endophytic Streptomyces cerevisiae of Sedum aizoon and its application, which can effectively increase yield, shorten the growth cycle, reduce or eliminate the use of chemical fertilizers, improve the growth quality of Sedum aizoon, and effectively promote the accumulation of total flavonoids and total triterpenes in Sedum aizoon.

[0006] To achieve the above objectives, this invention provides an endophytic Streptomyces coelicoflavus strain from Panax notoginseng. The strain is classified as *Streptomyces coelicoflavus*, deposited at the China General Microbiological Culture Collection Center (CGMCC), located at No. 3, Courtyard 1, Beichen West Road, Chaoyang District, Beijing, Institute of Microbiology, Chinese Academy of Sciences. The strain's deposit name is MEP0301, its accession number is CGMCCNo. 25778, and its deposit date is September 23, 2022.

[0007] Furthermore, its application promotes the increase of total flavonoids and total triterpenoids in Panax notoginseng.

[0008] A method for preparing an endophytic Streptomyces coelicoflavus inoculum of Sedum aizoon, comprising treating the Streptomyces coelicoflavus as follows:

[0009] The strain was inoculated into fresh Gao's No. 1 liquid medium and cultured in a shaker at 28°C and 200 rpm / min for 5 days to activate the strain.

[0010] The activated bacterial culture was transferred to Gao's No. 1 liquid medium at a ratio of 1% and cultured at 28°C and 200 rpm / min for 5 days.

[0011] Collect bacterial cells by centrifugation, wash the collected bacterial cells twice with sterile water, centrifuge again to collect bacterial cells, resuspend the bacterial cells in sterile water, and adjust the bacterial concentration to OD0.05. 600 =0.5.

[0012] Furthermore, the roots of Sedum aizoon plants were treated with bacterial solution, with 1 mL applied to each seedling, thereby promoting the increase of total flavonoids and total triterpenoids in Sedum aizoon.

[0013] Furthermore, the microbial agent is a microbial fertilizer.

[0014] A composition comprising Streptomyces coelicoflavus, an endophytic fungus of Sedum aizoon.

[0015] The strain MEP0301 of this invention was isolated from the roots of wild Sedum aizoon collected in Peixian County, Xuzhou, using endophytic bacteria isolation and purification technology. Molecular identification confirmed that it is *Streptomyces coelicoflavus*, and it was designated MEP0301. The specific isolation and purification method is as follows:

[0016] 1) Wild Sedum sarmentosum roots collected from Peixian County, Xuzhou, were used as the mycelial material. 1g of root material was weighed and rinsed with running water for 20 minutes to remove dust and sand. The rinsed sample was then ultrasonically cleaned for 30 minutes to further remove residual dust and sand from the root surface. Afterwards, in a clean bench, the rinsed sample was first soaked in 75% ethanol for 90 seconds, rinsed once with sterile water, then soaked in 2% sodium hypochlorite for 8 minutes, and rinsed three times with sterile water. The surface-sterilized sample was placed in a mortar, ground evenly with 9mL of sterile water, and the grinding solution was serially diluted with sterile water (10...). -1 10 -2 Then, the grinding stock solution and diluent (10) -1 10 -2 Spread them separately on Gao's No. 1 solid medium and incubate at 28℃ for 5-7 days until single colonies grow;

[0017] 2) Select a single colony and streak it onto the same Gao's No. 1 plate for purification, and incubate at 28℃ for 5-7 days;

[0018] 3) Repeat step 2) and select single bacteria until a pure culture is obtained.

[0019] The culture medium used in this invention includes:

[0020] Gao's No. 1 liquid culture medium: KNO3 1g; K2HPO4 0.5g; MgSO4·7H2O 0.5g; NaCl 0.5g; FeSO4·7H2O 0.01g; soluble starch 20g; distilled water 1000mL, pH=7.4-7.6.

[0021] Gao's No. 1 solid culture medium: KNO3 1g; K2HPO4 0.5g; MgSO4·7H2O 0.5g; NaCl 0.5g; FeSO4·7H2O 0.01g; soluble starch 20g; agar 20g; distilled water 1000mL, pH=7.4-7.6.

[0022] All of the above culture media are conventional culture media and require routine sterilization before use.

[0023] Beneficial effects: Compared with the prior art, the advantages of the present invention are:

[0024] (1) The Streptomyces coelicoflavus MEP0301 isolated by this invention can significantly increase the content of total flavonoids and total triterpenes in Panax notoginseng. This strain can be used as a microbial fertilizer for the artificial cultivation of Panax notoginseng, improving the quality of the medicinal material, ensuring the safety and effectiveness of the medicinal material, and at the same time, it can also alleviate the environmental pollution caused by the overuse of chemical fertilizers to a certain extent.

[0025] (2) The culture process of Streptomyces coelicoflavus MEP0301 of the present invention is simple, genetically stable, and easy to industrialize, and has good development and application prospects.

[0026] This invention, from the perspective of plant endophytic bacteria, utilizes the endophytic bacterium MEP0301 of Sedum sarmentosum to effectively increase the content of secondary metabolites of Sedum sarmentosum. The strain provided by this invention can be used as a microbial fertilizer to replace or reduce the use of traditional chemical fertilizers and other fertilizers for artificially cultivated Sedum sarmentosum, thereby improving the quality of artificially cultivated Sedum sarmentosum medicinal materials and reducing the environmental pollution problems caused by the overuse of chemical fertilizers. Attached Figure Description

[0027] Figure 1 The image shows the morphology of strain MEP0301 described in this invention on Gao's No. 1 solid medium. A is a front view of the plate, and B is a back view of the plate.

[0028] Figure 2 This is a comparative diagram showing the effect of strain MEP0301 described in this invention on the accumulation of total flavonoids in Panax notoginseng.

[0029] Figure 3 The effect of strain MEP0301 described in this invention on the accumulation of total triterpenes in Panax notoginseng. Detailed Implementation

[0030] The present invention will be further described below with reference to specific embodiments to help to better understand the present invention, but the scope of protection of the present invention is not limited thereto:

[0031] Example 1: Isolation, purification and identification of strain MEP0301

[0032] (1) The root tissue of wild Sedum sarmentosum collected from Peixian County, Xuzhou was used as the material for isolating endophytic bacteria. 1g of root tissue was weighed and rinsed with running water for 20min to remove dust and sand. Then the rinsed sample was placed in an ultrasonic cleaner for ultrasonic cleaning for 30min.

[0033] (2) The cleaned samples were surface disinfected in a clean bench. First, they were soaked in 75% ethanol solution for 90 seconds, rinsed once with sterile water, then soaked in 2% sodium hypochlorite solution for 8 minutes, and rinsed three times with sterile water. The sterile water after the third rinse was retained for subsequent reliability testing. The surface-disinfected samples were placed on sterile filter paper to absorb the moisture for later use.

[0034] (3) Place the surface-sterilized sample into a sterile mortar, add 9 mL of sterile water and grind evenly. Then, serially dilute the grinding solution with sterile water (10 mL / min). -1 10 -2 Then take the original grinding solution and the diluent (10). -1 10 -2 100 μL of each sample was spread onto Gao's No. 1 solid culture medium and incubated at 28°C for 5-7 days until a single colony grew. At the same time, the sterile water used for the last rinse in step (3) was subjected to routine sterility testing to ensure that the sample surface was thoroughly disinfected.

[0035] (4) Pick a single colony that grows on Gao's No. 1 plate and inoculate it into the same culture medium for streak purification and isolation. Incubate at 28℃ for 5-7 days. Continue to pick single colonies and repeat the isolation and purification culture multiple times until a pure culture is obtained.

[0036] (5) The single colonies obtained in step (4) above were subjected to 16S rRNA gene amplification (conventional PCR method) and sequencing. The sequencing results are shown in SEQ ID NO.1. The sequencing results were compared with the NCBI database (https: / / blast.ncbi.nlm.nih.gov / Blast.cgi). The comparison results showed that the similarity between strain MEP0301 and Streptomyces coelicoflavus strain NBRC 15399 was 99.86%. Combined with morphological observation, strain MEP0301 was identified as Streptomyces coelicoflavus.

[0037] The colony, morphological, and physiological and biochemical characteristics are as follows:

[0038] (1) Morphological characteristics of the strain

[0039] The strain grew well on Gao's No. 1 solid medium. The mycelium in the colony substrate was blue, while the aerial mycelium was white, with a dry surface and relatively neat edges (e.g., Figure 1 ).

[0040] (2) Physiological and biochemical characteristics of the strain

[0041] Strain MEP0301 can utilize glucose, hydrolyze starch, urea, xylose, and gelatin. It is negative for oxidase and VP reactions. It can solubilize phosphorus, degrade cellulose, produce indoleacetic acid, and produce protease. It grows in 12% NaCl but not in 14% NaCl. The optimal growth temperature is 28℃.

[0042] Example 2: Strain MEP0301 promotes the accumulation of total flavonoids in Panax notoginseng, such as... Figure 2 As shown:

[0043] (1) Activation of bacterial strains and preparation of bacterial solutions

[0044] Strain MEP0301 was inoculated into fresh Gao's No. 1 liquid medium and cultured at 28℃ and 200 rpm for 5 days to activate the strain. Then, the activated bacterial solution was transferred to Gao's No. 1 liquid medium at a ratio of 1% and cultured at 28℃ and 200 rpm for 5 days. The bacterial cells were collected by centrifugation, washed twice with sterile water, centrifuged again, and resuspended in sterile water. The bacterial concentration was adjusted to OD0.05. 600 =0.5 available.

[0045] (2) Treatment of Sedum aizoon plants with MEP0301 bacterial solution

[0046] The nutrient soil was sterilized by high temperature and high pressure. Sedum plants of uniform size, thickness and growth status were selected and planted in the sterilized nutrient soil. Three plants were planted in each pot, for a total of 15 pots. They were placed in a greenhouse for cultivation (12h / d light, 25℃). After 14 days of adaptation, the Sedum potted seedlings were treated with root irrigation. Each plant was irrigated with 1mL of the MEP0301 bacterial solution mentioned in (1) above. Sterile water was also irrigated into the plants in the same way as a control group. Seven Sedum plants were treated in each of the experimental and control groups. After 48h of inoculation with the bacterial solution, the same volume of sterile water was added to each group every 2 days. On the 7th day after inoculation with the bacterial solution, the roots of the plants in each group were removed to detect the components.

[0047] (3) Detection of total flavonoids in Panax notoginseng

[0048] The specific testing methods are as follows:

[0049] a. Take fresh root samples of Panax notoginseng, wash and cut them into small pieces, grind them thoroughly with liquid nitrogen, weigh 2g into a 50mL centrifuge tube, add 80% ethanol at a material-to-liquid ratio of 1:25 (g / mL), mix well and extract by ultrasonication at 35℃ for 15min.

[0050] b. Take an appropriate amount of the sonicated solution into a 50mL centrifuge tube, add 1mL of 5% NaNO2 solution and mix well. After standing for 6 minutes, add 1mL of 10% Al(NO3)3 solution and shake well. After standing for 6 minutes, add 8mL of 1mol / L NaOH, and then add deionized water to the mark. Mix well and take 0.3mL of the sample solution. OD 510 Detect the absorbance value.

[0051] c. Creating a standard curve:

[0052] Dry the rutin standard to constant weight. Weigh 0.012 g of the dried standard into a 50 mL volumetric flask and dilute to the mark with methanol. Mix well. Then, take 0, 0.5, 1.0, 2.0, 4.0, and 6.0 mL of the rutin standard solution into 25 mL volumetric flasks, add 6 mL of deionized water to each flask, and then perform the colorimetric experiment according to step b. OD 510 Detect the absorbance value.

[0053] With OD 510 With absorbance values ​​on the ordinate and standard concentrations on the x-axis, a standard curve was plotted, yielding the linear regression equation y = 0.0132x + 0.002, R0. 2 =0.9991, indicating a good linear relationship. Based on the standard curve, the total flavonoid content of the experimental and control groups was calculated, and the results are as follows: Figure 2 As shown, the total flavonoid content in the CK group was 16.78±0.68 mg / g, while the total flavonoid content in the experimental group was 29.57±0.68 mg / g. The total flavonoid content in the experimental group was approximately 1.8 times that of the CK group, indicating that strain MEP0301 can effectively promote the accumulation of total flavonoids in Panax notoginseng.

[0054] Example 3: Strain MEP0301 promotes the accumulation of total triterpenes in Panax notoginseng, such as... Figure 3 As shown:

[0055] The activation of the bacterial strain, preparation of the bacterial solution, and treatment of the Sedum aizoon plants with MEP0301 bacterial solution were all the same as in Example 2 (steps (1) and (2) in Example 2). The total triterpenoid content of Sedum aizoon was detected by the following methods:

[0056] a. Take fresh root samples of Panax notoginseng, wash and chop them, then grind them thoroughly with liquid nitrogen. Weigh 0.2g of the sample into a 50mL centrifuge tube, add 10mL of methanol and mix well. Extract by sonication at 35℃ for 2h, then let stand overnight.

[0057] b. Transfer 0.1 mL of the ultrasonically extracted solution to a 50 mL centrifuge tube, add 0.2 mL of vanillin solution and 0.8 mL of perchloric acid solution, shake well, and heat in a 70°C water bath for 15 min; after cooling to room temperature, transfer 0.2 mL from each group, add 5.0 mL of glacial acetic acid, and OD... 545Detect the absorbance value.

[0058] c. Creating a standard curve:

[0059] Weigh 0.02g of ursolic acid and add it to a 100mL volumetric flask. Dilute to the mark with methanol and mix well.

[0060] Vanillin-glacial acetic acid standard: Weigh 5g of vanillin powder into a 100mL volumetric flask, dilute to the mark with glacial acetic acid and mix well.

[0061] Take 0.1, 0.2, 0.4, 0.6, 0.8, 1.0, and 1.2 mL of ursolic acid standard solution, respectively, with 1.2 mL of methanol as the control group. Add 0.2 mL of vanillin-glacial acetic acid standard and 0.8 mL of perchloric acid solution to each solution, mix well, and heat in a 70°C water bath for 15 min. Cool to room temperature, then add 5.0 mL of glacial acetic acid. OD 545 Detect the absorbance value.

[0062] With OD 545 With absorbance values ​​on the ordinate and standard concentrations on the x-axis, a standard curve was plotted, yielding the linear regression equation y = 4.0649x + 0.0374, R0. 2 =0.9935, indicating a good linear relationship. Based on the standard curve, the total triterpenoid content in the experimental and control groups was calculated, and the results are as follows: Figure 3 As shown, the total triterpenoid content in the CK group was 21.9±0.78 mg / g, and the total triterpenoid content in the experimental group was 46.6±4.57 mg / g. The results indicate that strain MEP0301 can significantly increase the total triterpenoid content of Panax notoginseng.

[0063] In summary, the strain MEP0301 provided by this invention can effectively promote the accumulation of total flavonoids and total triterpenoids in Panax notoginseng, and can be used to improve the quality of artificially cultivated Panax notoginseng, which is of great significance for ensuring the effectiveness and safety of its medicinal use.

[0064] Finally, it should be noted that the above examples are merely some specific embodiments of the present invention. Obviously, the present invention is not limited to the above embodiments and many variations are possible. All variations that can be directly derived or conceived by those skilled in the art from the disclosure of the present invention should be considered within the scope of protection of the present invention.

Claims

1. An endophytic Streptomyces cyanobacterium in Sedum sarmentosum ( Streptomyces coelicoflavus ), characterized by: The classification of the endophytic Streptomyces cyanobacterium in Sedum sarmentosum is clearly named Streptomyces cyanobacterium (Streptomyces cyanobacterium). Streptomyces coelicoflavus The depositary institution is the China General Microbiological Culture Collection Center (CGMCC), located at No. 3, Courtyard 1, Beichen West Road, Chaoyang District, Beijing, Institute of Microbiology, Chinese Academy of Sciences. The strain name is MEP0301, the accession number is CGMCC No. 25778, and the deposit date is September 23, 2022.

2. The endophytic Streptomyces cyanobacterium described in claim 1 for Sedum aizoon ( Streptomyces coelicoflavus The application of ) is characterized by: Promotes the increase of total flavonoids and total triterpenoids in Panax notoginseng.

3. A method for preparing endophytic Streptomyces cerevisiae of Panax notoginseng ( Streptomyces coelicoflavus The method for using microbial agents is characterized by, The Streptomyces cyanidin described in claim 1 ( Streptomyces coelicoflavus Perform the following processing: The strain was inoculated into fresh Gao's No. 1 liquid medium and cultured at 28℃ and 200 rpm / min for 5 days to activate the strain. The activated bacterial culture was transferred to Gao's No. 1 liquid medium at a ratio of 1% and cultured at 28°C and 200 rpm / min for 5 days. Collect bacterial cells by centrifugation, wash the collected bacterial cells twice with sterile water, centrifuge again to collect bacterial cells, resuspend the bacterial cells in sterile water, and adjust the bacterial concentration to OD0.

05. 600 =0.

5.

4. The endophytic Streptomyces cyanobacterium described in claim 3 of *Sedum aizoon* (… Streptomyces coelicoflavus The application of microbial agents is characterized by: The fungal agent described in claim 3 was used to drench the roots of Sedum aizoon plants, with 1 mL of the agent per seedling, thereby promoting the increase of total flavonoids and total triterpenoids content in Sedum aizoon.

5. The endophytic Streptomyces cyanobacterium as described in claim 4 of *Sedum aizoon* (… Streptomyces coelicoflavus The application of microbial agents is characterized by: Gao's No. 1 liquid culture medium: KNO3 1g; K2HPO4 0.5g; MgSO4·7H2O 0.5g; NaCl 0.5g; FeSO4·7H2O 0.01g; soluble starch 20g; distilled water 1000mL, pH=7.4-7.

6.

6. The endophytic Streptomyces cyanobacterium as described in claim 4 of *Sedum aizoon* (… Streptomyces coelicoflavus The application of microbial agents is characterized by: Gao's No. 1 solid culture medium: KNO3 1g; K2HPO4 0.5g; MgSO4·7H2O 0.5g; NaCl 0.5g; FeSO4·7H2O 0.01g; soluble starch 20g; agar 20g; distilled water 1000mL, pH=7.4-7.

6.

7. The endophytic Streptomyces cyanobacterium as described in claim 4 of Sedum aizoon ( Streptomyces coelicoflavus The application of microbial agents is characterized by: The inoculant is a microbial fertilizer.

8. A composition comprising the *Streptomyces cyanobacterium* endophyte of *Sedum aizoon* as described in claim 1. Streptomyces coelicoflavus ).

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