A strain of Heterophytum rhizogenes for improving the growth and effective component content of Salvia miltiorrhiza and its application

Abstract

The present invention discloses a root heterostem mold and its application for improving the growth and effective ingredient content of salvia miltiorrhiza, belonging to the field of microbial technology. The strain is deposited in the China Center for Type Culture Collection on May 31, 2024, and the deposit number is CCTCC NO: M 20241118. The strain obtained by the present invention is root heterostem mold (Paraphoma radicina), which is isolated from the root system of Ammopiptanthus mongolicus, and exhibits excellent ability to dissolve phosphorus, potassium, and secrete indoleacetic acid (IAA). It can effectively promote the growth of salvia miltiorrhiza plants, increase the aboveground biomass and underground biomass of salvia miltiorrhiza plants, and effectively improve the content of effective ingredients in salvia miltiorrhiza, increase the content of tanshinone I, tanshinone II A, cryptotanshinone and salvianolic acid B, and has important application value in the quality improvement cultivation of salvia miltiorrhiza.

Description

Technical Field

[0001] The invention relates to the technical field of microorganisms, and in particular to a root heterophyll sp. for improving the growth of salvia miltiorrhiza and the content of effective components and an application thereof. Background Art

[0002] Salvia miltiorrhiza Bunge, also known as red root and blood ginseng root, belongs to the genus Salvia of the family Lamiaceae. It is a commonly used traditional Chinese medicine in my country. Its dried roots and rhizomes are used as medicine. The active ingredients of Salvia miltiorrhiza have the effects of removing blood stasis and relieving pain, promoting blood circulation and menstruation, and clearing the heart and relieving troubles. It is clinically used to treat coronary heart disease, angina pectoris, peptic ulcer, cirrhosis and other diseases. The main active ingredients of Salvia miltiorrhiza are water-soluble phenolic acids and fat-soluble tanshinone compounds. These two types of compounds are the main pharmacological basis for the pharmacological effects of Salvia miltiorrhiza. Tanshinones mainly include tanshinone I, tanshinone II A, and cryptotanshinone; phenolic acids mainly include rosmarinic acid and tanshinone B.

[0003] At present, due to the scarcity of wild resources, the harsh wild cultivation environment and the demand for standardization of medicinal materials in the modern Chinese medicinal materials industry, the salvia miltiorrhiza in the market is mainly cultivated artificially; however, its distribution is not concentrated, the resource renewal is long, and the quality varies. The content fluctuation of the main active ingredient tanshinone is particularly obvious, which is the main reason for the unqualified quality of salvia miltiorrhiza. On the other hand, the chemical synthesis of tanshinone and tanshinolic acid components is difficult to achieve industrialization. Therefore, improving the growth of salvia miltiorrhiza and the accumulation of its medicinal ingredients, especially the content of tanshinone compounds, is a major problem that needs to be solved urgently. Summary of the invention

[0004] In order to solve the above technical problems, the purpose of the present invention is to provide a root-heteromorpha that improves the growth of Salvia miltiorrhiza and the content of effective ingredients and its application, so as to solve the problems of poor quality of existing artificial cultivation of Salvia miltiorrhiza, low content of main medicinal ingredients, and unqualified quality of Salvia miltiorrhiza.

[0005] The technical solution of the present invention to solve the above technical problems is as follows:

[0006] A Paraphoma radicina strain that improves the growth and active ingredient content of Salvia miltiorrhiza, the strain is named AM-PR, and is deposited in the China Center for Type Culture Collection on May 31, 2024. The deposit number is CCTCC NO: M 20241118. The depository address is Wuhan University, Wuhan, China.

[0007] The beneficial effects of the present invention are as follows: Dark septate endophytic fungi (DSE) are a class of ascomycetes that are widely colonized in the intracellular and intercellular spaces of plant root cells in various ecosystems, and are characterized by typical dark septate hyphae and microsclerotia. In particular, DSE is more common in abiotic stress environments such as drought, salinity, and heavy metal pollution, which means that the interaction between DSE and host plants is crucial to plant survival, especially survival in adverse environments. Studies have found that DSE can secrete auxins and improve plant quality traits. In addition, the benefits of DSE on plants also include improving plant nutrient absorption. DSE can have a positive effect on plant nutrient absorption by increasing soil nutrient availability. At the same time, DSE has been reported to increase the content of medicinal ingredients in a variety of medicinal plants. These effects of DSE are of great significance to the development and protection of medicinal plant resources, as well as the accumulation of effective ingredients in medicinal plants.

[0008] The invention provides a dark septate endophytic fungus Paraphoma radicina AM-PR isolated from the root system of Ammopiptanthus mongolicus. The strain exhibits excellent abilities of dissolving phosphorus and potassium and secreting indoleacetic acid (IAA), can effectively promote the growth of salvia miltiorrhiza plants, increase the aboveground biomass and underground biomass of salvia miltiorrhiza plants, effectively improve the content of effective ingredients in the roots of salvia miltiorrhiza, increase the content of tanshinone I, tanshinone II A, cryptotanshinone and salvianolic acid B, and has important application value in the quality improvement cultivation of salvia miltiorrhiza.

[0009] A microbial preparation comprises the above-mentioned Heterophytum rhizogenes.

[0010] Application of the above-mentioned Rhizoctonia solani or microbial preparation in improving the growth of Salvia miltiorrhiza and the content of effective ingredients.

[0011] Furthermore, the fermentation liquid or microbial preparation of Heterostyma rhizogenes is applied to Salvia miltiorrhiza.

[0012] The present invention has the following beneficial effects:

[0013] The invention provides a Paraphoma radicina AM-PR isolated from the root system of Ammopiptanthus mongolicus. The strain exhibits excellent phosphorus solubilizing ability, with a phosphorus solubilizing index of 661.1%; excellent potassium solubilizing ability, with a potassium solubilizing index of 322.2%; and excellent IAA secreting ability, with a secreted IAA concentration of 8.12 μg / mL in a King's liquid shake flask; the growth of salvia miltiorrhiza plants can be significantly improved, with the aboveground biomass and underground biomass increased by 4.9% and 295.4% respectively; the content of effective components can be significantly improved, with the contents of tanshinone I, tanshinone II A, cryptotanshinone and salvianolic acid B increased by 381.3%, 39.9%, 284.5% and 3.13% respectively, and the fungus has important application value in the quality improvement cultivation of salvia miltiorrhiza. BRIEF DESCRIPTION OF THE DRAWINGS

[0014] Figure 1 This is the morphological picture of Heterophytum radix AM-PR on potato dextrose agar (PDA) plate medium;

[0015] Figure 2 This is a microscope image of the root-heterostyma AM-PR in the PDA plate culture medium, where Hy is hyphae, S is spores, and the scale bar is 50 μm;

[0016] Figure 3 is the phylogenetic tree of AM-PR of Heterophytum rhizogenes;

[0017] Figure 4 The morphological pictures of Heterophytum radix AM-PR on phosphate-soluble and potassium-soluble plate culture media, where A is the phosphate-soluble plate picture and B is the potassium-soluble plate picture;

[0018] Figure 5 The growth of Salvia miltiorrhiza plants 90 days after being treated with Radix Heterostyma AM-PR;

[0019] Figure 6 The morphological indexes of the plants after AM-PR acted on Salvia miltiorrhiza for 90 days, where Control is the control group and AM-PR is the experimental group;

[0020] Figure 7 The biomass of Salvia miltiorrhiza plants after AM-PR acted on them for 90 days, where Control was the control group and AM-PR was the experimental group;

[0021] Figure 8 The content of available phosphorus and available potassium in the matrix soil after AM-PR acted on Salvia miltiorrhiza for 90 days, where Control is the control group and AM-PR is the experimental group;

[0022] Fig. 9 The percentage of tanshinones and the content of salvianolic acid B after AM-PR acted on Salvia miltiorrhiza for 90 days, where Control is the control group and AM-PR is the experimental group. DETAILED DESCRIPTION

[0023] The principles and features of the present invention are described below in conjunction with the accompanying drawings. The examples are only used to explain the present invention and are not used to limit the scope of the present invention. If specific conditions are not specified in the embodiments, they are carried out according to normal conditions or conditions recommended by the manufacturer. If the manufacturer is not specified for the reagents or instruments used, they are all conventional products that can be purchased commercially.

[0024] Example 1: Isolation and identification of strains

[0025] (1) Isolation of strains

[0026] The roots of Ammopiptanthus mongolicus collected from Dengkou sampling site in Inner Mongolia were washed with distilled water, disinfected with 75% alcohol for 5 minutes, washed with sterile water for 30 seconds, disinfected with 5% sodium hypochlorite for 2 minutes, and rinsed with sterile water 3 times. The surface-sterilized root segments were placed in PDA medium supplemented with ampicillin and streptomycin sulfate, and the hyphae grown at the incision of the plant tissue were transferred to a new PDA plate. After the plate was sealed, it was inverted and cultured in a constant temperature incubator at 27°C in the dark. Figure 1 The colonies shown were observed and photographed under a 400x BX51 optical microscope to record the microscopic characteristics of the hyphae. Figure 2 shown.

[0027] (2) Identification of strains

[0028] The DNA of the colony obtained in step (1) was extracted using a genomic DNA extraction kit (SolarBio). The colony DNA was then amplified using primers ITS3: 5'-TCCTCCGCTTATTGATATGC-3' (SEQ ID NO. 1) and ITS4: 5'-GGAAGTAAAAGTCGTAACAAGG-3' (SEQ ID NO. 2), and the amplified product was sequenced.

[0029] The sequencing sequence of the strain is: (SEQ ID NO.3).

[0030] The sequences were aligned using the NCBI BLAST tool, and the phylogenetic tree was constructed using the MEGA6 software. Figure 3 The results showed that the strain was Paraphoma radicina, named AM-PR.

[0031] AM-PR was placed on PDA culture medium and cultured at 27°C for 14 days for activation in preparation for subsequent experiments.

[0032] Example 2: AM-PR growth promoting activity assay

[0033] (1) Determination of phosphorus and potassium dissolving capacity

[0034] The National Botanical Research Institute's phosphate growth medium (NBRIP) and potassium-dissolving plates were used to test the conversion effect of AM-PR on poorly soluble phosphorus and potassium.

[0035] NBPIR medium: 10 g glucose, 0.1 g (NH4)2SO4, 5 g MgCl2·6H2O, 0.25 g MgS O4·7H2O, 0.2 g KCl, 5 g Ca2(PO4)2, 16 g agar, pH = 7, dissolved in 1 L distilled water, and the medium was sterilized by high pressure at 121°C for 30 min.

[0036] Potassium-solubilizing culture medium: 10 g glucose, 5 g MgCl2·6H2O, 2.5 g potassium feldspar powder, 0.25 g MgSO4·7H2O, 0.1 g (NH4)2SO4, 2 g Na2HPO4, 18 g agar, dissolved in 1 L distilled water, and the culture medium was sterilized by high pressure at 121°C for 30 min.

[0037] The AM-PR bacterial cake with a radius of 9 mm from the purified culture was inoculated in the center of NBPIR medium and potassium-dissolving medium respectively. Each treatment was repeated 3 times and cultured inverted at 27°C. After 7 days, the diameter of the dissolution zone and the colony diameter were measured, and the dissolution index of the strain was calculated.

[0038] Dissolution index = dissolution zone diameter / colony diameter.

[0039] The growth of AM-PR on NBRIP plates is shown in Figure 4 As shown in Figure A, AM-PR can form an obvious transparent zone of phosphorus dissolution, and the phosphorus dissolution index is 661.1%.

[0040] The growth of AM-PR on potassium-dissolving plates is as follows Figure 4 As shown in Figure B, AM-PR can form an obvious transparent zone, and the potassium-solubilizing index is 322.2%.

[0041] (2) IAA secretion capacity determination

[0042] The IAA content was determined by the Salkowsky method to verify whether AM-PR secreted IAA.

[0043] The specific process is as follows:

[0044] The AM-PR cake with a radius of 9 mm from the purified culture was inoculated into 120 mL of King's liquid medium (250 mL conical flask) supplemented with L-tryptophan (100 μg / mL) and cultured for 72 h.

[0045] King's liquid medium: 20 g of peptone, 1.5 g of potassium dihydrogen phosphate, 1.5 g of magnesium sulfate, and 10 mL of glycerol were dissolved in 1 L of distilled water and sterilized by high pressure at 121°C for 30 min.

[0046] The culture was centrifuged at 6000r / min for 15min and the supernatant was collected. 2mL of Salkow sky reagent and 2mL of supernatant were mixed and reacted at 28±2℃ in the dark for 30min. Preparation of Salkowsky reagent: 300mL of H2SO4 was added to 500mL of distilled water, cooled, and then 15mL of 0.5mol / L ferric chloride was added and stored in the dark. After the reaction, the solution turned pink, indicating the presence of IAA. The absorbance was measured at 530nm with a spectrophotometer, and the concentration of IAA in the culture was calculated using the IAA standard as the standard curve.

[0047] The results showed that AM-PR had the ability to secrete IAA, and the IAA concentration in the supernatant obtained from the AM-PR shake flask was 8.12 μg / mL.

[0048] Example 3: Potted experiment on improving the quality of Salvia miltiorrhiza by AM-PR

[0049] (1) Preparation of inoculum

[0050] Take 3 AM-PR cakes with a radius of 9 mm, inoculate them into a 250 mL conical flask containing 120 mL of potato dextrose (PD) medium, and culture them at 25 °C and 180 r / min for 7 days. Use a vacuum filtration pump to collect the mycelium, weigh 2 g of mycelium and add 200 mL of sterile water to mix thoroughly to obtain the AM-PR inoculum.

[0051] (2) Seedling cultivation and AM-PR inoculation

[0052] Select plump and mold-free Salvia miltiorrhiza seeds, rinse them with tap water 2-3 times, and soak them in distilled water for 24 hours. Place the soaked seeds in a seedling tray covered with moist filter paper and culture them in the dark for 72 hours until the seeds germinate. The cultivation soil matrix is ​​a mixture of farmland soil and fine river sand in a volume ratio of 2:1. Weigh 1300g of matrix into a flowerpot (13.8cm in diameter, 7cm in diameter at the bottom of the pot, and 12.4cm in height) and water it until it is completely saturated, then select seedlings with good growth and move them to the flowerpot, with 4 seedlings per pot. After culturing for 45 days at room temperature of 27℃ / 22℃ (day / night) and 12h of sunlight, take 5mL of AM-PR inoculation solution and inoculate it at the root of a single seedling using the root irrigation method, set up 4 replicates, and set up 4 pots of single plants inoculated with 5mL of sterile water as a blank control, and continue to cultivate for 60 days before harvesting.

[0053] (3) Determination of morphology and soil indexes

[0054] When the seedlings were harvested, the height and number of leaves of the seedlings were recorded. The aboveground part and the root system were separated from the rhizome connection point. The fresh weight of the aboveground and underground parts was measured with an electronic balance with a precision of one ten-thousandth, and then they were placed in sampling plastic bags. The roots were cleaned and placed in a glass tank filled with clean water to fully unfold. The root system pictures were obtained by scanning with a scanner (EPSON V800), and the average root volume, root surface area and total root length were calculated by WinRHIZO image analysis system. The aboveground part and the root system were placed in an oven at 80°C to dry to constant weight, and the aboveground and root biomass were calculated. The growth medium of Salvia miltiorrhiza was harvested and air-dried. The available phosphorus (AP) content in the medium was determined by sodium bicarbonate extraction-molybdenum antimony colorimetry, and the available potassium (AK) content was determined by sodium tetraphenylborate turbidimetry.

[0055] The experimental results are as follows Figure 5-Figure 8 shown.

[0056] like Figure 6 As shown in the figure, AM-PR inoculation can improve the morphological indexes of Salvia miltiorrhiza. The plant height of the control Salvia miltiorrhiza plant was 3.53 cm and the number of leaves was 8, while the plant height of the AM-PR inoculated Salvia miltiorrhiza plant was 3.72 cm and the number of leaves was 9, which were 5.3% and 16.6% higher than the blank control, respectively. The root length of the control Salvia miltiorrhiza plant was 4.12 cm and the root surface area was 87.54 cm 2 The average root volume is 0.67 cm 3 ; The root length of the Salvia miltiorrhiza plant inoculated with AM-PR was 4.58 cm and the root surface area was 99.03 cm 2 The average root volume is 0.71 cm 3 , respectively increased by 11.0%, 13.1% and 6.3% compared with the blank control.

[0057] like Figure 7 As shown, AM-PR inoculation can increase the biomass of Salvia miltiorrhiza plants. The aboveground biomass and underground biomass of Salvia miltiorrhiza in the control treatment were 0.29g and 0.12g respectively; the aboveground biomass and underground biomass of Salvia miltiorrhiza in the AM-PR inoculation treatment were 0.31g and 0.49g respectively, which were 4.9% and 295.4% higher than those in the blank control, and the AM-PR inoculation treatment significantly increased the underground biomass of Salvia miltiorrhiza.

[0058] like Figure 8 As shown, AM-PR inoculation can effectively increase the content of available phosphorus and available potassium in the inoculated soil. The content of available phosphorus and available potassium in the control soil was 2.53μg / g and 30.4μg / g, respectively; the content of available phosphorus and available potassium in the AM-PR inoculated soil was 3.78μg / g and 99.8μg / g, respectively, which was significantly increased by 49.4% and 228.2% compared with the blank control.

[0059] Example 4: Determination of the content of tanshinones and salvianolic acids in the roots of Salvia miltiorrhiza plants

[0060] High performance liquid chromatography (HPLC) was used to analyze the contents of tanshinones and salvianolic acids, and the contents of tanshinone Ⅰ, tanshinone ⅡA, cryptotanshinone and salvianolic acid B were quantitatively detected.

[0061] The specific process is as follows:

[0062] (1) Tanshinone detection

[0063] Preparation of HPLC injection samples of tanshinones: Accurately weigh 0.25 g of salvia miltiorrhiza root powder dried to constant weight, add methanol to make up to 25 mL, ultrasonically extract for 30 minutes, and pass the obtained extract solution through a 0.22 μm microporous filter membrane to obtain the HPLC injection sample solution.

[0064] HPLC chromatographic analysis conditions: Chromatographic column: Symmetry C18 column (6mm×250mm, 5μm); mobile phase: acetonitrile (A)-mass fraction 0.05% phosphoric acid (B), gradient elution (0-6min A 61%, B 39%; 6-20min A 61-90%, B 39-10%; 20-20.5min, A 90-61%, B 10-39%; 20.5-25min, A 61%, B 39%), calculated by volume ratio. Chromatographic conditions: flow rate 1.0mL / min; column temperature 25°C; injection volume 10μL; detection wavelength 270nm.

[0065] (2) Detection of salvianolic acids

[0066] Preparation of HPLC injection samples of salvianolic acids: Accurately weigh 0.075 g of Salvia miltiorrhiza root powder dried to constant weight, add 80% methanol to make up to 25 mL, ultrasonically extract for 30 min, and pass the obtained extract solution through a 0.22 μm microporous filter membrane to obtain the HPLC injection sample solution.

[0067] HPLC chromatographic analysis conditions: Symmetry C18 column (6mm×250mm, 5μm); mobile phase: acetonitrile (A)-mass fraction 0.05% phosphoric acid (B), gradient elution (0-15min A 17-23%, B 88-77%; 15-30min A 23-25%, B 77-75%; 30-40min, A 25-90%, B75-10%; 40-50min, A 90-17%, B 10-83%), calculated by volume ratio. Chromatographic conditions: flow rate 1.0mL / min; column temperature 25℃; injection volume 10μL; detection wavelength 286nm.

[0068] The experimental results are as follows Fig. 9 As shown, AM-PR inoculation can effectively increase the content of medicinal components of Salvia miltiorrhiza. The percentage contents of tanshinone Ⅰ, tanshinone ⅡA, cryptotanshinone and the content of salvianolic acid B in the root of Salvia miltiorrhiza in the control treatment were 1.0%, 3.8%, 11.1% and 0.0053 mg / g, respectively; the percentage contents of tanshinone Ⅰ, tanshinone ⅡA, cryptotanshinone and the content of salvianolic acid B in the root of Salvia miltiorrhiza in the AM-PR inoculation treatment were 5.2%, 5.4%, 42.7% and 0.0055 mg / g, respectively; compared with the blank control treatment, they increased by 381.3%, 39.9%, 284.5% and 3.13%, and the AM-PR inoculation treatment significantly increased the percentage contents of tanshinone Ⅰ, tanshinone ⅡA and cryptotanshinone in the roots of Salvia miltiorrhiza plants.

[0069] The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. Any modifications, equivalent substitutions, improvements, etc. made within the spirit and principle of the present invention should be included in the protection scope of the present invention.

Claims

1. A Paraphoma radicina for improving the growth and active ingredient content of Salvia miltiorrhiza, characterized in that: The strain was named AM-PR and deposited in the China Center for Type Culture Collection on May 31, 2024, with the deposit number CCTCC NO: M20241118.

2. A microbial preparation, characterized in that: The invention comprises the root heterophyll as described in claim 1.

3. Use of the Rhizoctonia solani described in claim 1 or the microbial preparation described in claim 2 in improving the growth of Salvia miltiorrhiza and the content of active ingredients.

4. The use according to claim 3, characterized in that: The fermentation liquid of Heterophytum rhizogenes according to claim 1 or the microbial preparation according to claim 2 is applied to Salvia miltiorrhiza.

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