Method for increasing content of medicinal components of liquorice

Abstract

The invention discloses a method for increasing the content of medicinal components of liquorice, and belongs to the technical field of liquorice planting. The method comprises the following steps: inoculating a piriformospora indica hypha suspension into a carrier matrix, carrying out fermentation culture to obtain a piriformospora indica colonization carrier, mixing the piriformospora indica colonization carrier with liquorice seeds, sowing or applying the piriformospora indica colonization carrier to liquorice rhizosphere soil, and colonizing piriformospora indica at the roots of liquorice in the cultivation process, the two establish a symbiotic relationship. According to the invention, piriformospora indica is applied to liquorice for the first time, and a method for colonizing piriformospora indica on liquorice is provided. The piriformospora indica is colonized at the root of the liquorice, so that the medicinal component content of the liquorice is remarkably increased, the quality of the liquorice is improved, and a technical support is provided for cultivation of high-quality liquorice. The method is simple to operate and beneficial to popularization and application in production.

Description

Technical Field

[0001] This invention relates to the field of licorice cultivation technology, and specifically to a method for increasing the content of medicinal components in licorice. Background Technology

[0002] Licorice is a medicinal plant belonging to the genus Glycyrrhiza in the legume family. It has the functions of tonifying the spleen and replenishing qi, relieving cough and moistening the lungs, and relieving spasms and detoxifying. Licorice contains a variety of medicinal components, among which glycyrrhizic acid has various pharmacological effects such as anti-inflammatory, antipyretic, antitumor, anti-allergic and hepatoprotective effects; glycyrrhizin has antioxidant, anti-inflammatory and antibacterial effects (Zhou Dingding et al., Research progress on biosynthesis and regulation of glycyrrhizic acid and glycyrrhizin. Biotechnology Bulletin, 2023. 39(05): p. 44-53.). These components endow licorice with a wide range of pharmacological effects and clinical application value.

[0003] Licorice is primarily used medicinally for its roots and rhizomes, while its stems and leaves can also serve as excellent livestock feed. Its extremely wide range of applications is immeasurable. Medicinal licorice includes licorice root (Glycyrrhiza uralensis). Glycyrrhiza uralensis Fisch.), licorice root ( Glycyrrhiza inflata Batal. and Licorice glabra (Batal.) Glycyrrhiza glabra Licorice (L.), mainly distributed in Asia, Europe, and Mediterranean countries. Due to over-harvesting at the end of the last century, wild licorice resources became endangered, and medicinal licorice gradually shifted from wild to cultivated. Currently, cultivated licorice generally suffers from low levels of medicinal active ingredients, resulting in low medicinal value and seriously affecting the development of my country's traditional Chinese medicine industry. Therefore, increasing the content of medicinal components in cultivated licorice is an urgent problem to be solved in current licorice cultivation.

[0004] Indian piriformis ( Piriformospora indica *Pyrethrum indicum* is a fungus belonging to the genus *Pyrethrum* of the phylum Basidiomycota. It can colonize the roots of some plants, improving their stress resistance and yield, and playing a beneficial role in plants. For example, studies have found that *Pyrethrum indicum* colonization significantly increases the yield of plants such as Chinese cabbage, tobacco, rapeseed, rice, and sesame. There are relatively few studies on the application of *Pyrethrum indicum* in the cultivation of Chinese medicinal herbs. Mu Deyu et al. found that the drought resistance of honeysuckle was significantly enhanced after treatment with *Pyrethrum indicum* (Mu Deyu, Gao Shengyu, Yao Junxiu, et al. Effects of *Pyrethrum indicum* on the growth and physiology of honeysuckle under drought stress [J]. Journal of Qingdao Agricultural University (Natural Science Edition), 2024, 41(04):235-242). Cao Xingxing found that the growth and germination and growth of lateral buds of *Dendrobium officinale* were significantly increased after treatment with *Pyrethrum indicum* (Cao Xingxing. Isolation and identification of pathogenic fungi of *Dendrobium officinale* and study on the growth-promoting effect of *Pyrethrum indicum* [D]. Zhejiang University, 2015).

[0005] At present, the colonization of India pear-shaped fungus on crops mainly focuses on the laboratory stage, and the colonization is carried out by co-culturing India pear-shaped fungus and aseptic seedlings on a culture medium and then transplanting the seedlings. Some plants can be colonized by directly pouring India pear-shaped fungus into the soil of the roots of the plants. At present, there is no report on the colonization of India pear-shaped fungus on licorice. SUMMARY

[0006] The purpose of the present application is to provide a licorice cultivation method capable of increasing the content of medicinal ingredients in licorice, thereby providing a feasible scheme for improving the quality of cultivated licorice in the future.

[0007] To achieve the above-mentioned purpose, the present application adopts the following technical scheme: The present application provides the application of India pear-shaped fungus (Piriformospora indica) in increasing the content of medicinal ingredients in cultivated licorice, which comprises the following steps: Piriformospora indica The application includes: during the cultivation of licorice, India pear-shaped fungus is colonized in the licorice plants; and the medicinal ingredients include at least one of glycyrrhizin and glycyrrhizic acid.

[0008] The present application researches and finds that after India pear-shaped fungus is colonized on licorice, the content of medicinal ingredients such as glycyrrhizin and glycyrrhizic acid in licorice is significantly increased, thereby significantly improving the quality of cultivated licorice.

[0009] In the present application, the India pear-shaped fungus applied to the colonization of licorice can be the India pear-shaped fungus which has been reported to successfully colonize and be applied to other crops.

[0010] Further, the application includes: inoculating India pear-shaped fungus mycelium suspension into carrier substrate to culture and obtain India pear-shaped fungus colonization carrier; then mixing the India pear-shaped fungus colonization carrier with licorice seeds and sowing or applying the India pear-shaped fungus colonization carrier to the rhizosphere soil of licorice, so that the India pear-shaped fungus is colonized in the roots of licorice and a symbiotic relationship is established between them.

[0011] During the research of the present application, it is found that India pear-shaped fungus cannot be colonized on licorice through conventional irrigation, and after inoculating India pear-shaped fungus mycelium into carrier substrate to produce spores through aerobic fermentation culture, the carrier substrate loaded with India pear-shaped fungus is co-cultured with licorice, so that India pear-shaped fungus can be colonized on licorice. After India pear-shaped fungus and licorice establish a symbiotic relationship, the quality of licorice can be significantly improved, and the content of medicinal ingredients is significantly increased.

[0012] Another purpose of the present application is to provide a method for colonizing India pear-shaped fungus on licorice to increase the content of medicinal ingredients in licorice, which comprises the following steps: (1) expanding the culture of activated India pear-shaped fungus, collecting mycelium, and diluting and dispersing the mycelium with a suspension matrix to prepare mycelium suspension; (2) inoculating the mycelium suspension into carrier substrate for fermentation culture, and drying to obtain India pear-shaped fungus colonization carrier; (3) The Indian pear-shaped spores are inoculated into the carrier substrate or mixed with the licorice seeds, and then the licorice is planted, so that the Indian pear-shaped spores are colonized on the roots of the licorice during the cultivation process, and a symbiotic relationship is established between the Indian pear-shaped spores and the licorice, thereby increasing the content of medicinal ingredients in the licorice.

[0013] By using the above method, the Indian pear-shaped spores can be colonized on the roots of the licorice within 30 days, and the colonization rate reaches 100%. After 3 months of colonization, the content of glycyrrhizin and glycyrrhizin in the licorice is significantly increased, thereby providing technical support for improving the quality of licorice.

[0014] In step (1), the Indian pear-shaped spores are activated and cultured to obtain seed liquid of the Indian pear-shaped spores with the best mycelial activity. Then, the mycelium is collected by centrifugation, and the mycelium is cut into mycelial fragments to form a mycelial suspension after adding a suspension medium.

[0015] Preferably, the Indian pear-shaped spores are first activated and cultured on the activation medium at 25-28 DEG C for 5-10 days to make the mycelial activity reach the best state. Then, the mycelial cake is taken from the colony edge where the mycelial division is vigorous, and inoculated into the liquid culture medium for culture at 25-28 DEG C for 3-10 days, so that the mycelium enters the logarithmic growth phase and the mycelial activity is the strongest. Then, the mycelium is collected and diluted to prepare the mycelial suspension according to the mass ratio of the mycelium to the suspension medium of 1:100-500.

[0016] In the present application, the culture medium for culturing the Indian pear-shaped spores can be MMN medium, Kafer medium, M4N medium, MS medium, MMNC medium, MYP medium, PDA medium or maltose-yeast extract medium.

[0017] Preferably, the activation medium is PDA medium, the liquid culture medium is PDB medium, and the suspension medium is PDB medium or sterile water.

[0018] Preferably, the culture time of the Indian pear-shaped spores in the PDA medium and the PDB medium is 5 days.

[0019] Preferably, the mass ratio of the mycelium to the sterile water is 1:100.

[0020] In step (2), the mycelial suspension is inoculated into the carrier substrate for aerobic fermentation, and the Indian pear-shaped spore colonization carrier is obtained after sterile and dry.

[0021] In the present application, the carrier substrate is grain, legume or tuber crop food. Specifically, the carrier substrate can be, but is not limited to, barley, wheat, oat, sorghum, corn, rice, millet, mung bean, soybean, broad bean, pea, potato, sweet potato, cassava and products thereof.

[0022] The carrier substrate is sterilized, and the preparation method comprises the following steps: sterilizing the carrier substrate once or twice by high-pressure wet heat sterilization. Specifically, the sterilization conditions are as follows: 121 DEG C for 30 minutes, and the first sterilization is followed by the second sterilization after 24 hours, so as to completely kill spore bacillus and fungal spores. The carrier substrate after sterilization is dried under a sterile condition.

[0023] If the carrier substrate is barley, wheat, oat, sorghum, corn, rice, millet, mung bean, soybean, broad bean, pea, the seed coat should be softened by soaking in water for 12-24 hours, so as to facilitate the growth of India pear-shaped spores thereon.

[0024] As a specific embodiment of the present application, the carrier substrate is wheat grain, which is soaked in water for 12-24 hours and then sterilized by high-pressure wet heat sterilization.

[0025] As preferred, the sterilized carrier substrate is mixed with India pear-shaped spore mycelium suspension at a mass ratio of 5-50:1, and aerobic fermentation is carried out at 25-28 DEG C for 10-30 days; after the fermentation is completed, the mixture is dried under a sterile condition at room temperature.

[0026] More preferably, the sterilized wheat grain is mixed with India pear-shaped spore mycelium suspension at a mass ratio of 15-30:1, and aerobic fermentation is carried out at 25-28 DEG C for 20-30 days.

[0027] In step (3), the India pear-shaped spore colonization carrier is mixed with licorice seeds and then sowed, or the India pear-shaped spore colonization carrier is applied to the rhizosphere soil of licorice, and after the licorice is grown for one month under the condition that the daily average temperature is above 16 DEG C, the India pear-shaped spore is colonized in the licorice roots, and a symbiotic relationship is established between the two.

[0028] For the licorice seeds, the licorice seeds are mixed with the India pear-shaped spore colonization carrier at a mass ratio of 1:0.5-5 before sowing, and the sowing depth is 1-2 cm.

[0029] For the licorice plants, a ditch or a hole is dug in the cultivation substrate where the rhizosphere of the licorice is located, the India pear-shaped spore colonization carrier is applied, and then the ditch or hole is covered with soil, the distance between the ditch or hole and the main root of the licorice is less than 10 cm, and the application amount is 4-12 g per plant. The depth of the ditch or hole is 5-10 cm.

[0030] As preferred, the distance between the India pear-shaped spore colonization carrier and the licorice seeds or the main root of the licorice is less than or equal to 5 cm.

[0031] The present application has the following beneficial effects: (1) The present application first applies the India pear-shaped spore to the licorice, and provides a method for colonizing the India pear-shaped spore on the licorice, which solves the technical problem that the India pear-shaped spore cannot be colonized on the licorice by irrigation in the soil.

[0032] (2) The present application significantly increases the medicinal ingredient content of licorice and improves the quality of licorice by colonizing Indian pear-shaped spores in the roots of licorice, thereby providing technical support for cultivating high-quality licorice.

[0033] (3) The method of the present application is simple to operate and is conducive to popularization and application in production. BRIEF DESCRIPTION OF DRAWINGS

[0034] Figure 1 Biomass determination of Indian pear-shaped spores cultured in liquid medium for 5 days for different solid culture times.

[0035] Figure 2 Fluorescent quantitative PCR determination of the amount of Indian pear-shaped spores colonized on the carrier for different liquid culture times, the smaller the Ct value, the higher the biomass.

[0036] Figure 3 Spore quantity of Indian pear-shaped spores colonized on the carrier for different fermentation times under a microscope, the upper left graph is fermented for 5 days, the upper right graph is fermented for 10 days, the lower left graph is fermented for 20 days, and the lower right graph is fermented for 30 days.

[0037] Figure 4 Activation of the colonization carrier fermented for different times on PDA medium, the left graph is the colonization carrier fermented for 5 days, and the right graph is the colonization carrier fermented for 10 days.

[0038] Figure 5 Observation of Indian pear-shaped spores colonized on the roots of licorice under a microscope, the upper left graph is licorice obtained in Example 2, the upper right graph is licorice obtained in Example 3, the lower left graph is licorice obtained in Example 4, and the lower right graph is licorice obtained in Comparative Example 1.

[0039] Figure 6 HPLC detection results of licorice in Example 2, the peak indicated by the arrow is the target peak.

[0040] Figure 7 HPLC detection results of licorice in Example 3, the peak indicated by the arrow is the target peak.

[0041] Figure 8 HPLC detection results of licorice in Example 4, the peak indicated by the arrow is the target peak.

[0042] Figure 9 HPLC detection results of licorice in Example 5, the peak indicated by the arrow is the target peak.

[0043] Figure 10 HPLC detection results of licorice in Comparative Example 1, the peak indicated by the arrow is the target peak.

[0044] Figure 11To detect the colonization of Indian pyriformis in licorice roots by PCR, 1 in the figure represents Indian pyriformis, 2 represents licorice without Indian pyriformis colonization as a control, 3-5 represent licorice obtained in Examples 6-8 respectively, and 6 represents licorice obtained in Comparative Example 2. Detailed Implementation

[0045] To better explain the purpose, technical solution, and advantages of this invention, specific embodiments are described below. These embodiments are for illustrative purposes only and are not intended to limit the scope of the invention. Any modifications or substitutions made to the methods, steps, or conditions of this invention without departing from its spirit and essence are within the scope of this invention.

[0046] Unless otherwise specified, the experimental methods used in the following examples are conventional methods; the materials and reagents used are commercially available unless otherwise specified.

[0047] The Indian pyriformis used in the following examples was kindly provided by Professor Ralf Oelmüller of the University of Jena, Germany (see reference: Chen Youyuan et al. Preliminary study on the mechanism of Indian pyriformis-induced drought resistance in rapeseed [J]. Journal of Agricultural Biotechnology, 2013, 21(03):272-281). Licorice seeds were purchased from the Ningxia Yanchi Traditional Chinese Medicine Trading Market and underwent sulfuric acid dormancy breaking treatment.

[0048] The PDA culture medium formula used in the following examples is as follows: 200 g peeled potatoes, 20 g glucose, 15 g agar, distilled water to a final volume of 1000 mL, and sterilized at 121°C for 20 minutes.

[0049] The PDB culture medium formula used in the following examples is as follows: 200 g peeled potatoes, 20 g glucose, distilled water to a final volume of 1000 mL, and sterilized by moist heat at 121°C for 20 minutes.

[0050] Example 1 1. Determination of the optimal time for solid culture of *Pyrrosia lingua* The mycelial blocks of *Pyrophyllus indicum* were taken from the glycerol storage tube and inoculated on PDA medium for 3, 5, 10, and 15 days. The mycelial blocks were broken into 5 mm diameter discs along the edge of the colony. Five mycelial discs were inoculated into 200 mL of PDB medium and cultured at 28°C and 180 rpm for 5 days. The mycelial cells were collected by centrifugation and the biomass was determined by weighing the wet weight.

[0051] The results are as follows Figure 1 As shown, the biomass was highest after 5 days of solid culture followed by liquid culture after 10 days of solid culture. The biomass was lower after 3 and 15 days of solid culture. Therefore, solid culture for 5-10 days is suitable for liquid culture, with 5 days being the optimal time.

[0052] 2. Optimal time determination of Pyricularia grisea liquid culture Pyricularia grisea cultured on PDA medium for 5 days was punched into 5mm diameter plugs along the edge of the colony, 5 plugs were inoculated into 200 mL PDB medium respectively, cultured at 28℃, 180 rpm for 3 / 5 / 10 / 15 days, centrifuged to collect the mycelium, added sterile water at 1:100 mass ratio, and broken into mycelium suspension with a wall breaker.

[0053] 100 g wheat grains were weighed, soaked in 50 g water for 24 hours, autoclaved at 121℃ for 30 minutes, and autoclaved again after 12 hours. The wheat grains were dried in a sterile environment, 5 g mycelium suspension was added and stirred evenly, and then put into a fermentation tank for aerobic fermentation at 28℃ for 5 days. 50 wheat grains were randomly taken from the fermentation tank to extract fungal DNA, and the relative growth of mycelium was detected by fluorescent quantitative PCR.

[0054] The detection primers were: Forward: 5'-ACATCTGACGCACGAATAGC-3', Reverse: 5'-ACTGGGACCGTACCAATACC-3'.

[0055] The detection system was: Green Taq Mix 10 μL, 0.5 μL of forward and reverse primers respectively, 2 μL of DNA, and dd H2O to 20 μL.

[0056] The detection program was: 95℃ pre-denaturation for 3 minutes; 95℃ denaturation for 20 seconds, 56℃ annealing for 20 seconds, 72℃ extension for 20 seconds, a total of 40 cycles; 72℃ extension for 5 minutes.

[0057] The results are shown in Figure 2 The Ct values of liquid culture for 3 days and 5 days were significantly lower than those for 10 days and 15 days, indicating that the biomass of Pyricularia grisea colonized on the carrier was the highest, the viability was the best, and there was no significant difference between 3 days and 5 days, followed by 10 days, and 15 days was the lowest.

[0058] 3. Optimal time determination of Pyricularia grisea aerobic fermentation The Indian pear-shaped along the colony edge of the fungus cake with a diameter of 5 mm was punched in PDA medium for 5 days, 5 pieces of fungus cake were inoculated into 200 mL PDB medium, and the culture was carried out at 28℃, 180 rpm for 5 days. The mycelium was collected by centrifugation, and 100 g of wheat grains were added with 50 g of water for 24 hours, then sterilized at 121℃ for 30 minutes, and sterilized again after 12 hours. The wheat grains were dried in a sterile environment, 5 g of mycelium suspension was added and stirred evenly, and then put into a fermentation tank for aerobic fermentation at 28℃ for 5, 10, 20 and 30 days. After fermentation, the inoculation carrier was dried completely under sterile conditions, 1 g of the inoculation carrier was washed with 5 mL of sterile water, and the number of Indian pear-shaped spores was detected under a microscope. The inoculation carrier was placed on PDA medium to detect the activity of Indian pear-shaped spores.

[0059] The results are shown in Table 1. Figure 3 The number of Indian pear-shaped spores from large to small is in the order of fermentation for 30 days, 20 days, 10 days, and basically no spores for 5 days.

[0060] The results are shown in Table 2. Figure 4 The inoculation carriers fermented for 30 days, 20 days and 10 days all formed colonies on PDA medium, and the inoculation carrier fermented for 5 days had no mycelial growth on PDA medium.

[0061] The above three experimental results show that the Indian pear-shaped spores cultured on solid medium and liquid medium for 5 days and then transferred to wheat grains for 20-30 days have more spores and better activity, which are suitable for later colonization application.

[0062] Example 2 1. Preparation of inoculation carrier The Indian pear-shaped fungus cake with a diameter of 5 mm was punched along the colony edge in PDA medium for 5 days, 5 pieces of fungus cake were inoculated into 200 mL PDB medium, and the culture was carried out at 28℃, 180 rpm for 5 days. The mycelium was collected by centrifugation, and 100 g of wheat grains were added with 50 g of water for 24 hours, then sterilized at 121℃ for 30 minutes, and sterilized again after 12 hours. The wheat grains were dried in a sterile environment, 5 g of mycelium suspension was added and stirred evenly, and then put into a fermentation tank for aerobic fermentation at 28℃ for 5, 10, 20 and 30 days. After fermentation, the inoculation carrier was dried completely under sterile conditions, 1 g of the inoculation carrier was washed with 5 mL of sterile water, and the number of Indian pear-shaped spores was detected under a microscope. The inoculation carrier was placed on PDA medium to detect the activity of Indian pear-shaped spores.

[0063] 2. Co-culture with licorice seeds The prepared Indian pear-shaped spore inoculation carrier 1 kg was mixed with licorice seeds 200 g, and then sown in the soil with a hole seeder, the sowing depth was about 1 cm, the sowing plant distance was 15 cm, and the row spacing was 15 cm.

[0064] 3. Colonization status detection Take tissue samples from the base of seedlings (0-5 cm) 30 days after sowing, rinse them in running water for 30 minutes, soak them in 10% KOH overnight, wash them three times with water, soak them in 1% HCl for 5 minutes, stain them with 0.05% trypan blue for 5 minutes, wash them three times with water again, and observe their colonization status under an optical microscope at 400x magnification.

[0065] The results are as follows Figure 5 As shown in the upper left image, Indian piriformis colonizes the roots of licorice.

[0066] 4. Analysis of medicinal components Licorice root colonized by Indian piriformis for 3 months was dried at 50℃ and ground into powder. 200 mg of the powder was extracted with 100 mL of 70% ethanol by ultrasonication for 30 min. The contents of glycyrrhizin and glycyrrhizic acid were detected by HPLC.

[0067] HPLC detection conditions: A Poroshell 120 SB-C18 column was used, with acetonitrile (B) and 0.05% phosphoric acid (A) as the mobile phase. Gradient elution was performed at a flow rate of 1 mL / min. The elution conditions were: isocratic elution with 19% acetonitrile for 0-8 min, and gradient elution with 19%-50% acetonitrile for 8-35 min.

[0068] The results are shown in Table 1 and Figure 6 As shown.

[0069] Example 3 1. Preparation of colonization carriers Five *Pyrhodotorula praecox* mycelia, cultured on PDA medium for 5 days, were broken into 5 mm diameter mycelial discs along the colony edge. Five discs were inoculated into 200 mL of PDB medium and cultured at 28°C and 180 rpm for 5 days. The mycelial cells were collected by centrifugation, and sterile water was added at a mass ratio of 1:200. The mixture was then blended until no obvious mycelial clumps remained, forming a mycelial suspension. 100 g of wheat grains were weighed, soaked in 50 g of water for 24 hours, and autoclaved at 121°C for 30 minutes. The mixture was then autoclaved again after a 12-hour interval. The wheat grains were dried under sterile conditions, and 10 g of the mycelial suspension was added and stirred thoroughly. The mixture was then placed in a fermenter and fermented aerobically at 28°C for 20 days. After fermentation, the mixture was dried completely at room temperature before being stored in a dry place.

[0070] 2. Co-cultivation with licorice seeds 900 g of the prepared Indian piriformis colonization vector was mixed with 300 g of licorice seeds and then sown in the soil using a seeder at a depth of about 1 cm, with a plant spacing of 15 cm and a row spacing of 15 cm.

[0071] 3. Colonization status detection Method same as example 2, results see Figure 5 The upper right graph of Figure 1 shows that the roots of liquorice have colonization of M. phaseolina.

[0072] 4. Analysis of medicinal ingredients Method same as example 2, results see Table 1 and Figure 7 .

[0073] Example 4 1. Preparation of colonization carrier M. phaseolina which has been cultured on PDA medium for 5 days is cut into 5 mm diameter pieces along the edge of the colony, 5 pieces are inoculated into 200 mL PDB medium, and cultured at 28°C, 180 rpm for 5 days. The bacterial body is collected by centrifugation, and 100 g of wheat grains are weighed, 50 g of water is added to soak for 24 hours, and then autoclaved at 121°C for 30 minutes. The wheat grains are dried in a sterile environment to remove surface moisture, 20 g of the mycelium suspension is added and stirred evenly, and then placed in a fermentation tank for aerobic fermentation at 28°C for 20 days. After fermentation, the fermentation tank is dried at room temperature until completely dry, and then stored.

[0074] 2. Co-cultivation with liquorice seeds The prepared M. phaseolina colonization carrier 500 g is mixed with liquorice seeds 500 g, and then sown in the soil using a hole seeder, with a sowing depth of about 1 cm, a sowing plant distance of 15 cm, and a row distance of 15 cm.

[0075] 3. Detection of colonization status Method same as example 2, results see Figure 5 The lower left graph of Figure 1 shows that the roots of liquorice have colonization of M. phaseolina.

[0076] 4. Analysis of medicinal ingredients Method same as example 2, results see Table 1 and Figure 8 .

[0077] Example 5 1. Preparation of colonization carrier Method same as example 4.

[0078] 2. Co-cultivation with liquorice seeds The prepared M. phaseolina colonization carrier 400 g is mixed with liquorice seeds 800 g, and then sown in the soil using a hole seeder, with a sowing depth of about 1 cm, a sowing plant distance of 15 cm, and a row distance of 15 cm.

[0079] 3. Detection of colonization status Method same as example 2, the roots of liquorice have colonization of M. phaseolina.

[0080] 4. Analysis of medicinal components The method is the same as in Example 2, and the results are shown in Table 1 and 2. Figure 9 .

[0081] Comparative Example 1 Mix 400 g of carrier substrate (wheat grains) that does not grow Indian piriformis with 800 g of licorice seeds and sow them in the soil using a seeder at a depth of about 1 cm, with a plant spacing of 15 cm and a row spacing of 15 cm.

[0082] Microscopic observation of colonization of *Pyrophyllum indicum* on licorice roots, using the same method as in Example 2, results are shown below. Figure 5 The lower right image shows that there is no Indian piriformis colonization on the licorice root.

[0083] The content of medicinal components in licorice root was determined by HPLC, using the same method as in Example 2. The results are shown in Table 1 and 2. Figure 10 .

[0084] Table 1. Content of medicinal components in licorice root colonized with Indian piriformis Note: The letters to the right of the content in each column of the table indicate that there is a significant difference (p < 0.05) between different groups in the same column. As can be seen from Table 1, the contents of glycyrrhizin and glycyrrhizic acid in the licorice roots of Examples 2-5 were significantly higher than those in Comparative Example 1.

[0085] Example 6 1. Ural licorice planted for 3 months in sandy soil in Xiabulong Team 1, Puhui Township, Korla City, Xinjiang, was planted with holes 2 cm in diameter and 10 cm deep, 3 cm away from the root zone. 4 g of the Indian piriformis colonization carrier prepared in Example 2 was inserted, and after covering with soil, the soil was used for regular field management. The soil was then thoroughly watered once, and 2 kg / mu of nitrogen fertilizer was applied with the water. No insecticides or fungicides were applied.

[0086] 2. After 3 months, take 10 cm of licorice root from the base of the root, rinse it in running water for 30 minutes, peel off the cortex and extract the genome using a genome extraction kit, and use PCR to detect the colonization of Indian piriformis using the same primer sequence as in Example 1.

[0087] The results are as follows Figure 11 As shown in lane 3, Indian pyriformis was detected in the roots of licorice, indicating that this method can be used for the colonization of Indian pyriformis in the roots of licorice.

[0088] Example 7 1. In the sandy soil of the first team of Xibu village in Korla city of Xinjiang, 6 months old roots of Uralian licorice were planted at a distance of 5 cm from the rhizosphere. A hole with a diameter of 2 cm and a depth of 10 cm was drilled, and 8 g of the colonization carrier of P. indica prepared in Example 3 was applied. After covering the soil, the water was poured once according to the conventional field management method, and 2 kg / mu of nitrogen fertilizer was applied with the water. No pesticides and fungicides were applied.

[0089] 2. After 3 months, the licorice roots below the base of the licorice roots were taken, washed in flowing water for 30 minutes, and the cortex was peeled off. The genome was extracted using a genome extraction kit, and the colonization of P. indica was detected using PCR. The primer sequence was the same as in Example 1.

[0090] The results are shown in lane 4 of Figure 11 The P. indica was detected in the licorice roots, indicating that this method can be used for the colonization of P. indica in licorice roots.

[0091] Example 8 1. In the sandy soil of the first team of Xibu village in Korla city of Xinjiang, 3 months old roots of Uralian licorice were planted at a distance of 10 cm from the rhizosphere. A hole with a diameter of 2 cm and a depth of 10 cm was drilled, and 12 g of the colonization carrier of P. indica prepared in Example 4 was applied. After covering the soil, the water was poured once according to the conventional field management method, and 2 kg / mu of nitrogen fertilizer was applied with the water. No pesticides and fungicides were applied.

[0092] 2. After 3 months, the licorice roots below the base of the licorice roots were taken, washed in flowing water for 30 minutes, and the cortex was peeled off. The genome was extracted using a genome extraction kit, and the colonization of P. indica was detected using PCR. The primer sequence was the same as in Example 1.

[0093] The results are shown in lane 5 of Figure 11 The P. indica was detected in the licorice roots, indicating that this method can be used for the colonization of P. indica in licorice roots.

[0094] Comparative Example 2 1. In the sandy soil of the first team of Xibu village in Korla city of Xinjiang, 3 months old roots of Uralian licorice were planted, and 100 mL of 100 g / L P. indica suspension was injected at a depth of 5 cm from the soil surface. The preparation method of the bacterial suspension was the same as in Example 2. After injection, the water was poured once according to the conventional field management method, and 2 kg / mu of nitrogen fertilizer was applied with the water. No pesticides and fungicides were applied.

[0095] 2. After 3 months, the licorice roots below the base of the licorice roots were taken, washed in flowing water for 30 minutes, and the cortex was peeled off. The genome was extracted using a genome extraction kit, and the colonization of P. indica was detected using PCR. The primer sequence was the same as in Example 1.

[0096] The results are shown in lane 5 ofFigure 11 As shown in lane number 6, no G. indica was detected on the roots of G. glabra, indicating that G. indica was unable to colonize G. glabra through routine watering.

Claims

1. Indian piriformis ( Piriformospora indica Its application in increasing the medicinal component content of cultivated licorice is characterized by, The application includes: during the cultivation of licorice, Piriformospora indica is colonized in licorice plants; and the medicinal components include at least one of glycyrrhizin and glycyrrhizic acid.

2. Use according to claim 1, wherein The application includes: Piriformospora indica mycelium suspension is inoculated in a carrier substrate to obtain a Piriformospora indica colonization carrier; and then the Piriformospora indica colonization carrier is mixed with licorice seeds for sowing or is applied to the rhizosphere soil of licorice, so that Piriformospora indica is colonized in the roots of licorice and a symbiotic relationship is established.

3. A method of increasing the content of medicinal ingredients of liquorice, characterized in that, The method comprises the following steps: (1) the activated Piriformospora indica is subcultured, mycelium is collected, and a mycelium suspension is prepared by dilution with a suspension medium; (2) the mycelium suspension is inoculated in a carrier substrate for fermentation culture, and a Piriformospora indica colonization carrier is prepared after drying; (3) the Piriformospora indica colonization carrier is mixed with licorice seeds for sowing, or is applied to the cultivation substrate in which the rhizosphere of licorice is located, so that Piriformospora indica is colonized in the roots of licorice and a symbiotic relationship is established, thereby increasing the content of medicinal components in licorice.

4. The method of claim 3, wherein, In step (1), Piriformospora indica is first activated and cultured on an activation medium at 25-28°C for 5-10 days, then a bacterial cake is taken from the colony edge where mycelial division is vigorous and is inoculated into a liquid medium for culture at 25-28°C for 3-10 days, and then mycelium is collected and diluted at a mass ratio of mycelium to suspension medium of 1:100-500 to prepare a mycelium suspension.

5. The method of claim 4, wherein, The activation medium is PDA medium, the liquid medium is PDB medium, and the suspension medium is PDB medium or sterile water.

6. The method of claim 5, wherein, The culture time of Piriformospora indica in the PDA medium and the PDB medium is 5 days.

7. The method of claim 4, wherein, In step (2), the carrier substrate is grain food of cereal, legume or tuber crops; the sterilized carrier substrate is mixed with the Piriformospora indica mycelium suspension at a mass ratio of 5-50:1, and aerobic fermentation is carried out at 25-28°C for 10-30 days.

8. The method of claim 7, wherein, The carrier substrate is wheat grain, which is soaked in water for 12-24 hours and then subjected to high-pressure wet heat sterilization.

9. The method of claim 3, wherein, In step (3), licorice seeds are mixed with the Piriformospora indica colonization carrier at a mass ratio of 1:0.5-5.

10. The method of claim 3, wherein, In step (3), a ditch or a hole is opened in the cultivation substrate in which the rhizosphere of licorice is located, the Piriformospora indica colonization carrier is applied, and then the ditch or hole is covered with soil; the distance between the ditch or hole and the main root of licorice is less than 10 cm, and the application amount is 4-12 g per plant.

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