CN117965393A - Endophytic pseudomonas bacterium C145 and application thereof - Google Patents
Endophytic pseudomonas bacterium C145 and application thereof Download PDFInfo
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- Agricultural Chemicals And Associated Chemicals (AREA)
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Abstract
The invention discloses an endophytic pseudomonas bacterium C145 which is preserved in China general microbiological culture Collection center (China Committee for culture Collection of microorganisms) with a preservation date of 2023, 11 and 22 days and a preservation number of CGMCC NO.29111. The invention also discloses application of the red sage root extract in promoting the growth of red sage root and improving the quality of red sage root. The invention also discloses the application of the prepared microbial inoculum in cultivating high-quality seedlings of the red sage root and improving the yield and the content of medicinal components of the red sage root. The endophytic pseudomonas bacteria C145 screened by the method has the effect of promoting the growth of the red sage root, can improve the quality of the red sage root, and has guiding significance for the production of the red sage root and the development of special bio-organic fertilizer.
Description
Technical Field
The invention relates to the technical field of agricultural microorganisms, in particular to endophytic pseudomonas bacteria C145 for effectively promoting the growth of red sage root and improving the quality of the red sage root and application thereof.
Background
Pseudomonas is a straight or slightly curved gram-negative bacillus that is widely distributed in nature, such as soil, water, food, and air. At present, the application of pseudomonas in the agricultural field and the research on the influence of the pseudomonas on plant growth are more, but the research on the effect of the pseudomonas on Chinese medicinal materials is relatively lacking.
China is one of the most abundant countries of medicinal plant resources, and the quality of Chinese medicinal materials is influenced by factors such as medicinal plant varieties, soil, climate factors, agronomic measures, harvesting, processing, storage and the like. The root of red sage is a perennial upright herb of Salvia of Labiatae, and contains tanshinone, and has effects of dispelling blood stasis and promoting blood circulation. Under the condition of artificial cultivation, the biological communities in the ecological environment of the medicine field have poor diversity and low abundance, the dominant population of diseases and insect pests is prominent, serious diseases and insect pests are often generated, and the yield of the medicine materials is reduced, and the quality is deteriorated. And along with the increase of the demand of the medical industry for the red sage root, the planting area of the red sage root is continuously enlarged. However, due to the fact that the environmental factors have certain restriction on the growth of medicinal plants, the land area suitable for the cultivation of the red sage root is very limited, continuous cropping obstacles gradually appear in the area of the road, and the yield of medicinal materials is reduced and the quality of the medicinal materials is deteriorated.
Endophytes of plants refer to non-pathogenic microorganisms present in the plant body during the life cycle of the plant, and endophytes that have been found to have more than 200 genera, about 100 more than ten thousand species. The endophyte can not have adverse effect on a host, and plays an important role in plant disease control, secondary metabolite synthesis, plant growth regulation, stress resistance and the like. Endophytes can promote plant growth through a series of measures such as absorbing moisture and nutrients, inducing to generate hormone, siderophores and antibacterial secondary metabolites, regulating the content of proline, improving the activity of antioxidant enzymes and the like. Therefore, the invention researches the influence of the functional bacterial pseudomonas screened from healthy root of the red sage on the growth of the red sage seedling and the growth of the red sage in the field, and provides technical support for improving the yield and the quality of the cultivated red sage, improving the ecological environment of the cultivated land, solving the problems of continuous cropping obstacle of the red sage and the like.
Disclosure of Invention
The invention aims at providing endophytic pseudomonas bacteria C145 for effectively promoting the growth of red sage root and improving the quality of the red sage root and application thereof, aiming at practical problems and demands in production practice.
The aim of the invention can be achieved by the following technical scheme:
the first aspect of the invention provides an endophytic pseudomonas bacterium C145 which is preserved in China general microbiological culture Collection center (China Committee for culture Collection of microorganisms) with a preservation date of 2023, 11 and 22 days and a preservation number of CGMCC NO.29111.
In a second aspect, the invention provides a microbial inoculum prepared from endophytic Pseudomonas bacteria C145 as described above.
Further, the microbial inoculum is prepared by the following method: and (3) carrying out liquid fermentation on endophytic pseudomonas bacteria C145 with the preservation number of CGMCC NO.29111, centrifuging after fermentation to obtain thalli, and re-suspending the thalli with sterile water to obtain bacterial suspension.
Further, the microbial inoculum is prepared by the following method: inoculating endophytic pseudomonas bacteria C145 with the preservation number of CGMCC NO.29111 into a liquid culture medium for liquid fermentation, wherein the liquid culture medium comprises a TSB liquid culture medium, an LB liquid culture medium or an R2A liquid culture medium, and the liquid fermentation conditions are as follows: the temperature is 28-30 ℃, the rotating speed is 170-200 r.min -1, and the fermentation time is 1-2d; after fermentation, the bacteria are obtained by centrifugation, and the bacteria are resuspended by sterile water to obtain bacterial suspension, wherein the bacterial content in the bacterial suspension is more than or equal to 1 multiplied by 10 9 CFU/mL.
The third aspect of the invention provides the application of the endophytic pseudomonas bacterium C145 in promoting the growth of the red sage root and improving the quality of the red sage root, wherein the promotion of the growth of the red sage root comprises the improvement of the seedling quality of the red sage root and the improvement of the yield of the red sage root; improving quality of Saviae Miltiorrhizae radix comprises improving content of Saviae Miltiorrhizae radix medicinal components including tanshinone I, tanshinone IIA, tanshinone, salvianolic acid A and salvianolic acid B.
The fourth aspect of the invention provides the application of the microbial inoculum in cultivating high-quality seedlings of red sage root.
Further, when the method is applied, the red sage seeds are buried in the seedling raising matrix, and after the red sage seeds germinate for a period of time, the microbial inoculum is applied twice, wherein the application amount of each time is more than or equal to 1X 10 7 CFU/g dry weight seedling raising matrix, and the interval between the two applications is 10-14 days.
The fifth aspect of the invention provides the use of the microbial inoculum described above for increasing yield of salvia miltiorrhiza and content of drug-resistant components including tanshinone I, tanshinone IIA, tanshinone, salvianolic acid A, salvianolic acid B.
Further, when the microbial inoculum is applied, the decomposed organic fertilizer is used as a base fertilizer, and the microbial inoculum is applied to the red sage root for two times after the red sage root is transplanted for a period of time, wherein the application amount of each time is more than or equal to 1 multiplied by 10 10 CFU/plant of red sage root, and the interval between the two times of application is 15-20 days.
The invention has the beneficial effects that:
The invention collects the red sage root with strong growth vigor and long potential difference in large scale in the area of the red sage root, extracts DNA in the root of the red sage root and carries out high throughput sequencing to obtain a sequencing result, and then analyzes the microbial community difference of the red sage root with strong growth vigor and the red sage root with long potential difference to find that the pseudomonas bacteria is one of key bacteria in the microbial community of the red sage root with strong growth vigor. The bacteria are separated from the root of the red sage root by a coating separation method, and after molecular biological identification, a plurality of pseudomonas bacteria and other strains are obtained, and experiments prove that one endophytic pseudomonas bacteria can effectively promote the growth of the red sage root and is named as C145.
The endophytic pseudomonas bacteria C145 screened by the invention can effectively promote the growth of the red sage root and improve the quality of the red sage root. Compared with the fresh water control, after 1X 10 7 CFU of endophytic pseudomonas bacteria C145 is added into each gram of dry weight seedling substrate, the growth of the red sage root seedlings is obviously promoted, the growth index measurement (leaf length and width, stem thickness and plant height) is obviously reflected, and the quality of the red sage root seedlings is improved. A field test shows that the yield and the content of medicinal components (tanshinone I, tanshinone IIA, tanshinone, salvianolic acid A and salvianolic acid B) of the red sage root are effectively improved by adding the endophytic pseudomonas bacteria C145 microbial inoculum.
The endophytic pseudomonas bacteria C145 screened by the method has the effect of promoting the growth of the red sage root, can improve the quality of the red sage root, and has guiding significance for the production of the red sage root and the development of special bio-organic fertilizer.
Drawings
FIG. 1 is a plate photograph of an endophytic Pseudomonas bacterium C145 strain.
FIG. 2 is a 16S rRNA phylogenetic tree of endophytic Pseudomonas bacteria C145.
FIG. 3 shows the effect of different strain inoculation treatments on the seedling height of Salvia Miltiorrhiza.
FIG. 4 shows the effect of different strain treatments on leaf length of red sage seedlings.
FIG. 5 shows the effect of different strain treatments on leaf width of red sage seedlings.
FIG. 6 shows the effect of different strains on the stem thickness of red sage seedlings.
FIG. 7 shows the effect of inoculation with endophytic Pseudomonas bacteria C145 on the height of Salvia miltiorrhiza.
FIG. 8 is the effect of inoculating endophytic Pseudomonas bacteria C145 on leaf area of Salvia miltiorrhiza.
FIG. 9 is the effect of inoculation with endophytic Pseudomonas bacteria C145 on the stem thickness of Salvia miltiorrhiza.
FIG. 10 is the effect of inoculation with endophytic Pseudomonas bacteria C145 on yield of Salvia miltiorrhiza.
FIG. 11 shows the composition of the drug properties of the seed of Pseudomonas endophytosis bacterium C145 against Salvia Miltiorrhiza: effects of tanshinone I content.
FIG. 12 shows the composition of the drug properties of the seed of Pseudomonas endophytosis bacterium C145 against Salvia Miltiorrhiza: effects of tanshinone IIA content.
FIG. 13 shows the composition of the drug against Salvia Miltiorrhiza in the field inoculated with endophytic Pseudomonas bacteria C145: influence of tanshinone content.
FIG. 14 shows the composition of the drug properties of endophytic Pseudomonas bacteria C145 against Salvia Miltiorrhiza in field: influence of the content of salvianolic acid A.
FIG. 15 shows the composition of the drug properties of the seed of Pseudomonas endophytosis bacterium C145 against Salvia Miltiorrhiza: influence of the content of salvianolic acid B.
The different letters above the histogram are significant (P < 0.05) differences defined by one-way anova.
Biological material preservation information
And C145, namely Pseudomonas sp, which is classified and named as Pseudomonas sp and is preserved in the China general microbiological culture Collection center, wherein the preservation address is the China academy of sciences of China, no. 3, the Korean area North Star, the West road No.1, and the preservation date is 2023, 11 and 22 days, and the preservation number is CGMCC No.29111.
Detailed Description
The following examples facilitate a better understanding of the present invention, but are not intended to limit the same. The experimental methods in the following examples are conventional methods unless otherwise specified. The test materials used in the examples described below, unless otherwise specified, were purchased from conventional biochemical reagent stores.
Unless otherwise specified, the raw materials used in the following examples are as follows:
The seedling raising substrate is a special substrate for raising seedlings of Xingxing-nong cards produced by Jiangsu Xingjing substrate technology Co., ltd, and the product number is: 161102G0097N.
The organic fertilizer is Devoduo brand gardening fermented pig manure, and the organic matter content is more than or equal to 25%.
TSB solid medium, i.e., tryptone soy broth agar medium (1L): 30g of tryptone soybean broth, deionized water to a volume of 1L,20g of agar powder, and sterilizing at 115 ℃ for 30min. The 1/10TSB solid culture medium is prepared by diluting the TSB solid culture medium with clear water 10 times.
TSB broth, tryptone soy broth (1L): tryptone soybean broth 30g, deionized water to a volume of 1L, and sterilizing at 115℃for 30min.
Example 1 differential analysis of the microbial communities of Salvia Miltiorrhiza
The large-scale collection of the red sage root plants with strong growth vigor and long potential difference comprises the following steps: red sage plants with healthy growth and poor growth are collected in the areas of the provinces of Henan, fangcheng county, yuzhou, mianchi county and Shanxi province, ruijun county, hejin, quwa county, xiangfen county, gu county. After the healthy growth red sage plants are mixed, one part is used for the differential analysis of microbial communities in the roots of the red sage roots with different growth conditions in the embodiment, and the other part is used for the separation and identification of the functional strain in the embodiment 2. The red sage root plants with long potential difference are mixed and used for the differential analysis of microbial communities in red sage roots with different growth vigor in the embodiment. Extracting DNA in roots of red sage roots with different growth vigor, and carrying out high-throughput sequencing. The comparison of microorganisms OTUs from red sage of different vigor was performed using the edgeR differential analysis. As a result, it was found that 21 bacteria OTUs in the root-grown and robust red sage root were significantly enriched, and that 1 OTU was classified as Pseudomonas (Pseudomonas) bacteria, which is one of the key bacteria in the root-grown and robust red sage root.
Example 2 isolation and identification of functional Strain
Grinding a proper amount of root of red sage root with a mortar, weighing 1g of ground root sample of red sage root, placing the root sample in a centrifuge tube filled with 9mL of sterile water, oscillating for 30min at 30 ℃ for 170 r.min -1, taking out, carrying out gradient dilution, absorbing 100 mu L of different concentration gradient suspensions, placing the suspension on a 1/10TSB solid culture medium for coating, placing the coated flat plate in a 30 ℃ incubator for culturing for 2d, after bacterial colony grows out, selecting differential bacterial colony for purification, inoculating the purified bacterial strain on the 1/10TSB solid flat plate, placing the bacterial strain on a refrigerator at 4 ℃ for standby, and storing the purified single bacterial colony in the refrigerator at-80 ℃ by a glycerin tube.
Over 180 strains of bacteria are obtained from root of red sage root through co-screening, and through molecular biological identification, 3 strains of pseudomonas bacteria are found, and the serial numbers are C12, C90 and C145 respectively; 3 strains of microbacterium bacteria numbered C59, C73 and D112 respectively; 1 Streptomyces bacteria, numbered C70.
Example 3 liquid propagation culture of functional Strain
Taking out Pseudomonas bacteria C145 glycerol obtained from root of radix Salviae Miltiorrhizae from refrigerator at-80deg.C, placing on ice, dipping with sterile inoculating loop on ice residue in glycerol, streaking and activating on 1/10TSB solid culture medium, placing in 30 deg.C incubator for overnight culture, inoculating single colony into TSB liquid culture medium after colony grows out, and placing on 30deg.C 170 r.min -1 shaker for shaking for 1d to obtain fermentation broth of endophytic Pseudomonas bacteria C145. The fermentation broth is centrifuged for 10min at 9000rpm, the supernatant is discarded, and the thalli are resuspended in sterile water, so that the bacterial content of the resuspended bacterial liquid is 1X 10 9 CFU/mL.
The culture method of the other strains is the same as that of the other strains.
Example 4 test of growth promotion of functional bacteria on Salvia Miltiorrhiza Miq seedlings
The seedling substrate is added into a seedling tray with 50 holes, the single hole diameter of the seedling tray is 4.5cm, the depth is 4.5cm, the bottom diameter is 2cm, and about 8g of seedling substrate is added into each hole. Burying Saviae Miltiorrhizae radix seeds in seedling matrix, watering thoroughly with water, shading, and placing in greenhouse at 30deg.C with humidity of 40%. After about 7 days, the red sage seeds gradually bud, turning on the plant light supplement lamp of the greenhouse, and at night 22:00 to the next morning 6:00 plant light supplement lamp is automatically turned off (temperature and humidity conditions are the same as above).
After about 7 days, in order to facilitate the subsequent experiments and enable the red sage root seedlings to have more sufficient growth space, transplanting the red sage root seedlings with consistent growth vigor to a 21-hole seedling tray, adding about 22g of seedling substrate into each hole, watering thoroughly, transplanting the red sage root seedlings to the 21-hole seedling tray, and transplanting one red sage root seedling in each hole.
The red sage seedling is set with eight treatments of T1 (CK, clean water (sterile water)), T2 (C12), T3 (C90), T4 (C145), T5 (C59), T6 (C73), T7 (D112) and T8 (C70), and each treatment is set with 9 repetitions. After the red sage root seedling is transplanted to a 21-hole seedling tray for 10 days (the temperature, humidity and illumination conditions are the same as above), re-suspended and diluted bacterial liquid (namely, bacterial liquid in the embodiment 3 is diluted by 100 times by sterile water) is inoculated, and the inoculation amount is 1X 10 7 CFU/g dry weight seedling substrate (directly poured into the seedling substrate and near the root of the red sage root seedling). The bacteria were co-watered 2 times at intervals of 2 weeks.
After 2 times of fungus watering are completed, the red sage root seedlings are allowed to continue to grow for 3 weeks (the temperature, the humidity and the illumination conditions are the same as above). 3 weeks later, measuring the plant height of the upper part of each 9 red sage root seedlings by using a ruler, and taking an average value; selecting the top leaf of each root of red-rooted salvia, measuring the length and width of the leaf by using a ruler, and taking an average value; the stem thickness of the junction of the overground part and the underground part of each root of red-rooted salvia is measured by a vernier caliper, and an average value is obtained.
As shown in Table 1 and FIGS. 3 to 6, the T4 treatment with endophytic Pseudomonas bacteria C145 had higher growth targets than CK. The plant height, leaf length, leaf width and stem thickness of the T4 treatment are significantly different from those of the CK treatment, and are respectively improved by 39.97%, 33.26%, 28.10% and 69.39% compared with the CK treatment. Compared to CK treatment, T6 treatment significantly increased stem thickness, T8 treatment significantly increased leaf width and stem thickness, but the effect was less pronounced than T4 treatment. None of the treatments T2, T3, T5 and T7 significantly promoted the growth of the red sage seedlings compared to CK. T2, T3 treated test bacteria were also Pseudomonas bacteria, but did not show growth promotion in the validation test, whereas T4 treated endophytic Pseudomonas bacteria C145 had significant growth promotion. Taken together, it is shown that endophytic pseudomonas bacteria C145 can promote the growth of the red sage root seedling and improve the quality of the red sage root seedling.
EXAMPLE 5 Effect of functional bacteria on yield and content of drug-Properties of Danshen in field
The field was divided into 9 cells, each 3 m long and 1.8 m wide, three OF which were applied with Organic Fertilizer (OF), three OF which were not applied with fertilizer (CK), and the other three OF which were applied with organic fertilizer, and then the endophytic pseudomonas bacteria C145 bacterial liquid (of+c145) was poured in. That is, the experiment had 3 treatments in total, three replicates each, T1 (CK), T2 (OF), T3 (OF+C145), respectively. The application amount of the organic fertilizer was 6000kg/hm 2, i.e. 3.24kg of organic fertilizer was applied per cell. After one week of fertilization, root seedlings of the red sage root (purchased from a Shandong Weifang) are transplanted, the planting density is 25 multiplied by 30cm, soil is loosened and weeding is performed in time after the transplanting, and the water drainage is kept smooth during the whole growth period, so that the red sage root is prevented from waterlogging.
30 Days and 45 days after the red sage root is transplanted, C145 bacterial liquid (the concentration is 1X 10 8 CFU/mL, the bacterial liquid is obtained by diluting the bacterial liquid in example 3 by 10 times with clean water (tap water)) is respectively added into the T3 treated field, and the inoculation amount of each inoculation is 100mL of red sage root (directly poured into the soil and near the root of the red sage root). 4 months after the red sage root is transplanted, 3 red sage roots are selected from each cell of each treatment, and the plant height, leaf area (leaf length x leaf width x 0.75) and stem thickness (the measurement method is the same as in example 4) are measured. Half a year after the red sage root is transplanted, taking out the underground part of the whole red sage root, measuring the fresh weight of the whole red sage root, and taking the average value of the fresh weight of each treated three cells as the yield of each treated red sage root. As shown in Table 2 and FIGS. 7-10, compared with T1 (CK) treatment, T3 (OF+C145) treatment significantly improved the plant height, leaf area, stem thickness and yield (P < 0.05) OF Salvia Miltiorrhiza, by 65.74%, 56.97%, 35.14% and 78.78%, respectively. Compared with T2 (OF) treatment, the T3 treatment improves the plant height, the leaf area and the yield OF the red sage root, wherein the difference between the plant height and the yield OF the red sage root reaches significance, and the plant height and the yield OF the red sage root are respectively improved by 5.34 percent and 32.56 percent. The test result shows that the endophytic pseudomonas bacteria C145 can promote the growth of the red sage root and help to improve the yield.
Air-drying the harvested red sage root, mixing and grinding 5 thicker red sage roots and 5 thinner red sage roots in each cell, and simultaneously measuring the chemical component content in the red sage root by adopting ultra-high performance liquid chromatography (UPLC) and triple quadrupole mass spectrometry (UPLC-QQQ-MS). As shown in table 3 and fig. 11 to 15, the content OF tanshinone iia in three treatments OF T1 (CK), T2 (OF), and T3 (of+c145) was sequentially increased, but there was no significant difference between the three treatments; compared with T1 (CK) treatment, the T3 (OF+C145) treatment obviously improves the contents OF tanshinone I, tanshinone, salvianolic acid A and salvianolic acid B OF the salvia miltiorrhiza, and respectively improves the contents by 71.60%, 170.97%, 91.38% and 223.12%; compared with T2 (OF) treatment, T3 (OF+C145) treatment improves the contents OF tanshinone I, tanshinone, salvianolic acid A and salvianolic acid B OF the salvia miltiorrhiza, wherein the difference OF tanshinone I, tanshinone and salvianolic acid B OF the salvia miltiorrhiza reaches significance, and the contents OF 86.58%, 58.49% and 36.09% are respectively improved. The test result shows that the endophytic pseudomonas bacteria C145 remarkably improves the content of medicinal components of the red sage root, thereby improving the quality of the red sage root.
TABLE 1 Effect of different Strain treatments on the growth of Danshen seedlings (leaf Length, plant height, stem thickness)
TABLE 2 Effect of inoculating endophytic Pseudomonas bacterium C145 on growth and yield of Salvia Miltiorrhiza
TABLE 3 Effect of inoculation of endophytic Pseudomonas bacterium C145 on the content of drug-Properties of Salvia Miltiorrhiza
Example 6 identification of Strain C145
As shown in FIG. 1, endophytic Pseudomonas bacteria C145 are white oval in shape, smooth in surface, moist and easy to pick up on a plate. By aligning the 16s rRNA gene sequence of the strain C145 with similar sequences, a developmental tree is constructed. The results in FIG. 2 show that the homology of strain C145 with Pseudomonas Pseudomonas kairouanensis KC is high. The strain C145 is preserved in China general microbiological culture Collection center (CGMCC) with the preservation number of CGMCC NO.29111.
Claims (9)
1. The endophytic pseudomonas bacteria C145 are preserved in China general microbiological culture Collection center (China Committee for culture Collection of microorganisms) with a preservation date of 2023, 11 and 22 days, and a preservation number of CGMCC NO.29111.
2. The microbial inoculum prepared by endophytic pseudomonas bacteria C145 of claim 1.
3. The microbial agent according to claim 2, wherein the microbial agent is prepared by the following method: and (3) carrying out liquid fermentation on endophytic pseudomonas bacteria C145 with the preservation number of CGMCC NO.29111, centrifuging after fermentation to obtain thalli, and re-suspending the thalli with sterile water to obtain bacterial suspension.
4. A microbial agent according to claim 3, wherein the microbial agent is prepared by the following method: inoculating endophytic pseudomonas bacteria C145 with the preservation number of CGMCC NO.29111 into a liquid culture medium for liquid fermentation, wherein the liquid culture medium comprises a TSB liquid culture medium, an LB liquid culture medium or an R2A liquid culture medium, and the liquid fermentation conditions are as follows: the temperature is 28-30 ℃, the rotating speed is 170-200 r.min -1, and the fermentation time is 1-2d; after fermentation, the bacteria are obtained by centrifugation, and the bacteria are resuspended by sterile water to obtain bacterial suspension, wherein the bacterial content in the bacterial suspension is more than or equal to 1 multiplied by 10 9 CFU/mL.
5. The use of endophytic pseudomonas bacteria C145 as set forth in claim 1 for promoting the growth of salvia miltiorrhiza, improving the quality of salvia miltiorrhiza, including improving the quality of salvia miltiorrhiza seedlings and improving the yield of salvia miltiorrhiza; improving quality of Saviae Miltiorrhizae radix comprises improving content of Saviae Miltiorrhizae radix medicinal components including tanshinone I, tanshinone IIA, tanshinone, salvianolic acid A and salvianolic acid B.
6. Use of the microbial inoculum of any one of claims 2-4 for cultivating high quality seedlings of red sage root.
7. The use according to claim 6, wherein, in the application, the red sage seeds are buried in the seedling raising matrix, and after the red sage seeds germinate for a period of time, the microbial inoculum is applied twice, wherein the application amount of each application is not less than 1 x 10 7 CFU/g dry weight seedling raising matrix, and the interval between the two applications is 10-14 days.
8. Use of the microbial inoculum of any one of claims 2-4 for increasing yield of salvia miltiorrhiza and content of drug-resistant components including tanshinone i, tanshinone iia, tanshinone, salvianolic acid a, salvianolic acid B.
9. The use according to claim 8, wherein, in the application, the decomposed organic fertilizer is used as a base fertilizer, the microbial inoculum is applied twice for a period of time after the red sage root is transplanted, the application amount of each time is not less than 1 x 10 10 CFU/plant of red sage root, and the interval between the two applications is 15-20 days.
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