Crocus rhizosphere microorganism and application thereof

Abstract

The application discloses a crocus rhizosphere microorganism and application thereof, and belongs to the technical field of traditional Chinese medicinal material quality and quality improvement. The rhizosphere microorganism is obtained by using traditional bacterial separation and purification technology from a crocus rhodora plant crocus living body, belongs to the actinobacteria phylum, mycobacterium order, mycobacterium family and mycolicibacterium genus bacteria, is classified and named as (Mycolicibacterium frederiksbergense) SRB560, and the strain preservation number is CGMCC No. 29735. The application also discloses the application of the rhizosphere microorganism. The rhizosphere microorganism can effectively promote the growth and effective component content accumulation of radix scrophulariae, improves the medicinal material yield by 50.08% (P<0.05), and improves the total amount of harpagide and harpagoside by 25.56% (P<0.05). The rhizosphere microorganism has obvious effects on promoting the growth of radix scrophulariae and improving the quality of radix scrophulariae, and brings wide application prospects for the development of the radix scrophulariae industry.

Description

Technical Field

[0001] This invention relates to the field of improving the yield and quality of Chinese medicinal materials. Specifically, it relates to a rhizosphere microorganism of saffron and its application in promoting the growth and accumulation of effective components in the Chinese medicinal herb Scrophularia. Background Technology

[0002] High-quality, stable, and safe medicinal materials are prerequisites for ensuring the effectiveness of traditional Chinese medicine (TCM) clinical applications. Studies have found that the quality of medicinal materials directly impacts the clinical efficacy of TCM. If the quality of medicinal materials is not adequately guaranteed, the therapeutic effect on patients cannot be effectively achieved, and it will significantly affect their recovery. Poor-quality medicinal materials not only lack efficacy but may also produce toxic side effects, harming human health. Therefore, high-quality and stable medicinal materials are essential for ensuring the efficacy and safety of TCM clinical applications.

[0003] Scrophularia, also known as Yuan Shen, Zhe Xuan Shen, and Wu Xuan (Yuan) Shen, is the dried root of Scrophularia ningpoensis and Scrophularia buergeriana, perennial herbaceous plants of the Scrophulariaceae family and Scrophularia genus, native to my country. It is one of the "Eight Treasures of Zhejiang". It tastes sweet, bitter, and salty, and is slightly cold in nature. It enters the lung, spleen, stomach, and kidney meridians. Its effects include clearing heat and cooling blood, nourishing the kidneys and yin, moistening dryness and generating fluids, reducing fire and relieving irritability, detoxifying and dispersing nodules, benefiting the throat, and anti-inflammatory and antibacterial properties. In Traditional Chinese Medicine, it is commonly used for febrile diseases where heat enters the blood level, damaging yin and depleting blood, manifesting as fever, thirst, crimson tongue, febrile rashes, hematemesis, epistaxis, spontaneous sweating, night sweats, constipation due to fluid depletion, diabetes, dry cough, consumptive cough, bone steaming fever, five-center heat (heat in the palms, soles, and chest), insomnia due to deficiency, restlessness at night, red and dry eyes, blurred vision, sore throat, diphtheria, scrofula, carbuncles, and boils.

[0004] Due to long-term over-harvesting, wild resources of Scrophularia ningpoensis are nearing depletion. However, with breakthroughs in artificial cultivation techniques, the Scrophularia ningpoensis industry has developed rapidly. Research has found that the characteristic secondary metabolites of Scrophularia ningpoensis are susceptible to instability in quality due to both environmental and genetic factors, affecting the quality of Scrophularia ningpoensis-based traditional Chinese medicines and the safety of clinical use. Furthermore, asexual propagation through budding is commonly used in actual production of Scrophularia ningpoensis. While this method shortens the production cycle, it easily leads to bud degeneration, resulting in a decline in both yield and quality.

[0005] Rhizosphere microorganisms refer to the microorganisms tightly attached to soil particles in the rhizosphere, primarily bacteria, with Gram-negative bacteria being the most prevalent, the most common being *Pseudomonas*, *Bacillus*, and *Mycobacterium*. A symbiotic relationship exists between rhizosphere microorganisms and plants; they interact and promote each other. Microorganisms congregate in large numbers around the roots, converting organic matter into inorganic matter, providing effective nutrients for the plant. Simultaneously, microorganisms secrete vitamins and growth stimulants, promoting plant growth. During plant growth, dead roots and root abscissions (root hairs, epidermal cells, root cap, etc.), as well as inorganic and organic matter secreted by the roots, are important sources of nutrition and energy for microorganisms. Due to the interpenetration of the root system, the aeration and moisture conditions in the rhizosphere are better than those outside the rhizosphere, thus creating an ecological environment conducive to microorganisms. With the deepening research into the diversity and function of microorganisms, increasing evidence shows that plant rhizosphere microorganisms have a significant impact on crop growth and development. With the help of rhizosphere microorganisms, plants can better utilize nutrients and water in the soil and resist various adversities. For example, rice rhizosphere microorganisms can decompose organic matter and fix nitrogen, providing nutrients and growth hormones to promote rice growth and development. At the same time, rice rhizosphere microorganisms can also degrade harmful substances such as pesticide residues and heavy metals, protecting the healthy growth of rice.

[0006] However, there are currently no reports on the application of using rhizosphere microorganisms of saffron to enhance the yield and accumulation of active ingredients in Scrophularia ningpoensis. Summary of the Invention

[0007] The purpose of this invention is to address the shortcomings of existing technologies by providing an application of saffron rhizosphere microorganisms to enhance the yield and accumulation of effective components in Scrophularia ningpoensis.

[0008] In a first aspect, this invention provides a rhizosphere microorganism of saffron, belonging to the phylum Actinobacteria, order Mycobacteriales, family Mycobacteraceae, and genus Mycolicibacterium, classified as Mycolicibacterium frederiksbergense SRB560, with the strain preservation number CGMCC No. 29735. This rhizosphere microorganism was isolated from the rhizosphere soil of saffron (Crocus sativus L.), a plant of the genus Mycolicibacterium in the family Iridaceae, using traditional bacterial isolation and purification techniques. Microbiological taxonomy identification confirmed it as Mycolicibacterium frederiksbergense SRB560. This strain has been deposited, with the strain preservation number CGMCC No. 29735, the deposit date being January 23, 2024, at the China General Microbiological Culture Collection Center (CGMCC), located at No. 3, Courtyard 1, Beichen West Road, Chaoyang District, Beijing, 100101, China.

[0009] The solid culture characteristics of the endophytic fungi described in this invention are as follows:

[0010] When cultured on LB solid medium at 28°C, growth is rapid, with moist, milky-yellow colonies that are smooth-edged and round. Figure 1 Under a microscope, the bacteria appear as short rods, slightly twisted, with some cells having septa and no branching. Figure 2 As shown.

[0011] The 16S amplification electrophoresis image of the rhizosphere microorganisms described in this invention is shown below. Figure 3 The 16S molecular sequence is shown in SEQ ID NO.1.

[0012] Sequence alignment was performed on the NCBI website (http: / / blast.ncbi.nlm.nih.gov / Blast.cgi). The sequence homology with *Mycolicibacterium frederiksbergense* (MW578385), *Mycolicibacterium frederiksbergense* (MT634615), and *Mycolicibacterium frederiksbergense* (KR476406) was 99.71%, 99.43%, and 99.22%, respectively. Multiple sequence alignment was performed using MEGA 7.0, and neighbor-joining (NJ) analysis was used. One sequence was randomly selected and the alignment was repeated 1000 times. The phylogenetic tree of the endophytic fungus described in this invention is shown below. Figure 4 .

[0013] In a second aspect, the present invention provides the application of the above-mentioned plant rhizosphere microorganisms in promoting the growth of Scrophularia ningpoensis and the accumulation of effective components.

[0014] Specifically, the saffron rhizosphere microorganisms are prepared into a bio-fertilizer and applied to Scrophularia ningpoensis to promote its growth and the accumulation of effective components.

[0015] The bio-fertilizer is prepared by inoculating a bacterial suspension fermented in LB liquid medium into a microbial fertilizer substrate. The microbial fertilizer substrate is prepared by mixing wheat bran, rice straw powder, rice husk, and corn grits in a mass ratio of 15:10:10:3, and then adding water and mixing thoroughly. The saffron rhizosphere microbial suspension is concentrated at OD... 660 ≥0.5.

[0016] The formula for LB liquid medium is: 10g peptone, 5g yeast extract, 1000mL pure water, and pH adjusted to 7.0-7.4.

[0017] The preparation method of bio-fertilizer is as follows:

[0018] (1) Take an SRB560 cryopreservation tube, and under aseptic conditions, pipette 5 μL of bacterial solution and streak it onto LB solid medium (LB medium formula is 10 g tryptone, 10 g sodium chloride, 5 g yeast extract, 15 g agar, 1 L pure water, pH = 7.0-7.2). Incubate in the dark at 28℃ and 65% humidity for 2 days.

[0019] (2) Pick single-clone bacterial cells and inoculate them into LB liquid medium (formulation: 10g peptone, 5g yeast extract, 1000mL pure water, pH adjusted to 7.0-7.4), place in a shaker, and incubate at 28℃ and 180rpm for 2 days. Adjust to a uniform concentration (OD) with sterile water. 660 ≥0.5), obtain bacterial suspension, see Figure 5 ;

[0020] (3) Select wheat bran: rice straw powder: rice husk: corn residue = 15:10:10:3 as the substrate for microbial fertilizer. With a water content of 50% (w / v, g / mL) as the standard, add water and mix well. Place it in a 2L conical flask, seal it with 4 layers of gauze and 1 layer of sealing film, and sterilize it at 126℃ for 1 hour. Cool it to room temperature.

[0021] (4) Inoculate the bacterial suspension into the fermentation substrate at an inoculum rate of 20 vol%, and culture at 28°C in the dark for 7 days to obtain bio-fertilizer. See [link to bio-fertilizer]. Figure 6 .

[0022] Furthermore, the application rate of bio-fertilizer is 3000 kg / hm². 2 / time, for a total of 4 applications.

[0023] Furthermore, the application of bio-fertilizers can increase the yield of the traditional Chinese medicine Scrophularia ningpoensis and increase the total amount of harpagoside and harpagoside.

[0024] Furthermore, harbazoside and harbazoside were determined by high performance liquid chromatography (HPLC).

[0025] The advantages of this invention are:

[0026] The plant rhizosphere microorganism (Mycolicibacterium frederiksbergense) SRB560 described in this invention, when formulated into a bio-fertilizer, can promote the growth of Scrophularia ningpoensis, increasing the yield of Scrophularia ningpoensis medicinal materials by 50.08% (P<0.05) and the total amount of harpagoside and harpagoside by 25.56% (P<0.05). This presents broad application prospects for improving the high-yield and high-quality cultivation of Chinese medicinal herbs. Attached Figure Description

[0027] The present invention will be further described below with reference to the accompanying drawings and embodiments;

[0028] Figure 1 Colony morphology of (Mycolicibacterium frederiksbergense)SRB560;

[0029] Figure 2 The bacterial morphology of (Mycolicibacterium frederiksbergense)SRB560 under a microscope;

[0030] Figure 3 Gel electrophoresis image of Mycolicibacterium frederiksbergense SRB560 amplified using 16S primers;

[0031] Figure 4 Phylogenetic tree of Mycolicibacterium frederiksbergense SRB560 based on 16S (NJ method);

[0032] Figure 5 Photograph of the prepared (Mycolicibacterium frederiksbergense) SRB560 bacterial suspension;

[0033] Figure 6 Photograph of the prepared (Mycolicibacterium frederiksbergense) SRB560 bio-fertilizer;

[0034] Figure 7 The results of SRB560 bio-fertilizer improving the yield of Scrophularia ningpoensis and the total amount of harpagoside and harpagoside (different letters for the same indicator indicate that there is a significant difference between groups at P<0.05). Detailed Implementation

[0035] The rhizosphere microorganisms of this invention are strains isolated from the rhizosphere soil of saffron produced in Jiande, Hangzhou, Zhejiang Province.

[0036] In the following embodiments, unless otherwise specified, % refers to volume percentage.

[0037] Example 1:

[0038] The rhizosphere microorganisms were isolated and obtained according to the following steps: Fresh saffron bulbs were taken, and the soil tightly bound to the bulbs was gently brushed away with a brush. The soil was then passed through a 200-mesh sieve to remove plant tissue, thus obtaining a rhizosphere soil sample. 1.0 g of rhizosphere soil and sterilized glass beads were added to an Erlenmeyer flask containing 9.0 mL of sterile pure water, and the flask was shaken at 160 rpm for 30 minutes to prepare a solution of 10... -1 A rhizosphere soil suspension at a concentration (w / v). Take 1000 μL of 10 -1 Dilute the rhizosphere soil suspension to a concentration of 10 with 9.0 mL of sterile pure water, mix well and dilute to 100%. -2 A concentration of rhizosphere soil suspension was diluted using the above process to obtain 10. -3 10 -4 10 -5 A 100 μL suspension of rhizosphere soil at a specific concentration was spread onto LB medium to isolate rhizosphere microorganisms (LB medium formulation: 10 g tryptone, 10 g sodium chloride, 5 g yeast extract, 15 g agar, 1 L pure water, pH 7.0-7.2). The medium was then placed in a mold incubator and incubated at 28°C. Incubation was continued for 2–5 days (depending on colony quantity and growth). The resulting colonies were transferred to a fresh medium, and this process was repeated until the rhizosphere microorganism SRB560 of this invention was obtained. Figure 1 As shown, the colonies are moist, milky yellow, with smooth, round edges. Under a microscope, the bacteria appear as short rods, slightly twisted, with some cells having septa and no branching. Figure 2 As shown.

[0039] SRB560 strain, preserved at low temperature, was used. Under aseptic conditions, 5 μL of bacterial culture was pipetted and streaked onto LB agar (LB medium formulation: 10 g tryptone, 10 g sodium chloride, 5 g yeast extract, 15 g agar, 1 L pure water, pH 7.0-7.2). It was incubated in the dark at 28℃ and 65% humidity until mature, and then used for 16S sequence amplification and molecular identification. The ITS sequence was amplified by PCR using primers 1492R and 27F. The PCR reaction cycle parameters were as follows: 1. 95℃, 3 min, initial denaturation; 2. 94℃, 40 s, denaturation; 3. 52℃, 50 s, annealing; 4. 72℃, 1 min, extension; 5. Cycles 2-4 were repeated 35 times; 6. 72℃, 10 min, expansion. The PCR product, after agarose gel electrophoresis, was sent to Shanghai Sangon Biotech Co., Ltd. for sequencing. The sequence is shown in SEQ ID NO.1. Using the PCR product sequence as the target sequence, homologous sequences were searched in the GenBank database of NCBI. The reference sequence most similar to the morphological sequence was downloaded, and neighbor-joining (NJ) was used for phylogenetic analysis to determine the phylogenetic position of the strain to be identified. Figure 3 ,4 As shown. The rhizosphere microorganism of the present invention has sequence homology of 99.71%, 99.43%, and 99.22% with Mycolicibacterium frederiksbergense (MW578385), Mycolicibacterium frederiksbergense (MT634615), and Mycolicibacterium frederiksbergense (KR476406), respectively. It belongs to the phylum Actinobacteria, order Mycobacteriales, family Mycobacteriales, and genus Mycolicibacterium, and is classified as (Mycolicibacterium frederiksbergense) SRB560, with the strain preservation number CGMCC No. 29735.

[0040] Example 2:

[0041] Under aseptic conditions, a small amount of bacterial cells is picked up with an inoculation needle and placed in freshly prepared LB solid medium for activation. The cells are then cultured at 28°C in the dark until mature. The culture is then inoculated into 1L of LB liquid medium and cultured at 180 rpm until mature to obtain the bacterial suspension. Figure 5 As shown. Wheat bran, rice straw powder, rice husks, and corn grits were mixed in a mass ratio of 15:10:10:3. Water was added and mixed thoroughly to create a microbial fertilizer substrate with a moisture content of 50% (w / v, g / mL). The bacterial suspension was inoculated into the substrate at a rate of 20 vol%. The substrate was then incubated at 28℃ in the dark for 7 days to allow for microbial fermentation, yielding the bio-fertilizer. Figure 6 As shown. After being processed into bio-fertilizer, it was applied to the field of Scrophularia ningpoensis. A compound fertilizer group and a sterilized substrate group (prepared with wheat bran, rice straw powder, rice husk, and corn residue in a mass ratio of 15:10:10:3) were set up as controls. The application rate for each group was 3000 kg / hm². 2 The fertilizer was applied four times, in early March, late April, early June, and early September. The yield per acre was compared between the bio-fertilizer group, the compound fertilizer group, and the sterilized substrate group. Figure 7 As shown, the yield of the bio-fertilizer treatment group was 1338.47±189.03 kg / mu, the yield of the compound fertilizer treatment group was 891.84±81.03 kg / mu, and the yield of the sterilized substrate treatment group was 937.10±108.15 kg / mu. The study indicates that the application of bio-fertilizer can promote the growth of Scrophularia ningpoensis, increasing yield by 50.08% compared to the compound fertilizer group (P<0.05) and by 42.83% compared to the sterilized substrate group (P<0.05).

[0042] Slices of the washed Scrophularia root were dried in a 60℃ oven to constant weight, pulverized, and passed through a No. 3 sieve. 0.5 g of the Scrophularia sample powder was accurately weighed and placed in a stoppered conical flask. 10 mL of 50 vol% methanol was added, the flask was sealed tightly, shaken well, and the mass was measured. The flask was allowed to stand at room temperature for 1 h, then ultrasonically extracted (300 W, 40 kHz) for 45 min. After cooling, the mass was measured again, and the amount lost was replenished with 50 vol% methanol. The flask was shaken well and centrifuged (12,000 r / min, 5 min). The supernatant was collected and filtered through a 0.45 μm microporous membrane. The filtrate was collected as the final product. Preparation of reference solutions: Appropriate amounts of each reference standard were accurately weighed and added to methanol to prepare a mixed reference solution of harpagoside and harpagoside with mass concentrations of 1.56 mg / mL and 0.90 mg / mL, respectively. The reference solutions were diluted with methanol to prepare different mass concentrations and injected under the following chromatographic conditions: Symmetry C18 column (250 mm × 4.6 mm, 5 μm); mobile phase: acetonitrile (A) - 0.03% phosphoric acid water (B); gradient elution: 0–8 min, 5% A; 8–20 min, 5%–16% A; 20–28 min, 16%–24% A; 28–38 min, 24%–44% A; 38–39 min, 44%–5% A; 39–44 min, 5% A; flow rate: 1.00 mL / min; column temperature: 35 ℃; injection volume: 10 μL; programmed wavelength: 0–19 min, 210 nm; 19–44 min, 280 nm. A linear regression was performed with the mass concentration of the reference solution as the abscissa (X) and the peak area as the ordinate (Y). The linear equation for harbazoside was obtained as Y1 = 3236228.24X1 - 55303.66, r 2 =0.9999, with a linear range of 0.0487–1.5600 mg / mL; the linear equation for harbazoside is Y² = 23645778.75X² + 4109.30, r 2 =0.9999, with a linear range of 0.0017–0.9000 mg / mL. Based on the linear equation, the content of active ingredients in Scrophularia ningpoensis under different treatments was calculated. It was found that the total content of harpagoside and harpagoside in the SRB560 bio-fertilizer treatment group was 16.36 ± 0.04 mg / g, the total content of harpagoside and harpagoside in the sterilized matrix treatment group was 13.03 ± 0.05 mg / g, and the total content of harpagoside and harpagoside in the compound fertilizer treatment group was 14.06 ± 0.05 mg / g. Compared with the sterilized matrix treatment group, the total content of harpagoside and harpagoside in the SRB560 bio-fertilizer treatment group increased by 25.56% (P<0.05), and compared with the compound fertilizer treatment group, it increased by 16.34% (P<0.05). Figure 7 ).

[0043] The preferred embodiments of the present invention have been described in detail above, but the present invention is not limited to the embodiments described. Those skilled in the art can make various equivalent modifications or substitutions without departing from the spirit of the present invention, and these equivalent modifications or substitutions are all included within the scope defined by the claims of this application.

Claims

1. A rhizosphere microorganism of saffron, characterized in that, The rhizosphere microorganisms were isolated from the rhizosphere soil of saffron (Saffron saffron, Iridaceae family), belonging to the phylum Actinobacteria, order Mycobacteriales, family Mycobacteraceae, and genus Mycobacterium. They were deposited at the China General Microbiological Culture Collection Center on January 23, 2024, and classified as Mycobacterium spp. (…). Mycolicibacterium frederiksbergense The strain has the preservation number CGMCC No. 29735.

2. An application of the saffron rhizosphere microorganisms as described in claim 1, characterized in that, The plant rhizosphere microorganisms are used to promote the growth of Scrophularia ningpoensis and the accumulation of effective components. The promoting effect is manifested in the increase of tuber yield and the total amount of harpagoside and harpagoside.

3. The application according to claim 2, characterized in that, The saffron rhizosphere microorganisms were prepared into a bio-fertilizer and applied to Scrophularia ningpoensis to promote its growth and the accumulation of effective components.

4. The application according to claim 3, characterized in that, The bio-fertilizer comprises a saffron rhizosphere microbial suspension and a fertilizer substrate, wherein the fertilizer substrate is prepared by mixing wheat bran, rice straw powder, rice husk, and corn grits in a mass ratio of 15:10:10:3, and then adding water and mixing thoroughly; the saffron rhizosphere microbial suspension has a concentration of OD... 660 ≥0.

5.

5. The application according to claim 4, characterized in that, The saffron rhizosphere microbial suspension was prepared by culturing in LB liquid medium.

6. The application according to claim 2, characterized in that, The application rate is 3000 kg / hm. 2 / time, for a total of 4 applications.

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