Crocus sativus rhizosphere microorganism and application thereof

Abstract

The invention discloses a saffron rhizosphere microorganism and application thereof, and belongs to the technical field of quality and quality improvement of traditional Chinese medicinal materials. The rhizosphere microorganism is separated from a crocus sativus living body of an orris crocus plant by adopting a traditional bacterium separation and purification technology, belongs to bacteria of actinomycetes, mycobacteria, mycobacteriaceae and mycobacterial actinomycetes, and is classified and named as (Mycolicbacteria frederiksbergense) SRB560, and the preservation number of the strain is CGMCC (China General Microbiological Culture Collection Center) No.29735. The invention further discloses a preparation method of the rhizosphere microorganism. The rhizosphere microorganism can effectively promote the growth of radix scrophulariae and the accumulation of the content of effective components, and the yield of medicinal materials is increased by 50.08% (Plt; 0.05), the total amount of harpagide and harpagoside is increased by 25.56% (Plt; 0.05) of the method. The rhizosphere microorganism has obvious effects of promoting the growth of the radix scrophulariae and improving the quality of the radix scrophulariae medicinal material, and brings a wide application prospect for the industrial development of the radix scrophulariae.

Description

Technical Field

[0001] The invention relates to the technical field of improving the yield and quality of Chinese medicinal materials, and specifically relates to a saffron rhizosphere microorganism and an application thereof in promoting the growth of the Chinese medicinal material Scrophularia ningpoensis and the accumulation of effective components. Background Art

[0002] High-quality, stable and safe medicinal materials are the prerequisite for ensuring the clinical use of traditional Chinese medicine. Studies have found that the quality of medicinal materials has a direct impact on the clinical effect of traditional Chinese medicine. If the quality of medicinal materials is not sufficiently guaranteed, the patient's treatment effect cannot be effectively exerted, and it will also have a great impact on the patient's recovery. Poor-quality Chinese medicinal materials not only have no therapeutic effect, but may even produce some toxic side effects and endanger human health. Therefore, good and stable Chinese medicinal materials are the prerequisite for ensuring the efficacy and safety of clinical use of traditional Chinese medicine.

[0003] Scrophulariaceae is also known as Yuan Shen, Zhejiang Scrophulariaceae, Wu Xuan (Yuan) Shen, etc. It is the dried root of the perennial herbaceous plant Scrophulariaceae and Northern Scrophulariaceae, which is a specialty of my country. It is one of the "Eight Flavors of Zhejiang". It tastes sweet, bitter and salty, is slightly cold in nature, and enters the lung, spleen, stomach and kidney meridians. It has the effects of clearing heat and cooling blood, nourishing the kidney and nourishing yin, moistening dryness and producing fluid, reducing fire and relieving restlessness, detoxifying, dispersing knots and relieving sore throat, and anti-inflammatory and sterilizing. Traditional Chinese medicine is often used for febrile diseases with heat entering the blood, damaging yin and consuming blood, with fever and thirst, red tongue and fever, rashes caused by warm toxins, vomiting blood, epistaxis, spontaneous sweating, night sweats, constipation caused by intestinal dryness, thirst, cough caused by dry lungs, cough caused by fatigue, hot flashes caused by bone steaming, hot and humid heat in the five hearts, insomnia caused by deficiency, restlessness at night, red and dry eyes, blurred vision, sore throat, diphtheria, scrofula, phlegm nuclei, carbuncle and sore.

[0004] Due to long-term indiscriminate mining, wild resources of Scrophularia are on the verge of exhaustion, but with the breakthrough of artificial cultivation technology of Scrophularia, the Scrophularia industry has developed rapidly. However, studies have found that the characteristic secondary metabolites of Scrophularia are affected by both environmental and genetic factors, which can easily lead to instability in the quality of medicinal materials, which will affect the quality of Scrophularia Chinese patent medicine and clinical drug safety. At the same time, in actual production of Scrophularia, asexual buds are often used for reproduction. Although this reproduction method shortens the production cycle of Scrophularia, it is easy to cause the problem of bud degeneration, resulting in a decrease in the yield and quality of Scrophularia.

[0005] Rhizosphere microorganisms refer to microorganisms that are tightly attached to rhizosphere soil particles, mainly bacteria, among which Gram-negative bacteria are dominant, and the most common ones are Pseudomonas, Bacillus, Mycobacterium, etc. There is a mutualistic relationship between rhizosphere microorganisms and plants, and they interact and promote each other with plant roots. Microorganisms gather around the root system in large numbers, converting organic matter into inorganic matter, providing effective nutrients for plants; at the same time, microorganisms can also secrete vitamins, growth stimulants, etc. to promote plant growth. During the growth of plants, dead roots and root debris (root hairs, epidermal cells, root caps, etc.), as well as inorganic and organic matter secreted by the roots to the outside of the roots are important sources of nutrition and energy for microorganisms; due to the interlacing of the roots, the ventilation conditions and water conditions in the rhizosphere are better than those outside the rhizosphere, thus forming an ecological environment that is conducive to microorganisms. With the continuous deepening of research on microbial diversity and function, more and more evidence shows that plant rhizosphere microorganisms have a significant impact on the growth and development of crops. 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, provide nutrients and growth hormones for rice, and promote the growth and development of rice. At the same time, rice rhizosphere microorganisms can also degrade harmful substances, such as pesticide residues and heavy metals, to protect the healthy growth of rice.

[0006] However, there are no reports on the application of saffron rhizosphere microorganisms to improve the yield and accumulation of effective ingredients of Scrophularia ningpoensis. Summary of the invention

[0007] The purpose of the present invention is to provide an application of saffron rhizosphere microorganisms to improve the yield of Scrophularia ningpoensis and the accumulation of effective ingredient content in view of the deficiencies in the prior art.

[0008] In the first aspect of the present invention, a saffron rhizosphere microorganism is provided, belonging to the actinomycetes, mycobacteriales, mycobacteriaceae, mycolic acid rod-shaped bacteria, classified and named (Mycolicibacteriumfrederiksbergense) SRB560, and the strain collection number is CGMCC No.29735. The rhizosphere microorganism is obtained by separating and purifying the rhizosphere soil of Crocus sativus L., a plant of the genus Crocus in the family Iridaceae, using traditional bacterial separation and purification technology, and is identified as mycolic acid rod-shaped bacteria (Mycolicibacteriumfrederiksbergense) SRB560 by microbial taxonomy. The strain has been preserved, the strain collection number is CGMCC No.29735, the preservation date is January 23, 2024, and the preservation unit is the China General Microbial Culture Collection Management Center (CGMCC), the address is No. 3, Yard 1, Beichen West Road, Chaoyang District, Beijing, and the zip code is 100101.

[0009] The solid culture characteristics of the endophytic fungi of the present invention are:

[0010] Cultured on LB solid medium at 28°C, it grows faster, the colonies are moist milky yellow, with smooth and round edges, and can be seen Figure 1 Under the microscope, the bacteria are short rod-shaped, slightly twisted, and some cells have septa but no branches. Figure 2 shown.

[0011] The 16S amplification electrophoresis diagram of the rhizosphere microorganisms of the present invention is shown in Figure 3 , the 16S molecular sequence is shown in SEQ ID NO.1.

[0012] The sequencing results were compared on the NCBI website (http: / / blast.ncbi.nlm.nih.gov / Blast.cgi), and the sequence homology with Mycolicibacterium frederiksbergense (MW578385), Mycolicibacteriumfrederiksbergense (MT634615), and Mycolicibacterium frederiksbergense (KR476406) was 99.71%, 99.43%, and 99.22%, respectively. Multiple sequence alignment was performed using MEGA7.0, and neighbor-joining (NJ) analysis was performed. One sequence was randomly selected and the alignment was repeated 1000 times. The phylogenetic tree of the endophytic fungi of the present invention is shown in Figure 4 .

[0013] The second aspect of the present invention provides the use of the above-mentioned plant rhizosphere microorganisms in promoting the growth of Scrophularia ningpoensis and the accumulation of effective ingredient content.

[0014] Specifically, the saffron rhizosphere microorganisms are prepared into biological fertilizer and applied to Scrophularia ningpoensis to promote the growth of Scrophularia ningpoensis and the accumulation of effective ingredients.

[0015] The biological fertilizer is prepared by inoculating a bacterial suspension fermented in LB liquid culture medium into a fertilizer matrix, wherein the fertilizer matrix is ​​mixed in a mass ratio of wheat bran: rice straw powder: rice husk: corn residue = 15:10:10:3, and water is added and mixed well; the saffron rhizosphere microbial suspension concentration OD 660 ≥0.5.

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

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

[0018] (1) Take a SRB560 cryopreservation tube, and aseptically pipette 5 μL of bacterial solution, spread it on 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), and culture it in the dark at 28°C and 65% humidity for 2 days;

[0019] (2) Pick a single clone and inoculate it into LB liquid medium (formula: 10 g peptone, 5 g yeast extract, 1000 mL pure water, pH adjusted to 7.0-7.4), place it in a shaker, culture at 28°C, 180 rpm for 2 days, and adjust the concentration to a uniform value (OD 660 ≥0.5), obtain bacterial suspension, see Figure 5 ;

[0020] (3) Wheat bran: rice straw powder: rice husk: corn residue = 15:10:10:3 was selected as the fertilizer matrix, with a water content of 50% (w / v, g / mL) as the standard, water was added and mixed well, placed in a 2L conical flask, sealed with 4 layers of gauze and 1 layer of sealing film, sterilized at 126°C for 1 h, and cooled to room temperature;

[0021] (4) The suspension was inoculated into the fermentation matrix at an inoculum rate of 20 vol%, and cultured at 28°C in the dark for 7 days to obtain the biological fertilizer. Figure 6 .

[0022] Furthermore, the application rate of biofertilizer is 3000kg / hm 2 / times, for a total of 4 times.

[0023] Furthermore, the application of biological fertilizer can increase the yield of the Chinese medicinal herb Scrophularia ningpoensis and increase the total amount of harpagoside and harpagoside.

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

[0025] The advantages of the present invention are:

[0026] The plant rhizosphere microorganism (Mycolicibacterium frederiksbergense) SRB560 of the present invention can promote the growth of Scrophularia ningpoensis by being made into a biological fertilizer and applied, and the yield of Scrophularia ningpoensis medicinal materials is increased by 50.08% (P<0.05), and the total amount of harpagoside and harpagoside is increased by 25.56% (P<0.05), which brings broad application prospects to the field of improving the high-yield and high-quality cultivation of Chinese medicinal materials. BRIEF DESCRIPTION OF THE DRAWINGS

[0027] The present invention is further described below in conjunction with the accompanying drawings and embodiments;

[0028] Figure 1 :(Mycolicibacterium frederiksbergense)SRB560 colony morphology;

[0029] Figure 2 :(Mycolicibacterium frederiksbergense)SRB560 bacterial morphology under microscope;

[0030] Figure 3 : Gel electrophoresis of SRB560 of Mycolicibacterium frederiksbergense amplified based on 16S primers;

[0031] Figure 4 :16S-based phylogenetic tree of Mycolicibacteriumfrederiksbergense SRB560 (NJ method);

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

[0033] Figure 6 : Photo of prepared (Mycolicibacterium frederiksbergense) SRB560 biological fertilizer;

[0034] Figure 7 : The result diagram of (Mycolicibacterium frederiksbergense) SRB560 biological fertilizer improving the yield of Scrophularia ningpoensis, harpagoside and the total amount of harpagoside (different letters for the same indicator indicate significant differences between groups at P<0.05). DETAILED DESCRIPTION

[0035] The rhizosphere microorganism of the invention is a strain separated from the rhizosphere soil of saffron produced in Jiande, Hangzhou, Zhejiang.

[0036] In the following examples, unless otherwise indicated, % refers to volume percentage.

[0037] Embodiment 1:

[0038] The rhizosphere microorganisms were isolated and obtained by the following steps: fresh saffron bulbs were taken, soil tightly bound to the bulbs was gently brushed with a brush, and plant tissue was removed from the soil through a 200-mesh sieve to obtain a rhizosphere soil sample. 1.0 g of rhizosphere soil and sterilized glass beads were added to a conical flask containing 9.0 mL of sterile pure water, and the mixture was placed on a shaker at 160 rpm for 30 min to prepare 10 -1 Concentration (w / v) of rhizosphere soil suspension. Take 1000 μL10 -1 The concentration of rhizosphere soil suspension was added with 9.0 mL of sterile pure water, mixed and diluted to 10 -2 The above process was repeated to dilute the rhizosphere soil suspension to 10 -3 , 10 -4 , 10 -5 The concentration of rhizosphere soil suspension was taken, 100 μL was applied on LB medium to separate rhizosphere microorganisms (LB medium formula is 10g tryptone, 10g sodium chloride, 5g yeast extract, 15g agar, 1L pure water, pH = 7.0-7.2), and placed in a mold incubator at 28°C for 2-5 days (depending on the number of colonies and growth conditions), and the grown colonies were picked into new culture medium, and the process was repeated continuously to finally obtain the rhizosphere microorganism SRB560 of the present invention, such as Figure 1 As shown in the figure, the colony is moist and creamy yellow, with smooth and rounded edges. Under the microscope, the bacteria are short rod-shaped, slightly twisted, and some cells have septa but no branches. Figure 2 shown.

[0039] Take the SRB560 strain stored at low temperature, and under sterile conditions, take 5 μL of bacterial liquid with a pipette, streak and spread it on LB solid medium (LB medium formula is 10g tryptone, 10g sodium chloride, 5g yeast extract, 15g agar, 1L pure water, pH=7.0-7.2), and culture it in the dark at 28℃ and 65% humidity until it matures, which is used for 16S sequence amplification and molecular identification. Primers 1492R and 27F are used to amplify its ITS sequence by PCR. PCR reaction cycle parameters amplification steps: 1. 95℃, 3min, initial denaturation; 2. 94℃, 40s, denaturation; 3. 52℃, 50s, annealing; 4. 72℃, 1min, extension; 5. 2-4 repeated cycles 35 times; 6. 72℃, 10min, extension. The PCR product detected by agarose gel electrophoresis was sent to Shanghai Shenggong Company for sequence determination, and the sequence is shown in SEQ ID NO.1. The sequence obtained from the PCR product was used as the target sequence, and the homologous sequence was searched in the GenBank database in NCBI. The reference sequence most similar to the morphological sequence was downloaded, and the neighbor-joining method (NJ) was used for phylogenetic analysis to determine the phylogenetic status of the strain to be identified, such as Figure 3 ,4 The rhizosphere microorganism of the present invention has sequence homology of 99.71%, 99.43% and 99.22% with Mycolicibacterium frederiksbergense (MW578385), Mycolicibacteriumfrederiksbergense (MT634615) and Mycolicibacterium frederiksbergense (KR476406), respectively, and belongs to Actinobacteria, Mycobacteriales, Mycobacteriaceae and Mycolicobacterium, and is classified and named (Mycolicibacterium frederiksbergense) SRB560, and the strain collection number is CGMCC No.29735.

[0040] Embodiment 2:

[0041] Under sterile conditions, use an inoculation needle to pick up a small amount of bacteria and place them in a freshly prepared LB solid medium for activation. Culture them at 28°C in the dark until they mature, then inoculate them into 1L of LB liquid medium and expand and culture them at 180rpm until they mature to obtain a bacterial suspension. Figure 5 Wheat bran: rice straw powder: rice husk: corn residue were prepared in a mass ratio of 15:10:10:3, and then water was added to a standard of 50% (w / v, g / mL) of water content, and mixed well as a bacterial fertilizer matrix. The suspended bacterial liquid was inoculated into the bacterial fertilizer matrix at an inoculation rate of 20 vol%, and cultured at 28°C in a dark environment for 7 days for bacterial fertilizer fermentation to obtain biological bacterial fertilizer, such as Figure 6 After being made into biological fertilizer, it was applied to the field of Scrophularia ningpoensis. A compound fertilizer group and a sterilized matrix group (wheat bran: rice straw powder: rice husk: corn residue prepared at a mass ratio of 15:10:10:3) were set up as controls. The application rate of each group was 3000 kg / hm 2 / times, a total of 4 times, fertilization time is early March, late April, early June, early September. The per mu yield of the biological fertilizer application group and the compound fertilizer group and the sterilized matrix group was compared. Figure 7 As shown, the yield of the biological fertilizer treatment group was 1338.47±189.03kg / mu, the yield of the compound fertilizer treatment group was 891.84±81.03kg / mu, and the yield of the sterilized matrix treatment group was 937.10±108.15. The study showed that the application of biological fertilizer can promote the growth of Scrophularia ningpoensis, increasing the yield by 50.08% (P<0.05) compared with the compound fertilizer group and 42.83% (P<0.05) compared with the sterilized matrix group.

[0042] The washed root of Scrophularia ningpoensis was sliced ​​and placed in a 60°C oven to dry to constant weight, crushed and passed through a No. 3 sieve. Accurately weigh 0.5 g of Scrophularia ningpoensis sample powder, placed in a stoppered conical flask, added 10 mL of 50 vol% methanol, sealed, shaken, weighed, left standing at room temperature for 1 hour, ultrasonically extracted (300 W, 40 kHz) for 45 minutes, cooled, weighed again, supplemented with 50 vol% methanol to make up the loss, shaken, centrifuged (12 000 r / min, 5 minutes), took the supernatant, filtered through a 0.45 μm microporous filter membrane, and took the filtrate. Preparation of reference solution: Accurately weigh an appropriate amount of each reference substance, add methanol to prepare a mixed reference solution of harpagine and harpagine with a mass concentration of 1.56 and 0.90 mg / mL, respectively. The reference solutions of different mass concentrations were diluted with methanol and injected according to the following chromatographic conditions: Symmetry C18 chromatographic column (250mm×4.6mm, 5μm); the mobile phase was acetonitrile (A)-0.03% phosphoric acid water (B), gradient elution: 0-8min, 5% A; 8-20min, 5%-16% A; 20-28min, 16%-24% A; 28-38min, 24%-44% A; 38-39min, 44%-5% A; 39-44min, 5% A; volume flow rate 1.00mL / min; column temperature 35℃; injection volume 10μL; programmed wavelength change: 0-19min, 210nm; 19-44min, 280nm. 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 of harpagide was Y1=3236228.24X1-55303.66, r 2 =0.9999, the linear range is 0.0487~1.5600mg / mL; the linear equation of harpagine is Y2=23645778.75X2+4109.30, r 2 =0.9999, the linear range is 0.0017~0.9000mg / mL. According to the linear equation, the content of active ingredients in Scrophularia ningpoensis under different treatments was calculated. It was found that the total content of Scrophularia ningpoensis harpagine and harpagine in the SRB560 biological fertilizer treatment group was 16.36±0.04mg / g, the total content of Scrophularia ningpoensis harpagine and harpagine in the sterilized matrix treatment group was 13.03±0.05mg / g, and the total content of Scrophularia ningpoensis harpagine and harpagine in the compound fertilizer treatment group was 14.06±0.05mg / g. Compared with the sterilized matrix treatment group, the total content of harpagine and harpagine in the SRB560 biological fertilizer increased by 25.56% (P<0.05), and increased by 16.34% (P<0.05) compared with the compound fertilizer treatment group ( Figure 7 ).

[0043] The preferred embodiments of the present invention have been specifically described above, but the present invention is not limited to the embodiments. Those skilled in the art may make various equivalent modifications or substitutions without violating the spirit of the present invention. These equivalent modifications or substitutions are all included in the scope defined by the claims of this application.

Claims

1. A saffron rhizosphere microorganism, characterized in that: The rhizospheric microorganisms are isolated from the rhizospheric soil of saffron, a plant of the genus Crocus in the family Iridaceae. They belong to the phylum Actinomycetes, the order Mycobacteriales, the family Mycobacterium, and the genus Mycolicibacterium. They were deposited in the China General Microbiological Culture Collection Center on January 23, 2024, and were classified and named (Mycolicibacterium frederiksbergense) SRB560, and the culture collection number is CGMCC No.29735.

2. An application of saffron rhizosphere microorganisms according to claim 1, characterized in that: Used to promote the growth of Scrophularia and the accumulation of effective ingredients.

3. The use according to claim 2, characterized in that: The saffron rhizosphere microorganisms are prepared into biological fertilizer and applied to Scrophularia ningpoensis to promote the growth of Scrophularia ningpoensis and the accumulation of effective components.

4. The use according to claim 3, characterized in that: The biological fertilizer comprises a saffron rhizosphere microbial suspension and a fertilizer matrix, wherein the fertilizer matrix is ​​mixed in a mass ratio of wheat bran: rice straw powder: rice husk: corn residue = 15:10:10:3, and water is added and mixed to obtain the biological fertilizer; the saffron rhizosphere microbial suspension has an OD concentration of 660 ≥0.

5.

5. The use according to claim 4, characterized in that: The saffron rhizosphere microbial suspension is prepared by culturing in LB liquid culture medium.

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

7. The use according to claim 2, characterized in that: The promoting effect of the plant rhizosphere microorganisms is manifested in the increase of root tuber yield and the total amount of harpagoside and harpagoside.

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