Method for promoting growth of houttuynia cordata and increasing content of medicinal ingredients

Abstract

The present invention discloses a method for promoting the growth of houttuynia cordata and increasing the content of medicinal ingredients, belonging to the technical field of houttuynia cordata cultivation. The present invention discloses a Rhizobium sp. bacterium BH46, whose deposit number is GDMCC No.: 63513; and a method for inoculating houttuynia cordata with the Rhizobium sp. bacterium BH46. The present invention uses a suspension of the Rhizobium sp. bacteria as a biological fertilizer and sprays it directly on houttuynia cordata plants. The houttuynia cordata seedlings cultivated by this method have a high survival rate, vigorous growth, and strong adaptability. During harvest, the yield of houttuynia cordata is increased and the content of medicinal ingredients is improved. The method is safe, simple, convenient, easy to implement, and has significant effects, which is conducive to promotion and application.

Description

Technical Field

[0001] The invention relates to the technical field of houttuynia cordata cultivation, in particular to a method for promoting the growth of houttuynia cordata and increasing the content of medicinal ingredients. Background Art

[0002] As a traditional Chinese medicine, Herba Houttuyniae has the effects of clearing away heat and toxic substances, eliminating carbuncle and draining pus, promoting diuresis and relieving stranguria, and is rich in substances such as flavonoids and polyphenols. It has anti-oxidation, anti-leukocyte, anti-mutation, anti-inflammatory effects and biological activities such as promoting the immune system. Epidemiological studies have shown that regular consumption of Herba Houttuyniae can effectively reduce the incidence and mortality of diseases such as cardiovascular, myocardial infarction and cancer. With the improvement of living standards, people have a higher pursuit of the yield and quality of Herba Houttuyniae. However, due to the shortcomings of traditional selective cultivation technology and the limitation of land area, improving the yield of Herba Houttuyniae and improving the content of medicinal ingredients have become problems to be solved urgently. For this reason, the present invention proposes a cultivation method for improving the yield and content of medicinal ingredients of Herba Houttuyniae by inoculating bacteria. Summary of the Invention

[0003] The purpose of the present invention is to provide a method for promoting the growth of Houttuynia cordata and increasing the content of medicinal ingredients, so as to solve the problems existing in the above-mentioned prior art. The present invention uses a suspension of Rhizobium bacteria as a biological fertilizer and directly sprays it on Houttuynia cordata plants, thereby increasing the yield of Houttuynia cordata and improving the content of medicinal ingredients.

[0004] To achieve the above object, the present invention provides the following solutions:

[0005] The present invention provides a Rhizobium sp. bacterium BH46, which was deposited in the Guangdong Provincial Microbial Culture Collection Center (GDMCC) on June 1, 2023, with a deposit number of GDMCC No: 63513, and the deposit address is the Institute of Microbiology, Guangdong Academy of Sciences, 5th Floor, Building 59, Compound 100 Xianlie Middle Road, Guangzhou.

[0006] The present invention also provides a method for promoting the growth of houttuynia cordata and increasing the content of medicinal ingredients, comprising the step of inoculating the rhizobium bacteria BH46 into houttuynia cordata.

[0007] Further, the following steps are included:

[0008] Choose yellow soil with loose texture, sufficient nutrients, good drainage, appropriate organic matter content and low sand and gravel ratio as seedling soil;

[0009] The roots of Houttuynia cordata seedlings are placed in a suspension of Rhizobium bacteria BH46 to obtain Houttuynia cordata seedlings with bacteria;

[0010] Transplanting the infected Houttuynia cordata seedlings into nursery soil to cultivate Houttuynia cordata cultivated seedlings;

[0011] The Houttuynia cordata seedlings were transferred to natural soil, sprayed with a suspension of Rhizobium bacteria BH46, and cultured according to conventional water and fertilizer management methods until harvest.

[0012] Furthermore, the organic matter content of the seedling soil is 400-2200 g / kg, the clay silicon-aluminum ratio is 1.5 to 3.0, and the silicon-iron-aluminum ratio is 2 to 2.5.

[0013] Furthermore, the concentration of Rhizobium bacteria BH46 in the suspension is 10 3 -10 6 CFU / mL.

[0014] Furthermore, the roots of the Houttuynia cordata seedlings were placed in a suspension of Rhizobium bacteria BH46 and cultured at 28° C. with shaking for 30 minutes.

[0015] Furthermore, the spraying method is: repeat the spraying once every 14 days until harvest.

[0016] The present invention also provides a use of the Rhizobium bacteria BH46 in preparing a biological fertilizer for promoting the growth of Houttuynia cordata and increasing the content of medicinal ingredients.

[0017] The present invention also provides a biofertilizer for promoting the growth of Houttuynia cordata and increasing the content of medicinal ingredients, which comprises an effective dose of Rhizobium bacteria BH46.

[0018] Furthermore, the effective dose of the Rhizobium bacteria BH46 is 10 3 -10 6 CFU / mL.

[0019] The present invention discloses the following technical effects:

[0020] The present invention uses a suspension of Rhizobium bacteria as a biofertilizer and sprays it directly on Houttuynia cordata plants. This method cultivates Houttuynia cordata seedlings with a high survival rate, vigorous growth, and strong adaptability. During harvest, the yield of Houttuynia cordata and the content of its active ingredients are increased. The method is safe, simple, convenient, and effective, and is conducive to widespread application. BRIEF DESCRIPTION OF THE DRAWINGS

[0021] In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the following briefly introduces the drawings required for use in the embodiments. Obviously, the drawings described below are only some embodiments of the present invention. For ordinary technicians in this field, other drawings can be obtained based on these drawings without paying any creative work.

[0022] Figure 1Flowchart of the method for promoting the growth of Houttuynia cordata and increasing the content of medicinal ingredients according to Example 2;

[0023] Figure 2 Flow chart for cultivating infected Houttuynia cordata seedlings into cultivated seedlings;

[0024] Figure 3 The phylogenetic tree of BH46 strain is Rhizobium sp.;

[0025] Figure 4 This is the bacterial morphology of the BH46 strain. DETAILED DESCRIPTION

[0026] Various exemplary embodiments of the present invention will now be described in detail. This detailed description should not be considered as limiting the present invention, but rather as a more detailed description of certain aspects, features, and embodiments of the present invention.

[0027] It should be understood that the terms described herein are intended only to describe particular embodiments and are not intended to limit the present invention. In addition, for numerical ranges herein, it should be understood that each intermediate value between the upper and lower limits of the range is also specifically disclosed. The intermediate value within any stated value or stated range, and each smaller range between any other stated value or intermediate value within the stated range, is also encompassed within the present invention. The upper and lower limits of these smaller ranges may be independently included or excluded within the scope.

[0028] Unless otherwise indicated, all technical and scientific terms used herein have the same meaning as commonly understood by those skilled in the art. Although only preferred methods and materials are described herein, any methods and materials similar or equivalent to those described herein may also be used in the practice or testing of the present invention. All documents mentioned in this specification are incorporated by reference to disclose and describe the methods and / or materials associated with the documents. In the event of any conflict with any incorporated document, the contents of this specification shall prevail.

[0029] It will be apparent to those skilled in the art that various modifications and variations may be made to the specific embodiments described herein without departing from the scope or spirit of the invention. Other embodiments will be apparent to those skilled in the art from the description of the invention. The description and examples are intended to be illustrative only.

[0030] The words “include,” “including,” “have,” “contain,” etc. used in this document are open-ended terms, meaning including but not limited to.

[0031] Example 1 Isolation, purification and identification of Rhizobium bacteria

[0032] Purification: Fresh, healthy rhizomes of Houttuynia cordata were collected, cleaned of fibrous roots and attached soil, and rinsed under running water for 4 hours. The cleaned rhizomes were cut into 2-3 cm segments, surface-sterilized by soaking in 75% ethanol (v / v), shaken for 3 minutes, then shaken in 0.2% HgCl₂ (w / v) solution for 5 minutes. Rinse with sterile water five times, 2 minutes each time. The sterilized segments were blotted dry with sterile filter paper on a laminar flow hood, the ends removed, and cut into 5 mm segments along the growth direction. Five to six segments of Houttuynia cordata rhizomes were placed in an Erlenmeyer flask containing 50 ml of beef extract peptone broth (NA, 3 g / L beef extract, 5 g / L peptone, 5 g / L NaCl) and incubated in the dark at 28°C on a shaker at 80 rpm for 3 days. To check whether the surface disinfection of the rhizomes is thorough, the sterilized rhizomes were directly rinsed with sterile phosphate buffer (PH=7.4) and the rinse was cultured on NA. The culture conditions were the same as above. No bacterial colonies were detected, indicating that the sterilized rhizomes were sterile explants. The obtained bacterial solution was diluted to 10 -5 -10 -7 The strain was spread on a plate, and a single colony was selected and streaked three times to purify the strain numbered BH46 for storage.

[0033] Identification: BH46 bacterial strain was selected for DNA extraction. DNA was extracted using the Ezup column-based bacterial genomic DNA extraction kit (Sangon Biotech). Primers used for strain identification were: 27F: 5'-AGAGTTTGATCMTGGCTCAG-3' (SEQ ID NO: 1); 1492R: 5'-GGTTACCTTGTTACGACTT-3' (SEQ ID NO: 2). Polymerase chain reaction (PCR) was performed in a 25 μL reaction mixture containing 0.2 μL enzyme, 0.5 μL template DNA, 0.5 μL (10 μM) forward primer, 0.5 μL (10 μM) reverse primer, and 2.5 μL 10× buffer (containing Mg). 2+ ) and 1 μL of DNTP, and double-distilled water was added to a total volume of 25 μL. The PCR amplification procedure included: initial denaturation at 94°C for 4 minutes; denaturation at 94°C for 45 seconds, annealing at 55°C for 45 seconds, and extension at 72°C for 1 minute, followed by 30 cycles of denaturation-annealing-extension; a final extension at 7°C for 10 minutes, followed by termination at 4°C; and visualization using 1% agarose gel electrophoresis at 150 V and 100 mA for 20 minutes. The 16S rDNA sequence was obtained and compared with sequences submitted to the NCBI database using the BLAST search program (http: / / www.ncbi.nlm.nih.gov). Phylogenetic analysis was performed using the neighbor-joining method using MEGA7 software. Figure 3 ).

[0034] Identification results: BH46 is a Rhizobium sp. bacterium with accession number: JQ697680.1:2-1386. The bacterial morphology of BH46 strain is as follows: Figure 4 shown.

[0035] The BH46 strain was classified as Rhizobium sp. BH46 and was deposited in the Guangdong Provincial Microbial Culture Collection (GDMCC) on June 1, 2023. The deposit number is GDMCC No: 63513. The deposit address is the Institute of Microbiology, Guangdong Academy of Sciences, 5th Floor, Building 59, Compound 100 Xianlie Middle Road, Guangzhou.

[0036] Example 2

[0037] The cultivated seedlings described in this embodiment mainly refer to mature Houttuynia cordata plant seedlings that have a certain plant height and can be cultivated in natural soil.

[0038] The Houttuynia cordata described in this embodiment belongs to the plant Houttuynia cordata Thunb. of the Saururaceae family.

[0039] Figure 1 The figure is a flow chart of the method for promoting the growth of Houttuynia cordata and increasing the content of medicinal ingredients according to the method of Example 2.

[0040] A method for promoting the growth of houttuynia cordata and increasing the content of medicinal ingredients, comprising the following steps:

[0041] S1: Preparation and sterilization of high-quality seedling soil

[0042] Select texture to be loose, nutrient is sufficient, drainage is good, organic content is suitable, the yellow soil with low sand and gravel ratio, grind after natural air drying, after removing plant residue, meeting the yellow soil that organic content is 400-2200g / kg, clay silicon aluminum ratio 1.5 to 3.0, silicon iron aluminum ratio 2 to 2.5 is seedling soil, the seedling soil organic content of the present embodiment is 1000g / kg, clay silicon aluminum ratio is 1.8, silicon iron aluminum ratio is 2, through 121 ℃ of high temperature steam sterilization 60min, be sub-packed into black seedling bag after cooling;

[0043] S2: Tissue culture of Houttuynia cordata seedlings

[0044] Houttuynia cordata seeds were sterilized with 75% alcohol and sterilized seedlings were cultured on MS medium until the plant height reached 5-7 cm.

[0045] The stem tips of Houttuynia cordata can also be used for tissue culture seedling cultivation. The plant cells in these parts have strong differentiation ability, which is conducive to subsequent cultivation to form complete seedlings.

[0046] S3: Preparation of Rhizobium bacterial suspension

[0047] BH46 Rhizobium bacterial suspension was prepared using beef extract peptone agar medium (NA). Specifically, the strain was inoculated into beef extract peptone agar medium (NA) to culture Rhizobium bacteria. After activation for 3 days, the bacterial strain was picked up with an inoculation loop and placed in a 50 mL conical flask containing 20 mL of sterile water to prepare a bacterial suspension. The bacterial suspension was placed on a shaker at 28 ° C and 180 rpm for 15 min. The resulting Rhizobium bacterial suspension concentration was 10 5 CFU / mL.

[0048] S4: Bacterial inoculation of Houttuynia cordata seedlings

[0049] The roots of the sterile Houttuynia cordata seedlings were placed in a suspension of Rhizobium bacteria and cultured with shaking at 28°C for 30 minutes. This time setting can avoid the probability of damage to the Houttuynia cordata seedlings, and finally obtain the infected Houttuynia cordata seedlings.

[0050] S5: Transplanting infected Houttuynia cordata seedlings

[0051] Transplant the infected Houttuynia cordata tissue culture seedlings into the seedling soil at a depth of 2-3 cm and cover with fine soil for compaction; water thoroughly every other day for the first 14 days, spray water on the leaves appropriately, avoid direct sunlight and high temperatures, and acclimate to the natural environment to obtain Houttuynia cordata cultivated seedlings;

[0052] The purpose of transplanting infected seedlings into sterile culture soil is to cultivate the seedlings into seedlings with a certain plant height, high bacterial activity and cultivation survival rate, and be able to adapt to growing into mature plants in natural soil.

[0053] Figure 2 Flowchart for cultivating infected Houttuynia cordata seedlings into cultivated seedlings.

[0054] S6: Houttuynia cordata cultivation

[0055] Planting the Houttuynia cordata seedlings together with the seedling soil in natural soil at a depth sufficient to cover the soil in the seedling bag, spraying the bacterial suspension solution every 14 days, cultivating the Houttuynia cordata in a natural environment, and supplementing water and fertilizer every 30 days until harvest;

[0056] The purpose of regularly spraying the Rhizobium bacteria suspension is to prevent the dominance of the Rhizobium bacteria in Houttuynia cordata from being affected in the natural environment during its growth.

[0057] Effect verification

[0058] Three Houttuynia cordata plants obtained by the method of Example 2 were randomly selected and labeled as A, B, and C. Three Houttuynia cordata plants that were not inoculated with bacteria under the same culture conditions were selected as controls. The fresh weight, total rhizome length, leaf area, chlorogenic acid, rutin, chelidonin, and quercetin contents were measured. The indices measured for the fresh Houttuynia cordata produced by inoculating bacteria were compared with the average values ​​of the various indices of Houttuynia cordata obtained without inoculating bacteria. The increase ratio of each indicator after Houttuynia cordata was inoculated with Rhizobium bacteria was calculated. The increase in each indicator of Houttuynia cordata after inoculation compared with the control group is shown in Table 1:

[0059] Table 1 Statistical results of Houttuynia cordata indicators in each group

[0060] Ratio of increase in indicators A B C Fresh weight 63.6% 83.6% 54.7% Total rhizome length 85.5% 122.6% 61.1% Total leaf area 109.0% 182.6% 136.3% Chlorogenic acid 101.2% 89.3% 121.0% Rutin 76.2% 91.5% 62.7% cyperus foetida 168.0% 148.9% 120.1% Quercetin 265.2% 241.2% 187.8%

[0061] The results in Table 1 indicate that the fresh weight, total rhizome length, and total leaf area of ​​Houttuynia cordata cultivated using this method were significantly increased. Furthermore, the content of the active ingredients chlorogenic acid and rutin in Houttuynia cordata increased by more than 60%, and the content of scutellarin and calciferol increased by more than 100%. This demonstrates that the yield of Houttuynia cordata was significantly increased and the content of its active ingredients was significantly improved after inoculation with Rhizobium bacteria. Furthermore, this method is simple to operate, convenient, and easy to implement, making it suitable for widespread application.

[0062] The embodiments described above are merely descriptions of preferred embodiments of the present invention and are not intended to limit the scope of the present invention. Without departing from the spirit of the present invention, various modifications and improvements made to the technical solutions of the present invention by persons skilled in the art should fall within the scope of protection defined by the claims of the present invention.

Claims

1. A Rhizobium sp. bacterium BH46, characterized in that It was deposited in the Guangdong Provincial Microbial Culture Collection on June 1, 2023, with the deposit number GDMCC No: 63513, and the deposit address is the Institute of Microbiology, Guangdong Academy of Sciences, 5th Floor, Building 59, No. 100 Xianlie Middle Road, Guangzhou.

2. A method for promoting the growth of Houttuynia cordata and increasing the content of medicinal ingredients, characterized in that: The method comprises the step of inoculating the Rhizobium bacteria BH46 described in claim 1 into Houttuynia cordata; the medicinal ingredients are chlorogenic acid, rutin, chlorophyllin and quercetin.

3. The method according to claim 2, characterized in that The following steps are involved: Choose yellow soil with loose texture, sufficient nutrients, good drainage, appropriate organic matter content and low sand and gravel ratio as seedling soil; The roots of Houttuynia cordata seedlings are placed in a suspension of Rhizobium bacteria BH46 to obtain Houttuynia cordata seedlings with bacteria; Transplanting the infected Houttuynia cordata seedlings into nursery soil to cultivate Houttuynia cordata cultivated seedlings; The Houttuynia cordata seedlings were transferred to natural soil, sprayed with a suspension of Rhizobium bacteria BH46, and cultured according to conventional water and fertilizer management methods until harvest.

4. The method according to claim 3, characterized in that The clay-silicon-aluminum ratio of the seedling soil is 1.5-3.0, and the silicon-iron-aluminum ratio is 2-2.

5.

5. The method according to claim 3, characterized in that The concentration of Rhizobium bacteria BH46 in the suspension is 10 3 -10 6 CFU / mL.

6. The method according to claim 3, characterized in that The roots of the Houttuynia cordata seedlings were placed in a suspension of Rhizobium bacteria BH46 and cultured with shaking at 28° C. for 30 min.

7. The method according to claim 3, characterized in that The spraying method is: spraying is repeated once every 14 days until harvest.

8. A use of the Rhizobium bacteria BH46 as claimed in claim 1 in the preparation of a biofertilizer for promoting the growth of Houttuynia cordata and increasing the content of medicinal ingredients, characterized in that: The medicinal ingredients are chlorogenic acid, rutin, clematisin and quercetin.

9. A biofertilizer for promoting the growth of Houttuynia cordata and increasing the content of medicinal ingredients, characterized in that: The invention comprises an effective dose of Rhizobium bacteria BH46; the deposit number of the Rhizobium bacteria BH46 is GDMCC No: 63513.

10. The biological fertilizer according to claim 9, characterized in that The effective dose of the Rhizobium bacteria BH46 is 10 3 -10 6 CFU / mL.

Images