Leonurus growth-promoting microbial inoculum based on stenotrophomonas and preparation method and application of leonurus growth-promoting microbial inoculum

Abstract

The invention relates to the field of agricultural planting, and provides a leonurus growth-promoting microbial agent based on stenotrophomonas, which comprises stenotrophomonas and an LB liquid culture medium. The invention also provides a preparation method and application of the growth-promoting microbial agent. Meanwhile, the invention also provides a method for promoting the growth of motherwort seedlings, namely applying the growth-promoting microbial agent to the motherwort seedlings. According to the method, the leonurus growth-promoting microbial agent based on the stenotrophomonas is applied in the growth of the leonurus seedlings, so that the leonurus seedlings are promoted to adapt to the environment, the absorption of the leonurus seedlings to soil nutrients and moisture is promoted, the development and growth of the seedlings are accelerated, and the problems of low growth rate and poor growth vigor of the seedlings in the existing leonurus planting are solved; the method has important significance for planting motherwort and providing the benefits of pharmacy by taking motherwort as a raw material.

Description

Technical Field

[0001] The present invention relates to the field of agricultural planting, and in particular to a motherwort growth-promoting bacterial agent based on Stenotrophomonas, and a preparation method and application thereof. Background Art

[0002] Motherwort, the tender stems and leaves of the Lamiaceae plant, promotes blood circulation, regulates menstruation, and promotes diuresis and swelling. It is used to treat irregular menstruation, dysmenorrhea, amenorrhea, lochia retention, edema, oliguria, and edema caused by acute nephritis. It is a common, bulk medicinal ingredient in clinical prescriptions and a key raw material for pharmaceutical companies. Research shows that over 500 traditional Chinese medicine ingredients marketed use motherwort as an ingredient, demonstrating significant market demand. Therefore, large-scale cultivation of motherwort is crucial.

[0003] However, during the cultivation of motherwort, it was found that the seedlings often experienced slow growth rates and poor growth. Analysis showed that the reason for this is that the growing seedlings cannot quickly and effectively utilize substances in the soil environment, resulting in slowed growth and poor growth. The existence of these problems will greatly reduce the benefits of using motherwort as a raw material for pharmaceutical production, resulting in significant economic losses.

[0004] Symbiosis between plants and rhizosphere-promoting bacteria is a mutually beneficial relationship between plant roots and soil bacteria, which has a significant impact on plant growth and ecological function. These bacteria activate soil nutrients, improve soil physical and chemical properties, increase soil fertility, antagonize pathogens, reduce plant diseases, improve disease resistance, enhance salt tolerance, cold tolerance, and heavy metal toxicity resistance, promote crop growth and development, increase crop yield, and improve quality. Therefore, the symbiotic relationship between plants and rhizosphere-promoting bacteria could be explored to cultivate motherwort, potentially addressing the low seedling growth rate and poor growth seen in current motherwort cultivation. Summary of the Invention

[0005] The technical problem to be solved by the present invention is to provide a motherwort growth-promoting agent based on Stenotrophomonas, as well as its preparation method and application. By applying the growth-promoting agent during the growth of motherwort seedlings, the motherwort seedlings are promoted to adapt to the environment, their absorption of soil nutrients and water is promoted, and the development and growth of the seedlings are accelerated, thereby solving the problems of low seedling growth rate and poor seedling growth in the current motherwort planting.

[0006] The present invention adopts the following technical solutions to solve the above technical problems:

[0007] A motherwort growth-promoting bacterial agent based on Stenotrophomonas comprises Stenotrophomonas and LB liquid culture medium.

[0008] As one of the preferred embodiments of the present invention, the Stenotrophomonas is specifically Stenotrophomonas geniculata.

[0009] As one of the preferred embodiments of the present invention, the composition of the LB liquid culture medium ( / L) is as follows: 5 g YEASTEXTRACT, 10 g NaCl, and 10 g peptone.

[0010] As one of the preferred embodiments of the present invention, the OD of Stenotrophomonas spp. in the Leonurus japonicus growth-promoting agent is 600 The value is 0.95 to 1.05, and more preferably 1.00.

[0011] A preparation method of the above-mentioned motherwort growth-promoting bacterial agent based on Stenotrophomonas is as follows: Stenotrophomonas is inoculated into the culture medium at a volume ratio of 1:(100-200) between Stenotrophomonas and LB liquid culture medium, and finally the culture is dark cultured at 25°C-28°C.

[0012] As one of the preferred embodiments of the present invention, the volume ratio of the Stenotrophomonas to the LB liquid culture medium is 1:200.

[0013] An application of the above-mentioned motherwort growth-promoting bacterial agent based on Stenotrophomonas in promoting the growth of motherwort seedlings.

[0014] A method for promoting the growth of motherwort seedlings: applying the motherwort growth-promoting bacterial agent based on Stenotrophomonas to the motherwort seedlings.

[0015] As one of the preferred embodiments of the present invention, the specific steps are as follows:

[0016] (1) Prepare a planting pit using black soil from Zingiber officinale;

[0017] (2) Sow motherwort seeds in the planting pit, cover with soil, and water;

[0018] (3) After the motherwort seeds germinate and the motherwort seedlings grow, the motherwort growth-promoting agent based on Stenotrophomonas is poured into the motherwort.

[0019] As one of the preferred embodiments of the present invention, in step (1), the length × width × depth of the planting pit is (1.5-2.5 cm) × (1.5-2.5 cm) × (1-3 cm), more preferably 2 cm × 2 cm × 1.5 cm; the pit distance is 8-10 cm, more preferably 8 cm.

[0020] As one of the preferred embodiments of the present invention, in step (2), the thickness of the covering soil is 1 to 3 cm, more preferably 1.5 cm.

[0021] As one of the preferred embodiments of the present invention, in step (2), watering is to pour clean water after covering the soil to allow water to penetrate, and cover the ground with a film to prevent the surface soil from drying out. Watering is done once a day, and each time watering is done until the soil surface is moist.

[0022] As one of the preferred embodiments of the present invention, in step (3), the amount of the motherwort growth-promoting agent based on Stenotrophomonas is poured in is 8 to 12 mL / time, more preferably 10 mL / time.

[0023] The advantages of the present invention over the prior art are:

[0024] (1) The present invention promotes the establishment of a symbiotic relationship between motherwort and plant rhizosphere growth-promoting bacteria by applying a motherwort growth-promoting agent based on Stenotrophomonas during the growth of motherwort seedlings, which is beneficial to the physiological and ecological functions of plant rhizosphere growth-promoting bacteria, promotes the absorption of soil nutrients and water by seedlings, accelerates the development and growth of seedlings, and solves the problems of low seedling growth rate and poor growth of seedlings in the current motherwort planting. It is of great significance to the planting of motherwort and the benefits of using motherwort as a raw material for pharmaceutical production.

[0025] (2) The method of the motherwort growth-promoting agent based on Stenotrophomonas adopted in the present invention is easy to operate and has a simple process, which is more convenient for agricultural and forestry workers to implement;

[0026] (3) The present invention uses green and pollution-free products, which do not pollute the environment and produce beneficial mycorrhizal symbionts, which play an important role in maintaining ecological balance. BRIEF DESCRIPTION OF THE DRAWINGS

[0027] Figure 1 This is a comparison of the growth of Leonurus japonicus in the blank control group (CK), experimental group (N) and commercially available Bacillus subtilis control group (K) in Experimental Example 1;

[0028] Figure 2 This is a comparison chart of the total fresh weight results of Leonurus japonicus in the blank control group (CK), experimental group (N) and commercially available Bacillus subtilis control group (K) in Experimental Example 1;

[0029] Figure 3 This is a comparison chart of the fresh weight of the aboveground part of Leonurus japonicus in the blank control group (CK), experimental group (N) and commercially available Bacillus subtilis control group (K) in Experimental Example 1;

[0030] Figure 4 This is a comparison chart of the fresh weight of the underground part of Leonurus japonicus in the blank control group (CK), experimental group (N) and commercially available Bacillus subtilis control group (K) in Experimental Example 1;

[0031] Figure 5 This is a comparison chart of the total dry weight results of Leonurus japonicus in the blank control group (CK), experimental group (N) and commercially available Bacillus subtilis control group (K) in Experimental Example 1;

[0032] Figure 6 This is a comparison chart of the dry weight results of the aboveground part of Leonurus japonicus in the blank control group (CK), experimental group (N) and commercially available Bacillus subtilis control group (K) in Experimental Example 1;

[0033] Figure 7 This is a comparison chart of the dry weight results of the underground part of Leonurus japonicus in the blank control group (CK), experimental group (N) and commercially available Bacillus subtilis control group (K) in Experimental Example 1;

[0034] Figure 8 This is a comparison chart of the total length results of Leonurus japonicus in the blank control group (CK), experimental group (N) and commercially available Bacillus subtilis control group (K) in Experimental Example 1;

[0035] Figure 9 This is a comparison chart of the plant height results of Leonurus japonicus in the blank control group (CK), experimental group (N) and commercially available Bacillus subtilis control group (K) in Experimental Example 1;

[0036] Figure 10 This is a comparison chart of the root length results of Leonurus japonicus in the blank control group (CK), experimental group (N) and commercially available Bacillus subtilis control group (K) in Experimental Example 1;

[0037] Figure 11 This is a comparison chart of the leaf area results of Leonurus japonicus in the blank control group (CK), experimental group (N) and commercially available Bacillus subtilis control group (K) in Experimental Example 1. DETAILED DESCRIPTION

[0038] The following examples of the present invention are described in detail. These examples are based on the technical solutions of the present invention and provide detailed implementation methods and specific operating procedures. However, the scope of protection of the present invention is not limited to the following examples. At the same time, the experimental methods used in the following examples are conventional methods unless otherwise specified. The materials, reagents, etc. used in the following examples are all commercially available unless otherwise specified.

[0039] Example 1

[0040] The motherwort growth-promoting bacterial agent based on Stenotrophomonas geniculata of this embodiment includes Stenotrophomonas geniculata and LB liquid culture medium.

[0041] Among them, Stenotrophomonas geniculata is recorded in the document "Omomowo, Olawale I., Babalola, Olubukola O. Genomic Insights into Two Endophytic Strains: Stenotrophomonas geniculata NWUBe21 and Pseudomonas carnis NWUBe30 from Cowpea with Plant Growth-Stimulating Attributes[J]. Applied Sciences" (corresponding to "S. geniculata NWUBe21" in the document). The public can obtain the bacterial material from the applicant within twenty years from the date of application, only for use in repeating experiments related to the present invention.

[0042] LB liquid medium was a homemade medium prepared as follows: 5 g YEAST EXTRACT, 10 g NaCl, 10 g peptone, dilute to 1 L with distilled water, and sterilize at 121°C for 21 min.

[0043] Preparation method:

[0044] (1) Take the tube containing the test strain, Stenotrophomonas genuformis, out of the ultra-low temperature freezer and dissolve it at room temperature for 5 minutes.

[0045] (2) In an ultra-clean workbench, inoculate Stenotrophomonas genuformis into the culture medium at a volume ratio of Stenotrophomonas genuformis liquid to LB liquid culture medium of "1:200".

[0046] (3) The culture medium inoculated with the strain was placed in a dark incubation at 25°C for 2 days, and the OD of the bacterial solution was measured. 600 value;

[0047] (4) Dilute the bacterial solution with distilled water to an OD of 0. 600 A value of 1.0 indicates that the target desired motherwort growth-promoting agent is obtained.

[0048] Example 2

[0049] The motherwort growth-promoting bacterial agent based on Stenotrophomonas in this embodiment is basically the same as that in Example 1, except that:

[0050] (1) Take the tube containing the test strain, Stenotrophomonas genuformis, out of the ultra-low temperature freezer and dissolve it at room temperature for 5 minutes.

[0051] (2) In a clean bench, inoculate the culture medium with Stenotrophomonas genuformis at a volume ratio of 1:150 between the bacterial liquid of Stenotrophomonas genuformis and the LB liquid culture medium.

[0052] (3) The culture medium inoculated with the strain was placed in a dark incubation at 27°C for 2 days, and the OD of the bacterial solution was measured. 600 value.

[0053] (4) Dilute the bacterial solution with distilled water to an OD of 0. 600 A value of 1.0 indicates that the target desired motherwort growth-promoting agent is obtained.

[0054] Example 3

[0055] The motherwort growth-promoting bacterial agent based on Stenotrophomonas in this embodiment is basically the same as that in Example 1, except that:

[0056] (1) Take the tube containing the test strain, Stenotrophomonas genuformis, out of the ultra-low temperature freezer and dissolve it at room temperature for 5 minutes.

[0057] (2) In an ultra-clean workbench, inoculate Stenotrophomonas genuformis into the culture medium at a volume ratio of Stenotrophomonas genuformis liquid to LB liquid culture medium of "1:100".

[0058] (3) The culture medium inoculated with the strain was placed in a dark incubation at 25°C for 2 days, and the OD of the bacterial solution was measured. 600 value.

[0059] (4) Dilute the bacterial solution with distilled water to an OD of 0. 600 The value is 0.95, which means the motherwort growth-promoting agent required for the target is obtained.

[0060] Example 4

[0061] The motherwort growth-promoting bacterial agent based on Stenotrophomonas in this embodiment is basically the same as that in Example 1, except that:

[0062] (1) Take the tube containing the test strain, Stenotrophomonas genuformis, out of the ultra-low temperature freezer and dissolve it at room temperature for 5 minutes.

[0063] (2) In an ultra-clean workbench, inoculate Stenotrophomonas genuformis into the culture medium at a volume ratio of Stenotrophomonas genuformis liquid to LB liquid culture medium of "1:200".

[0064] (3) The culture medium inoculated with the strain was placed in a dark incubation at 28°C for 2 days, and the OD of the bacterial solution was measured. 600 value.

[0065] (4) Dilute the bacterial solution with distilled water to an OD of 0. 600 The value is 1.05, which means the motherwort growth-promoting agent required for the target is obtained.

[0066] Example 5

[0067] A method for growing motherwort in this embodiment:

[0068] (1) Prepare planting pits using black soil of sand ginger. The length × width × depth of the planting pits are 2 cm × 2 cm × 1.5 cm; the pit spacing is 8 cm.

[0069] (2) Sow motherwort seeds in the planting pit.

[0070] (3) After planting the motherwort seeds, cover with 1.5 cm of soil and water them; pour 200 mL of clean water per plant to allow water to penetrate. Cover with mulch to prevent the topsoil from drying out. Remove the mulch after germination. Thereafter, water once a day, 50 mL each time, to keep the soil moist. During this period, the temperature and light intensity should be based on the natural conditions in the greenhouse (the greenhouse temperature is maintained at 26°C and the average daily light intensity is 11.5 hours).

[0071] (4) After 7 days of germination, the motherwort growth-promoting agent based on Stenotrophomonas described in the above example was poured into the motherwort seedlings at a rate of 10 mL per time, and the amount of watering was once every 3 days.

[0072] Example 6

[0073] A method for growing motherwort in this embodiment:

[0074] (1) Planting pits were prepared using black soil from sand ginger. The length × width × depth of the planting pits were 1.5 cm × 1.5 cm × 1 m, more preferably 2 cm × 2 cm × 1.5 cm. The pit spacing was 9 cm.

[0075] (2) Sow motherwort seeds in the planting pit.

[0076] (3) After planting the motherwort seeds, cover with 1 cm of soil and water them; pour 200 mL of clean water per plant to allow water to penetrate. Cover with mulch to prevent the topsoil from drying out. Remove the mulch after germination. Thereafter, water once a day, 50 mL each time, to keep the soil moist. During this period, the temperature and light intensity should be based on the natural conditions in the greenhouse (the greenhouse temperature should be maintained at 26°C and the average daily light intensity should be 11.5 hours).

[0077] (4) After 7 days of germination, the motherwort growth-promoting agent based on Stenotrophomonas described in the above example was poured into the motherwort seedlings at a rate of 8 mL per time, and the amount of watering was once every 3 days.

[0078] Example 7

[0079] A method for growing motherwort in this embodiment:

[0080] (1) Prepare planting pits using black soil of sand ginger; the length × width × depth of the planting pits are 2.5 cm × 2.5 cm × 3 cm, and the pit distance is 10 cm.

[0081] (2) Sow motherwort seeds in the planting pit.

[0082] (3) After planting the motherwort seeds, cover with 3 cm of soil and water them; pour 200 mL of clean water per plant to allow water to penetrate. Cover with mulch to prevent the topsoil from drying out. Remove the mulch after germination. Thereafter, water once a day, 50 mL each time, to keep the soil moist. During this period, the temperature and light intensity should be based on the natural conditions in the greenhouse (the greenhouse temperature is maintained at 26°C and the average daily light intensity is 11.5 hours).

[0083] (4) After 7 days of germination, the motherwort growth-promoting agent based on Stenotrophomonas described in the above example was poured into the motherwort seedlings at a rate of 12 mL per time, and the amount of watering was once every 3 days.

[0084] Experimental Example 1

[0085] This experimental example is used to verify the growth-promoting effect of the growth-promoting bacteria agent of the present invention on the growth of Leonurus japonicus seedlings.

[0086] 1. Experimental methods:

[0087] (1) Prepare nine pots of black soil for sand ginger, set up three planting holes in each pot, the length × width × depth of the planting hole is 2cm × 2cm × 1.5cm, and the pit distance is 8cm.

[0088] (2) Plant one motherwort seed in each planting hole.

[0089] (3) After planting the motherwort seeds, cover with 1.5 cm of soil and water them; pour 200 mL of clean water per plant to allow water to penetrate. Cover with mulch to prevent the topsoil from drying out. Remove the mulch after germination. Thereafter, water once a day, 50 mL each time, to keep the soil moist. During this period, the temperature and light intensity should be based on the natural conditions in the greenhouse (the greenhouse temperature is maintained at 26°C and the average daily light intensity is 11.5 hours).

[0090] (4) After 7 days of germination, the motherwort plants were divided into a blank control group (CK), an experimental group (N), and a commercially available Bacillus subtilis control group (K) for separate treatments.

[0091] Experimental group (3 pots): The Leonurus japonicus seedlings of the corresponding group were poured with the Leonurus japonicus growth-promoting agent of the present invention (taking the growth-promoting agent of Example 1 as an example) at an injection volume of 10 mL / time, once every 3 days.

[0092] Commercially available Bacillus subtilis control group (3 pots): Commercially available Bacillus subtilis was poured into the Leonurus japonicus seedlings of the corresponding group at a rate of 10 mL / time, once every 3 days.

[0093] Blank control group (3 pots): no growth-promoting agents were applied.

[0094] (5) One month later, the fresh weight (including total fresh weight, fresh weight of aboveground parts, and fresh weight of underground parts), dry weight (including total dry weight, dry weight of aboveground parts, and dry weight of underground parts), total length, plant height, root length, and leaf area of ​​Leonurus japonicus in the blank control group (CK), experimental group (N), and commercially available Bacillus subtilis control group (K) were statistically analyzed.

[0095] 2. Experimental results:

[0096] The growth of motherwort in the blank control group (CK), experimental group (N) and commercial Bacillus subtilis control group (K) is shown in Figure 2. Figure 1 The results of total fresh weight, above-ground fresh weight and underground fresh weight are shown in Figures 2-4 The results of total dry weight, above-ground dry weight and underground dry weight are shown in Figures 5-7 The results of total length, plant height and root length are shown in Figures 8-10 The leaf area results are shown as Figure 11 shown.

[0097] The above results showed that: (1) compared with the blank control group (CK), the total fresh weight, aboveground fresh weight and underground fresh weight of the experimental group (N) increased by an average of 46.16%, 62.84% and 62.60%, respectively; (2) compared with the blank control group (CK), the total dry weight, aboveground dry weight and underground dry weight of the experimental group (N) increased by an average of 57.05%, 62.08% and 35.29%, respectively; (3) compared with the blank control group (CK), the total length, plant height and root length of the experimental group (N) increased by an average of 40.01%, 49.00% and 41.00%, respectively; (4) compared with the control group, the leaf area of ​​the experimental group (N) increased by an average of 25.42%; (5) compared with the commercially available Bacillus subtilis control group (K), the experimental group (N) had better effect.

[0098] In summary, inoculation of the motherwort growth-promoting agent based on Stenotrophomonas of the present invention can significantly promote the growth of motherwort seedlings, increase the plant height, leaf length and leaf width of motherwort, and the effect is significantly better than that of commercially available Bacillus subtilis.

[0099] The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. Any modifications, equivalent substitutions and improvements made within the spirit and principles of the present invention should be included in the scope of protection of the present invention.

Claims

1. A motherwort growth-promoting agent based on Stenotrophomonas, characterized in that: Includes Stenotrophomonas and LB liquid medium.

2. The motherwort growth-promoting agent based on Stenotrophomonas according to claim 1, characterized in that The Stenotrophomonas is specifically Stenotrophomonas geniculata.

3. The motherwort growth-promoting agent based on Stenotrophomonas according to claim 1, characterized in that In the motherwort growth-promoting bacteria agent, the OD of Stenotrophomonas 600 The value is 0.95~1.

05.

4. A method for preparing a motherwort growth-promoting agent based on Stenotrophomonas according to any one of claims 1 to 3, characterized in that: Stenotrophomonas was inoculated into the culture medium at a volume ratio of 1:(100-200) between the volume of Stenotrophomonas and the volume of the LB liquid culture medium, and the culture medium was finally placed in a dark environment at 25° C. to 28° C.

5. Use of the motherwort growth-promoting agent based on Stenotrophomonas as claimed in any one of claims 1 to 3 in promoting the growth of motherwort seedlings.

6. A method for promoting the growth of motherwort seedlings, characterized in that: Apply the motherwort growth-promoting bacterial agent based on Stenotrophomonas according to any one of claims 1 to 3 to motherwort seedlings.

7. The method for promoting the growth of motherwort seedlings according to claim 6, characterized in that: The specific steps are as follows: (1) Prepare a planting pit using black soil from Zingiber officinale; (2) Sow motherwort seeds in the planting pit, cover with soil, and water; (3) After the motherwort seeds germinate and the motherwort seedlings grow, the motherwort growth-promoting agent based on Stenotrophomonas is poured into the motherwort.

8. The method for promoting the growth of motherwort seedlings according to claim 7, characterized in that: In the step (1), the length×width×depth of the planting pit is (1.5-2.5 cm)×(1.5-2.5 cm)×(1-3 cm), and the pit distance is 8-10 cm.

9. The method for promoting the growth of motherwort seedlings according to claim 7, characterized in that: In the step (2), the thickness of the covering soil is 1 to 3 cm.

10. The method for promoting the growth of Leonurus japonicus seedlings according to claim 7, characterized in that: In the step (3), the amount of the motherwort growth-promoting agent based on Stenotrophomonas is poured in is 8 to 12 mL per time.

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