Microbial complex microbial inoculant for promoting growth of leaf vegetables and application of microbial complex microbial inoculant

Abstract

The invention discloses a microbial complex microbial inoculant for promoting the growth of leaf vegetables, which comprises bacillus subtilis, rhodopseudomonas palustris and bradyrhizobium japonicum, and the viable count ratio of the bacillus subtilis, the rhodopseudomonas palustris and the bradyrhizobium japonicum is 3: 1: 1. The microbial complex microbial inoculant for promoting the growth of the leaf vegetables comprises bacillus subtilis, rhodopseudomonas palustris and bradyrhizobium japonicum, and the viable count ratio of bacillus subtilis, rhodopseudomonas palustris and bradyrhizobium japonicum is 3: 1: 1. The preparation method of the microbial inoculum comprises the following steps: S1, respectively culturing the three bacteria to a logarithmic phase to obtain 109CFU / ml bacterial liquid; s2, mixing the fermentation broth according to a ratio, and ensuring that the viable count is accurately proportioned; s3, adding a carrier and auxiliary materials, and controlling the water content to be 35%; s4, drying at low temperature, protecting thallus activity, crushing, sieving and packaging to obtain a finished product. The three functional bacteria are scientifically proportioned, so that the synergistic growth promoting effect on the lettuce and the leaf mustard is realized, and the yield and the quality are remarkably improved.

Description

Technical Field

[0001] This invention relates to the field of agricultural production, and in particular to a microbial compound inoculant for promoting the growth of leafy vegetables and its application. Background Technology

[0002] With the development of green agriculture, microbial fertilizers have attracted much attention due to their environmental friendliness and outstanding effects. Plant growth-promoting bacteria (PGPR) can directly promote crop growth through phosphorus and potassium solubilization and the secretion of plant growth hormones; photosynthetic bacteria can improve the soil microecological environment and enhance crop resistance; although rhizobia mainly fix nitrogen in symbiosis with leguminous plants, recent studies have shown that some rhizobia strains also have growth-promoting effects on non-leguminous plants.

[0003] Most microbial inoculants currently on the market are single-species or simple combinations of two species, with relatively limited functions. Although a few compound inoculant products exist, they are mainly targeted at field crops or aquaculture, with few compound microbial inoculants specifically designed for leafy vegetables. In particular, there are no reports on the application of rhizobia to non-leguminous leafy vegetables such as lettuce and mustard greens, and their synergistic effects with other functional bacteria. Summary of the Invention

[0004] To overcome the shortcomings of the existing technologies, a microbial compound inoculant is provided, which combines plant growth-promoting bacteria, photosynthetic bacteria and rhizobia in a specific ratio to achieve a synergistic growth-promoting effect on lettuce and mustard greens, significantly improving yield and quality.

[0005] To achieve the above objectives, the present invention adopts the following technical solution:

[0006] One of the objectives of this invention is to provide a microbial compound inoculant for promoting the growth of leafy vegetables, comprising Bacillus subtilis, Rhodopseudomonas palustris, and Bradyrhizobium japonicum, with a viable count ratio of 3:1:1.

[0007] In one embodiment of the present invention, the Bacillus subtilis, with accession number CGMCC 1.3358, has strong phosphorus and potassium solubilizing abilities and can secrete gibberellins and indoleacetic acid.

[0008] In one embodiment of the present invention, the *Rhodopseudomonas palustris*, with accession number ATCC 17003, can produce coenzyme Q10 and carotenoids.

[0009] In one embodiment of the present invention, the slow-growing soybean rhizobium (Bradyrhizobium japonicum), with accession number ATCC 11927, has a cross-species growth-promoting effect on non-leguminous plants.

[0010] In one embodiment of the present invention, the total number of viable bacteria in the microbial compound agent is ≥5×10⁻⁶. 9 CFU / g.

[0011] The second objective of this invention is to provide a method for preparing the aforementioned microbial compound inoculant, comprising the following steps:

[0012] S1. Culture the three bacteria separately to the logarithmic growth phase to obtain 10 9 CFU / ml bacterial solution;

[0013] S2. Mix the fermentation broth according to the specified ratio to ensure an accurate proportion of live bacteria;

[0014] S3. Add carrier and excipients, and control the moisture content to 35%;

[0015] S4. Low-temperature drying to protect bacterial activity, followed by pulverization, sieving, and packaging to obtain the finished product.

[0016] In one embodiment of the present invention, the carrier is humic acid and diatomaceous earth in a mass ratio of 3:2, providing a protective matrix and a slow-release effect.

[0017] In one embodiment of the present invention, the excipients are 3.5 wt% trehalose and 3 wt% maltodextrin, serving as a protective agent and nutritional supplement.

[0018] In one embodiment of the present invention, the drying temperature is ≤40°C, and the material is pulverized through an 80-mesh sieve.

[0019] A third objective of this invention is to provide the application of the aforementioned microbial compound agent in at least one of the following:

[0020] 1) Soil ecological restoration, improving soil enzyme activity to promote the cycling of N, P, and S elements;

[0021] 2) Promote the yield and quality of non-leguminous leafy vegetables.

[0022] The above technical solution has the following beneficial effects:

[0023] 1. Synergistic effect: The scientific ratio of three functional bacteria achieves a "1+1+1>3" effect, and the growth-promoting effect is better than that of a single bacterial agent.

[0024] 2. Cross-border application: For the first time, rhizobia were successfully applied to non-leguminous leafy vegetables such as lettuce and mustard greens, which broadened the application scope of rhizobia.

[0025] 3. Environmentally friendly: It can reduce the use of chemical fertilizers by 20-30%, which is in line with the development direction of green agriculture.

[0026] 4. Enhanced stress resistance: Enhances crop resistance to disease and cold, reducing pesticide use. Attached Figure Description

[0027] Figure 1 Figures showing lettuce cultivation in different treatment groups according to this application;

[0028] Figure 2 This application shows mustard cultivation diagrams for different treatment groups.

[0029] Figure 3 Bar chart showing the chlorophyll content of lettuce after harvest in different treatment groups according to this application;

[0030] Figure 4 Bar chart showing the chlorophyll content of mustard greens after harvest in different treatment groups of this application;

[0031] Figure 5 Bar chart showing the post-harvest plant weight of mustard in different treatment groups of this application;

[0032] Figure 6 Bar chart showing the post-harvest plant weight of lettuce from different treatment groups in this application;

[0033] Figure 7 Bar graphs showing the root length of mustard after harvest for different treatment groups in this application;

[0034] Figure 8 Bar graphs showing root lengths of lettuce after harvest from different treatment groups in this application;

[0035] Figure 9 This is a bar chart showing the plant height of mustard after harvest for different treatment groups in this application;

[0036] Figure 10 This is a bar chart showing the plant height of lettuce after harvest in different treatment groups according to this application. Detailed Implementation

[0037] The present invention will be further illustrated below with reference to specific embodiments. It should be understood that these embodiments are for illustrative purposes only and are not intended to limit the scope of the invention. Experimental methods in the following embodiments, unless otherwise specified, are generally performed under conventional conditions or as recommended by the manufacturer.

[0038] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as are familiar to those skilled in the art. The reagents and raw materials used in this invention are readily available through conventional means, and all three strains were purchased from their respective depositary institutions. Unless otherwise specified, the reagents and raw materials used in this invention are used in accordance with conventional methods in the art or according to the product instructions. Furthermore, any methods or materials similar to or equivalent to those described herein may be applied to the methods of this invention. The preferred embodiments and materials described herein are for illustrative purposes only.

[0039] Example 1

[0040] Preparation of microbial agents

[0041] 1. Activation of strains: The three preserved strains were inoculated into LB medium (growth-promoting bacteria: Bacillus subtilis), RCVBN medium (photosynthetic bacteria: Rhodopseudomonas palustris), and YMA medium (rhizobium: slow-growing soybean rhizobium) and cultured at 30°C for 24 hours, respectively.

[0042] 2. Large-scale culture: Inoculate the fermenter with 5% inoculum, and culture the growth-promoting bacteria and rhizobia for 36 hours and the photosynthetic bacteria for 48 hours;

[0043] 3. Mixing: Mix the fermentation broth at a ratio of 3:1:1 for effective live bacteria;

[0044] 4. Adsorption: Add three times the mass of the carrier (humic acid: diatomaceous earth = 3:2) to the mixed bacterial solution and stir well;

[0045] 5. Add protective agents: Add 3.5 wt% trehalose and 3 wt% maltodextrin;

[0046] 6. Drying: Dry in a fluidized bed at 38℃ until the moisture content is 8%;

[0047] 7. Detection: The final viable count determined by plate count method was 6.2 × 10⁻⁶. 9 CFU / g

[0048] Example 2

[0049] Lettuce pot experiment

[0050] Experimental location: Shenzhen Kus Biotechnology Co., Ltd., Guangming District, Guangdong Province; Treatment design:

[0051] Control group: Water group

[0052] Experimental Group 1: Rhizobium basal application (diluted 600 times, 5 kg / ha)

[0053] Experimental Group 2: Photosynthetic bacteria basal application (diluted 600 times, 5 kg / ha)

[0054] Experimental Group 3: Bacillus subtilis applied as a base dressing (diluted 600 times, 5 kg / ha)

[0055] Experimental group 4: The inoculant of this invention was applied as a base fertilizer (diluted 600 times, 5 kg / ha).

[0056] Example 3: Field Experiment of Mustard Greens

[0057] Experimental site: Shenzhen Kusi Biotechnology Co., Ltd., Guangming District, Guangdong Province; Treatment: The inoculant was diluted 600 times and applied at a rate of 5 kg / ha, 7 days after transplanting.

[0058] The results of the above experiments are detailed in Table 1 and Table 2.

[0059] Table 1. Phenotypes of different treatment groups after lettuce harvest

[0060]

[0061] Table 2. Phenotypes of different treatment groups after mustard harvest

[0062]

[0063]

[0064] Combination Figures 1 to 10 Tables 1 and 2 show that the microbial fertilizer prepared by this invention can promote the growth and yield of plants, such as lettuce and mustard greens. The three functional bacteria work synergistically, and the growth-promoting effect is significantly better than that of a single bacterial agent.

[0065] It should be noted that, in this document, relational terms such as "first" and "second" are used only to distinguish one entity or operation from another, and do not necessarily require or imply any such actual relationship or order between these entities or operations. Furthermore, the terms "comprising," "including," or any other variations thereof are intended to cover non-exclusive inclusion, such that a process, method, article, or terminal device that comprises a list of elements includes not only those elements but also other elements not expressly listed, or elements inherent to such a process, method, article, or terminal device. Unless otherwise specified, an element defined by the phrase "comprising..." or "including..." does not exclude the presence of additional elements in the process, method, article, or terminal device that includes said element. Additionally, in this document, "greater than," "less than," "exceeding," etc., are understood to exclude the stated number; "above," "below," "within," etc., are understood to include the stated number.

[0066] Although the above embodiments have been described, those skilled in the art, once they understand the basic inventive concept, can make other changes and modifications to these embodiments. Therefore, the above descriptions are merely embodiments of the present invention and do not limit the scope of patent protection of the present invention. Any equivalent structural or procedural transformations made using the content of the present invention's specification and drawings, or direct or indirect applications in other related technical fields, are similarly included within the scope of patent protection of the present invention.

Claims

1. A microbial complex microbial inoculant for promoting growth of leafy vegetables, characterized in that, The three strains include Bacillus subtilis, Rhodopseudomonas palustris and Bradyrhizobium japonicum, and the ratio of the viable cell count of the three strains is 3:1:

1.

2. The microbial complexing agent according to claim 1, wherein The Bacillus subtilis has a preservation number of CGMCC 1.3358.

3. The microbial complexing agent according to claim 2, wherein the Bacillus subtilis is a strain of Bacillus subtilis var. amyloliquefaciens. The Rhodopseudomonas palustris has a preservation number of ATCC 17003.

4. The microbial complexing agent according to claim 3, wherein The Bradyrhizobium japonicum has a preservation number of ATCC 11927.

5. The microbial complexing agent according to claim 1, wherein The total number of effective viable bacteria in the microbial complex microbial agent is ≥5×10 9 CFU / g.

6. The preparation method of the microbial complex microbial agent according to any one of claims 1-5, characterized in that, The method comprises the following steps: S1. Respectively cultivate three kinds of bacteria to logarithmic growth phase, obtain 10 9 CFU / ml bacterial solution; S2. Mixing the fermentation broth in proportion to ensure that the viable cell count is accurately proportioned; S3. Adding carriers and adjuvants to control the water content to 35%; S4. Low-temperature drying to protect the activity of the bacterial cells, crushing and sieving, and packaging the finished product.

7. The production method according to claim 6, wherein The carrier is humic acid and diatomite, and the mass ratio of the two is 3:

2.

8. The production method according to claim 6, wherein The adjuvant is 3.5wt% trehalose and 3wt% malt dextrin.

9. The production method according to claim 6, wherein The drying temperature is ≤40℃, and the crushing is passed through an 80-mesh sieve.

10. The use of the microbial complexing agent according to any one of claims 1-5 in at least one of the following: 1) Soil ecological restoration, improving soil enzyme activity, and promoting the circulation of N, P and S elements; 2) Promoting the yield and quality of non-legume leaf vegetables.

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