A bacterial agent for promoting alfalfa growth and its application

Abstract

The invention provides a bacterial agent for promoting the growth of alfalfa and an application thereof, and relates to the technical field of agriculture. The bacterial agent comprises Sinorhizobium meliloti MCSWL1: Sinorhizobium meliloti MCSD1: nitrogen-fixing rhizobium (Rhizobium sp.) MCDC3 in a mass ratio of 1:1:2. The invention provides a bacterial agent for promoting the growth of alfalfa, which can form a close symbiotic relationship with alfalfa (especially Grassland No. 3), provide sufficient nitrogen nutrition for the growth of alfalfa, thereby effectively improving soil fertility and promoting the healthy growth of plants.

Description

Technical Field

[0001] The invention relates to the technical field of agriculture, and in particular to a bacterial agent for promoting the growth of alfalfa and application thereof. Background Art

[0002] Alfalfa (Medicago sativa) is a high-quality legume forage that is widely planted around the world, especially in animal husbandry. It is rich in protein, vitamins and minerals, and has good palatability and grazing resistance, making it an indispensable feed source in animal husbandry production. However, the yield and quality of alfalfa are affected by many factors, among which soil fertility, water management, and pest and disease control are the key links.

[0003] In order to improve the yield and quality of alfalfa, agricultural researchers are constantly exploring new planting techniques and fertilizer application methods. In recent years, microbial agents have received widespread attention due to their environmental protection and high efficiency. Microbial agents improve the soil microecological environment, promote plant root growth, and improve the plant's absorption and utilization rate of nutrients, thereby achieving the purpose of increasing production and efficiency.

[0004] In the cultivation process of alfalfa, rhizobia play a vital role. Rhizobia can form a symbiotic relationship with alfalfa, fix nitrogen in the air and convert it into nitrogen that can be used by plants, thereby significantly improving the nitrogen nutrition level of alfalfa. However, under natural conditions, the number and activity of rhizobia in the soil are often difficult to meet the needs of alfalfa growth. Therefore, artificial inoculation of high-efficiency rhizobia has become an effective way to improve the yield and quality of alfalfa.

[0005] At present, although there are some microbial agents for alfalfa on the market, most of them have problems such as single strain, poor effect or high cost. Therefore, it is of great significance to develop a microbial agent that is efficient, low-cost and suitable for alfalfa. Summary of the invention

[0006] In order to solve the above technical problems, the present invention provides a microbial agent containing multiple high-efficiency rhizobia, which improves the nitrogen fixation ability and growth performance of alfalfa by optimizing the combination and ratio of bacterial species, and provides a new technical approach for high-yield and high-quality cultivation of alfalfa, as follows:

[0007] A bacterial agent for promoting the growth of alfalfa, comprising Sinorhizobium meliloti MCSWL1, Sinorhizobium meliloti MCSD1, and nitrogen-fixing rhizobium (Rhizobium sp.) MCDC3.

[0008] Nitrogen-fixing rhizobium (Rhizobium sp.) MCDC3:

[0009] Deposit name: Rhizobium sp. MCDC3;

[0010] Depository: China Center for Type Culture Collection;

[0011] Deposit address: Wuhan University, Wuhan, China;

[0012] Accession number: CCTCC NO:M20231118, 16S rDNA sequence is shown in SEQ ID NO:1;

[0013] Date of preservation: June 28, 2023.

[0014] Sinorhizobium meliloti MCSD1:

[0015] Deposit name: Sinorhizobium meliloti MCSD1;

[0016] Depository: China Center for Type Culture Collection;

[0017] Deposit address: Wuhan University, Wuhan, China;

[0018] Accession number: CCTCC NO:M20231119, 16S rDNA sequence is shown in SEQ ID NO:2;

[0019] Date of preservation: June 28, 2023.

[0020] Sinorhizobium meliloti MCSWL1:

[0021] Deposit name: Sinorhizobium meliloti MCSWL1;

[0022] Depository: China Center for Type Culture Collection;

[0023] Deposit address: Wuhan University, Wuhan, China;

[0024] Accession number: CCTCC NO:M20231120, 16S rDNA sequence is shown in SEQ ID NO:3;

[0025] The preservation date is June 28, 2023.

[0026] The mass ratio of Sinorhizobium meliloti MCSWL1: Sinorhizobium meliloti MCSD1: nitrogen-fixing rhizobium (Rhizobium sp.) MCDC3 is 1:1:2.

[0027] Moreover, the Sinorhizobium meliloti MCSWL1 is isolated and screened from the rhizosphere of alfalfa.

[0028] Moreover, the Sinorhizobium meliloti MCSD1 is isolated and screened from the rhizosphere of alfalfa.

[0029] Moreover, the nitrogen-fixing rhizobium (Rhizobium sp.) MCDC3 is isolated and screened from the rhizosphere of alfalfa.

[0030] The specific method for isolating Sinorhizobium meliloti MCSWL1, Sinorhizobium meliloti MCSD1, and Rhizobium sp. MCDC3 is as follows:

[0031] 1. Preparation and dilution of alfalfa root soil stock solution

[0032] The alfalfa root soil was dissolved in sterile water to prepare a soil stock solution. The prepared soil stock solution was diluted to 10 -1 , 10 -2 , 10 -3 Serial dilution;

[0033] 2. Soil stock solution inoculation culture

[0034] Inoculate the diluted soil stock solution on YMA medium;

[0035] 3. Strain Purification

[0036] Select a single rhizobium colony from the culture medium of the initial inoculation culture and streak it onto a new culture medium for strain purification;

[0037] 4. Secondary purification of strains

[0038] Repeatedly streak the purified rhizobium strains on new YMA medium to improve the purity of the strains;

[0039] 5. Bacterial culture

[0040] Prepare YMA liquid medium, inoculate the purified rhizobium strains into the YMA liquid medium and culture in a shaking incubator for 1-2 days;

[0041] 6. Identification and preservation of bacterial strains

[0042] Rhizobium culture liquid was used for strain sequencing. According to the sequencing results, Sinorhizobium meliloti MCSWL1, Sinorhizobium meliloti MCSD1, and Rhizobium sp. MCDC3 were selected for strain preservation. The main laboratory strain preservation methods include glycerol preservation, test tube slant preservation, and freeze-drying preservation.

[0043] Moreover, the alfalfa is Grassland No. 3.

[0044] On the other hand, the present invention provides the above-mentioned bacterial agent for promoting the growth of alfalfa and its application, which are used to promote the growth of alfalfa. The specific method is: after the bacterial agent including Sinorhizobium meliloti MCSWL1, Sinorhizobium meliloti MCSD1, and nitrogen-fixing rhizobium (Rhizobium sp.) MCDC3 is activated, it is diluted 3-5 times after liquid culture for 3-5 days, and the mass ratio of Sinorhizobium meliloti MCSWL1: Sinorhizobium meliloti MCSD1: nitrogen-fixing rhizobium (Rhizobium sp.) MCDC3 is 1:1:2, and then it is sprayed on the roots of alfalfa.

[0045] Moreover, the alfalfa root spraying amount is 1 L per mu of land, and it is sprayed in the soil around the seedlings once a week for three consecutive weeks.

[0046] Compared with the prior art, the present invention has the following beneficial effects:

[0047] 1. The present invention provides a bacterial agent for promoting the growth of alfalfa. Sinorhizobium meliloti MCSWL1, MCSD1 and nitrogen-fixing rhizobium (Rhizobium sp.) MCDC3 can form a close symbiotic relationship with alfalfa, provide sufficient nitrogen nutrition for the growth of alfalfa, thereby effectively improving soil fertility and promoting healthy growth of plants.

[0048] 2. Through scientific research and experimental verification, the present invention has determined that the optimal mass ratio of alfalfa Sinorhizobium MCSWL1, MCSD1 and nitrogen-fixing rhizobium MCDC3 is 1:1:2. This ratio can give full play to the synergistic effect of each strain, maximize the growth rate and biomass of alfalfa, and improve its nutritional quality and stress resistance.

[0049] 3. The microbial agent of the present invention is composed entirely of natural microbial strains, does not contain any chemical synthetic substances, is environmentally friendly, safe and harmless. Its application can not only effectively improve the yield and quality of alfalfa, but also promote the balance and stability of the soil ecosystem, providing strong support for achieving sustainable agricultural development.

[0050] 4. The microbial agent of the present invention not only promotes the growth of alfalfa, but also improves the soil microecological environment, increases the number and activity of beneficial microorganisms in the soil, thereby enhancing the resistance of alfalfa to adverse conditions such as drought and salinity, and improves its adaptability and stability.

[0051] 5. Compared with the use of traditional fertilizers and pesticides, the bacterial agent of the present invention has the advantages of low cost, no pollution, and easy operation. By spraying it on the roots of alfalfa, it not only saves costs, but also reduces environmental pollution, improves the sustainability of agricultural production, and brings higher economic benefits to farmers. BRIEF DESCRIPTION OF THE DRAWINGS

[0052] Figure 1 This is the growth of alfalfa plant height;

[0053] Figure 2 This is the branching situation of alfalfa plants with a height of 50 cm. DETAILED DESCRIPTION

[0054] Example 1 Acquisition of MCSWL1 strain

[0055] A Sinorhizobium meliloti MCSWL1 has a deposit number of CCTCC NO:M20231120, a 16S rDNA sequence as shown in SEQ ID NO:3, a deposit date of June 28, 2023, and is isolated and screened from the rhizosphere of alfalfa. The details are as follows:

[0056] 1. Preparation and dilution of alfalfa root soil stock solution

[0057] The alfalfa root soil was dissolved in sterile water to prepare a soil stock solution. The prepared soil stock solution was heated to 10 -1 , 10 -2 , 10 -3 Serial dilution;

[0058] 2. Soil stock solution inoculation culture

[0059] Inoculate the diluted soil stock solution on YMA medium;

[0060] 3. Strain Purification

[0061] Select a single colony from the medium of the initial inoculation culture and streak it onto a new medium for strain purification;

[0062] 4. Secondary purification of strains

[0063] Repeatedly streak the purified strains in new culture medium to improve the purity of the strains;

[0064] 5. Bacterial culture and identification

[0065] Prepare YMA liquid culture medium, inoculate the purified strain into the liquid culture medium and culture it in a shaker for 1-2 days. The bacterial liquid can be used for strain sequencing and strain preservation;

[0066] 6. Preservation of bacterial strains

[0067] The main methods of preserving laboratory strains include glycerol preservation, test tube slant preservation, and freeze-drying preservation.

[0068] Example 2 Acquisition of MCSD1 strain

[0069] A Sinorhizobium meliloti MCSD1 has a deposit number of CCTCC NO:M20231119, a 16S rDNA sequence as shown in SEQ ID NO:2, a deposit date of June 28, 2023, and is isolated and screened from the rhizosphere of alfalfa. The details are as follows:

[0070] 1. Preparation and dilution of alfalfa root soil stock solution

[0071] The alfalfa root soil was dissolved in sterile water to prepare a soil stock solution. The prepared soil stock solution was diluted to 10 -1 , 10 -2 , 10 -3 Serial dilution;

[0072] 2. Soil stock solution inoculation culture

[0073] Inoculate the diluted soil stock solution on YMA medium;

[0074] 3. Strain Purification

[0075] Select a single colony from the medium of the initial inoculation culture and streak it onto a new medium for strain purification;

[0076] 4. Secondary purification of strains

[0077] Repeatedly streak the purified strains in new culture medium to improve the purity of the strains;

[0078] 5. Bacterial culture and identification

[0079] Prepare YMA liquid culture medium, inoculate the purified strain into the liquid culture medium and culture it in a shaker for 1-2 days. The bacterial liquid can be used for strain sequencing and strain preservation;

[0080] 6. Preservation of bacterial strains

[0081] The main methods of preserving laboratory strains include glycerol preservation, test tube slant preservation, and freeze-drying preservation.

[0082] Example 3 Acquisition of MCDC3 strain

[0083] A nitrogen-fixing rhizobium (Rhizobium sp.) MCDC3 has a deposit number of CCTCC NO:M20231118, a 16SrDNA sequence as shown in SEQ ID NO:1, a deposit date of June 28, 2023, and is isolated and screened from the rhizosphere of alfalfa, as follows:

[0084] 1. Preparation and dilution of alfalfa root soil stock solution

[0085] The alfalfa root soil was dissolved in sterile water to prepare a soil stock solution. The prepared soil stock solution was heated to 10 -1 , 10 -2 , 10 -3 Serial dilution;

[0086] 2. Soil stock solution inoculation culture

[0087] Inoculate the diluted soil stock solution on YMA medium;

[0088] 3. Strain Purification

[0089] Select a single colony from the medium of the initial inoculation culture and streak it onto a new medium for strain purification;

[0090] 4. Secondary purification of strains

[0091] Repeatedly streak the purified strains in new culture medium to improve the purity of the strains;

[0092] 5. Bacterial culture and identification

[0093] Prepare YMA liquid culture medium, inoculate the purified strain into the liquid culture medium and culture it in a shaker for 1-2 days. The bacterial liquid can be used for strain sequencing and strain preservation;

[0094] 6. Preservation of bacterial strains

[0095] The main methods of preserving laboratory strains include glycerol preservation, test tube slant preservation, and freeze-drying preservation.

[0096] Example 4 Preparation of bacterial agent

[0097] The MCSWL1, MCDC3 and MCSWL1 obtained in Examples 1-3 are activated, cultured in liquid for 3-5 days, diluted 3-5 times, and mixed evenly in a mass ratio of MCSWL1:MCSD1:MCDC3=1:1:2 to obtain the product.

[0098] Example 5 Application of bacterial agent

[0099] The bacterial agent prepared in Example 4 was sprayed on the roots of Grassland No. 3 alfalfa, with a spraying amount of 1 L per mu of land, and sprayed in the soil around the seedlings once a week for three consecutive weeks.

[0100] Comparative Example 1

[0101] The difference between this comparative example and Example 4 is that tap water is used instead of the bacterial agent (CK).

[0102] Comparative Example 2

[0103] The difference between this comparative example and Example 4 is that only one strain, MCDC3, is used.

[0104] Comparative Example 3

[0105] The difference between this comparative example and Example 4 is that only one strain, MCSD1, is used.

[0106] Comparative Example 4

[0107] The difference between this comparative example and Example 4 is that only one strain, MCSWL1, is used.

[0108] Comparative Example 5

[0109] The difference between this comparative example and Example 4 is that MCSWL1:MCSD1:MCDC3=1:1:1.

[0110] Comparative Example 6

[0111] The difference between this comparative example and Example 4 is that MCSWL1:MCSD1:MCDC3=2:1:1.

[0112] Comparative Example 7

[0113] The difference between this comparative example and Example 4 is that MCSWL1:MCSD1:MCDC3=1:2:1.

[0114] Experimental Section

[0115] Experiment 1

[0116] The same farmland was divided into 8 identical experimental plots, each of which was planted with alfalfa (Grassland No. 3). The bacterial agents prepared in Example 4 and Comparative Examples 1-7 were sprayed on the roots of Grassland No. 3 alfalfa. The spraying amount was 1 L per mu of land. It was sprayed in the soil around the seedlings once a week. After spraying 3 times, water was applied normally. Ten plants were randomly selected every half a month to measure the plant height once. The plant height was measured 5 times and the average value was taken. The specific results are shown in Tables 1 and Figure 1 .

[0117] Table 1 Plant height growth

[0118]

[0119] From Table 1 and Figure 1 It can be seen that MCDC3, MCSD1, MCSWL1 and their mixture can all increase the plant height of Grassland No. 3 alfalfa, but the best effect is MCSWL1:MCSD1:MCDC3=1:1:2.

[0120] Experiment 2

[0121] After the end of experiment 1, water was applied normally. After the alfalfa continued to grow for 15 days, 10 plants were collected and the number of 50 cm branches was counted. The results are shown in Table 2 and Figure 2 .

[0122] Table 2 50cm branching

[0123]

[0124] From Table 2 and Figure 2 It can be seen that MCDC3, MCSD1, MCSWL1 and their mixtures can promote the branching of grassland 3 alfalfa, and the number of 50 cm branches of all mixed treatments is higher than that of any single treatment, which indicates that the mixed treatment of MCDC3, MCSD1 and MCSWL1 has a stronger effect in promoting branching, which may be due to the complementary effect between different treatments or varieties; when mixed, the ratio of MCSWL1:MCSD1:MCDC3 is 1:1:2, with the best effect, and the number of 50 cm branches reaches a maximum of 93.

[0125] Experiment 3

[0126] After harvesting the alfalfa in Experiment 2, the soil in the experimental field was sampled and analyzed to determine indicators such as the nitrogen content and organic matter content in the soil.

[0127] Table 3 Effects of microbial agents on soil fertility

[0128]

[0129] As can be seen from Table 3, compared with the CK group, all treatments with the addition of MCDC3, MCSD1, MCSWL1 or their combination significantly increased the nitrogen content in the soil. Among the strains used alone, MCDC3 had the most obvious effect on increasing the soil nitrogen content, reaching 235.6 mg / kg. When these strains were used in combination in different proportions, the soil nitrogen content was further increased. The best effect was achieved with MCSWL1:MCSD1:MCDC3=1:1:2, and the soil nitrogen content reached the highest 293.4 mg / kg.

[0130] Compared with the control group, except for MCSD1 which had no obvious increase in soil organic matter content when used alone, all other treatments significantly increased the soil organic matter content. When the strains were used in combination, the increase in soil organic matter content was more significant, among which MCSWL1:MCSD1:MCDC3=1:1:2 had the best effect, with the organic matter content reaching the highest 5.3%.

[0131] Compared with using a single strain, the combined use of strains can more effectively increase the soil nitrogen and organic matter content. Among all the combinations, Example 4 (MCSWL1: MCSD1: MCDC3 = 1: 1: 2) has the highest soil nitrogen and organic matter content, indicating that MCSWL1: MCSD1: MCDC3 = 1: 1: 2 performs best in improving soil fertility.

Claims

1. A bacterial agent for promoting the growth of alfalfa, characterized in that: The bacterial agents include Sinorhizobium meliloti MCSWL1, Sinorhizobium meliloti MCSD1, and nitrogen-fixing rhizobium (Rhizobium sp.) MCDC3; The accession number of the nitrogen-fixing rhizobium (Rhizobium sp.) MCDC3 is CCTCC NO:M20231118, the 16S rDNA sequence is shown in SEQ ID NO:1, and the deposit date is June 28, 2023; The deposit number of the Sinorhizobium meliloti MCSD1 is CCTCC NO:M20231119, the 16S rDNA sequence is shown in SEQ ID NO:2, and the deposit date is June 28, 2023; The deposit number of the Sinorhizobium meliloti MCSWL1 is CCTCC NO:M20231120, the 16S rDNA sequence is shown in SEQ ID NO:3, and the deposit date is June 28, 2023; The mass ratio of Sinorhizobium meliloti MCSWL1: Sinorhizobium meliloti MCSD1: nitrogen-fixing rhizobium (Rhizobium sp.) MCDC3 is 1:1:

2.

2. The use of the bacterial agent for promoting the growth of alfalfa as claimed in claim 1, characterized in that: Used to promote the growth of alfalfa.

3. The use of the bacterial agent for promoting the growth of alfalfa as claimed in claim 2, characterized in that: The specific method is: after activating the bacterial agents including Sinorhizobium meliloti MCSWL1, Sinorhizobium meliloti MCSD1, and nitrogen-fixing rhizobium (Rhizobium sp.) MCDC3, they are diluted 3-5 times after liquid culture for 3-5 days, mixed evenly in a mass ratio of Sinorhizobium meliloti MCSWL1: Sinorhizobium meliloti MCSD1: nitrogen-fixing rhizobium (Rhizobium sp.) MCDC3 of 1:1:2, and then sprayed on the roots of alfalfa.

4. The use of the bacterial agent for promoting the growth of alfalfa as claimed in claim 3, characterized in that: The alfalfa root spraying amount is 1 L per mu of land, and it is sprayed in the soil around the seedlings, once a week for three consecutive weeks.

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