Soybean bradyrhizobium and application thereof

Abstract

The invention relates to the technical field of agricultural microorganisms, and particularly discloses bradyrhizobium japonicum and application thereof. The bradyrhizobium japonicum is preserved in the China General Microbiological Culture Collection Center (CGMCC) on December 15, 2023, and the preservation number is CGMCC No.29321. The bradyrhizobium japonicum is named as Bradyrhizobium japonicum. The invention also provides a culture, a fermentation broth, a bacterial suspension, a bacterial agent and bacterial powder of the strain. The invention further discloses application of the bacterial strain and the culture, the fermentation liquor, the bacterial suspension, the bacterial agent and the bacterial powder of the bacterial strain in increasing the number of soybean nodules and promoting soybean plant growth. The bradyrhizobium japonicum provided by the invention has a relatively good nodulation effect and can effectively promote plant growth.

Description

Technical Field

[0001] The present application relates to the technical field of agricultural microorganisms, and more specifically, to a soybean bradyrhizobium and its application. Background Art

[0002] As one of the important food crops in my country, soybeans are limited in yield due to their lack of ability to use atmospheric nitrogen. Soybean rhizobia, as nitrogen-fixing bacteria, can convert nitrogen in the air into ammonia required by plants to make up for the lack of soybean crops' ability to use atmospheric nitrogen, thereby increasing soybean crop yields. Compared with soybean crops fertilized with traditional fertilizers, soybean crops using soybean rhizobia can increase yields by about 15%.

[0003] Therefore, in order to enrich the types of soybean rhizobia and further increase the yield of soybean crops, it is necessary to find new soybean rhizobia. Summary of the invention

[0004] The present application provides a soybean bradyrhizobium and its application. The soybean bradyrhizobium has a good nodulation effect and can effectively promote plant growth.

[0005] In a first aspect, the present application provides a soybean bradyrhizobium, using the following technical solution:

[0006] The soybean bradyrhizobium sp. provided in this application was deposited in the General Microbiology Center of the China Microorganism Culture Collection Administration on December 15, 2023, with the deposit number CGMCC No. 29321, and the deposit address is: Institute of Microbiology, Chinese Academy of Sciences, No. 1 Beichen West Road, Chaoyang District, Beijing.

[0007] The strain was isolated from a soil sample with nodules in Tieli City, Heilongjiang Province. The vegetative cells of the strain have smooth and protruding colony surfaces on YMA solid culture medium, and are milky white or white colonies.

[0008] The soybean bradyrhizobium provided in the present application has a good nodulation effect, and the majority of the nodules are effective nodules. In particular, the number of soybean nodules can be increased by more than 200%, and the number of nodules of soybean plants can be increased by more than 50%. The soybean bradyrhizobium provided in the present application can effectively promote plant growth. In particular, the root dry weight of soybean plants can be increased by more than 20%.

[0009] In a second aspect, the present application provides a culture comprising the above-mentioned Bradyrhizobium japonicum.

[0010] In a third aspect, the present application provides a fermentation broth, which includes the above-mentioned soybean bradyrhizobium.

[0011] In a fourth aspect, the present application provides a bacterial suspension comprising the above-mentioned soybean bradyrhizobium.

[0012] In a fifth aspect, the present application provides a bacterial agent, which includes the above-mentioned soybean bradyrhizobium.

[0013] In a sixth aspect, the present application provides a bacterial powder, which includes the above-mentioned soybean bradyrhizobium.

[0014] In a seventh aspect, the present application provides a use of a culture, fermentation liquid, bacterial suspension, bacterial agent or bacterial powder of the above-mentioned soybean bradyrhizobium in increasing the number of soybean nodules and promoting the growth of soybean plants.

[0015] Optionally, the soybean nodulation number can be increased by more than 200%.

[0016] Optionally, the number of nodules of the soybean plant can be increased by more than 50%.

[0017] Optionally, the root dry weight of the soybean plant can be increased by more than 20%.

[0018] In summary, this application has the following beneficial effects:

[0019] 1. The present application provides a soybean bradyrhizobium, which was deposited in the General Microbiology Center of the China Culture Collection Administration on December 15, 2023, with the deposit number CGMCC No.29321.

[0020] 2. The soybean bradyrhizobium provided in the present application has a good nodulation effect, and most of the nodules are effective nodules. In particular, the number of soybean nodules can be increased by more than 200%, and the number of nodules of soybean plants can be increased by more than 50%.

[0021] 3. The soybean bradyrhizobium provided in the present application can effectively promote plant growth, especially increase the root dry weight of soybean plants by more than 20%. BRIEF DESCRIPTION OF THE DRAWINGS

[0022] Figure 1 This is the phylogenetic tree of the strains isolated and purified in this application.

[0023] Figure 2 It is the comparison result of the above-ground parts of the test group (the strain isolated and purified in this application) and the control group in the field test.

[0024] Figure 3 It is the comparison result of the underground part of the test group (the strain isolated and purified in this application) and the control group in the field test.

[0025] Figure 4This is a cross-sectional view of the nodules formed by the test group (the strain isolated and purified in this application) in the field test.

[0026] Figure 5 The nodulation results of the strain isolated and purified in this application and different varieties of soybeans. DETAILED DESCRIPTION

[0027] Before describing the embodiments of the present application in detail, it should be understood that the terms used herein are only used for the purpose of describing specific embodiments. Unless otherwise defined, all technical and scientific terms used herein have the same meaning as those generally understood by ordinary technicians in the field to which the terms belong.

[0028] The endpoints and any values ​​of the ranges disclosed in this application are not limited to the precise ranges or values, and these ranges or values ​​should be understood to include values ​​close to these ranges or values. For numerical ranges, the endpoint values ​​of each range, the endpoint values ​​of each range and the individual point values, and the individual point values ​​can be combined with each other to obtain one or more new numerical ranges, which should be considered as specifically disclosed in this article.

[0029] In the present application, the terms "comprise" or "include" are open expressions, that is, including the contents specified in the present application but not excluding other contents.

[0030] The present application provides a Bradyrhizobium sp., which is deposited in the General Microbiological Center of China Microbiological Culture Collection Committee with a deposit number of CGMCC No. 29321. In addition, the present application also provides a method for isolating, purifying and culturing the strain. It also provides a culture, a fermentation broth, a bacterial suspension, a bacterial powder and a bacterial agent comprising the strain.

[0031] The present application also provides the soybean bradyrhizobium and the use of the culture, fermentation liquid, bacterial suspension, bacterial powder and bacterial agent comprising the strain in increasing the number of soybean nodules and promoting the growth of soybean plants.

[0032] In order to make the purpose, technical scheme and advantages of the present application clearer, the technical scheme in the embodiments of the present application will be clearly and completely described below. Based on the embodiments of the present application, all other embodiments obtained by those skilled in the art without making creative work belong to the scope of protection of the present application. The embodiments described below are exemplary and are only used to explain the present application, and cannot be interpreted as limiting the present application.

[0033] If no specific techniques or conditions are specified in the examples, the techniques or conditions described in the literature in the field or the product instructions are used. If no manufacturer is specified for the reagents or instruments used, they are all conventional products that can be purchased commercially.

[0034] The present application is further described in detail below in conjunction with the embodiments and test results.

[0035] Example

[0036] Example 1

[0037] This example provides a new bacterial strain separation and purification process.

[0038] The details are as follows:

[0039] (1) Soil sample collection: Soil samples with nodules were collected from Tieli City, Heilongjiang Province using a multi-point soil sampling method for potted plant experiments.

[0040] (2) Pot test: 3 kg of soil sample was placed in a round-bottomed flower pot with a diameter of 25 cm, and soybean seeds were sown at a sowing rate of 10 seeds per pot, and the soybean seeds were cultivated according to the conventional cultivation method in the art. After 30-40 days of cultivation, when the soybean plants grew to the flowering stage, the roots of the soybean plants were dug out and the nodules in the roots were removed.

[0041] (3) Isolation of target bacteria: After disinfection with alcohol and mercuric chloride, cut with a scalpel and place the cut surface on the surface of YMA solid culture medium (formula: mannitol 10 g / L, yeast 1 g / L, potassium dihydrogen phosphate 0.25 g / L, potassium dihydrogen phosphate 0.25 g / L, magnesium sulfate 0.2 g / L, sodium chloride 0.1 g / L, agar 18 g / L; pH 6.8-7.2) for batch culture. Place it at 28°C and invert for 7 days. Use an inoculation needle to pick up single colonies with different colonies and perform streak culture. When there is only one colony morphology in the YMA solid culture medium, the separation and purification is complete. Store it for future use.

[0042] (4) Preliminary identification: The isolated and purified strain was examined and observed under a microscope. Based on the results of the microscopic examination, it was preliminarily determined that the strain was a Bradyrhizobium genus.

[0043] Example 2

[0044] This example provides the properties of the strain isolated and purified in Example 1 and the results of 16S rDNA sequencing. The details are as follows:

[0045] (I) Strain properties

[0046] (1) Morphological characteristics

[0047] The vegetative cells of the strain have smooth and protruding colony surfaces on the YMA solid culture medium, and are milky white or white colonies.

[0048] (2) Culture characteristics

[0049] The optimal growth conditions for the strain are: pH=6.8-7.2, temperature of 28-30°C, and rotation speed of 160-200r / min.

[0050] (3) Functional characteristics

[0051] The soybean bradyrhizobium provided in the present application has a good nodulation effect, and most of the nodules are effective nodules. In particular, the number of soybean nodules can be increased by more than 200%, and the number of nodules of soybean plants can be increased by more than 50%.

[0052] The soybean bradyrhizobium provided in the present application can effectively promote plant growth, and in particular, the root dry weight of soybean plants can be increased by more than 20%.

[0053] (4) The strain was identified by 16S rDNA sequencing technology, and the 16S rDNA sequence of the strain was obtained, such as the nucleotide sequence shown in SEQ ID NO: 1, and the sequence was compared with the Gen Bank by Blast sequence comparison. The phylogenetic tree was constructed by the neighbor-joining method using MEGA5.0 software (such as Figure 1 The strain was identified as Bradyrhizobium sp. and named as Bradyrhizobium sp. AMSD1.

[0054] Bradyrhizobium sp. was deposited in the General Microbiology Center of China Microorganism Culture Collection on December 15, 2023, with the deposit number CGMCC No.29321, and the deposit address is: Institute of Microbiology, Chinese Academy of Sciences, No. 1 Beichen West Road, Chaoyang District, Beijing.

[0055] Example 3

[0056] This embodiment provides a fermentation broth of Bradyrhizobium japonicum.

[0057] The preparation method of the above fermentation liquid is as follows:

[0058] The strain isolated and purified in Example 1 was activated and transferred to YMA liquid culture medium with a pH of 6.8-7.2 (formula: 10 g / L mannitol, 1 g / L yeast, 0.25 g / L dipotassium hydrogen phosphate, 0.25 g / L potassium dihydrogen phosphate, 0.2 g / L magnesium sulfate, 0.1 g / L sodium chloride), and cultured at 28°C for 7 days to obtain the fermentation broth of soybean bradyrhizobium.

[0059] Example 4

[0060] This embodiment provides a fermentation supernatant of Bradyrhizobium japonicum.

[0061] The preparation method of the above fermentation supernatant is as follows:

[0062] The strain isolated and purified in Example 1 was activated and transferred to a YMA liquid culture medium with a pH of 6.8-7.2, and cultured at 28°C for 7 days to obtain a fermentation broth of Bradyrhizobium sojae, in which the number of bacteria in the fermentation broth was 4 billion / ml; the fermentation broth was centrifuged and the supernatant was retained, which was the fermentation supernatant of Bradyrhizobium sojae.

[0063] Example 5

[0064] This embodiment provides a bacterial suspension of Bradyrhizobium japonicum.

[0065] The preparation method of the above bacterial suspension is as follows:

[0066] The strain isolated and purified in Example 1 was activated and transferred to a YMA liquid culture medium with a pH of 6.8-7.2, and cultured at 28° C. for 7 days to obtain a fermentation broth of soybean bradyrhizobium; the fermentation broth was centrifuged to retain the precipitate, and the precipitate was resuspended with sterile water to obtain a bacterial suspension of soybean bradyrhizobium.

[0067] Example 6

[0068] This embodiment provides a bacterial agent of soybean Bradyrhizobium.

[0069] The preparation process of the above-mentioned microbial agent is as follows:

[0070] The activated strain plate was used to inoculate the soybean bradyrhizobium isolated and purified in Example 1 into a 500 mL conical flask with liquid fermentation seed liquid culture medium, and cultured in a constant temperature shaker at 28° C. and 200 rpm for 7 days to obtain cultured seed liquid for standby use.

[0071] The cultured seed liquid is inoculated into a 100L fermentation tank, the fermentation liquid volume of the fermentation tank is 70% of the total volume of the fermentation tank, the fermentation inoculation amount is 1%, the rotation speed is 200rpm, the temperature is 28℃, the ventilation volume is 1vvm, and the culture is 7d before the tank is removed. This is the soybean bradyrhizobium bacterial agent.

[0072] Example 7

[0073] This embodiment provides a bacterial powder of Bradyrhizobium japonicum.

[0074] The specific preparation process of the above bacterial powder is as follows: the bacterial agent prepared in Example 6 is spray-dried to obtain bacterial powder, that is, the bacterial powder of soybean bradyrhizobium is obtained.

[0075] Example 8

[0076] This embodiment provides a microbial agent, which includes an agent prepared by using Bradyrhizobium japonicum.

[0077] The preparation process of the above-mentioned microbial agent is as follows:

[0078] The microbial agent prepared in Example 6 was compounded with zeolite powder, and evenly mixed at a weight ratio of 1:40 between the microbial powder and the zeolite powder. The mixed microbial agent product contained 500 million bacteria / g.

[0079] Performance test 1

[0080] The strain isolated and purified in this application was used for field trials.

[0081] Test scale: round-bottomed flower pots with a diameter of 25 cm.

[0082] The test method is as follows:

[0083] After mixing the seeds with 10 mL of rhizobium solution or sterile water per kilogram of soybean seeds, the seeds were dried in the shade. 6 kg of Pinggu base greenhouse soil samples were placed in round-bottomed flower pots, and soybean seeds were sown at a sowing rate of 10 seeds per pot, and the soybean seeds were cultivated according to conventional cultivation methods in the field.

[0084] The test groups were sterile water (blank group), fermentation broth of rhizobium with the preservation number of CGMCC No. 10994, with the number of bacteria in the fermentation broth being 4 billion / ml (control group), and fermentation broth of soybean bradyrhizobium isolated and purified in the present application, with the number of bacteria in the fermentation broth being 4 billion / ml (test group). Three parallels were set for each group.

[0085] After 30-40 days of cultivation, when the soybean plants grow to the flowering stage, the roots of the soybean plants are dug out. The various parameters of the aboveground and underground parts of the soybean plants are tested respectively. The test parameters are shown in Table 1-2. The test results are shown in Table 1-2 and Figure 2-3 The nodules formed by the roots in the test group were taken out, and the cross-section of the nodules was as shown in Figure 4 shown.

[0086] Table 1 Detection parameters and test results of aboveground parts

[0087]

[0088] Table 2 Underground part test parameters and test results

[0089]

[0090] From Table 1-2 and Figure 2-3 It can be seen that the plant height, stem thickness, root length and root dry weight of the test group were significantly increased compared with the blank group and the control group, indicating that the soybean bradyrhizobium provided by the present application has a better growth-promoting effect. In particular, the root dry weight of soybean plants can be increased by more than 20%.

[0091] As shown in Table 2, no nodules appeared in the blank group. Compared with the control group, the number of nodules, the weight of nodules and the number of nodules per plant in the test group increased significantly, indicating that the soybean bradyrhizobium provided by the present application has a good nodulation effect and a large number of nodules. In particular, the number of soybean nodules can be increased by more than 200%, and the number of nodules of soybean plants can be increased by more than 50%.

[0092] Depend on Figure 4 It can be seen that the nodules formed by the roots in the test group are all red inside after being cut open, and can be determined to be effective nodule sections, which indicates that the soybean bradyrhizobium provided in the present application not only has a large number of nodules, but also all of them are effective nodules.

[0093] Performance test 2

[0094] The strain isolated and purified in the present application was used to conduct nodulation tests on different varieties of soybeans.

[0095] Test scale: 500mL triangular flask hydroponics.

[0096] The test method is as follows:

[0097] 300 mL of culture solution was added to a triangular flask, and soybean seeds were sown at a sowing rate of 4 seeds / bottle, and the soybean seeds were cultivated according to the conventional culture method in the art. The soybean seeds were: Heinong 88, Heihe 53, Mengdou 15, Jiuyan 13. Three parallels were set for each group.

[0098] After 15-20 days of cultivation, the soybean plants were taken out. The nodulation of each type of soybean plant was tested. The test results are shown in Table 3 and Figure 5 shown.

[0099] Table 3 Nodulation of various soybean plants

[0100] Soybean category Number of nodules Total weight of nodules (g) Single nodule weight (g) Number of nodules per plant Black Farmer 88 59 1.82 0.030 11 Black River 53 46 0.91 0.019 12 Mengdou15 55 1.33 0.026 11 Jiuyan 13 36 0.52 0.014 10

[0101] From Table 3 and Figure 5It can be seen that the soybean bradyrhizobium provided in the present application can produce nodules with four soybean varieties, namely Heinong 88, Heihe 53, Mengdou 15, and Jiuyan 13.

[0102] In the description of this specification, the description with reference to the terms "one embodiment", "some embodiments", "example", "specific example" or "some examples" etc. means that the specific features, structures, materials or characteristics described in conjunction with the embodiment or example are included in at least one embodiment or example of the present application. In this specification, the schematic representations of the above terms do not necessarily refer to the same embodiment or example. Moreover, the specific features, structures, materials or characteristics described may be combined in any one or more embodiments or examples in a suitable manner. In addition, those skilled in the art may combine and combine the different embodiments or examples described in this specification and the features of the different embodiments or examples, without contradiction.

[0103] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present application, rather than to limit it. Although the present application has been described in detail with reference to the aforementioned embodiments, those skilled in the art should understand that they can still modify the technical solutions described in the aforementioned embodiments, or make equivalent replacements for some of the technical features therein. However, these modifications or replacements do not deviate the essence of the corresponding technical solutions from the spirit and scope of the technical solutions of the embodiments of the present application.

Claims

1. A soybean bradyrhizobium, characterized in that: The soybean bradyrhizobium ( Bradyrhizobium sp. ) is deposited in the General Microbiology Center of China Microorganism Culture Collection Administration with the deposit number CGMCC No. 29321.

2. A culture, characterized in that The culture comprises the Bradyrhizobium japonicum of claim 1 .

3. A fermentation broth, characterized in that: The fermentation broth comprises the soybean bradyrhizobium according to claim 1.

4. A bacterial suspension, characterized in that The bacterial suspension comprises the soybean bradyrhizobium according to claim 1.

5. A bacterial agent, characterized in that The bacterial agent includes the soybean bradyrhizobium according to claim 1.

6. A bacterial powder, characterized in that: The bacterial powder comprises the soybean bradyrhizobium according to claim 1.

7. Use of the soybean bradyrhizobium according to claim 1, the culture according to claim 2, the fermentation liquid according to claim 3, the bacterial suspension according to claim 4, the bacterial agent according to claim 5, and the bacterial powder according to claim 6 in increasing the number of soybean nodules and promoting the growth of soybean plants.

8. The use according to claim 7, characterized in that: The soybean nodule quantity can be increased by more than 200%.

9. The use according to claim 7, characterized in that: The number of nodules of the soybean plant can be increased by more than 50%.

10. The use according to claim 7, characterized in that: The root dry weight of the soybean plant can be increased by more than 20%.

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