Method for increasing oil content of soybeans by utilizing rhizosphere marssonia

Abstract

The invention provides a method for increasing the oil content of soybeans by utilizing marssonia rhizosphere. The method for increasing the oil content of the soybeans by utilizing the marssonia rhizosphere comprises the following steps: separating and identifying microorganisms, analyzing the oil content and fatty acid of seeds, and detecting the plant height, the root length and the nitrogen and oil content. The microbial separation and identification comprises the following steps: putting 1g of an unplanted fresh soil sample and 2ml of filtered and disinfected Hengfeng 52 root exudates into a 5ml centrifugal tube, culturing for 24 hours under the oscillation conditions of 28 DEG C and 180r / min, and diluting the sample in a 10-fold gradient manner. According to the method for improving the oil content of the soybeans by utilizing the rhizosphere massinia, provided by the invention, two strains of massinia capable of promoting the oil content of the soybeans are provided, the bacteria of the genus can be enriched by rhizospheres of high-oil soybean varieties, and the growth of the soybeans can be promoted and the oil content can be improved; the quality of the soybeans can be efficiently and conveniently improved, and the national grain and oil productivity requirement is met.

Description

Technical Field

[0001] The invention belongs to the technical field of agricultural applied microorganisms, and in particular relates to a method for improving soybean oil content by utilizing rhizosphere Masseillaris. Background Art

[0002] Soybean is one of the most important oil crops, accounting for more than 50% of the total global edible oil consumption. It is also the world's main source of plant protein. Due to the growing demand for oil in the global oilseed market, the development of high-oil soybean varieties has become a priority for soybean breeding programs. To date, many improved varieties with high yield and high oil quality have been developed through breeding programs to meet the rapidly growing demand for global soybean production, but there are still significant gaps. Identification of determinants associated with seed oil quality traits is crucial to improving soybean quality traits. Recent studies have shown that plant domestication and genetic improvement of agronomic traits (such as stress resistance and disease resistance) change the structure and function of the rhizosphere microbial community. However, it is still unclear whether seed quality breeding affects the rhizosphere microbial community and what the contribution of the rhizosphere microbial community to seed quality is.

[0003] Soil is the habitat of plants and their associated microorganisms and the medium for their interactions. To a certain extent, host plants drive microbial selection by depositing specific secretions (root exudates) at the soil-root interface. Root exudates are a mixture of soluble organic matter, including sugars, amino acids, organic acids, fatty acids, proteins and other secondary metabolites, which can act as nutrients, antimicrobial substances, chemotaxis or signaling molecules to coordinate changes in the composition of rhizosphere microorganisms. The composition and quantity of plant root exudates are species or variety specific and vary significantly during the life cycle and in response to environmental changes. These changes exert selective pressure on the rhizosphere microbial community. However, current knowledge on the relationship between quality traits, root exudation and rhizosphere microbial assembly is very limited. Microbial colonization of the plant rhizosphere can confer a variety of biological functions to the host. For example, nitrogen-fixing rhizobia can form symbiotic nodules to provide nitrogen sources for the growth of legume hosts, which represents the highest level of mutualistic relationship between host plants and rhizosphere bacteria. Many Pseudomonas and Bacillus species are able to secrete siderophores and antimicrobial substances to provide plants with iron nutrition and confer resistance to pathogens. Recently, increasing evidence has shown that the microbiota and its group effects have regulatory functions in plant development and environmental adaptation. Studies have found that in indica rice, synthetic communities enriched in the roots can improve the efficiency of organic nitrogen utilization. In addition, a single bacterial genus can completely reverse the severe inhibition of root growth by complex microbial communities by degrading auxins. The microbiota and root endodermis can support the homeostasis of plant mineral nutrition by inhibiting the abscisic acid signaling pathway. These results indicate that rhizosphere microorganisms have evolved functions that strongly affect plant growth and development. It is not yet clear whether and how the microbiota affects seed oil content and quality.

[0004] Therefore, it is necessary to provide a new method for increasing soybean oil content by using rhizospheric Masseilli bacteria to solve the above technical problems. Summary of the invention

[0005] The technical problem solved by the present invention is to provide a method for increasing the oil content of soybeans by using rhizosphere Marseillariae, which can be enriched in the rhizosphere of high-oil soybean varieties and can promote the growth of soybeans and increase the oil content, thereby helping to efficiently and conveniently improve the quality of soybeans and meet the national grain and oil production capacity needs.

[0006] In order to solve the above technical problems, the method for improving soybean oil content by using rhizosphere Massimo bacteria provided by the present invention comprises: microbial isolation and identification, seed oil content and fatty acid analysis, and plant height, root length, nitrogen and oil content detection;

[0007] The microbial separation and identification comprises the following steps:

[0008] 1) Place 1 gram of fresh unplanted soil sample and 2 ml of filtered and sterilized Hefeng 52 root exudates into a 5 ml centrifuge tube, incubate at 28°C and 180 r / min for 24 hours, then dilute the sample 10-fold, apply it to the minimum culture medium, and incubate it in a 28°C incubator for 2-3 days;

[0009] 2) Select representative clones based on morphology and color, purify them and store them in glycerol at -80°C;

[0010] 3) After overnight culture of the purified bacteria, DNA was extracted, and 16S rRNA gene was amplified and sequenced using primers 27F and 1492R to identify the isolated strains.

[0011] As a further embodiment of the present invention, the seed oil content and fatty acid analysis comprises the following steps:

[0012] 1) Plant the chlorine-sterilized seeds in a mixture of soil and nutrient soil and grow them for 4 days;

[0013] 2) Inoculate the plants with 30 ml of mixed Marseille suspension (OD 600 =0.1), and then the same volume of water was used as a control, the plants were grown at 20°C / 28°C, a photoperiod of 16 h night / day, normal water and fertilizer treatments were performed during the growth period, and the same concentration of bacterial suspension was inoculated again during flowering;

[0014] 3) After the seeds matured, the oil content of the seeds treated with and without the treatment of Massillaria was measured, the seeds were ground with n-hexane to extract lipids, incubated at 37°C for 5 h, and centrifuged at 11,000 r / min for 10 min at room temperature;

[0015] 4) The supernatant was evaporated in a pre-weighed 1.5 ml centrifuge tube under vacuum until a constant weight was reached. This process was repeated twice, and the centrifuge tube was weighed again. The lipid content in the tube was divided by the seed weight to determine the seed lipid content. The seed fatty acid content was then determined using the gas phase method.

[0016] As a further embodiment of the present invention, the plant height, root length and nitrogen content detection comprises the following steps:

[0017] 1) Soybean seeds sterilized with chlorine were germinated on sterile water agar for 3 days, and then the plants were transplanted into a suspension of Massey M16 or M117 (OD 600 =0.1) in vermiculite pots to ensure one seedling per pot, with sterile water as control;

[0018] 2) Plants were grown under 16h light at 20℃ / 28℃ and watered with sterilized water as needed;

[0019] 3) After 15 days of growth, the seedlings were treated with nitrogen-free (NF), low nitrogen (LN, 0.25 mM) and normal nitrogen (NN, 7.5 mM) BD nutrient solutions;

[0020] 4) Each treatment included 15 seedlings, and after 15 days of growth, the plants were sampled to determine their height and length;

[0021] 5) The sample was then dried at 105°C for 0.5 h and then at 70°C until constant weight was reached, and the nitrogen content was determined by the Kjeldahl method.

[0022] As a further embodiment of the present invention, the minimal culture medium comprises 0.5 g glucose, 0.5 g polypeptide, 0.5 g sodium glutamate, 0.5 g yeast extract, 0.44 g K2HPO4, 0.1 g (NH4)2SO4, 0.1 g MgSO4 and pH 7.0.

[0023] As a further embodiment of the present invention, the volume ratio of the chlorine-sterilized seeds to the mixture is 3:1, and the chlorine-sterilized seeds are Jilin Chalihua and Hefeng 52.

[0024] As a further embodiment of the present invention, the soil can be treated with root exudates of high-oil varieties and two candidate strains can be isolated.

[0025] As a further solution of the present invention, the >M117 is as follows:

[0026] >M117

[0027] CGGGGATGCTTTACCTGCAGTCGAACGGCAGCGCGGGGCAACCTGGCGGCGAGTGGCGAACGGGTGAGTAATATATCGGAACGTACCCAAGAGTGGGGGATAACGTAGCGAAAGTTACGCTAATACCGCATACGATCTAAGGATGAAAGTGGGGGATCGCAAGACCTCATGCTCCTGGAGCGGCCGATATCTGATTAGCTAGTTGGTGAGGTAAAGGCTCACCAAGGCGACGATCAGTAGCTGGTCTGAGAGGACGACCAGCCACACTGGGACTGAGACACGGCCCAGACTCCTACGGGAGGCAGCAGTGGGGAATTTTGGACAATGGGCGCAAGCCTGATCCAGCAATGCCGCGTGAGTGAAGAAGGCCTTCGGGTTGTAAAGCTCTTTTGTCAGGGAAGAAACGGCTCCGGCTAATATCCGGGGCTAATGACGGTACCTGAAGAATAAGCACCGGCTAACTACGTGCCAGCAGCCGCGGTAATACGTAGGGTGCAAGCGTTAATCGGAATTACTGGGCGTAAAGCGTGCGCAGGCGGTTTTGTAAGTCTGTCGTGAAAGCCCCGGGCTTAACCTGGGAATTGCGATGGAGACTGCAAGGCTTGAATCTGGCAGAGGGGGGTAGAATTCCACGTGTAGCAGTGAAATGCGTAGAGATGTGGAGGAACACCGATGGCGAAGGCAGCCCCCTGGGTCAAGATTGACGCTCATGCACGAAAGCGTGGGGAGCAAACAGGATTAGATACCCTGGTAGTCCACGCCCTAAACGATGTCTACTAGTTGTCGGGTCTTAATTGACTTGGTAACGCAGCTAACGCGTGAAGTAGACCGCCTGGGGAGTACGGTCGCAAGATTAAAACTCAAAGGAATTGACGGGGACCCGCACAAGCGGTGGATGATGTGGATTATTCGATGCACCGCGAAAACCTTACCTCCCTTGACTGTCAGGACCTCCGAAAATTGGAGGTGCCCAAAGGAACCTGAACCAGTGCTGCTGGC。

[0028] As a further solution of the present invention, the >M16 is as follows:

[0029] >M16

[0030] AAACGATGCTTTACCTGCAGTCGAACGGCAGCGCGGGGCAACCTGGCGGCGAGTGGCGAACGGGTGAGTAATATATCGGAACGTACCCAGAAGTGGGGGATAACGTAGCGAAAGTTACGCTAATACCGCATACGATCTACGGATGAAAGTGGGGGACCTTCGGGCCTCATGCTTTTGGAGCGGCCGATATCTGATTAGCTAGTTGGTGAGGTAAAGGCTCACCAAGGCGACGATCAGTAGCTGGTCTGAGAGGACGACCAGCCACACTGGGACTGAGACACGGCCCAGACTCCTACGGGAGGCAGCAGTGGGGAATTTTGGACAATGGGCGCAAGCCTGATCCAGCAATGCCGCGTGAGTGAAGAAGGCCTTCGGGTTGTAAAGCTCTTTTGTCAGGGAAGAAACGGCCTCAGCTAATATCTGGGGCTAATGACGGTACCTGAAGAATAAGCACCGGCTAACTACGTGCCAGCAGCCGCGGTAATACGTAGGGTGCAAGCGTTAATCGGAATTACTGGGCGTAAAGCGTGCGCAGGCGGTTTTGTAAGTCTGTCGTGAAAGCCCCGGGCTTAACCTGGGAATTGCGATGGAGACTGCAAGGCTTGAATCTGGCAGAGGGGGGTAGAATTCCACGTGTAGCAGTGAAATGCGTAGAGATGTGGAGGAACACCGATGGCGAAGGCAGCCCCCTGGGTCAAGATTGACGCTCATGCACGAAAGCGTGGGGAGCAAACAGGATTAGATACCCTGGTAGTCCACGCCCTAAACGATGTCTACTAGTTGTCGGGTCTTAATTGACTTGGTAACGCAGCTAACGCGTGAAGTAGACCGCCTGGGGAGTACGGTCGCAAGATTAAAACTCAAAGGAATTGACGGGGACCCGCACAAGCGGTGGATGATGTGGATTAATTCGATGCAACGCGAAAAACCTTACCTACCCTTGACATGTCAGGAACCTCCGAGAGATTGGAGGGTGCCCGAAAGGGAACCTGAACCCAG。

[0031] As a further embodiment of the present invention, the Masseella M117 and M16 are used to promote soybean growth and increase soybean oil content.

[0032] As a further solution of the present invention, the Masseella is used in the preparation of microbial agents, microbial fertilizers, biological fertilizers and other products for increasing soybean yield and oil content.

[0033] Compared with the related art, the method for increasing soybean oil content by using rhizosphere Masseillaris provided by the present invention has the following beneficial effects:

[0034] 1. The present invention provides two strains of Masseillicum that can promote the oil content of soybeans. The strains can be enriched in the rhizosphere of high-oil soybean varieties, and can promote the growth of soybeans and increase the oil content, which helps to efficiently and conveniently improve the quality of soybeans and meet the national grain and oil production capacity needs. BRIEF DESCRIPTION OF THE DRAWINGS

[0035] In order to facilitate understanding by those skilled in the art, the present invention is further described below with reference to the accompanying drawings.

[0036] Figure 1 Schematic diagram of identification of isolated strains of the present invention;

[0037] Figure 2 This is a schematic diagram of the method for treating soil with root exudates of the present invention;

[0038] Figure 3 A schematic diagram of increasing the weight, oil content and fatty acid content of seeds of the present invention is provided;

[0039] Figure 4 The Massillarism of the present invention promotes the growth and nitrogen content of plant roots. DETAILED DESCRIPTION

[0040] Please refer to Figures 1 to 4 ,in, Figure 1 Schematic diagram of identification of isolated strains of the present invention; Figure 2 This is a schematic diagram of the method for treating soil with root exudates of the present invention; Figure 3 A schematic diagram of increasing the weight, oil content and fatty acid content of seeds of the present invention is provided; Figure 4 The method of using rhizosphere Massillaria to increase soybean oil content includes: microbial isolation and identification, seed oil content and fatty acid analysis, and plant height, root length, nitrogen and oil content detection;

[0041] The microbial separation and identification comprises the following steps:

[0042] 1) Place 1 gram of fresh unplanted soil sample and 2 ml of filtered and sterilized Hefeng 52 root exudates into a 5 ml centrifuge tube, incubate at 28°C and 180 r / min for 24 hours, then dilute the sample 10-fold, apply it to the minimum culture medium, and incubate it in a 28°C incubator for 2-3 days;

[0043] 2) Select representative clones based on morphology and color, purify them and store them in glycerol at -80°C;

[0044] 3) After overnight culture of the purified bacteria, DNA was extracted, and 16S rRNA gene was amplified and sequenced using primers 27F and 1492R to identify the isolated strains.

[0045] The seed oil content and fatty acid analysis comprises the following steps:

[0046] 1) Plant the chlorine-sterilized seeds in a mixture of soil and nutrient soil and grow them for 4 days;

[0047] 2) Inoculate the plants with 30 ml of mixed Marseille suspension (OD 600 =0.1), and then the same volume of water was used as a control, the plants were grown at 20°C / 28°C, a photoperiod of 16 h night / day, normal water and fertilizer treatments were performed during the growth period, and the same concentration of bacterial suspension was inoculated again during flowering;

[0048] 3) After the seeds matured, the oil content of the seeds treated with and without the treatment of Massillaria was measured, the seeds were ground with n-hexane to extract lipids, incubated at 37°C for 5 h, and centrifuged at 11,000 r / min for 10 min at room temperature;

[0049] 4) The supernatant was evaporated in a pre-weighed 1.5 ml centrifuge tube under vacuum until a constant weight was reached. This process was repeated twice, and the centrifuge tube was weighed again. The lipid content in the tube was divided by the seed weight to determine the seed lipid content. The seed fatty acid content was then determined using the gas phase method.

[0050] The plant height, root length and nitrogen content detection comprises the following steps:

[0051] 1) Soybean seeds sterilized with chlorine were germinated on sterile water agar for 3 days, and then the plants were transplanted into a suspension of Massey M16 or M117 (OD 600 =0.1) in vermiculite pots to ensure one seedling per pot, with sterile water as control;

[0052] 2) Plants were grown under 16h light at 20℃ / 28℃ and watered with sterilized water as needed;

[0053] 3) After 15 days of growth, the seedlings were treated with nitrogen-free (NF), low nitrogen (LN, 0.25 mM) and normal nitrogen (NN, 7.5 mM) BD nutrient solutions;

[0054] 4) Each treatment included 15 seedlings, and after 15 days of growth, the plants were sampled to determine their height and length;

[0055] 5) The sample was then dried at 105°C for 0.5 h and then at 70°C until constant weight was reached, and the nitrogen content was determined by the Kjeldahl method.

[0056] The minimal medium comprises 0.5 g glucose, 0.5 g polypeptide, 0.5 g sodium glutamate, 0.5 g yeast extract, 0.44 g K2HPO4, 0.1 g (NH4)2SO4, 0.1 g MgSO4 and pH 7.0.

[0057] The volume ratio of the chlorine-sterilized seeds to the mixture is 3:1, and the chlorine-sterilized seeds are Jilin Chalihua and Hefeng 52.

[0058] The soil can be treated with root exudates of high-oil varieties and 2 candidate strains can be isolated.

[0059] The>M117 is as follows:

[0060] >M117

[0061] CGGGGATGCTTTACCTGCAGTCGAACGGCAGCGCGGGGCAACCTGGCGGCGAGTGGCGA

[0062] ACGGTGAGTAATATATCGGAACGTACCCAAGAGTGGGGGATAACGTAGCGAAAGTTAC

[0063] GCTAATACCGCATACGATCTAAGGATGAAAGTGGGGGATCGCAAGACCTCATGTCCCTG

[0064] GAGCGGCCGATATCTGATTAGCTAGTTGGTGAGGTAAAGGCTCACCAAGGCGACGATCA

[0065] GTAGCTGGTCTGAGAGGACGACCAGCCACACTGGGACTGAGACACGGCCCAGACTCCTA

[0066] CGGGAGGCAGCAGTGGGGAATTTTGGACAATGGGCGCAAGCCTGATCCAGCAATGCCGC

[0067] GTGAGTGAAGAAGGCCTTCGGGTTGTAAAGCTCTTTTGTCAGGGAAGAAACGGCTCCGG

[0068] CTAATATCCGGGGCTAATGACGGTACCTGAAGAATAAGCACCGGCTAACTACGTGCCAG

[0069] CAGCCGCGGTAATACGTAGGGTGCAAGCGTTAATCGGAATTACTGGGCGTAAAGCGTGC

[0070] GCAGGCGGTTTTGTAAGTCTGTCGTGAAAGCCCCGGGCTTAACCTGGGAATTGCGATGG

[0071] AGACTGCAAGGCTTGAATCTGGCAGAGGGGGGTAGAATTCCACGTGTAGCAGTGAAATG

[0072] CGTAGAGATGTGGAGGAACACCGATGGCGAAGGCAGCCCCCTGGGTCAAGATTGACGCT

[0073] CATGCACGAAAGCGTGGGGAGCAAACAGGATTAGATACCCTGGTAGTCCACGCCCTAAA

[0074] CGATGTCTACTAGTTGTCGGGTCTTAATTGACTTGGTAACGCAGCTAACGCGTGAAGTA

[0075] GACCGCCTGGGGAGTACGGTCGCAAGATTAAAACTCAAAGGAATTGACGGGGACCCGCA

[0076] CAAGCGGTGGATGATGTGGATTATTCGATGCACCGCGAAAACCTTACCTCCCTTGACTGTCAGGACCTCCGAAAATTGGAGGTGCCCAAAGGAACCTGAACCAGTGCTGCTGGC。

[0077] The said >M16 is as follows:

[0078] >M16

[0079] AAACGATGCTTTACCTGCAGTCGAACGGCAGCGCGGGGCAACCTGGCGGCGAGTGGCGA

[0080] ACGGGTGAGTAATATATCGGAACGTACCCAGAAGTGGGGGATAACGTAGCGAAAGTTAC

[0081] GCTAATACCGCATACGATCTACGGATGAAAGTGGGGGACCTTCGGGCCTCATGCTTTTG

[0082] GAGCGGCCGATATCTGATTAGCTAGTTGGTGAGGTAAAGGCTCACCAAGGCGACGATCA

[0083] GTAGCTGGTCTGAGAGGACGACCAGCCACACTGGGACTGAGACACGGCCCAGACTCCTA

[0084] CGGGAGGCAGCAGTGGGGAATTTTGGACAATGGGCGCAAGCCTGATCCAGCAATGCCGC

[0085] GTGAGTGAAGAAGGCCTTCGGGTTGTAAAGCTCTTTTGTCAGGGAAGAAACGGCCTCAG

[0086] CTAATATCTGGGGCTAATGACGGTACCTGAAGAATAAGCACCGGCTAACTACGTGCCAG

[0087] CAGCCGCGGTAATACGTAGGGTGCAAGCGTTAATCGGAATTACTGGGCGTAAAGCGTGC

[0088] GCAGGCGGTTTTGTAAGTCTGTCGTGAAAGCCCCGGGCTTAACCTGGGAATTGCGATGG

[0089] AGACTGCAAGGCTTGAATCTGGCAGAGGGGGGTAGAATTCCACGTGTAGCAGTGAAATG

[0090] CGTAGAGATGTGGAGGAACACCGATGGCGAAGGCAGCCCCCTGGGTCAAGATTGACGCT

[0091] CATGCACGAAAGCGTGGGGAGCAAACAGGATTAGATACCCTGGTAGTCCACGCCCTAAA

[0092] CGATGTCTACTAGTTGTCGGGTCTTAATTGACTTGGTAACGCAGCTAACGCGTGAAGTA

[0093] GACCGCCTGGGGAGTACGGTCGCAAGATTAAAACTCAAAGGAATTGACGGGGACCCGCA

[0094] CAAGCGGTGGATGATGTGGATTAATTCGATGCAACGCGAAAAACCTTACCTACCCTTGACATGTCAGGAACCTCCGAGAGATTGGAGGGTGCCCGAAAGGGAACCTGAACCCAG.

[0095] The application of the Masseella M117 and M16 in promoting soybean growth and increasing soybean oil content.

[0096] The Massella is used in the preparation of microbial agents, microbial fertilizers, biological fertilizers and other products for improving soybean yield and oil content.

[0097] Two strains of Marseille bacteria that can promote the oil content of soybeans are provided. The bacteria can be enriched in the rhizosphere of high-oil soybean varieties, and can promote the growth of soybeans and increase the oil content, which helps to efficiently and conveniently improve the quality of soybeans and meet the national grain and oil production capacity needs.

[0098] Through NCBI sequence alignment, the genus Masseria Figure 1 .

[0099] The research on the ability of Marseille bacteria to increase the oil content and the number of oil bodies in seeds is as follows:

[0100] A low-oil cultivar (Jilincharihua) and a high-oil cultivar (Hefeng 52) were grown in a greenhouse. Under normal water and fertilizer conditions, four mixed Marseille suspensions were treated twice at the seedling stage and flowering stage. After treatment with the mixed suspensions, the seed weight of the low-oil cultivar Jilincharihua increased significantly, while the seed weight of the high-oil cultivar Hefeng 52 did not increase ( Figure 3a), In addition, it was found that the treatment with Marseille bacteria significantly increased the oil content of seeds of the two varieties (14.9% for Jilin Chalihua and 15.7% for Hefeng 52), but had no effect on the protein content ( Figure 3 a), Transmission electron microscopy (TEM) showed that after treatment with Massillaria, the oil bodies of seeds of both varieties were more densely distributed ( Figure 3 b), and then the contents of 19 major fatty acids were determined by gas chromatography (GC). In the Jilinchali flower, the contents of all 19 fatty acids increased significantly after treatment with Marseille bacteria, among which C18:2n6c increased the most, followed by C18:1n9t, C20:0 and C22:0 ( Figure 3 c);

[0101] The research on the effect of Massillarism on promoting plant growth and nitrogen accumulation is as follows:

[0102] As rhizospheric bacteria, Massillaria can promote lateral root growth and nitrogen accumulation in the lateral root defective mutant lrt1 of maize under nitrogen deficiency conditions. Similarly, under low nitrogen (LN) conditions, Massillaria can also increase soybean plant height and leaf nitrogen accumulation. In contrast, under low nitrogen and normal nitrogen (NN) conditions, the leaf nitrogen content of plants treated with strain M117 was significantly higher than that of plants treated with the control and M16. In addition, it was found that Massillaria can promote root length ( Figure 4 ).

[0103] The present invention is further described below in conjunction with specific embodiments to provide a better understanding of the present invention.

[0104] Example 1

[0105] Isolation and cultivation of Marseilles M16 and M117:

[0106] In this embodiment, the plant growth-promoting rhizosphere bacteria M16 and M117 were isolated from the rhizosphere of soybeans, and the soybeans were collected from the Jilin Academy of Agricultural Sciences in Gongzhuling City;

[0107] To isolate Marseille bacteria that respond positively to root exudates from high-oil cultivars, we used root exudates from Hefeng 52 to treat soil as follows:

[0108] 1) Transfer 1 gram of fresh unplanted soil sample and 2 ml of filtered and sterilized root exudate or sterile distilled water (control) into a 5 ml centrifuge tube. Incubate at 28°C and 180 r / min for 24 hours, centrifuge at 10,000 r / min in a refrigerated centrifuge for 5 minutes, collect the soil sample, and store it at -80°C ( Figure 2 );

[0109] 2), gradient dilutions were prepared and cultured in minimal medium (MM; 0.5 g glucose, 0.5 g polypeptide, 0.5 g sodium glutamate, 0.5 g yeast extract, 0.44 g K2HPO4, 0.1 g (NH4)2SO4, 0.1 g MgSO4; pH 7.0), representative clones were selected based on morphology and color, purified and stored in MM containing 20% ​​glycerol at -80°C until further use, and 16S rRNA gene sequencing was performed using 27F and 1492R primers to classify the isolates at the genus level.

[0110] Embodiment 2:

[0111] The method for cultivating and producing seeds of Marseille bacteria M16 and M117 is carried out according to the following steps:

[0112] Massimo CCTCC M 20241739 (M16) and CCTCC M20241740 (M117) stored at -80°C were inoculated into TY liquid medium (tryptone 10 g / L, yeast extract 5 g / L, calcium chloride 10 mM / L), and cultured at 28°C with shaking for 24-36 h to obtain the OD 600 The fermentation broth is in the logarithmic growth phase with a concentration of about 2.0.

[0113] Embodiment 3:

[0114] The method of inoculating soybean with Marseille bacteria to increase oil content is carried out according to the following steps:

[0115] 1) Chlorine-sterilized seeds (Jilin Chalihua and Hefeng 52) were planted in pots (15 × 15 cm) filled with a mixture of soil and vermiculite (vol:vol = 3:1) and grown for 4 days, after which the plants were inoculated with 30 ml of a mixed Marseille suspension (OD 600 =0.1), with the same volume of water as control;

[0116] 2) Plants were grown at 20°C / 28°C and a 16-h night / day photoperiod, with normal water and fertilizer treatment during the growth period, and inoculated with the same concentration of bacterial suspension again during flowering;

[0117] 3) After maturity, the oil content of the seeds of plants treated with and without Marseille bacteria was measured according to the method. The seeds were ground with n-hexane to extract lipids. After incubation at 37°C for 5 hours, they were centrifuged at 11,000 r / min for 10 minutes at room temperature. The supernatant was vacuum evaporated in a pre-weighed 1.5 ml centrifuge tube until a constant weight was reached. This process was repeated twice, and then the weight of the centrifuge tube was weighed again. The amount of lipid in the test tube was divided by the seed weight to determine the seed lipid content, and then the seed fatty acid content was determined according to the method.

[0118] Embodiment 4:

[0119] The method of promoting soybean growth and nitrogen accumulation by inoculation with Massillaria spp. is carried out in the following steps:

[0120] Chlorine-sterilized soybean seeds were germinated on sterile water agar for 3 days, and then the plants were transplanted into a suspension of Massey M16 or M117 (OD 600 =0.1) in vermiculite pots (13×13 cm) to ensure one seedling per pot, with sterile water as control. The plants were grown at 20℃ / 28℃ under 16h light and watered with sterilized water as needed. After growing for 15 days, the seedlings were treated with nitrogen-free (NF), low nitrogen (LN, 0.25mM) and normal nitrogen (NN, 7.5mM) BD nutrient solutions, with each treatment including 15 seedlings. After growing for 15 days, the plants were sampled to determine their height and length, and the samples were subsequently dried at 105℃ for 0.5h and then at 70℃ until constant weight was reached. The nitrogen content was determined by the Kjeldahl method.

[0121] Two strains of Marseille bacteria capable of promoting the oil content of soybeans are provided. The bacteria can be enriched in the rhizosphere of high-oil soybean varieties, and can promote the growth of soybeans and increase the oil content, thereby helping to efficiently and conveniently improve the quality of soybeans.

[0122] The above description is only a preferred specific implementation manner of the present invention, but the protection scope of the present invention is not limited thereto. Any technician familiar with the technical field can make equivalent replacements or changes according to the technical scheme and inventive concept of the present invention within the technical scope disclosed by the present invention, which should be covered by the protection scope of the present invention.

Claims

1. A method for increasing soybean oil content by using rhizosphere Massimo bacteria, characterized in that: include: Isolation and identification of microorganisms, analysis of seed oil content and fatty acids, and testing of plant height, root length, and nitrogen and oil content; The microbial separation and identification comprises the following steps: 1) Place 1 gram of fresh unplanted soil sample and 2 ml of filtered and sterilized Hefeng 52 root exudates into a 5 ml centrifuge tube, incubate at 28°C and 180 r / min for 24 hours, then dilute the sample 10-fold, apply it to the minimum culture medium, and incubate it in a 28°C incubator for 2-3 days; 2) Select representative clones based on morphology and color, purify them and store them in glycerol at -80°C; 3) After overnight culture of the purified bacteria, DNA was extracted, and 16S rRNA gene was amplified and sequenced using primers 27F and 1492R to identify the isolated strains.

2. The method for increasing soybean oil content by using rhizobacteria according to claim 1, characterized in that: The seed oil content and fatty acid analysis comprises the following steps: 1) Plant the chlorine-sterilized seeds in a mixture of vermiculite and nutrient soil and grow them for 4 days; 2) Inoculate the plants with 30 ml of mixed Marseille suspension (OD 600 =0.1), and then the same volume of water was used as a control, the plants were grown at 28°C / 20°C, day / night (16h / 8h) photoperiod, normal water and fertilizer treatment was performed during the growth period, and the same concentration of bacterial suspension was inoculated again during flowering; 3) After the seeds matured, the oil content of the seeds treated with and without the treatment of Massillaria was measured, the seeds were ground with n-hexane to extract lipids, incubated at 37°C for 5 h, and centrifuged at 11,000 r / min for 10 min at room temperature; 4) The supernatant was evaporated in a pre-weighed 1.5 ml centrifuge tube under vacuum until a constant weight was reached. This process was repeated twice, and the centrifuge tube was weighed again. The lipid content in the tube was divided by the seed weight to determine the seed lipid content. The seed fatty acid content was then determined using the gas phase method.

3. The method for increasing soybean oil content by using rhizobacteria according to claim 1, characterized in that: The plant height, root length and nitrogen content detection comprises the following steps: 1) Soybean seeds sterilized with chlorine were germinated on sterile water agar for 3 days, and then the plants were transplanted into a suspension of Massey M16 or M117 (OD 600 =0.1) in vermiculite pots to ensure one seedling per pot, with sterile water as control; 2) Plants were grown under 16h light at 20℃ / 28℃ and watered with sterilized water as needed; 3) After 15 days of growth, the seedlings were treated with nitrogen-free (NF), low nitrogen (LN, 0.25 mM) and normal nitrogen (NN, 7.5 mM) BD nutrient solutions; 4) Each treatment included 15 seedlings, and after 15 days of growth, the plants were sampled to determine their height and length; 5) The sample was then dried at 105°C for 0.5 h and then at 70°C until constant weight was reached, and the nitrogen content was determined by the Kjeldahl method.

4. The method for increasing soybean oil content by using rhizobacteria according to claim 1, characterized in that: The minimal medium comprises 0.5 g glucose, 0.5 g polypeptide, 0.5 g sodium glutamate, 0.5 g yeast extract, 0.44 g K2HPO4, 0.1 g (NH4)2SO4, 0.1 g MgSO4 and pH 7.

0.

5. The method for increasing soybean oil content by using rhizobacteria according to claim 3, characterized in that: The volume ratio of the chlorine-sterilized seeds to the mixture is 3:1, and the chlorine-sterilized seeds are Jilin Chalihua and Hefeng 52.

6. The method for increasing soybean oil content by using rhizobacteria according to claim 1, characterized in that: The soil can be treated with root exudates of high-oil varieties and 2 candidate strains can be isolated.

7. The method for increasing soybean oil content by using rhizospheric Massimo bacteria according to claim 3, characterized in that: The>M117 is as follows: >M117 CGGGGATGCTTTACCTGCAGTCGAACGGCAGCGCGGGGCAACCTGGCGGCGAGTGGCGAACGGGTGAGTAATATATCGGAACGTACCCAAGAGTGGGGGATAACGTAGCGAAAGTTACGCTAATACCGCATACGATCTAAGGATGAAAGTGGGGGATCGCAAGACCTCATGCTCCTGGAGCGGCCGATATCTGATTAGCTAGTTGGTGAGGTAAAGGCTCACCAAGGCGACGATCAGTAGCTGGTCTGAGAGGACGACCAGCCACACTGGGACTGAGACACGGCCCAGACTCCTACGGGAGGCAGCAGTGGGGAATTTTGGACAATGGGCGCAAGCCTGATCCAGCAATGCCGCGTGAGTGAAGAAGGCCTTCGGGTTGTAAAGCTCTTTTGTCAGGGAAGAAACGGCTCCGGCTAATATCCGGGGCTAATGACGGTACCTGAAGAATAAGCACCGGCTAACTACGTGCCAGCAGCCGCGGTAATACGTAGGGTGCAAGCGTTAATCGGAATTACTGGGCGTAAAGCGTGCGCAGGCGGTTTTGTAAGTCTGTCGTGAAAGCCCCGGGCTTAACCTGGGAATTGCGATGGAGACTGCAAGGCTTGAATCTGGCAGAGGGGGGTAGAATTCCACGTGTAGCAGTGAAATGCGTAGAGATGTGGAGGAACACCGATGGCGAAGGCAGCCCCCTGGGTCAAGATTGACGCTCATGCACGAAAGCGTGGGGAGCAAACAGGATTAGATACCCTGGTAGTCCACGCCCTAAACGATGTCTACTAGTTGTCGGGTCTTAATTGACTTGGTAACGCAGCTAACGCGTGAAGTAGACCGCCTGGGGAGTACGGTCGCAAGATTAAAACTCAAAGGAATTGACGGGGACCCGCACAAGCGGTGGATGATGTGGATTATTCGATGCACCGCGAAAACCTTACCTCCCTTGACTGTCAGGACCTCCGAAAATTGGAGGTGCCCAAAGGAACCTGAACCAGTGCTGCTGGC。 8. The method for increasing soybean oil content by using rhizobacteria according to claim 3, characterized in that: The M16 is as follows: >M16 AAACGATGCTTTACCTGCAGTCGAACGGCAGCGCGGGGCAACCTGGCGGCGAGTGGCGAACGGGTGAGTAATATATCGGAACGTACCCAGAAGTGGGGGATAACGTAGCGAAAGTTACGCTAATACCGCATACGATCTACGGATGAAAGTGGGGGACCTTCGGGCCTCATGCTTTTGGAGCGGCCGATATCTGATTAGCTAGTTGGTGAGGTAAAGGCTCACCAAGGCGACGATCAGTAGCTGGTCTGAGAGGACGACCAGCCACACTGGGACTGAGACACGGCCCAGACTCCTACGGGAGGCAGCAGTGGGGAATTTTGGACAATGGGCGCAAGCCTGATCCAGCAATGCCGCGTGAGTGAAGAAGGCCTTCGGGTTGTAAAGCTCTTTTGTCAGGGAAGAAACGGCCTCAGCTAATATCTGGGGCTAATGACGGTACCTGAAGAATAAGCACCGGCTAACTACGTGCCAGCAGCCGCGGTAATACGTAGGGTGCAAGCGTTAATCGGAATTACTGGGCGTAAAGCGTGCGCAGGCGGTTTTGTAAGTCTGTCGTGAAAGCCCCGGGCTTAACCTGGGAATTGCGATGGAGACTGCAAGGCTTGAATCTGGCAGAGGGGGGTAGAATTCCACGTGTAGCAGTGAAATGCGTAGAGATGTGGAGGAACACCGATGGCGAAGGCAGCCCCCTGGGTCAAGATTGACGCTCATGCACGAAAGCGTGGGGAGCAAACAGGATTAGATACCCTGGTAGTCCACGCCCTAAACGATGTCTACTAGTTGTCGGGTCTTAATTGACTTGGTAACGCAGCTAACGCGTGAAGTAGACCGCCTGGGGAGTACGGTCGCAAGATTAAAACTCAAAGGAATTGACGGGGACCCGCACAAGCGGTGGATGATGTGGATTAATTCGATGCAACGCGAAAAACCTTACCTACCCTTGACATGTCAGGAACCTCCGAGAGATTGGAGGGTGCCCGAAAGGGAACCTGAACCCAG。 9. The method for increasing soybean oil content by using rhizobacteria according to claim 3, characterized in that: The application of the Masseella M117 and M16 in promoting soybean growth and increasing soybean oil content.

10. The method for increasing soybean oil content by using rhizobacterium spp. according to claim 9, characterized in that: The Massella is used in the preparation of microbial agents, microbial fertilizers, biological fertilizers and other products for improving soybean yield and oil content.

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