A peanut composite rhizobium agent and its preparation method and application

Abstract

The present invention provides a peanut composite rhizobium agent and its preparation method and application, belonging to the technical field of microbial agents. The peanut composite rhizobium agent of the present invention comprises the following components: a mixed bacterial solution, K2HPO4, NaCl, and sucrose; the mixed bacterial solution comprises a Bradyrhizobium WD-1 bacterial solution and a Bacillus subtilis LX13 bacterial solution, and the number of viable bacteria in the Bradyrhizobium WD-1 bacterial solution and the Bacillus subtilis LX13 bacterial solution is 4 to 6×10 9 cfu / mL. The present invention shortens the preparation cycle of a peanut composite rhizobium agent by optimizing the culture medium of Bradyrhizobium WD-1. Furthermore, Bradyrhizobium WD-1 and Bacillus subtilis LX13 have significant synergistic effects in preventing and controlling peanut root rot and increasing the number of peanut nodules, and can be widely used in the field of peanut cultivation.

Description

Technical Field

[0001] The present invention relates to the technical field of microbial agents, and in particular to a peanut composite rhizobium agent and a preparation method and application thereof. Background Art

[0002] Rhizobium is a type of Gram-negative bacteria that can form nodules symbiotically with legumes and reduce nitrogen in the air to ammonia, providing plant nutrition. Rhizobium cells are rod-shaped, possess flagella, and capsules, but do not produce spores. They stain Gram-negative. The bacteria living in nodules are pear-shaped, stick-shaped, or in shapes such as "T," "X," or "Y." These deformed bacteria are called bacteroids. Each rhizobium species can only form nodules on one or several legumes, establishing a symbiotic relationship and demonstrating its specificity. Rhizobia invade the host root and stimulate certain cells in the root cortex and pericycle, causing them to grow vigorously, resulting in localized swelling of the root and forming nodules. Once the rhizobia have established themselves within the root, the plant provides the rhizobia with mineral nutrients and energy, while the rhizobia fix free nitrogen in the atmosphere, providing nitrogen nutrients for the plant. The two maintain a balanced antagonistic parasitic relationship, resulting in a symbiotic phenomenon.

[0003] The preparation of rhizobium inoculants generally involves several stages: seed activation, solid culture in test tubes, liquid shaking culture in triangular flasks, and fermentation. However, peanut rhizobia suffer from a slow growth rate. For example, patent application number 201310643826.5, entitled "A Bradyrhizobium for Peanuts and Its Use," discloses a Bradyrhizobium strain, WD-1, which requires 5-7 days for slant culture, 4-5 days for seed culture, and 4-5 days for fermentation, for a total of 9-17 days, resulting in a relatively long culture cycle. Furthermore, while rhizobia can fix nitrogen and promote growth, they lack the ability to prevent and control crop diseases, hindering their widespread application. Summary of the Invention

[0004] The purpose of the present invention is to provide a peanut composite rhizobium agent and a preparation method and application thereof, so as to solve the problems of long culture cycle and single action of peanut rhizobia.

[0005] In order to achieve the above-mentioned object of the invention, the present invention provides the following technical solutions:

[0006] The present invention provides a peanut composite rhizobium agent, comprising the following components in parts by weight: 90-95 parts of a mixed bacterial solution, 0.6-1.2 parts of K2HPO4, 0.1-0.5 parts of NaCl, and 0.5-1.0 parts of sucrose; the mixed bacterial solution comprises a Bradyrhizobium WD-1 bacterial solution and a Bacillus subtilis LX13 bacterial solution, and the number of viable bacteria in the Bradyrhizobium WD-1 bacterial solution and the Bacillus subtilis LX13 bacterial solution is 4-6×10 9 cfu / mL.

[0007] Preferably, the volume ratio of the Bradyrhizobium WD-1 bacterial solution to the Bacillus subtilis LX13 bacterial solution is 1:5 to 5:1.

[0008] The present invention also provides a method for preparing the peanut composite rhizobium agent, comprising the following steps:

[0009] S1. Bradyrhizobium WD-1 and Bacillus subtilis LX13 were inoculated into slant culture medium, respectively, and cultured at 28-30°C for 24-48 hours to obtain slant cultures;

[0010] S2. The slant culture was inoculated into the seed culture medium, and cultured at 28-30°C, 150-200 rpm for 48-72 hours to obtain a seed solution;

[0011] S3. The seed solution was inoculated into the fermentation medium at an inoculum rate of 6-12%, and the mixture was incubated at 28-30°C, 150-200 rpm, and a ventilation rate of 0.2-0.3 m 3 / h for 60 to 80 hours to obtain Bradyrhizobium WD-1 and Bacillus subtilis LX13 bacterial liquids;

[0012] S4. Mix the Bradyrhizobium WD-1 bacterial solution, the Bacillus subtilis LX13 bacterial solution, K2HPO4, NaCl and sucrose to obtain the peanut composite rhizobium agent.

[0013] Preferably, the seed culture medium and fermentation medium of Bradyrhizobium WD-1 include the following components: 8-12 g of fructose, 0.8-1.2 g of yeast extract, 1-2 g of γ-polyglutamic acid, 0.1-0.3 g of MgSO4·7H2O, 0.4-0.6 g of K2HPO4, 0.08-0.12 g of NaCl, 0.04-0.06 g of CaCl2, 3-5 mL of 0.5% sodium molybdate solution, 3-5 mL of 0.5% boric acid solution, 1000 mL of distilled water, and a pH of 6.8-7.0.

[0014] Preferably, the seed culture medium and fermentation medium of the Bacillus subtilis LX13 bacterial liquid are LB medium, comprising the following components: 8-12 g of tryptone, 4-6 g of yeast powder, 8-12 g of NaCl, 1000 mL of distilled water, and pH 7.0-7.2.

[0015] The present invention also provides the use of the peanut composite rhizobium agent in promoting peanut growth and / or increasing peanut yield.

[0016] The present invention also provides application of the peanut composite rhizobium agent in preventing and treating peanut root rot.

[0017] The present invention also provides a method for using the peanut composite rhizobium agent, which comprises mixing the rhizobium agent with peanut seeds, drying in the shade, and sowing the seeds within 12 hours.

[0018] Preferably, the usage of the peanut composite rhizobium agent is 20 to 30 mL / kg peanut seeds.

[0019] By adopting the above technical scheme, the present invention has the following beneficial effects: the present invention shortens the preparation cycle of the peanut composite rhizobium agent by optimizing the culture medium of Bradyrhizobium WD-1, and Bradyrhizobium WD-1 and Bacillus subtilis LX13 have significant synergistic effects in preventing and controlling peanut root rot and increasing the number of peanut nodules, and can be widely used in the field of peanut cultivation. DETAILED DESCRIPTION

[0020] The present invention provides a peanut composite rhizobium agent, comprising the following components in parts by weight: 90-95 parts of a mixed bacterial solution, 0.6-1.2 parts of K2HPO4, 0.1-0.5 parts of NaCl, and 0.5-1.0 parts of sucrose; the mixed bacterial solution comprises a Bradyrhizobium WD-1 bacterial solution and a Bacillus subtilis LX13 bacterial solution, and the number of viable bacteria in the Bradyrhizobium WD-1 bacterial solution and the Bacillus subtilis LX13 bacterial solution is 4-5×10 9 cfu / mL.

[0021] In the peanut composite rhizobium agent of the present invention, the weight proportion of the mixed bacterial solution is 90 to 95 parts, more preferably 91 to 94 parts, and more preferably 92 parts; the mixed bacterial solution includes a Bradyrhizobium WD-1 bacterial solution and a Bacillus subtilis LX13 bacterial solution;

[0022] The weight portion of K2HPO4 is 0.6 to 1.2 parts, more preferably 0.8 to 1.0 parts, and more preferably 0.9 parts;

[0023] The weight portion of NaCl is 0.1 to 0.5 parts, more preferably 0.2 to 0.4 parts, and more preferably 0.3 parts;

[0024] The mass fraction of the sucrose is 0.5 to 1.0 parts, more preferably 0.6 to 0.9 parts, and more preferably 0.8 parts;

[0025] The number of viable bacteria in the Bradyrhizobium WD-1 bacterial solution and the Bacillus subtilis LX13 bacterial solution is 4 to 6×10 9 cfu / mL, more preferably 4.5 to 5.5×10 9 cfu / mL, more preferably 5×10 9 cfu / mL.

[0026] The volume ratio of the bradyrhizobium WD-1 bacterial solution to the Bacillus subtilis LX13 bacterial solution of the present invention is 1:5 to 5:1, more preferably 2:4 to 4:2, and more preferably 1:1.

[0027] The present invention also provides a method for preparing the peanut composite rhizobium agent, comprising the following steps:

[0028] S1. Bradyrhizobium WD-1 and Bacillus subtilis LX13 were inoculated into slant culture medium, respectively, and cultured at 28-30°C for 24-48 hours to obtain slant cultures;

[0029] S2. The slant culture was inoculated into the seed culture medium, and cultured at 28-30°C, 150-200 rpm for 48-72 hours to obtain a seed solution;

[0030] S3. The seed solution was inoculated into the fermentation medium at an inoculum rate of 6-12%, and the mixture was incubated at 28-30°C, 150-200 rpm, and a ventilation rate of 0.2-0.3 m 3 / h for 60 to 80 hours to obtain Bradyrhizobium WD-1 and Bacillus subtilis LX13 bacterial liquids;

[0031] S4. Mix the Bradyrhizobium WD-1 bacterial solution, the Bacillus subtilis LX13 bacterial solution, K2HPO4, NaCl and sucrose to obtain the peanut composite rhizobium agent.

[0032] The present invention first inoculates Bradyrhizobium WD-1 into a slant culture medium for cultivation to obtain a slant culture; preferably, inoculation is performed using an inoculating loop, and the slant culture medium is preferably prepared in a test tube. The culture temperature is 28 to 30° C., more preferably 28.5 to 29.5° C., and more preferably 29° C.; the culture time is 24 to 48 hours, more preferably 30 to 40 hours, and more preferably 35 hours.

[0033] In the present invention, the slant culture medium is inoculated into the seed culture medium for cultivation to obtain a seed solution; the seed solution is preferably cultured in a 250 mL shake flask; the inoculation amount is 3 to 4 inoculation loops / flask; the culture temperature is 28 to 30°C, more preferably 28.5 to 29.5°C, and more preferably 29°C; the culture rotation speed is 150 to 200 rpm, more preferably 170 to 190 rpm, and more preferably 180 rpm; the culture time is 48 to 72 h, more preferably 55 to 65 h, and more preferably 60 h.

[0034] In the present invention, the seed liquid is inoculated into the fermentation medium for cultivation to obtain Bradyrhizobium WD-1 bacterial solution and Bacillus subtilis LX13 bacterial solution, the inoculation amount is 6-12%, more preferably 8-10%, more preferably 9%; the culture temperature is 28-30°C, more preferably 28.5-29.5°C, more preferably 29°C; the culture speed is 150-200 rpm, more preferably 170-190 rpm, more preferably 180 rpm; the ventilation volume during cultivation is 0.2-0.3 m 3 / h, more preferably 0.22 to 0.28 m 3 / h, more preferably 0.25m 3 / h, the culture time is 60 to 80h, more preferably 65 to 75h, and more preferably 70h. The number of viable bacteria in the fermented Bradyrhizobium WD-1 bacterial solution and the Bacillus subtilis LX13 bacterial solution is independently 4 to 6×10 9 cfu / mL, more preferably 4.5 to 5.5×10 9 cfu / mL, more preferably 5×10 9 cfu / mL.

[0035] The seed culture medium and fermentation medium of Bradyrhizobium WD-1 are the same, including the following ingredients: 8-12 g fructose, 0.8-1.2 g yeast extract, 1-2 g γ-polyglutamic acid, 0.1-0.3 g MgSO4·7H2O, 0.4-0.6 g K2HPO4, 0.08-0.12 g NaCl, 0.04-0.06 g CaCl2, 3-5 mL 0.5% sodium molybdate solution, 3-5 mL 0.5% boric acid solution, 1000 mL distilled water, pH 6.8-7.0; in the culture medium, fructose is further preferably 9-11 g, more preferably 10 g, yeast extract is further preferably 0.9-1.1 g, more preferably 1 g, γ-polyglutamic acid is further preferably 1.2-1.8 g, more preferably 1.5 g, MgSO4·7H2O is further preferably 0.15-0.25 g, more preferably 0.2 g, K2HPO4 is further preferably 0.45-0.55 g, more preferably 0.5 g, NaCl is further preferably 0.09-0.11 g, more preferably 0.1 g, CaCl2 is more preferably 0.056 g, 0.5% sodium molybdate solution is further preferably 3.5-4.5 mL, more preferably 4 mL, and 0.5% boric acid solution is further preferably 3.5-4.5 mL, more preferably 4 mL.

[0036] The slant culture medium of Bradyrhizobium WD-1 is supplemented with 10-20 g agar on the basis of the seed culture medium, and the other components are the same as those of the seed culture medium; the amount of agar added is further preferably 13-17 g, more preferably 15 g.

[0037] The seed culture medium and fermentation medium of Bacillus subtilis LX13 are LB medium, comprising the following components: 8-12 g of tryptone, 4-6 g of yeast powder, 8-12 g of NaCl, 1000 mL of distilled water, and a pH of 7.0-7.2; the tryptone content is further preferably 9-11 g, more preferably 10 g; the amount of yeast powder added is further preferably 4.5-5.5 g, more preferably 5 g, and the NaCl content is further preferably 9-11 g, more preferably 10 g.

[0038] The slant culture medium of Bacillus subtilis LX13 is supplemented with 10-20 g agar on the basis of the seed culture medium, and the other components are the same as those of the seed culture medium; the amount of agar added is further preferably 13-17 g, more preferably 15 g.

[0039] The invention mixes Bradyrhizobium WD-1 bacterial solution, Bacillus subtilis LX13 bacterial solution, K2HPO4, NaCl and sucrose to obtain the peanut composite rhizobium agent.

[0040] The present invention also provides the use of the peanut composite rhizobium agent in promoting peanut growth and / or increasing peanut yield.

[0041] The present invention also provides application of the peanut composite rhizobium agent in preventing and treating peanut root rot.

[0042] The present invention also provides a method for using the peanut composite rhizobium agent, which comprises mixing the rhizobium agent with peanut seeds, drying in the shade, and sowing within 12 hours; the usage amount of the peanut composite rhizobium agent is 20 to 30 mL / kg of peanut seeds, more preferably 23 to 28 mL / kg of peanut seeds, and even more preferably 25 mL / kg of peanut seeds.

[0043] The technical solutions provided by the present invention are described in detail below with reference to the embodiments, but they should not be construed as limiting the scope of protection of the present invention.

[0044] The Bradyrhizobium WD-1 described in the present invention was purchased from the China General Microbiological Culture Collection Center with a collection number of CGMCC No. 8386.

[0045] The Bacillus subtilis LX13 of the present invention was purchased from China General Microbiological Culture Collection Center with a collection number of GMCC No.8805.

[0046] Example 1

[0047] Preparation of slant culture medium for Bradyrhizobium WD-1: Dissolve 8 g of fructose, 0.8 g of yeast extract, 1 g of γ-polyglutamic acid, 0.1 g of MgSO4·7H2O, 0.4 g of K2HPO4, 0.08 g of NaCl, 0.04 g of CaCl2, 3 mL of 0.5% sodium molybdate solution, 3 mL of 0.5% boric acid solution, and 10 g of agar in 1000 mL of distilled water, adjust the pH to 6.8, sterilize at 121°C and 0.1 MPa for 30 min, and cool.

[0048] Preparation of seed culture medium for Bradyrhizobium WD-1: Dissolve 8 g of fructose, 0.8 g of yeast extract, 1 g of γ-polyglutamic acid, 0.1 g of MgSO4·7H2O, 0.4 g of K2HPO4, 0.08 g of NaCl, 0.04 g of CaCl2, 3 mL of 0.5% sodium molybdate solution, and 3 mL of 0.5% boric acid solution in 1000 mL of distilled water, adjust the pH to 6.8, sterilize at 121°C and 0.1 MPa for 30 min, and cool.

[0049] The fermentation medium of Bradyrhizobium WD-1 has the same composition as the seed culture medium.

[0050] Preparation of slant culture medium for Bacillus subtilis LX13: Dissolve 8 g of tryptone, 4 g of yeast powder, 8 g of NaCl, and 10 g of agar in 1000 mL of distilled water, adjust the pH to 7.0, sterilize at 121°C and 0.1 MPa for 30 min, and cool.

[0051] Preparation of seed culture medium for Bacillus subtilis LX13: Dissolve 8 g of tryptone, 4 g of yeast powder, and 8 g of NaCl in 1000 mL of distilled water, adjust the pH to 7.0, sterilize at 121°C and 0.1 MPa for 30 min, and cool.

[0052] The fermentation medium of Bacillus subtilis LX13 has the same composition as the seed medium.

[0053] Bradyrhizobium WD-1 and Bacillus subtilis LX13 were picked up with an inoculation loop and inoculated into the slant culture medium in the test tube, and cultured in a constant temperature incubator at 28° C. for 48 h to obtain slant cultures of the two bacteria.

[0054] Use an inoculating loop to pick up the slant culture of Bradyrhizobium WD-1 and Bacillus subtilis LX13 respectively into a 250 mL shake flask containing 100 mL of seed culture medium, inoculate 3 inoculating loops into each flask, and culture at 28°C, 150 rpm and shaking for 72 hours to obtain the seed liquid of the two bacteria respectively.

[0055] The seed liquid was inoculated into the fermentation medium and cultured under the following conditions: 28°C, 150 rpm, and aeration volume of 0.22 m3 / h, and cultured for 80h to obtain Bradyrhizobium WD-1 and Bacillus subtilis LX13 bacterial liquids. The number of viable bacteria in the Bradyrhizobium WD-1 bacterial liquid was 4.8×10 9 cfu / mL, and the number of viable bacteria in the Bacillus subtilis LX13 bacterial solution was 5.2×10 9 cfu / mL.

[0056] A Bradyrhizobium WD-1 bacterial solution and a Bacillus subtilis LX13 bacterial solution were mixed at a volume ratio of 1:5 to obtain a mixed bacterial solution. 90 kg of the mixed bacterial solution, 0.6 kg of K2HPO4, 0.1 kg of NaCl, and 0.5 kg of sucrose were mixed and stirred uniformly to obtain a peanut composite rhizobium agent.

[0057] Example 2

[0058] Preparation of slant culture medium for Bradyrhizobium WD-1: Dissolve 10 g of fructose, 1 g of yeast extract, 1.5 g of γ-polyglutamic acid, 0.2 g of MgSO4·7H2O, 0.5 g of K2HPO4, 0.08-0.12 g of NaCl, 0.05 g of CaCl2, 4 mL of 0.5% sodium molybdate solution, 4 mL of 0.5% boric acid solution, and 15 g of agar in 1000 mL of distilled water, adjust the pH to 6.9, sterilize at 121°C and 0.1 MPa for 30 min, and cool.

[0059] Preparation of seed culture medium for Bradyrhizobium WD-1: Dissolve 10 g of fructose, 1 g of yeast extract, 1.5 g of γ-polyglutamic acid, 0.2 g of MgSO4·7H2O, 0.5 g of K2HPO4, 0.08-0.12 g of NaCl, 0.05 g of CaCl2, 4 mL of 0.5% sodium molybdate solution, and 4 mL of 0.5% boric acid solution in 1000 mL of distilled water, adjust the pH to 6.9, sterilize at 121°C and 0.1 MPa for 30 min, and cool.

[0060] The fermentation medium of Bradyrhizobium WD-1 has the same composition as the seed culture medium.

[0061] Preparation of slant culture medium for Bacillus subtilis LX13: Dissolve 10 g of tryptone, 5 g of yeast powder, 10 g of NaCl, and 15 g of agar in 1000 mL of distilled water, adjust the pH to 7.1, sterilize at 121°C and 0.1 MPa for 30 min, and cool.

[0062] Preparation of seed culture medium for Bacillus subtilis LX13: Dissolve 10 g of tryptone, 5 g of yeast powder, and 10 g of NaCl in 1000 mL of distilled water, adjust the pH to 7.0-7.2, sterilize at 121°C and 0.1 MPa for 30 min, and cool.

[0063] The fermentation medium of Bacillus subtilis LX13 has the same composition as the seed medium.

[0064] Bradyrhizobium WD-1 and Bacillus subtilis LX13 were picked up with an inoculation loop and inoculated into the slant culture medium in the test tube, and cultured in a constant temperature incubator at 29° C. for 36 h to obtain slant cultures of the two bacteria.

[0065] Use an inoculating loop to pick up the slant culture of Bradyrhizobium WD-1 and Bacillus subtilis LX13 respectively into a 250 mL shake flask containing 100 mL seed culture medium, inoculate 3 inoculating loops into each flask, and culture at 29°C, 180 rpm, and shake for 60 hours to obtain the seed liquid of the two bacteria.

[0066] The seed liquid was inoculated into the fermentation medium and cultured under the following conditions: 29°C, 180 rpm, and aeration of 0.25 m 3 / h, and cultured for 70h to obtain Bradyrhizobium WD-1 and Bacillus subtilis LX13 bacterial liquids. The number of viable bacteria in the Bradyrhizobium WD-1 bacterial liquid was 5.5×10 9 cfu / mL, and the number of viable bacteria in the Bacillus subtilis LX13 bacterial solution was 5.8×10 9 cfu / mL.

[0067] A Bradyrhizobium WD-1 bacterial solution and a Bacillus subtilis LX13 bacterial solution were mixed at a volume ratio of 1:1 to obtain a mixed bacterial solution. 92 kg of the mixed bacterial solution, 0.8 kg of K2HPO4, 0.3 kg of NaCl, and 0.8 kg of sucrose were mixed and stirred uniformly to obtain a peanut composite rhizobium agent.

[0068] Example 3

[0069] Preparation of slant culture medium for Bradyrhizobium WD-1: Dissolve 12 g of fructose, 1.2 g of yeast extract, 2 g of γ-polyglutamic acid, 0.3 g of MgSO4·7H2O, 0.6 g of K2HPO4, 0.12 g of NaCl, 0.06 g of CaCl2, 5 mL of 0.5% sodium molybdate solution, 5 mL of 0.5% boric acid solution, and 20 g of agar in 1000 mL of distilled water, adjust the pH to 7.0, sterilize at 121°C and 0.1 MPa for 30 min, and cool.

[0070] Preparation of seed culture medium for Bradyrhizobium WD-1: Dissolve 12 g of fructose, 1.2 g of yeast extract, 2 g of γ-polyglutamic acid, 0.3 g of MgSO4·7H2O, 0.6 g of K2HPO4, 0.12 g of NaCl, 0.06 g of CaCl2, 5 mL of 0.5% sodium molybdate solution, and 5 mL of 0.5% boric acid solution in 1000 mL of distilled water, adjust the pH to 7.0, sterilize at 121°C and 0.1 MPa for 30 min, and cool.

[0071] The fermentation medium of Bradyrhizobium WD-1 has the same composition as the seed culture medium.

[0072] Preparation of slant culture medium for Bacillus subtilis LX13: Dissolve 12 g of tryptone, 6 g of yeast powder, 12 g of NaCl, and 20 g of agar in 1000 mL of distilled water, adjust the pH to 7.2, sterilize at 121°C and 0.1 MPa for 30 min, and cool.

[0073] Preparation of seed culture medium for Bacillus subtilis LX13: Dissolve 12 g of tryptone, 6 g of yeast powder, and 12 g of NaCl in 1000 mL of distilled water, adjust the pH to 7.2, sterilize at 121°C and 0.1 MPa for 30 min, and cool.

[0074] The fermentation medium of Bacillus subtilis LX13 has the same composition as the seed medium.

[0075] Bradyrhizobium WD-1 and Bacillus subtilis LX13 were picked up with an inoculation loop and inoculated into the slant culture medium in the test tube, and cultured in a constant temperature incubator at 30°C for 24 hours to obtain slant cultures of the two bacteria.

[0076] Use an inoculating loop to pick up the slant culture of Bradyrhizobium WD-1 and Bacillus subtilis LX13 respectively into a 250 mL shake flask containing 100 mL seed culture medium, inoculate 3 inoculating loops into each flask, and culture at 30°C, 200 rpm, and shake for 48 hours to obtain the seed liquid of the two bacteria.

[0077] The seed liquid was inoculated into the fermentation medium and cultured under the following conditions: 30°C, 200 rpm, and aeration volume of 0.28 m 3 / h, and cultured for 60h to obtain Bradyrhizobium WD-1 and Bacillus subtilis LX13 bacterial liquids. The number of viable bacteria in the Bradyrhizobium WD-1 bacterial liquid was 5.1×10 9 cfu / mL, and the number of viable bacteria in the Bacillus subtilis LX13 bacterial solution was 4.8×10 9 cfu / mL.

[0078] A Bradyrhizobium WD-1 bacterial solution and a Bacillus subtilis LX13 bacterial solution were mixed at a volume ratio of 5:1 to obtain a mixed bacterial solution. 95 kg of the mixed bacterial solution, 1.2 kg of K2HPO4, 0.5 kg of NaCl, and 1.0 kg of sucrose were mixed and stirred uniformly to obtain a peanut composite rhizobium agent.

[0079] Comparative Example 1

[0080] The difference from Example 1 is that the fructose in the Bradyrhizobium WD-1 slant culture medium, seed culture medium and fermentation culture medium is replaced by mannitol, and the other components and contents are the same as those in Example 1.

[0081] Comparative Example 2

[0082] Different from Example 1, the slant culture medium, seed culture medium and fermentation culture medium of Bradyrhizobium WD-1 do not contain γ-polyglutamic acid, and the other components and contents are the same as those in Example 1.

[0083] Comparative Example 3

[0084] The difference from Example 1 is that the peanut composite rhizobium agent does not contain Bacillus subtilis LX13 bacterial solution, and is replaced by an equal amount of Bradyrhizobium WD-1 bacterial solution. The other components and contents are the same as those in Example 1.

[0085] Comparative Example 4

[0086] The difference from Example 1 is that the peanut composite rhizobium agent does not contain Bradyrhizobium WD-1 bacterial solution, but is replaced by an equal amount of Bacillus subtilis LX13 bacterial solution. The other components and contents are the same as those in Example 1.

[0087] Experimental Example 1

[0088] After the fermentation was completed, the plate count method was used to count the bacterial cultures of Bradyrhizobium WD-1 prepared in Examples 1 to 3 and Comparative Examples 1 and 2. The results are shown in Table 1.

[0089] Table 1 The number of live bacteria in the Bradyrhizobium WD-1 bacterial solution in different groups

[0090]

[0091]

[0092] As shown in Table 1, the culture medium of Bradyrhizobium WD-1 of the present invention uses fructose as a carbon source and simultaneously adds γ-polyglutamic acid, which can effectively promote the growth and reproduction of Bradyrhizobium WD-1 and shorten the fermentation cycle.

[0093] Experimental Example 2

[0094] "Luhua No. 9" peanuts were selected as the experimental variety, and large, plump, and pest-free peanut seeds were selected for seed dressing. The experiment was divided into six groups, each of which was treated with the peanut composite rhizobium agent prepared in Examples 1 to 3 and Comparative Examples 3 and 4. The dosage of the peanut composite rhizobium agent was 25 mL / kg of peanut seeds. After drying in the shade, the seeds were sown within 12 hours. Peanut seeds that did not receive any treatment served as a control.

[0095] The experiment was conducted at the base of Shandong Academy of Agricultural Sciences in Huangsanjiao Agricultural High-tech Zone, Guangrao County, Dongying City, Shandong Province. The experimental field was divided into 10×10m 2 Peanut seeds treated with each compound rhizobium agent were sown in separate plots. Each seed group was sown in a separate plot, and two plots were sown for each seed group. One plot was used to count nodules and root rot, and the other plot was used to count peanut yield. Each group of peanut seeds was planted in a randomized block design and labeled. Water, fertilizer, and field management for peanuts were the same across all plots. On the 45th day after sowing, 10 peanut plants were randomly selected from each plot for testing. Each peanut plant was carefully dug out and the soil attached to the roots was carefully washed with clean water. During this operation, care was taken to avoid damaging the nodules and plant integrity. The number of nodules per peanut plant was counted, and root rot was also counted. Ten points were set in each plot, and 10 plants were surveyed at each point. The number of diseased plants and the severity level were recorded. The diseased plant rate, disease index, and control efficacy were calculated. After the peanuts matured, the peanut yield was measured. The results are shown in Tables 2 and 3.

[0096] Root rot grading standard: Level 0: No lesions on the stem base and main fibrous roots; Level 1: A small number of lesions on the stem base and main root; Level 3: More lesions on the stem base and main root, and the area of ​​lesions accounts for 1 / 4 to 1 / 2 of the total area of ​​the stem base and roots; Level 5: There are many and large lesions on the stem base and main root, and the area of ​​lesions accounts for 1 / 2 to 3 / 4 of the total area of ​​the stem base and roots; Level 7: The lesions on the stem base and main root are connected, forming a stem-circling phenomenon, but the root system is not dead; Level 9: The root system is necrotic, and the aboveground part of the plant wilts or dies.

[0097] Diseased plant rate = number of diseased plants / total number of plants × 100%;

[0098] Disease index = ∑(representative value of disease level × number of diseased plants at each level) × 100 / (total number of plants surveyed × representative value of the highest disease level);

[0099] Control effect = [(control disease index - treatment disease index) / control disease index] × 100%.

[0100] Table 2 Occurrence of peanut white rot in each group

[0101] Group Incidence (%) Disease index (%) Control effect Example 1 35 19.8 69.8 Example 2 34 18.6 71.6 Example 3 38 20.3 69.1 Comparative Example 3 73 62.5 -- Comparative Example 4 55 32.7 50.2 control group 79 65.6 --

[0102] As can be seen from Table 2, the use of the composite rhizobium agent of the present invention to treat peanut seeds has a control effect of 70.6% on peanut root rot, which is significantly higher than that of the control group and significantly higher than the control effect of a single agent of Bradyrhizobium WD-1 and Bacillus subtilis LX13 fermentation bacteria liquid. This shows that Bradyrhizobium WD-1 can improve the control effect of Bacillus subtilis LX13 on peanut root rot, and the two have a synergistic effect.

[0103] Table 3 Nodule number and yield of peanuts in each group

[0104] Group Number of nodules per plant Output (kg) Example 1 306.1±19.7 115.3 Example 2 289.3±20.6 112.9 Example 3 295.7±18.2 109.5 Comparative Example 3 210.4±15.3 62.8 Comparative Example 4 18.6±2.1 75.1 control group 20.3±1.8 40.3

[0105] As can be seen from Table 3, when the composite rhizobium agent of the present invention is used to treat peanut seeds, the number of rhizobia per peanut plant reaches a maximum of 306.1±19.7, and the yield is 115.3 kg, which is significantly higher than that of the control group and the experimental group using only single agents of Bradyrhizobium WD-1 and Bacillus subtilis LX13 fermentation liquid. This shows that Bacillus subtilis LX13 can improve the efficacy of Bradyrhizobium WD-1, and the two have a synergistic effect in increasing the number of peanut rhizobia.

[0106] As can be seen from the above examples, the present invention provides a peanut composite rhizobium agent, its preparation method, and application. The peanut composite rhizobium agent of the present invention has a short preparation cycle, and Bradyrhizobium WD-1 and Bacillus subtilis LX13 have significant synergistic effects in preventing and controlling peanut root rot and increasing the number of peanut nodules, and can be applied to the field of peanut cultivation.

[0107] The above is only a preferred embodiment of the present invention. It should be pointed out that for ordinary technicians in this technical field, several improvements and modifications can be made without departing from the principles of the present invention. These improvements and modifications should also be regarded as within the scope of protection of the present invention.

Claims

1. A peanut composite rhizobium agent, characterized in that: The invention comprises the following components in parts by weight: 90-95 parts of a mixed bacterial solution, 0.6-1.2 parts of K2HPO4, 0.1-0.5 parts of NaCl, and 0.5-1.0 parts of sucrose; the mixed bacterial solution comprises a Bradyrhizobium WD-1 bacterial solution and a Bacillus subtilis LX13 bacterial solution, and the number of viable bacteria in the Bradyrhizobium WD-1 bacterial solution and the Bacillus subtilis LX13 bacterial solution is 4-6×10 9 cfu / mL; The preparation method of the peanut composite rhizobium agent comprises the following steps: S1. Inoculate Bradyrhizobium WD-1 and Bacillus subtilis LX13 onto slant culture medium, respectively, and culture at 28-30°C for 24-48 hours to obtain slant cultures. S2. The slant cultures were inoculated into seed culture medium, respectively, and cultured at 28-30°C and 150-200 rpm for 48-72 hours to obtain seed solution; S3. Inoculate the seed solution into the fermentation medium at an inoculum rate of 6-12% at 28-30°C, 150-200 rpm, and aeration of 0.2-0.3 m 3 / h for 60-80h to obtain Bradyrhizobium WD-1 and Bacillus subtilis LX13 bacterial solutions; S4. The Bradyrhizobium WD-1 bacterial solution, Bacillus subtilis LX13 bacterial solution, K2HPO4, NaCl and sucrose were mixed to obtain the peanut composite rhizobium agent; The seed culture and fermentation medium of Bradyrhizobium WD-1 are as follows: fructose 8-12 g, yeast extract 0.8-1.2 g, γ-polyglutamic acid 1-2 g, MgSO4·7H2O 0.1-0.3 g, K2HPO4 0.4-0.6 g, NaCl 0.08-0.12 g, CaCl2 0.04-0.06 g, 0.5% sodium molybdate solution 3-5 mL, 0.5% boric acid solution 3-5 mL, distilled water 1000 mL, pH 6.8-7.0; The deposit number of the Bradyrhizobium WD-1 is CGMCC No. 8386, and the deposit number of the Bacillus subtilis LX13 is GMCC No. 8805.

2. The peanut composite rhizobium agent according to claim 1, characterized in that The volume ratio of the bradyrhizobium WD-1 bacterial solution to the Bacillus subtilis LX13 bacterial solution is 1:5 to 5:

1.

3. The method for preparing the peanut composite rhizobium agent according to claim 1 or 2, characterized in that: The following steps are involved: S1. Inoculate Bradyrhizobium WD-1 and Bacillus subtilis LX13 onto slant culture medium, respectively, and culture at 28-30°C for 24-48 hours to obtain slant cultures. S2. The slant cultures were inoculated into seed culture medium, respectively, and cultured at 28-30°C and 150-200 rpm for 48-72 hours to obtain seed solution; S3. Inoculate the seed solution into the fermentation medium at an inoculum rate of 6-12% at 28-30°C, 150-200 rpm, and aeration of 0.2-0.3 m 3 / h for 60-80h to obtain Bradyrhizobium WD-1 and Bacillus subtilis LX13 bacterial solutions; S4. The Bradyrhizobium WD-1 bacterial solution, Bacillus subtilis LX13 bacterial solution, K2HPO4, NaCl and sucrose were mixed to obtain the peanut composite rhizobium agent; The seed culture medium and fermentation medium of Bradyrhizobium WD-1 are as follows: fructose 8–12 g, yeast extract 0.8–1.2 g, γ-polyglutamic acid 1–2 g, MgSO₄·7H₂O 0.1–0.3 g, K₂HPO₄ 0.4–0.6 g, NaCl 0.08–0.12 g, CaCl₂ 0.04–0.06 g, 0.5% sodium molybdate solution 3–5 mL, 0.5% boric acid solution 3–5 mL, and distilled water 1000 mL, pH 6.8–7.

0.

4. The preparation method according to claim 3, characterized in that The seed culture medium and fermentation medium of Bacillus subtilis LX13 culture liquid were LB medium, which included the following components: 8-12 g of tryptone, 4-6 g of yeast powder, 8-12 g of NaCl, 1000 mL of distilled water, and pH 7.0-7.

2.

5. The preparation method according to claim 3, characterized in that The number of viable bacteria in Bradyrhizobium WD-1 and Bacillus subtilis LX13 solutions were 4-6×10 9 cfu / mL.

6. Use of the peanut composite rhizobium agent according to claim 1 or 2 in promoting peanut growth and / or increasing peanut yield.

7. Use of the peanut composite rhizobium agent according to claim 1 or 2 in preventing and treating peanut root rot.

8. The method for using the peanut composite rhizobium agent according to claim 1 or 2, characterized in that: Mix the rhizobium agent with peanut seeds, dry them in the shade and sow them within 12 hours.

9. The method of use according to claim 8, characterized in that: The usage amount of the peanut composite rhizobium agent is 20-30 mL / kg peanut seeds.

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