Composite rhizobium inoculant for peanuts as well as preparation method and application of composite rhizobium inoculant

Abstract

The invention provides a peanut composite rhizobium inoculant and a preparation method and application thereof, and belongs to the technical field of microbial inoculants. The peanut composite rhizobium inoculant comprises the following components: a mixed bacteria solution, K2HPO4, NaCl and cane sugar, the mixed bacterial liquid comprises a slow-growing rhizobium WD-1 bacterial liquid and a bacillus subtilis LX13 bacterial liquid, and the viable count in the slow-growing rhizobium WD-1 bacterial liquid and the bacillus subtilis LX13 bacterial liquid is (4-6) * 10 < 9 > cfu / mL. According to the peanut compound rhizobium inoculant and the preparation method thereof, the preparation period of the peanut compound rhizobium inoculant is shortened by optimizing a culture medium of the slow-growing rhizobium WD-1, and the slow-growing rhizobium WD-1 and the bacillus subtilis LX13 have a remarkable synergistic effect in the aspects of preventing and treating peanut root rot and increasing the number of peanut root nodules and can be widely applied to the field of peanut cultivation.

Description

Technical Field

[0001] The 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

[0003] Rhizobium is a type of Gram-negative bacteria that can symbiotically form nodules with legumes and reduce nitrogen in the air to ammonia to provide plant nutrition. Rhizobium cells are rod-shaped, have flagella and capsules, and do not produce spores. Gram staining is negative. The bacteria living in the nodules are pear-shaped, stick-shaped, or "T", "X", "Y" and other shapes. Such deformed bacteria are called bacteroids. Each type of rhizobium can only form nodules on one or several legumes, establish a symbiotic relationship, and show their own specificity. Rhizobium invades the host root, stimulates certain cells in the cortex and pericycle of the root, causes these cells to grow vigorously, and causes local swelling of the root to form nodules; rhizobia settle in the roots, the plant provides rhizobia with mineral nutrients and energy, and the rhizobia fixes free nitrogen in the atmosphere to provide nitrogen nutrients for the plant. The two are in a state of equilibrium in the antagonistic parasitic relationship and show symbiosis.

[0004] The preparation of rhizobium inoculants generally includes several stages: original seed activation → test tube solid culture → triangular flask liquid shaking culture → fermentation culture, but peanut rhizobium has the defect of slow growth. For example, the application number "201310643826.5" and the invention name "a peanut bradyrhizobium and its use" discloses a bradyrhizobium WD-1, which has a slant culture time of 5 to 7 days, a seed culture time of 4 to 5 days, and a fermentation culture time of 4 to 5 days, which is a total of 9 to 17 days, and a long culture cycle. In addition, although rhizobia have the function of nitrogen fixation and growth promotion, they have no ability to prevent and control crop diseases, which restricts the promotion and application of rhizobia. Summary of the invention

[0005] 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 period and single action of peanut rhizobium.

[0006] In order to achieve the above-mentioned object of the invention, the present invention provides the following technical solutions:

[0007] The present invention provides a peanut composite rhizobium agent, comprising the following components in parts by weight: 90 to 95 parts of a mixed bacterial solution, K 2 HPO 4 0.6-1.2 parts, 0.1-0.5 parts of NaCl, and 0.5-1.0 parts of sucrose; the mixed bacterial solution includes 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 4-6×109 cfu / mL.

[0008] Preferably, the volume ratio of the brassinolide WD-1 bacterial solution to the Bacillus subtilis LX13 bacterial solution is 1:5 to 5:1.

[0009] The present invention also provides a method for preparing the peanut composite rhizobium agent, comprising the following steps:

[0010] S1. Bradyrhizobium WD-1 and Bacillus subtilis LX13 were inoculated into slant medium respectively, and the slant culture was carried out at 28-30° C. for 24-48 hours to obtain slant culture;

[0011] 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;

[0012] S3. The seed solution was inoculated into the fermentation medium at an inoculum rate of 6-12%, and the fermentation medium was inoculated at 28-30°C, 150-200 rpm, and a ventilation rate of 0.2-0.3 m 3 / h for 60-80h to obtain Bradyrhizobium WD-1 bacterial solution and Bacillus subtilis LX13 bacterial solution;

[0013] S4. The bacterial solution of bradyrhizobium WD-1, Bacillus subtilis LX13, K 2 HPO 4 , NaCl and sucrose are mixed to obtain the peanut composite rhizobium agent.

[0014] Preferably, the seed culture medium and fermentation medium of Bradyrhizobium WD-1 include the following ingredients: 8-12 g of fructose, 0.8-1.2 g of yeast extract, 1-2 g of γ-polyglutamic acid, MgSO 4 7H 2 O 0.1~0.3g, K 2 HPO 4 0.4~0.6g, NaCl 0.08~0.12g, CaCl 2 0.04~0.06g, 0.5% sodium molybdate solution 3~5mL, 0.5% boric acid solution 3~5mL, distilled water 1000mL, pH 6.8~7.0.

[0015] 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.

[0016] The present invention also provides the use of the peanut composite rhizobium agent in promoting peanut growth and / or increasing peanut yield.

[0017] The invention also provides application of the peanut composite rhizobium agent in preventing and controlling peanut root rot.

[0018] The invention also provides a method for using the composite peanut rhizobium agent, which comprises mixing the rhizobium agent with peanut seeds, drying in the shade and then sowing within 12 hours.

[0019] Preferably, the usage of the peanut composite rhizobium agent is 20-30 mL / kg peanut seeds.

[0020] 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 the bradyrhizobium WD-1, and the 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

[0021] The present invention provides a peanut composite rhizobium agent, comprising the following components in parts by weight: 90 to 95 parts of a mixed bacterial solution, K 2 HPO 4 0.6-1.2 parts, NaCl 0.1-0.5 parts, sucrose 0.5-1.0 parts; the mixed bacterial solution includes bradyrhizobium WD-1 bacterial solution and Bacillus subtilis LX13 bacterial solution, and the number of live bacteria in the bradyrhizobium WD-1 bacterial solution and the Bacillus subtilis LX13 bacterial solution is 4-5×10 9 cfu / mL.

[0022] 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;

[0023] The K 2 HPO 4 The weight portion is 0.6 to 1.2 parts, more preferably 0.8 to 1.0 parts, and more preferably 0.9 parts;

[0024] 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;

[0025] 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;

[0026] The number of live 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.

[0027] The volume ratio of the slow-growing soybean rhizobium 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.

[0028] The present invention also provides a method for preparing the peanut composite rhizobium agent, comprising the following steps:

[0029] S1. Bradyrhizobium WD-1 and Bacillus subtilis LX13 were inoculated into slant medium respectively, and the slant culture was carried out at 28-30° C. for 24-48 hours to obtain slant culture;

[0030] 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;

[0031] S3. The seed solution was inoculated into the fermentation medium at an inoculum rate of 6-12%, and the fermentation medium was inoculated at 28-30°C, 150-200 rpm, and a ventilation rate of 0.2-0.3 m 3 / h for 60-80h to obtain Bradyrhizobium WD-1 bacterial solution and Bacillus subtilis LX13 bacterial solution;

[0032] S4. The bacterial solution of bradyrhizobium WD-1, Bacillus subtilis LX13, K 2 HPO 4 , NaCl and sucrose are mixed to obtain the peanut composite rhizobium agent.

[0033] The present invention first inoculates Bradyrhizobium WD-1 into a slant culture medium for cultivation to obtain a slant culture; preferably, an inoculation loop is used for inoculation, and the slant culture medium is preferably prepared in a test tube, and 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.

[0034] In the present invention, the slant culture medium is inoculated into the seed culture medium for culture 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.

[0035] In the present invention, the seed liquid is inoculated into the fermentation medium for culture, respectively, to obtain the bradyrhizobium WD-1 bacterial liquid and the Bacillus subtilis LX13 bacterial liquid, 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 150-200rpm, more preferably 170-190rpm, more preferably 180rpm; the ventilation volume during culture is 0.2-0.3m 3 / h, more preferably 0.22 to 0.28 m 3 / h, more preferably 0.25m 3 / h, the culture time is 60 to 80 hours, more preferably 65 to 75 hours, and more preferably 70 hours. 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.

[0036] The seed culture medium of bradyrhizobium WD-1 is the same as the fermentation medium, including the following ingredients: 8-12 g fructose, 0.8-1.2 g yeast extract, 1-2 g γ-polyglutamic acid, MgSO 4 7H 2 O 0.1~0.3g, K 2 HPO 4 0.4~0.6g, NaCl0.08~0.12g, CaCl 2 0.04-0.06 g, 3-5 mL of 0.5% sodium molybdate solution, 3-5 mL of 0.5% boric acid solution, 1000 mL of 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 7H 2 O is more preferably 0.15 to 0.25 g, more preferably 0.2 g, and K 2 HPO 4 More preferably, it is 0.45 to 0.55 g, more preferably 0.5 g. More preferably, NaCl is 0.09 to 0.11 g, more preferably 0.1 g. More preferably, CaCl 2 A more preferred amount is 0.056 g, a 0.5% sodium molybdate solution is further preferably 3.5 to 4.5 mL, and a 0.5% boric acid solution is further preferably 3.5 to 4.5 mL, and a 4 mL.

[0037] 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 the seed culture medium; the amount of agar added is further preferably 13-17 g, and more preferably 15 g.

[0038] 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 pH 7.0-7.2; the content of tryptone 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 content of NaCl is further preferably 9-11 g, more preferably 10 g.

[0039] 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 the seed culture medium; the addition amount of agar is further preferably 13-17 g, and more preferably 15 g.

[0040] The present invention uses a bacterial solution of bradyrhizobium WD-1, a bacterial solution of Bacillus subtilis LX13, and a K 2 HPO 4 , NaCl and sucrose are mixed to obtain the peanut composite rhizobium agent.

[0041] The present invention also provides the use of the peanut composite rhizobium agent in promoting peanut growth and / or increasing peanut yield.

[0042] The invention also provides application of the peanut composite rhizobium agent in preventing and controlling peanut root rot.

[0043] The present invention also provides a method for using the composite rhizobium agent for peanuts, which comprises mixing the rhizobium agent with peanut seeds, drying in the shade and sowing within 12 hours; the usage amount of the composite rhizobium agent for peanuts is 20 to 30 mL / kg of peanut seeds, more preferably 23 to 28 mL / kg of peanut seeds, and more preferably 25 mL / kg of peanut seeds.

[0044] The technical solutions provided by the present invention are described in detail below in conjunction with the embodiments, but they should not be construed as limiting the protection scope of the present invention.

[0045] The bradyrhizobium WD-1 described in the present invention was purchased from China General Microbiological Culture Collection Center, with the collection number of CGMCC No.8386;

[0046] 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.

[0047] Example 1

[0048] Preparation of slant medium for bradyrhizobium WD-1: 8 g fructose, 0.8 g yeast extract, 1 g γ-polyglutamic acid, MgSO 4 7H 2 O 0.1g, K 2 HPO 4 0.4g, NaCl 0.08g, CaCl 2 0.04g, 3mL of 0.5% sodium molybdate solution, 3mL of 0.5% boric acid solution, 10g of agar is dissolved in 1000mL of distilled water, the pH is adjusted to 6.8, sterilized at 121℃ and 0.1MPa pressure for 30min, and cooled.

[0049] Preparation of seed culture medium for bradyrhizobium WD-1: 8 g fructose, 0.8 g yeast extract, 1 g γ-polyglutamic acid, MgSO 4 7H 2 O 0.1g, K 2 HPO 4 0.4g, NaCl 0.08g, CaCl 2 0.04g, 3mL of 0.5% sodium molybdate solution, 3mL of 0.5% boric acid solution, dissolved in 1000mL of distilled water, adjusted the pH to 6.8, sterilized at 121℃ and 0.1MPa for 30min, and cooled to obtain.

[0050] The fermentation medium of Bradyrhizobium WD-1 has the same composition as the seed culture medium.

[0051] Preparation of slant culture medium of 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 pressure for 30 min, and cool to obtain the result.

[0052] Preparation of seed culture medium of 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 pressure for 30 min, and cool to obtain the culture medium.

[0053] The fermentation medium of Bacillus subtilis LX13 has the same composition as the seed medium.

[0054] 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, respectively.

[0055] Use an inoculation 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 inoculation loops into each flask, and culture at 28°C and 150 rpm for 72 h to obtain the seed liquid of the two bacteria respectively.

[0056] The seed solution was inoculated into the fermentation medium and cultured under the following conditions: 28°C, 150 rpm, and aeration volume of 0.22 m 3 / h, and cultured for 80h to obtain Bradyrhizobium WD-1 and Bacillus subtilis LX13 bacterial liquids. According to plate counting, the number of live bacteria in the Bradyrhizobium WD-1 bacterial liquid was 4.8×10 9 cfu / mL, the number of viable bacteria of Bacillus subtilis LX13 culture liquid was 5.2×10 9 cfu / mL.

[0057] The Bradyrhizobium WD-1 bacterial solution and the 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, K 2 HPO 4 0.6kg of sodium iodide, 0.1kg of NaCl and 0.5kg of sucrose are mixed and stirred evenly to obtain a peanut composite rhizobium agent.

[0058] Example 2

[0059] Preparation of slant medium for bradyrhizobium WD-1: 10 g fructose, 1 g yeast extract, 1.5 g γ-polyglutamic acid, MgSO 4 7H 2 O 0.2g, K 2HPO 4 0.5g, NaCl 0.08~0.12g, CaCl 2 0.05g, 4mL of 0.5% sodium molybdate solution, 4mL of 0.5% boric acid solution, 15g of agar are dissolved in 1000mL of distilled water, the pH is adjusted to 6.9, sterilized at 121℃ and 0.1MPa pressure for 30min, and cooled.

[0060] Preparation of seed culture medium for bradyrhizobium WD-1: 10 g fructose, 1 g yeast extract, 1.5 g γ-polyglutamic acid, MgSO 4 7H 2 O 0.2g, K 2 HPO 4 0.5g, NaCl 0.08~0.12g, CaCl 2 0.05g, 4mL of 0.5% sodium molybdate solution, 4mL of 0.5% boric acid solution, dissolved in 1000mL of distilled water, adjusted the pH to 6.9, sterilized at 121℃ and 0.1MPa pressure for 30min, and cooled to obtain.

[0061] The fermentation medium of Bradyrhizobium WD-1 has the same composition as the seed culture medium.

[0062] 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 to obtain the culture medium.

[0063] Preparation of seed culture medium of Bacillus subtilis LX13: 10 g of tryptone, 5 g of yeast powder, and 10 g of NaCl are dissolved in 1000 mL of distilled water, and the pH is adjusted to 7.0-7.2. The mixture is sterilized at 121° C. and 0.1 MPa for 30 min, and cooled.

[0064] The fermentation medium of Bacillus subtilis LX13 has the same composition as the seed medium.

[0065] 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 hours to obtain slant cultures of the two bacteria.

[0066] Use an inoculation loop to pick up the slant culture of Bradyrhizobium WD-1 and Bacillus subtilis LX13 respectively into a 250 mL shake flask filled with 100 mL seed culture medium, inoculate 3 inoculation loops into each flask, and culture at 29°C and 180 rpm for 60 h to obtain the seed liquid of the two bacteria respectively.

[0067] The seed solution was inoculated into the fermentation medium and the culture conditions were: 29°C, 180 rpm, and aeration volume of 0.25 m 3 / h, and cultured for 70h to obtain Bradyrhizobium WD-1 and Bacillus subtilis LX13 bacterial liquids. According to plate counting, the number of live bacteria in the Bradyrhizobium WD-1 bacterial liquid was 5.5×10 9 cfu / mL, the number of viable bacteria of Bacillus subtilis LX13 culture liquid was 5.8×10 9 cfu / mL.

[0068] The Bradyrhizobium WD-1 bacterial solution and the Bacillus subtilis LX13 bacterial solution were mixed in a volume ratio of 1:1 to obtain a mixed bacterial solution. 92 kg of the mixed bacterial solution, K 2 HPO 4 0.8kg of sodium iodide, 0.3kg of NaCl and 0.8kg of sucrose are mixed and stirred evenly to obtain the peanut composite rhizobium agent.

[0069] Example 3

[0070] Preparation of slant medium for bradyrhizobium WD-1: 12 g fructose, 1.2 g yeast extract, 2 g γ-polyglutamic acid, MgSO 4 7H 2 O 0.3g, K 2 HPO 4 0.6g, NaCl 0.12g, CaCl 2 0.06g, 5mL of 0.5% sodium molybdate solution, 5mL of 0.5% boric acid solution, 20g of agar are dissolved in 1000mL of distilled water, the pH is adjusted to 7.0, sterilized at 121℃ and 0.1MPa pressure for 30min, and cooled.

[0071] Preparation of seed culture medium for bradyrhizobium WD-1: 12 g fructose, 1.2 g yeast extract, 2 g γ-polyglutamic acid, MgSO 4 7H 2 O 0.3g, K 2 HPO 4 0.6g, NaCl 0.12g, CaCl 2 0.06g, 5mL of 0.5% sodium molybdate solution, 5mL of 0.5% boric acid solution, dissolved in 1000mL of distilled water, adjusted the pH to 7.0, sterilized at 121℃ and 0.1MPa for 30min, and cooled to obtain.

[0072] The fermentation medium of Bradyrhizobium WD-1 has the same composition as the seed culture medium.

[0073] 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 to obtain the culture medium.

[0074] Preparation of seed culture medium of 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 pressure for 30 min, and cool to obtain the culture medium.

[0075] The fermentation medium of Bacillus subtilis LX13 has the same composition as the seed medium.

[0076] 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.

[0077] Use an inoculation 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 inoculation 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 respectively.

[0078] The seed solution was inoculated into the fermentation medium and cultured at 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. According to plate counting, the number of live bacteria in the Bradyrhizobium WD-1 bacterial liquid was 5.1×10 9 cfu / mL, the number of viable bacteria of Bacillus subtilis LX13 culture liquid was 4.8×10 9 cfu / mL.

[0079] The Bradyrhizobium WD-1 bacterial solution and the 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, K 2 HPO 4 1.2kg of sodium iodide, 0.5kg of NaCl and 1.0kg of sucrose were mixed and stirred evenly to obtain a peanut composite rhizobium agent.

[0080] Comparative Example 1

[0081] The difference from Example 1 is that the fructose in the slant culture medium, seed culture medium and fermentation culture medium of Bradyrhizobium WD-1 is replaced by mannitol, and the other components and contents are the same as those in Example 1.

[0082] Comparative Example 2

[0083] 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.

[0084] Comparative Example 3

[0085] The difference from Example 1 is that the peanut composite rhizobium agent does not contain Bacillus subtilis LX13 bacterial solution, but 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.

[0086] Comparative Example 4

[0087] 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.

[0088] Experimental Example 1

[0089] After the fermentation was completed, the plate count method was used to count the Bradyrhizobium WD-1 bacterial solution prepared in Examples 1 to 3 and Comparative Examples 1 and 2. The results are shown in Table 1.

[0090] Table 1 Number of live bacteria in the bacterial solution of bradyrhizobium WD-1 in different groups

[0091]

[0092]

[0093] 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.

[0094] Experimental Example 2

[0095] "Luhua No. 9" peanuts were selected as the test variety, and peanut seeds with large, plump and no pests and diseases were selected for seed dressing. The experiment was divided into 6 groups, and the peanut composite rhizobium agents prepared in Examples 1 to 3 and Comparative Examples 3 and 4 were used for seed dressing, respectively. The use amount of the peanut composite rhizobium agent was 25 mL / kg peanut seeds, and the seeds were sown within 12 hours after being dried in the shade. Peanut seeds without any treatment were used as the control.

[0096] 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 2In the plots, the peanut seeds sown with each group of composite rhizobium agents were sown in the plots, and each group of seeds was sown in one plot alone. Each group of seeds was sown in two plots, one plot was used to count the number of nodules and root rot, and the other plot was used to count the peanut yield. Each group of peanut seeds was designed with random blocks and marked. The water and fertilizer management and field management of peanuts in each plot were the same. On the 45th day after sowing, 10 peanut plants were randomly selected from each plot for measurement, each peanut plant was carefully dug out, and the soil attached to the roots was carefully washed off with clean water. During the operation, care was taken to avoid damaging the nodules and plant integrity. The number of nodules of each peanut was counted, and the root rot was counted at the same time. 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, and the disease rate, disease index and prevention and control effect were calculated. After the peanuts matured, the peanut yield was measured. The results are shown in Tables 2 and 3.

[0097] Root rot 10-level 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: There are 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-wrapped 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.

[0098] Disease rate = number of diseased plants / total number of plants × 100%;

[0099] 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);

[0100] Control effect = [(control disease index - treatment disease index) / control disease index] × 100%.

[0101] Table 2 Occurrence of peanut white rot in each group

[0102] 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 --

[0103] As can be seen from Table 2, the composite rhizobium agent of the present invention is used to treat peanut seeds, and the control effect of peanut root rot is 70.6%, which is significantly higher than that of the control group, and is also significantly higher than the control effect of the single agent of the fermentation liquid of the bradyrhizobium WD-1 and the Bacillus subtilis LX13, indicating that the bradyrhizobium WD-1 can improve the control effect of Bacillus subtilis LX13 on peanut root rot, and the two have a synergistic effect.

[0104] Table 3 Nodule number and yield of peanuts in each group

[0105] 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

[0106] 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 is as high as 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, indicating 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.

[0107] It can be seen from the above embodiments that the present invention provides a peanut composite rhizobium agent and a preparation method and application thereof. The peanut composite rhizobium agent of the present invention has a short preparation cycle, and the 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.

[0108] 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 principle of the present invention. These improvements and modifications should also be regarded as 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 to 95 parts of mixed bacterial solution, 0.6 to 1.2 parts of K2HPO4, 0.1 to 0.5 parts of NaCl, and 0.5 to 1.0 parts of sucrose; the mixed bacterial solution comprises a bacterial solution of bradyrhizobium WD-1 and a bacterial solution of Bacillus subtilis LX13, and the number of viable bacteria in the bacterial solution of bradyrhizobium WD-1 and the bacterial solution of Bacillus subtilis LX13 is 4 to 6×10 9 cfu / mL.

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

1.

3. The method for preparing the composite rhizobium agent for peanut according to claim 1 or 2, characterized in that: The following steps are involved: S1. Bradyrhizobium WD-1 and Bacillus subtilis LX13 were inoculated into slant medium respectively, and the slant culture was carried out at 28-30° C. for 24-48 hours to obtain slant culture; 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. The seed solution was inoculated into the fermentation medium at an inoculum rate of 6-12%, and the fermentation medium was inoculated at 28-30°C, 150-200 rpm, and a ventilation rate of 0.2-0.3 m 3 / h for 60-80h to obtain Bradyrhizobium WD-1 bacterial solution and Bacillus subtilis LX13 bacterial solution; S4. Mix the bacterial solution of bradyrhizobium WD-1, the bacterial solution of Bacillus subtilis LX13, K2HPO4, NaCl and 0.5-1.0 of sucrose to obtain the peanut composite rhizobium agent.

4. The preparation method according to claim 3, characterized in that: The seed culture medium and fermentation culture medium of bradyrhizobium WD-1 include the following components: 8-12g of fructose, 0.8-1.2g of yeast extract, 1-2g of gamma-polyglutamic acid, 0.1-0.3g of MgSO4·7H2O, 0.4-0.6g of K2HPO4, 0.08-0.12g of NaCl, 0.04-0.06g of CaCl2, 3-5mL of 0.5% sodium molybdate solution, 3-5mL of 0.5% boric acid solution, 1000mL of distilled water, and pH value of 6.8-7.

0.

5. The preparation method according to claim 3, characterized in that: The seed culture medium and fermentation medium of the Bacillus subtilis LX13 bacterial liquid are LB medium, which includes 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.

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

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

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

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

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

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