Microbial preparation for enhancing disease resistance of soybeans as well as preparation method and application of microbial preparation

Abstract

The invention discloses a microbial preparation for enhancing disease resistance of soybeans as well as a preparation method and application of the microbial preparation. Belongs to the technical field of microbial preparations. According to the invention, the bacillus subtilis ACCC 60383 and the aspergillus usamii ACCC 30122 are synergistically compatible to prepare the microbial preparation, compared with the single bacillus subtilis ACCC 60383, the control effect is greatly improved, meanwhile, the overall technical scheme is optimized by accurately regulating and controlling the proportioning relation and the fermentation method of the two strains, the treatment effect and the yield of soybean crops are improved, and the application prospect is wide. And the application potential is wide.

Description

Technical Field

[0001] The present invention relates to the technical field of microbial agents, and more particularly to a microbial agent for enhancing the disease resistance of soybeans, its preparation method and application. Background Art

[0002] Soybeans, commonly known as yellow soybeans, are annual herbs. Originating in China, they are cultivated throughout China and are also widely cultivated around the world. With a cultivation history of 5,000 years, they were anciently called shu. The main producing area in China is Northeast China. It is a crop whose seeds are rich in plant protein. Soybeans are one of the main economic crops in China, promoting the economic development of the producing areas. However, diseases frequently occur during soybean cultivation, seriously affecting the soybean yield and reducing the economic income. Common soybean diseases include: target spot, fusarium wilt, pod blight, stem blight, powdery mildew, bacterial leaf spot, rust, damping-off, root rot, and soybean mosaic virus disease, etc.

[0003] The conventional control method is chemical control. However, long-term reliance on a single chemical agent for disease control easily leads to the generation of drug resistance in pathogenic bacteria. For example, certain fungi such as Botrytis cinerea and Fusarium spp. have developed drug resistance to a variety of commonly used fungicides. In order to achieve the control effect, the dosage is often increased. However, the use of a large dose of chemical agents will further enhance the drug resistance of pathogenic bacteria, and the use of a large dose of chemical agents will pollute the environment. Therefore, at present, biological control means have attracted much attention. However, the control effect of a single microorganism on soybean pests and diseases is not good, and it is unable to effectively prevent and control the occurrence of soybean pests and diseases.

[0004] Therefore, how to provide a microbial agent for effectively preventing and controlling the occurrence of soybean pests and diseases, its preparation method and application is an urgent technical problem to be solved by those skilled in the art. Summary of the Invention

[0005] In view of this, the present invention provides a microbial agent for enhancing the disease resistance of soybeans, its preparation method and application. Bacillus subtilis ACCC 60383 and Aspergillus usamii ACCC 30122 are used in combination. After Bacillus subtilis and Aspergillus usamii are respectively fermented and cultured, mixed culture is carried out in sequence to prepare a microbial fermentation agent, significantly improving the biological control effect of the microbial agent.

[0006] In order to achieve the above object, the present invention adopts the following technical scheme:

[0007] A microbial agent for enhancing the disease resistance of soybeans, comprising Bacillus subtilis ACCC 60383 and Aspergillus usamii ACCC 30122.

[0008] As a preferred technical solution, the viable bacteria content of the microbial preparation is 10 4 -10 5 cfu / g.

[0009] As a preferred technical solution, the microbial preparation further comprises preparation excipients.

[0010] Another object of the present invention is to provide a preparation method of the above-mentioned microbial preparation for enhancing the disease resistance of soybeans, comprising the following steps:

[0011] (1) Inoculate the activated Bacillus subtilis ACCC 60383 into a fermentation medium and ferment at 35-38°C. After 20-25 hours of fermentation, lower the temperature to 28-30°C and continue to ferment for 16-48 hours to obtain a Bacillus subtilis fermentation broth;

[0012] (2) Inoculate the activated Aspergillus usamii ACCC 30122 into a fermentation medium and ferment for 56-72 hours to obtain an Aspergillus usamii fermentation broth;

[0013] (3) Inoculate the Bacillus subtilis ACCC 60383 fermentation broth obtained in step (1) into a mixed fermentation medium. After 1-2 hours of fermentation, add the Aspergillus usamii ACCC 30122 fermentation broth and preparation excipients obtained in step (2), and further ferment for 3-4 hours to obtain a mixed fermentation broth;

[0014] (4) Dry the mixed fermentation broth obtained in step (3) to obtain a microbial preparation for enhancing the disease resistance of soybeans.

[0015] As a preferred technical solution, the fermentation medium of Bacillus subtilis ACCC 60383 in step (1) comprises: 10-15 g / L of tryptone, 5-8 g / L of yeast extract, 5-6 g / L of sodium chloride, 1-5 g / L of L-glutamic acid, 1-2 g / L of glucose, and distilled water is made up to 1 L; the pH of the fermentation medium of Bacillus subtilis ACCC 60383 is 7.0-7.5.

[0016] As a preferred technical solution, the fermentation medium of Aspergillus usamii ACCC 30122 in step (2) comprises: 15-20 g / L of soluble starch, 5-6 g / L of yeast extract, 1-3 g / L of NH4NO3, 0.5-1 g / L of KH2PO4, 0.1-0.5 g / L of MgSO4·7H2O, and distilled water is made up to 1 L; the fermentation temperature of Aspergillus usamii ACCC 30122 is 25-28°C and the pH is 4.0-7.0.

[0017] As a preferred technical solution, the mixed fermentation medium in step (3) comprises: 8 - 10 g / L of glucose, 3 - 5 g / L of yeast extract, 2 - 5 g / L of peptone, 5 - 10 g / L of wheat bran, 0.1 - 2 g / L of KH2PO4, 0.1 - 0.5 g / L of MgSO4·7H2O, 0.1 - 0.5 g / L of NaCl, 0.5 - 1 g / L of (NH4)2SO4, and distilled water is added to make up to 1 L;

[0018] The temperature for fermentation culture when the Bacillus subtilis ACCC 60383 fermentation broth in step (3) is inoculated into the mixed fermentation medium is 35 - 38 °C, and the pH is 7.0 - 7.5. After adding Aspergillus usamii ACCC 30122, the fermentation temperature is adjusted to 25 - 28 °C, and the pH is adjusted to 4.0 - 7.0 for fermentation;

[0019] The Bacillus subtilis ACCC 60383 fermentation broth and Aspergillus usamii ACCC 30122 fermentation broth in step (3) are inoculated into the mixed fermentation medium according to the viable bacteria ratio of 6 - 8:2 - 3.

[0020] Another object of the present invention is to provide the application of the microbial preparation prepared by the above method in soybean disease resistance and / or improving soybean disease resistance.

[0021] As a preferred technical solution, the disease resistance types include cercospora leaf spot, root rot, phytophthora root rot, powdery mildew, anthracnose and soybean mosaic virus disease.

[0022] As a preferred technical solution, the specific application methods include but are not limited to seed treatment, soil application or foliar spraying.

[0023] It can be seen from the above technical solutions that compared with the prior art, the present invention has the following beneficial effects:

[0024] The present invention uses Bacillus subtilis ACCC 60383 and Aspergillus usamii ACCC 30122 to carry out fermentation culture in a specific ratio and specific fermentation method to prepare a microbial preparation. By synergistically combining Aspergillus usamii and Bacillus subtilis, the resistance of Bacillus subtilis to cercospora leaf spot, root rot, phytophthora root rot, powdery mildew, anthracnose and soybean mosaic virus disease can be significantly improved. At the same time, the yield of soybeans is increased, the incidence rate is reduced, providing technical support for the biological control of soybeans. Specific embodiments

[0025] Next, in combination with the embodiments of the present invention, the technical solutions in the embodiments of the present invention will be clearly and completely described. Obviously, the described embodiments are only a part of the embodiments of the present invention, rather than all the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those of ordinary skill in the art without creative efforts shall fall within the protection scope of the present invention.

[0026] The commercial number of Bacillus subtilis used in the embodiments of the present invention is ACCC 60383; the commercial number of Aspergillus usamii used is ACCC 30122.

[0027] Example 1

[0028] A microbial agent for enhancing the disease resistance of soybeans, comprising Bacillus subtilis ACCC 60383 and Aspergillus usamii ACCC 30122.

[0029] The specific preparation method is as follows:

[0030] (1) The activated Bacillus subtilis ACCC 60383 was inoculated into a fermentation medium (tryptone 10 g / L, yeast extract 5 g / L, sodium chloride 5 g / L, L-glutamic acid 1 g / L, glucose 1 g / L, made up to 1 L with distilled water, adjusted to pH 7.0), fermented at 35°C. After 20 h of fermentation, the temperature was lowered to 28°C and fermentation continued for 48 h to prepare a Bacillus subtilis fermentation broth.

[0031] (2) The activated Aspergillus usamii ACCC 30122 was inoculated into a fermentation medium (soluble starch 15 g / L, yeast extract 5 g / L, NH4NO3 1 g / L, KH2PO4 0.5 g / L, MgSO4·7H2O 0.1 g / L, made up to 1 L with distilled water, adjusted to pH 4.0), fermented and cultured at 25°C for 72 h to prepare an Aspergillus usamii fermentation broth.

[0032] (3) Inoculate the Bacillus subtilis ACCC 60383 fermentation broth obtained in step (1) into a mixed fermentation medium (glucose 8 g / L, yeast extract 3 g / L, peptone 2 g / L, wheat bran 5 g / L, KH₂PO₄ 0.1 g / L, MgSO₄·7H₂O 0.1 g / L, NaCl 0.1 g / L, (NH₄)₂SO₄ 0.5 g / L, made up to 1 L with distilled water), adjust the fermentation temperature to 35 °C, adjust the pH to 7.0, after fermenting for 1 h, add the Aspergillus usamii ACCC 30122 fermentation broth, antioxidant and carrier material obtained in step (2), adjust the fermentation temperature to 25 °C, adjust the pH to 4.0, and further ferment for 3 h to prepare a mixed fermentation broth; the Bacillus subtilis ACCC 60383 fermentation broth and the Aspergillus usamii ACCC 30122 fermentation broth are inoculated into the mixed fermentation medium according to the viable bacteria ratio of 6:2;

[0033] (4) Dry the mixed fermentation broth obtained in step (3) to prepare a microbial preparation for enhancing the disease resistance of soybeans, and the viable bacteria content of the microbial preparation is 3.12×10 4 cfu / g.

[0034] Example 2

[0035] A microbial preparation for enhancing the disease resistance of soybeans, comprising Bacillus subtilis ACCC 60383 and Aspergillus usamii ACCC 30122;

[0036] The specific preparation method is as follows:

[0037] (1) Inoculate the activated Bacillus subtilis ACCC 60383 into a fermentation medium (tryptone 15 g / L, yeast extract 8 g / L, sodium chloride 6 g / L, L-glutamic acid 5 g / L, glucose 2 g / L, made up to 1 L with distilled water, adjust the pH to 7.5), place it at 38 °C for fermentation, after fermenting for 25 h, lower the temperature to 30 °C and continue to ferment for 16 h to prepare a Bacillus subtilis fermentation broth;

[0038] (2) Inoculate the activated Aspergillus usamii ACCC 30122 into a fermentation medium (soluble starch 20 g / L, yeast extract 6 g / L, NH₄NO₃ 3 g / L, KH₂PO₄ 1 g / L, MgSO₄·7H₂O 0.5 g / L, made up to 1 L with distilled water, adjust the pH to 7.0), place it at 28 °C for fermentation and culture for 56 h to prepare an Aspergillus usamii fermentation broth;

[0039] (3) Inoculate the Bacillus subtilis ACCC 60383 fermentation broth obtained in step (1) into a mixed fermentation medium (10 g / L of glucose, 5 g / L of yeast extract, 5 g / L of peptone, 10 g / L of wheat bran, 2 g / L of KH2PO4, 0.5 g / L of MgSO4·7H2O, 0.5 g / L of NaCl, 1 g / L of (NH4)2SO4, made up to 1 L with distilled water). Adjust the fermentation temperature to 38°C and the pH to 7.5. After 2 h of fermentation culture, add the Aspergillus usamii ACCC 30122 fermentation broth, antioxidant, and carrier material obtained in step (2). Adjust the fermentation temperature to 28°C and the pH to 7.0, and further ferment for 4 h to obtain a mixed fermentation broth; inoculate the Bacillus subtilis ACCC 60383 fermentation broth and the Aspergillus usamii ACCC 30122 fermentation broth into the mixed fermentation medium according to a viable bacteria ratio of 8:3;

[0040] (4) Dry the mixed fermentation broth obtained in step (3) to prepare a microbial agent for enhancing the disease resistance of soybeans; the viable bacteria content of the microbial agent is 3.48×10 4 cfu / g.

[0041] Example 3

[0042] A microbial agent for enhancing the disease resistance of soybeans, comprising Bacillus subtilis ACCC 60383 and Aspergillus usamii ACCC 30122;

[0043] The specific preparation method is as follows:

[0044] (1) Inoculate the activated Bacillus subtilis ACCC 60383 into a fermentation medium (12 g / L of tryptone, 6 g / L of yeast extract, 5 g / L of sodium chloride, 4 g / L of L-glutamic acid, 1 g / L of glucose, made up to 1 L with distilled water, adjust the pH to 7.2), place it at 37°C for fermentation. After 22 h of fermentation, lower the temperature to 29°C and continue fermentation for 36 h to obtain a Bacillus subtilis fermentation broth;

[0045] (2) Inoculate the activated Aspergillus usamii ACCC 30122 into a fermentation medium (18 g / L of soluble starch, 5 g / L of yeast extract, 2 g / L of NH4NO3, 0.8 g / L of KH2PO4, 0.3 g / L of MgSO4·7H2O, made up to 1 L with distilled water, adjust the pH to 6.5), place it at 26°C for fermentation culture for 68 h to obtain an Aspergillus usamii fermentation broth;

[0046] (3) Inoculate the Bacillus subtilis ACCC 60383 fermentation broth obtained in step (1) into a mixed fermentation medium (glucose 9 g / L, yeast extract 4 g / L, peptone 3 g / L, wheat bran 6 g / L, KH2PO4 1 g / L, MgSO4·7H2O 0.2 g / L, NaCl 0.2 g / L, (NH4)2SO4 0.6 g / L, made up to 1 L with distilled water), adjust the fermentation temperature to 37 °C, adjust the pH to 7.2, after fermenting for 1.5 h, add the Aspergillus usamii ACCC 30122 fermentation broth, antioxidant and carrier material obtained in step (2), adjust the fermentation temperature to 26 °C, adjust the pH to 6.5, and further ferment for 3 h to prepare a mixed fermentation broth; the Bacillus subtilis ACCC 60383 fermentation broth and the Aspergillus usamii ACCC 30122 fermentation broth are inoculated into the mixed fermentation medium according to a viable bacteria ratio of 5:2;

[0047] (4) Dry the mixed fermentation broth obtained in step (3) to prepare a microbial preparation for enhancing the disease resistance of soybeans, and the viable bacteria content of the microbial preparation is 3.56×10 4 cfu / g.

[0048] Comparative Example 1

[0049] It is basically the same as Example 3, except that:

[0050] In step (3), the Bacillus subtilis ACCC 60383 fermentation broth and the Aspergillus usamii ACCC 30122 fermentation broth are inoculated into the mixed fermentation medium according to a viable bacteria ratio of 1:1, and the viable bacteria content of the microbial preparation is 8.74×10 3 cfu / g.

[0051] Comparative Example 2

[0052] It is basically the same as Example 3, except that:

[0053] In step (3), the Bacillus subtilis ACCC 60383 fermentation broth and the Aspergillus usamii ACCC 30122 fermentation broth are inoculated into the mixed fermentation medium according to a viable bacteria ratio of 5:1, and the viable bacteria content of the microbial preparation is 8.96×10 3 cfu / g.

[0054] Comparative Example 3

[0055] It is basically the same as Example 3, except that:

[0056] Step (1): Inoculate the activated Bacillus subtilis ACCC 60383 into a fermentation medium (tryptone 12 g / L, yeast extract 6 g / L, sodium chloride 5 g / L, L-glutamic acid 4 g / L, glucose 1 g / L, made up to 1 L with distilled water, adjust the pH to 7.2), and ferment at 37°C for 58 h to obtain a Bacillus subtilis fermentation broth. The viable count of the microbial preparation is 3.71×10 4 cfu / g.

[0057] Comparative Example 4

[0058] It is basically the same as Example 3, except that:

[0059] Step (1): Inoculate the activated Bacillus subtilis ACCC 60383 into a fermentation medium (tryptone 12 g / L, yeast extract 6 g / L, sodium chloride 5 g / L, glucose 1 g / L, made up to 1 L with distilled water, adjust the pH to 7.2), and ferment at 37°C. After 22 h of fermentation, lower the temperature to 29°C and continue to ferment for 36 h to obtain a Bacillus subtilis fermentation broth. The viable count of the microbial preparation is 3.54×10 4 cfu / g.

[0060] Comparative Example 5

[0061] It is basically the same as Example 3, except that:

[0062] Step (3): Simultaneously inoculate the Bacillus subtilis ACCC 60383 fermentation broth obtained in step (1), the Aspergillus usamii ACCC 30122 fermentation broth obtained in step (2), an antioxidant, and a carrier material into a mixed fermentation medium (glucose 9 g / L, yeast extract 4 g / L, peptone 3 g / L, wheat bran 6 g / L, KH2PO4 1 g / L, MgSO4·7H2O 0.2 g / L, NaCl 0.2 g / L, (NH4)2SO4 0.6 g / L, made up to 1 L with distilled water), adjust the fermentation temperature to 30°C, adjust the pH to 7.0, and ferment for 4.5 h to obtain a mixed fermentation broth; inoculate the Bacillus subtilis ACCC 60383 fermentation broth and the Aspergillus usamii ACCC 30122 fermentation broth into the mixed fermentation medium at a viable count ratio of 5:2. The viable count of the obtained microbial preparation is 3.12×10 4 cfu / g.

[0063] Comparative Example 6

[0064] It is basically the same as Example 3, except that:

[0065] A microbial agent for enhancing the disease resistance of soybeans, comprising Bacillus subtilis ACCC 60383 and Aspergillus clavatus ACCC 30579; the fermentation broth of Bacillus subtilis ACCC 60383 and the fermentation broth of Aspergillus clavatus ACCC 30579 are inoculated into a mixed fermentation medium according to a viable bacteria ratio of 5:2, and the viable bacteria content of the microbial agent is 2.16×10 4 cfu / g.

[0066] Control Example 7

[0067] It is basically the same as Example 3, except that:

[0068] A microbial agent for enhancing the disease resistance of soybeans, comprising Bacillus subtilis ACCC 60383,

[0069] (1) The activated Bacillus subtilis ACCC 60383 is inoculated into a fermentation medium (tryptone 12 g / L, yeast extract 6 g / L, sodium chloride 5 g / L, L-glutamic acid 4 g / L, glucose 1 g / L, made up to 1 L with distilled water, adjust the pH to 7.2), and fermented at 37°C. After 22 h of fermentation, the temperature is lowered to 29°C and fermentation continues for 36 h to prepare the fermentation broth of Bacillus subtilis;

[0070] (3) The fermentation broth of Bacillus subtilis ACCC 60383 obtained in step (1) is inoculated into a mixed fermentation medium (glucose 9 g / L, yeast extract 4 g / L, peptone 3 g / L, wheat bran 6 g / L, KH2PO4 1 g / L, MgSO4·7H2O 0.2 g / L, NaCl 0.2 g / L, (NH4)2SO4 0.6 g / L, made up to 1 L with distilled water), adjust the fermentation temperature to 37°C, adjust the pH to 7.2, and ferment for 4.5 h to prepare a fermentation broth; the viable bacteria content of the obtained microbial agent is 3.32×10 4 cfu / g.

[0071] Verification of antibacterial effect

[0072] An antibacterial experiment was carried out on the microbial agents prepared in Examples 1-3 and Control Examples 1-7. The specific process is as follows:

[0073] Test strains: Cercospora kikuchii, Fusarium oxysporum, Phytophthora sojae, Erysiphe diffusa, Colletotrichum truncatum, and Soybean Mosaic Virus (SMV).

[0074] Using a punch with a diameter of 5 mm, cut out a mycelial cake of the above-mentioned test strain along the outer edge of the mycelium and inoculate it into a medium containing a microbial inoculant. Incubate it under suitable conditions for 5 days. At the same time, set a blank control: add an equal amount of double-distilled water to the medium. Set three replicates for each group, measure the colony diameter, and take the average value of the results. Calculate the inhibition rate by comparing with the blank control. The calculation formula is as follows:

[0075] Inhibition rate (%) = [(colony diameter of the blank control group - colony diameter of the treatment group) / (colony diameter of the blank control group - diameter of the mycelial cake)] × 100%. The experimental results are shown in Table 1.

[0076] Table 1 Inhibition rates of different groups against different strains

[0077] Group Cercospora sojina Fusarium oxysporum Phytophthora sojae Erysiphe cichoracearum Colletotrichum gloeosporioides Soybean mosaic virus Example 1 88.7％ 92.4％ 85.4％ 87.9％ 92.6％ 70.2％ Example 2 89.2％ 91.3％ 84.6％ 88.4％ 93.1％ 71.3％ Example 3 89.6％ 94.6％ 86.8％ 88.5％ 94.8％ 71.5％ Comparative Example 1 58.2％ 60.3％ 52.4％ 57.6％ 61.2％ 40.5％ Comparative Example 2 61.35 61.5％ 53.8％ 58.4％ 62.5％ 42.6％ Comparative Example 3 80.4％ 82.6％ 78.5％ 79.3％ 81.3％ 64.8％ Comparative Example 4 82.6％ 84.5％ 79.6％ 80.1％ 82.4％ 65.2％ Comparative Example 5 63.5％ 67.3％ 62.9％ 64.1％ 65.8％ 46.2％ Comparative Example 6 60.4％ 63.4％ 60.1％ 61.1％ 63.4％ 41.3％ Comparative Example 7 59.6％ 62.1％ 58.4％ 60.3％ 61.9％ 40.5％

[0078] Result analysis: According to the content of Table 1, it can be seen that the antibacterial performance of the microbial inoculants prepared in Examples 1-3 of the present invention is significantly better than that of the microbial inoculants prepared in Comparative Examples 1-7. By comparing Examples 1-3 with Comparative Examples 6 and 7, it can be known that when Bacillus subtilis ACCC 60383 and Aspergillus usamii ACCC 30122 are co-fermented to prepare a microbial inoculant, the antibacterial effect against pathogenic bacteria of soybeans can be significantly improved. Most importantly, the control effect of Bacillus subtilis on soybean mosaic virus of ACCC 60383 is relatively low, but when Bacillus subtilis ACCC 60383 and Aspergillus usamii ACCC 30122 are combined, the control effect on soybean mosaic virus is significantly improved.

[0079] Pot experiment for control effect

[0080] (1) Verification of control effect against soybean frog-eye leaf spot

[0081] Select 220 soybean seedlings and divide them into 11 groups. After inoculating with Cercospora sojina for 36 hours, dilute the microbial inoculants prepared in Examples 1-3 and Control Groups 1-7 with water by 100 times and spray them on the soybean seedlings in the form of mist. Then place the soybean seedlings under suitable conditions for 10 days. At the same time, set a blank control group with clear water, observe and grade the disease condition. The grading criteria are as follows:

[0082] Grade 0: No disease spots, no visible disease spots on the leaves;

[0083] Grade 1: Slightly diseased, with a small number of disease spots on the leaves, less than 5 disease spots on each leaf, and the disease spot area is small, with a diameter generally not exceeding 3 mm;

[0084] Grade 2: Mildly diseased, with the number of disease spots on each leaf between 5 and 10, the disease spot area has increased, but the overall disease spot coverage area does not exceed 1 / 4 of the total leaf area;

[0085] Level 3: Moderate disease incidence, with the number of lesions on each leaf exceeding 10. The lesion area is relatively large, and some lesions may fuse. The area covered by lesions accounts for 1 / 4 to 1 / 2 of the total leaf area.

[0086] Level 4: Severe disease incidence, with a very large number of lesions on the leaves. The lesions almost cover the entire leaf, and the area covered by lesions exceeds 1 / 2 of the total leaf area, even reaching more than 75%.

[0087] Level 5: Extremely severe disease incidence, with almost the entire leaf completely covered by lesions. The leaf shows a withered yellow or dead state, seriously affecting the photosynthesis function.

[0088] Disease index = [Σ(disease level × number of samples at that level)] / (total number of samples × highest disease level) × 100;

[0089] Control effect = (disease index of blank group - disease index of treatment group) / disease index of blank group × 100%.

[0090] Table 2 Control effect results of different groups on soybean frog-eye leaf spot

[0091]

[0092]

[0093] Result analysis: As can be seen from Table 2, the microbial inoculants prepared in Examples 1-3 of the present invention can significantly improve the control effect of soybean frog-eye leaf spot.

[0094] (2) Verification of the control effect on soybean powdery mildew

[0095] Select 220 soybean seedlings and divide them into 11 groups. After inoculating with soybean powdery mildew for 24 hours, dilute the microbial inoculants prepared in Examples 1-3 and Control Groups 1-7 with water by 100 times and spray them on the soybean seedlings in the form of mist. Then cultivate the soybean seedlings under suitable conditions for 10 days. At the same time, set up a blank control group with clear water, observe and grade the disease conditions. The grading criteria are as follows:

[0096] Level 0: No lesions, and no visible symptoms of soybean powdery mildew on the leaves.

[0097] Level 1: Slight disease incidence, with a small number of scattered small lesions on the leaves. The number of lesions on each leaf is less than 5, and the lesion area is relatively small, with a diameter generally not exceeding 3 mm.

[0098] Level 2: Mild disease incidence, with the number of lesions on each leaf between 5 and 10. The lesion area has increased, but the overall area covered by lesions does not exceed 1 / 4 of the total leaf area.

[0099] Level 3: Moderate incidence, with the number of lesions on each leaf exceeding 10. The lesion area is relatively large, and some lesions may merge. The area covered by lesions accounts for 1 / 4 to 1 / 2 of the total leaf area.

[0100] Level 4: Severe incidence, with a very large number of lesions on the leaves. The lesions almost cover the entire leaf, and the area covered by lesions exceeds 1 / 2 of the total leaf area, even reaching more than 75%.

[0101] Level 5: Extremely severe incidence, with almost the entire leaf completely covered by lesions. The leaf shows a withered yellow or dead state, seriously affecting the photosynthesis function.

[0102] Disease index = [Σ(disease level × number of samples at that level)] / (total number of samples × highest disease level) × 100;

[0103] Control effect = (disease index of the blank group - disease index of the treatment group) / disease index of the blank group × 100%.

[0104] Table 3 Control effect results of different groups on soybean powdery mildew

[0105]

[0106]

[0107] Result analysis: As can be seen from the content of Table 2, the microbial inoculum prepared in Examples 1 - 3 of the present invention can significantly improve the control effect of soybean powdery mildew.

[0108] Field experiment

[0109] In order to verify the effect of the microbial preparation prepared by the present invention, a field experiment was carried out in 2021. The application rate of the microbial inoculum was 0.5 g / plant. After diluting 100 times, it was sprayed on the leaf surface. At the same time, a blank control group without applying the microbial preparation was set up. During the planting process, the incidence of soybean was observed and recorded, and the yield was measured. The test results are shown in Table 4.

[0110] Table 4 Field experiment results of different groups

[0111] Group Yield (kg) Growth rate (%) Incidence rate (%) Blank control group 256 - 10.59 Example 3 318 24.22 2.41 Comparative Example 1 284 10.94 5.89 Comparative Example 2 291 13.67 5.76 Comparative Example 3 301 17.58 4.24 Comparative Example 4 304 18.75 4.21 Comparative Example 5 289 12.89 5.30 Comparative Example 6 267 4.30 5.46 Comparative Example 7 264 3.13 6.73

[0112] Result analysis: As can be seen from Table 4, the microbial inoculum of the present invention can increase the yield of soybeans, reduce the disease prevention rate, and has a good control effect compared with other groups.

[0113] In this specification, each embodiment is described in a progressive manner. The key point of each embodiment is to illustrate the differences from other embodiments. For the same or similar parts among the embodiments, reference can be made to each other.

[0114] The foregoing description of the disclosed embodiments enables those skilled in the art to practice or use the present invention. Various modifications to these embodiments will be readily apparent to those skilled in the art, and the general principles defined herein may be implemented in other embodiments without departing from the spirit or scope of the present invention. Thus, the present invention is not intended to be limited to the embodiments shown herein but is to be accorded the widest scope consistent with the principles and novel features disclosed herein.

Claims

1. A microbial preparation for enhancing the disease resistance of soybeans, characterized in that, It includes Bacillus subtilis ACCC 60383 and Aspergillus usamii ACCC 30122.

2. The microbial agent for enhancing the disease resistance of soybeans according to claim 1, wherein The viable bacteria content in the microbial preparation is 10 4 -10 5 cfu / g.

3. The microbial preparation for enhancing the disease resistance of soybeans according to claim 1 or 2, characterized in that, The microbial preparation also includes preparation excipients.

4. The preparation method of the microbial agent for enhancing the disease resistance of soybeans according to any one of claims 1-3, characterized in that, It includes the following steps: (1) Inoculate the activated Bacillus subtilis ACCC 60383 into a fermentation medium and ferment at 35 - 38 °C. After 20 - 25 h of fermentation, lower the temperature to 28 - 30 °C and continue to ferment for 16 - 48 h to obtain a Bacillus subtilis fermentation broth. (2) Inoculate the activated Aspergillus usamii ACCC 30122 into a fermentation medium and ferment for 56 - 72 h to obtain an Aspergillus usamii fermentation broth. (3) Inoculate the Bacillus subtilis ACCC 60383 fermentation broth obtained in step (1) into a mixed fermentation medium. After 1 - 2 h of fermentation, add the Aspergillus usamii ACCC 30122 fermentation broth and preparation excipients obtained in step (2), and further ferment for 3 - 4 h to obtain a mixed fermentation broth. (4) Dry the mixed fermentation broth obtained in step (3) to obtain a microbial preparation for enhancing the disease resistance of soybeans.

5. The preparation method of the microbial preparation according to claim 4, wherein, The fermentation medium of the Bacillus subtilis ACCC 60383 in step (1) includes: 10 - 15 g / L of tryptone, 5 - 8 g / L of yeast extract, 5 - 6 g / L of sodium chloride, 1 - 5 g / L of L - glutamic acid, 1 - 2 g / L of glucose, and make up the volume to 1 L with distilled water; the pH of the fermentation medium of Bacillus subtilis ACCC 60383 is 7.0 - 7.

5.

6. The preparation method of the microbial preparation according to claim 4, characterized in that, The fermentation medium of the Aspergillus usamii ACCC 30122 in step (2) includes: 15 - 20 g / L of soluble starch, 5 - 6 g / L of yeast extract, 1 - 3 g / L of NH4NO3, 0.5 - 1 g / L of KH2PO4, 0.1 - 0.5 g / L of MgSO4·7H2O, and make up the volume to 1 L with distilled water; the fermentation temperature of Aspergillus usamii ACCC 30122 is 25 - 28 °C, and the pH is 4.0 - 7.

0.

7. The preparation method of the microbial preparation according to claim 4, wherein The mixed fermentation medium in step (3) includes: 8 - 10 g / L of glucose, 3 - 5 g / L of yeast extract, 2 - 5 g / L of peptone, 5 - 10 g / L of wheat bran, 0.1 - 2 g / L of KH2PO4, 0.1 - 0.5 g / L of MgSO4·7H2O, 0.1 - 0.5 g / L of NaCl, 0.5 - 1 g / L of (NH4)2SO4, and make up the volume to 1 L with distilled water; The temperature for fermenting and culturing after inoculating the Bacillus subtilis ACCC 60383 fermentation broth into the mixed fermentation medium in step (3) is 35 - 38 °C, and the pH is 7.0 - 7.

5. After adding Aspergillus usamii ACCC 30122, adjust the fermentation temperature to 25 - 28 °C and adjust the pH to 4.0 - 7.0 for fermentation; The Bacillus subtilis ACCC 60383 fermentation broth and Aspergillus usamii ACCC 30122 fermentation broth in step (3) are inoculated into the mixed fermentation medium according to the viable bacteria ratio of 6 - 8:2 - 3.

8. Use of the microbial agent prepared by the method according to any one of claims 4-7 in soybean disease resistance and / or enhancing soybean disease resistance.

9. The application according to claim 8, characterized in that, The types of diseases against which resistance is provided include frogeye leaf spot, root rot, Phytophthora root rot, powdery mildew, anthracnose, and soybean mosaic virus disease.

10. The application according to claim 8, wherein Specific application methods include, but are not limited to, seed treatment, soil application, or foliar spraying.

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