CN110938559A - Compound microbial agent for preventing and treating soil-borne diseases, preparation method and application thereof in summer corn cultivation - Google Patents
Compound microbial agent for preventing and treating soil-borne diseases, preparation method and application thereof in summer corn cultivation Download PDFInfo
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- UWVQIROCRJWDKL-UHFFFAOYSA-N oxadixyl Chemical compound CC=1C=CC=C(C)C=1N(C(=O)COC)N1CCOC1=O UWVQIROCRJWDKL-UHFFFAOYSA-N 0.000 description 1
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N1/00—Microorganisms, e.g. protozoa; Compositions thereof; Processes of propagating, maintaining or preserving microorganisms or compositions thereof; Processes of preparing or isolating a composition containing a microorganism; Culture media therefor
- C12N1/20—Bacteria; Culture media therefor
-
- A—HUMAN NECESSITIES
- A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
- A01G—HORTICULTURE; CULTIVATION OF VEGETABLES, FLOWERS, RICE, FRUIT, VINES, HOPS OR SEAWEED; FORESTRY; WATERING
- A01G13/00—Protection of plants
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Abstract
The invention relates to the technical field of microorganisms, in particular to a preparation method of a compound microbial agent for preventing and treating soil-borne diseases and application of the compound microbial agent in summer corn cultivation. Comprises Bacillus megaterium BMJBN02, Bacillus methylotrophicus KNK-1, Bacillus cereus BCJB01 and Shewanella sp.A0B14 (the viable bacteria number ratio is 4:3:2: 1-3: 3:3:1), and the strains are separated and stored in the laboratory. The most remarkable characteristic of the invention is the application of the compound microbial agent in preventing and controlling soil-borne corn diseases, and simultaneously has the function of promoting the growth of corn. The relative prevention effect on the corn stalk basal rot of the soil-borne corn disease can reach 73 percent, and the relative prevention effect on the corn downy mildew can reach 44 percent; in addition, the compound microbial inoculum can improve the strong seedling index of corn in the three-leaf stage by more than 50%, is beneficial to improving the lodging resistance of corn in the seedling stage, and has great application value for the cultivation and production of corn.
Description
Technical Field
The invention relates to the technical field of microorganisms, in particular to a preparation method of a compound microbial agent for preventing and treating soil-borne diseases and application of the compound microbial agent in summer corn cultivation.
Background
The traditional soil-borne disease control method is to apply chemical agents, but the chemical agents have the problems of drug residues, environmental pollution, ecological damage and the like, so that the application of the chemical agents does not meet the requirements of sustainable and healthy agricultural development. In order to reduce the harm of soil-borne diseases to crops and maintain ecological balance, the control strategy of 'controlling bacteria with bacteria' gradually becomes a research hotspot at home and abroad. The strategy refers to the use of one or more beneficial microorganisms to inhibit the proliferation of pathogenic bacteria.
The microorganism has the advantages of rich resources, no pollution, no residue, prevention and treatment, low cost, ecological balance maintenance, long-acting effect and the like. Firstly, microorganisms can induce plants to generate system resistance through planting plant rhizosphere to improve the capability of resisting pathogenic bacteria, or reduce diseases caused by pathogenic bacteria by competing with soil-borne pathogenic bacteria for ecological sites; in addition, the microorganism regulates the contents and activities of three major elements, namely nitrogen, phosphorus and potassium, and more than ten trace elements in the soil through life activities, and promotes the growth of plants; finally, the plant hormones produced by the microorganisms can regulate plant metabolism and improve the quality of the plant and/or its fruit. The compound microbial agent is obtained by compounding microorganisms with disease prevention and/or disease resistance and microorganisms with the functions of regulating the contents and activities of major elements and trace elements in soil, and has become a trend of effectively supplying modern green agriculture.
The Shandong area is divided into corn planting areas in China and belongs to a spring and summer corn sowing area in the plain of Huang-Huai-Hai. The growth cycle of the spring corn is harvested from 3 months to 9 months or 10 months; summer corns are sown in 6 months and can be harvested from the bottom of 9 months to the beginning of 10 months, so that summer corn planting is mainly used for corn planting in the area. The summer corn sowing area is continuously increased along with the adjustment of the planting industry and the breeding industry structure in agriculture in China. In recent years, the planting area of summer corn is rapidly enlarged along with the reduction of the planting area of cotton, but the increase of the total yield of corn mainly depends on the increase of the planting area and the application of chemical pesticides to control diseases and protect yield, and the yield per unit is not remarkably improved. The invention provides a compound microbial agent for preventing and treating soil-borne corn diseases and an application method thereof, aiming at improving the yield of summer corn and food safety in the area.
Disclosure of Invention
The invention aims at providing a preparation method of a compound microbial agent for preventing and treating soil-borne diseases and an application of the compound microbial agent in summer corn cultivation aiming at preventing and treating the damage of long-term excessive application of chemical pesticides to the environment for the soil-borne corn diseases, wherein the compound microbial agent comprises Bacillus megaterium BMJBN02, Bacillus methylotrophicus KNK-1, Bacillus cereus BCJB01 and Bacillus Shewanella sp.A0B14. The compound microbial agent has the characteristics of preventing and treating corn stalk base rot caused by the infection of soil-borne disease fusarium graminearum and corn downy mildew caused by the infection of downy mildew, and has the characteristics of promoting the growth of corn and enhancing the lodging resistance of corn, so that the compound microbial agent has obvious effects of preventing and treating corn diseases and promoting the growth of corn during the cultivation of corn.
The technical scheme of the invention is as follows:
a compound microbial agent comprises Bacillus megaterium BMJBN02, Bacillus methylotrophicus KNK-1, Bacillus cereus BCJB01 and Bacillus Shewanella sp.A0B14, wherein the strains are separated and stored in the laboratory.
Wherein, the strain of the bacillus megaterium BMJBN02 is preserved in the common microorganism center of China Committee for culture Collection of microorganisms, the preservation number is CGMCC NO.10478, the preservation date is 2015, 1 month and 30 days, and the strain is recorded in the application document with the application number of 201510082296.0; the strain Bacillus methylotrophicus KNK-1 is preserved in China general microbiological culture Collection center (CGMCC) with the preservation number of CGMCC NO.16842 and the preservation date of 2018, 1 month and 30 days; the strain (consistent with the published patent and with modified name) bacillus cereus BCJB01 has been preserved in China general microbiological culture Collection center (CGMCC) with the preservation number of CGMCC NO.10477 and the preservation date of 2015 for 1 month and 30 days, and is recorded in the application document with the application number of 201510081973.7; the Shewanella sp.A0B14 strain has been preserved in the China general microbiological culture Collection center with the preservation number of CGMCC NO.16843 and the preservation date of 2018, 11 and 30, and is recorded in the application document with the application number of 201910049412.7.
A preparation method of a compound microbial agent comprises the following steps:
(1) preserved Bacillus megaterium BMJBN02, Bacillus methylotrophicus KNK-1, Bacillus cereus BCJB01 and ShewaneRespectively streaking and inoculating the lla sp.A0B14 to a solid LB culture medium, performing light-shielding culture at 30 +/-1 ℃, selecting a single colony after 18-24 hours, inoculating the single colony into a liquid LB culture medium, performing culture at 30 +/-1 ℃ until the logarithmic growth phase is reached, wherein the OD value is 0.6-0.8, and the concentration of a seed solution is not lower than 1.0 multiplied by 108cfu/mL, respectively obtaining a seed solution A, a seed solution B, a seed solution C and a seed solution D to be used by inoculating into a fermentation culture medium;
(2) fermentation medium formula a: corn flour 2%, soybean meal 1%, yeast powder 0.3%, CaCl20.1％，MgSO4·7H2O 0.05％，K2HPO·3H2O 0.3％，KH2PO40.2％，FeSO4·7H20.0008 percent of O (all are mass percent), and the balance of water; carrying out high-pressure sterilization on the fermentation medium A and a fermentation tank at 121 ℃ for 60min under the conditions of pH 7.2-7.5 and 0.1MPa, cooling to normal pressure and normal temperature under reduced pressure for later use, and fermenting the seed solution A and the seed solution C by using the fermentation medium A;
fermentation medium formula B: molasses (sugar degree 48%) 10%, carbonamide 0.5%, K2HPO4·3H2O 0.3％，KH2PO40.2 percent of potassium chloride, 0.1 percent of potassium chloride (all in mass percent), and the balance of water; carrying out high-pressure sterilization on the fermentation medium B and a fermentation tank at the temperature of 115 ℃ for 60min under the conditions of pH 7.0-7.2 and 0.1MPa, cooling to normal pressure and normal temperature under reduced pressure for later use, and fermenting the seed liquid B and the seed liquid D by adopting the fermentation medium B;
(3) respectively inoculating seed liquid A, seed liquid B, seed liquid C and seed liquid D which correspond to the strains Bacillus megaterium BMJBN02, Bacillus methylotrophicus KNK-1, Bacillus cereus BCJB01 and Shewanella sp.A0B14 which grow to the logarithmic phase in the step (1) into corresponding fermentation culture media, and ensuring that the initial concentration of thalli in each fermentation culture system is not less than 1.0 x 107cfu/mL; wherein the fermentation conditions of the Bacillus megatherium BMJBN02 and the Bacillus cereus BCJB01 are as follows: under the condition of 30 +/-1 ℃, the tank pressure is kept at 0.05-0.10 KPa, oxygen is introduced to ensure the dissolved oxygen content to be 20-22 percent, and then the operation is carried outThe speed is 180r/min, the pH value of the fermentation liquor is 7.0-7.2 in 12 hours before fermentation, the pH value of the fermentation liquor is 7.5-8.0 in 12 hours after fermentation, and the fermentation is cultured for 24-36 hours; the fermentation conditions of Bacillus methylotrophicus KNK-1 and Shewanella sp.A0B14 are as follows: under the condition of 30 +/-1 ℃, the tank pressure is kept at 0.05-0.10 KPa, oxygen is introduced to ensure the dissolved oxygen content to be 20-22%, the rotating speed is 180r/min, the pH value is controlled to be 7.0-7.5, and the culture is carried out for 36-48 h; obtaining fermentation liquor A, fermentation liquor B, fermentation liquor C and fermentation liquor D which correspond to strains of Bacillus megaterium BMJBN02, Bacillus methylotrophicus KNK-1 and Bacillus cereus BCJB01 and Bacillus Shewanella sp.A0B14 after the fermentation is finished, wherein the sporulation rate reaches more than 90%, and the number of the viable bacteria in the fermentation liquor respectively reaches 8.0 multiplied by 109cfu/mL、1.5×109cfu/mL、6.0×109cfu/mL and 1.0X 109cfu/mL；
(4) Mixing and compounding the strains obtained in the step (3) such as Bacillus megaterium BMJBN02, Bacillus methylotrophicus KNK-1, Bacillus cereus BCJB01 and fermentation broth A, fermentation broth B, fermentation broth C and fermentation broth D corresponding to Shewanella sp.A0B14, and obtaining mixed fermentation broth according to the viable bacteria number ratio of 4:3:2: 1-3: 3:3: 1;
(5) adding turfy soil or medical stone into the mixed fermentation liquor obtained in the step (4) according to the mass-volume ratio of 20%, fully and uniformly mixing, carrying out spray drying to obtain a mixed fermentation liquor powdery prefabricated agent containing bacterial strains of Bacillus megaterium BMJBN02, Bacillus methylotrophicus KNK-1, Bacillus cereus BCJB01 and Shewanella sp.A0B14, and adding an agriculturally acceptable auxiliary agent to obtain a compound microbial agent, wherein the total viable bacteria number in the microbial agent is not less than 1.0 multiplied by 1011cfu/g, wherein the number ratio of viable bacteria of a strain Bacillus megaterium BMJBN02, Bacillus methylotrophicus KNK-1, Bacillus cereus BCJB01 and Shewanella sp.A0B14 is 4:3:2: 1-3: 3:3: 1.
Further preferably, the compound microbial agent comprises wettable powder, water dispersible granules, a suspending agent, a suspoemulsion or an emulsion in water.
Preferably, the preparation method of the wettable powder comprises the steps of taking 94-97 parts of the mixed fermentation liquid powdery prefabricated agent obtained in the step (5), fully mixing 1-2 parts of turfy soil, 1-2 parts of sodium carboxymethyl cellulose, 1-2 parts of chitosan and 1-2 parts of sodium lignosulfonate, and crushing the mixture to phi less than 45 microns through an airflow crusher to obtain the wettable powder of the compound microbial agent.
The compound microbial agent disclosed by the invention is applied to cultivation and production of summer corn.
The compound microbial agent disclosed by the invention is applied to prevention and treatment of summer corn soil-borne diseases and growth promotion of corn.
Furthermore, the compound microbial agent disclosed by the invention is applied to preventing and treating basal rot of corn caused by infection of soil-borne disease fusarium graminearum and downy mildew of corn caused by infection of downy mildew.
Furthermore, the compound microbial agent disclosed by the invention is applied to promoting the growth of corns and enhancing the lodging resistance of corns.
The invention has the beneficial effects that:
the invention provides a compound microbial agent containing Bacillus megaterium BMJBN02, Bacillus methylotrophicus KNK-1, Bacillus cereus BCJB01 and Shewanella Shewanella sp.A0B14 and a preparation method thereof.
The compound microbial agent is applied to preventing and treating corn stalk base rot caused by soil-borne disease fusarium graminearum infection and corn downy mildew caused by downy mildew infection, and is applied to promoting corn growth and enhancing corn lodging resistance, so that a new way is developed for the research and development of biological pesticides.
Compared with the chemical pesticide which is commonly used in the market for preventing and treating corn diseases caused by fusarium graminearum and/or downy mildew infection, the compound microbial agent has good prevention and treatment effect, the relative prevention effect on the soil-borne corn disease corn stalk basal rot can reach 73 percent, and the relative prevention effect on the corn downy mildew can reach 44 percent; in addition, the microbial inoculum can improve the strong seedling index of the corn in the seedling stage by more than 50 percent.
Detailed Description
For better understanding of the present invention, the technical solution of the present invention will be described in detail with specific examples, but the present invention is not limited thereto.
Example 1 (wettable powder)
A preparation method of a compound microbial agent comprises the following steps:
(1) respectively streak-inoculating the preserved Bacillus megaterium BMJBN02, Bacillus methylotrophicus KNK-1, Bacillus cereus BCJB01 and Shewanella Shewanella sp.A0B14 to a solid LB culture medium, carrying out light-shielding culture at 30 +/-1 ℃, picking out a single colony after 18-24 hours, inoculating the single colony to a liquid LB culture medium, and culturing at 30 +/-1 ℃ to a logarithmic growth phase, wherein the OD value is 0.6-0.8, and the concentration of a seed solution is not lower than 1.0 multiplied by 108cfu/mL, respectively obtaining a seed solution A, a seed solution B, a seed solution C and a seed solution D to be used by inoculating into a fermentation culture medium;
(2) fermentation medium formula a: corn flour 2%, soybean meal 1%, yeast powder 0.3%, CaCl20.1％，MgSO4·7H2O 0.05％，K2HPO·3H2O 0.3％，KH2PO40.2％，FeSO4·7H20.0008 percent of O (all are mass percent), and the balance of water; carrying out high-pressure sterilization on the fermentation medium A and a fermentation tank at 121 ℃ for 60min under the conditions of pH 7.2-7.5 and 0.1MPa, cooling to normal pressure and normal temperature under reduced pressure for later use, and fermenting the seed solution A and the seed solution C by using the fermentation medium A;
fermentation medium formula B: molasses (sugar degree 48%) 10%, carbonamide 0.5%, K2HPO4·3H2O 0.3％，KH2PO40.2 percent of potassium chloride, 0.1 percent of potassium chloride (all in mass percent), and the balance of water; the fermentation medium B is sterilized under high pressure at 115 ℃ for 60min together with a fermentation tank under the conditions of pH7.0-7.2 and 0.1MPa,cooling to normal pressure and normal temperature under reduced pressure for later use, and fermenting the seed liquid B and the seed liquid D by adopting a fermentation culture medium B;
(3) respectively inoculating seed liquid A, seed liquid B, seed liquid C and seed liquid D which correspond to the strains Bacillus megaterium BMJBN02, Bacillus methylotrophicus KNK-1, Bacillus cereus BCJB01 and Shewanella sp.A0B14 which grow to the logarithmic phase in the step (1) into corresponding fermentation culture media, and ensuring that the initial concentration of thalli in each fermentation culture system is not less than 1.0 x 107cfu/mL; wherein the fermentation conditions of the Bacillus megatherium BMJBN02 and the Bacillus cereus BCJB01 are as follows: under the condition of 30 +/-1 ℃, the tank pressure is kept at 0.05-0.10 KPa, oxygen is introduced to ensure the dissolved oxygen content to be 20-22%, the rotating speed is 180r/min, the pH value of the fermentation liquor is 7.0-7.2 in 12 hours before fermentation, the pH value of the fermentation liquor is 7.5-8.0 in 12 hours after fermentation, and the fermentation liquor is cultured for 24-36 hours; the fermentation conditions of Bacillus methylotrophicus KNK-1 and Shewanella sp.A0B14 are as follows: under the condition of 30 +/-1 ℃, the tank pressure is kept at 0.05-0.10 KPa, oxygen is introduced to ensure the dissolved oxygen content to be 20-22%, the rotating speed is 180r/min, the pH value is controlled to be 7.0-7.5, and the culture is carried out for 36-48 h; obtaining fermentation liquor A, fermentation liquor B, fermentation liquor C and fermentation liquor D which correspond to strains of Bacillus megaterium BMJBN02, Bacillus methylotrophicus KNK-1 and Bacillus cereus BCJB01 and Bacillus Shewanella sp.A0B14 after the fermentation is finished, wherein the sporulation rate reaches more than 90%, and the number of the viable bacteria in the fermentation liquor respectively reaches 8.0 multiplied by 109cfu/mL、1.5×109cfu/mL、6.0×109cfu/mL and 1.0X 109cfu/mL；
(4) Mixing and compounding the strains obtained in the step (3) such as Bacillus megaterium BMJBN02, Bacillus methylotrophicus KNK-1, Bacillus cereus BCJB01 and fermentation broth A, fermentation broth B, fermentation broth C and fermentation broth D corresponding to Shewanella sp.A0B14, and obtaining mixed fermentation broth according to the viable bacteria number ratio of 4:3:2: 1;
(5) adding turfy soil or medical stone into the mixed fermentation liquor obtained in the step (4) according to a mass-volume ratio of 20%, fully and uniformly mixing, and performing spray drying to obtain a mixed fermentation liquor powdery prefabricated agent containing bacterial strains of Bacillus megaterium BMJBN02, Bacillus methylotrophicus KNK-1, Bacillus cereus BCJB01 and Shewanella sp.A0B14;
(6) taking 94-97 parts of the mixed fermentation liquid powdery prefabricated agent obtained in the step (5), 1-2 parts of turfy soil, 1-2 parts of sodium carboxymethylcellulose, 1-2 parts of chitosan and 1-2 parts of sodium lignosulfonate, fully mixing, and crushing the mixture to phi less than 45 mu m by using a jet mill to obtain a powdery compound microbial agent, wherein the number of total viable bacteria in the microbial agent is not less than 1.0 multiplied by 1011cfu/g, wherein the number ratio of viable bacteria of the strain Bacillus megaterium BMJBN02, Bacillus methylotrophicus KNK-1, Bacillus cereus BCJB01 and Shewanella sp.A0B14 is 4:3:2: 1.
The compound microbial agents in the following examples 2, 3, 4 and 5 are all prepared in example 1; the percentage content of the product is mass/volume percentage.
Example 2 application of compound microbial agent in prevention and treatment of corn stalk rot caused by fusarium graminearum infection
The application of the compound microbial agent in preventing and treating the corn stalk basal rot caused by fusarium graminearum infection is carried out by the following steps:
(1) adding 10g of compound microbial agent and 0.5g of fusarium graminearum into per liter of nutrient soil (the nutrient soil is purchased from Pidmann company, the formula of the nutrient soil is that the content of organic matters is more than or equal to 45 percent, the content of humic acid is more than or equal to 15 percent, the pH value is 5.5-6.8, and the content of free water is less than or equal to 20 percent), mixing uniformly and fully to obtain a culture substrate I, sowing and conventionally managing the 'Zhengdan 958' of corn according to a conventional method; control group: adding 0.5g of fusarium graminearum into each liter of nutrient soil, mixing to obtain a culture substrate II, sowing and conventionally managing corns 'Zhengdan 958' according to a conventional method, treating 30 corns in each group, repeating for 3 times, observing and counting the morbidity and disease grade of the infected corns after 14 days. The test results are shown in Table 1.
(2) The corn stem basal rot grading standard reference GB 1353-2018-corn national standard is as follows:
level 0: the whole plant has no disease attack; level 1: the whole plant grows normally, the middle and lower leaves have the symptoms of bacterial wilt/bacterial yellow withering, and the normal stem base growth disease accounts for 0.1 to 5.0 percent; and 3, level: the leaf of the whole plant has a withered symptom, and the normal growth of the stem base accounts for 5.1 to 10.0 percent; and 5, stage: the typical withered symptom appears on the whole leaf, the color of the stem base part is changed, and the disease is slightly soaked in water and accounts for 10.1 to 30.0 percent; and 7, stage: the typical withered symptom appears on the whole leaf, and the stem base part is obviously softened but the disease is 30.1 to 40.0 percent; and 9, stage: the whole plant withers and falls down, the vascular bundles at the base of the stem are broken, and the disease accounts for 40.1 to 100 percent.
The calculation formula is as follows:
incidence (%) is ═ (number of diseased plants/number of investigated plants) × 100%
Disease index of 100 × Σ (number of disease leaves at each stage × relative disease stage)/(total number of survey leaves × 9)
Relative control effect (%) < control area index of disease-treatment area index of disease)/control area index of disease 100%
Data statistical analysis: statistical analysis was performed using EXCEL2007 and SPSS19.0 software, and multiple comparisons were performed using duncan's new range test.
TABLE 1 test for preventing and treating corn stalk rot caused by Fusarium graminearum with compounded microbial inoculum
| Treatment of | The incidence of disease% | Index of disease grade | Relative control effect% |
| Compound microorganismBiological agent group | 1.52±0.09 | 0.27±0.01 | 52.04±1.09 |
| Control group | 3.79±0.67 | 0.54±0.05 | -- |
The result shows that compared with the control, the incidence of the corn plant using the compound microbial inoculum is only 1.52 percent, the relative prevention effect reaches more than 52 percent, and the incidence of the corn stalk basal rot caused by fusarium graminearum can be obviously inhibited.
Example 3 application of Compound microbial Agents in controlling corn downy mildew caused by downy mildew infection
The application of the compound microbial agent in preventing and treating corn downy mildew caused by downy mildew infection comprises the following steps:
(1) adding 10g of compound microbial agent and 0.5g of downy mildew thalli into each liter of nutrient soil, mixing uniformly to obtain a culture substrate I, sowing and conventionally managing the corn Zhengdan 958 according to a conventional method; control group: adding 0.5g fusarium graminearum into each liter of nutrient soil, mixing to obtain a culture substrate II, sowing and conventionally managing corn Zhengdan 958 according to a conventional method, treating 30 plants in each group, repeating for 3 times, observing and counting the morbidity and disease grade of the corn after 14 days. The test results are shown in Table 2.
(2) The corn downy mildew grading standard refers to the following GB 1353-2018-corn national standard:
level 0: the whole plant has no disease attack; level 1: the whole plant grows normally, the leaf has a light green or yellow green alternate stripe symptom, and the disease of the white frost-like mildew layer on the back of the leaf is 0.1 to 5.0 percent; and 3, level: the leaf has the light green or yellow green alternate stripe symptom, and the white frost-like mildew layer on the back of the leaf is 5.1 to 10.0 percent; and 5, stage: the leaf has the light green or yellow green alternate stripe symptom, and the white frost-like mildew layer on the back of the leaf is 10.1 to 30.0 percent; and 7, stage: the leaf has the light green or yellow green alternate stripe symptom, and the white frost-like mildew layer on the back of the leaf is 30.1 to 40.0 percent; and 9, stage: the leaf has the alternate stripe symptom of light green or yellow green, and the disease of the white frost-like mildew layer on the back of the leaf is 40.1 to 100 percent.
The calculation formula is as follows:
incidence (%) is ═ (number of diseased plants/number of investigated plants) × 100%
Disease index of 100 × Σ (number of disease leaves at each stage × relative disease stage)/(total number of survey leaves × 9)
Relative control effect (%) < control area index of disease-treatment area index of disease)/control area index of disease 100%
Data statistical analysis: statistical analysis was performed using EXCEL2007 and SPSS19.0 software, and multiple comparisons were performed using duncan's new range test.
TABLE 2 prevention and control test of compound microbial inoculum for corn downy mildew
| Treatment of | The incidence of disease% | Index of disease grade | Relative control effect% |
| Compound microbial agent group | 1.23±0.21 | 0.33±0.05 | 35.85±0.77 |
| Control group | 2.04±0.39 | 0.52±0.06 | ---- |
The result shows that compared with the control, the incidence rate of the corn plants using the compound microbial inoculum is only 1.23 percent, the relative prevention effect reaches 35.85 percent, and the attack of the corn downy mildew can be obviously inhibited.
Example 4 application of compound microbial inoculum in corn growth promotion
The application of the compound microbial agent in promoting the growth of the corn comprises the following steps:
(1) adding a compound microbial agent into a seedling culture substrate according to 10g/L, fully and uniformly mixing to obtain a culture substrate, sowing and conventionally managing corn 'zhengdan 958' according to a conventional method, directly using the seedling culture substrate as the culture substrate to cultivate the corn 'zhengdan 958' as a blank control, and using the corn 'zhengdan 958' cultivated group added with a nitrogen-phosphorus-potassium compound fertilizer (nitrogen: phosphorus: potassium is 1: 0.5: 1.5) in the culture substrate as the nitrogen-phosphorus-potassium compound fertilizer control (the using amount is 2.5-2.7 kg of nitrogen (N), phosphorus (P) (P2O5) 1.1-1.4 kg potassium (K)2O) 3.7-4.2 kg), treating 30 plants in each group, repeating for 3 times, observing and counting the plant height, the stem thickness, the fresh weight of the whole plant and the strong seedling index after 21 days. The test results are shown in Table 3.
Strong seedling index (stem thickness (cm)/plant height (cm) × whole plant dry weight (g)
TABLE 3 growth promotion test of Complex microbial Agents on corn
| Treatment of | Blank control | Compound fertilizer control | Treatment with compounded spore fungicide |
| Plant height (cm) | 51.23±0.94 | 57.35±1.17 | 59.72±1.81 |
| Stem diameter (mm) | 8.33±0.08 | 10.78±0.09 | 11.91±0.08 |
| Whole plant Dry weight (g) | 6.92±1.23 | 8.07.±1.99 | 8.77±1.21 |
| Seedling strengthening index | 0.112±0.0023 | 0.150±0.0027 | 0.175±0.0021 |
The results show that compared with a blank control, the plant height, stem thickness, whole plant dry weight and strong seedling index of 'Zhengdan 958' using the compound microbial inoculum are obviously increased, the biomass accumulation is 29 percent, and particularly the strong seedling index is increased by 56.25 percent; in addition, the growth promoting effect (plant height and stem thickness) of the compound microbial agent on 'Zhengdan 958' is obviously better than that of a nitrogen-phosphorus-potassium compound fertilizer (nitrogen, phosphorus and potassium are 1: 0.5: 1.5). This shows that the compound microbial inoculum has good effect of promoting the growth of corn.
(2) And after sowing for 21d, detecting the contents of available nitrogen, available phosphorus and available potassium in the corn root soil of the compound microbial agent treatment group and the blank control group. The effective nitrogen detection adopts an alkaline hydrolysis diffusion method, the effective phosphorus detection adopts a 0.5M sodium bicarbonate leaching-molybdenum-antimony colorimetric method and the effective potassium detection adopts NH4OAc leaching-flame photometry. The method for detecting the contents of available nitrogen, available phosphorus and available potassium is referred to soil agriculture analysis and environmental monitoring (edited by Yangtze Sword iris, Wangcheng, Binheng forest) of the Ministry of China, Ministry of Japan, 2008. The test results are shown in Table 4.
TABLE 4 influence of the Complex microbial Agents on the content of available Nitrogen, phosphorus and Potassium in corn root systems
| Treatment of | Available nitrogen (mg/kg) | Available phosphorus (mg/kg) | Effective potassium (mg/kg) |
| Compound microbial agent group | 1012.39±2.88 | 227.41±3.46 | 846.71±3.76 |
| Blank control | 143.27±3.77 | 105.55±5.58 | 110.71±3.32 |
The results show that compared with a blank control, the contents of available nitrogen, available phosphorus and available potassium in the corn root system soil using the compound microbial inoculum are obviously improved by 607.70%, 116.19% and 669.09%, respectively, which shows that the compound microbial inoculum realizes the effect of promoting the growth of the corn by improving the contents of the available nitrogen, the available phosphorus and the available potassium in the corn root system soil.
Example 5 application of Compound microbial Agents in summer corn
The application of the compound microbial agent in summer corn comprises the following steps:
the cell test was conducted in the Dieryue district of Taian city in 6 months in 2019. The soil of the test field is the yellow brown soil, the soil fertility is moderate, the topography is flat, and the test field has irrigation conditions. In the area, summer corns are planted in 2016-2018, and the corn downy mildew and the corn stalk base rot are caused in the summer corn planting process in 2017-2018, so that the tested variety is 'Zhengdan 958' of the corn. The fertilizer using amount, the application method and the field cultivation management are carried out according to the local best measures.
The test is designed according to the field test criteria of the drug inspection institute of the ministry of agriculture, and the total number of the tests is 5: compounded with microbial preparation, azophoska compound fertilizer (nitrogen, phosphorus and potassium in the ratio of 1 to 0.5 to 1.5), chlorothalonil powder 75%, hymexazol powder 70% and clear water as reference. The compound microbial inoculant (5.0-5.5 kg/mu) is applied to a nitrogen-phosphorus-potassium compound fertilizer (nitrogen, phosphorus and potassium are 1: 0.5: 1.5, and the nitrogen-phosphorus-potassium compound fertilizer is 80 kg/mu), 75% chlorothalonil powder (2.0 kg/mu) and 70% hymexazol powder (2.0 kg/mu) by a base fertilizer method, the application is carried out along with a sowing period, each treatment is carried out for 0.04 mu, the treatment is repeated for 3 times, the total area of 15 districts is 0.6 mu, and 2 rows of protective rows are reserved in each district. And (5) designing a random block, and selecting 21d for field investigation. Each cell adopts a double diagonal line to fix 5 points for sampling, 6 plants are surveyed at each point, and the growth condition, the morbidity condition and the disease grade of a surveyed object are recorded. The temperature during the test was not lower than 28 ℃. The results are shown in tables 5 and 6.
Grading index of disease severity: the grading standard refers to the grading standard of corn downy mildew and corn stalk base rot in the standard GB 1353-2018-corn national standard, and the grading is carried out by taking the whole plant as a unit. Level 0: no disease spots; level 1: 0.1 to 5.0 percent of scab; and 3, level: 5.1 to 10.0 percent of scab; and 5, stage: 10.1 to 30.0 percent of scab; and 7, stage: 30.1 to 40.0 percent of scab; and 9, stage: the disease spot is 40.1-100%, and the plant is seriously malformed and even dies.
The calculation formula is as follows:
incidence (%) is ═ (number of diseased plants/number of investigated plants) × 100%
Disease index of 100 × Σ (number of disease leaves at each stage × relative disease stage)/(total number of survey leaves × 9)
Relative control effect (%) < control area index of disease-treatment area index of disease)/control area index of disease 100%
Data statistical analysis: statistical analysis was performed using EXCEL2007 and SPSS19.0 software, and multiple comparisons were performed using duncan's new range test.
TABLE 5 comparison of agronomic traits of corn by Compound microbial Agents
| Treatment of | Plant height (cm) | Stem diameter (cm) | Growth vigor in field |
| Compound microbial inoculum (5.0-5.5 kg/mu) | 59.78±1.81 | 1.14±0.08 | Good effect |
| NPK compound fertilizer (80 kg/mu) | 57.39±1.18 | 1.01±0.09 | Good effect |
| 75% chlorothalonil powder (2.0 kg/mu) | 52.17±1.39 | 0.88±0.07 | Good effect |
| 70% hymexazol powder (2.0 kg/mu) | 51.23±0.99 | 0.81±0.08 | Good effect |
| Clear water control | 50.49±1.21 | 0.81±0.133 | Good effect |
As can be seen from Table 5, the growth promoting effect of the compound microbial agent on the corns in the seedling stage is remarkable, the stem thickness growth promoting effect is improved to 40.74 percent, the growth promoting effect is close to that of a control medicament nitrogen-phosphorus-potassium compound fertilizer, but the dosage is far lower than that of the nitrogen-phosphorus-potassium compound fertilizer.
TABLE 6 Effect of different treatments on maize stalk rot and maize downy mildew
As can be seen from Table 6, the compound microbial agent has significant relative control effect on basal stem rot and downy mildew of corn. The relative prevention effect of the compound microbial inoculum on the basal stem rot and the downy mildew of the corn is equal to or superior to the relative prevention effect of a reference chemical protective agent (75 percent of chlorothalonil powder and 70 percent of oxadixyl); the statistics of the total disease rate in the reference table shows that the number of diseased strains is obviously reduced after the compound microbial agent is applied.
Claims (9)
1. A compound microbial agent is characterized by comprising Bacillus megaterium BMJBN02, Bacillus methylotrophicus KNK-1, Bacillus cereus BCJB01 and Bacillus Shewanella sp.A0 B14.
2. The compound microbial inoculant according to claim 1, wherein the strain bacillus megaterium BMJBN02 has been preserved in the common microorganism center of the china committee for culture collection of microorganisms with the preservation number of CGMCC No.10478 and the preservation date of 2015 for 1 month and 30 days; the strain Bacillus methylotrophicus KNK-1 is preserved in China general microbiological culture Collection center (CGMCC) with the preservation number of CGMCC NO.16842 and the preservation date of 2018, 1 month and 30 days; the strain bacillus cereus BCJB01 has been preserved in China general microbiological culture Collection center (CGMCC) with the preservation number of CGMCC NO.10477 and the preservation date of 2015, 1 month and 30 days; the Shewanella sp.A0B14 strain has been preserved in the China general microbiological culture Collection center with the preservation number of CGMCC No.16843 and the preservation date of 2018, 11 months and 30 days.
3. A preparation method of a compound microbial agent is characterized by comprising the following steps:
(1) respectively streak-inoculating the preserved Bacillus megaterium BMJBN02, Bacillus methylotrophicus KNK-1, Bacillus cereus BCJB01 and Shewanella Shewanella sp.A0B14 to a solid LB culture medium, carrying out light-shielding culture at 30 +/-1 ℃, picking out a single colony after 18-24 hours, inoculating the single colony to a liquid LB culture medium, and culturing at 30 +/-1 ℃ to a logarithmic growth phase, wherein the OD value is 0.6-0.8, and the concentration of a seed solution is not lower than 1.0 multiplied by 108cfu/mL, respectively obtaining a seed solution A, a seed solution B, a seed solution C and a seed solution D to be used by inoculating into a fermentation culture medium;
(2) fermentation medium formula a: corn flour 2%, soybean meal 1%, yeast powder 0.3%, CaCl20.1％，MgSO4·7H2O0.05％，K2HPO·3H2O 0.3％，KH2PO40.2％，FeSO4·7H20.0008 percent of O (all are mass percent), and the balance of water; sterilizing the fermentation medium A together with the fermentation tank at 121 deg.C under high pressure of 0.1MPa and pH 7.2-7.5 for 60min, cooling to normal pressure and temperature under reduced pressure, and collecting seed liquidFermenting the A and the seed liquid C by adopting a fermentation medium A;
fermentation medium formula B: molasses (sugar degree 48%) 10%, carbonamide 0.5%, K2HPO4·3H2O 0.3％，KH2PO40.2 percent of potassium chloride, 0.1 percent of potassium chloride (all in mass percent), and the balance of water; carrying out high-pressure sterilization on the fermentation medium B and a fermentation tank at the temperature of 115 ℃ for 60min under the conditions of pH 7.0-7.2 and 0.1MPa, cooling to normal pressure and normal temperature under reduced pressure for later use, and fermenting the seed liquid B and the seed liquid D by adopting the fermentation medium B;
(3) respectively inoculating seed liquid A, seed liquid B, seed liquid C and seed liquid D which correspond to the strains Bacillus megaterium BMJBN02, Bacillus methylotrophicus KNK-1, Bacillus cereus BCJB01 and Shewanella sp.A0B14 which grow to the logarithmic phase in the step (1) into corresponding fermentation culture media, and ensuring that the initial concentration of thalli in each fermentation culture system is not lower than 1.0 multiplied by 107cfu/mL; wherein the fermentation conditions of the Bacillus megatherium BMJBN02 and the Bacillus cereus BCJB01 are as follows: under the condition of 30 +/-1 ℃, the tank pressure is kept at 0.05-0.10 KPa, oxygen is introduced to ensure the dissolved oxygen content to be 20-22%, the rotating speed is 180r/min, the pH value of the fermentation liquor is 7.0-7.2 in 12 hours before fermentation, the pH value of the fermentation liquor is 7.5-8.0 in 12 hours after fermentation, and the fermentation liquor is cultured for 24-36 hours; the fermentation conditions of Bacillus methylotrophicus KNK-1 and Shewanella sp.A0B14 are as follows: under the condition of 30 +/-1 ℃, the tank pressure is kept at 0.05-0.10 KPa, oxygen is introduced to ensure the dissolved oxygen content to be 20-22%, the rotating speed is 180r/min, the pH value is controlled to be 7.0-7.5, and the culture is carried out for 36-48 h; obtaining fermentation liquor A, fermentation liquor B, fermentation liquor C and fermentation liquor D which correspond to strains of Bacillus megaterium BMJBN02, Bacillus methylotrophicus KNK-1 and Bacillus cereus BCJB01 and Bacillus Shewanella sp.A0B14 after the fermentation is finished, wherein the sporulation rate reaches more than 90%, and the number of the viable bacteria in the fermentation liquor respectively reaches 8.0 multiplied by 109cfu/mL、1.5×109cfu/mL、6.0×109cfu/mL and 1.0X 109cfu/mL；
(4) Mixing and compounding the strains obtained in the step (3) such as Bacillus megaterium BMJBN02, Bacillus methylotrophicus KNK-1, Bacillus cereus BCJB01 and fermentation broth A, fermentation broth B, fermentation broth C and fermentation broth D corresponding to Shewanella sp.A0B14, and obtaining mixed fermentation broth according to the viable bacteria number ratio of 4:3:2: 1-3: 3:3: 1;
(5) adding turfy soil or medical stone into the mixed fermentation liquor obtained in the step (4) according to the mass-volume ratio of 20%, fully and uniformly mixing, carrying out spray drying to obtain a mixed fermentation liquor powdery prefabricated agent containing bacterial strains of Bacillus megaterium BMJBN02, Bacillus methylotrophicus KNK-1, Bacillus cereus BCJB01 and Shewanella sp.A0B14, and adding an agriculturally acceptable auxiliary agent to obtain a compound microbial agent, wherein the total viable bacteria number in the microbial agent is not less than 1.0 multiplied by 1011cfu/g, wherein the number ratio of viable bacteria of a strain Bacillus megaterium BMJBN02, Bacillus methylotrophicus KNK-1, Bacillus cereus BCJB01 and Shewanella sp.A0B14 is 4:3:2: 1-3: 3:3: 1.
4. The preparation method of the compound microbial agent according to claim 3, wherein the compound microbial agent comprises wettable powder, water dispersible granules, a suspending agent, a suspoemulsion or an emulsion in water.
5. The preparation method of the compound microbial agent according to claim 4, wherein the wettable powder is prepared by taking 94-97 parts of the mixed fermentation liquid powdery prefabricated agent obtained in the step (5), 1-2 parts of turfy soil, 1-2 parts of sodium carboxymethylcellulose, 1-2 parts of chitosan and 1-2 parts of sodium lignosulfonate, fully mixing, and crushing the mixture to phi less than 45 μm by using an airflow crusher to obtain the wettable powder of the compound microbial agent.
6. The use of the compound microbial agent of claims 1-5 in summer corn cultivation production.
7. The application of claim 6, wherein the compound microbial agent is applied to prevention and treatment of soil-borne diseases of summer corn and growth promotion of corn.
8. The application of the compound microbial agent as claimed in claim 6, wherein the compound microbial agent is applied to prevention and treatment of basal rot of corn caused by infection of soil-borne disease fusarium graminearum and downy mildew of corn caused by infection of downy mildew.
9. The use of claim 6, wherein the use of the compounded microbial agent is for promoting the growth of corn and enhancing the lodging resistance of corn.
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