CN113999837B - Agricultural microbial agent and preparation process thereof - Google Patents
Agricultural microbial agent and preparation process thereof Download PDFInfo
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Abstract
The present application provides an agricultural microbial inoculant comprising a microbial carrier material. The diatomite is added into the agricultural microbial agent, so that the effective viable count of phosphorus-dissolving microorganisms is improved, the stable long-term fertilizer efficiency is obtained, and good benefits are obtained in crop production and application.
Description
Technical Field
The application belongs to the field of microbial fertilizers, and particularly provides an agricultural microbial inoculant and a preparation process thereof, and application of the agricultural microbial inoculant in tomato planting and phosphorus-deficient soil improvement.
Background
According to statistics, 8 more cultivated lands in China are in phosphorus deficiency at different degrees, phosphate fertilizer is required to be used for achieving high yield required by modern agriculture, and common crops such as tomatoes, soybeans, sweet potatoes and the like are all high-phosphorus-demand varieties. The first consumption of the phosphate fertilizer in the world is large, and the consumption of the phosphate fertilizer per year is more than 1000 ten thousand tons. The method is not suitable for the land condition, the phosphorite resources in China are lack, the reserve of 151.98 hundred million tons is reserved, the high-quality phosphorite resources are completely exploited in decades, and the phosphate fertilizer becomes a bottleneck for limiting the sustainable development of agriculture in China.
On the other hand, most of the phosphate fertilizer is fixed by the soil after being applied to the soil: in calcareous soil available phosphorus is converted into apatite with very low solubility; iron phosphate and aluminum phosphate are formed in acidic soil. In China, phosphorus fertilizer/phosphorite is deficient, and meanwhile, phosphorus in soil is accumulated due to the use of phosphorus fertilizer for many years, and most accumulated phosphorus exists in a form of indissolvable inorganic phosphorus, is slowly released, and is difficult to be utilized by crops, namely, closed phosphorus or high-stability phosphorus.
The activation and release of the phosphorus in the soil are solved, and the utilization efficiency of the phosphorus and the phosphate fertilizer in the soil is improved, so that the sustainable development of agriculture in China is realized. The phosphorus-dissolving microorganism is a beneficial microorganism with the capability of activating soil insoluble inorganic phosphorus, and the application of the phosphorus-dissolving microorganism to activate the soil insoluble phosphorus is a worldwide recognized safe, economic and effective biological measure. At present, most of the phosphorus-dissolving bacterial agents are bacterial agents of single bacterial strains, and the phosphorus-dissolving effect and the adaptive soil type are limited.
A great deal of researches show that the microbial fertilizer is a good method for protecting the ecological environment and improving the yield and quality of crops. The microbial fertilizer is a microbial agent prepared from one or more beneficial living microorganisms, and the metabolic process or the metabolic product of the microbial agent is used for improving the plant growth condition so as to achieve the effect of promoting the plant growth. Therefore, maintaining microbial activity is a key to preparing microbial fertilizers, and the selection and addition of carrier materials have a key effect on the quality of microbial fertilizers. Under the condition of the same dosage, the adsorption capacities of different carrier materials to the thalli are different. Meanwhile, due to the different characteristics of various microorganisms, the same carrier material has different storage periods for different bacteria. Therefore, determining a suitable microbial carrier material in a microbial agent is of great importance for improving microbial activity in a microbial agent and for extending the shelf life of a microbial agent.
Disclosure of Invention
The invention aims to solve the problems of small effective viable count, unstable fertilizer efficiency and short shelf life of an agricultural microbial agent by providing the agricultural microbial agent containing a microbial carrier material. According to the invention, diatomite is added into the microbial inoculum, so that the effective viable count of phosphorus-dissolving microorganisms is improved, stable long-term fertilizer efficiency is obtained, and good benefits are obtained in crop production and application.
In order to achieve the above object, the present invention provides the following technical solutions:
an agricultural microbial inoculant comprising a microbial carrier material.
Preferably, the microbial carrier material is diatomaceous earth.
Preferably, the agricultural microbial agent comprises 8-12% of phosphorus-dissolving microorganisms, 4-6% of microbial carrier materials, 20-30% of peptone, 4-8% of potassium dihydrogen phosphate, 2-6% of glucose and 50-60% of water according to mass ratio, wherein the microbial carrier materials are diatomite.
Preferably, the agricultural microbial agent comprises 10% of phosphorus-dissolving microorganisms, 5% of microbial carrier materials, 25% of peptone, 6% of potassium dihydrogen phosphate, 4% of glucose and the balance of water according to mass ratio.
Preferably, the number of mould sundry fungus in the agricultural microbial agent is less than or equal to 3.0x10 6 The mixed bacteria rate is less than or equal to 30 percent, the fineness is more than or equal to 80 percent, the pH value is 6.5-7.5, and the moisture is less than or equal to 20 percent.
Preferably, the phosphorus-solubilizing microorganism comprises Pantoea ananatis and Bacillus pumilus.
Preferably, the ratio of the effective viable count of the pantoea ananatis to the bacillus cavitating is 2:1.
Further, the application provides a preparation process of the agricultural microbial agent, which comprises the following steps:
1) Performing activation and expansion culture on the original strain;
2) Cleaning the fermentation tank, purifying and sterilizing air, and sterilizing the seed tank, the fermentation tank and raw materials;
3) Inoculating, fermenting, detecting bacterial count and qualified indexes of the bacterial liquid after fermentation, and then pumping the bacterial liquid into a storage tank and inputting the storage tank into a spray dryer for drying and pulverizing;
4) And (3) conveying the bacterial powder into a granulator to form particles, sampling and detecting various indexes of products with qualified sizes, and packaging the products into finished products and warehousing.
Further, the application provides application of the agricultural microbial agent and the preparation process thereof in tomato planting.
Further, the application provides application of the agricultural microbial agent and the preparation process thereof in phosphorus-deficient soil improvement.
The invention has the beneficial effects that: different microbial carrier materials, such as common turf, diatomite, talcum powder and bagasse, are added into the microbial agent, and diatomite is selected from the microbial carrier materials which have activity for phosphorus-dissolving microorganisms in the microbial agent and have good quality guarantee period prolonging effect. The agricultural microbial agent of the invention well adsorbs phosphorus-dissolving microorganisms through the addition and use of diatomite, maintains the activity of the phosphorus-dissolving microorganisms, realizes stable long-term fertilizer efficiency in application, has great promotion effect on the production of crops, and obtains good economic benefits.
Detailed Description
Strain and culture medium:
pantoea ananatis (Pantoea ananatis): applicant purchased from chinese academy of agricultural sciences and deposited and passaged by itself, initially with ATCC 19321;
bacillus cavitating (bacillus depressus): the applicant purchases from China national academy of agricultural science and then self-maintains and passages, and the initial source is 1.15124 of CGMCC;
aspergillus niger (Aspergillus niger): the applicant purchases from China national academy of agricultural science and then self-stores and passages, and the initial source is ACCC 32579;
trichoderma harzianum (Trichoderma harzianum): applicant purchased from the national academy of agricultural science and then deposited and passaged by itself, the initial source being ACCC 32517.
During passage, pantoea ananatis and Bacillus subtlis use respective special culture media, and in experiments, the same modified beef extract peptone culture media (0.1 g of monopotassium phosphate, 0.1g of dipotassium phosphate, 2.5g of ammonium sulfate, 10g of calcium carbonate and 0.005g of ferrous sulfate are added per liter).
Other raw materials:
peptone, glucose: tobacco stand Weidan biotechnology Co., ltd;
potassium dihydrogen phosphate: sichuan Cheng Feng chemical Co., ltd;
diatomaceous earth was purchased from commercial products.
The main equipment comprises:
| main production facility name | Brand model | Quantity of | Production unit name |
| Seed pot | RSB-350 | 1 number of | Wenzhou city Wanfu machinery manufacturing plant |
| Fermentation tank | GJB-2.0 | 1 number of | Wenzhou food machinery Co Ltd |
| Storage tank | 1000L | 2 pieces of | Qingyun county trust Chengzhuan Co., ltd |
| Spray dryer | TPG type | 1 table | JIANGSU XIANFENG DRYING ENGINEERING Co.,Ltd. |
| Screw elevator | FQ610 | 1 table | HENAN LIWANG MACHINERY Co.,Ltd. |
| Extrusion granulator | WNJ-300-1Q | 1 table | Shandong province Weifang city agricultural machinery institute |
| Automatic packing machine | WHFBZJ-01 | 1 table | SHANGHAI MACWELL PACKAGING MACHINERY Co.,Ltd. |
The main experimental method comprises the following steps:
phosphorus dissolution experiment:
using insoluble phosphorus solid medium: glucose 10g, mgSO 4 ·7H 2 O0.4 g, phosphorus source 5g, (NH 4) 2 SO 4 0.5g、NaCl 0.2g、KCl 0.3g、MnSO 4 0.03g、FeSO 4 ·7H 2 0.01g of O, 20g of agar, 0.5g of yeast extract,1000mL of water. The phosphorus source is respectively calcium phosphate, hydroxyapatite and ferric phosphate.
The strain to be tested is prepared into bacterial suspension with similar OD, 200 mu L of bacterial suspension is taken and evenly coated in a 6cm culture dish. Culturing at 25deg.C for 1 day, punching with 6mm puncher to obtain bacterial cake, inoculating bacterial cake onto the culture medium plate, culturing at 25deg.C for 3 days, and measuring the ratio (D/D) of diameter (D) of phosphorus dissolving ring to colony diameter (D) to determine phosphorus dissolving capacity of the bacterial strain.
Field experiment:
experiment site 1: two bridge warmhouse booth of inner Mongolia Hangzhou brocade last flag
Test soil: the test land is an 8-meter span greenhouse, the soil is silt-filled soil, the land is flat, the irrigation and drainage are good, the fertility is moderate, and the soil is light saline soil (table 1).
Table 1 test soil nutrient status
Test fertilizer: the microbial agent produced by Zhejiang Fengya ecological technology and technology Co., ltd.) is composed of Pantoea ananatis and Bacillus pumilus Cytobacillus depressus, and the preparation is granule, and the technical index is that the effective viable count is not less than 2.0 hundred million/g.
Test varieties and cultivation modes: the tomato variety is Provence. Adopts a ridging film-covering and vine-hanging three-dimensional cultivation mode. 2. 6-7 true leaves of seedlings are planted in 7 days of the month. The large row spacing is 60-70 cm, the small row spacing is 40 cm, the plant spacing is 50-55 cm, and 2500 plants are reserved in each mu.
Test design and fertilization method:
and (3) test design: conventional fertilization+base application test granule (T), conventional fertilization+base Shi Miehuo matrix (CK 1), conventional fertilization (CK 2). Repeating for 4 times, the cell area is 30m 2 The protection rows are arranged in random groups, 1 meter are set up, and other management is consistent.
T1: on the basis of conventional fertilization, applying 8kg of granular microbial agent on a soil preparation time base Shi Muji;
CK1: applying 8kg of equivalent inactivated matrix during soil preparation on the basis of conventional fertilization;
CK2: i.e. conventional fertilization. Soil preparation is carried out 15 days before field planting in 1 month and 22 days in 2019, sufficient base fertilizer is applied, 5000kg of fully decomposed farmyard manure, 80kg of triple superphosphate, 35kg of potassium sulfate, deep ploughing for 30 cm, deep watering is carried out, and soil moisture conservation and alkali pressing are carried out. When the first fruit is grown to the large walnut, critical water and additional fertilizer are poured, and 20kg of compound fertilizer is applied to each mu; and watering every 10 days later, and topdressing with water after each fruit is stable.
Experiment site 2: agricultural office greenhouse for sunflower and blojord in inner Mongolia pentayuan county
Test soil: the test land is an 8-meter span greenhouse, the soil is silt-filled soil, the land is flat, the irrigation and drainage are good, the fertility is moderate, and the soil is light saline soil (table 2).
Table 2 tested soil nutrient status
Test fertilizer: the microbial agent produced by Zhejiang Fengya ecological technology and technology Co., ltd.) is composed of Pantoea ananatis and Bacillus pumilus Cytobacillus depressus, and the preparation is granule, and the technical index is that the effective viable count is not less than 2.0 hundred million/g.
Test varieties and cultivation modes: the tomato variety is Provence. Adopts a ridging film-covering and vine-hanging three-dimensional cultivation mode. 2. 6-7 true leaves of seedlings are planted in 5 days of the month. The large row spacing is 60-70 cm, the small row spacing is 40 cm, the plant spacing is 50-55 cm, and 2500 plants are reserved in each mu.
Test design and fertilization method
And (3) test design: conventional fertilization+base application test granule (T), conventional fertilization+base Shi Miehuo matrix (CK 1), conventional fertilization (CK 2), and three treatments. Repeating for 4 times, the cell area is 30m 2 The protection rows are arranged in random groups, 1 meter are set up, and other management is consistent.
T1: on the basis of conventional fertilization, applying 8kg of granular microbial agent on a soil preparation time base Shi Muji;
CK1: applying 8kg of equivalent inactivated matrix during soil preparation on the basis of conventional fertilization;
CK2: i.e. conventional fertilization. Soil preparation is carried out 15 days before field planting in 1 month and 20 days in 2019, sufficient base fertilizer is applied, 5000kg of fully decomposed farmyard manure, 80kg of triple superphosphate, 35kg of potassium sulfate, deep ploughing for 30 cm, deep watering is carried out, and soil moisture conservation and alkali pressing are carried out. When the first fruit is grown to the large walnut, critical water and additional fertilizer are poured, and 20kg of compound fertilizer is applied to each mu; and watering every 10 days later, and topdressing with water after each fruit is stable.
Example 1 selection of species in microbial organic fertilizer
According to the research of the prior art, a large amount of pantoea ananatis has been found to have different degrees of phosphorus dissolving capability, and the inventor tries to obtain various pantoea ananatis strains from different ways when researching microbial organic fertilizer, and the problems of insufficient dissolving performance on indissolvable phosphorus, poor application effect in actual soil and the like generally exist. To address this problem, the inventors tried the phosphorus solubilizing effect of different combinations of species, with the results shown in the following table (more species actually validated, not fully shown for many combinations with antagonistic effects and privacy and continuation-in-service reasons):
TABLE 3 phosphorus solubilizing Effect of Single species and Strain combinations
| Bacterial strain | Calcium phosphate D/D | Hydroxyapatite D/D | Iron phosphate D/D |
| Pantoea ananatis (L.) Var. Azakii | 2.37 | 2.54 | — |
| Aspergillus niger | 2.21 | 1.57 | — |
| Bacillus pumilus | 1.82 | 1.43 | 1.27 |
| Ha Cimu mould | — | 1.76 | — |
| Pantoea ananatis plus Aspergillus niger | 2.26 | 2.62 | 1.21 |
| Pantoea ananatis+bacillus cereus | 4.62 | 4.29 | 2.85 |
| Pantoea ananatis + Ha Cimu mould | 2.56 | 3.09 | 1.43 |
200. Mu.l of bacterial suspension on the plate at the time of combination of the species consisted of 100. Mu.l of each of the two bacterial suspensions.
"-" no soluble phosphate ring or no measurable apparent soluble phosphate ring.
The result shows that the Pantoea ananatis and the Aspergillus niger to be detected have better indissolvable phosphorus dissolving capacity, and Ha Cimu mould and bacillus caviae have obvious defects in indissolvable phosphorus dissolving capacity although showing a certain phosphorus dissolving capacity in screening. Compared with the combination of the pantoea ananatis, the bacillus caving and the pantoea ananatis and the Ha Cimu mould, the single strain obviously improves the phosphorus dissolving capacity of indissolvable phosphorus, in particular to ferric phosphate.
Subsequently, the inventors further verified the phosphate solubilizing effect of the combination of pantoea ananatis + bacillus caviae and pantoea ananatis + Ha Cimu mould in different proportions. The results are shown in Table 4:
TABLE 4 phosphorus dissolution effects of different ratio strain combinations
"-" no soluble phosphate ring or no measurable apparent soluble phosphate ring.
The result shows that the pantoea ananatis and bacillus caviae with the viable count of 2:1 have the best phosphorus dissolving effect.
Example 2 preparation Process of actual microbial agent
The pantoea ananatis and bacillus subtlis (the ratio of the viable count is 2:1, and the effective viable count in the actual fertilizer detection is more than or equal to 2.0 hundred million/g), peptone, potassium dihydrogen phosphate, glucose, diatomite and water are prepared into the granular microbial agent.
The basic production process of the agricultural microbial agent comprises the following steps: according to the formula, the original strain is activated, expanded and cultivated, the fermentation tank is cleaned, air purification and disinfection are carried out, the seed tank, the fermentation tank and raw materials are sterilized, inoculated and fermented, the bacterial liquid after fermentation is detected that the bacterial count and various indexes are qualified, and then the bacterial liquid is put into a storage tank and is input into a spray dryer for drying and pulverizing, and the bacterial powder is conveyed into a granulator to form granulesAnd (5) sampling products with qualified sizes, detecting each index to be qualified, and packaging the products into finished products for warehousing. The mould and mixed bacteria number is less than or equal to 3.0 multiplied by 10 6 The mixed bacteria rate is less than or equal to 30 percent, the fineness is more than or equal to 80 percent, the pH value is 6.5-7.5, and the moisture is less than or equal to 20 percent.
Example 3 detection of effective microbial Release Rate and survival Rate of agricultural microbial agent
Different microbial carrier materials are selected, and the agricultural microbial agents are added according to the same mass ratio, and the following detection method is adopted for experiments. The invention selects common microorganism carrier materials such as talcum powder, diatomite, turf, bagasse and the like.
The method for detecting the release rate of the thalli comprises the following steps: the microbial inoculum is placed in a shade for preservation, 10g of sample is taken after two days, 100mL of physiological saline is added for shaking for 2 hours at 200r/min, and then the released microbial count is immediately determined by a dilution plate counting method. The release rate was calculated using the number of inoculated bacteria compared with the number of released bacteria (the actual number of active bacteria of the carrier at the time of measurement). The results are shown in Table 5:
TABLE 5 microbial Carrier material release rates
The survival rate detection method comprises the following steps: the microbial inoculum placed at room temperature was subjected to shaking at 200r/min for 2 hours by adding 100mL of physiological saline to 10g of samples taken at 2, 10, 20, 40, 80 and 100d, respectively, and the sampling method was the same as above. The number of viable bacteria released at different times was measured by dilution plate counting, and the ratio of the number of viable bacteria to the initial number of viable bacteria was used as the cell survival rate. The results are shown in Table 6:
TABLE 6 bacterial viability of different microbial Carrier materials
As can be seen from the experimental results of the thallus release rate and the survival rate, the effective viable count in the microbial agent with diatomite as the microbial carrier material is higher.
Example 4 results of field experiments
The agricultural microbial agent prepared in example 2 was used in actual field experiments with the same microbial agent of Pantoea ananatis (effective viable count: 2.0 hundred million/g in actual fertilizer detection) as that prepared previously, and the results are shown in the following table:
hangjingjingyangqi):
TABLE 7 Effect of application of particulate microbial inoculants on tomato growth conditions
TABLE 8 Effect of application of particulate microbial inoculants on tomato yield
TABLE 9 analysis of variance table
As shown by experimental investigation results, compared with conventional fertilization, the application of the granular microbial agent on tomatoes has the advantages of thick stems, increased plant fruit number and single fruit weight, reduced split fruits, reduced incidence of navel rot and capability of improving the nutritional growth and reproductive growth conditions of tomatoes.
As can be seen from comparison of test yields, the yield of tomatoes can be remarkably improved by applying the granular microbial agent, the yield increase rate reaches 9.1%, the income increase is 3300 yuan/mu, and the yield increase and efficiency increase are realized by applying the granular microbial agent produced by Zhejiang Fengya ecological technology and technology Co-Ltd.
Five-source county:
table 10 effect of application of particulate microbial inoculant on tomato growth conditions
Table 11 effect of applying particulate microbial inoculant on tomato yield
Table 12 analysis of variance table
As shown by experimental investigation results, compared with conventional fertilization, the application of the granular microbial agent on tomatoes has the advantages of thick stems, increased plant fruit number and single fruit weight, reduced split fruits, reduced incidence of navel rot and capability of improving the nutritional growth and reproductive growth conditions of tomatoes.
As can be seen from comparison of test yields, the tomato yield can be obviously improved by applying the granular microbial agent, the yield increase rate reaches 6.3%, the income increase is 2154 yuan/mu, and the yield increase and efficiency increase are realized by applying the granular microbial agent of Zhejiang Fengya ecological technology Co Ltd.
Claims (6)
1. The agricultural microbial agent is characterized by comprising, by mass, 8% -12% of phosphorus-dissolving microorganisms, 4% -6% of microbial carrier materials, 20% -30% of peptone, 4% -8% of potassium dihydrogen phosphate, 2% -6% of glucose and 50% -60% of water;
the microbial carrier material is diatomite; the phosphorus-dissolving microorganism comprises pantoea ananatis and bacillus caving, and the ratio of the effective viable count of the pantoea ananatis to the bacillus caving is 2:1; pantoea ananatis is ATCC 19321, and bacillus subtlis is 1.15124 of CGMCC.
2. The agricultural microbial agent according to claim 1, wherein the microbial agent comprises 10% of phosphorus-dissolving microorganisms, 5% of microbial carrier materials, 25% of peptone, 6% of potassium dihydrogen phosphate, 4% of glucose and the balance of water according to mass ratio.
3. The agricultural microbial agent according to claim 1, wherein the number of mold impurities in the agricultural microbial agent is not more than 3.0x10 6 The mixed bacteria rate is less than or equal to 30 percent, the fineness is more than or equal to 80 percent, the pH value is 6.5-7.5, and the moisture is less than or equal to 20 percent.
4. A process for preparing an agricultural microbial agent as claimed in claim 1, characterized in that the process comprises the following steps:
1) Performing activation and expansion culture on the original strain;
2) Cleaning the fermentation tank, purifying and sterilizing air, and sterilizing the seed tank, the fermentation tank and raw materials;
3) Inoculating, fermenting, detecting bacterial count and qualified indexes of the bacterial liquid after fermentation, and then pumping the bacterial liquid into a storage tank and inputting the storage tank into a spray dryer for drying and pulverizing;
4) And (3) conveying the bacterial powder into a granulator to form particles, sampling and detecting various indexes of the product, and packaging the product into a finished product and warehousing.
5. Use of an agricultural microbial inoculant according to any one of claims 1-3 in tomato planting.
6. Use of the agricultural microbial inoculant according to any one of claims 1-3 in phosphorus-deficient soil remediation.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN202111313019.8A CN113999837B (en) | 2021-11-08 | 2021-11-08 | Agricultural microbial agent and preparation process thereof |
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