CN113999837A - Agricultural microbial agent and preparation process thereof - Google Patents
Agricultural microbial agent and preparation process thereof Download PDFInfo
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Abstract
An agricultural microbial inoculant is provided comprising a microbial carrier material. The agricultural microbial agent improves the effective viable count of phosphorus-dissolving microorganisms by adding the diatomite, obtains stable and long-term fertilizer efficiency, and obtains good benefits in production and application of crops.
Description
Technical Field
The application belongs to the field of microbial fertilizers, and particularly provides an agricultural microbial agent, a preparation process thereof and application thereof in tomato planting and phosphorus-deficient soil improvement.
Background
According to statistics, the cultivated land with more than 8 crops in China is in phosphorus deficiency with different degrees, phosphate fertilizers are required to be used for realizing the high yield required by modern agriculture, and common crops such as tomatoes, soybeans, sweet potatoes and the like are all high in phosphorus demand. China is the first major country of world phosphate fertilizer consumption, and the annual phosphate fertilizer consumption is over 1000 million tons. What is not suitable for the situation of land is that the phosphorite resource in China is lack, the reserve of the phosphorite resource is kept at 151.98 hundred million tons, the high-quality phosphorite resource is used up in tens of years, and the phosphate fertilizer increasingly becomes the bottleneck 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 into the soil: in calcareous soil the available phosphorus is converted into apatite with extremely low solubility; iron phosphate and aluminum phosphate are formed in acidic soil. While the phosphate fertilizer/phosphate rock is deficient in China, the phosphorus fertilizer used for many years causes the accumulation of phosphorus in soil, the accumulated phosphorus exists in a form of insoluble inorganic phosphorus, is slowly released, and is difficult to utilize by crops, namely, the so-called closed phosphorus or high-stability phosphorus.
The solution of the activation and release of the soil phosphorus is a necessary way to improve the utilization efficiency of the soil phosphorus and the phosphate fertilizer and further realize the sustainable development of agriculture in China. The phosphorus-dissolving microorganism is a beneficial microorganism with the capability of activating the insoluble inorganic phosphorus in the soil, and the application of the phosphorus-dissolving microorganism to activate the insoluble phosphorus in the soil is a world-recognized safe, economic and effective biological measure. At present, most of phosphorus-dissolving bactericides are single-strain bactericides, and the phosphorus-dissolving effect and the applicable soil type are limited.
A large number of researches show that the microbial fertilizer is a good method for protecting the ecological environment and improving the yield and the quality of crops. The microbial fertilizer is a microbial inoculum prepared from one or more beneficial living microorganisms, and the metabolic process or metabolic products of the microbial inoculum are used for improving the growth conditions of plants so as to achieve the effect of promoting the growth of the plants. Therefore, the maintenance of microbial activity is the key to the preparation of microbial fertilizers, and the selection and addition of carrier materials have a key influence on the quality of the microbial fertilizers. Under the condition of the same dosage, the adsorption capacity of different carrier materials to the thalli is different. Meanwhile, due to different characteristics of various microorganisms, the retention periods of the same carrier material for different thalli are also different. Therefore, the determination of the proper microbial carrier material in the microbial inoculum has important significance for improving the microbial activity in the microbial inoculum and prolonging the shelf life of the microbial inoculum.
Disclosure of Invention
The invention aims to solve the problems of few effective viable bacteria, 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, the diatomite is added into the microbial inoculum, so that the effective viable count of phosphorus-dissolving microorganisms is increased, the stable long-term fertilizer efficiency is obtained, and good benefits are obtained in the production and application of crops.
In order to achieve the above object, the present invention provides the following technical solutions:
an agricultural microbial agent, which comprises 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 monopotassium phosphate, 2-6% of glucose and 50-60% of water by 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 in percentage by mass.
Preferably, the number of the mold and the sundry fungus in the agricultural microbial agent is less than or equal to 3.0 multiplied by 106The rate of mixed bacteria 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 water content is less than or equal to 20 percent.
Preferably, the phosphate solubilizing microorganism comprises Pantoea ananatis and Bacillus atrophaeus.
Preferably, the ratio of the effective viable count of the pantoea ananatis to the effective viable count of the bacillus cuprapae 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 the air, and sterilizing the seed tank, the fermentation tank and the raw materials;
3) inoculating, fermenting, detecting bacterial count and various indexes of the fermented bacterial liquid, injecting the bacterial liquid into a storage tank, and inputting the bacterial liquid into a spray dryer for drying and milling;
4) and conveying the bacterial powder into a granulator to form granules, sampling products with qualified sizes, detecting various indexes to be qualified, and packaging the products into finished products to be put in storage.
Further, the application provides the application of the agricultural microbial agent and the preparation process thereof in tomato planting.
Further, the application provides the application of the agricultural microbial agent and the preparation process thereof in the transformation of phosphorus-deficient soil.
The invention has the beneficial effects that: different microbial carrier materials such as common grass peat, diatomite, talcum powder and bagasse are added into the microbial inoculum, and the diatomite is selected from the microbial carrier materials which have activity maintaining effect on phosphorus-dissolving microbes in the microbial inoculum and have good quality guarantee period prolonging effect. The agricultural microbial agent well adsorbs phosphorus-dissolving microorganisms, maintains the activity of the phosphorus-dissolving microorganisms, realizes stable and long-term fertilizer efficiency in application, has a great promotion effect on the production of crops, and obtains good economic benefits by adding and using the diatomite.
Detailed Description
Strains and culture medium:
pantoea ananatis (Pantoea ananatis): the applicant purchased from Chinese academy of agricultural sciences and then self-preserved and passaged, and the initial source is ATCC 19321;
bacillus cuprinus (bacillus depression): the applicant purchases the product from Chinese academy of agricultural sciences and then self-preserves the product and passes the product, and the initial source of the product is 1.15124 of CGMCC;
aspergillus niger (Aspergillus niger): the applicant purchased from Chinese academy of agricultural sciences and then self-preserved and passaged, the initial source being ACCC 32579;
trichoderma harzianum (Trichoderma harzianum): the applicant purchased from the Chinese academy of agricultural sciences and then self-preserved and passaged, the initial source being ACCC 32517.
The Pantoea ananatis and the Bacillus cavendiculatus use respective special culture mediums during passage, and the two bacteria use the same improved beef extract peptone culture medium (potassium dihydrogen phosphate 0.1g, dipotassium hydrogen phosphate 0.1g, ammonium sulfate 2.5g, calcium carbonate 10g, ferrous sulfate 0.005g are added per liter) in the experiment.
And (3) other raw materials:
peptone, glucose: nicotianweidan biotechnology limited;
potassium dihydrogen phosphate: sichuan Shengfeng chemical Co., Ltd;
diatomaceous earth was purchased from a commercially available product.
The main equipment is as follows:
| name of main production equipment | Brand model | Number of | Name of production unit |
| Seed tank | RSB-350 | 1 is provided with | Wenzhou Wanfu machinery manufacturing plant |
| Fermentation tank | GJB-2.0 | 1 is provided with | Wenzhou food machinery, Inc |
| Storage tank | 1000L | 2 are provided with | Qingyun county Credit plastics Co Ltd |
| Spray drier | TPG type | 1 table | JIANGSU XIANFENG DRYING ENGINEERING Co.,Ltd. |
| Spiral elevator | FQ610 | 1 table | HENAN LIWANG MACHINERY Co.,Ltd. |
| Extrusion granulator | WNJ-300-1Q | 1 table | Weifang city agricultural machinery research institute in Shandong province |
| Automatic packaging machine | WHFBZJ-01 | 1 table | SHANGHAI MACWELL PACKAGING MACHINERY Co.,Ltd. |
The main experimental method comprises the following steps:
phosphorus dissolution experiment:
using a hardly soluble phosphorus solid culture medium: glucose 10g, MgSO4·7H2O0.4 g, phosphorus source 5g, (NH4)2SO4 0.5g、NaCl 0.2g、KCl 0.3g、MnSO4 0.03g、FeSO4·7H20.01g of O, 20g of agar, 0.5g of yeast extract and 1000mL of water. The phosphorus source is calcium phosphate, hydroxyapatite and iron phosphate.
Preparing bacterial suspension with similar OD from the bacterial strain to be detected, and uniformly coating 200 mu L of bacterial suspension in a 6cm culture dish. And (3) culturing at 25 ℃ for 1 day, then beating the strain into a bacterial cake by using a 6mm puncher, inoculating the bacterial cake onto the culture medium plate, and measuring the ratio (D/D) of the diameter (D) of a phosphorus-dissolving ring to the diameter (D) of a bacterial colony after culturing at 25 ℃ for 3 days to determine the phosphorus-dissolving capacity of the strain.
Field experiment:
site of experiment 1: two-bridge greenhouse with Hangzhou brocade inner Mongolia
Test soil: the test field is a greenhouse with 8-meter span, the soil is silt irrigation soil, the terrain is flat, the irrigation and drainage is better, the fertility is moderate, and the soil is light saline soil (Table 1).
TABLE 1 nutrient status of the soil tested
Fertilizer to be tested: the microbial agent is produced by Zhejiang Fengyu ecological science and technology Limited company, the strains are Pantoea ananatis (Pantoea ananatis) and Bacillus dentus (Cytobacillus Depressus), the dosage form is particles, and the technical index is that the effective viable count is more than or equal to 2.0 hundred million/g.
Test varieties and cultivation modes: the tomato variety is Provence. Adopts a ridging, film-covering and vine-hanging type three-dimensional cultivation mode. 6-7 true leaves of the seedlings are planted in the period of 2 months and 7 days. The large row spacing is 60-70 cm, the small row spacing is 40 cm, the plant spacing is 50-55 cm, and the mu reserved seedling is 2500 strains.
The test design and fertilization method comprises the following steps:
and (3) experimental design: conventional fertilization + basal application of the test granular inoculant (T), conventional fertilization + basal application of the inactivation matrix (CK1) and conventional fertilization (CK 2). Repeating for 4 times, the cell area is 30m2And random block arrangement, setting up a protection row for 1 meter, and keeping other management consistent.
T1: on the basis of conventional fertilization, 8kg of granular microbial agent is applied to the ground of each mu in land preparation;
CK 1: on the basis of conventional fertilization, 8kg of equivalent inactivation substrate is applied during soil preparation;
CK 2: namely conventional fertilization. Soil preparation is carried out 15 days before field planting in 22 days 1 month in 2019, sufficient base fertilizer is applied, 5000kg of fully decomposed farmyard manure, 80kg of heavy calcium superphosphate and 35kg of potassium sulfate are applied per mu in combination of land turning, deep ploughing is carried out for 30 cm, water is deeply poured, soil moisture is preserved, and alkali is pressed. When the first set of fruits grows to be large, critical water and topdressing are poured, and 20kg of compound fertilizer is topdressed per mu each time; watering every 10 days, and topdressing with water after each set of stable fruit seat.
Site of experiment 2: greenhouse of agriculture bureau of prefecture county sunflower Boyuan of inner Mongolia
Test soil: the test field is a greenhouse with 8-meter span, the soil is silt irrigation soil, the terrain is flat, the irrigation and drainage is better, the fertility is moderate, and the soil is light saline soil (Table 2).
TABLE 2 nutrient status of the soil tested
Fertilizer to be tested: the microbial agent is produced by Zhejiang Fengyu ecological science and technology Limited company, the strains are Pantoea ananatis (Pantoea ananatis) and Bacillus dentus (Cytobacillus Depressus), the dosage form is particles, and the technical index is that the effective viable count is more than or equal to 2.0 hundred million/g.
Test varieties and cultivation modes: the tomato variety is Provence. Adopts a ridging, film-covering and vine-hanging type three-dimensional cultivation mode. 6-7 true leaves of the seedlings are planted in 2 months and 5 days. The large row spacing is 60-70 cm, the small row spacing is 40 cm, the plant spacing is 50-55 cm, and the mu reserved seedling is 2500 strains.
Test design and fertilization method
And (3) experimental design: conventional fertilization + basal application of test granular inoculant (T), conventional fertilization + basal application of inactivation matrix (CK1), conventional fertilization (CK2) and three treatments. Repeating for 4 times, the cell area is 30m2And random block arrangement, setting up a protection row for 1 meter, and keeping other management consistent.
T1: on the basis of conventional fertilization, 8kg of granular microbial agent is applied to the ground of each mu in land preparation;
CK 1: on the basis of conventional fertilization, 8kg of equivalent inactivation substrate is applied during soil preparation;
CK 2: namely conventional fertilization. Soil preparation is carried out 15 days before field planting for 20 days in 1 month and 20 days in 2019, sufficient base fertilizer is applied, 5000kg of fully decomposed farmyard manure, 80kg of heavy calcium superphosphate and 35kg of potassium sulfate are applied per mu in combination of field turning, deep ploughing is carried out for 30 cm, water is deeply poured, soil moisture is preserved, and alkali is pressed. When the first set of fruits grows to be large, critical water and topdressing are poured, and 20kg of compound fertilizer is topdressed per mu each time; watering every 10 days, and topdressing with water after each set of stable fruit seat.
Example 1 selection of species in microbial organic fertilizer
According to the research of the prior art, a large number of pantoea ananatis have been found to have different degrees of phosphorus dissolving capacity, and when the inventor researches a microbial organic fertilizer, the inventor tries to obtain a plurality of pantoea ananatis strains from different ways, which generally has the problems of insufficient dissolving performance of insoluble phosphorus, poor application effect in actual soil and the like. In response to this problem, the inventors tried the phosphate solubilizing effect of different combinations of strains, and the results are shown in the following table (actually verified strains are more, and are not shown fully for many combinations with antagonistic effect and for reasons of confidentiality and continued application):
TABLE 3 phosphorus solubilization Effect of Individual strains and combinations of strains
| Bacterial strain | Calcium phosphate D/D | Hydroxyapatite D/D | Iron phosphate D/D |
| Pantoea ananatis | 2.37 | 2.54 | — |
| Aspergillus niger | 2.21 | 1.57 | — |
| Bacillus cuprinus | 1.82 | 1.43 | 1.27 |
| Trichoderma harzianum | — | 1.76 | — |
| Pantoea ananatis + Aspergillus niger | 2.26 | 2.62 | 1.21 |
| Pantoea ananatis + Bacillus sagittiformis | 4.62 | 4.29 | 2.85 |
| Pantoea ananatis + Trichoderma harzianum | 2.56 | 3.09 | 1.43 |
". bacterial combinations 200. mu.l of bacterial suspension on the plate consisted of 100. mu.l each of the two bacterial suspensions.
"-" has no soluble phosphorus ring or no measurable and obvious soluble phosphorus ring.
The results show that the Pantoea ananatis and Aspergillus niger to be detected both have good insoluble phosphorus dissolving capacity, and Trichoderma harzianum and Bacillus sagittiformis have obvious deficiency in the insoluble phosphorus dissolving capacity although the Trichoderma harzianum and Bacillus sagittiformis show certain phosphorus dissolving performance in screening. Compared with a single strain, the combination of the pantoea ananatis and the bacillus cuprapae and the trichoderma harzianum obviously improves the phosphorus dissolving capacity of insoluble phosphorus, particularly the phosphorus dissolving capacity of iron phosphate.
Subsequently, the inventors further verified the phosphate solubilizing effect of the combination of pantoea ananatis + bacillus caveolus and pantoea ananatis + trichoderma harzianum in different proportions. The results are shown in Table 4:
TABLE 4 phosphorus solubilization Effect of different ratio strain combinations
"-" has no soluble phosphorus ring or no measurable and obvious soluble phosphorus ring.
The result shows that the pantoea ananatis and bacillus caveolus with the viable count of 2:1 have the best phosphorus dissolving effect.
EXAMPLE 2 preparation of actual microbial inoculum
Pantoea ananatis and Bacillus pumilus (viable count ratio is 2:1, effective viable count is more than or equal to 2.0 hundred million/g in actual fertilizer detection) are mixed with peptone, potassium dihydrogen phosphate, glucose, diatomite and water to prepare 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 subjected to activation and expanded culture, the fermentation tank is cleaned and disinfected, the seed tank, the fermentation tank and the raw materials are sterilized, inoculated and fermented, after the bacterial liquid after fermentation is qualified through detecting the bacterial number and various indexes, the bacterial liquid is injected into the storage tank and is input into a spray dryer to be dried and powdered, the bacterial powder is conveyed into a granulator to form particles, after the qualified products are sampled and detected, various indexes are qualified, the particles are packaged into finished products to be put in storage. The number of the mould mixed bacteria is less than or equal to 3.0 multiplied by 106The rate of mixed bacteria 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 water content is less than or equal to 20 percent.
Example 3 effective cell Release Rate and survival Rate detection of agricultural microbial Agents
Different microbial carrier materials are selected, 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, grass carbon, bagasse and the like.
The method for detecting the thallus release rate comprises the following steps: the microbial inoculum is placed in a shade for storage, 10g of sample is taken after two days, 100mL of physiological saline is added into the sample, the sample is oscillated for 2 hours at 200r/min, and then the number of released bacteria is immediately measured by a dilution plate counting method. The number of inoculated bacteria was compared with the number of released bacteria (the actual effective viable count of the carrier at the time of measurement) to calculate the release rate. The results are shown in Table 5:
TABLE 5 bacterial Release rates for different microbial Carrier materials
The survival rate detection method comprises the following steps: the bacterial preparation is placed at room temperature, 10g of samples are respectively taken at 2 days, 10 days, 20 days, 40 days, 80 days and 100 days, 100mL of physiological saline is added, and the samples are shaken for 2 hours at 200r/min, and the sampling method is the same as the above. And (4) measuring the number of the viable bacteria released in different periods by using a dilution plate counting method, and taking the ratio of the number of the viable bacteria to the initial number of the viable bacteria as the survival rate of the thallus. The results are shown in Table 6:
TABLE 6 bacterial survival rates for different microbial carrier materials
The experimental results of the thallus release rate and the survival rate show that the effective viable count of the microbial agent added with the diatomite as the microbial carrier material is higher.
Example 4 field test results
The agricultural microbial agent prepared in example 2 and the previously prepared microbial agent of the Pantoea ananatis (the number of effective viable bacteria in actual fertilizer detection is more than or equal to 2.0 hundred million/g) with similar properties are used for an actual field experiment, and the results are as follows:
hangjin houqi:
TABLE 7 Effect of administration of particulate microbial Agents on tomato growth status
TABLE 8 Effect of administration of particulate microbial Agents on tomato yield
TABLE 9 ANOVA TABLE
According to the test investigation result, compared with the conventional fertilization, the granular microbial agent applied to the tomatoes shows that the tomatoes have thick and strong stems, the number of the plant fruits and the weight of a single fruit are increased, the fruit cracking is reduced, the incidence rate of the navel rot is reduced, and the vegetative growth and reproductive growth conditions of the tomatoes can be improved.
The comparison of the test yield shows that the yield of the tomatoes can be remarkably improved by applying the granular microbial agent, the yield is increased by 9.1 percent, the income is increased by 3300 yuan/mu, and the yield and the efficiency are increased by applying the granular microbial agent produced by Zhejiang Feng yoga ecological science and technology limited company.
Wuyuan county:
TABLE 10 Effect of administration of particulate microbial Agents on tomato growth status
TABLE 11 Effect of administration of particulate microbial Agents on tomato yield
TABLE 12 ANOVA TABLE
According to the test investigation result, compared with the conventional fertilization, the granular microbial agent applied to the tomatoes shows that the tomatoes have thick and strong stems, the number of the plant fruits and the weight of a single fruit are increased, the fruit cracking is reduced, the incidence rate of the navel rot is reduced, and the vegetative growth and reproductive growth conditions of the tomatoes can be improved.
The comparison of the test yield shows that the yield of the tomatoes can be obviously improved by applying the granular microbial agent, the yield is increased by 6.3 percent, the income is increased by 2154 yuan/mu, and the yield and the efficiency are increased by applying the granular microbial agent of Zhejiang Feng yoga ecological science and technology limited company.
Claims (10)
1. An agricultural microbial agent, which is characterized by comprising a microbial carrier material.
2. An agricultural microbial inoculant according to claim 1 wherein the microbial carrier material is diatomaceous earth.
3. The agricultural microbial agent according to claim 1, which 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 by mass ratio.
4. The agricultural microbial inoculant according to claim 3, wherein the inoculant comprises 10% of phosphorus-solubilizing microorganisms, 5% of microbial carrier material, 25% of peptone, 6% of potassium dihydrogen phosphate, 4% of glucose, and the balance of water by mass ratio.
5. The agricultural microbial agent according to claim 3, wherein the number of fungi and fungi in the agricultural microbial agent is less than or equal to 3.0 x 106The rate of mixed bacteria 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 water content is less than or equal to 20 percent.
6. The agricultural microbial inoculant of claim 3, wherein said phosphate solubilizing microorganisms comprise Pantoea ananatis and Bacillus atrophaeus.
7. The agricultural microbial agent according to claim 3, wherein the ratio of the effective viable count of Pantoea ananatis to that of Bacillus sagittiformis is 2: 1.
8. A process for preparing an agricultural microbial inoculant according to claim 1, wherein the process comprises the steps of:
1) performing activation and expansion culture on the original strain;
2) cleaning the fermentation tank, purifying and sterilizing the air, and sterilizing the seed tank, the fermentation tank and the raw materials;
3) inoculating, fermenting, detecting bacterial count and various indexes of the fermented bacterial liquid, injecting the bacterial liquid into a storage tank, and inputting the bacterial liquid into a spray dryer for drying and milling;
4) conveying the fungus powder into a granulator to form granules, sampling the product, detecting each index to be qualified, and packaging the product into a finished product to be put in storage.
9. The agricultural microbial agent and the preparation process thereof according to any one of claims 1 to 8, wherein the agricultural microbial agent is applied to tomato planting.
10. The agricultural microbial agent and the preparation process thereof according to any one of claims 1 to 8, for application in phosphorus-deficient soil improvement.
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| CN114907981A (en) * | 2022-06-06 | 2022-08-16 | 福建农林大学 | Preservation method of Pantoea ananatis solid microbial inoculum |
| WO2023108595A1 (en) * | 2021-12-17 | 2023-06-22 | 浙江丰瑜生态科技股份有限公司 | Microbial organic fertilizer |
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