Special bacterial fertilizer for onobrychis viciaefolia and preparation method thereof

Abstract

The application discloses a special bacterial fertilizer for red bean grass and belongs to the technical field of microorganisms. The special bacterial fertilizer for red bean grass comprises the following components: a bacterial strain Rhizobium yanglingense GAU-00008, a bacterial strain Bacillus sp. CHO87, Bacillus subtilis LSH11 and a carrier. Through the synergistic effect of the three bacterial strains, the special bacterial fertilizer for red bean grass can make the red bean grass roots form sufficient and effective nodules, activate the soil, promote the absorption of other nutrient elements by crops, improve the nutrient structure of plants, regulate and promote the growth of plants and enhance the disease resistance of plants.

Description

TECHNICAL FIELD

[0001] The present application relates to the technical field of microorganisms, in particular to a special bacterial fertilizer for Onobrychis viciaefolia and a preparation method thereof. BACKGROUND

[0002] Onobrychis viciaefolia is a perennial herb of the genus Onobrychis in the family Fabaceae, which is a very important forage and green manure crop. It has high feeding value and can be comparable to alfalfa. It has a certain ornamental value due to its beautiful flowers, and is therefore known as the "Queen of Forage". Onobrychis viciaefolia has a developed root system, a thick main root and abundant lateral roots, and has high efficiency in absorbing and utilizing nutrients and water in the soil. It has very outstanding environmental adaptability and strong resistance to adverse factors such as drought, cold, early frost and deep autumn rainfall.

[0003] Although Onobrychis viciaefolia has very wide adaptability to soil and climate, and its root nodules can provide it with certain nitrogen nutrition, its productivity level varies greatly under different cultivation and management conditions. In the existing technology, the production of Onobrychis viciaefolia at home and abroad relies excessively on chemical fertilizers, which reduces the utilization efficiency of chemical fertilizers, causes ecological pollution problems, and leads to poor safety and high production cost. SUMMARY

[0004] In order to solve the problems in the prior art, the present application provides a special bacterial fertilizer for Onobrychis viciaefolia and a preparation method thereof. The technical solution is as follows:

[0005] On the one hand, a special bacterial fertilizer for Onobrychis viciaefolia is provided, which comprises: a strain of Rhizobium yanglingense GAU-00008, a strain of Bacillus sp. CHO87, Bacillus subtilis LSH11 and a carrier.

[0006] Further, the carrier comprises: charcoal powder, flower soil and humic acid.

[0007] Further, the strain of Rhizobium yanglingense GAU-00008 was preserved in the China Center for Type Culture Collection in 2019, with the preservation number CCTCC NO: M 2019564; the strain of Bacillus sp. CHO87 was preserved in the China Center for Type Culture Collection in 2010, with the preservation number CCTCC NO: M 2010230; and the Bacillus subtilis LSH11 was preserved in the China Center for Type Culture Collection in 2017, with the preservation number CCTCC NO: M 2017180.

[0008] In another aspect, a preparation method of a special bacterial fertilizer for Indigofera genus is provided, and the preparation method comprises the following steps:

[0009] Step (1), preparation of a carrier: uniformly mixing charcoal powder, flower soil and humic acid to obtain the carrier, and then sterilizing the carrier;

[0010] Step (2), selection of strains: selecting the strain Rhizobium yanglingense GAU-00008, the strain Bacillus sp. CHO87 and the strain Bacillus subtilis LSH11.

[0011] Step (3), preparation of a mixed bacterial liquid: liquid culturing the strain Rhizobium yanglingense GAU-00008, the strain Bacillus sp. CHO87 and the strain Bacillus subtilis LSH11, and mixing to obtain the mixed bacterial liquid.

[0012] Step (4), preparation of the special bacterial fertilizer for Indigofera genus: uniformly mixing the mixed bacterial liquid and the sterilized carrier, and then culturing to obtain the special bacterial fertilizer for Indigofera genus.

[0013] Further, the step (1) comprises the following steps: uniformly mixing the charcoal powder, the flower soil and the humic acid according to a mass ratio of 2:1:1 to obtain the carrier, the pH of the uniformly mixed carrier is 7.0±0.2, and then high-pressure steam sterilization (103.4 KPa, 121℃, 30 minutes) or γ-ray sterilization is performed on the uniformly mixed carrier.

[0014] Further, the step (2) comprises the following steps: obtaining 50 strains of plant rhizosphere growth-promoting bacteria from rhizobia and root surface of Indigofera genus in different habitats; screening excellent rhizobia through rhizobium back-inoculation and nitrogenase activity detection, screening excellent phosphorus-dissolving bacteria through the combination of PKO and Menkina medium and molybdenum-antimony anti-colorimetric method, and screening excellent biocontrol strains through plate confrontation experiment with common plant pathogenic fungi such as Sclerotium, Heterobasidion, Fusarium and Septoria; then, the strain Rhizobium yanglingense GAU-00008, the strain Bacillus sp. CHO87 and the strain Bacillus subtilis LSH11 are selected according to the results of the antagonistic experiment between strains.

[0015] Further, the step (3) comprises: culturing the strain Rhizobium yanglingense GAU-00008 in YMA culture medium, culturing the strain Bacillus sp. CHO87 and the Bacillus subtilis LSH11 in LB culture medium respectively, 28℃, 180r / min, culturing for 48-72h respectively, until the content of the living growth-promoting bacteria in the culture solution reaches 1×10 9 cfu / ml, respectively obtaining the culture solution of the above three strains, and then mixing the culture solution of the above three strains in a volume ratio of 2:1:1 to obtain the mixed bacteria solution.

[0016] Further, the step (4) comprises: mixing the mixed bacteria solution and the carrier after sterilization in a volume-mass ratio of 1:4, and then culturing at 28℃ for 1 week.

[0017] The technical scheme provided by the embodiment of the present application has the beneficial effects that: the three strains in the special bacterial fertilizer for Potentilla atufolia synergistically form sufficient and effective root nodules on the roots of Potentilla atufolia, activate the soil, promote the absorption of other nutrient elements by crops, improve the nutrient structure of plants, regulate and promote the growth of plants, and enhance the disease resistance of plants; in addition, the special bacterial fertilizer for Potentilla atufolia can improve the soil structure, increase the organic matter content of the soil, and improve saline-alkali land; furthermore, the special bacterial fertilizer for Potentilla atufolia has the advantages of low cost, safe use, good sustained effect, less consumption of non-renewable energy, high economic benefit, no environmental and food pollution, etc. BRIEF DESCRIPTION OF DRAWINGS

[0018] In order to more clearly illustrate the technical scheme in the embodiments of the present application, the drawings needed in the embodiment description will be briefly introduced. Obviously, the drawings in the following description are only some embodiments of the present application, and other drawings can be obtained by those skilled in the art without creative labor.

[0019] Figure 1 is a flow chart of a method for preparing a special bacterial fertilizer for Potentilla atufolia provided by the embodiment of the present application;

[0020] Figure 2 is a flow chart of another method for preparing a special bacterial fertilizer for Potentilla atufolia provided by the embodiment of the present application. DETAILED DESCRIPTION

[0021] In order to make the purpose, technical scheme and advantages of the present application more clear, the embodiments of the present application will be further described in detail below with reference to the drawings.

[0022] The red bean grass has important value for the development of national economy and the protection of ecological environment. The yield of red bean grass is different due to different regions and growth years. In the arid region with poor water and fertilizer conditions, the average yield of dry grass per mu is 250-500 kg; in the region with better water and heat conditions, the yield of fresh grass per mu is 1400 kg, and the yield of fresh grass per mu in the ditch slope land is 950 kg; in the irrigation area with good water, fertilizer and heat conditions, the yield of fresh grass per mu is 1500 kg in the seeding year, 2500 kg in the second year, and the highest yield is 3500 kg in 2-4 years; in the region with altitude of more than 2600 meters and insufficient heat, the red bean grass can grow to the flowering stage in the seeding year, the average yield of dry grass per mu is 143 kg, the yield per mu in the second year is 248 kg, and the highest yield per mu in the fourth year is 663 kg; the yield of the fifth year and later decreases, and the yield per mu is 433 kg. From the yield composition of 1-4 years, the yield in the seeding year accounts for 9.2%, the yield in the second year accounts for 16%, the yield in the third year accounts for 32.2%, the yield in the fourth year accounts for 42.6%, and the yield increases year by year, and the increase in the third year is the largest. In Hexi, which is cut 4 times a year, the yield composition of multiple harvests in the second year is: the first harvest accounts for 30-35%, the second harvest accounts for 25-30%, the third harvest accounts for 20-25%, and the fourth harvest accounts for 10-15%, and the yield decreases gradually, and the decrease in the fourth harvest is the largest. The general utilization period of red bean grass is 5-7 years, the yield decreases year by year from the fifth year, and the yield gradually declines, and the utilization period can be 8-10 years in good conditions, and the life is 15-20 years. As can be seen from the above, although the red bean grass has very wide adaptability to soil and climate, and the root nodule can provide certain nitrogen nutrition for the red bean grass, but under different cultivation and management conditions, the productivity level is very different, especially the dependence on phosphorus is larger, and phosphorus is the necessary element for the nodule formation of legume forage grass rhizobia, which directly affects the number of root nodules and the nitrogen fixation efficiency. In addition, phosphorus also affects the yield, quality and stress resistance (such as disease resistance) of red bean grass and the absorption of potassium. Therefore, with the expansion of planting area and the improvement of industrialization degree, it is of great significance to improve the phosphorus content and utilization efficiency of grassland for the production of red bean grass. At the same time, in recent years, due to the rapid increase of continuous single planting area, some diseases, especially soil-borne diseases, have caused great harm and potential harm to red bean grass.

[0023] Studies at home and abroad have shown that there are a large number of phosphorus-solubilizing microorganisms (phosphorus-solubilizing bacteria) in the rhizosphere of plants, which can convert phosphorus in the soil that is difficult for plants to directly absorb and utilize into a form that can be absorbed by plants. Some phosphorus-solubilizing bacteria have the characteristics of self-nitrogen fixation and secretion of plant hormones (such as auxin, gibberellin, etc.). Therefore, it is of great practical significance to use biotechnology to develop efficient phosphorus-solubilizing bacteria, rhizobia and other growth-promoting bacteria in the rhizosphere of plants, and to develop and develop composite inoculants (or bacterial manure).

[0024] Example 1

[0025] In order to explain the technical content of the present application, the purposes and effects achieved are described below in conjunction with the embodiments.

[0026] A special bacterial fertilizer for red bean grass, comprising: a strain of Rhizobium yanglingense GAU-00008, a strain of Bacillus sp. CHO87, Bacillus subtilis LSH11 and a carrier.

[0027] It should be noted that Rhizobium is a kind of gram-negative bacteria existing in soil which can infect the roots of legume plants and form root nodules, and almost only forms a symbiotic system with legume plants, the strain of Rhizobium yanglingense has a high nodule formation rate and the nodules formed have a high nitrogenase activity; Bacillus has a high ability to dissolve inorganic phosphorus and organic phosphorus; Bacillus subtilis has strong and extensive biocontrol characteristics for pathogenic fungi of common red bean grass soil-borne diseases.

[0028] Further, the carrier comprises: charcoal powder, flower soil and humic acid.

[0029] It should be noted that charcoal powder can not only store the water required by plant roots, but also improve the air permeability and drainage of soil, providing a good living space for beneficial microorganisms; flower soil has sufficient nutrients for plant absorption, and can also enhance plant photosynthesis, and has strong air permeability, water and fertilizer retention capacity, and no diseases and pests; humic acid is the remains of animals and plants, mainly the remains of plants, which is a kind of organic matter accumulated through microbial decomposition and transformation, and a series of chemical processes, it is a high molecular organic acid composed of aromatic and various functional groups, and has good physiological activity and absorption, complexation, exchange and other functions.

[0030] As can be seen from the above, the use of Rhizobium yanglingense with high nodule formation rate and strong nitrogen fixation ability, combined with Bacillus with strong phosphorus dissolving ability and Bacillus subtilis with outstanding biocontrol ability, reduces the application amount of chemical fertilizers and pesticides, and can prevent the occurrence of red bean grass soil-borne diseases; the carrier prepared by charcoal powder, flower soil and humic acid provides a good living environment for the three strains.

[0031] Further, the strain of Rhizobium yanglingense GAU-00008 was preserved in China Center for Type Culture Collection in 2019, with the preservation number of CCTCC NO: M 2019564; the strain of Bacillus sp. CHO87 was preserved in China Center for Type Culture Collection in 2010, with the preservation number of CCTCC NO: M 2010230; Bacillus subtilis LSH11 was preserved in China Center for Type Culture Collection in 2017, with the preservation number of CCTCC NO: M 2017180.

[0032] Example two

[0033] The application discloses a preparation method of a special bacterial fertilizer for red bean grass. Figure 1 The method comprises the following steps:

[0034] Step (1), preparing a carrier: uniformly mixing charcoal powder, flower soil and humic acid to obtain the carrier, and then sterilizing the carrier.

[0035] Step (2), selecting strains: selecting strains Rhizobium yanglingense GAU-00008, Bacillus sp. CHO87 and Bacillus subtilis LSH11.

[0036] Step (3), preparing a mixed bacterial liquid: liquid culturing the strains Rhizobium yanglingense GAU-00008, Bacillus sp. CHO87 and Bacillus subtilis LSH11, and mixing to obtain the mixed bacterial liquid.

[0037] It should be noted that the determination of the compound strain system after mixing shows that there is no antagonistic reaction among the three strains, and the strains maintain the original growth-promoting characteristics.

[0038] Step (4), preparing the special bacterial fertilizer for red bean grass: uniformly mixing the mixed bacterial liquid and the sterilized carrier, and then culturing to obtain the special bacterial fertilizer for red bean grass.

[0039] Example three

[0040] The application discloses a preparation method of a special bacterial fertilizer for red bean grass. Figure 2 The application discloses a preparation method of a special bacterial fertilizer for red bean grass. Figure 1 The application discloses a preparation method of a special bacterial fertilizer for red bean grass.

[0041] Step (101), uniformly mixing charcoal powder, flower soil and humic acid according to a mass ratio of 2:1:1 to obtain a carrier, and then sterilizing the uniformly mixed carrier by high-pressure steam sterilization (103.4 KPa, 121 DEG C, 30 min) or gamma-ray sterilization, wherein the pH of the uniformly mixed carrier is 7.0±0.2.

[0042] It should be noted that high-pressure steam sterilization, high temperature and high pressure sterilization can kill general bacteria, fungi and other microorganisms, and is the most reliable and most widely used physical sterilization method. Gamma-ray sterilization is a method of destroying the structure of bacteria by using the characteristics of rays to kill bacteria. In the embodiments of the present application, high-pressure steam sterilization (103.4 KPa, 121℃, 30 minutes) or gamma-ray sterilization of the carrier is taken as an example for illustration, and other methods can also be used for sterilization in actual implementation.

[0043] Step (102): 50 strains of plant growth promoting rhizobacteria were obtained from rhizobium and root surface of different habitats.

[0044] Plant growth promoting rhizobacteria (PGPR) refers to bacteria living around, on and in the roots of plants, which secrete special metabolites to promote the growth of plants during their life cycle. Studies have shown that such bacteria generally have the ability of nitrogen fixation, phosphorus solubilization, plant hormone secretion and antibiotic production. In addition, PGPR strains can also improve soil structure, increase soil organic matter content, improve saline-alkali land and repair heavy metal contaminated land.

[0045] Step (103): Excellent rhizobium was screened by rhizobium back inoculation and nitrogenase activity detection, and excellent phosphorus-solubilizing bacteria were screened by using PKO combined with Menkina medium and molybdenum-antimony anti-colorimetric method. Excellent biocontrol strains were screened by plate confrontation experiment with common plant pathogenic fungi such as sclerotium, filamentous fungus, fusarium and alternaria.

[0046] It should be noted that nitrogenase is an enzyme that can reduce molecular nitrogen to ammonia. Nitrogenase is composed of two proteins: one contains iron, called ferritin, and the other contains iron and molybdenum, called molybdenum-iron protein. Only when molybdenum-iron protein and ferritin exist at the same time, nitrogenase has the function of nitrogen fixation (because these two substances can act as electron carriers to transfer electrons). The principle of biological nitrogen fixation: biological nitrogen fixation is a special physiological function of nitrogen-fixing microorganisms, which is carried out under the catalysis of nitrogenase; molybdenum-antimony anti-colorimetric method is a process of quantitative analysis of phosphorus in soil, water and other samples.

[0047] Step (104): Strains Rhizobium yanglingense GAU-00008, strain Bacillus sp. CHO87 and Bacillus subtilis LSH11 were selected in combination with the results of antagonistic experiment between strains.

[0048] It should be noted that the excellent rhizobium screened in step (103) is the strain Rhizobium yanglingense; since Bacillus has a high ability of dissolving inorganic phosphorus and organic phosphorus, the excellent phosphorus-dissolving bacteria screened in step (103) is the strain Bacillus; since Bacillus subtilis has a strong and extensive biocontrol characteristics for the pathogenic fungi of common Atkinsonia filipes soil-borne diseases, the excellent biocontrol strain screened in step (103) is Bacillus subtilis. Thus, one strain of Rhizobium yanglingense GAU-00008, one strain of Bacillus sp. CHO87 and one strain of Bacillus subtilis LSH11 can be selected from the excellent rhizobium, the excellent phosphorus-dissolving bacteria and the excellent biocontrol strain screened in step (103) according to the results of the antagonistic experiment between the strains.

[0049] Step (105): the strain Rhizobium yanglingense GAU-00008 is inoculated into YMA culture medium for culture, and the strains Bacillus sp. CHO87 and Bacillus subtilis LSH11 are respectively inoculated into LB culture medium for culture, 28°C, 180r / min, respectively for 48-72h, until the content of the living growth-promoting bacteria in the culture solution reaches 1×10 9 cfu / ml, and the culture solutions of the above three strains are obtained.

[0050] Step (106): the culture solutions of the above three strains are mixed in a volume ratio of 2:1:1 to obtain a mixed bacteria solution.

[0051] Step (107): the mixed bacteria solution and the carrier subjected to sterilization are mixed in a volume-mass ratio of 1:4, and then 28°C static culture is carried out for 1 week.

[0052] It should be noted that the special bacteria fertilizer for Atkinsonia filipes is stored at room temperature, and the effective period of the bacteria agent is more than 6 months.

[0053] Example Four

[0054] A field test of a special bacterial fertilizer for red grass (the test site is Jingyuan County, Gansu Province), before sowing, the red grass seeds are placed on the plastic cloth, and a proper amount of sterile water is sprayed on the seeds to make the seeds wet, then the special bacterial fertilizer for red grass is scattered on the seeds, and stirred while scattering until evenly mixed, the usage amount is 2.0 kg per mu, and after drying, it can be sown, at the same time, 30%-40% of chemical fertilizer is reduced, this group is the test group; in addition, the same amount of red grass seeds without special bacterial fertilizer for red grass are sown, as the control group, for reference, see Table 1 below:

[0055] Table 1: Comparison of growth and quality of red grass field test

[0056]

[0057] The results in Table 1 show that the dry grass yield of red grass treated by using the special bacterial fertilizer for red grass is increased by 10.5% compared with the control; the crude protein and crude fat of red grass are increased by 16.5% and 11.8% respectively compared with the control; and the relative feeding value is increased by 7.6%, and the root rot incidence is reduced by 8.1%, it can be seen that the use of the special bacterial fertilizer for red grass can regulate and promote plant growth, and can enhance plant disease resistance.

[0058] It is worth noting that the three strains in the special bacterial fertilizer for red grass can work together to form a sufficient amount of effective root nodules on the roots of red grass, activate the soil, promote the absorption of other nutrients by crops, improve the nutritional structure of plants, regulate and promote plant growth, and enhance plant disease resistance; secondly, it can also improve the soil structure, increase the soil organic matter content, and improve saline-alkali land; in addition, the special bacterial fertilizer for red grass has the advantages of low cost, safe use, good sustained effect, less non-renewable energy consumption, high economic benefit, no environmental and food pollution, etc.

[0059] The above-mentioned serial numbers of the embodiments of the present application are only for description, and do not represent the advantages and disadvantages of the embodiments.

[0060] The above-mentioned serial numbers of the embodiments of the present application are only for description, and do not represent the advantages and disadvantages of the embodiments. The above-mentioned serial numbers of the embodiments of the present application are only for description, and do not represent the advantages and disadvantages of the embodiments.

Claims

1. A special bacterial manure for red clover, characterized in that, The bacterial fertilizer comprises Rhizobium yanglingense GAU-00008, Bacillus sp. CHO87, Bacillus subtilis LSH11 and a carrier. The Rhizobium yanglingense GAU-00008 is preserved in the China Center for Type Culture Collection in 2019, and the preservation number is CCTCC NO: M 2019564; the Bacillus sp. CHO87 is preserved in the China Center for Type Culture Collection in 2010, and the preservation number is CCTCC NO: M 2010230; and the Bacillus subtilis LSH11 is preserved in the China Center for Type Culture Collection in 2017, and the preservation number is CCTCC NO: M 2017180. The preparation method of the special bacterial fertilizer for red clover comprises the following steps: Step (1), preparing a carrier: uniformly mixing charcoal powder, flower soil and humic acid to obtain the carrier, and then sterilizing the carrier; Step (2), selecting strains: selecting the Rhizobium yanglingense GAU-00008, the Bacillus sp. CHO87 and the Bacillus subtilis LSH11; Step (3), preparing a mixed bacterial liquid: liquid culturing the Rhizobium yanglingense GAU-00008, the Bacillus sp. CHO87 and the Bacillus subtilis LSH11, and mixing to obtain the mixed bacterial liquid; Step (4), preparing the special bacterial fertilizer for red clover: uniformly mixing the mixed bacterial liquid and the sterilized carrier, and then culturing to obtain the special bacterial fertilizer for red clover; The step (2) comprises the following steps: obtaining 50 plant rhizosphere growth-promoting bacteria from rhizobium and root surface of red clover in different habitats; screening excellent rhizobium through rhizobium back-inoculation and nitrogenase activity detection, screening excellent phosphorus-dissolving bacteria through the combination of PKO and Menkina medium and molybdenum-antimony anti-colorimetric method, and screening excellent biocontrol strains through plate confrontation experiment with common plant pathogenic fungi such as sclerotium, rhizoctonia, fusarium and alternaria; then, the Rhizobium yanglingense GAU-00008, the Bacillus sp. CHO87 and the Bacillus subtilis LSH11 are selected according to the results of the antagonistic experiment between strains.

2. The bacterial manure of claim 1, wherein the bacterial manure is characterized by, The carrier comprises charcoal powder, flower soil and humic acid.

3. The method for preparing the special bacterial manure for Chamaecistus acutifolius of claim 1, characterized in that, The preparation method comprises the following steps: Step (1), preparing a carrier: uniformly mixing charcoal powder, flower soil and humic acid to obtain the carrier, and then sterilizing the carrier; Step (2), selecting strains: selecting the strain Rhizobium yanglingense GAU-00008, the strain Bacillus sp. CHO87 and the Bacillus subtilis LSH11; Step (3), preparing a mixed bacterial liquid: liquid culturing the strain Rhizobium yanglingense GAU-00008, the strain Bacillus sp. CHO87 and the Bacillus subtilis LSH11, and mixing to obtain the mixed bacterial liquid; Step (4), preparing a special bacterial fertilizer for Onobrychis viciaefolia: uniformly mixing the mixed bacterial liquid and the sterilized carrier, and then culturing to obtain the special bacterial fertilizer for Onobrychis viciaefolia.

4. The method of claim 3, wherein, The step (1) comprises the following steps: uniformly mixing the charcoal powder, the flower soil and the humic acid according to a mass ratio of 2:1:1 to obtain the carrier, the pH of the uniformly mixed carrier is 7.0±0.2, and then the uniformly mixed carrier is sterilized by high-pressure steam sterilization at 103.4 KPa and 121 DEG C for 30 minutes or gamma-ray sterilization.

5. The method of claim 3, wherein, The step (3) comprises: inoculating the strain Rhizobium yanglingense GAU-00008 into YMA culture medium for culture, inoculating the strain Bacillus sp. CHO87 and the Bacillus subtilis LSH11 into LB culture medium respectively for culture, and culturing at 28 DEG C and 180 r / min for 48-72 h respectively, so as to obtain culture solutions of the above three strains respectively, and then mixing the culture solutions of the above three strains in a volume ratio of 2:1:1 to obtain the mixed bacterial solution. 9 The content of the viable growth-promoting bacteria in the culture solution reaches 1x10 9 6. The method of claim 3, wherein, The step (4) comprises the following steps: uniformly mixing the mixed bacterial liquid and the sterilized carrier according to a volume-mass ratio of 1:4, and then culturing at 28 DEG C for 1 week.

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