A method for preparing tetramycin fertilizer by using non-hygroscopic streptomyces and its application

Abstract

The invention discloses a method for preparing tetramycin fertilizer by using water-intolerant streptomyces and its application. By expanding and culturing the water-intolerant streptomyces Wuzhou subspecies, a solid fermentation product (i.e., tetramycin fertilizer) containing 0.01wt% to 0.5wt% tetramycin is obtained by fermentation culture on a solid fermentation medium, and then the obtained tetramycin fertilizer is mixed with fertilizers with different functions to prepare fertilizers with different functions. Compared with the prior art, the tetramycin fertilizer prepared by the invention has the advantages of good control effect on plant fungi, no large amount of fermentation waste liquid is generated during the fermentation process, and the bacterial yield is high. The obtained tetramycin fertilizer can also be mixed with alginic acid, humic acid, amino acids, trace elements, medium elements, macroelements, and adhesives to prepare fertilizers with different functions, which can effectively prevent and control soil-borne diseases of crops, improve the stress resistance of crops, and promote crop growth.

Description

Technical Field

[0001] The invention relates to the technical field of biological pesticide formulations, and in particular to a method for preparing tetramycin fertilizer by using non-hygroscopic streptomyces and application thereof. Background Art

[0002] Plant diseases and insect pests not only affect crop yields, but also reduce crop quality. In order to prevent and control diseases and insect pests and maintain the normal production of agricultural products, people have long used a large number of chemical pesticides to prevent and control diseases and insect pests. Although the use of chemical pesticides can ensure the normal production of crops and play an effective role, due to the high toxicity and difficult degradation of some chemical pesticides themselves and the inevitable production of many low environmental friendliness by-products in the process of synthesizing chemical pesticides, it will cause serious damage to the ecological environment in the long run, such as soil structure destruction, reduced enzyme activity in the soil, serious pollution of soil and water bodies, etc., and the toxic pesticide residues in almost all foods such as grains, vegetables, and fruits are seriously exceeded. There are also incidents of human and animal poisoning caused by eating fruits and vegetables with seriously exceeded chemical pesticide residues. Microbial pesticides are the most important biological pesticides. They are pesticides that use microorganisms themselves or their metabolites to prevent and control plant diseases, insects, weeds, etc. Among them, the metabolites to prevent and control plant diseases, insects, and weeds are the development and utilization of various agricultural antibiotics. Agricultural antibiotics are secondary metabolites produced by microorganisms such as bacteria, fungi and actinomycetes during the fermentation process. At low concentrations, they selectively inhibit or affect the growth and development of crop pathogens, pests, mites and weeds, and are relatively safe for humans, livestock and beneficial organisms.

[0003] Tetramycin is an antibiotic produced by the metabolism and secretion of Streptomyces wuzhouensis, which has a strong killing effect on 26 known pathogenic fungi in three major categories, including flagellates, ascomycetes and Ascomycetes, and is suitable for the prevention and treatment of various fungal diseases of various crops. Tetramycin is mostly used for the prevention and treatment of fungal diseases of vegetables and fruits, and is currently recognized as the best agent for preventing apple rot. However, tetramycin has a short shelf life and unsatisfactory biological efficacy, which limits its use and efficacy. It can be seen that in the preparation process of tetramycin, it is very important to improve the efficacy of tetramycin.

[0004] The invention patent with the publication number CN101633941B discloses a method for preparing a biological pesticide, oxadinomycin, from hygroscopic Streptomyces wuzhouensis and the use of the obtained product in the prevention and treatment of diseases such as apple leaf spot disease. The preparation method is to first prepare a seed fermentation culture medium, the seed fermentation culture medium contains soybean cake powder, starch, glucose, peptone, magnesium sulfate, ammonium sulfate, dipotassium hydrogen phosphate, calcium carbonate, sodium chloride, and vegetable oil, inoculate the hygroscopic Streptomyces wuzhouensis strain in the seed fermentation culture medium, and cultivate to obtain a seed fermentation liquid; then prepare a submerged fermentation medium, the submerged fermentation medium contains glucose, starch, cornmeal, soybean cake powder, peptone (or corn steep liquor), ammonium sulfate, magnesium sulfate, dipotassium hydrogen phosphate, calcium carbonate, sodium chloride, vegetable oil, and n-propanol, measure the seed fermentation liquid, and submerge it in the submerged fermentation medium for 64 to 72 hours to obtain oxadinomycin fermentation liquid. After adding 0.3-0.5% of n-propanol during the preparation process, the biological potency of the fermentation liquid increased by 15.5%. The tetromycin not only increases the effect of preventing and treating apple rot to more than 95%, but also has a good prevention and treatment effect on diseases such as apple leaf spot and tomato gray mold; tetromycin has an inhibitory effect on yeast, Gram-negative and Gram-positive bacteria, and fungi (including pathogenic fungi), has a broad spectrum, and is an environmentally friendly biological pesticide. However, a large amount of waste liquid is generated during the fermentation process of the tetromycin (tetramycin) prepared by this method.

[0005] The invention patent with publication number CN114107097A discloses a solid fermentation process of active spores of Streptomyces avermannii, comprising the following steps: Step 1, preparation of strains of Streptomyces avermannii: inoculating frozen and preserved strains of Streptomyces avermannii on eggplant bottle slant culture medium, culturing at 28-30 degrees Celsius for 7 days, and setting aside; Step 2, solid fermentation culture: Step 2-1, preparing solid fermentation culture medium and sterilizing it; Step 2-2, taking the eggplant bottle slant strains prepared in step 1, and making a strain suspension with sterile water ; Step 2-3, inoculate the bacterial suspension evenly in the solid fermentation medium prepared in step 2-1; Step 2-4, carry out solid fermentation culture in a horizontal solid bioreactor for 9 to 11 days, and stop fermentation when the number of live spores per gram of material is greater than or equal to 5 billion; Step 3, preparation of live spore powder: move the solid culture after the fermentation in step 2 into a double cone rotary blower dryer and dry it until the moisture content of the material is less than or equal to 10%, and then crush it into 60 to 80 mesh powder to obtain Averman live spore powder. The culture conditions of solid fermentation culture are: sterile air ventilation volume 1: 0.2 to 0.5 v / v / min (sterile air ventilation volume refers to the volume ratio of the culture medium to the sterile air introduced per minute); reactor pressure: 0.005 to 0.01 MPa, culture temperature 28 to 30 degrees Celsius, and culture humidity 50 to 60%. The solid fermentation medium includes the following raw materials in mass percentage: 60-70% of rice bran, 12-15% of bran, 3-5% of bean cake, 3-5% of peanut cake powder, 8-10% of corn flour, 0.5% of calcium carbonate, 0.5% of ammonium sulfate, 0.1-0.2% of hematuria, and natural pH, ensuring that the total percentage of each raw material is 100%; then water is added to adjust the humidity to 50-60%. The active spores of Streptomyces avermectin are prepared by fermentation of solid medium. The preparation method is green, efficient, and low in cost. In the preparation process, highly polluting reagents such as organic solvents are not used, which is safe and environmentally friendly, and there is no problem of waste liquid discharge, thereby avoiding a series of environmental hazards caused by the avermectin industry. During the solid fermentation process, soybean cake powder, peanut cake powder, corn flour and hematuria are added as carbon and nitrogen sources, supplemented with ammonium sulfate as a quick carbon source, and rice bran and bran are added to adjust the water content. In addition, the addition of hematuria in the culture medium also provides some trace elements for the growth and reproduction of Streptomyces avermannii. The reasonable combination of various culture medium components and their combined effect promote the growth and reproduction of Streptomyces avermannii and the production of live spores, but the bacterial production is not significant, and the effective period of direct use for plant antibacterial control is short. Summary of the invention

[0006] In view of the problems in the prior art that liquid culture medium uses organic solvents to produce a large amount of waste liquid and the efficacy of pesticide fertilizer is poor, the present invention inoculates non-hygroscopic Streptomyces on a solid culture medium for fermentation to obtain a fertilizer with good prevention and control effect, without a large amount of fermentation waste liquid, which can be directly made into tetramycin pesticide fertilizer.

[0007] The present invention provides a method for preparing tetramycin fertilizer by using non-hygroscopic streptomyces, and the method steps are as follows, and the parts are all parts by weight:

[0008] Step 1, seed expansion culture: mix the slant culture medium materials, pour them into the slant culture container, sterilize them with high-temperature steam, and obtain the slant culture medium after cooling; inoculate the refrigerated non-hygroscopic Streptomyces wuzhouensis subspecies on the slant culture medium, culture them at a temperature of 25 to 30° C. for 4 to 10 days, and set aside;

[0009] Step 2, preparing solid fermentation medium: 45-55 parts of carbon source, 35-40 parts of nitrogen source, 1-2 parts of inorganic salt, 20-40 parts of water are mixed evenly, pH is adjusted to 6.5-7.5, placed in a canning bottle, sterilized by high-temperature steam, cooled to 45-60° C., 2-5 parts of auxiliary agent are added, mixed evenly, cooled to 25-30° C., and solid fermentation medium is obtained for standby use;

[0010] Step 3, under sterile conditions, use an inoculation loop to scrape 5 to 10 loops of the expanded culture of the bacteria in step 1, disperse 2 to 10 mL into sterile water, determine the concentration by hemocytometer, and dilute to prepare a bacterial suspension with a concentration of 13 to 16 million / L;

[0011] Step 4: evenly inoculate the bacterial suspension prepared in step 3 on the solid fermentation medium prepared in step 2 to carry out solid fermentation culture to obtain a solid fermentation product containing 0.01wt% to 0.5wt% tetramycin, i.e., tetramycin fertilizer.

[0012] Preferably, the slant culture medium comprises the following raw materials in weight percentage: 0.4% to 0.7% peptone, 0.4% to 0.6% pea extract powder, 0.1% to 0.5% cassava residue, 1% to 2% agar, 0.02% to 0.05% calcium carbonate, 0.04% to 0.07% potassium dihydrogen phosphate, and the balance is water, and the pH is adjusted to 6.5 to 7.5.

[0013] Preferably, the carbon source is 10-30 parts of rice flour mixed with 10-30 parts by weight of cassava residue; the nitrogen source is 20-30 parts by weight of soybean meal powder mixed with 10-20 parts by weight of beef extract powder; the inorganic salt includes the following components in mass percentage: 24%-30% sodium chloride, 25%-30% dipotassium hydrogen phosphate, 33%-36% magnesium sulfate heptahydrate, 5%-8% calcium chloride, and 0.5%-1% ferrous sulfate heptahydrate.

[0014] Preferably, the preparation method of the auxiliary agent is as follows, and the parts are all parts by weight: 3 to 9 parts of acrylamide, 2 to 6 parts of potassium acrylate, and 1 to 2 parts of calcium bentonite are added to 100 to 300 parts of water, mixed evenly, 0.05 to 0.08 parts of initiator solution are added, reacted at 25 to 32° C. for 25 to 35 minutes under a nitrogen atmosphere, and then heated to 48 to 53° C., and continued to react under a nitrogen atmosphere for 6 to 8 hours, added anhydrous ethanol to precipitate and washed 2 to 4 times, vacuum dried at 45 to 55° C. for 6 to 8 hours, ground, and ultraviolet sterilized for 30 to 60 minutes to obtain an auxiliary agent.

[0015] More preferably, the initiating solution is a mixed aqueous solution obtained by dissolving 2 to 4 parts of urea and 9 to 18 parts of potassium persulfate in 220 to 450 parts of water.

[0016] Preferably, the high temperature steam sterilization in step 1 and step 2 has a sterilization temperature of 115 to 121° C. and a sterilization time of 20 to 40 minutes.

[0017] Preferably, the bacterial inoculation amount in step 4 is 5% to 15% mL / g.

[0018] Preferably, the solid fermentation culture conditions in step 4 are: fermentation environment temperature is 25-30° C., fermentation culture time is 3-5 days, and fermentation humidity is 45%-80%.

[0019] The invention also discloses the application of the method for preparing tetramycin medicinal fertilizer by adopting non-water-absorbing streptomyces in preparing functional medicinal fertilizer.

[0020] The functional fertilizer can be a solid granule or a powder. The preparation method of the functional fertilizer in the form of a solid granule is as follows:

[0021] (1) drying the tetramycin fertilizer by rotary blast drying until the moisture content of the material is less than or equal to 10%, crushing, passing through a 100-200 mesh sieve, and obtaining a tetramycin fertilizer dry basis for standby use;

[0022] (2) mixing a binder with water, stirring at 300 to 500 rpm for 10 to 40 min at 40 to 60° C. to obtain a viscous substance; mixing alginic acid, humic acid, amino acids, trace elements, secondary elements, macroelements, and the tetramycin fertilizer dry basis prepared in step (1) uniformly, adding the mixture to the viscous substance, stirring uniformly, extruding and forming, granulating, and drying at 45 to 60° C. for 10 to 16 hours to obtain a solid granular functional fertilizer.

[0023] More preferably, the binder is one or more of pregelatinized starch, dextrin, gum arabic, and sodium alginate.

[0024] Further preferably, the trace element is at least one of copper fertilizer, iron fertilizer, manganese fertilizer, zinc fertilizer, boron fertilizer and molybdenum fertilizer; the mass proportion of a single element in the solid granular fertilizer is: 0.05%≤molybdenum content≤0.5%, and the content of other single elements is ≥0.05%.

[0025] Further preferably, the secondary element is at least one of calcium fertilizer and magnesium fertilizer; the mass proportion of the single element in the solid functional medicinal fertilizer: the content of the single element is ≥0.1%.

[0026] Further preferably, the macro-elements are two or more of nitrogen fertilizer, phosphorus fertilizer and potassium fertilizer, and the mass proportion of the single elements in the functional fertilizer is: the content of the single elements is ≥2%.

[0027] The present invention is to carry out fermentation culture by uniformly inoculating the water-intolerant streptomyces wuzhouensis on a solid fermentation medium composed of a carbon source, a nitrogen source, an inorganic salt and an auxiliary agent after expansion culture, so as to obtain tetramycin fertilizer, which has a significant control effect on plant fungal diseases, and can also be mixed with alginic acid, humic acid, amino acids, trace elements, medium elements, macroelements and adhesives to prepare solid fertilizer. The carbon sources of the solid fermentation medium of the water-intolerant streptomyces wuzhouensis in the present invention are rice flour and cassava residues. Rice flour and cassava residues contain a large amount of carbon elements, and rice flour also contains protein, vitamins and minerals, which can provide trace elements for microbial growth. Cassava residue can maintain the water content in the solid fermentation medium, and some of the starch substances therein can also provide energy for the water-intolerant Streptomyces. At the same time, during the fermentation process, the fermented cassava residue can also promote the growth and metabolism of the water-intolerant Streptomyces Wuzhou subspecies and increase the yield of tetramycin; the nitrogen source of the solid fermentation medium is composed of soybean meal powder and beef extract powder, which provides an organic nitrogen source for the growth of the water-intolerant Streptomyces Wuzhou subspecies; the inorganic salt is composed of sodium chloride, dipotassium hydrogen phosphate, magnesium sulfate heptahydrate, calcium chloride, ferrous sulfate heptahydrate, and cobalt chloride hexahydrate, which can promote the growth and metabolism of the water-intolerant Streptomyces Wuzhou subspecies; the additive added to the solid fermentation medium is a copolymerization reaction between acrylamide and potassium acrylate. The calcium-based bentonite is added to the copolymer chain, the calcium-based bentonite enters the copolymer chain, increases the fluidity of the copolymer, makes it dispersed more evenly in the solid fermentation medium, and can promote the full mixing of the solid fermentation medium materials. At the same time, the copolymer and the calcium-based bentonite can also maintain the humidity of the solid fermentation medium due to their own water absorption and water retention properties. When the internal temperature of the solid fermentation medium is too high due to fermentation, it can also reduce the situation of the internal temperature of the solid fermentation medium being too high during the fermentation process due to its water retention effect, and provide a positive environment for the growth and metabolism of the non-water-absorbing Streptomyces wuzhou subspecies. The trace elements contained in the copolymer and the calcium-based bentonite, such as potassium, calcium, sodium, magnesium, etc., can promote the growth and metabolism of the non-water-absorbing Streptomyces wuzhou subspecies. The solid fermentation medium is mixed with humic acid, amino acids, trace elements, medium elements, and macroelements, and a viscous agent is added. The functional fertilizer is obtained by extrusion molding and granulation, which can prevent and control soil-borne diseases, improve the stress resistance of crops, and promote crop growth.

[0028] Due to the adoption of the above technical scheme, the advantages of the present invention are: 1) rice flour and cassava residue are used as carbon sources, which has the effect of maintaining the water content of the solid fermentation medium, not only improving carbon energy, but also providing other trace nutrients for the fermentation of the non-water-absorbing Streptomyces wuzhou subspecies. 2) adding auxiliary agents can make the solid fermentation medium nutrient material mixing more uniform, and provide additional trace elements for the growth of non-water-absorbing Streptomyces wuzhou subspecies, and can also maintain the humidity and temperature balance of the solid fermentation medium, promote growth metabolism, and provide sufficient space for the growth of bacteria, which can improve the prevention and control effect of solid fermentation products on plant infection fungi. 3) the nutrients and auxiliary agents of the culture medium assist each other, work together, promote the growth and metabolism of microorganisms, produce a large amount of tetramycin fertilizer, and improve the prevention and control effect of plant diseases. 4) the solid fermentation medium can be mixed with humic acid, amino acids, trace elements, medium elements, and macroelements to prepare functional fertilizers, which can prevent and control soil-borne diseases, have the effect of improving crop resistance and promoting crop growth. DETAILED DESCRIPTION

[0029] The sources of drugs used in the embodiments and comparative examples are:

[0030] Pea extract powder: Shanghai Yansheng Biochemical Reagent Co., Ltd., model: YS-PYJ0824.

[0031] Cassava residue: Jiangmen Pengjiang Zhongda Textile Co., Ltd., item number: ZD11.

[0032] Soybean paste powder: Shandong Futian Zhengda Biotechnology Co., Ltd., product number: FTZDD-0030.

[0033] Beef extract powder: Jinan Jinyuyuan Biotechnology Co., Ltd., item number: 20181008.

[0034] Calcium-based bentonite: Shijiazhuang Linyi Mineral Products Co., Ltd., particle size: 325 mesh, product number: BX31.

[0035] Non-hygroscopic Streptomyces wuzhouensis subspecies: CGMCC collection number: 4.7082.

[0036] Pregelatinized starch: Jining Huakai Resin Co., Ltd., viscosity: greater than 300mpa·s.

[0037] Superphosphate: Yunnan Mingcheng Fertilizer Co., Ltd., effective phosphorus (P2O5) content: 12-18wt%.

[0038] Example 1

[0039] The method steps for preparing solid granular functional fertilizer are as follows:

[0040] (1) 250 kg of tetramycin fertilizer obtained in Example 4 was dried by rotary blast drying until the moisture content of the material was less than or equal to 10%, then crushed and passed through a 150-mesh sieve to obtain a tetramycin fertilizer dry basis for standby use;

[0041] (2) adding 4 kg of pregelatinized starch to 60 kg of water, stirring at 400 rpm for 25 min at 50° C. to obtain a viscous substance; mixing 23.54 kg of alginic acid, 36 kg of humic acid, 47.19 kg of amino acids, 0.38 kg of molybdic acid, 0.59 kg of copper sulfate, 0.84 kg of ferric sulfate, 0.65 kg of manganese sulfate, 2.08 kg of borax, 9.66 kg of calcium nitrate, 11.69 kg of magnesium sulfate, 20.25 kg of urea, 59 kg of superphosphate, 24.17 kg of potassium bicarbonate, and 160 kg of the tetramycin fertilizer dry basis prepared in step (1) uniformly, adding the mixture to the viscous substance, stirring uniformly, extruding, granulating, and drying at 50° C. for 12 hours to obtain a functional fertilizer of solid particles.

[0042] Application of solid particle functional fertilizer:

[0043] Target of prevention and control: Wheat soil-borne mosaic disease

[0044] Field test: 4 wheat fields with similar soil were selected in Anhui rural area, 2 of which had serious soil-borne mosaic disease, as experimental group 1-1 and control group 1-1; the other 2 wheat fields had no soil-borne mosaic disease, as experimental group 1-2 and control group 1-2; the test area of ​​each wheat field was 0.5 mu. The functional fertilizer prepared by the solid particles of the present invention was applied respectively, and the application amount was 100 kg / mu.

[0045] Field test effect: compared with the control group 1-1, the soil-borne mosaic disease of wheat in the experimental group 1-1 was significantly reduced, and the wheat grew more vigorously; compared with the control group 1-2, the wheat in the experimental group 1-2 grew faster, and the wheat ears were larger and had more grains; applying the functional pesticide fertilizer prepared by the solid particles of the present invention can effectively prevent and control soil-borne diseases, improve the stress resistance of crops, and promote the growth of crops.

[0046] Example 2

[0047] The method steps for preparing solid granular functional fertilizer are as follows:

[0048] (1) 250 kg of tetramycin fertilizer obtained in Example 4 was dried by rotary blast drying until the moisture content of the material was less than or equal to 10%, then crushed and passed through a 150-mesh sieve to obtain a tetramycin fertilizer dry basis for standby use;

[0049] (2) adding 4 kg of pregelatinized starch to 60 kg of water, stirring at 400 rpm for 25 min at 50° C. to obtain a viscous substance; mixing 27.58 kg of alginic acid, 42 kg of humic acid, 55.2 kg of amino acids, 0.44 kg of molybdic acid, 0.69 kg of copper sulfate, 0.99 kg of ferric sulfate, 0.76 kg of manganese sulfate, 2.43 kg of borax, 11.3 kg of calcium nitrate, 13.66 kg of magnesium sulfate, 23.68 kg of urea, 69 kg of P2O5, 28.27 kg of K2O, and 120 kg of the tetramycin fertilizer dry basis prepared in step (1) uniformly, adding the mixture to the viscous substance, stirring uniformly, extruding, granulating, and drying at 50° C. for 12 hours to obtain a functional fertilizer of solid particles.

[0050] Application of solid particle functional fertilizer:

[0051] Target of prevention and control: Cotton Verticillium wilt

[0052] Field test: Two cotton planting fields with similar soil and cotton wilt were selected in rural Hubei, which were counted as experimental group 1 and control group 1. The test area of ​​each cotton field was 0.5 mu, and the cotton seedlings were in the middle and late stages of growth. The functional fertilizer of solid particles prepared by the present invention was applied respectively, and the application amount was 150 kg / mu. After the cotton was harvested, cotton was planted in the cotton field of experimental group 1 in the second year, and the functional fertilizer of solid particles prepared by the present invention was not applied.

[0053] Field test results: compared with control group 1, the leaves of cotton seedlings in experimental group 1 turned green, and the problem of bud and boll shedding was solved; cotton planted in the second year did not suffer from cotton wilt. This shows that the functional fertilizer with solid particles prepared by the present invention can effectively prevent and control cotton wilt.

[0054] Example 3

[0055] The solid granular functional fertilizers obtained in Examples 1 and 2 were respectively ground into 120-mesh powder fertilizers, which were respectively recorded as powder fertilizer 1 and powder fertilizer 2, and field tests were carried out.

[0056] Target of prevention and control: Wheat soil-borne mosaic disease

[0057] Field test: Four wheat fields with similar soil were selected in Anhui rural areas. Two wheat fields had serious soil-borne mosaic disease, which were used as experimental group 1-1 and control group 1-1; the other two wheat fields had no soil-borne mosaic disease, which were used as experimental group 1-2 and control group 1-2; the test area of ​​each wheat field was 0.5 mu. Powder fertilizer 1 was applied at a rate of 100 kg / mu.

[0058] Target of prevention and control: Cotton Verticillium wilt

[0059] Field test: Two cotton fields with similar soil and Verticillium wilt were selected in rural Hubei, which were counted as experimental group 1 and control group 1. The experimental area of ​​each cotton field was 0.5 mu, and the cotton seedlings were in the middle and late stages of growth. Powder fertilizer 2 was applied at a rate of 150 kg / mu. After the cotton was harvested, cotton was planted in the cotton field of experimental group 1 in the second year without applying powder fertilizer 2.

[0060] Field test results: After applying powder fertilizer 1, wheat soil-borne mosaic disease was significantly improved, and wheat grew more luxuriantly; after applying powder fertilizer 2, the leaves of cotton seedlings turned green, the problem of bud and boll shedding was solved, and cotton planted in the second year did not suffer from cotton wilt. It can be seen that the solid fertilizer prepared by the present invention is directly ground into powder for use, and its effect of preventing soil-borne diseases is not affected, and it can effectively prevent and control wheat soil-borne mosaic disease and cotton wilt.

[0061] Example 4

[0062] A method for preparing tetramycin fertilizer by using non-hygroscopic streptomyces, the method steps are as follows:

[0063] Step 1, seed expansion culture: first, add 1.1kg of peptone, 1.0kg of pea powder, 0.6kg of cassava residue, 3.0kg of agar, 0.06kg of calcium carbonate, and 0.1kg of potassium dihydrogen phosphate into 200kg of water, stir at 500 rpm for 45 minutes, adjust the pH to between 6.5 and 7.5, and prepare a culture medium mixture; steam sterilize at 120° C. in a high-pressure steam sterilizer for 30 minutes, then take out, pour into a sterilized eggplant slant culture container, and obtain an eggplant slant culture medium after cooling; inoculate the refrigerated non-hygroscopic Streptomyces Wuzhou subspecies on the eggplant slant culture medium, culture at 28° C. for 8 days, and set aside;

[0064] Step 2, prepare solid fermentation medium: 434.193kg rice flour, 434.193kg cassava residue, 347.354kg soybean meal, 277.883kg beef powder, 6.947kg sodium chloride, 6.947kg dipotassium hydrogen phosphate, 8.684kg magnesium sulfate heptahydrate, 1.737kg calcium chloride, 0.174kg ferrous sulfate heptahydrate, 0.0695kg cobalt chloride hexahydrate, 521.031kg water are mixed uniformly, pH is adjusted between 6.5 and 7.5, poured into a 3500L fermentor, steam is introduced to make the temperature in the fermentor reach 120°C, the tank pressure is controlled between 0.09 and 0.11Mpa, steam sterilized for 30 minutes, then cooled to 50°C, 60.787kg auxiliary agent is added, mixed uniformly, and then cooled to 27°C to obtain a solid fermentation medium;

[0065] Step 3: Under sterile conditions, use an inoculation loop to scrape 8 loops of the expanded culture of the strain in step 1, disperse them into 6 mL of sterile water, determine the concentration by hemocytometer, and dilute to prepare a strain suspension with a concentration of 140 million / L;

[0066] Step 4: The bacterial suspension prepared in step 3 is evenly inoculated in the solid fermentation tank prepared in step 2 at an inoculation amount of 10% mL / g, and fermented under sterile conditions at a temperature of 28° C. and a humidity of 50% for 4 days to obtain a solid fermentation product containing 0.45wt% tetramycin, i.e., tetramycin fertilizer.

[0067] The preparation method of the auxiliary agent is as follows: 104.206 kg of acrylamide, 69.471 kg of potassium acrylate, and 26.052 kg of calcium-based bentonite are added to 3473.543 kg of water, mixed evenly, and 1.042 kg of initiator solution (a mixed aqueous solution obtained by dissolving 0.05 kg of urea and 0.225 kg of potassium persulfate in 5.5 kg of water) is added, reacted at 30° C. for 30 minutes under a nitrogen atmosphere, and then heated to 50° C., and continued to react under a nitrogen atmosphere for 7 hours, anhydrous ethanol is added for precipitation and washed 3 times, vacuum dried at 50° C. for 7 hours, ground, and ultraviolet sterilized for 45 minutes to obtain an auxiliary agent.

[0068] Comparative Example 1

[0069] A method for preparing tetramycin fertilizer by using non-hygroscopic streptomyces, the method steps are as follows:

[0070] Step 1, seed expansion culture: first, add 1.1kg of peptone, 1.0kg of pea powder, 0.6kg of cassava residue, 3.0kg of agar, 0.06kg of calcium carbonate, and 0.1kg of potassium dihydrogen phosphate into 200kg of water, stir at 500 rpm for 45 minutes, adjust the pH to between 6.5 and 7.5, and prepare a culture medium mixture; steam sterilize at 120° C. in a high-pressure steam sterilizer for 30 minutes, then take out, pour into a sterilized eggplant slant culture container, and obtain an eggplant slant culture medium after cooling; inoculate the refrigerated non-hygroscopic Streptomyces Wuzhou subspecies on the eggplant slant culture medium, culture at 28° C. for 8 days, and set aside;

[0071] Step 2, prepare solid fermentation medium: 434.193kg rice flour, 434.193kg bran, 347.354kg soybean meal powder, 277.883kg beef powder, 6.947kg sodium chloride, 6.947kg dipotassium hydrogen phosphate, 8.684kg magnesium sulfate heptahydrate, 1.737kg calcium chloride, 0.174kg ferrous sulfate heptahydrate, 0.0695kg cobalt chloride hexahydrate, 521.031kg water are mixed uniformly, pH is adjusted between 6.5 and 7.5, poured into a 3500L fermentation tank, steam is introduced to make the temperature in the fermentation tank reach 120°C, the tank pressure is controlled between 0.09 and 0.11Mpa, steam sterilized for 30 minutes, then cooled to 50°C, 60.787kg auxiliary agent is added, mixed uniformly, and then cooled to 27°C to obtain a solid fermentation medium;

[0072] Step 3: Under sterile conditions, use an inoculation loop to scrape 8 loops of the expanded culture of the strain in step 1, disperse them into 6 mL of sterile water, determine the concentration by hemocytometer, and dilute to prepare a strain suspension with a concentration of 140 million / L;

[0073] Step 4: The bacterial suspension prepared in step 3 is evenly inoculated on the solid fermentation medium prepared in step 2 at an inoculum amount of 10% mL / g, and fermented under sterile conditions at a temperature of 28° C. and a humidity of 50% for 4 days to obtain a solid fermentation product containing 0.3wt% tetramycin, i.e., tetramycin fertilizer.

[0074] The preparation method of the auxiliary agent is the same as that of Example 4.

[0075] Test Example 1

[0076] Bacterial production test:

[0077] The determination of bacterial count after solid fermentation was carried out according to the double-layer plate culture counting test method, and the steps are as follows:

[0078] 1) drying a tetramycin fertilizer (solid fermentation product containing tetramycin) sample with rotary blast drying until the moisture content of the material is less than or equal to 10%, crushing the sample, and passing the sample through an 80-mesh sieve to obtain a solid fermentation dry basis;

[0079] 2) Mix 1g of peptone, 2g of agar, 0.6g of beef extract, 1g of glucose, and 200g of water, sterilize them in a high pressure steam sterilizer at 120°C for 30 minutes, and take them out for later use;

[0080] 3) Take 1 g of the solid fermentation product in step 1) and add 100 mL of sterile water to obtain a 10 -2 The bacterial suspension was then diluted with sterile water until a dilution of magnitude of 10 -8 Take 1mL of the order of magnitude of 10-8 The gradient bacterial suspension is added into the semi-solid culture medium to prepare a bacteria-containing semi-solid culture medium;

[0081] 4) 1g of peptone, 4.2g of agar, 0.6g of beef extract, 1g of glucose and 200g of water were mixed uniformly, and steam sterilized at 120°C for 30 minutes in a high pressure steam autoclave. The mixture was taken out and poured into a culture dish. After cooling and solidification, the semi-solid culture medium containing bacteria prepared in step 3) was poured into the culture dish. After the culture medium solidified, the culture medium was fermented and cultured for 3 days in a sterile environment at 28°C and 50% humidity. The number of single viable spore colonies in the culture dish was recorded. The number of colonies was measured in colony forming units. Three parallels were made for each sample, and the average value was taken. The number of bacteria produced after solid fermentation (cfu / g dry basis) was calculated according to Formula 1. The results are shown in Table 1.

[0082] Solid fermentation bacterial count = single live spore colony count × gradient dilution multiple formula 1

[0083] Test Example 2

[0084] Test on the control of apple black rot peel and Ralstonia solanacearum by tetramycin fertilizer (solid fermentation product containing tetramycin) (apple black rot peel: deposit number: ACCC 36137; Ralstonia solanacearum: deposit number: ACCC 01473, each strain was cultured on solid culture medium for 5 days before use):

[0085] The bioassay of tetramycin fertilizer (solid fermentation product containing tetramycin) against black rot of apple and Ralstonia solanacearum was carried out by the following steps:

[0086] 1) drying a tetramycin fertilizer (solid fermentation product containing tetramycin) sample with rotary blast drying until the moisture content of the material is less than or equal to 10%, crushing and grinding, and passing through a 90-mesh sieve to obtain a solid fermentation dry basis;

[0087] 2) diluting the solid fermentation product dry base with water 800 times to obtain a medicine;

[0088] 3) Boil 200 g peeled potato chunks in 1000 g water for 40 minutes, remove the potato chunks and leave the liquid, add 18 g agar and cook until dissolved, then add 15 g glucose and dissolve, then add water until the weight of the mixture is 1000 g, and steam sterilize at 120° C. in a high pressure steam sterilizer for 30 minutes, pour into a petri dish while hot, cool to 45° C., and set aside;

[0089] 4) taking a ring of apple black rot peel and Ralstonia solanacearum cultured on a solid culture medium for 5 days, respectively, dispersing them in 100 mL of sterile water, and then diluting them 50 times with sterile water to obtain bacterial suspensions; taking 1 mL of the bacterial suspension on the culture medium prepared in step 3), gently rotating the culture dish to mix the culture medium and the bacterial solution, and letting it stand to solidify into a plate;

[0090] 5) Use sterile tweezers to place a sterile filter paper with a diameter of 2 cm into the drug prepared in step 2) and soak it. When taking it out, stop it at the edge of the container to filter out excess drug solution. Then place the filter paper in the center of the bacterial culture medium prepared in step 4) so ​​that it is fully in close contact with the culture medium. Culture it in a sterile environment at 28°C for 5 days, use a ruler or vernier caliper to measure the size of the inhibition zone, and calculate the inhibition rate. Perform 3 parallel tests on each bacterial species, and use filter paper soaked in sterile water as a blank control. The inhibition rate is calculated according to Formula 2.

[0091]

[0092] Table 1 Test results of samples

[0093]

[0094] By comparing Example 4 with Comparative Examples 1 to 3, it can be found that the bacterial yield and antibacterial property of Example 4 are better than those of the comparative examples. This is because cassava residues and additives are added to the fermentation solid fermentation medium of the non-hydroscopic Streptomyces wuzhou subspecies, which provides effective nutrients, inorganic salts, trace elements required for growth, etc. for the growth metabolism of the non-hydroscopic Streptomyces wuzhou subspecies. At the same time, the addition of additives also maintains the humidity balance of the solid fermentation medium, helps stabilize the temperature inside the solid fermentation medium, promotes the growth metabolism of the non-hydroscopic Streptomyces wuzhou subspecies, and increases the effective content in the solid fermentation product and the prevention and control effect of the solid fermentation product on plant fungal infections. At the same time, the solid fermentation product can also be directly prepared into granular fertilizer for use, and the shelf life of the fertilizer can also be extended.

Claims

1. A method for preparing tetramycin fertilizer by using non-hygroscopic streptomyces, characterized in that: The steps include: Step 1, seed expansion culture: mix the slant culture medium materials, pour them into the slant culture container, sterilize them with high-temperature steam, and obtain the slant culture medium after cooling; inoculate the refrigerated non-hygroscopic Streptomyces wuzhouensis subspecies on the slant culture medium, culture them at a temperature of 25-30° C. for 4-10 days, and set aside; Step 2, preparing a solid fermentation medium: by weight, 45-55 parts of a carbon source, 35-40 parts of a nitrogen source, 1-2 parts of an inorganic salt, and 20-40 parts of water are mixed evenly, the pH is adjusted to 6.5-7.5, sterilized by high-temperature steam, cooled to 45-60° C., 2-5 parts of an auxiliary agent are added, mixed evenly, and then cooled to 25-35° C. to obtain a solid fermentation medium for standby use; Step 3: Under sterile conditions, use an inoculation loop to scrape 5 to 10 loops of the expanded culture of the bacteria in step 1, disperse 2 to 10 mL into sterile water, determine the concentration by hemocytometer, and dilute to prepare a bacterial suspension with a concentration of 13 to 16 million / L; Step 4, the bacterial suspension prepared in step 3 is evenly inoculated on the solid fermentation medium prepared in step 2, and solid fermentation culture is performed to obtain a solid fermentation product containing 0.01wt%~0.5wt% tetramycin, i.e. tetramycin fertilizer; the bacterial inoculation amount is 5%~15% mL / g; the solid fermentation culture conditions are: temperature 25~30°C, fermentation culture time is 3~5 days, and fermentation humidity is 45%~80%; The slant culture medium comprises the following raw materials in weight percentage: 0.4% to 0.7% peptone, 0.4% to 0.6% pea extract powder, 0.1% to 0.5% cassava residue, 1% to 2% agar, 0.02% to 0.05% calcium carbonate, 0.04% to 0.07% potassium dihydrogen phosphate, and the balance is water; The carbon source is 10-30 parts by weight of rice flour mixed with 10-30 parts by weight of cassava residue; The preparation method of the auxiliary agent is as follows: in parts by weight, 3-9 parts of acrylamide, 2-6 parts of potassium acrylate, and 1-2 parts of calcium-based bentonite are added to 100-300 parts of water, mixed evenly, 0.05-0.08 parts of initiating solution are added, reacted at 25-32° C. for 25-35 minutes under a nitrogen atmosphere, heated to 48-53° C., continued to react under a nitrogen atmosphere for 6-8 hours, added with anhydrous ethanol for precipitation and washed 2-4 times, vacuum dried at 45-55° C. for 6-8 hours, ground, and ultraviolet sterilized for 30-60 minutes to obtain the auxiliary agent; the initiating solution is a mixed aqueous solution obtained by dissolving 2-4 parts of urea and 9-18 parts of potassium persulfate in 220-450 parts of water.

2. A method for preparing tetramycin fertilizer by using non-hygroscopic streptomyces as claimed in claim 1, characterized in that: The nitrogen source is 20-30 parts by weight of soybean meal powder mixed with 10-20 parts by weight of beef extract powder.

3. A method for preparing tetramycin fertilizer by using non-hygroscopic Streptomyces as claimed in claim 1, characterized in that: The inorganic salt includes the following components in mass percentage: 24% to 30% sodium chloride, 25% to 30% dipotassium hydrogen phosphate, 33% to 36% magnesium sulfate heptahydrate, 5% to 8% calcium chloride, and 0.5% to 1% ferrous sulfate heptahydrate.

4. The use of a method for preparing tetramycin fertilizer by using non-hygroscopic streptomyces as claimed in any one of claims 1 to 3 in preparing functional fertilizer, characterized in that: The functional medicinal fertilizer is a solid granule or powder; the preparation method of the functional medicinal fertilizer in the form of a solid granule is as follows: (1) drying the tetramycin fertilizer prepared by the method according to any one of claims 1 to 3 by rotary blast drying until the moisture content of the material is less than or equal to 10%, crushing, passing through a 100-200 mesh sieve, obtaining a tetramycin fertilizer dry basis, and setting aside; (2) Mixing a binder with water, stirring at 40-60° C. for 10-40 minutes to obtain a viscous substance; uniformly mixing alginic acid, humic acid, amino acids, trace elements, medium elements, macroelements, and the tetramycin-containing fertilizer prepared in step (1) on a dry basis, adding the mixture to the viscous substance, stirring uniformly, extruding and forming, granulating, and drying at 45-60° C. for 10-16 hours to obtain a solid granular functional fertilizer; The function refers to the prevention and control of soil-borne diseases.

5. The use according to claim 4, characterized in that The binder is one of pregelatinized starch, dextrin, gum arabic and sodium alginate.

6. The use according to claim 4, characterized in that The trace element is at least one of copper fertilizer, iron fertilizer, manganese fertilizer, zinc fertilizer, boron fertilizer, and molybdenum fertilizer; the medium element is at least one of calcium fertilizer and magnesium fertilizer; the macroelement is a mixture of two or more of nitrogen fertilizer, phosphorus fertilizer, and potassium fertilizer.

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