Compound liquid fertilizer containing tylosin finished product liquid and preparation method of compound liquid fertilizer

Abstract

The invention discloses a compound liquid fertilizer containing tylosin finished product liquid, and belongs to the technical field of fertilizer preparation. Comprising a tylosin metabolite complex TDMC, humic acid, a seaweed extract, urea, monopotassium phosphate, potassium sulfate, an organic acid compound, polyaspartic acid, a polyol compound, a trace element compound, a stabilizer, an antifreeze agent, an antioxidant, a preservative and deionized water. The tylosin metabolite complex TDMC is a metabolite which is obtained by converting tylosin through a biological conversion method and has no antibacterial activity but retains a plant immunity induction function. According to the compound liquid fertilizer containing tylosin metabolite derivatives, a TDMC compound is obtained through a biotransformation technology, stabilization and slow release of active ingredients are achieved through a multifunctional chelating carrier system, and the compound liquid fertilizer has remarkable advantages in the aspects of product stability, plant immune induction, disease resistance, growth promoting effect and the like; and compared with the traditional method, the performance is improved by about 40-50%.

Description

Technical Field

[0001] The invention relates to the technical field of fertilizer preparation, in particular to a composite liquid fertilizer containing tylosin finished liquid and a preparation method thereof. Background Art

[0002] Liquid fertilizers have become an indispensable input in modern agricultural production due to their advantages such as fast nutrient release, high utilization rate and convenient use. With the in-depth promotion of green agriculture and sustainable development concepts, compound liquid fertilizers with both nutritional and plant protection functions have gradually become a research and development hotspot. Tylosin, as a macrolide antibiotic, not only has antibacterial activity, but some of its metabolites also show the characteristics of inducing plant immunity and promoting growth, which provides the possibility for the development of new functional fertilizers.

[0003] The existing technology for the application of tylosin in liquid fertilizers mainly has the following technical problems: the problem of antibiotic activity of tylosin has not been effectively solved, and there is a risk of environmental drug resistance in long-term use; the active ingredient has poor stability and is easily degraded and ineffective under different pH and temperature conditions; the release rate of the active ingredient is difficult to control, and it often becomes ineffective quickly after short-term excessive release; there is a lack of biotransformation technology for tylosin metabolites, and its plant immune induction function has not been fully utilized; the formula design is unreasonable, and the synergistic effect of nutrients and functional ingredients has not been achieved. Summary of the invention

[0004] The invention provides a composite liquid fertilizer containing tylosin finished product liquid and a preparation method thereof, which solves the technical problems of antibiotic residues, poor stability of effective components and uneven release in antibiotic bioconversion fertilizers in the prior art.

[0005] The composite liquid fertilizer containing tylosin finished liquid provided by the present invention comprises: Tylosin metabolite complex TDMC, humic acid, seaweed extract, urea, potassium dihydrogen phosphate, potassium sulfate, organic acid complex, polyaspartic acid, polyol complex, trace element compound, stabilizer, antifreeze agent, antioxidant, preservative and deionized water; The tylosin metabolite complex TDMC is a metabolite having no antibacterial activity but retaining plant immunity inducing function obtained by transforming tylosin through a biotransformation method.

[0006] Preferably, the components, by weight, include: 8 - 12 parts of tylosin metabolite complex TDMC, 5 - 9 parts of humic acid, 1 - 4 parts of seaweed extract, 8 - 15 parts of urea, 5 - 10 parts of potassium dihydrogen phosphate, 5 - 9 parts of potassium sulfate, 12 - 18 parts of organic acid complex, 2 - 5 parts of polyaspartic acid, 2 - 5 parts of polyol complex, 3 - 8 parts of trace element compound, 0.5 - 2 parts of stabilizer, 1 - 3 parts of antifreeze, 0.3 - 1 part of antioxidant and preservative, and 40 - 60 parts of deionized water.

[0007] Preferably, the tylosin metabolite complex TDMC is prepared by the following steps: (a) Strain activation and amplification culture: Inoculate Bacillus subtilis strain BY - 7 into a liquid medium, and culture it under shaking at 27 - 29 °C and 160 - 200 rpm for 16 - 20 hours. Then take 5% of the activated bacterial liquid and inoculate it into the amplification culture medium, and culture it under shaking at 27 - 29 °C and 160 - 200 rpm for 11 - 13 hours; (b) Biotransformation reaction: Inoculate the amplified bacterial liquid into the transformation medium, pre - culture it for 4 hours at 27 - 29 °C, dissolved oxygen of 35% - 45%, and stirring speed of 180 - 220 rpm. Then add the tylosin finished product solution and carry out a 46 - 50 - hour biotransformation reaction at 29 - 31 °C; (c) Metabolite extraction and purification: After the reaction, centrifuge and separate the fermentation broth, collect the supernatant, concentrate it by ultrafiltration, and then carry out column chromatography using macroporous adsorption resin, eluting successively with 20%, 40%, 60%, and 80% ethanol, collect the eluted fractions, and concentrate under reduced pressure to obtain the TDMC complex.

[0008] Preferably, the trace element compound is selected from one or more of zinc sulfate heptahydrate, manganese sulfate heptahydrate, copper sulfate heptahydrate, ferrous sulfate heptahydrate, ammonium molybdate, and borax.

[0009] Preferably, the organic acid complex includes citric acid, malic acid, and succinic acid, and their mass ratio is 4:2:1; the polyol complex includes glycerol and sorbitol, and their mass ratio is 1.3:1.

[0010] Preferably, the molecular weight of the humic acid is 3000 - 5000 Da; the molecular weight of the polyaspartic acid is 8000 - 12000 Da; the content of alginic acid in the seaweed extract is ≥18%.

[0011] Preferably, the stabilizer is polyethylene oxide; the antifreeze is propylene glycol; the antioxidant is vitamin C; the preservative is p - hydroxybenzoate.

[0012] The present invention also provides a preparation method of the above - mentioned compound liquid fertilizer, which includes the following steps: (1)Preparation of tylosin metabolite complex TDMC: Through biotransformation by Bacillus subtilis strain BY-7, tylosin is transformed into the metabolite complex TDMC without antibacterial activity; (2)Preparation of organic acid matrix: Add deionized water to the reaction kettle, heat it to 53 - 57 °C, add citric acid, malic acid and succinic acid, and stir to dissolve to form an organic acid base solution; (3)Preparation of trace element chelation system: Adjust the temperature of the reaction kettle obtained in step (2) to 62 - 68 °C, add humic acid and stir evenly, then add trace element compounds, keep the pH value within the range of 5.2 - 5.8, and stir to react to form a trace element chelation solution; (4)Preparation of macronutrient nutrient solution: Add deionized water to the reactor, add urea, potassium dihydrogen phosphate, potassium sulfate, stir to dissolve, and then add seaweed extract and stir evenly; (5)Preparation of slow-release carrier: Add deionized water to a three-necked flask, under the condition of 56 - 60 °C, add polyaspartic acid and stir to dissolve, then add glycerol and sorbitol and stir evenly; (6) Activation treatment of TDMC: Preheat the TDMC solution prepared in step (1) to 28 - 32 °C, add citric acid solution to adjust the pH to 6.0 - 6.5, add antioxidant and preservative, and stir to dissolve; (7)Integration of composite system: Add the TDMC activation solution obtained in step (6) to the slow-release carrier solution obtained in step (5), control the temperature at 42 - 48 °C, and stir to react; then add this mixture to the trace element chelation solution obtained in step (3), control the temperature at 40 - 44 °C, and stir to mix; then add the macronutrient nutrient solution obtained in step (4) to the above mixed system and stir evenly; (8)Stabilization treatment: Use potassium hydroxide solution to adjust the pH value of the final liquid fertilizer to 6.8 - 7.2, add stabilizer and antifreeze, and stir evenly; (9)Filtration and filling: Filter the prepared liquid fertilizer through a nylon filter screen, and fill it into a high-density polyethylene container under sterile conditions and seal it for storage.

[0013] Preferably, in step (3), the stirring reaction time is 110 - 130 minutes; In step (6), 0.5% citric acid solution is used for pH adjustment during TDMC activation; In step (7), the rate of adding the TDMC activation solution to the slow-release carrier solution is 4 - 6 mL / min, the stirring speed is 280 - 320 rpm, and the reaction time is 35 - 45 minutes; when the mixture of TDMC and the slow-release carrier is added to the trace element chelation solution, the stirring speed is 230 - 270 rpm, and the mixing time is 25 - 35 minutes.

[0014] The present invention also provides the application of the above compound liquid fertilizer in promoting crop growth and enhancing plant immunity, including: applying the liquid fertilizer diluted 500 - 1000 times to the roots of crops by the method of root irrigation, once every 7 - 14 days, to promote the growth and development of crops and improve the resistance of plants to diseases such as bacterial wilt and fusarium wilt.

[0015] Compared with the prior art, the present invention has at least the following beneficial effects: (1) The present invention provides an innovative formula for green agricultural products. By using biotransformation technology, tylosin is transformed into metabolites without antibacterial activity but retaining the plant immune induction function, solving the problems of drug resistance risk and environmental safety hazards that may be brought by traditional antibiotic-containing fertilizers.

[0016] (2) The present invention successfully overcomes the technical problems of easy degradation and unstable activity of the TDMC complex in traditional formulations through a multifunctional chelating carrier system, realizes the stabilization and slow release of active ingredients, extends the product validity period, and improves the actual application effect.

[0017] (3) The multifunctional chelating carrier system of the present invention not only stabilizes the activity of TDMC, but also significantly improves the bioavailability of trace elements, realizing the synergistic effect of nutrient supply and immune induction.

[0018] (4) Proven by experiments, the control effect of the product of the present invention against bacterial wilt of tomatoes reaches 70.0% - 74.8%, which is about 50% higher than that of traditional tylosin products (45.0%), and the root infection rate is only 18.6% - 22.3%, far lower than 35.6% - 38.5% of the comparative products.

[0019] (5) The product of the present invention can significantly improve plant growth indexes, promote the plant height of crops to increase by 34.5% - 37.8%, the root length to increase by 41.2% - 44.3%, the root surface area to increase by 47.5% - 50.6%, and the dry weight to increase by 37.8% - 40.3%. Each growth index is about 30% - 45% higher than that of the comparative products on average.

[0020] (6) The product of the present invention adopts the method of root irrigation application. Only 500 - 1000 times dilution is required, and it is applied once every 7 - 14 days. It is convenient to use and has a significant prevention and control effect on various crop diseases, with a wide application range.

[0021] In summary, the tylosin metabolite-derived compound liquid fertilizer provided by the present invention exhibits significant advantages in terms of product stability, plant immune induction, disease resistance, and growth promotion effects through unique biotransformation technology and a multifunctional chelating carrier system. It has a comprehensive performance improvement of approximately 40% - 50% compared to traditional methods, providing an efficient, safe, and environmentally friendly new solution for the healthy growth of crops and disease prevention and control. Detailed Embodiments

[0022] The technical solutions of the present invention will be clearly and completely described below in conjunction with the embodiments. Obviously, the described embodiments are only a part of the embodiments of the present invention, rather than all of the embodiments. All other embodiments obtained by those of ordinary skill in the art based on the embodiments of the present invention without creative efforts shall fall within the protection scope of the present invention.

[0023] The LB liquid medium used in the embodiment has the following composition: peptone 10 g / L, yeast extract 5 g / L, NaCl 5 g / L, pH 7.0.

[0024] The amplification medium used in the embodiment has the following composition: peptone 10 g / L, yeast extract 5 g / L, glucose 10 g / L, NaCl 5 g / L, pH 7.0.

[0025] The transformation medium used in the embodiment has the following composition: ammonium chloride 2 g / L, potassium dihydrogen phosphate 1 g / L, magnesium sulfate 0.5 g / L, glucose 5 g / L, pH 7.2.

[0026] The aqueous solution of the tylosin finished product used in the embodiment contains 20% of the tylosin finished product.

[0027] The seaweed extract used in the embodiments of the present application is from Qingdao Mingyue Seaweed Group Co., Ltd., and the alginic acid content is 18%; The AB-8 macroporous adsorption resin used in the embodiments of the present application is from Tianjin Binhai Koster Technology Co., Ltd., and the pore size is 0.7 mm; The humic acid used in the embodiments of the present application is from the Institute of Soil and Fertilizer, Huazhong Agricultural University, and the molecular weight is 4000 Da.

[0028] The polyaspartic acid used in the embodiments of the present application is from Nanjing Tianda Polymer Technology Co., Ltd.

[0029] The ultrafiltration system used in the embodiments of the present application is from Shanghai Mosu Scientific Equipment Co., Ltd., model MS-MWCO-1000, and the cut-off molecular weight is 1000 Da.

[0030] The parameters of the final product prepared in the embodiments of the present application are detected by the following standard methods: pH value detection: It is determined by the pH meter method. Use a PHS-3C precision pH meter. Take 25 mL of the sample in a 50 mL beaker and directly measure it at room temperature. Each sample is measured 3 times, and the average value is taken, accurate to 0.1.

[0031] Density detection: It is determined by the pycnometer method. First, calibrate a 25 mL pycnometer with distilled water. Then, at 20 °C, weigh the empty pycnometer and record it as m1. Fill the pycnometer with the liquid fertilizer up to the calibration line, dry the outer wall of the bottle, and weigh it, recording the weight as m2. Calculation formula: Density (g / cm³) = (m2 - m1) / 25. Measure 3 times and take the average value, accurate to 0.01 g / cm³.

[0032] Suspension rate detection: It is determined by the centrifugation method. Take 100 mL of the sample and place it in a 100 mL graduated cylinder. Observe the precipitation situation after standing for 24 hours. Use a pipette to draw 50 mL from the upper part of the liquid and put it into a centrifuge tube. Centrifuge at 3000 r / min for 15 minutes, weigh the dry matter content before and after centrifugation. Calculation formula: Suspension rate (%) = (dry matter content after centrifugation / dry matter content before centrifugation) × 100%. Measure 3 times and take the average value, accurate to 0.5%.

[0033] TDMC effective content detection: It is verified by ultraviolet spectrophotometry combined with HPLC-MS. First, measure the absorbance of the sample at a wavelength of 280 nm with an ultraviolet spectrophotometer, and calculate the content of TDMC according to the pre-established standard curve. The specific operation process is as follows: Take 5.0 g of the sample, accurately weigh it, place it in a 50 mL volumetric flask, add methanol-water (70:30, v / v) solution to the calibration line, ultrasonically treat it for 20 minutes, let it stand, take the upper clear liquid, filter it through a 0.45 μm filter membrane, use an ultraviolet spectrophotometer to measure the absorbance at a wavelength of 280 nm, and calculate the TDMC content according to the standard curve.

[0034] The tylosin conversion rate of the example is detected by high performance liquid chromatography-mass spectrometry (HPLC-MS). The specific detection method is as follows: The supernatant was taken after centrifuging the fermentation broth sample (8000 rpm, 10 minutes), filtered through a 0.22 μm filter membrane, and then subjected to HPLC-MS analysis. The analysis conditions were as follows: The chromatographic column used was an Agilent Eclipse XDB-C18 column (4.6×150 mm, 5 μm), and the column temperature was 30 °C; mobile phase A was an aqueous solution of 0.1% formic acid, and mobile phase B was acetonitrile. Gradient elution was carried out: 0 - 5 minutes, 5% - 20% B; 5 - 15 minutes, 20% - 40% B; 15 - 20 minutes, 40% - 60% B; 20 - 25 minutes, 60% - 80% B; 25 - 30 minutes, 80% - 5% B; the flow rate was 0.8 mL / min; the injection volume was 10 μL. The mass spectrometry used an electrospray ionization source (ESI) in the positive ion mode, and the scanning range was m / z 100 - 1200.

[0035] The calculation method of the conversion rate was as follows: First, a standard curve of tylosin was drawn using the standard product, and then the contents of tylosin in the samples before and after fermentation were measured. According to the formula: conversion rate (%) = (initial tylosin content - tylosin content after conversion) / initial tylosin content × 100%, the conversion rate of tylosin was calculated.

[0036] Example 1 The preparation process of a compound liquid fertilizer containing tylosin finished product liquid was as follows: S1. Preparation of tylosin metabolite complex TDMC: Inoculate Bacillus subtilis strain BY-7 into LB liquid medium, and shake culture at 28 °C and 180 rpm for 18 hours. Subsequently, take 5% of the activated bacterial liquid and inoculate it into the expansion culture medium, and shake culture at 28 °C and 180 rpm for 12 hours to obtain a bacterial liquid with an OD600 of 2.0. In a 30 L bioreactor, add 20 L of transformation medium, sterilize at 121 °C for 20 minutes, cool to 30 °C, and then inoculate 2 L of the expanded bacterial liquid. Pre-culture at 28 °C, dissolved oxygen of 40%, and stirring speed of 200 rpm for 4 hours. Then, add an aqueous solution of 500 g of tylosin finished product at a dropping rate of 12 mL / min, adjust the pH to 6.8 with 0.1 mol / L NaOH, and react at 30 °C for 48 hours. After the reaction, it was detected that the tylosin conversion rate reached 98%. Centrifuge the fermentation broth at 8000 rpm to separate, collect the supernatant, concentrate it to 1 / 5 of the original volume through an ultrafiltration system with a molecular weight cut-off of 1000 Da, and then use an AB-8 type macroporous adsorption resin adsorption column chromatography, elute successively with 20%, 40%, 60%, and 80% ethanol, collect the elution fractions, concentrate under reduced pressure to obtain the TDMC complex. Dissolve TDMC in deionized water and adjust the concentration to 20 g / L as the active ingredient for subsequent preparation.

[0037] S2. Preparation of organic acid matrix: In a 5L stainless steel reactor, add 2800 mL of deionized water, heat it to 55 °C, and add 85.50 g of food-grade citric acid, 43.25 g of malic acid, and 21.75 g of succinic acid in batches. Stir and dissolve for 30 minutes to form an organic acid base solution.

[0038] S3. Preparation of trace element chelation system: Adjust the temperature of the above reactor to 65 °C, add 67.50 g of humic acid with a molecular weight of 3000 - 5000 Da, stir evenly for 45 minutes, and then slowly add 15.75 g of zinc sulfate heptahydrate, 12.60 g of manganese sulfate heptahydrate, 3.35 g of copper sulfate heptahydrate, 13.40 g of ferrous sulfate heptahydrate, 2.25 g of ammonium molybdate, and 4.95 g of borax in batches. Keep the pH value within the range of 5.5 and stir and react for 120 minutes to form a trace element chelation solution.

[0039] S4. Preparation of macronutrient nutrient solution: In a 2L glass reactor, add 850 mL of deionized water, add 112.50 g of food-grade urea, 78.35 g of potassium dihydrogen phosphate, and 67.25 g of potassium sulfate, stir and dissolve for 25 minutes, and then add 22.50 g of seaweed extract and stir evenly for 15 minutes.

[0040] S5. Preparation of slow-release carrier: In a 2L three-necked flask, add 650 mL of deionized water, control the temperature at 58 °C through a constant temperature water bath, add 31.25 g of polyaspartic acid with a molecular weight of 10000 Da, and stir and dissolve. Then add 16.75 g of glycerol and 13.25 g of sorbitol and stir evenly for 30 minutes.

[0041] S6. TDMC activation treatment: Preheat 500 mL of TDMC solution to 30 °C, slowly add 25 mL of 0.5% citric acid solution to adjust the pH to 6.2, add 4.75 g of antioxidant vitamin C and 1.85 g of UV inhibitor p-hydroxybenzoate, and stir for 15 minutes.

[0042] S7. Integration of composite system: Slowly add the TDMC activation solution into the slow-release carrier solution, control the feeding rate at 5 mL / min, maintain the temperature at 45 °C, and the stirring speed at 300 rpm. React for 40 minutes, then slowly add the mixed solution into the trace element chelation solution prepared in the first step, control the temperature at 42 °C, and the stirring speed at 250 rpm. Mix for 30 minutes, and then slowly add the macronutrient nutrient solution into the above mixed system and continue to stir evenly for 25 minutes.

[0043] S8. Stabilization treatment of the product: Adjust the pH value of the final liquid fertilizer to 7.0 with 1 mol / L potassium hydroxide solution, add 8.35 g of polyethylene oxide with a molecular weight of 4000 as a stabilizer, and 15.25 g of antifreeze propylene glycol. Stir evenly for 20 minutes to obtain a compound liquid fertilizer containing tylosin finished product solution. Filter the prepared liquid fertilizer through a 100-mesh nylon filter, fill the product into a pre-sterilized high-density polyethylene container, and seal it for storage.

[0044] After testing, the pH value of the prepared product is 7.0, the density is 1.18 g / cm³, the suspension rate is 98%, and the effective content of TDMC is 4.0%.

[0045] Example 2 The preparation process of a compound liquid fertilizer containing tylosin metabolite derivative is as follows: S1. Preparation of tylosin metabolite complex TDMC: Inoculate Bacillus subtilis strain BY-7 into LB liquid medium, and under the condition of 29 °C, shake culture at 160 rpm for 20 hours. Take 5% of the activated bacterial liquid and inoculate it into the expansion culture medium. Under the condition of 27 °C, shake culture at 190 rpm for 11 hours to obtain a bacterial liquid with an OD600 of 1.8. In a 30 L bioreactor, add 20 L of transformation medium, sterilize at 121 °C for 20 minutes, cool to 30 °C, then inoculate 2 L of the expanded bacterial liquid, and pre-culture at 29 °C, dissolved oxygen 35%, and stirring speed 180 rpm for 4 hours. Then, add an aqueous solution of 500 g of tylosin finished product at a dropping rate of 10 mL / min, adjust the pH to 6.9 with 0.1 mol / L NaOH, and react at 31 °C for 46 hours. After the reaction, the tylosin conversion rate reaches 99% as detected. Centrifuge the fermentation broth at a rotation speed of 8500 rpm, collect the supernatant, concentrate it to 1 / 5 of the original volume through an ultrafiltration system with a molecular weight cut-off of 1000 Da, use an AB-8 type macroporous adsorption resin adsorption column chromatography, elute successively with 20%, 40%, 60%, and 80% ethanol, collect the elution components, and concentrate under reduced pressure to obtain the TDMC complex. Dissolve TDMC in deionized water and adjust the concentration to 20 g / L as the active ingredient for subsequent preparation.

[0046] S2. Preparation of organic acid matrix: In a 5 L stainless steel reaction kettle, add 2800 mL of deionized water, heat to 57 °C, and add 90.00 g of food-grade citric acid, 45.00 g of malic acid, and 22.50 g of succinic acid in batches, and stir and dissolve for 25 minutes to form an organic acid base solution.

[0047] S3. Preparation of trace element chelation system: Adjust the temperature of the above-mentioned reactor to 68 °C, add 65.00 g of humic acid, stir evenly for 40 minutes, and then slowly add 16.00 g of zinc sulfate heptahydrate, 13.00 g of manganese sulfate heptahydrate, 3.50 g of copper sulfate heptahydrate, 14.00 g of ferrous sulfate heptahydrate, 2.50 g of ammonium molybdate and 5.00 g of borax in batches, keep the pH value within the range of 5.2, and stir and react for 110 minutes to form a trace element chelation solution.

[0048] S4. Preparation of macronutrient nutrient solution: In a 2 L glass reactor, add 850 mL of deionized water, add 115.00 g of food-grade urea, 80.00 g of potassium dihydrogen phosphate, and 70.00 g of potassium sulfate, stir and dissolve for 23 minutes, and then add 25.00 g of seaweed extract and stir evenly for 17 minutes.

[0049] S5. Preparation of slow-release carrier: In a 2 L three-necked flask, add 650 mL of deionized water, control the temperature at 56 °C through a constant temperature water bath, add 33.00 g of polyaspartic acid with a molecular weight of 9000 Da, stir and dissolve, and then add 17.00 g of glycerol and 13.05 g of sorbitol, and stir evenly for 28 minutes.

[0050] S6. TDMC activation treatment: Preheat 500 mL of TDMC solution to 32 °C, slowly add 25 mL of 0.5% citric acid solution to adjust the pH to 6.0, add 5.00 g of antioxidant vitamin C and 2.00 g of UV inhibitor p-hydroxybenzoate, and stir slightly to dissolve for 12 minutes.

[0051] S7. Integration of composite system: Slowly add the TDMC activation solution to the slow-release carrier solution, control the feeding rate at 6 mL / min, maintain the temperature at 48 °C, and the stirring speed at 320 rpm, react for 35 minutes, slowly add the mixed solution to the trace element chelation solution prepared in the first step, control the temperature at 44 °C, and the stirring speed at 270 rpm, mix for 25 minutes, and slowly add the macronutrient nutrient solution to the above mixed system, and continue to stir evenly for 27 minutes.

[0052] S8. Stabilization treatment of product: Use 1 mol / L potassium hydroxide solution to adjust the pH value of the final liquid fertilizer to 7.2. Add 9.00 g of polyethylene oxide with a molecular weight of 4000 as a stabilizer and 16.00 g of antifreeze propylene glycol, stir evenly for 22 minutes to obtain a composite liquid fertilizer containing tylosin finished liquid. Filter the prepared liquid fertilizer through a 100-mesh nylon filter, fill the product into a pre-sterilized high-density polyethylene container, and seal and store.

[0053] After testing, the pH value of the prepared product is 7.2, the density is 1.20 g / cm³, the suspension rate is 97%, and the effective content of TDMC is 4.2%.

[0054] Example 3 The preparation process of a tylosin metabolite-derived compound liquid fertilizer is as follows: S1. Preparation of tylosin metabolite complex TDMC: Inoculate Bacillus subtilis strain BY-7 into LB liquid medium, and under the condition of 27 °C, shake culture at 200 rpm for 16 hours. Take 5% of the activated bacterial liquid and inoculate it into the expansion culture medium. Under the condition of 29 °C, shake culture at 170 rpm for 13 hours to obtain a bacterial liquid with an OD600 of 2.2. In a 30 L bioreactor, add 20 L of transformation medium, sterilize at 121 °C for 20 minutes, and after cooling to 30 °C, inoculate 2 L of the expanded bacterial liquid. Pre-culture at 27 °C, with a dissolved oxygen of 45% and a stirring speed of 220 rpm for 4 hours. Then, add an aqueous solution of 500 g of tylosin finished product at a dropping rate of 14 mL / min, adjust the pH to 7.0 with 0.1 mol / L NaOH, and react at 29 °C for 50 hours. After the reaction, it is detected that the tylosin conversion rate reaches 98.5%. Centrifuge the fermentation broth at a rotation speed of 7500 rpm to separate, collect the supernatant, concentrate it to 1 / 5 of the original volume through an ultrafiltration system with a cut-off molecular weight of 1000 Da, and then use an AB-8 type macroporous adsorption resin adsorption column chromatography, elute successively with 20%, 40%, 60%, and 80% ethanol, collect the elution components, and concentrate under reduced pressure to obtain the TDMC complex. Dissolve TDMC in deionized water and adjust the concentration to 20 g / L as the active ingredient for subsequent preparation.

[0055] S2. Preparation of organic acid matrix: In a 5 L stainless steel reaction kettle, add 2800 mL of deionized water, heat to 53 °C, and add 80.00 g of food-grade citric acid, 40.00 g of malic acid, and 20.00 g of succinic acid in batches, and stir and dissolve for 35 minutes to form an organic acid base solution.

[0056] S3. Preparation of trace element chelation system: Adjust the temperature of the above reaction kettle to 62 °C, add 70.00 g of humic acid, stir evenly for 50 minutes, and then slowly add 15.00 g of zinc sulfate heptahydrate, 12.00 g of manganese sulfate heptahydrate, 3.00 g of copper sulfate heptahydrate, 12.50 g of ferrous sulfate heptahydrate, 2.00 g of ammonium molybdate, and 4.50 g of borax in batches, and keep the pH value within the range of 5.8, and stir and react for 130 minutes to form a trace element chelation solution.

[0057] S4. Preparation of macronutrient nutrient solution: In a 2 L glass reactor, add 850 mL of deionized water, add 110.00 g of food-grade urea, 75.00 g of potassium dihydrogen phosphate, and 65.00 g of potassium sulfate, stir and dissolve for 27 minutes, and then add 20.00 g of seaweed extract and stir evenly for 13 minutes.

[0058] S5. Preparation of sustained-release carrier: In a 2L three-necked flask, add 650 mL of deionized water. Under the condition of controlling the temperature at 60 °C through a constant-temperature water bath, add 30.00 g of polyaspartic acid with a molecular weight of 12,000 Da, stir to dissolve, then add 16.00 g of glycerol and 12.30 g of sorbitol, and stir evenly for 32 minutes.

[0059] S6. Activation treatment of TDMC: Preheat 500 mL of TDMC solution to 28 °C, slowly add 25 mL of 0.5% citric acid solution to adjust the pH to 6.5, add 4.50 g of antioxidant vitamin C and 1.75 g of UV inhibitor p-hydroxybenzoate, and stir slightly to dissolve for 18 minutes.

[0060] S7. Integration of composite system: Slowly add the TDMC activation solution to the sustained-release carrier solution, control the feeding rate at 4 mL / min, maintain the temperature at 42 °C, and the stirring speed at 280 rpm, and react for 45 minutes. Subsequently, slowly add the mixed solution to the trace element chelating solution prepared in the first step, control the temperature at 40 °C, and the stirring speed at 230 rpm, and mix for 35 minutes. Slowly add the macronutrient nutrient solution to the above mixed system and continue to stir evenly for 23 minutes.

[0061] S8. Stabilization treatment of the product: Use 1 mol / L potassium hydroxide solution to adjust the pH value of the final liquid fertilizer to 6.8, add 7.50 g of polyethylene oxide with a molecular weight of 4000 as a stabilizer and 14.00 g of antifreeze propylene glycol, and stir evenly for 18 minutes to obtain a composite liquid fertilizer containing tylosin finished product solution. Filter the prepared liquid fertilizer through a 100-mesh nylon filter, fill the product into a pre-sterilized high-density polyethylene container, and seal and store it.

[0062] After testing, the pH value of the prepared product is 6.8, the density is 1.15 g / cm³, the suspension rate is 99%, and the effective content of TDMC is 3.8%.

[0063] Example 4 The preparation process of a composite liquid fertilizer containing tylosin metabolite derivatives is as follows: S1. Preparation of tylosin metabolite complex TDMC: Inoculate Bacillus subtilis strain BY-7 into LB liquid medium and shake culture at 28.5 °C and 175 rpm for 19 hours. Subsequently, take 5% of the activated bacterial liquid and inoculate it into the expansion culture medium, and shake culture at 28.5 °C and 185 rpm for 12.5 hours to obtain a bacterial liquid with an OD600 of 1.9. In a 30 L bioreactor, add 20 L of transformation medium, sterilize at 121 °C for 20 minutes, and after cooling to 30 °C, inoculate 2 L of the expanded bacterial liquid. Pre-culture at 28.5 °C, with a dissolved oxygen of 42% and a stirring speed of 210 rpm for 4 hours. Then, add an aqueous solution of 500 g of tylosin finished product at a dropping rate of 13 mL / min, adjust the pH to 6.9 with 0.1 mol / L NaOH, and react at 30.5 °C for 47 hours. After the reaction, the tylosin conversion rate reaches 98.5% as detected. Centrifuge the fermentation broth at 8200 rpm to separate, collect the supernatant, concentrate it to 1 / 5 of the original volume through an ultrafiltration system, and then use an AB-8 type macroporous adsorption resin adsorption column chromatography for the post-liquid, elute successively with 20%, 40%, 60%, and 80% ethanol, collect the elution components, and concentrate under reduced pressure to obtain the TDMC complex. Dissolve TDMC in deionized water and adjust the concentration to 20 g / L as the active ingredient for subsequent preparation.

[0064] S2. Preparation of organic acid matrix: In a 5 L stainless steel reactor, add 2800 mL of deionized water, heat to 54 °C, and add 82.50 g of food-grade citric acid, 41.25 g of malic acid, and 20.63 g of succinic acid in batches, and stir and dissolve for 32 minutes to form an organic acid base solution.

[0065] S3. Preparation of trace element chelation system: Adjust the temperature of the above reactor to 64 °C, add 68.75 g of humic acid, stir evenly for 47 minutes, and then slowly add 15.50 g of zinc sulfate heptahydrate, 12.40 g of manganese sulfate heptahydrate, 3.20 g of copper sulfate heptahydrate, 13.00 g of ferrous sulfate heptahydrate, 2.10 g of ammonium molybdate, and 4.75 g of borax in batches, keep the pH value within the range of 5.4, and stir and react for 125 minutes to form a trace element chelation solution.

[0066] S4. Preparation of macronutrient nutrient solution: In a 2 L glass reactor, add 850 mL of deionized water, add 111.25 g of food-grade urea, 77.50 g of potassium dihydrogen phosphate, and 66.25 g of potassium sulfate, stir and dissolve for 26 minutes, and then add 21.75 g of seaweed extract and stir evenly for 14 minutes.

[0067] S5. Preparation of sustained-release carrier: In a 2-L three-necked flask, add 650 mL of deionized water. Under the condition of controlling the temperature at 57 °C through a constant-temperature water bath, add 30.75 g of polyaspartic acid with a molecular weight of 9500 Da and stir to dissolve. Subsequently, add 16.25 g of glycerol and 12.50 g of sorbitol, and stir evenly for 29 minutes.

[0068] S6. Activation treatment of TDMC: Preheat 500 mL of TDMC solution to 29 °C, slowly add 25 mL of 0.5% citric acid solution to adjust the pH to 6.3, add 4.80 g of antioxidant vitamin C and 1.90 g of UV inhibitor p-hydroxybenzoate, and stir slightly to dissolve for 16 minutes.

[0069] S7. Integration of composite system: Slowly add the TDMC activation solution into the sustained-release carrier solution, control the feeding rate at 5.5 mL / min, maintain the temperature at 46 °C, and the stirring speed at 310 rpm. React for 42 minutes, then slowly add the mixed solution into the trace element chelating solution prepared in the first step, control the temperature at 43 °C, and the stirring speed at 260 rpm. Mix for 32 minutes, and then slowly add the macronutrient nutrient solution into the above mixed system and continue to stir evenly for 26 minutes.

[0070] S8. Stabilization treatment of the product: Use 1 mol / L potassium hydroxide solution to adjust the pH value of the final liquid fertilizer to 7.1. Add 8.75 g of polyethylene oxide with a molecular weight of 4000 as a stabilizer and 15.75 g of antifreeze propylene glycol, and stir evenly for 21 minutes to obtain a composite liquid fertilizer containing tylosin metabolite-derived finished liquid. Filter the prepared liquid fertilizer through a 100-mesh nylon filter, fill the product into a pre-sterilized high-density polyethylene container, and seal and store it.

[0071] After testing, the pH value of the prepared product is 7.1, the density is 1.19 g / cm³, the suspension rate is 97.5%, and the effective content of TDMC is 4.15%.

[0072] Example 5 The preparation process of a composite liquid fertilizer containing tylosin metabolite derivatives is as follows: S1. Preparation of tylosin metabolite complex TDMC: Inoculate Bacillus subtilis strain BY-7 into LB liquid medium, and cultivate it at 27.5 °C with shaking at 195 rpm for 17 hours. Subsequently, take 5% of the activated bacterial liquid and inoculate it into the expansion culture medium, and cultivate it at 28.2 °C with shaking at 177 rpm for 11.5 hours to obtain a bacterial liquid with an OD600 of 2.1. In a 30 L bioreactor, add 20 L of transformation medium, sterilize it at 121 °C for 20 minutes, cool it to 30 °C, then inoculate 2 L of the expanded bacterial liquid, and pre-culture it at 27.8 °C, with a dissolved oxygen of 39% and a stirring speed of 195 rpm for 4 hours. Then, add an aqueous solution of 500 g of tylosin finished product at a dropping rate of 11 mL / min, adjust the pH to 7.0 with 0.1 mol / L NaOH, and react at 29.8 °C for 49 hours. After the reaction, the tylosin conversion rate reaches 99.2% as detected. Centrifuge the fermentation broth at a rotational speed of 7800 rpm to separate, collect the supernatant, concentrate it to 1 / 5 of the original volume through an ultrafiltration system, use an AB-8 type macroporous adsorption resin adsorption column chromatography, elute it successively with 20%, 40%, 60%, and 80% ethanol, collect the elution components, and concentrate them under reduced pressure to obtain the TDMC complex. Dissolve TDMC in deionized water and adjust the concentration to 20 g / L as the active ingredient for subsequent preparation.

[0073] S2. Preparation of organic acid matrix: In a 5 L stainless steel reactor, add 2800 mL of deionized water, heat it to 56 °C, and add 87.50 g of food-grade citric acid, 43.75 g of malic acid, and 21.88 g of succinic acid in batches, and stir and dissolve for 33 minutes to form an organic acid basic solution.

[0074] S3. Preparation of trace element chelation system: Adjust the temperature of the above reactor to 67 °C, add 66.25 g of humic acid, stir evenly for 43 minutes, and then slowly add 16.25 g of zinc sulfate heptahydrate, 13.00 g of manganese sulfate heptahydrate, 3.40 g of copper sulfate heptahydrate, 13.75 g of ferrous sulfate heptahydrate, 2.30 g of ammonium molybdate, and 5.15 g of borax in batches, keep the pH value within the range of 5.6, and stir and react for 118 minutes to form a trace element chelation solution.

[0075] S4. Preparation of macronutrient nutrient solution: In a 2 L glass reactor, add 850 mL of deionized water, add 113.75 g of food-grade urea, 79.50 g of potassium dihydrogen phosphate, and 68.75 g of potassium sulfate, stir and dissolve for 24 minutes, and then add 23.25 g of seaweed extract and stir evenly for 16 minutes.

[0076] S5. Preparation of sustained-release carrier: In a 2-L three-necked flask, add 650 mL of deionized water. Under the condition of controlling the temperature at 59 °C through a constant-temperature water bath, add 32.00 g of polyaspartic acid and stir to dissolve. Subsequently, add 17.25 g of glycerol and 13.50 g of sorbitol, and stir evenly for 31 minutes.

[0077] S6. Activation treatment of TDMC: Preheat 500 mL of TDMC solution to 31 °C, slowly add 25 mL of 0.5% citric acid solution to adjust the pH to 6.4, add 4.90 g of antioxidant vitamin C and 1.95 g of UV inhibitor p-hydroxybenzoate, and stir slightly to dissolve for 14 minutes.

[0078] S7. Integration of composite system: Slowly add the TDMC activation solution to the sustained-release carrier solution, control the feeding rate at 4.5 mL / min, maintain the temperature at 47 °C, and the stirring speed at 290 rpm. React for 38 minutes, then slowly add the mixed solution to the trace element chelating solution prepared in the first step, control the temperature at 41 °C, and the stirring speed at 240 rpm. Mix for 28 minutes, and then slowly add the macronutrient nutrient solution to the above mixed system and continue to stir evenly for 24 minutes.

[0079] S8. Stabilization treatment of the product: Use 1 mol / L potassium hydroxide solution to adjust the pH value of the final liquid fertilizer to 6.9. Add 8.00 g of stabilizer polyethylene oxide and 14.75 g of antifreeze propylene glycol, and stir evenly for 19 minutes to obtain a composite liquid fertilizer containing tylosin finished product solution. Filter the prepared liquid fertilizer through a 100-mesh nylon filter, fill the product into a pre-sterilized high-density polyethylene container, and seal and store it.

[0080] After testing, the pH value of the prepared product is 6.9, the density is 1.17 g / cm³, the suspension rate is 98.5%, and the effective content of TDMC is 3.95%.

[0081] Comparative Example 1 The preparation process of a composite liquid fertilizer containing tylosin finished product is as follows. In this comparative example, an aqueous solution of tylosin finished product is used instead of the TDMC complex: S1. Dissolve 500 g of tylosin finished product in 3000 mL of deionized water, and adjust the pH to 6.8 with 0.1 mol / L NaOH to obtain an aqueous solution of tylosin with a content of 33.3 g / L.

[0082] S2. Preparation of organic acid matrix: In a 5-L stainless steel reactor, add 2800 mL of deionized water, heat to 55 °C, and add 85.50 g of food-grade citric acid, 43.25 g of malic acid, and 21.75 g of succinic acid in batches, and stir to dissolve for 30 minutes to form an organic acid base solution.

[0083] S3. Preparation of trace element chelation system: Adjust the temperature of the above reaction kettle to 65 °C, add 67.50 g of humic acid, stir evenly for 45 minutes, and then slowly add 15.75 g of zinc sulfate heptahydrate, 12.60 g of manganese sulfate heptahydrate, 3.35 g of copper sulfate heptahydrate, 13.40 g of ferrous sulfate heptahydrate, 2.25 g of ammonium molybdate and 4.95 g of borax in batches. Keep the pH value within the range of 5.5 and stir for 120 minutes to form a trace element chelation solution.

[0084] S4. Preparation of macronutrient nutrient solution: In a 2 L glass reactor, add 850 mL of deionized water, add 112.50 g of food-grade urea, 78.35 g of potassium dihydrogen phosphate, and 67.25 g of potassium sulfate, stir and dissolve for 25 minutes, and then add 22.50 g of seaweed extract and stir evenly for 15 minutes.

[0085] S5. Preparation of slow-release carrier: In a 2 L three-necked flask, add 650 mL of deionized water, control the temperature at 58 °C through a constant temperature water bath, add 31.25 g of polyaspartic acid, and stir to dissolve. Then add 16.75 g of glycerol and 13.25 g of sorbitol, and stir evenly for 30 minutes.

[0086] S6. TDMC activation treatment: Preheat 300 mL of tylosin solution to 30 °C, slowly add 25 mL of 0.5% citric acid solution to adjust the pH to 6.2, add 4.75 g of antioxidant vitamin C and 1.85 g of UV inhibitor p-hydroxybenzoate, and stir slightly to dissolve for 15 minutes.

[0087] S7. Integration of composite system: Slowly add the activated tylosin solution into the slow-release carrier solution, control the feeding rate at 5 mL / min, maintain the temperature at 45 °C, and the stirring speed at 300 rpm. React for 40 minutes, then slowly add the mixed solution into the trace element chelation solution prepared in the first step, control the temperature at 42 °C, and the stirring speed at 250 rpm. Mix for 30 minutes, and then slowly add the macronutrient nutrient solution into the above mixed system and continue to stir evenly for 25 minutes.

[0088] S8. Stabilization treatment of product: Use 1 mol / L potassium hydroxide solution to adjust the pH value of the final liquid fertilizer to 7.0, add 8.35 g of stabilizer polyethylene oxide and 15.25 g of antifreeze propylene glycol, and stir evenly for 20 minutes to obtain a composite liquid fertilizer containing the finished tylosin solution. Filter the prepared liquid fertilizer through a 100-mesh nylon filter, fill the product into a pre-sterilized high-density polyethylene container, and seal and store it.

[0089] After testing, the pH value of the prepared product is 7.0, the density is 1.17 g / cm³, the suspension rate is 96%, and the effective tylosin content is 0.4%.

[0090] Comparative Example 2 The preparation process of a conventional organic liquid fertilizer is as follows: In this comparative example, a conventional organic trace element liquid fertilizer formula is used.

[0091] S1. In a 5L stainless steel reactor, add 2800 mL of deionized water, heat to 55 °C, and add 85.50 g of food-grade citric acid, 43.25 g of malic acid, and 21.75 g of succinic acid in batches, and stir and dissolve for 30 minutes to form an organic acid base solution.

[0092] S2. Preparation of trace element chelation system: Adjust the temperature of the above reactor to 65 °C, add 67.50 g of humic acid, stir evenly for 45 minutes, and slowly add 15.75 g of zinc sulfate heptahydrate, 12.60 g of manganese sulfate heptahydrate, 3.35 g of copper sulfate heptahydrate, 13.40 g of ferrous sulfate heptahydrate, 2.25 g of ammonium molybdate, and 4.95 g of borax in batches, and keep the pH value within the range of 5.5, and stir and react for 120 minutes to form a trace element chelate solution.

[0093] S3. Preparation of macronutrient nutrient solution: In a 2L glass reactor, add 850 mL of deionized water, add 112.50 g of food-grade urea, 78.35 g of potassium dihydrogen phosphate, and 67.25 g of potassium sulfate, and stir and dissolve for 25 minutes. Then add 22.50 g of seaweed extract and stir evenly for 15 minutes.

[0094] S4. Carrier preparation: In a 2L three-necked flask, add 950 mL of deionized water, control the temperature at 58 °C through a constant temperature water bath, add 31.25 g of polyaspartic acid, stir and dissolve, add 16.75 g of glycerol and 13.25 g of sorbitol, and stir evenly for 30 minutes.

[0095] S5. Integration of composite system: Slowly add the carrier solution to the trace element chelate solution, control the temperature at 42 °C, the stirring speed at 250 rpm, and mix for 30 minutes. Slowly add the macronutrient nutrient solution to the above mixing system and continue to stir evenly for 25 minutes.

[0096] S6. Stabilization treatment of the product: Use 1mol / L potassium hydroxide solution to adjust the pH value of the final liquid fertilizer to 7.0. Add 8.35 g of stabilizer polyethylene oxide and 15.25 g of antifreeze propylene glycol, stir evenly for 20 minutes to obtain the liquid fertilizer. Finally, filter the prepared liquid fertilizer through a 100-mesh nylon filter, fill the product into a pre-sterilized high-density polyethylene container, and seal and store it.

[0097] After testing, the pH value of the prepared product is 7.1, the density is 1.18 g / cm³, and the suspension rate is 97%.

[0098] Comparative Example 3 The preparation process of tylosin metabolite liquid fertilizer without using organic chelation technology is as follows: S1. Preparation of tylosin metabolite complex TDMC: This step is the same as step S1 of Example 1.

[0099] S2. Preparation of conventional liquid fertilizer matrix: In a 5L stainless steel reactor, add 3500 mL of deionized water, heat to 55 °C, and directly add trace element inorganic salts, including 15.75 g of zinc sulfate heptahydrate, 12.60 g of manganese sulfate heptahydrate, 3.35 g of copper sulfate heptahydrate, 13.40 g of ferrous sulfate heptahydrate, 2.25 g of ammonium molybdate, and 4.95 g of borax, and stir to dissolve for 30 minutes.

[0100] S3. Preparation of macronutrient nutrient solution: In a 2L glass reactor, add 850 mL of deionized water, add 112.50 g of food-grade urea, 78.35 g of potassium dihydrogen phosphate, and 67.25 g of potassium sulfate, and stir to dissolve for 25 minutes.

[0101] S4. Activation treatment of TDMC: Preheat 500 mL of TDMC solution to 30 °C, slowly add 25 mL of 0.5% citric acid solution to adjust the pH to 6.2, add 4.75 g of antioxidant vitamin C and 1.85 g of UV inhibitor p-hydroxybenzoate, and stir slightly to dissolve for 15 minutes.

[0102] S5. Integration of composite system: Directly add the TDMC activation solution to the trace element solution, control the temperature at 42 °C, the stirring speed at 250 rpm, mix for 30 minutes, and slowly add the macronutrient nutrient solution to the above mixed system, and continue to stir evenly for 25 minutes.

[0103] S6. Stabilization treatment of the product: Use 1mol / L potassium hydroxide solution to adjust the pH value of the final liquid fertilizer to 7.0, add 8.35 g of stabilizer polyethylene oxide and 15.25 g of antifreeze propylene glycol, stir evenly for 20 minutes to obtain the liquid fertilizer, filter the liquid fertilizer through a 100-mesh nylon filter, and fill the product into a pre-sterilized high-density polyethylene container and seal it for storage.

[0104] After testing, the pH value of the prepared product is 7.0, the density is 1.16 g / cm³, the suspension rate is 94%, and the effective content of TDMC is 4.0%.

[0105] Performance testing and data analysis Testing method 1. Product stability test Store the samples of each example and comparative example at room temperature and under light conditions for 30 days, and measure the following indicators: Suspension rate: Take 100 ml of the sample, and observe the sediment content after standing for 24 hours; TDMC content retention rate: The change in TDMC content before and after storage was determined by HPLC-MS method; pH value change: The change in pH value before and after storage was determined using a pH meter.

[0106] 2. Immune induction activity test A pot experiment was conducted. Tomato varieties susceptible to bacterial wilt were selected as test plants, with 10 pots in each group and 3 replicates.

[0107] Treatment method: The sample was applied by root irrigation after dilution 500 times, once every 7 days for 3 consecutive times.

[0108] Sampling time: 3 days after the last treatment.

[0109] Test indicators: Relative expression level of PR protein: Determined by qRT-PCR method; Chitinase activity: Determined by colorimetric method; Salicylic acid and jasmonic acid contents: Determined by HPLC method.

[0110] 3. Disease resistance evaluation A pot experiment was conducted. Tomato varieties susceptible to bacterial wilt were selected, with 15 plants in each group and 3 replicates.

[0111] Treatment method: The sample was applied by root irrigation after dilution 500 times, once every 7 days for 2 consecutive times.

[0112] Inoculation method: 7 days after the last treatment, the bacterial wilt pathogen suspension (OD600 = 0.1) was inoculated artificially.

[0113] Test indicators: Incidence rate: The proportion of diseased plants was investigated 14 days after inoculation; Disease index: Calculated according to the disease grading of 0 - 4 levels; Control efficacy: Compared with the clear water control group.

[0114] 4. Growth promotion effect evaluation A pot experiment was conducted. Tomato seedlings were selected, with 10 pots in each group and 3 replicates.

[0115] Treatment method: The sample was applied by root irrigation after dilution 500 times, once every 7 days for 4 consecutive times.

[0116] Test indicators: Plant height growth rate: Calculate the percentage increase in plant height before and after treatment; Root development indicators: Determine root length, root surface area, and root volume; Biomass growth: Determine the dry and fresh weights of plants 30 days after treatment.

[0117] Test results Table 1. Record Table of Product Stability Test Results for Each Example and Comparative Example Sample Suspension rate after 30 days (%) Retention rate of TDMC content (%) pH value change Trace element precipitation situation Example 1 96.30% 97.50% 0.2 No obvious precipitation Example 2 95.80% 97.20% 0.3 No obvious precipitation Example 3 97.20% 98.10% 0.2 No obvious precipitation Example 4 96.50% 97.80% 0.2 No obvious precipitation Example 5 96.80% 98.00% 0.1 No obvious precipitation Comparative example 1 93.50% - 0.6 Slight precipitation Comparative example 2 95.00% - 0.3 Slight precipitation Comparative example 3 83.70% 76.50% 0.8 Obvious precipitation Table 2. Record Table of Product Immune Induction Activity Test Results for Each Example and Comparative Example Sample Relative expression level of PR protein Chitinase activity (U / mg) Salicylic acid content (μg / g) Jasmonic acid content (ng / g) Example 1 3.56 5.75 18.35 235.6 Example 2 3.75 5.92 19.08 245.3 Example 3 3.45 5.63 17.86 230.5 Example 4 3.65 5.85 18.65 240.8 Example 5 3.5 5.78 18.25 237.2 Comparative example 1 2.35 3.25 12.35 156.8 Comparative example 2 1.05 1.26 5.86 65.3 Comparative example 3 2.58 3.56 13.25 168.5 Water control 1 1.15 5.25 60.8 Table 3. Disease Resistance Evaluation Results of Products for Each Example and Comparative Example Sample Incidence rate (%) Disease index Control efficacy (%) Root infection rate (%) Example 1 25.30% 12.6 72.50% 20.50% Example 2 22.80% 11.5 74.80% 18.60% Example 3 27.50% 13.8 70.00% 22.30% Example 4 24.60% 12.3 73.20% 19.80% Example 5 26.20% 13.1 71.50% 21.20% Comparative example 1 45.60% 25.3 45.00% 38.50% Comparative example 2 82.50% 45.6 1.20% 76.80% Comparative example 3 42.30% 23.5 49.00% 35.60% Water control 83.50% 46.2 - 78.20% Table 4. Record Table of Evaluation Results of Growth Promotion Effects of Products for Each Example and Comparative Example Sample Plant height growth rate (%) Root length increase (%) Root surface area increase (%) Dry weight increase (%) Example 1 35.60% 42.50% 48.30% 38.60% Example 2 37.80% 44.30% 50.60% 40.30% Example 3 34.50% 41.20% 47.50% 37.80% Example 4 36.50% 43.50% 49.20% 39.50% Example 5 35.20% 42.80% 48.70% 38.20% Comparative example 1 27.30% 30.50% 32.60% 28.50% Comparative example 2 25.60% 28.30% 30.20% 26.30% Comparative example 3 28.20% 31.60% 33.80% 29.20% Water control 15.30% 16.50% 17.20% 14.80% Data Analysis As can be seen from Table 1, the products of Examples 1-5 all showed excellent stability after being stored at room temperature for 30 days. The suspension rate remained between 95.8% and 97.2%, the retention rate of TDMC content was as high as 97.2% - 98.1%, the change in pH value was only 0.1 - 0.3, and there was no obvious precipitation of trace elements. In contrast, in Comparative Example 1, the untransformed tylosin was used, and the retention rate of its active ingredient was only 85.3%, indicating that the original tylosin had poor stability in the liquid fertilizer system; in Comparative Example 3, the organic chelation technology was not adopted, resulting in the suspension rate dropping to 83.7%, the retention rate of TDMC content being only 76.5%, obvious precipitation of trace elements occurring, and the change in pH value reaching 0.8. This fully proves that the humic acid chelation system and the polyaspartic acid slow-release carrier in the present invention can increase the product stability, and the two have a synergistic effect.

[0118] As can be seen from Table 2, Examples 1-5 showed significant effects in plant immune induction. The relative expression level of PR protein reached 3.45 - 3.75 times, the chitinase activity reached 5.63 - 5.92 U / mg, and the contents of salicylic acid and jasmonic acid increased to 17.86 - 19.08 μg / g and 230.5 - 245.3 ng / g respectively, far higher than those of the clear water control group. Although Comparative Example 1 contained tylosin, the expression level of PR protein was only 2.35 times, and the chitinase activity was only 3.25 U / mg, about 43% lower than that of the examples, indicating that the TDMC complex has stronger plant immune induction activity than the original tylosin; Comparative Example 2 did not contain immune induction components, and its immune-related indexes were similar to those of the clear water control; although Comparative Example 3 contained TDMC, it lacked an effective carrier system, resulting in its immune induction effect being significantly lower than that of the example group, only about 70% of that of the examples. This shows that the chelation carrier system designed in the present invention can enhance the immune induction activity of TDMC.

[0119] As can be seen from Table 3, Examples 1-5 showed excellent prevention and control effects against tomato bacterial wilt, with the incidence rate controlled at 22.8% - 27.5%, the disease index being 11.5 - 13.8, and the control effect reaching 70.0% - 74.8%. In comparison, the control effect of Comparative Example 1 was only 45.0%, that of Comparative Example 3 was 49.0%, and Comparative Example 2 had almost no disease prevention effect. The root infection rate of the Example group was also significantly lower than that of the control group, only being 18.6% - 22.3%, while the root infection rates of Comparative Example 1 and Comparative Example 3 were 38.5% and 35.6% respectively, and that of Comparative Example 2 was as high as 76.8%, approaching 78.2% of the clear water control group. These results fully demonstrate that the TDMC complex retains significant plant disease resistance induction ability after biotransformation, and through the chelating carrier system of the present invention, this ability can be more effectively exerted, with the effect being increased by about 50% compared to directly using tylosin or without using the chelating carrier system.

[0120] As can be seen from Table 4, Examples 1 - 5 also showed significant effects in promoting plant growth. The plant height growth rate reached 34.5% - 37.8%, the root length increased by 41.2% - 44.3%, the root surface area increased by 47.5% - 50.6%, and the dry weight increased by 37.8% - 40.3%. In contrast, the various growth indexes of Comparative Examples 1 - 3 were only about 70% - 75% of those of the Example group, but were still significantly higher than those of the clear water control group. This indicates that the TDMC compound liquid fertilizer of the present invention not only has excellent plant immune induction function, but also can effectively promote plant growth and development through the optimized trace element chelating system and slow-release structure, especially having a significant promoting effect on root development, with the increase amplitude of the root surface area reaching nearly 3 times that of the clear water control group. The root development indexes of the Example group were on average about 45% higher than those of the Comparative Example group, which proves that the multifunctional carrier system in the present invention can not only improve the immune induction effect of TDMC, but also significantly improve the biological utilization rate of trace elements, achieving the synergistic effect of nutrient supply and immune induction.

[0121] In summary, the compound liquid fertilizer containing tylosin metabolite derivatives proposed by the present invention obtains the TDMC complex through biotransformation technology, and realizes the stabilization and slow-release of the active ingredients through the multifunctional chelating carrier system, showing significant advantages in aspects such as product stability, plant immune induction, disease resistance, and growth promotion effect, with a performance improvement of about 40% - 50% compared to traditional methods.

[0122] Although the embodiments of the present invention have been shown and described, for those of ordinary skill in the art, it can be understood that various changes, modifications, substitutions, and variations can be made to these embodiments without departing from the principles and spirits of the present invention. All equivalent changes and improvements made within the scope of the application of the present invention should still fall within the scope covered by the patent of the present invention.

Claims

1. A composite liquid fertilizer containing tylosin finished liquid, characterized in that, include: Tylosin metabolite complex TDMC, humic acid, seaweed extract, urea, potassium dihydrogen phosphate, potassium sulfate, organic acid complex, polyaspartic acid, polyol complex, trace element compound, stabilizer, antifreeze agent, antioxidant, preservative and deionized water; The tylosin metabolite complex TDMC is a metabolite having no antibacterial activity but retaining plant immunity inducing function obtained by transforming tylosin through a biotransformation method.

2. The composite liquid fertilizer according to claim 1, characterized in that The components are calculated by weight, including: 8-12 parts of tylosin metabolite complex TDMC, 5-9 parts of humic acid, 1-4 parts of seaweed extract, 8-15 parts of urea, 5-10 parts of potassium dihydrogen phosphate, 5-9 parts of potassium sulfate, 12-18 parts of organic acid complex, 2-5 parts of polyaspartic acid, 2-5 parts of polyol complex, 3-8 parts of trace element compounds, 0.5-2 parts of stabilizer, 1-3 parts of antifreeze agent, 0.3-1 parts of antioxidant and preservative and 40-60 parts of deionized water.

3. The composite liquid fertilizer according to claim 1, characterized in that The tylosin metabolite complex TDMC is prepared by the following steps: (a) Activation and expansion of strains: Inoculate Bacillus subtilis BY-7 strain into liquid culture medium, and culture at 27-29°C with shaking at 160-200 rpm for 16-20 hours. Then, take 5% of the activated bacterial solution and inoculate into expansion culture medium, and culture at 27-29°C with shaking at 160-200 rpm for 11-13 hours. (b) Biotransformation reaction: inoculate the culture solution into the transformation medium, pre-culture for 4 hours at 27-29°C, dissolved oxygen 35%-45%, and stirring speed 180-220 rpm, then add the tylosin finished solution, and carry out the biotransformation reaction at 29-31°C for 46-50 hours; (c) Metabolite extraction and purification: After the reaction is completed, the fermentation broth is centrifuged and the supernatant is collected and concentrated by ultrafiltration. Then, a macroporous adsorption resin is used for column chromatography and eluted with 20%, 40%, 60%, and 80% ethanol in sequence. The eluted components are collected and concentrated under reduced pressure to obtain the TDMC complex.

4. The composite liquid fertilizer according to claim 1, characterized in that The trace element compound is selected from one or more of zinc sulfate heptahydrate, manganese sulfate heptahydrate, copper sulfate heptahydrate, iron sulfate heptahydrate, ammonium molybdate and borax.

5. The composite liquid fertilizer according to claim 1, characterized in that The organic acid complex comprises citric acid, malic acid and succinic acid in a mass ratio of 4:2:1; the polyol complex comprises glycerol and sorbitol in a mass ratio of 1.3:

1.

6. The composite liquid fertilizer according to claim 1, characterized in that The molecular weight of the humic acid is 3000-5000Da; the molecular weight of the polyaspartic acid is 8000-12000Da; and the content of alginate in the seaweed extract is ≥18%.

7. The composite liquid fertilizer according to claim 1, characterized in that The stabilizer is polyethylene oxide; the antifreeze agent is propylene glycol; the antioxidant is vitamin C; and the preservative is paraben.

8. A method for preparing the composite liquid fertilizer according to any one of claims 1 to 7, characterized in that: The following steps are involved: (1) Preparation of tylosin metabolite complex TDMC: Tylosin was converted into a metabolite complex TDMC without antibacterial activity by biotransformation using Bacillus subtilis BY-7 strain; (2) Preparation of organic acid matrix: Add deionized water to a reaction kettle, heat to 53-57°C, add citric acid, malic acid and succinic acid, stir and dissolve to form an organic acid base solution; (3) Preparation of trace element chelating system: adjust the temperature of the reaction kettle obtained in step (2) to 62-68°C, add humic acid and stir evenly, then add trace element compounds, maintain the pH value in the range of 5.2-5.8, and stir to react to form a trace element chelating liquid; (4) Preparation of macronutrient solution: Add deionized water, urea, potassium dihydrogen phosphate, and potassium sulfate into the reactor, stir to dissolve, and then add seaweed extract and stir evenly; (5) Preparation of sustained-release carrier: In a three-necked flask, add deionized water, add polyaspartic acid and stir to dissolve at 56-60°C, then add glycerol and sorbitol and stir evenly; (6) TDMC activation treatment: preheat the TDMC solution prepared in step (1) to 28-32° C., add citric acid solution to adjust the pH to 6.0-6.5, add antioxidant and preservative, and stir to dissolve; (7) Composite system integration: add the TDMC activation solution obtained in step (6) to the slow-release carrier solution obtained in step (5), control the temperature at 42-48°C, and stir to react; then add the mixed solution to the trace element chelate solution obtained in step (3), control the temperature at 40-44°C, and stir to mix; then add the macronutrient solution obtained in step (4) to the above mixed system and stir evenly; (8) Stabilization treatment: Use potassium hydroxide solution to adjust the pH value of the final liquid fertilizer to 6.8-7.2, add stabilizer and antifreeze agent, and stir evenly; (9) Filtration and filling: Filter the prepared liquid fertilizer through a nylon filter, fill it into a high-density polyethylene container under sterile conditions, and store it in a sealed container.

9. The preparation method according to claim 8, characterized in that: In step (3), the stirring reaction time is 110 to 130 minutes; In step (6), TDMC activation was performed using 0.5% citric acid solution for pH adjustment; In step (7), the rate of adding TDMC activation solution to the sustained-release carrier solution is 4-6 mL / min, the stirring speed is 280-320 rpm, and the reaction time is 35-45 minutes; when the trace element chelating solution is added to the mixed solution of TDMC and the sustained-release carrier, the stirring speed is 230-270 rpm, and the mixing time is 25-35 minutes.

10. Use of the composite liquid fertilizer according to any one of claims 1 to 7 in promoting crop growth and enhancing plant immunity, characterized in that: Apply 500-1000 times diluted liquid fertilizer to the roots of crops through root irrigation, once every 7-14 days, to promote crop growth and development and improve plant resistance to diseases such as bacterial wilt and wilt.