Microbial water-soluble fertilizer and preparation method thereof

Abstract

The application discloses a microbial water-soluble fertilizer and a preparation method thereof, and belongs to the technical field of bio-organic fertilizer.The water-soluble fertilizer comprises the following components: nutrient elements, fish hydrolyzed protein, low-temperature-resistant microorganisms, plant growth regulating components, a surfactant and water.The water-soluble fertilizer is added with nitrogen, phosphorus, potassium and other macronutrient elements and calcium, magnesium, zinc and other micronutrient elements, so that sufficient nutrient substances are provided for crops.The water-soluble fertilizer is added with plant growth regulating components composed of 5-aminoacetyl propionic acid, trehalose and amine ester, and the three components are used in combination, so that the adaptability of plants to a low-temperature environment can be effectively improved.Two low-temperature-resistant microorganisms, i.e., new cold-tolerant branch actinomyces and Tianshan branch actinomyces, are selected and added, and the two cold-tolerant fungi work together to improve the stress resistance of crops.The water-soluble fertilizer is used in combination with spraying and flushing, so that the low-temperature growth of tomatoes is effectively promoted, the stress resistance and quality of crops are improved, and the application prospect is wide.

Description

TECHNICAL FIELD

[0001] The application belongs to the technical field of bio-organic fertilizer, and particularly relates to a microbial water-soluble fertilizer and a preparation method thereof. BACKGROUND

[0002] Tomato (Lycopersicon esculentum Mill.) is an important economic crop with high nutritional value and wide application, and its cultivation area is expanding. However, in the development process of facility tomatoes, a series of problems have emerged, especially in recent years, with the frequent occurrence of extreme weather and the continuous deterioration of the living environment of crops, which has seriously affected the quality and yield of facility tomatoes. Facility tomatoes in northern winter and spring crops are always threatened by low temperature stress, which is the primary reason for limiting their growth and development. Low temperature leads to low temperature stress on plants, and their growth and development are seriously hindered. If the duration is too long, it will eventually lead to plant death. In addition, the damage to plant cells is also irreversible. Therefore, the current common means to alleviate low temperature stress is to achieve tomato seedling growth by applying new fertilizers, to regulate the physiological process in the plant body, to enhance the resistance of the plant itself, and to prevent or resist the harm of low temperature stress to the plant. The advantage of this method is that the fertilizer required by farmers is easy to obtain in agricultural production activities, and the operation is simple and fast.

[0003] For example, Chinese patent application CN201910636754.9 discloses a preparation method of a carbon-based bio-organic fertilizer for planting tomatoes, which comprises the following steps: (1) micro-carbonization treatment of tomato straw; (2) mixing the micro-carbonized straw with the above-ground stems and leaves of Glehnia littoralis, the above-ground stems and leaves of Platycodon grandiflorum, and livestock and poultry manure; (3) aerobic fermentation of the mixture; (4) addition of nutrient components to the fermented and decomposed material; and (5) low-temperature drying treatment. The positive effects of the present application are as follows: the organic matter content is high, the activity of beneficial microorganisms is good, the biomass carbon produced in the micro-carbonization process directly participates in the composting fermentation process, the porous structure fully absorbs nutrients and hosts a large number of beneficial microorganisms; the fully decomposed livestock and poultry manure, the above-ground stems and leaves of Glehnia littoralis and Platycodon grandiflorum are used as the main components of the fertilizer, which effectively alleviates the continuous cropping obstacles of tomatoes; reasonable nutrient ratio is more conducive to the growth of tomatoes and improves the fertilizer nutrient utilization rate. In addition, a large amount of above-ground stems and leaves produced during the local planting of Glehnia littoralis, Platycodon grandiflorum and tomatoes, and a large amount of excrement and urine produced in the cattle industry can be used, thereby avoiding pollution to the environment.

[0004] Similar fertilizer for improving the ability of crops to resist low temperature is also disclosed in Chinese patent application CN201911298313.9, which discloses a fertilizer for improving the low-temperature cold resistance of rice and a preparation method and application thereof. The preparation raw materials of the fertilizer include the following components by weight: shrimp and crab shell powder (25-28) parts, wood ash (30-35) parts, puerarin (5-8) parts, zinc gluconate (3-5) parts, ferric humate (2-5) parts, amino acid chelated rare earth (1-3) parts, boric acid (1-3) parts, and probiotic compound (7-10) parts. The fertilizer can not only provide cold resistance and low-temperature resistance of rice, but also promote rice rooting, increase thousand-grain weight, and achieve the effect of increasing yield and income.

[0005] However, the existing technologies at present are all solid fertilizers, which have slow and unstable effects after being applied to the soil, and poor specificity, complex fertilizer components, low activity, and low practical application value. SUMMARY

[0006] The present application provides a new type of microbial fertilizer to solve the problems in the prior art. The fertilizer can be used by spraying and flushing to enhance the absorption and utilization of nutrient elements by plants under low temperature conditions, effectively promote the growth of tomatoes under low temperature conditions, and improve the resistance and quality of crops.

[0007] To achieve the above technical purposes, the technical scheme adopted by the present application is as follows:

[0008] A microbial type water-soluble fertilizer, which comprises, by weight percentage, 12-25% of nutrient elements, 5-10% of fish hydrolyzed protein, 5-10% of low-temperature resistant microorganisms, 0.5-1.5% of plant growth regulating components, 0.1-0.5% of surfactants, and the balance of water, totaling 100%.

[0009] Further, the nutrient elements include macroelements and microelements; the macroelements include at least two of nitrogen, phosphorus, and potassium nutrient elements; the nitrogen element is ammonium nitrate; the phosphorus is potassium dihydrogen phosphate; and the potassium is potassium nitrate. The microelements are one or more of calcium nitrate, magnesium nitrate, copper sulfate, manganese sulfate, and EDTA-zinc.

[0010] Further, the preparation method of the fish hydrolyzed protein is as follows:

[0011] (1) Put the discarded fish skin into a container, add water to cover the fish skin, and homogenize to obtain fish skin slurry;

[0012] (2) Add 0.1-0.3% of alkaline protease based on the mass of the fish skin to the fish skin slurry, adjust the pH to 9-11, and the temperature to 40-55℃, and then perform enzymatic hydrolysis in a water bath for 8-12h, and filter with double-layer gauze to obtain an enzymatic hydrolysis liquid;

[0013] (3) the enzymatic hydrolysate obtained in step (2) is added with 5% ferrous sulfate and 2.5% ascorbic acid by weight of the enzymatic hydrolysate, adjusted to pH 4-5, and placed in a constant-temperature shaker at 50-60°C for 2-3h; then the reaction solution is centrifuged at 3000-5000 r / min for 10-20 min to remove the precipitate residue, and the filtrate is dried to obtain fish hydrolyzed protein.

[0014] Further, the waste fish skin in step (1) is one of tilapia skin, grass carp skin, crucian carp skin and salmon skin.

[0015] Amino acids are ideal nutrients and have complexing ability, for example, glycine can increase the content of chlorophyll in plants, and amino acids are not only raw materials for synthesizing proteins, but also precursor substances for synthesizing other nitrogen-containing compounds, including nucleotides, hormones, alkaloids and polyamines, which play a very important role in plants. Fe is an important component of plant cytochrome and metalloenzyme, and plays an indispensable role in chlorophyll synthesis, DNA replication, active oxygen removal and electron transfer.

[0016] The application uses waste tilapia skin, grass carp skin, crucian carp skin and salmon skin as raw materials to prepare small-molecule bioactive peptides by enzymolysis. The peptides are composed of amino acids, and the amino acids of the peptides after enzymolysis are combined with iron to form stable chelates, which are more easily absorbed and utilized in plants. Not only can the growth conditions of crops be improved, but also the disease resistance and stress resistance of crops can be enhanced.

[0017] Further, the plant growth regulating component is a mixture of 5-aminoacetylpropionic acid, trehalose and aminexil in a mass ratio of 1:2:0.5.

[0018] The application adds a plant growth regulating component composed of 5-aminoacetylpropionic acid, trehalose and aminexil. The 5-aminoacetylpropionic acid not only has the effects of color conversion and sweetness enhancement, but also can improve the activity of antioxidant enzymes in plants, such as superoxide dismutase (SOD), catalase (CAT) and peroxidase (POD), thereby removing active oxygen (ROS) caused by low temperature and reducing the damage of low temperature to plant cells. Trehalose can promote energy metabolism of plants and improve the adaptability of plants in low temperature environment. Aminexil can increase the content of chlorophyll, protein and nucleic acid in plants, thereby enhancing the photosynthesis and carbon-nitrogen metabolism capacity of plants. These physiological changes help plants to maintain normal growth activities under low temperature conditions and improve their cold resistance. The combination of the three can effectively improve the adaptability of plants to low temperature environment and improve the quality of crops.

[0019] Further, the low-temperature-resistant and stress-resistant microorganism comprises a new cold-tolerant branch fungus with strain number CGMCC No.3.18032.Cladosporium neopsychrotolerans ) and the strain numbered as CGMCC No. 3.18033 Cladosporium tianshanense Cladosporium tianshanense ), and the preparation method is as follows: the strain numbered as CGMCC No. 3.18032 Cladosporium psychrotolerans and the strain numbered as CGMCC No. 3.18033 Cladosporium tianshanense are respectively inoculated into PDA plates for activation, cultured in a mold incubator at 15-20 DEG C for 10 days, cut into small mycelium blocks, respectively inoculated into PDB culture medium, cultured in a shaker at 15-20 DEG C for 10 days at a rotating speed of 120 r / min, filtered to collect mycelium by double-layer sterilized gauze, washed with sterilized water for 3 times, squeezed dry, mixed according to a mass ratio of 1:1, ground by a homogenizer for 1 min, prepared into a mycelium suspension with a concentration of 1x10 9 CFU / mL by using sterile water, and a low-temperature resistant microorganism is obtained.

[0020] The strain numbered as CGMCC No. 3.18032 Cladosporium psychrotolerans is purchased from the China General Microbiological Culture Collection Center, and the original preservation time is September 7, 2016; the strain numbered as CGMCC No. 3.18033 Cladosporium tianshanense is purchased from the China General Microbiological Culture Collection Center, and the original preservation time is September 7, 2016; both of the two strains can be purchased from the preservation center, and do not need to be repeatedly biologically preserved.

[0021] The preparation method of the PDA plate is as follows: 250 g of peeled potato is cut into pieces, boiled in ultrapure water for 25 min, and slightly cooled. After being filtered for three times by using eight layers of gauze, 30 g of glucose and 20 g of agar are added, and the volume is made up to 1000 ml by using ultrapure water. The steam sterilizer is sterilized at 121 DEG C for 20 min, poured into 90 mm sterilized plates, sealed and stored in a 4 DEG C refrigerator for standby.

[0022] The composition and preparation method of the PDB culture medium are as follows: 250 g of peeled potato is cut into pieces, boiled in ultrapure water for 25 min, and slightly cooled. After being filtered for three times by using eight layers of gauze, 30 g of glucose is added, and the volume is made up to 1000 ml by using ultrapure water. The steam sterilizer is sterilized at 121 DEG C for 20 min, sealed and stored in a 4 DEG C refrigerator for standby.

[0023] The application screens two low-temperature resistant microorganisms, new cold-resistant branch actinomycete and Tianshan branch actinomycete, which have good low-temperature survival ability, and can regulate the response of plants to low temperature by affecting the synthesis and metabolism of plant hormones such as abscisic acid (ABA) after colonizing the root system and leaves of tomatoes. Abscisic acid is an important plant growth regulator that can enhance the cold resistance of plants. At the same time, it can induce the formation of ice crystal protein in the plant cell wall, thereby enhancing the stability of the cell wall, reducing cell rupture caused by low temperature, and helping plants maintain the integrity of cell structure in cold environments. Acting on the soil can promote root development, thereby improving the plant's ability to absorb nutrients, helping plants maintain normal growth and metabolic activities under low temperature conditions, and improving crop yield and quality. The two cold-resistant fungi work together to improve the stress resistance of crops and ultimately improve yield and quality.

[0024] Further, the surfactant is a fatty alcohol polyoxyethylene ether or sodium dodecyl sulfate.

[0025] A preparation method of a microbial type water-soluble fertilizer, comprising the steps of:

[0026] (1) preparing fish hydrolyzed protein;

[0027] (2) preparing low-temperature resistant microorganisms;

[0028] (3) uniformly dispersing the nutrient elements, fish hydrolyzed protein, low-temperature resistant microorganisms, plant growth regulating components, and surfactants in water, and storing in the dark to obtain the water-soluble fertilizer.

[0029] The use method of the water-soluble fertilizer is: the water-soluble fertilizer is used at a dosage of 5-8 kg per mu for irrigation, and 3-5 kg per mu for leaf spraying. The water-soluble fertilizer is used at a dilution of 200 times for irrigation and 500 times for leaf spraying at the seedling stage, early flowering stage, and full flowering stage of tomatoes.

[0030] In summary, the beneficial effects of the technical scheme of the application are:

[0031] (1) The application adds nitrogen, phosphorus, potassium and other macronutrients, and calcium, magnesium, zinc and other micronutrients. Appropriate nitrogen fertilizer can promote the stem and leaf growth of tomato plants and improve photosynthetic efficiency. Phosphorus is mainly involved in energy conversion, photosynthesis and cell division in plants. Appropriate phosphorus fertilizer can promote flower bud differentiation, root development and seedling growth, and improve the stress resistance and fruit quality of tomatoes. Potassium has a significant effect on the strengthening of plant stems, the improvement of fruit quality and the enhancement of stress resistance. Potassium can improve the disease resistance of tomatoes and promote fruit ripening, making the fruit more plump. The micronutrients have a significant positive effect on improving the single fruit weight and yield of tomatoes, and enhancing the flavor and nutritional content.

[0032] (2) The plant growth regulating component composed of 5-amino levulinic acid, trehalose and aminexil is added, the three are used together, the adaptability of the plant to low temperature environment can be effectively improved, and the crop quality is improved;

[0033] (3) The two low-temperature resistant microorganisms, new cold-tolerant Cladosporium and Tian Mountain Cladosporium, are added, the two cold-tolerant fungi cooperate, the stress resistance of crops is improved, and finally the yield and quality are improved.

[0034] (4) The application of the present application can strengthen the absorption and utilization of nutrients by plants under low temperature conditions, effectively promote the low temperature growth of tomatoes, improve the resistance and quality of crops, and has a wide application prospect. BRIEF DESCRIPTION OF DRAWINGS

[0035] Figure 1 It is the strain antagonism diagram of the new cold-tolerant Cladosporium and Tian Mountain Cladosporium of the present application;

[0036] Figure 2 It is the single tomato yield experiment comparison of the field experiment of example 1 and comparative examples 1-9 and blank control of the present application. DETAILED DESCRIPTION

[0037] The technical solutions of the present application will be further described below in combination with specific embodiments, but are not limited thereto.

[0038] Example 1

[0039] A microbial type water-soluble fertilizer, which comprises, by weight percentage: nutrient elements 12%, fish hydrolyzed protein 5%, low-temperature resistant microorganisms 5%, plant growth regulating component 0.5%, surfactant 0.1%, and the balance is water, totaling 100%.

[0040] The nutrient elements include macroelements and microelements; the macroelements include nitrogen, phosphorus and potassium; the nitrogen is ammonium nitrate; the phosphorus is potassium dihydrogen phosphate; the potassium is potassium nitrate; the mass ratio of ammonium nitrate, potassium dihydrogen phosphate and potassium nitrate is 1:1:2. The microelements are calcium nitrate, magnesium nitrate and copper sulfate, and the mass ratio of the three is 1:3:0.5.

[0041] The preparation method of the fish hydrolyzed protein is as follows:

[0042] (1) The waste fish skin is placed in a container, water is added to cover the fish skin, and fish skin slurry is obtained by homogenization;

[0043] (2) 0.1% of alkaline protease of the fish skin mass is added to the fish skin slurry, the pH is adjusted to 9-11, the temperature is 40-55 DEG C, and the enzyme is hydrolyzed in water bath for 8h, and double-layer gauze is filtered to obtain the enzyme hydrolysate;

[0044] (3) the enzymatic hydrolysate obtained in step (2) is added with 5% ferrous sulfate and 2.5% ascorbic acid by mass of the enzymatic hydrolysate, adjusted to pH 4-5, and placed in a constant-temperature shaker at 50-60°C for constant-temperature shaking reaction for 2h; then the reaction solution is centrifuged at 3000 r / min for 10 min to remove the precipitate residue, and the filtrate is dried to obtain fish hydrolyzed protein.

[0045] The waste fish skin in step (1) is tilapia skin.

[0046] The plant growth regulating component is a mixture of 5-aminolevulinic acid, trehalose and amixtral in a mass ratio of 1:2:0.5.

[0047] The low-temperature resistant microorganism comprises a new cold-tolerant Cladosporium strain numbered CGMCC No.3.18032 and a Tianshan Cladosporium strain numbered CGMCC No.3.18033, and the preparation method is as follows: the new cold-tolerant Cladosporium strain numbered CGMCC No.3.18032 and the Tianshan Cladosporium strain numbered CGMCC No.3.18033 are inoculated into PDA plates for activation, cultured in a mold incubator at 15-20°C for 10 days, cut into small mycelium blocks, inoculated into PDB culture medium, cultured in a shaker at 15-20°C and a rotation speed of 120 r / min for 10 days, filtered to collect mycelium, washed with sterile water for 3 times, squeezed to remove water, mixed the two kinds of mycelium in a mass ratio of 1:1, ground for 1 min by a homogenizer, prepared into a mycelium suspension with a concentration of 1×10 9 CFU / mL by using sterile water, and obtained the low-temperature resistant microorganism.

[0048] The new cold-tolerant Cladosporium strain numbered CGMCC No.3.18032 is purchased from the China General Microbiological Culture Collection Center, and the original preservation time is September 7, 2016; the Tianshan Cladosporium strain numbered CGMCC No.3.18033 is purchased from the China General Microbiological Culture Collection Center, and the original preservation time is September 7, 2016; both strains can be purchased from the preservation center without repeated biological preservation.

[0049] The antagonism test is performed on the two strains, as shown in FIG. 1, the left side is the new cold-tolerant Cladosporium strain, and the colony morphology is round, yellow and opaque; the right side is the Tianshan Cladosporium strain, and the colony morphology is round, white and opaque. The two strains are closely connected without antagonism. Figure 1

[0050] The surfactant is a fatty alcohol polyoxyethylene ether.

[0051] A preparation method of a microbial type water-soluble fertilizer, comprising the steps of: ​

[0052] (1) preparing fish hydrolyzed protein;

[0053] (2) preparing low-temperature resistant microorganism;

[0054] (3) uniformly dispersing the nutrient element, fish hydrolyzed protein, low-temperature resistant microorganism, plant growth regulating component and surfactant in water, storing in dark, and obtaining water-soluble fertilizer.

[0055] Example 2

[0056] A microbial type water-soluble fertilizer, which comprises, by weight percentage, nutrient element 18%, fish hydrolyzed protein 7%, low-temperature resistant microorganism 8%, plant growth regulating component 1%, surfactant 0.3%, and the balance is water, totaling 100%.

[0057] The nutrient element comprises macroelement and microelement; the macroelement comprises nitrogen, phosphorus and potassium; the nitrogen element is ammonium nitrate; the phosphorus is potassium dihydrogen phosphate; and the potassium is potassium nitrate. The mass ratio of ammonium nitrate, potassium dihydrogen phosphate and potassium nitrate is 2:1:3. The microelement is calcium nitrate, magnesium nitrate, copper sulfate, manganese sulfate and EDTA-zinc with a mass ratio of 3:1:1:1:1.

[0058] The preparation method of the fish hydrolyzed protein is as follows:

[0059] (1) placing discarded fish skin in a container, adding water to cover the fish skin, and homogenizing to obtain fish skin slurry;

[0060] (2) adding 0.2% alkaline protease based on the mass of the fish skin to the fish skin slurry, adjusting the pH to 9-11, and performing enzymatic hydrolysis at 40-55℃ for 10h in a water bath, and then filtering with double-layer gauze to obtain enzymatic hydrolysis liquid;

[0061] (3) adding 5% ferrous sulfate and 2.5% ascorbic acid based on the mass of the enzymatic hydrolysis liquid to the enzymatic hydrolysis liquid obtained in step (2), adjusting the pH to 4-5, and placing in a constant-temperature oscillator at 50-60℃ for constant-temperature oscillation reaction for 3h; then centrifuging the reaction liquid at 5000 r / min for 20min to remove the precipitate residue, and drying the filtrate to obtain fish hydrolyzed protein.

[0062] In step (1), the discarded fish skin is grass carp skin.

[0063] The plant growth regulating component is a mixture of 5-aminoacetylpropionic acid, trehalose and amixtral with a mass ratio of 1:2:0.5.

[0064] The low-temperature resistant microorganism comprises a strain numbered as CGMCC No. 3.18032, a new cold-tolerant Cladosporium and a strain numbered as CGMCC No. 3.18033, Tian Shan Cladosporium, and the preparation method of the low-temperature resistant microorganism is the same as that in Embodiment 1.

[0065] The surfactant is sodium dodecyl sulfate.

[0066] A preparation method of a microbial type water-soluble fertilizer, comprising the following steps:

[0067] (1) preparing fish hydrolyzed protein;

[0068] (2) preparing low-temperature resistant microorganism;

[0069] (3) uniformly dispersing the nutrient element, fish hydrolyzed protein, low-temperature resistant microorganism, plant growth regulating component and surfactant in water, and storing in the dark to obtain the water-soluble fertilizer.

[0070] Embodiment 3

[0071] A microbial type water-soluble fertilizer, comprising, by weight percentage, nutrient element 25%, fish hydrolyzed protein 10%, low-temperature resistant microorganism 10%, plant growth regulating component 1.5%, surfactant 0.5%, and the balance being water, totaling 100%.

[0072] The nutrient element comprises macroelement and microelement; the macroelement comprises nitrogen, phosphorus and potassium; the nitrogen element is ammonium nitrate; the phosphorus is potassium dihydrogen phosphate; and the potassium is potassium nitrate. The mass ratio of ammonium nitrate, potassium dihydrogen phosphate and potassium nitrate is 1:2:3. The microelement is a mixture of calcium nitrate, magnesium nitrate, copper sulfate, manganese sulfate and EDTA-zinc.

[0073] The preparation method of the fish hydrolyzed protein is as follows:

[0074] (1) placing discarded fish skin in a container, adding water to cover the fish skin, and homogenizing to obtain fish skin slurry;

[0075] (2) adding alkaline protease with a mass fraction of 0.3% of the fish skin to the fish skin slurry, adjusting the pH to 9-11, and performing enzymatic hydrolysis in a water bath at a temperature of 40-55°C for 12 h, and then filtering with double-layer gauze to obtain an enzymatic hydrolysis liquid;

[0076] (3) adding ferrous sulfate with a mass fraction of 5% of the enzymatic hydrolysis liquid and ascorbic acid with a mass fraction of 2.5% of the enzymatic hydrolysis liquid to the enzymatic hydrolysis liquid obtained in step (2), adjusting the pH to 4-5, and placing in a constant-temperature oscillator at a temperature of 50-60°C for constant-temperature oscillation reaction for 3 h; then centrifuging the reaction liquid at 5000 r / min for 20 min to remove the precipitate residue, and drying the filtrate to obtain fish hydrolyzed protein.

[0077] The waste fish skin in step (1) is crucian carp skin.

[0078] The low-temperature resistant microorganism comprises a strain numbered as CGMCC No. 3.18032, a new cold-tolerant Cladosporium, and a strain numbered as CGMCC No. 3.18033, Tianshan Cladosporium, both of which are the same as in Embodiment 1, and the preparation method of the low-temperature resistant microorganism is also the same as in Embodiment 1.

[0079] The plant growth regulating component is a mixture of 5-aminoacetylpropionic acid, trehalose, and amixtral in a mass ratio of 1:2:0.5.

[0080] Further, the surfactant is a fatty alcohol polyoxyethylene ether or sodium dodecyl sulfate.

[0081] A preparation method of a microbial type water-soluble fertilizer, comprising the following steps:

[0082] (1) preparing fish hydrolyzed protein;

[0083] (2) preparing low-temperature resistant microorganism;

[0084] (3) uniformly dispersing the nutrient elements, fish hydrolyzed protein, low-temperature resistant microorganism, plant growth regulating component, and surfactant in water, and storing in the dark to obtain the water-soluble fertilizer.

[0085] Comparative Example 1

[0086] In this comparative example, except that only the new cold-tolerant Cladosporium is used in the low-temperature resistant microorganism, the remaining raw materials and preparation method are the same as in Embodiment 1. That is:

[0087] A microbial type water-soluble fertilizer, comprising the following components in percentage by weight: nutrient elements 12%, fish hydrolyzed protein 5%, low-temperature resistant microorganism 5%, plant growth regulating component 0.5%, surfactant 0.1%, and the balance being water, totaling 100%.

[0088] The low-temperature resistant microorganism is a strain numbered as CGMCC No. 3.18032, a new cold-tolerant Cladosporium, and the preparation method is as follows: inoculating the strain numbered as CGMCC No. 3.18032, a new cold-tolerant Cladosporium, to a PDA plate for activation, culturing in a mold incubator at 15-20°C for 10 days, cutting into small pieces of mycelium, inoculating into a PDB culture medium, culturing in a shaker at 15-20°C and a rotation speed of 120 r / min for 10 days, filtering the mycelium with double layers of sterilized gauze, washing with sterilized water for 3 times, squeezing out the water, grinding the mycelium with a homogenizer for 1 min, and preparing a mycelium suspension with a concentration of 1×10 9 CFU / mL with sterilized water to obtain the low-temperature resistant microorganism.

[0089] Comparative Example 2

[0090] The present comparative example, except that only the strain of the low-temperature resistant microorganism is the strain of the Cladosporium tianschanicum, the other raw materials and the preparation method are the same as those in Example 1. Namely:

[0091] A microbial type water-soluble fertilizer, which comprises, by weight percentage: 12% of nutrient elements, 5% of fish hydrolyzed protein, 5% of low-temperature resistant microorganism, 0.5% of plant growth regulating component, 0.1% of surfactant, and the balance is water, totaling 100%.

[0092] The low-temperature resistant microorganism is the strain of the Cladosporium tianschanicum with the strain number of CGMCC No.3.18033, and the preparation method is as follows: the strain of the Cladosporium tianschanicum with the strain number of CGMCC No.3.18033 is inoculated into a PDA plate for activation, and then cultured in a mold incubator at 15-20℃ for 10 days, and then cut into small pieces of mycelium, inoculated into a PDB culture medium, and then cultured in a shaker at 15-20℃ and at a rotating speed of 120r / min for 10 days, and then filtered to collect the mycelium with double layers of sterilized gauze, washed with sterilized water for 3 times, squeezed dry, and then ground with a homogenizer for 1 min, and then prepared into a mycelium suspension with a concentration of 1×10 9 CFU / mL with sterilized water, to obtain the low-temperature resistant microorganism.

[0093] Comparative Example 3

[0094] The present comparative example, except that the mass ratio of the low-temperature resistant microorganism is changed to 1:2 between the strain of the Cladosporium novicolum and the strain of the Cladosporium tianschanicum, the other raw materials and the preparation method are the same as those in Example 1. Namely:

[0095] A microbial type water-soluble fertilizer, which comprises, by weight percentage: 12% of nutrient elements, 5% of fish hydrolyzed protein, 5% of low-temperature resistant microorganism, 0.5% of plant growth regulating component, 0.1% of surfactant, and the balance is water, totaling 100%.

[0096] The low-temperature resistant microorganism comprises the strain of the Cladosporium novicolum with the strain number of CGMCC No.3.18032 and the strain of the Cladosporium tianschanicum with the strain number of CGMCC No.3.18033, and the preparation method is as follows: the strain of the Cladosporium novicolum with the strain number of CGMCC No.3.18032 and the strain of the Cladosporium tianschanicum with the strain number of CGMCC No.3.18033 are respectively inoculated into PDA plates for activation, and then cultured in a mold incubator at 15-20℃ for 10 days, and then cut into small pieces of mycelium, respectively inoculated into PDB culture media, and then cultured in a shaker at 15-20℃ and at a rotating speed of 120r / min for 10 days, and then filtered to collect the mycelium with double layers of sterilized gauze, washed with sterilized water for 3 times, squeezed dry, and then mixed with a homogenizer for 1 min, and then prepared into a mycelium suspension with a concentration of 1×10 9CFU / mL of mycelium suspension, to obtain the low-temperature resistant microorganism.

[0097] Comparative Example 4

[0098] In this comparative example, except that the mass ratio of the new cold-tolerant Cladosporium and Tian Shan Cladosporium is changed to 2:1 in the low-temperature resistant microorganism, the remaining raw materials and preparation methods are the same as in Example 1. That is:

[0099] A microbial type water-soluble fertilizer, which comprises, by weight percentage: nutrient elements 12%, fish hydrolyzed protein 5%, low-temperature resistant microorganism 5%, plant growth regulating component 0.5%, surfactant 0.1%, and the balance is water, totaling 100%.

[0100] The low-temperature resistant microorganism comprises Cladosporium novum with strain number CGMCC No.3.18032 and Tian Shan Cladosporium with strain number CGMCC No.3.18033, and the preparation method is as follows: Cladosporium novum with strain number CGMCC No.3.18032 and Tian Shan Cladosporium with strain number CGMCC No.3.18033 are inoculated into PDA plates for activation, cultured in a mold incubator at 15-20℃ for 10 days, then cut into small pieces of mycelium, inoculated into PDB medium, cultured in a shaker at 15-20℃ and a rotation speed of 120r / min for 10 days, then filtered to collect mycelium with double-layer sterilized gauze, washed with sterilized water for 3 times, squeezed dry, mixed the two kinds of mycelium according to a mass ratio of 2:1, ground for 1 min with a homogenizer, and prepared into a mycelium suspension with a concentration of 1×10 9 CFU / mL of mycelium suspension, to obtain the low-temperature resistant microorganism.

[0101] Comparative Example 5

[0102] In this comparative example, except that no low-temperature resistant microorganism is added, the remaining raw materials and preparation methods are the same as in Example 1. That is:

[0103] A microbial type water-soluble fertilizer, which comprises, by weight percentage: nutrient elements 12%, fish hydrolyzed protein 5%, plant growth regulating component 0.5%, surfactant 0.1%, and the balance is water, totaling 100%.

[0104] Comparative Example 6

[0105] In this comparative example, except that 5-aminolevulinic acid is not used as the plant growth regulating component, the remaining raw materials and preparation methods are the same as in Example 1. That is:

[0106] The plant growth regulating component is a mixture of trehalose and aminexil according to a mass ratio of 2:0.5.

[0107] Comparative Example 7

[0108] The comparative example is the same as example 1 except that trehalose is not used in the plant growth regulating component. That is:

[0109] The plant growth regulating component is 5-aminoacetylpropionic acid and malate mixed at a mass ratio of 1:0.5.

[0110] Comparative example 8

[0111] The comparative example is the same as example 1 except that malate is not used in the plant growth regulating component. That is:

[0112] The plant growth regulating component is 5-aminoacetylpropionic acid and trehalose mixed at a mass ratio of 1:2.

[0113] Comparative example 9

[0114] The comparative example is the same as example 1 except that no plant growth regulating component is added. That is:

[0115] A microbial type water-soluble fertilizer, which includes, by weight percentage: nutrient elements 12%, fish hydrolyzed protein 5%, low-temperature resistant microorganisms 5%, surfactant 0.1%, and the balance being water, totaling 100%.

[0116] Anti-freezing performance test

[0117] Pot experiment:

[0118] Test material: Tunhe 4850, provided by Shandong Jinyangguang Seed Co., Ltd.

[0119] Experimental site: Shidani Agricultural Science and Technology Demonstration Garden, Linshu County, Linyi City, Shandong Province.

[0120] In late November 2022, seeds were sown in 10x10cm square nutrient pots, and the culture medium was commercially available nutrient soil. After germination, one seedling was left in each pot, and water was poured once every other day. When the seedlings grew to 3 leaves and 1 heart, they were used as test plants. The culture room temperature was 25°C, and the light cycle was 16L:8D.

[0121] Low temperature treatment:

[0122] Application method: the water-soluble fertilizer to be tested was sprayed and irrigated, 200 times dilution was used for irrigation, and 500 times dilution was used for leaf spraying. A handheld sprayer was used for spraying until the leaves were dripping with water; 20mL of fertilizer was irrigated per pot. Tap water was used as a blank control, and there were 5 pots for each treatment. The mean value of all indicators was taken. After application, the plants were cultured in a 25°C culture room for 1d, and then transferred to a light incubator for low temperature treatment for 3d. The low temperature treatment temperature was 5°C, the light intensity was 10000lx, and the light cycle was 16L:8D.

[0123] Treatment groups are as follows:

[0124] S1: Example 1 water-soluble fertilizer;

[0125] S2: Example 2 water-soluble fertilizer;

[0126] S3: Example 3 water-soluble fertilizer;

[0127] S4: Comparative Example 1 water-soluble fertilizer;

[0128] S5: Comparative Example 2 water-soluble fertilizer;

[0129] S6: Comparative Example 3 water-soluble fertilizer;

[0130] S7: Comparative Example 4 water-soluble fertilizer;

[0131] S8: Comparative Example 5 water-soluble fertilizer;

[0132] S9: Comparative Example 6 water-soluble fertilizer;

[0133] S10: Comparative Example 7 water-soluble fertilizer;

[0134] S11: Comparative Example 8 water-soluble fertilizer;

[0135] S12: Comparative Example 9 water-soluble fertilizer;

[0136] CK: equal amount of water control group.

[0137] Frost resistance index determination: After low temperature treatment, the frost resistance effect evaluation was carried out, and a total of 4 physiological indexes were determined, including relative conductivity, freezing point, plant height and fresh weight.

[0138] Conductivity determination: fresh first leaf was washed twice with deionized water, then dried with absorbent paper, 0.2 g of leaf was weighed, put into a 25 mL covered test tube with 20 mL distilled water, room temperature standing for 12 h, using conductivity meter to measure the conductivity after zeroing the distilled water, then boiling the soaking liquid in a water bath for 25 min, cooling to room temperature to determine the conductivity, then calculating the relative conductivity.

[0139] Relative conductivity (%) = treatment conductivity / boiling conductivity x 100%.

[0140] Freezing point determination: fresh second leaf was selected, the surface of the leaf was wiped with a paper towel, and then the supercooling point determination instrument was used for determination. The hot-wire resistance was completely wrapped with the leaf, so that it was in full contact and tightly adhered, and was placed in a -20℃ refrigerator. When the temperature dropped to about 5℃, the temperature was recorded. The temperature of the leaf decreased with the passage of time, and when it dropped to a certain temperature, the temperature of the leaf suddenly rose due to the release of heat during the phase change when the leaf froze. This temperature mutation point was the freezing point.

[0141] Plant height and aboveground fresh weight determination: Place a tape vertically on the ground and measure the height from the main stem to the highest point of the plant top, which is the plant height. Cut off the aboveground part of the plant, rinse with tap water, then dry with a paper towel, and weigh on a balance to obtain the fresh weight.

[0142] The experimental results are shown in Table 1;

[0143] Table 1 Anti-freezing performance of different treatment groups

[0144]

[0145] From the data in Table 1, we can see that the plant in the example group has low conductivity and low freezing point, showing good anti-freezing effect. The microbial composition of Comparative Examples 1-5 and the plant growth regulating component of Comparative Examples 5-9 are changed, and the synergistic balance between the strains and components is broken, the regulating effect is weakened, resulting in a decrease in the macro anti-freezing planting effect.

[0146] In order to further verify the influence of water-soluble fertilizer on the resistance of crops, field experiments were carried out. Indicators such as MDA content, plant SOD, POD enzyme activity, soluble sugar content, etc. can be used to describe and evaluate the ability of plants to resist low temperature stress, which are called stress resistance indicators. By calculating these indicators, the ability of plants to resist low temperature stress can be evaluated, and the general influence direction of the ability of plants to resist low temperature stress can be explored. The specific experimental and testing methods are as follows:

[0147] Scaling-up experiment: The test crop is tomato, and the variety is Lula. Seedlings were grown on August 2, and were planted on September 7. They grew over winter and matured in early January 2024, and were pulled in March.

[0148] The experiment was set up with 13 treatments, each repeated three times, and the results were averaged:

[0149] S1: Example 1 water-soluble fertilizer;

[0150] S2: Example 2 water-soluble fertilizer;

[0151] S3: Example 3 water-soluble fertilizer;

[0152] S4: Comparative Example 1 water-soluble fertilizer;

[0153] S5: Comparative Example 2 water-soluble fertilizer;

[0154] S6: Comparative Example 3 water-soluble fertilizer;

[0155] S7: Comparative Example 4 water-soluble fertilizer;

[0156] S8: Comparative Example 5 water-soluble fertilizer;

[0157] S9: Comparative Example 6 water-soluble fertilizer;

[0158] S10: Comparative Example 7 water-soluble fertilizer;

[0159] S11: Comparative Example 8 water-soluble fertilizer;

[0160] S12: Comparative Example 9 water-soluble fertilizer;

[0161] CK: equal amount of water control group.

[0162] The use of the water-soluble fertilizer of the examples and comparative examples is: the water-soluble fertilizer is applied at a dosage of 5-8 kg per mu, and the leaf spraying is applied at a dosage of 3-5 kg per mu. The water-soluble fertilizer is applied by irrigation at a dilution of 200 times and sprayed on the leaves at a dilution of 500 times at the seedling stage, early flowering stage and full flowering stage of the tomato. Randomized block arrangement is adopted. The ditches are isolated between plots, and protective rows are set outside the test area. The plot area is 10.5 m x 0.6 m. The cold resistance of tomato is mainly reflected by the indicators of soluble sugar, malondialdehyde (MDA), superoxide dismutase (SOD), etc. The tomato leaves are frozen on January 10 for testing. The yield per plant is counted after the tomato is harvested.

[0163] Plant MDA content determination method:

[0164] A 30 g of analytical pure trichloroacetic acid solid was dissolved in 300 ml of water in advance to prepare a 10% trichloroacetic acid solution, and 0.3 g of analytical pure TBA solid was dissolved in 50 ml of 10% trichloroacetic acid to prepare a 0.6% TBA solution. Both were stored in the dark for later use. Fresh plant leaves were collected, the surface moisture was absorbed with non-woven fabric, and 0.2 g was weighed as a sample. The sample was quickly frozen in liquid nitrogen and stored in a -80°C refrigerator. When tested, it was taken out and transported with liquid nitrogen. The sample was ground into a homogenate with a homogenizer. 2 ml of ultrapure water was added, and centrifugal force was used to centrifuge for 10 minutes. The supernatant was taken and placed in a flat-bottom test tube. 2 ml of 0.6% TBA solution was added to each flat-bottom test tube, and another set of flat-bottom test tubes were set up with 2 ml of ultrapure water and 2 ml of 0.6% TBA solution as a blank control. The holes were sealed with a punching film. After mixing evenly, it was placed in a boiling water bath for 25 minutes. After taking it out, it was cooled with running water, and then centrifuged again for 10 minutes. The supernatant was taken. A spectrophotometer was used, and the blank control was zeroed. The absorbance of each sample supernatant was measured at 532 nm, 600 nm and 450 nm. According to the empirical formula C=6.45*(A532-A600)-0.56*A450, the concentration of MDA in the supernatant was calculated, and the content of MDA in the sample was calculated accordingly.

[0165] Plant SOD activity determination method: SOD enzyme activity determination was performed using a Biyun Tian total SOD activity detection kit (WST-8 method) (item number S0101S). The soluble sugar content was determined by anthrone colorimetry. The VC content in tomato fruit was determined by molybdenum blue colorimetry.

[0166] Table 2 performance index of tomato fruit

[0167]

[0168] From Table 2 and Figure 2 We can see from the data that the experimental group treated by the embodiment of the present application has higher superoxide dismutase activity and lower malondialdehyde content. Higher SOD activity helps tomato plants resist oxidative damage caused by low temperature, and lower MDA content indicates that the cell membrane is less damaged by low temperature stress, which is better for enhancing the cold resistance of the plant. Therefore, whether it is the quality of the plant or the fruit, the embodiment shows good results. The comparative examples 1-9 change the technical features, and due to the change of microorganisms and the weakening of the function of plant regulating components, the overall regulation ability of the fertilizer on tomatoes is weakened, resulting in the decline of tomato yield and quality.

[0169] It should be noted that the above embodiments are only part of the preferred modes of implementing the present application, not all. Obviously, based on the above embodiments of the present application, all other embodiments obtained by those of ordinary skill in the art without creative labor should belong to the scope of protection of the present application.

Claims

1. A microbial water-soluble fertilizer, characterized in that, Its components, by weight percentage, include: 12-25% nutrients, 5-10% hydrolyzed fish protein, 5-10% low-temperature resistant microorganisms, 0.5-1.5% plant growth regulators, 0.1-0.5% surfactants, and the balance being water, totaling 100%. The plant growth regulators are 5-aminolevulinic acid, trehalose, and amino acid esters mixed in a mass ratio of 1:2:0.

5. The low-temperature resistant microorganisms include strain CGMCC 3.18032 (novel cold-resistant Cladosporium) and strain CGMCC 3.18033 (Cyclosporium tianshanense). The preparation method is as follows: [The text abruptly ends here, so the translation stops as well.] 3.18033 *Cladosporium tianshanense* was inoculated onto PDA plates for activation and cultured in a mold incubator at 15-20℃ for 10 days. Afterward, it was cut into small pieces and inoculated onto PDB medium. The pieces were then cultured on a shaker at 120 rpm at 15-20℃ for 10 days. Mycelia were collected by filtration through double-layered sterile gauze, washed three times with sterile water, and squeezed dry. The two types of mycelia were mixed at a 1:1 mass ratio and homogenized for 1 minute. A 1×10⁻⁶ concentration was prepared using sterile water. 9 Low-temperature resistant microorganisms were obtained by preparing a mycelial suspension of CFU / mL.

2. The microbial water-soluble fertilizer according to claim 1, characterized in that, The nutrients include macronutrients and micronutrients; the macronutrients include at least two of nitrogen, phosphorus, and potassium; the micronutrients are one or more of calcium nitrate, magnesium nitrate, copper sulfate, manganese sulfate, and EDTA-zinc.

3. The microbial water-soluble fertilizer according to claim 1, characterized in that, The method for preparing the hydrolyzed fish protein is as follows: (1) Place the waste fish skin in a container, add water to cover the fish skin, and homogenize to obtain fish skin slurry; (2) Add 0.1-0.3% alkaline protease by weight of fish skin to the fish skin slurry, adjust the pH to 9-11, set the temperature to 40-55℃, and enzymatically hydrolyze in a water bath for 8-12 hours. Filter the solution through double gauze to obtain the hydrolysate. (3) Take the enzymatic hydrolysate obtained in step (2) and add 5% ferrous sulfate and 2.5% ascorbic acid by mass of the enzymatic hydrolysate. Adjust the pH to 4-5 and place it in a constant temperature shaker at 50-60℃ for 2-3 hours. Then centrifuge the reaction solution at 3000-5000 r / min for 10-20 minutes, remove the precipitate residue, and dry the filtrate to obtain fish hydrolysate protein.

4. The microbial water-soluble fertilizer according to claim 1, characterized in that, The surfactant is a fatty alcohol polyoxyethylene ether or sodium dodecyl sulfate.

5. A method for preparing the microbial water-soluble fertilizer according to any one of claims 1-4, characterized in that, Includes the following steps: (1) Preparation of hydrolyzed fish protein; (2) Preparation of low-temperature resistant microorganisms; (3) Disperse the nutrients, fish hydrolysate, low-temperature resistant microorganisms, plant growth regulators and surfactants evenly in water and store them away from light to obtain water-soluble fertilizer.

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