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 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-resistant cladosporium and Tianshan cladosporium, are selected and added, and the two cold-resistant 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 invention belongs to the technical field of biological organic fertilizers, and in particular relates to a microbial water-soluble fertilizer and a preparation method thereof. Background Art

[0002] Tomato (Lycopersicon esculentum Mill.) is an important thermophilic economic crop with high nutritional value and wide application. The cultivated area is constantly expanding. However, a series of problems have emerged in the development of greenhouse tomatoes, especially in recent years, with the frequent occurrence of extreme weather and the continuous deterioration of the living environment of crops, which have seriously affected the quality and yield of greenhouse tomatoes. The greenhouse tomatoes in the winter and spring crops in the north are always facing the threat of low temperature stress, which is the primary reason for limiting their growth and development. The low temperature causes the plants to be subjected to low temperature stress, and their growth and development are seriously hindered. If the temperature lasts too long, the plants will eventually die. In addition, the damage to the plant cell level is irreversible. Therefore, the common means of alleviating low temperature stress is to achieve tomato seedling growth by applying new fertilizers, while regulating the physiological processes in the plant body, enhancing the stress resistance of the plant itself, and preventing or resisting the harm caused by low temperature stress to the plant. The advantage of this method is that the fertilizers needed by farmers are easy to obtain in agricultural production activities, and the operation is simple and effective.

[0003] For example, Chinese patent application CN201910636754.9 discloses a method for preparing charcoal-based bio-organic fertilizer for growing tomatoes, comprising the following steps: (1) micro-carbonization of tomato straw; (2) mixing micro-carbonized straw with aboveground stems and leaves of Glehnia littoralis, aboveground stems and leaves of Platycodon grandiflorum, and livestock and poultry manure; (3) aerobic fermentation of the mixture; (4) adding nutrients to the fermented and decomposed matter; and (5) low-temperature drying. The positive effects of the present invention are that the biochar produced by the micro-carbonization process has high organic matter content, good activity of beneficial microorganisms, and directly participates in the composting and fermentation process, so that its porous structure fully absorbs nutrients and hosts a large number of beneficial microorganisms; using fully decomposed livestock and poultry manure, Glehnia littoralis and aboveground stems and leaves of Platycodon grandiflorum as the main components of fertilizer, effectively alleviating the obstacles of continuous cropping of tomatoes; and reasonable nutrient ratio is more conducive to tomato growth and improves fertilizer nutrient utilization. In addition, a large amount of aboveground stems and leaves produced during the planting of local Glehnia littoralis, Platycodon grandiflorum, and tomatoes, and a large amount of feces and urine excrement produced by the cattle industry can be used to avoid pollution to the environment.

[0004] There are similar fertilizers that improve the ability of crops to resist low temperatures. Chinese patent application CN201911298313.9 discloses a fertilizer that improves the resistance of rice to low temperature damage and its preparation method and application. The raw materials for preparing the fertilizer include the following components by weight: shrimp and crab shell powder (25-28) parts, plant ash (30-35) parts, puerarin (5-8) parts, zinc gluconate (3-5) parts, humic acid iron (2-5) parts, amino acid chelated rare earth (1-3) parts, boric acid (1-3) parts and probiotic complex (7-10) parts. The fertilizer of this scheme can not only provide rice with cold resistance and low temperature resistance, but also promote rice rooting, increase thousand-grain weight, and achieve the effect of increasing production and income.

[0005] However, the existing technologies are all solid fertilizers. After being applied to the soil, the effects are slow and unstable. At the same time, the fertilizers have poor targeting, complex ingredients, low activity, and low practical application value. Summary of the invention

[0006] In view of the problems existing in the prior art, the present invention provides a new type of microbial fertilizer, which can enhance the absorption and utilization of nutrients by plants under low temperature conditions by spraying and flushing, and improve the crop resistance and quality while effectively promoting the low temperature growth of tomatoes.

[0007] In order to achieve the above technical objectives, the technical solution adopted by the present invention is: A microbial water-soluble fertilizer comprises the following components by weight: 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 ingredients, 0.1-0.5% of surfactants, and the balance is water, which is 100% in total.

[0008] Further, the nutrient elements include macroelements and trace elements; 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; the potassium is potassium nitrate. The trace elements are one or more of calcium nitrate, magnesium nitrate, copper sulfate, manganese sulfate and EDTA-zinc.

[0009] Furthermore, the preparation method of the fish protein hydrolysate is: (1) placing discarded fish skin in a container, adding water to cover the fish skin, and homogenizing to obtain fish skin slurry; (2) adding 0.1-0.3% alkaline protease by weight of fish skin to the fish skin slurry, adjusting the pH to 9-11 and the temperature to 40-55° C., performing enzymolysis in a water bath for 8-12 h, and filtering through a double-layer gauze to obtain an enzymolysis solution; (3) Add 5% by weight of ferrous sulfate and 2.5% by weight of ascorbic acid to the enzymatic hydrolyzate obtained in step (2), adjust the pH to 4-5, place the mixture in a constant temperature oscillator at 50-60° C., and oscillate at the constant temperature for 2-3 hours; then centrifuge the reaction mixture at 3000-5000 r / min for 10-20 minutes to remove the precipitate residue, and dry the filtrate to obtain fish hydrolyzed protein.

[0010] Furthermore, the waste fish skin in step (1) is one of tilapia skin, grass carp skin, crucian carp skin and salmon skin.

[0011] Amino acids have both chelating ability and are ideal nutrients. For example, glycine can increase the chlorophyll content of plants. Amino acids are not only the raw materials for synthesizing proteins, but also the precursors 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 cytochromes and metalloenzymes, and plays an indispensable role in chlorophyll synthesis, DNA replication, active oxygen scavenging and electron transfer.

[0012] The present invention uses discarded tilapia skin, grass carp skin, crucian carp skin, salmon skin and the like as raw materials, and prepares small-molecule bioactive polypeptides through enzymatic hydrolysis. The polypeptides are composed of amino acids, and the polypeptide amino acids after enzymatic hydrolysis are combined with iron to form stable chelates, which are more easily absorbed and utilized in plants, and can not only improve the growth conditions of crops, but also enhance the disease resistance and stress resistance of crops.

[0013] Furthermore, the plant growth regulating component is obtained by mixing 5-aminolevulinic acid, trehalose and diethylaminoethyl hexanoate in a mass ratio of 1:2:0.5.

[0014] The present invention adds a plant growth regulating component composed of 5-aminolevulinic acid, trehalose and diethylaminoethyl ester, wherein 5-aminolevulinic acid not only has the effect of color change and sweetening, but also can increase the activity of antioxidant enzymes in plants, such as superoxide dismutase (SOD), catalase (CAT) and peroxidase (POD), thereby removing reactive oxygen species (ROS) caused by low temperature and reducing the damage of low temperature to plant cells. Trehalose can promote the energy metabolism of plants and improve the adaptability of plants in low temperature environments. Diethylaminoethyl ester can increase the content of chlorophyll, protein and nucleic acid in plants, thereby enhancing the photosynthesis and carbon and nitrogen metabolism capabilities of plants. These physiological changes help plants maintain normal growth activities under low temperature conditions and improve their cold resistance. The three can be used together to effectively improve the adaptability of plants to low temperature environments and improve crop quality.

[0015] Further, the low temperature resistant microorganisms include a strain numbered CGMCC No.3.18032 New Psychrotrophic Cladosporium (Cladosporium neopsychrotolerans ) and the strain number is CGMCC No.3.18033 Tianshan Cladosporium ( Cladosporium tianshanense ), the preparation method is as follows: the strain numbered CGMCC No.3.18032 new cold-resistant Cladosporium and the strain numbered CGMCC No.3.18033 Tianshan Cladosporium were inoculated into PDA plates for activation, cultured in a 15-20℃ mold incubator for 10 days, cut into small fungal blocks, and inoculated into PDB culture medium respectively, cultured on a shaker at 15-20℃ and a speed of 120r / min for 10 days, filtered and collected mycelium with double-layer sterilized gauze, washed with sterile water for 3 times and squeezed dry, the two kinds of mycelium were mixed at a mass ratio of 1:1, ground with a homogenizer for 1 minute, and prepared with sterile water to a concentration of 1×10 9 CFU / mL of mycelial suspension to obtain low-temperature resistant microorganisms.

[0016] The strain number of the new cold-resistant Cladosporium of the present invention is CGMCC No.3.18032, which is purchased from the General Microbiology Center of the China National Microbiological Culture Collection Administration, and the original preservation time is September 7, 2016; the strain number of the Tianshan Cladosporium is CGMCC No.3.18033, which is purchased from the General Microbiology Center of the China National Microbiological Culture Collection Administration, and the original preservation time is September 7, 2016; both strains can be purchased through the preservation center without repeated biological preservation.

[0017] The preparation method of the PDA plate composition is as follows: 250g of potatoes with skin are peeled and cut into pieces, boiled in ultrapure water for 25 minutes, and then cooled down. After filtering three times with eight layers of gauze, 30g of glucose and 20g of agar are added, and the volume is adjusted to 1000ml with ultrapure water. Sterilize at 121℃ for 20 minutes in a steam sterilizer, pour into a 90mm sterilized plate, seal it, and place it in a 4℃ refrigerator for use.

[0018] The composition and preparation method of PDB medium are as follows: 250g of potatoes with skin are peeled and cut into pieces, boiled in ultrapure water for 25 minutes, and then cooled down. After filtering three times with eight layers of gauze, 30g of glucose is added, and the volume is adjusted to 1000ml with ultrapure water. Sterilize at 121℃ for 20 minutes in a steam sterilizer, seal and place in a 4℃ refrigerator for use.

[0019] The present invention screens two low-temperature-resistant microorganisms, Neopsychroclast and Tianshan Cladosporium, both of which have good low-temperature survival ability. At the same time, after colonizing the roots and leaves of tomatoes, they can regulate the response of plants to low temperatures by affecting the synthesis and metabolism of plant hormones, such as abscisic acid (ABA). 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 proteins in plant cell walls, thereby enhancing the stability of cell walls, reducing cell rupture caused by low temperatures, and helping plants maintain the integrity of cell structures in cold environments. Acting on the soil, it 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 synergistically to jointly achieve the improvement of crop stress resistance, and ultimately achieve an increase in yield and quality.

[0020] Furthermore, the surfactant is fatty alcohol polyoxyethylene ether or sodium lauryl sulfate.

[0021] A method for preparing a microbial water-soluble fertilizer comprises the following steps: (1) preparing fish protein hydrolysate; (2) preparing low-temperature resistant microorganisms; (3) The nutrient elements, fish hydrolyzed protein, low-temperature resistant microorganisms, plant growth regulating ingredients, and surfactants are uniformly dispersed in water and stored away from light to obtain a water-soluble fertilizer.

[0022] The method of using the water-soluble fertilizer of the present invention is as follows: 5-8 kg of water-soluble fertilizer is applied per mu by flushing, and 3-5 kg ​​of water-soluble fertilizer is applied per mu by spraying on leaves. 200 times diluted water-soluble fertilizer is applied by flushing with water, and 500 times diluted water-soluble fertilizer is applied by spraying on leaves at the seedling stage, early flowering stage and full flowering stage of tomatoes, respectively.

[0023] In summary, the beneficial effects of the technical solution of the present invention are: (1) The present invention adds macronutrients such as nitrogen, phosphorus, potassium, and trace elements such as calcium, magnesium, and zinc. An appropriate amount of nitrogen fertilizer can promote the growth of stems and leaves of tomato plants and improve photosynthesis efficiency; phosphorus is mainly involved in energy conversion, photosynthesis, and cell division in plants. An appropriate amount of 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 robustness of plant stems, the improvement of fruit quality, and the enhancement of stress resistance. Potassium can improve the disease resistance of tomatoes, promote fruit ripening, and make the fruit fuller. And trace elements have a significant positive effect on improving the quality and yield of single fruits of tomatoes, and enhancing the flavor and nutritional content; (2) The present invention adds a plant growth regulating component consisting of 5-aminolevulinic acid, trehalose and diethylaminoethyl ester, and the three are used together to effectively improve the adaptability of plants to low temperature environments and improve crop quality; (3) The present invention selects and adds two cold-resistant microorganisms, namely, Cladosporium neotropis and Cladosporium tianshanica. The two cold-resistant fungi work together to improve the stress resistance of crops, and ultimately achieve an increase in yield and quality.

[0024] (4) Application The present invention can enhance the absorption and utilization of nutrients by plants under low temperature conditions by combining spraying and flushing. It can effectively promote the low temperature growth of tomatoes while improving the crop resistance and quality, and has broad application prospects. BRIEF DESCRIPTION OF THE DRAWINGS

[0025] Figure 1 It is the strain antagonism diagram of the new psychrotrophic Cladosporium and Cladosporium tianshanica of the present invention; Figure 2 The single-plant tomato yield experiment of field experiment of Example 1 of the present invention, Comparative Examples 1-9 and blank control is compared. DETAILED DESCRIPTION

[0026] The technical solution of the present invention is further described below in conjunction with specific embodiments, but is not limited thereto.

[0027] Example 1 A microbial water-soluble fertilizer comprises, by weight percentage, 12% of nutrient elements, 5% of fish hydrolyzed protein, 5% of low-temperature-resistant microorganisms, 0.5% of plant growth regulating ingredients, 0.1% of surfactants, and the balance being water, which amounts to 100%.

[0028] The nutrient elements include macroelements and medium and trace elements; 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 medium and trace elements are calcium nitrate, magnesium nitrate and copper sulfate, and the mass ratio of the three is 1:3:0.5.

[0029] The preparation method of the fish protein hydrolysate is: (1) placing discarded fish skin in a container, adding water to cover the fish skin, and homogenizing to obtain fish skin slurry; (2) adding alkaline protease (0.1% by weight of fish skin) to the fish skin slurry, adjusting the pH to 9-11 and the temperature to 40-55° C., performing enzymolysis in a water bath for 8 h, and filtering through a double-layer gauze to obtain an enzymolysis solution; (3) Add 5% by weight of ferrous sulfate and 2.5% by weight of ascorbic acid to the enzymatic hydrolysate obtained in step (2), adjust the pH to 4-5, place in a constant temperature oscillator at 50-60° C., and oscillate at the constant temperature for 2 h; then centrifuge the reaction solution at 3000 r / min for 10 min, remove the precipitate residue, and dry the filtrate to obtain fish hydrolyzed protein.

[0030] The discarded fish skin in step (1) is tilapia skin.

[0031] The plant growth regulating component is obtained by mixing 5-aminolevulinic acid, trehalose and diethylaminoethyl hexanoate in a mass ratio of 1:2:0.5.

[0032] The low-temperature resistant microorganisms include a strain numbered CGMCC No.3.18032 new cold-resistant Cladosporium and a strain numbered CGMCC No.3.18033 Tianshan Cladosporium. The preparation method thereof is as follows: the strain numbered CGMCC No.3.18032 new cold-resistant Cladosporium and the strain numbered CGMCC No.3.18033 Tianshan Cladosporium are inoculated into a PDA plate for activation, cultured in a 15-20°C mold incubator for 10 days, cut into small bacterial blocks, and inoculated into PDB culture medium respectively, cultured on a shaker at 15-20°C with a rotation speed of 120 r / min for 10 days, filtered and collected mycelium with a double-layer sterilized gauze, washed with sterilized water for 3 times, and then squeezed dry, the two kinds of mycelium were mixed at a mass ratio of 1:1, ground with a homogenizer for 1 minute, and prepared with sterile water into a concentration of 1×10 9 CFU / mL of mycelial suspension to obtain low-temperature resistant microorganisms.

[0033] The strain number of the new cold-resistant Cladosporium in this example is CGMCC No.3.18032, which was purchased from the General Microbiological Center of the China National Microbiological Culture Collection Administration, and the original preservation time is September 7, 2016; the strain number of the Tianshan Cladosporium is CGMCC No.3.18033, which was purchased from the General Microbiological Center of the China National Microbiological Culture Collection Administration, and the original preservation time is September 7, 2016; both strains can be purchased through the collection center without the need for repeated biological preservation.

[0034] Antagonistic test was carried out on two bacterial strains. Figure 1 As shown, the new cold-resistant Cladosporium strain on the left has a round, yellow, and opaque colony morphology; the Tianshan Cladosporium strain on the right has a round, white, and opaque colony morphology. The two strains are tightly connected without antagonism.

[0035] The surfactant is fatty alcohol polyoxyethylene ether.

[0036] A method for preparing a microbial water-soluble fertilizer comprises the following steps: (1) preparing fish protein hydrolysate; (2) preparing low-temperature resistant microorganisms; (3) The nutrient elements, fish hydrolyzed protein, low-temperature resistant microorganisms, plant growth regulating ingredients, and surfactants are uniformly dispersed in water and stored away from light to obtain a water-soluble fertilizer.

[0037] Example 2 A microbial water-soluble fertilizer comprises, by weight percentage, 18% of nutrient elements, 7% of fish hydrolyzed protein, 8% of low-temperature-resistant microorganisms, 1% of plant growth regulating ingredients, 0.3% of surfactants, and the remainder of water, which is 100% in total.

[0038] The nutrient elements include macroelements and medium and trace elements; the macroelements include 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 medium and trace elements are calcium nitrate, magnesium nitrate, copper sulfate, manganese sulfate, and EDTA-zinc in a mass ratio of 3:1:1:1:1.

[0039] The preparation method of the fish protein hydrolysate is: (1) placing discarded fish skin in a container, adding water to cover the fish skin, and homogenizing to obtain fish skin slurry; (2) adding 0.2% alkaline protease by weight of fish skin to the fish skin slurry, adjusting the pH to 9-11 and the temperature to 40-55° C., performing enzymolysis in a water bath for 10 h, and filtering through double-layer gauze to obtain an enzymolysis solution; (3) Add 5% by weight of ferrous sulfate and 2.5% by weight of ascorbic acid to the enzymatic hydrolysate obtained in step (2), adjust the pH to 4-5, place the mixture in a constant temperature oscillator at 50-60° C., and oscillate at the constant temperature for 3 h; then centrifuge the reaction solution at 5000 r / min for 20 min, remove the precipitate residue, and dry the filtrate to obtain fish hydrolyzed protein.

[0040] The discarded fish skin in step (1) is grass carp skin.

[0041] The plant growth regulating component is obtained by mixing 5-aminolevulinic acid, trehalose and diethylaminoethyl hexanoate in a mass ratio of 1:2:0.5.

[0042] The cold-resistant microorganisms include a new cold-resistant Cladosporium with a strain number of CGMCC No.3.18032 and a Cladosporium tianshanense with a strain number of CGMCC No.3.18033, both of which are the same as in Example 1. The preparation method of the cold-resistant microorganisms is also the same as in Example 1.

[0043] The surfactant is sodium lauryl sulfate.

[0044] A method for preparing a microbial water-soluble fertilizer comprises the following steps: (1) preparing fish protein hydrolysate; (2) preparing low-temperature resistant microorganisms; (3) The nutrient elements, fish hydrolyzed protein, low-temperature resistant microorganisms, plant growth regulating ingredients, and surfactants are uniformly dispersed in water and stored away from light to obtain a water-soluble fertilizer.

[0045] Example 3 A microbial water-soluble fertilizer comprises, by weight percentage, 25% of nutrient elements, 10% of fish hydrolyzed protein, 10% of low-temperature-resistant microorganisms, 1.5% of plant growth regulating ingredients, 0.5% of surfactants, and the remainder of water, which is 100% in total.

[0046] The nutrient elements include macroelements and medium and trace elements; the macroelements include 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 medium and trace elements are a mass mixture of calcium nitrate, magnesium nitrate, copper sulfate, manganese sulfate, EDTA-zinc and the like.

[0047] The preparation method of the fish protein hydrolysate is: (1) placing discarded fish skin in a container, adding water to cover the fish skin, and homogenizing to obtain fish skin slurry; (2) adding 0.3% alkaline protease by weight of fish skin to the fish skin slurry, adjusting the pH to 9-11 and the temperature to 40-55° C., performing enzymolysis in a water bath for 12 h, and filtering through a double-layer gauze to obtain an enzymolysis solution; (3) Add 5% by weight of ferrous sulfate and 2.5% by weight of ascorbic acid to the enzymatic hydrolysate obtained in step (2), adjust the pH to 4-5, place the mixture in a constant temperature oscillator at 50-60° C., and oscillate at the constant temperature for 3 h; then centrifuge the reaction solution at 5000 r / min for 20 min, remove the precipitate residue, and dry the filtrate to obtain fish hydrolyzed protein.

[0048] The discarded fish skin in step (1) is crucian carp skin.

[0049] The cold-resistant microorganisms include a new cold-resistant Cladosporium with a strain number of CGMCC No.3.18032 and a Cladosporium tianshanense with a strain number of CGMCC No.3.18033, both of which are the same as in Example 1. The preparation method of the cold-resistant microorganisms is also the same as in Example 1.

[0050] The plant growth regulating component is obtained by mixing 5-aminolevulinic acid, trehalose and diethylaminoethyl hexanoate in a mass ratio of 1:2:0.5.

[0051] Furthermore, the surfactant is fatty alcohol polyoxyethylene ether or sodium lauryl sulfate.

[0052] A method for preparing a microbial water-soluble fertilizer comprises the following steps: (1) preparing fish protein hydrolysate; (2) preparing low-temperature resistant microorganisms; (3) The nutrient elements, fish hydrolyzed protein, low-temperature resistant microorganisms, plant growth regulating ingredients, and surfactants are uniformly dispersed in water and stored away from light to obtain a water-soluble fertilizer.

[0053] Comparative Example 1 In this comparative example, except that only the new psychrotrophic Cladosporium was used as the low-temperature resistant microorganism, the other raw materials and preparation methods were the same as those in Example 1. That is: A microbial water-soluble fertilizer comprises, by weight percentage, 12% of nutrient elements, 5% of fish hydrolyzed protein, 5% of low-temperature-resistant microorganisms, 0.5% of plant growth regulating ingredients, 0.1% of surfactants, and the balance being water, which amounts to 100%.

[0054] The low-temperature resistant microorganism is a new cold-resistant Cladosporium with a strain number of CGMCC No.3.18032. The preparation method thereof is as follows: the new cold-resistant Cladosporium with a strain number of CGMCC No.3.18032 is inoculated into a PDA plate for activation, cultured in a 15-20°C mold incubator for 10 days, cut into small bacterial blocks, inoculated into a PDB culture medium, cultured on a shaker at 15-20°C with a rotation speed of 120 r / min for 10 days, filtered and collected mycelium with a double-layer sterilized gauze, washed with sterilized water for 3 times and then squeezed dry, the mycelium was ground with a homogenizer for 1 minute, and sterilized water was used to prepare a concentration of 1×10 9 CFU / mL of mycelial suspension to obtain low-temperature resistant microorganisms.

[0055] Comparative Example 2 In this comparative example, except that only Cladosporium tianshanica was used as the low-temperature resistant microorganism, the other raw materials and preparation methods were the same as those in Example 1. That is: A microbial water-soluble fertilizer comprises, by weight percentage, 12% of nutrient elements, 5% of fish hydrolyzed protein, 5% of low-temperature-resistant microorganisms, 0.5% of plant growth regulating ingredients, 0.1% of surfactants, and the balance being water, which amounts to 100%.

[0056] The low-temperature resistant microorganism is a strain numbered CGMCC No.3.18033 Tianshan Cladosporium, and the preparation method thereof is as follows: the strain numbered CGMCC No.3.18033 Tianshan Cladosporium is inoculated into a PDA plate for activation, cultured in a 15-20°C mold incubator for 10 days, cut into small bacterial blocks, inoculated into a PDB culture medium, cultured on a shaker at 15-20°C with a rotation speed of 120 r / min for 10 days, filtered and collected mycelium with a double-layer sterilized gauze, washed with sterilized water for 3 times and then squeezed dry, the mycelium was ground with a homogenizer for 1 minute, and sterilized water was used to prepare a concentration of 1×10 9 CFU / mL of mycelial suspension to obtain low-temperature resistant microorganisms.

[0057] Comparative Example 3 In this comparative example, except that the mass ratio of the new psychrotrophic Cladosporium to the tianshanica Cladosporium in the low-temperature resistant microorganisms is changed to 1:2, the other raw materials and preparation methods are the same as those in Example 1. That is: A microbial water-soluble fertilizer comprises, by weight percentage, 12% of nutrient elements, 5% of fish hydrolyzed protein, 5% of low-temperature-resistant microorganisms, 0.5% of plant growth regulating ingredients, 0.1% of surfactants, and the balance being water, which amounts to 100%.

[0058] The low-temperature resistant microorganisms include a strain numbered CGMCC No.3.18032 new cold-resistant Cladosporium and a strain numbered CGMCC No.3.18033 Tianshan Cladosporium. The preparation method thereof is as follows: the strain numbered CGMCC No.3.18032 new cold-resistant Cladosporium and the strain numbered CGMCC No.3.18033 Tianshan Cladosporium are inoculated into a PDA plate for activation, cultured in a 15-20°C mold incubator for 10 days, cut into small bacterial blocks, and inoculated into PDB culture medium respectively, cultured on a shaker at 15-20°C with a rotation speed of 120 r / min for 10 days, filtered and collected mycelium with a double-layer sterilized gauze, washed with sterilized water for 3 times, and then squeezed dry, the two kinds of mycelium were mixed in a mass ratio of 1:2, ground with a homogenizer for 1 minute, and prepared with sterile water into a concentration of 1×10 9 CFU / mL of mycelial suspension to obtain low-temperature resistant microorganisms.

[0059] Comparative Example 4 In this comparative example, except that the mass ratio of the new psychrotrophic Cladosporium and the tianshanica Cladosporium in the low-temperature resistant microorganisms is changed to 2:1, the other raw materials and preparation methods are the same as those in Example 1. That is: A microbial water-soluble fertilizer comprises, by weight percentage, 12% of nutrient elements, 5% of fish hydrolyzed protein, 5% of low-temperature-resistant microorganisms, 0.5% of plant growth regulating ingredients, 0.1% of surfactants, and the balance being water, which amounts to 100%.

[0060] The low-temperature resistant microorganisms include a strain numbered CGMCC No.3.18032 new cold-resistant Cladosporium and a strain numbered CGMCC No.3.18033 Tianshan Cladosporium. The preparation method thereof is as follows: the strain numbered CGMCC No.3.18032 new cold-resistant Cladosporium and the strain numbered CGMCC No.3.18033 Tianshan Cladosporium are inoculated into a PDA plate for activation, cultured in a 15-20°C mold incubator for 10 days, cut into small bacterial blocks, and inoculated into PDB culture medium respectively, cultured on a shaker at 15-20°C with a rotation speed of 120 r / min for 10 days, filtered and collected mycelium with a double-layer sterilized gauze, washed with sterilized water for 3 times, and then squeezed dry, the two kinds of mycelium were mixed at a mass ratio of 2:1, ground with a homogenizer for 1 minute, and prepared with sterile water into a concentration of 1×10 9 CFU / mL of mycelial suspension to obtain low-temperature resistant microorganisms.

[0061] Comparative Example 5 In this comparative example, except that no low-temperature resistant microorganisms are added, the other raw materials and preparation methods are the same as those in Example 1. That is: A microbial water-soluble fertilizer comprises, by weight percentage, 12% of nutrient elements, 5% of fish hydrolyzed protein, 0.5% of plant growth regulating ingredients, 0.1% of surfactants, and the balance being water, which amounts to 100%.

[0062] Comparative Example 6 In this comparative example, except that 5-aminolevulinic acid is not used in the plant growth regulating component, the remaining raw materials and preparation method are the same as those in Example 1. That is: The plant growth regulating component is obtained by mixing trehalose and diethylaminoethyl ester according to a mass ratio of 2:0.5.

[0063] Comparative Example 7 In this comparative example, except that trehalose is not used in the plant growth regulating component, the other raw materials and preparation methods are the same as those in Example 1. That is: The plant growth regulating component is obtained by mixing 5-aminolevulinic acid and diethylaminoethyl ester in a mass ratio of 1:0.5.

[0064] Comparative Example 8 In this comparative example, except that diethylaminoethyl hexanoate is not used in the plant growth regulating component, the other raw materials and preparation method are the same as those in Example 1. That is: The plant growth regulating component is obtained by mixing 5-aminolevulinic acid and trehalose in a mass ratio of 1:2.

[0065] Comparative Example 9 In this comparative example, except that no plant growth regulating component is added, the remaining raw materials and preparation method are the same as those in Example 1. That is: A microbial water-soluble fertilizer comprises, by weight percentage, 12% of nutrient elements, 5% of fish hydrolyzed protein, 5% of low-temperature-resistant microorganisms, 0.1% of surfactant, and the balance of water, which is 100% in total.

[0066] Antifreeze performance test Pot experiment: Test material: Tunhe 4850, provided by Shandong Golden Sunshine Seed Co., Ltd.

[0067] Experimental location: Stanley Agricultural Science and Technology Demonstration Park, Linshu County, Linyi City, Shandong Province.

[0068] In late November 2022, the seeds were sown in 10×10 cm square nutrient pots. The culture medium was commercially available nutrient soil. After the seedlings emerged, one seedling was left in the pot and watered every other day. When the seedlings grew to 3 leaves and 1 heart, they were used as experimental plants. The temperature in the culture room was 25°C and the photoperiod was 16 L:8D.

[0069] Low temperature treatment: Application method: Spray and root irrigate the water-soluble fertilizer to be tested. Use 200 times dilution for root irrigate and 500 times dilution for leaf spray. Use a handheld sprayer to spray until the leaves drip water; 20mL fertilizer is irrigated per pot for root irrigate treatment. Use clean water as a blank control, 5 for each treatment, and take the average of all indicators. After application, culture in a 25℃ culture room for 1 day, and then transfer to a light culture box for low temperature treatment for 3 days. The temperature of low temperature treatment is 5℃, the light intensity is 10000lx, and the photoperiod is 16L:8D.

[0070] The treatment groups were: S1: water-soluble fertilizer of Example 1; S2: water-soluble fertilizer of Example 2; S3: water-soluble fertilizer of embodiment 3; S4: water-soluble fertilizer of comparative example 1; S5: Comparative Example 2 water-soluble fertilizer; S6: water-soluble fertilizer of comparative example 3; S7: water-soluble fertilizer of comparative example 4; S8: water-soluble fertilizer of comparative example 5; S9: water-soluble fertilizer of comparative example 6; S10: water-soluble fertilizer of comparative example 7; S11: water-soluble fertilizer of comparative example 8; S12: water-soluble fertilizer of comparative example 9; CK: control group with equal volume of water.

[0071] Determination of antifreeze indexes: After low temperature treatment, the antifreeze effect was evaluated, and a total of four physiological indexes were measured, including relative conductivity, freezing point, plant height and fresh weight.

[0072] Conductivity determination: Rinse the first fresh true leaf with deionized water twice, then dry it with absorbent paper, cut it into pieces and weigh 0.2g of leaves, put it into a 25mL covered test tube with 20mL of distilled water, and let it stand at room temperature for 12h. Use a conductivity meter to measure the conductivity of the distilled water after it returns to zero, then boil the soaking solution in a boiling water bath for 25min, cool it to room temperature and measure its conductivity again, and then calculate the relative conductivity.

[0073] Relative conductivity (%) = treatment conductivity / boiling conductivity × 100%.

[0074] Freezing point determination: Select a fresh second true leaf, wipe the leaf surface with a paper towel, and then use a supercooling point tester to measure. Use the leaf to completely fit the thermistor so that it is fully in contact and tightly bonded, and place it in a -20℃ refrigerator. When the temperature drops to about 5℃, start recording the temperature, and record the temperature change every 1s. The leaf temperature continues to drop with the passage of freezing time. When it drops to a certain temperature, the leaf temperature suddenly rises due to the phase change that releases heat when the leaf freezes. This temperature mutation point is the freezing point.

[0075] Plant height and aboveground fresh weight determination: Place the tape measure vertically on the ground and measure the height of the highest point of the seedling along the main stem, which is the plant height. Cut the aboveground part of the plant, rinse it with tap water, wipe it dry with a paper towel, and weigh it on a scale to obtain the fresh weight.

[0076] The experimental results are shown in Table 1; Table 1 Freeze resistance of different treatment groups From the data in Table 1, we can see that the plants in the embodiment group of the present invention have low conductivity and low freezing point, showing good antifreeze effect. However, in the comparative examples 1-5 with changed microbial composition and comparative examples 5-9 with plant growth regulating components, the synergistic balance between the strains and the components was broken, and the regulating effect was weakened, thus resulting in a decrease in the macroscopic antifreeze planting effect.

[0077] In order to further verify the effect of water-soluble fertilizer on crop resistance, field experiments were conducted. Indicators such as MDA content, plant SOD, POD enzyme activity, soluble sugar content, etc. that can be used to describe and evaluate the ability of plants to resist low temperature stress are called stress resistance indicators. By measuring these indicators, the ability of plants to resist low temperature stress can be evaluated, and the general direction of the impact of the ability of plants to resist low temperature stress can be explored. The specific experimental and testing methods are as follows: Amplification experiment: The test crop is tomato, the variety is Rolla. Seedlings were raised on August 2, transplanted on September 7, grown over the winter, matured in early January 2024, and transplanted in March.

[0078] The experiment set up 13 treatments, each treatment was repeated three times, and the results were averaged: S1: water-soluble fertilizer of Example 1; S2: water-soluble fertilizer of Example 2; S3: water-soluble fertilizer of embodiment 3; S4: water-soluble fertilizer of comparative example 1; S5: Comparative Example 2 water-soluble fertilizer; S6: water-soluble fertilizer of comparative example 3; S7: water-soluble fertilizer of comparative example 4; S8: water-soluble fertilizer of comparative example 5; S9: water-soluble fertilizer of comparative example 6; S10: water-soluble fertilizer of comparative example 7; S11: water-soluble fertilizer of comparative example 8; S12: water-soluble fertilizer of comparative example 9; CK: control group with equal volume of water.

[0079] The usage of water-soluble fertilizer in the embodiment and comparative example is: 5-8 kg per mu of water-soluble fertilizer is applied by flushing, and 3-5 kg ​​per mu of water-soluble fertilizer is applied by spraying on leaves. At the seedling stage, early flowering stage and full flowering stage of tomatoes, 200 times diluted water-soluble fertilizer is applied with water flushing, and 500 times diluted water-soluble fertilizer is sprayed on leaves. Random block arrangement. Ditches are opened between the plots for isolation, and protective rows are set outside the test area. The plot area is 10.5m×0.6m. The cold resistance of tomatoes is mainly reflected by indicators such as soluble sugar, malondialdehyde (MDA), and superoxide dismutase (SOD). Tomato leaves were frozen on January 10 for testing. The yield per plant was counted after the tomatoes were harvested.

[0080] Plant MDA content determination method: Weigh 30g of analytically pure trichloroacetic acid solid and dissolve it in 300ml of water to make a 10% trichloroacetic acid solution. Weigh 0.3g of analytically pure TBA solid and dissolve it in 50ml of 10% trichloroacetic acid to make a 0.6% TBA solution. Both are kept away from light for later use. Collect fresh plant leaves, absorb surface moisture with non-woven fabric, weigh 0.2g as a sample, freeze it with liquid nitrogen, store it in a -80℃ refrigerator, take it out when it is tested, transfer it with liquid nitrogen, and grind it into a homogenate with a homogenizer. Add 2ml of ultrapure water to it, centrifuge it for 10 minutes, take the supernatant, and place it in a flat-bottomed test tube. Add 2ml of 0.6% TBA solution to each flat-bottomed test tube, set up a group of flat-bottomed test tubes, add 2ml of ultrapure water and 2ml of 0.6% TBA solution as a blank control, and seal it with a perforated film. After mixing evenly, boil it in a boiling water bath for 25 minutes, take it out and rinse it with running water to cool it down, centrifuge it for 10 minutes, and take the supernatant. Using a spectrophotometer, the blank control was used to zero and the absorbance of the supernatant of each sample was measured at three wavelengths: 532nm, 600nm and 450nm. The concentration of MDA in the supernatant was calculated according to the empirical formula C=6.45*(A532-A600)-0.56*A450 and the content of MDA in the sample was deduced accordingly.

[0081] Plant SOD activity determination method: SOD enzyme activity was determined using the total SOD activity detection kit (WST-8 method) (Cat. No. S0101S) of Biyuntian Company, and the soluble sugar content was determined by anthrone colorimetry. The VC content in tomato fruit was determined by molybdenum blue colorimetry.

[0082] Table 2 Tomato fruit performance indicators From Table 2 and Figure 2 From the data, we can see that the activity of superoxide dismutase in the experimental group treated with the embodiments of the present invention was increased, and the content of malondialdehyde was lower. Higher SOD activity helps tomato plants resist oxidative damage caused by low temperature, and low MDA content indicates that the cell membrane is less damaged by low temperature stress, which has a better effect on enhancing the cold resistance of the plant. Therefore, the embodiments have shown good results in terms of both plant and fruit quality. However, in Comparative Examples 1-9, which have changed the technical features, the overall regulation ability of the fertilizer on tomatoes is weakened due to changes in microorganisms and weakening of the functions of plant regulatory components, resulting in a decrease in tomato yield and quality.

[0083] It should be noted that the above embodiments are only partial embodiments of the preferred methods of implementing the present invention, rather than all embodiments. Obviously, based on the above embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without creative work should fall within the scope of protection of the present invention.

Claims

1. A microbial water-soluble fertilizer, characterized in that: The components thereof include, 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 ingredients, 0.1-0.5% of surfactants, and the balance being water, which totals 100%.

2. The microbial water-soluble fertilizer according to claim 1, characterized in that: The nutrient elements include macroelements and trace elements; the macroelements include at least two of nitrogen, phosphorus and potassium nutrient elements; the trace elements 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 preparation method of the fish protein hydrolysate is: (1) placing discarded fish skin in a container, adding water to cover the fish skin, and homogenizing to obtain fish skin slurry; (2) adding 0.1-0.3% alkaline protease by weight of fish skin to the fish skin slurry, adjusting the pH to 9-11 and the temperature to 40-55° C., performing enzymolysis in a water bath for 8-12 h, and filtering through a double-layer gauze to obtain an enzymolysis solution; (3) Add 5% by weight of ferrous sulfate and 2.5% by weight of ascorbic acid to the enzymatic hydrolyzate obtained in step (2), adjust the pH to 4-5, place the mixture in a constant temperature oscillator at 50-60° C., and oscillate at the constant temperature for 2-3 hours; then centrifuge the reaction mixture at 3000-5000 r / min for 10-20 minutes to remove the precipitate residue, and dry the filtrate to obtain fish hydrolyzed protein.

4. The microbial water-soluble fertilizer according to claim 1, characterized in that: The plant growth regulating component is obtained by mixing 5-aminolevulinic acid, trehalose and diethylaminoethyl hexanoate in a mass ratio of 1:2:0.

5.

5. The microbial water-soluble fertilizer according to claim 1, characterized in that: The low-temperature resistant microorganisms include a strain numbered CGMCC No. 3.18032 new cold-resistant Cladosporium and a strain numbered CGMCC No. 3.18033 Tianshan Cladosporium. The preparation method thereof is as follows: the strain numbered CGMCC No. 3.18032 new cold-resistant Cladosporium and the strain numbered CGMCC No. 3.18033 Tianshan Cladosporium are inoculated into a PDA plate for activation, cultured in a 15-20° C. mold incubator for 10 days, cut into small bacterial blocks, and inoculated into PDB culture medium respectively, cultured on a shaker at 15-20° C. with a rotation speed of 120 r / min for 10 days, filtered and collected mycelium with a double-layer sterilized gauze, washed with sterilized water for 3 times, and then squeezed dry, the two kinds of mycelium were mixed at a mass ratio of 1:1, ground with a homogenizer for 1 minute, and prepared with sterile water into a concentration of 1×10 9 CFU / mL of mycelial suspension to obtain low-temperature resistant microorganisms.

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

7. A method for preparing the microbial water-soluble fertilizer according to any one of claims 1 to 6, characterized in that: The steps include: (1) preparing fish protein hydrolysate; (2) preparing low-temperature resistant microorganisms; (3) The nutrient elements, fish hydrolyzed protein, low-temperature resistant microorganisms, plant growth regulating ingredients, and surfactants are uniformly dispersed in water and stored away from light to obtain a water-soluble fertilizer.

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