Nitrogen-phosphorus-potassium complex microbial inoculant as well as preparation method and application thereof

Abstract

The invention relates to a nitrogen-phosphorus-potassium complex microbial inoculant as well as a preparation method and application thereof. The bacterial strain of the complex microbial inoculant consists of pseudomonas sp. 1416X3, Pristeria sp. ST2 and Burkholderia sp. YHN3-13, and the bacterial strain of the complex microbial inoculant consists of the following components: Pseudomonas sp. 1416X3, Pristeria sp. ST2 and Burkholderia sp. YHN3-13. Three specific strains of different types are compounded, potential interaction exists among the three strains, and the three strains can be matched with one another to improve the physical and chemical properties of soil, increase nitrogen, phosphorus, potassium, calcium and organic matter nutrients of the soil, increase the biological effective state content of mineral elements in the soil, improve the soil nutrition and improve the soil quality. And sufficient nutrients are provided for crop growth. The plant growth can be promoted, the plant height, the root length, the dry weight and the fresh weight of plants are obviously increased, the content of mineral elements such as calcium, iron, zinc and selenium in the plants is effectively increased, and the nutrition and health quality of the crops is remarkably improved.

Description

Technical Field

[0001] This invention relates to the field of microbial technology, specifically to a nitrogen-phosphorus-potassium compound bacterial agent, its preparation method, and its application. Background Technology

[0002] With the increasing demand for sustainable agricultural development globally, the soil degradation, environmental pollution, and agricultural product safety issues caused by the long-term use of chemical fertilizers and pesticides are becoming increasingly prominent. Soil health is the cornerstone of the nutritional health of agricultural products and human health. Microbial inoculants, due to their eco-friendly, low-cost, and easy-to-produce characteristics, have become an important research direction for the development of green agriculture.

[0003] Microorganisms can achieve multiple growth-promoting effects through their own metabolic activities, such as converting and supplying nutrients like nitrogen, phosphorus, and potassium; producing plant growth regulators like auxins (IAA) and gibberellins (GA); generating antibacterial substances to inhibit pathogens; and alleviating biotic stress. However, current microbial inoculants and their applications still suffer from problems such as limited strain functionality, poor environmental adaptability, and unstable effects. Furthermore, research on microbial inoculants specifically designed to activate soil micronutrients is relatively weak. Micronutrients play an irreplaceable role in key physiological processes such as plant enzyme system activation, photosynthesis, carbohydrate transport, and nitrogen fixation and metabolism, and are transmitted to the human body through crops and fruits, influencing human health. However, most micronutrients in the soil exist in a relatively fixed state with low solubility, making them difficult for plants to directly absorb and utilize, thus limiting the improvement of crop yield and nutritional quality.

[0004] Compound microbial agents, through the scientific combination of various functional strains, enhance environmental adaptability while performing multiple functions such as nitrogen fixation, phosphorus solubilization, potassium solubilization, and auxin secretion, thereby improving nutrient utilization efficiency and demonstrating significant advantages.

[0005] Reference 1: Chinese patent document with publication number CN116814457A.

[0006] Reference 1 describes a growth-promoting bacteria, a compound bacteria, a microbial agent, a microbial fertilizer, and their applications, including *Burkholderia pyrrocinia* and *Pseudomonas atagonensis*. The *Burkholderia pyrrocinia* mentioned above is *Burkholderia pyrrocinia* FJS-3, deposited at the Guangdong Provincial Microbial Culture Collection Center on July 20, 2022, with accession number GDMCC No. 62432. The *Pseudomonas atagonensis* mentioned above is *Pseudomonas atagonensis* FJS-16, also deposited at the Guangdong Provincial Microbial Culture Collection Center on July 20, 2022, with accession number GDMCC No. 62433. This growth-promoting compound bacteria has a good growth-promoting effect on plants.

[0007] Existing microbial agents primarily focus on addressing the supply of macronutrients such as nitrogen, phosphorus, and potassium in the soil, resulting in a relatively singular functional focus. Their effectiveness in comprehensively and systematically improving soil fertility, particularly in simultaneously activating micronutrients (such as calcium, iron, zinc, manganese, molybdenum, and selenium), is limited or unreported. Micronutrients are crucial for crop quality, stress resistance, and human health, and their low bioavailability has become a key bottleneck restricting the production of high-quality agricultural products.

[0008] Therefore, addressing the shortcomings of existing microbial agents in terms of functional comprehensiveness, quality improvement potential, and broad application scope, this invention aims to provide a novel compound microbial agent and its applications. This agent not only effectively increases the content of nitrogen, phosphorus, potassium, and organic matter in the soil, achieving fertilizer reduction and yield increase, but its core advantage lies in its ability to significantly enhance the bioavailability of various key micronutrients (calcium, iron, zinc, manganese, molybdenum, and selenium) in the soil, thereby fundamentally improving crop nutrition. While increasing yield, it also significantly improves the nutritional quality of agricultural products and demonstrates good applicability across various crops. Summary of the Invention

[0009] To address the shortcomings of existing technologies, the present invention aims to provide a nitrogen-phosphorus-potassium compound microbial agent, its preparation method, and its application, which can improve the bioavailability of nutrients and mineral elements in soil, improve soil health, and enhance crop growth and quality.

[0010] To achieve this objective, the present invention adopts the following technical solution: In a first aspect, the present invention provides a nitrogen-phosphorus-potassium compound bacterial agent, wherein the bacterial strains in the nitrogen-phosphorus-potassium compound bacterial agent are composed of Pseudomonas sp. 1416X3, Priestia sp. ST2 and Burkholderia sp. YHN3-13.

[0011] The Pseudomonas sp. 1416X3 strain was obtained through autonomous isolation and screening from aquatic sediments and was deposited at the China General Microbiological Culture Collection Center on April 11, 2019, with accession number CGMCC No. 17557.

[0012] The Priestia sp. ST2 strain was obtained through autonomous isolation and screening from freshwater lake interface sediments and was deposited at the China General Microbiological Culture Collection Center on September 18, 2025, with accession number CGMCC No. 36011.

[0013] The Burkholderia sp. YHN3-13 was independently isolated and screened from tobacco planting soil and deposited at the China General Microbiological Culture Collection Center on September 18, 2025, with the accession number CGMCC No. 36012.

[0014] Preferably, the ratio of viable counts of Pseudomonas sp. 1416X3, Priestia sp. ST2 and Burkholderia sp. YHN3-13 is (1~3):(1~3):(1~3).

[0015] Secondly, the present invention provides a method for preparing the above-mentioned nitrogen, phosphorus, and potassium compound bacterial agent, wherein Pseudomonas 1416X3, Prestella ST2, and Burkholderia YHN3-13 are respectively inoculated into liquid culture medium and cultured to the logarithmic growth phase, and their OD values ​​are adjusted with sterile physiological saline. 600 The nitrogen, phosphorus and potassium compound bacterial agent is prepared by mixing the sample with a volume ratio of (1~3):(1~3):(1~3) to 0.9-1.0.

[0016] Preferably, the liquid culture medium is LB liquid culture medium, and the culture conditions are: temperature 28-30 ℃, stirring speed 150-180 r / min, and culture for 12 h.

[0017] Preferably, the pH of the LB medium is 7.0-7.2, and the components of the LB medium are: 10 g / L tryptone, 10 g / L NaCl and 5 g / L yeast extract.

[0018] Thirdly, the present invention provides the application of the above-mentioned nitrogen, phosphorus and potassium compound bacterial agent in promoting plant growth.

[0019] Fourthly, the present invention provides the application of the above-mentioned nitrogen, phosphorus, and potassium compound bacterial agent in increasing the nitrogen, phosphorus, potassium, and organic matter content in soil.

[0020] Fifthly, the present invention provides the application of the above-mentioned nitrogen, phosphorus and potassium compound bacterial agent in improving the bioavailability of calcium, iron, zinc, manganese, molybdenum and selenium in soil.

[0021] Sixthly, the present invention provides the application of the above-mentioned nitrogen, phosphorus and potassium compound microbial agent in improving the bioavailability of calcium, iron, zinc, manganese, molybdenum and selenium elements in the soil while increasing the content of nitrogen, phosphorus, potassium and organic matter in the soil.

[0022] The beneficial effects of this invention are as follows: The nitrogen-phosphorus-potassium compound microbial agent of this invention is composed of three different functional microorganisms, possessing multiple functions such as phosphorus solubilization, nitrogen fixation, and potassium solubilization. This avoids the limitations of single-incident microbial agents and adapts to more complex and varied application scenarios. This compound microbial agent not only simultaneously completes basic nutrient activation tasks such as phosphorus solubilization, potassium solubilization, and nitrogen fixation, but also enhances the bioavailability of key mineral elements such as calcium, iron, manganese, zinc, molybdenum, and selenium in the soil through microbial metabolic activities. This significantly improves the absorption and accumulation efficiency of these elements by crops, thereby improving the nutritional health of the soil and providing an efficient biological solution to the common problem of mineral element deficiency in crops. Attached Figure Description

[0023] Figure 1 Phylogenetic tree of Primate ST2 based on 16S rRNA gene sequence; Figure 2 Phylogenetic tree of Burkholderia YHN3-13 based on 16S rRNA gene sequence. Detailed Implementation

[0024] To better illustrate the technical solutions and implementation effects of the present invention, the present invention will be further described below in conjunction with the embodiments, but is not limited to the following embodiments.

[0025] Example 1: Isolation and screening of nitrogen, phosphorus, and potassium functional strains Phospholytic strain *Pseudomonas* 1416X3 was isolated from freshwater sediments using an inorganic phosphate medium (10 g glucose, 0.2 g KCl, 0.1 g (NH4)2SO4, 5 g MgCl2·6H2O, 0.25 g MgSO4·7H2O, 5 g Ca3(PO4)2, 15 g agar, 1.0 L distilled water, pH 7.0) via a tenfold serial dilution spreader method. The initial phosphate-solubilizing capacity of strain 1416X3 was 646.04 mg / L when formulated with a compound inoculum.

[0026] The potassium-solubilizing strain *Primateriel* ST2 was isolated from freshwater lake interface sediments using a potassium-solubilizing bacterial selection medium (5 g sucrose, 0.5 g MgSO4·7H2O, 2.0 g Na2HPO4, 0.1 g CaCO3, 0.005 g FeCl3, 3.0 g potassium feldspar powder, 2 g Ca3(PO4)2, 15 g agar, 1.0 L distilled water, pH 7.0). Separation was performed using a tenfold serial dilution plating method based on the size of the clear zone, and the potassium-solubilizing ability was determined by flame spectrophotometry. The initial potassium-solubilizing ability of strain ST2 was 1.31 mg / L.

[0027] The nitrogen-fixing strain Burkholderia sp. YHN3-13 was isolated from the topsoil of a tobacco field. A nitrogen-fixing selection medium (KH₂PO₄ 0.1 g, MgSO₄·7H₂O 0.2 g, K₂HPO₄ 0.9 g, Na₂MoO₄·2H₂O 0.005 g, FeSO₄·7H₂O 0.01 g, CaCO₃ 5 g, glucose 5 g, lactose 5 g, CaCl₂·2H₂O 0.1 g, agar 15 g, distilled water 1.0 L, pH 7.0) was used for isolation and qualitative screening via a tenfold serial dilution plating method combined with the size of the clear zone. The Kjeldahl method was used to determine the nitrogen-fixing capacity. The initial nitrogen-fixing capacity of strain YHN3-13 was 24.33 mg / L.

[0028] The taxonomic position of all strains was determined by phylogenetic analysis of their 16S rRNA genes combined with physiological and biochemical characteristics. Specifically, genomic DNA was extracted from the strains using the SDS-alkali lysis method. The 16S rRNA gene was amplified by PCR using universal primers E. coli 27F and 1492R. After 1% agarose gel electrophoresis, the nucleotide sequence was determined using the Sanger dideoxy chain termination method. The obtained sequences were compared with those in the GenBank database, and a phylogenetic tree was constructed using the nearest neighbor method with MEGA software. The 16S rRNA sequence of strain 1416X3, with GenBank accession number MK280701.1, is *Pseudomonas* sp., a species already published in existing literature (see Chinese patent document CN110669704A). The 16S rRNA gene sequence of strain ST2, as shown in SEQ ID NO.1, was identified as *Priestiasp.*, and its phylogenetic tree is attached. Figure 1 The 16S rRNA gene sequence of strain YHN3-13 is shown in SEQ ID NO.2, and it has been identified as Burkholderia sp. The phylogenetic tree is attached. Figure 2 .

[0029] Example 2: Compound formulation of nitrogen, phosphorus, and potassium bacterial agents (1) Freshly activated strains 1416X3, ST2, and YHN3-13 were picked and inoculated into LB medium, and cultured at 28 ℃ for 12 h to prepare seed culture. After centrifugation and resuspending, the OD was adjusted. 600 =1.0, and obtained bacterial suspensions of Pseudomonas 1416X3, Priscilla ST2 and Burkholderia YHN3-13.

[0030] (2) The volume ratio of Pseudomonas 1416X3, Priscilla ST2 and Burkholderia YHN3-13 was (1~3): (1~3): (1~3), and all of them had good activity. To determine the optimal compounding ratio within this range, for ten specific ratios (1:1:1, 2:1:1, 1:2:1, 1:1:2, 3:1:1, 1:3:1, 1:1:3, 2:2:1, 2:1:2, 1:2:2), the bacterial agent was inoculated at 5% (v / v) on inorganic phosphorus fermentation medium and cultured at 28 ℃ and 150 r / min for 7 days. The phosphorus-solubilizing ability of the bacterial agent was determined using the molybdenum-antimony colorimetric method according to the "General Technical Requirements for Quality Evaluation of Microbial Fertilizer Production Strains" (NY / T 1847-2010). The bacterial agent was also inoculated at 5% (v / v) on potassium-solubilizing medium and cultured at 28 ℃ and 150 r / min for 7 days. The potassium-solubilizing ability was determined using the flame spectrophotometric method according to the "Silicate Bacterial Strains" (NY 882-2004). Finally, the bacterial agent was inoculated at 5% (v / v) on Ashby nitrogen-free medium and cultured at 28 ℃ and 150 r / min for 7 days. The nitrogen fixation capacity was determined by the Kjeldahl method after culturing at ℃ and 150 r / min for 7 days. The results are shown in Appendix Table 1.

[0031] The inorganic phosphorus medium formula is as follows: glucose 10 g / L, KCl 0.2 g / L, (NH4)2SO4 0.1 g / L, MgCl2·6H2O 5 g / L, MgSO4·7H2O 0.25 g / L, Ca3(PO4)2 5 g / L, pH 7.0. The potassium-solubilizing medium formula is as follows: sucrose 10 g / L, MgSO4·7H2O 0.5 g / L, Na2HPO4 2.0 g / L, CaCO3 1.0 g / L, FeCl3 0.005 g / L, yeast extract 0.5 g / L, (NH4)2SO4 1.0 g / L, NaCl 0.1 g / L, potassium feldspar powder 10.0 g / L, pH 7.0. Ashby's nitrogen-free medium formula is: glucose 10 g / L, KH2PO4 0.2 g / L, NaCl 0.2 g / L, MgSO4·7H2O 0.2 g / L, CaCO3 5 g / L, CaSO4 0.1 g / L, pH 7.0.

[0032] Appendix Table 1. Phosphorus-, potassium-, and nitrogen-fixing capabilities of NPK compound bacterial agents with different formulation ratios Note: The data in the table above are mean ± standard error. Different lowercase letters 'a' and 'b' in the upper right corner of the same column indicate significant differences between different treatments (p < 0.05).

[0033] The results showed that, because the total inoculum of the three bacteria in the compound system was the same as that of the single-strain control system, the initial biomass of each functional bacteria in the compound system was much lower than that of the single-strain system. Therefore, the individual functional activities (such as phosphorus solubilization and nitrogen fixation) were close to but slightly lower than those of the corresponding single-strain controls. However, the compound system allowed for good coexistence of the bacteria, significantly improved potassium solubilization, and simultaneously achieved multiple functions of phosphorus solubilization, potassium solubilization, and nitrogen fixation, exhibiting good performance in various compound ratios. Among these, when the volume ratio of strain 1416X3: strain ST2: strain YHN3-13 was 3:1:1, the compound system showed the strongest overall performance in phosphorus solubilization, potassium solubilization, and nitrogen fixation, at 555.59 mg / L, 3.32 mg / L, and 21.27 mg / L, respectively, representing the optimal compound ratio.

[0034] Example 3: Growth-promoting effect of nitrogen, phosphorus, and potassium compound microbial agent on corn A seedling growth-promoting experiment was conducted using the compound microbial agent prepared according to this invention, with an experimental period of 21 days. 50 mL of the compound microbial agent (strain 1416X3: strain ST2: strain YHN3-13, volume ratio 3:1:1) was inoculated into the rhizosphere, and a sterile water system and a single-strain system were set up as controls. The microbial agent was replenished every 7 days. Corn plant height, root length, fresh weight, and dry weight were measured regularly. Phosphorus content was determined using the national standard method "Determination of Total Phosphorus Content in Plants - Molybdenum Antimony Colorimetric Method" (NY / T 2421-2013), potassium content was determined using the national standard method "Determination of Total Potassium Content in Plants - Flame Photometric Method" (NY / T 2420-2013), nitrogen content was determined using the national standard method "Determination of Total Nitrogen Content in Plants" (NY / T 2419-2013), and calcium and trace element content were determined using the national standard method "Determination of Multiple Elements in Food" (GB 5009.268-2016). The results are shown in Appendix Table 2.

[0035] Appendix Table 2 Effects of NPK Compound Microbial Agent on Nutrient Content in Maize Plants Compared to the control group, the corn plant height increased by 37.59% under the action of the nitrogen, phosphorus and potassium compound microbial agent; the root length increased by 9.31%; the fresh weight per plant increased by 76.92%; and the dry weight increased by 61.29%. This compound microbial agent has a good plant growth promoting effect.

[0036] In terms of nutritional composition, under the action of the nitrogen, phosphorus, and potassium compound microbial agent, the phosphorus content in corn seedlings increased by 32.45%, nitrogen content by 60.60%, potassium content by 24.35%, and calcium content by 14.27%. Among the trace elements, the iron content increased by 43.85%, zinc content by 12.83%, manganese content by 41.54%, molybdenum content by 2.59%, and selenium content by 108.15%.

[0037] Compared with treatments using single strains of phosphate-solubilizing bacteria, nitrogen-fixing bacteria, and potassium-solubilizing bacteria, this NPK compound microbial agent effectively increased the levels of nitrogen, phosphorus, potassium, calcium, iron, zinc, manganese, and selenium in crops. The efficiency of increase in the content of each mineral element was 4.72-29.18%, 18.18-31.95%, 10.80-22.89%, 8.10-9.69%, 54.35-82.82%, 9.58-24.28%, 27.78-48.39%, and 56.11-121.26%, respectively. The comprehensive enhancement of mineral elements by the compound microbial agent avoids the nutrient imbalance that may be caused by single microbial agents, providing crops with more balanced and comprehensive mineral nutrition.

[0038] Example 4: Effect of NPK compound microbial agent on soil nutrient improvement The effects of nitrogen, phosphorus, and potassium compound microbial agents on soil nutrition during crop growth promotion were investigated using the same experimental setup as in Example 3. Soil rhizosphere samples were collected periodically for index testing. Soil pH testing followed the method specified in "Soil pH Determination" (NY / T1121.2-2006); organic matter content testing followed the method specified in "Determination of Solid Organic Matter by Loss on Ignition Method" (HJ 761-2015); nitrogen content testing followed the method specified in "Determination of Total Nitrogen in Soil" (NY / T 1121.24-2012); available phosphorus content testing followed the method specified in "Determination of Available Phosphorus in Soil" (NY / T 1121.7-2014); available potassium content testing followed the method specified in "Determination of Available Potassium in Soil" (NY / T 889-2004); and available calcium content testing followed the method specified in "Determination of Exchangeable Calcium and Magnesium in Soil" (NY / T 1121.12-2006). The available content of soil trace elements was tested using inductively coupled plasma mass spectrometry. The results are shown in Appendix Table 3.

[0039] Appendix Table 3 Effects of NPK Compound Microbial Agent on the Available Content of Soil Nutrients Under the action of nitrogen, phosphorus and potassium compound bacterial agent, the soil pH decreased from 6.93 to 6.56, and the organic matter content increased from 31.6 g / kg to 34.3 g / kg.

[0040] The results showed that, compared with the blank control group, the nitrogen content increased from 0.65 g / kg to 0.88 g / kg, the available phosphorus content increased from 7.40 mg / kg to 10.93 mg / kg, the available potassium content increased from 74.33 mg / kg to 101.67 mg / kg, the available calcium content increased from 8677.32 mg / kg to 8777.52 mg / kg, the available iron content increased from 6.32 mg / kg to 6.54 mg / kg, the available zinc content increased from 0.39 mg / kg to 0.43 mg / kg, the available manganese content increased from 2.19 mg / kg to 2.31 mg / kg, the available molybdenum content increased from 0.064 mg / kg to 0.067 mg / kg, and the available selenium content increased from 0.074 mg / kg to 0.104 mg / kg. Compared with single-strain treatments, the nitrogen-phosphorus-potassium compound microbial agent of this invention can effectively increase the content of organic matter and nitrogen, phosphorus, and potassium nutrients in the soil, while also enhancing the bioavailability of various elements such as calcium, iron, zinc, manganese, molybdenum, and selenium. Through functional complementarity, the compound microbial agent overcomes the functional limitations of single-strain agents, achieving simultaneous activation of multiple mineral elements and significantly improving soil nutrient health.

[0041] Although the present invention has been described in detail above with general descriptions and specific embodiments, modifications or improvements can be made to it, which will be obvious to those skilled in the art. Therefore, all such modifications or improvements made without departing from the spirit of the present invention fall within the scope of protection claimed by the present invention.

Claims

1. A nitrogen-phosphorus-potassium compound bacterial agent, characterized in that, The strains in the nitrogen, phosphorus, and potassium compound bacterial agent consist of Pseudomonas sp. 1416X3 (CGMCC No. 17557), Priestia sp. ST2 (CGMCC No. 36011), and Burkholderia sp. YHN3-13 (CGMCC No. 36012).

2. The nitrogen-phosphorus-potassium compound bacterial agent according to claim 1, characterized in that, The ratio of viable counts of Pseudomonas sp. 1416X3, Priestia sp. ST2 and Burkholderia sp. YHN3-13 was (1~3):(1~3):(1~3).

3. The method for preparing the nitrogen-phosphorus-potassium compound bacterial agent according to claim 1 or 2, characterized in that, The *Pseudomonas* 1416X3, *Primate* ST2, and *Burkholderia* YHN3-13 were inoculated into liquid culture medium and cultured to the logarithmic growth phase. Their OD values ​​were then adjusted with sterile physiological saline. 600 The nitrogen, phosphorus and potassium compound bacterial agent is prepared by mixing the sample with a volume ratio of (1~3):(1~3):(1~3) to 0.9-1.

0.

4. The method for preparing the nitrogen-phosphorus-potassium compound bacterial agent according to claim 3, characterized in that, The liquid culture medium is LB liquid culture medium, and the culture conditions are: temperature 28-30 ℃, stirring speed 150-180 r / min, culture for 12 h.

5. The application of the nitrogen, phosphorus, and potassium compound microbial agent according to claim 1 or 2 in promoting plant growth.

6. The application of the nitrogen, phosphorus, and potassium compound microbial agent according to claim 1 or 2 in increasing the nitrogen, phosphorus, potassium, and organic matter content in soil.

7. The application of the nitrogen-phosphorus-potassium compound microbial agent according to claim 1 or 2 in improving the bioavailability of calcium, iron, zinc, manganese, molybdenum and selenium in soil.

8. The application of the nitrogen, phosphorus, and potassium compound microbial agent according to claim 1 or 2 in improving the bioavailability of calcium, iron, zinc, manganese, molybdenum, and selenium in the soil while increasing the content of nitrogen, phosphorus, potassium, and organic matter in the soil.

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