Efficient synergistic calcareous soil phosphorus activation preparation and application thereof

Abstract

The invention discloses an efficient synergistic calcareous soil phosphorus activation preparation and application thereof. The efficient synergistic calcareous soil phosphorus activation preparation comprises nano TiO2, phosphate solubilizing bacteria and yeast peptide. When the fertilizer is applied to calcareous soil crop planting, the phosphorus absorption of plants is improved. Through combined application of nano TiO2, phosphate solubilizing bacteria and yeast peptide, the pH value of calcareous soil can be reduced, fixed-state inorganic phosphorus in the soil is activated, the content of available phosphorus in the soil is increased, and supply of inorganic phosphorus in a soil-plant system is promoted. Through combined application of nano TiO2, phosphate solubilizing bacteria and yeast peptide, plants can obtain phosphorus nutrients in calcareous soil, and growth of crops is promoted.

Description

Technical Field

[0001] The present invention relates to the technical field of soil conditioners, and more particularly to a high-efficiency synergistic calcareous soil phosphorus activation preparation and application thereof. Background Art

[0002] Agricultural production relies primarily on the application of phosphorus fertilizers to ensure a stable phosphorus supply for crops and maintain stable yields. However, 80% of the phosphorus in phosphate fertilizers applied to calcareous soils is readily fixed, leaving only 10%-25% available for seasonal crop growth, reducing phosphorus availability. This is attributed to the high pH and calcium carbonate levels in the soil. Calcareous soils, widely distributed in arid and semi-arid regions, have high CaCO₃ contents and pH values, but low levels of available phosphorus and organic matter, making them a crucial resource for agricultural development. Long-term application of phosphate fertilizers leads to the gradual accumulation of Ca-P and organic matter in calcareous soils. Existing research suggests that if the availability and supply capacity of accumulated phosphorus in soils could be improved, it could be sufficient to support plant life for 100 years. Therefore, how to activate the fixed phosphorus in calcareous soils for bioavailability is of great theoretical and practical significance.

[0003] Phosphate-solubilizing bacteria (Phosphate-solubilizing bacteria) have the ability to convert insoluble phosphorus in the soil into phosphorus that can be directly absorbed and utilized by plants. They can lower soil pH by producing organic acids, such as 2-ketogluconic acid, thereby dissolving insoluble inorganic phosphorus into soluble phosphorus, increasing its availability. Application of the highly phosphate-solubilizing agent Burkholderia XQP35 significantly increased soil resin-Pi content by 67%, while application of Raoultella SQP80 significantly increased soil NaHCO3-Pi content. Furthermore, Phosphate-solubilizing bacteria secrete phosphatases, enhancing the ability to hydrolyze organic phosphorus in the soil and increasing its available phosphorus content. Studies have shown that the application of Phosphate-solubilizing bacteria can increase soil acid phosphatase activity, significantly increasing available phosphorus content in the rhizosphere soil of maize and boosting its phosphorus uptake. Under conditions of reduced phosphorus availability, application of the Pseudomonas aeruginosa (MK 764942.1) agent can effectively dissolve soil phosphorus, increasing both phosphorus content and peanut yield. However, there are a large number of indigenous microorganisms in the soil, and there may be antagonism between phosphate-solubilizing bacteria and other microorganisms, which will have a significant inhibitory effect on the growth and reproduction of phosphate-solubilizing bacteria and interfere with their effective phosphate-solubilizing effect in the soil.

[0004] Yeast peptides, a byproduct of brewer's yeast production, serve as a nutrient source for the growth of soil rhizosphere microorganisms, enriching plant-beneficial microorganisms and activating insoluble phosphorus in the soil by dissolving and hydrolyzing phosphorus. Yeast peptides can improve plant nutrient utilization and increase crop yield and quality. However, the synergistic effects of yeast peptides with other soil phosphorus activation measures (such as microbial agents) have not been fully studied. These synergistic effects may have a greater promoting effect on soil phosphorus activation and plant growth.

[0005] Nanoparticles have shown great potential and advantages in soil phosphorus activation. Among them, nano-TiO2 has a significant impact on soil nutrient conversion / availability, soil microbial population and plant physiological characteristics. Studies have found that 20mg·kg -1 Nano-TiO2 particles can significantly increase the activity of soil phosphatase and microbial biomass, 80 mg·kg -1 Nano-TiO2 particles can significantly change the community structure of soil microorganisms, and 100 mg·kg -1 The addition of nano-TiO2 significantly reduced the activity of soil acid and alkaline phosphatase and the biomass of microorganisms. -1 When the concentration of nano-TiO2 particles increases, it will produce a significant inhibitory effect. As the concentration of applied nano-TiO2 particles increases, the pH value of the rhizosphere soil shows a downward trend, while the effectiveness of phosphorus in the rhizosphere soil is significantly improved. TiO2 nanomaterials can not only stimulate soil enzyme activity, but also increase the absorption and utilization of phosphorus by plants, thereby improving crop yield and quality. For example, nano-TiO2 particles help rhizobia attach and grow in the root system, and promote the biomass of wheat seedlings. At the same time, the application of nano-TiO2 and phosphate-solubilizing bacteria regulates the low mobility of phosphorus by increasing the number of root hairs and improving the activity of phosphatase and dehydrogenase in the soil, thereby promoting phosphorus absorption by plants.

[0006] However, excessively high nanoparticle doses can easily cause negative effects. For example, studies have found that 1000 mg kg -1 The addition of nano-TiO2 particles inhibited the growth of wheat and also inhibited the development of leaves and roots in tobacco seedlings. Furthermore, the combined application of nano-TiO2 with other phosphorus enhancement technologies should also reduce its dosage.

[0007] Previously, most studies have focused on the effects of single nanoparticles, phosphate-solubilizing bacteria, or peptides, or their combination, on soil enzyme activity or pH. There has been a lack of systematic research examining the combined effects of these three factors on soil phosphorus form transformation, plant phosphorus nutrient absorption, and utilization. Therefore, developing a rationale for improving the quality of nano-TiO2 combined with phosphate-solubilizing bacteria and yeast peptides has been a pressing challenge for those skilled in the art. Summary of the Invention

[0008] In view of this, the present invention provides a high-efficiency synergistic calcareous soil phosphorus activation preparation and its application, which uses nano-TiO2, phosphate-solubilizing bacteria and yeast peptides as main components, and optimizes the composition ratio to achieve the best effect on calcareous soil phosphorus activation and vegetable phosphorus nutrient absorption and growth.

[0009] To achieve the above object, the present invention adopts the following technical solutions:

[0010] First, the present invention provides a highly efficient, synergistic calcareous soil phosphorus activation agent comprising nano-TiO2, phosphate-solubilizing bacteria, and yeast peptide. The phosphate-solubilizing bacteria is Pseudomonas protegens LSOJM27, which has been deposited with the China Center for Type Culture Collection under the CCTCC No. M20222101 and on December 29, 2022. Both nano-TiO2 and yeast peptide are commercially available, with the yeast peptide being produced by Angel Micro.

[0011] Preferably, the application amount of nano-TiO2 is 25 μg / kg-25 mg / kg based on the weight of dry soil.

[0012] Furthermore, the nano-TiO2 content is greater than 99.8%, the crystal form is anatase, the average particle size is 30nm, and the specific surface area is 78m 2 / g.

[0013] Preferably, the amount of phosphate-solubilizing bacteria applied is 6×10 7 CFU / g.

[0014] Preferably, the administration amount of the yeast peptide is 0-10 mg / kg.

[0015] The present invention also provides the use of the high-efficiency synergistic calcareous soil phosphorus activation preparation described in the above technical solution in the cultivation of crops in calcareous soil.

[0016] Furthermore, the crop is bok choy.

[0017] It can be seen from the above technical solutions that, compared with the prior art, the present invention discloses a highly efficient synergistic calcareous soil phosphorus activation preparation and its application, which has the following beneficial effects:

[0018] Nano-TiO2, combined with phosphate-solubilizing bacteria and yeast peptides, can lower the pH of calcareous soils, activate fixed inorganic phosphorus in the soil, increase the available phosphorus content in the soil, and promote the supply of inorganic phosphorus in the soil-plant system. The combination of nano-TiO2, phosphate-solubilizing bacteria, and yeast peptides can help plants obtain phosphorus nutrients in calcareous soils and promote crop growth. BRIEF DESCRIPTION OF THE DRAWINGS

[0019] In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the following briefly introduces the drawings required for use in the embodiments or the description of the prior art. Obviously, the drawings described below are merely embodiments of the present invention. For ordinary technicians in this field, other drawings can be obtained based on the provided drawings without paying any creative work.

[0020] Figure 1 is the available phosphorus content in the soil in the experimental case (different lowercase letters indicate significant differences among treatments, P < 0.05, the same below);

[0021] Figure 2 The effects of different treatments on soil pH in the experimental case;

[0022] Figure 3 represents the soil P transformation-related enzyme activities in the experimental example, where A: acid phosphatase activity B: alkaline phosphatase activity C: phosphodiesterase activity D: phytase activity;

[0023] Figure 4 It is the absorption of Shanghai Qing P in the experimental example;

[0024] Figure 5 The fresh weight and dry weight of the bok choy in the experiment, where A: fresh weight of bok choy and B: dry weight of bok choy;

[0025] Figure 6 This is the Pearson correlation analysis between plant indicators and soil indicators in the experimental example, where AcP: soil acid phosphatase activity; AlP: soil alkaline phosphatase activity; PD: phosphodiesterase activity; Phytase: phytase activity; AP: soil available phosphorus content; P uptake: plant phosphorus uptake; FW: plant fresh weight; DW: plant dry weight. DETAILED DESCRIPTION

[0026] The following will clearly and completely describe the technical solutions of the present invention in conjunction with the embodiments and experimental examples of the present invention. Obviously, the embodiments described are only part of the embodiments of the present invention, not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without making creative efforts are within the scope of protection of the present invention.

[0027] Experimental example

[0028] 1. Overview of the test soil

[0029] The test soil was collected from Taohuatu, Shuangta District, Liaoning Province (41°43'N, 120°36'E). Located in a low mountainous and hilly area, the area has a temperate semi-arid, semi-humid continental monsoon climate with an average annual temperature of 8.4°C, an average annual precipitation of 483.1 mm, and a frost-free period of approximately 160 days. The soil type is calcareous brown soil. Soil samples were collected from multiple locations within the 0-20 cm depth and prepared as composites for future use. The soil contained 14.1 g / kg organic matter, 0.86 g / kg total nitrogen, 0.63 g / kg total phosphorus, 0.38 g / kg inorganic phosphorus, 0.25 g / kg organic phosphorus, 5.98 mg / kg available phosphorus extracted with NaHCO3, and a pH of 7.38.

[0030] 2. Experimental Design

[0031] Before incubation, soil was sieved through a 2 mm sieve to remove roots, shoots, and other material. The soil moisture content was then adjusted to approximately 40% of its field capacity and incubated in the dark at 25°C for two weeks to restore soil microbial activity. After pre-incubation, 300 g of dry soil was assigned to each of the following nine treatments: control (no phosphorus activation); low-concentration nanoparticle groups: L (25 μg / kg nano-TiO2); LP (25 μg / kg nano-TiO2 and phosphate-solubilizing bacteria); LY (25 μg / kg nano-TiO2 and yeast peptide); and LPY (25 μg / kg TiO2, phosphate-solubilizing bacteria, and yeast peptide); and high-concentration nanoparticle groups: H (25 mg / kg TiO2); HP (25 mg / kg TiO2 and phosphate-solubilizing bacteria); HY (25 mg / kg TiO2 and yeast peptide); and HPY (25 mg / kg TiO2, phosphate-solubilizing bacteria, and yeast peptide). All treatments were simultaneously treated with an amount equivalent to 200 kg N / hm-3 of nitrogen per liter. 2 Urea and 73kg K / hm 2 The amount of phosphate-solubilizing bacteria added was 6×10 7 CFU / g dry soil, yeast peptide addition was 10mg / kg dry soil. Soil moisture was adjusted to 60% of field capacity, and soil thickness was approximately 15cm. The experiment was conducted according to a completely randomized design, with three replicates per treatment. The test crop was Shanghai Qing, sown in five holes per pot, with two seeds per hole, at a sowing depth of approximately 1-1.5cm. During the cultivation period, water was applied every three to five days according to the growth requirements of the Shanghai Qing.

[0032] Nano-TiO2 (VK-TA30D), content> 99.8%, crystal form is anatase, average particle size is 30nm, specific surface area is 78m 2 / g, purchased from Xuancheng Jingrui New Materials Co., Ltd. Shanghai Qing seeds were purchased from Cangzhou Heshuo Agricultural Technology Co., Ltd., Hebei Province. The phosphate-solubilizing strain Pseudomonas protegens LSOJM27 were isolated and identified in our laboratory and deposited in the China Center for Type Culture Collection, Wuhan University, with the identification number CCTCC No: M20222101. Yeast peptides were provided by Angel Micro.

[0033] 3. Soil Sample Analysis

[0034] After the cultivation, the soil around the roots of the green vegetables was collected and divided into two parts after passing through a 2 mm sieve. One part was stored in a refrigerator at 4 ° C for the determination of soil enzyme activity; the other part was air-dried under natural conditions for the determination of soil pH and available phosphorus content.

[0035] Soil pH was measured using a pH meter at a soil-water ratio of 1:2.5. Available soil phosphorus (resin-Pi and NaHCO3-Pi) was determined using a modified Hedley continuous extraction fractionation method: resin-Pi was extracted with anion exchange resin and deionized water, shaken for 16 hours, removed from the resin, shaken for 1 hour with 0.25 M sulfuric acid, and assayed using the molybdenum blue colorimetric method. Soil samples were centrifuged at 10,000 g for 10 minutes, and NaHCO3-Pi was extracted with 30 mL of 0.5 M NaHCO3 (pH 8.5). After shaking for 16 hours and centrifuging, the supernatant was collected and assayed using the malachite green method. In this study, the sum of resin-phosphorus and inorganic phosphorus extracted with NaHCO3 was defined as available soil phosphorus. Soil acid phosphatase (AcP) and alkaline phosphatase (AlP) activities were measured using sodium p-nitrophenylphosphate (ρNPP, Sigma-Aldrich, USA) as substrates, and phosphodiesterase (PD) activity was measured using bis-p-nitrophenyl phosphate (Sigma-Aldrich, USA) as substrates. The activities of these three enzymes were characterized by the amount of p-nitrophenol produced when 1.00 g of fresh soil was incubated with universal buffer solutions of different pH values (6.5, 11.0, and 8.0) at 37°C for 1 h. Phytase (PhA) activity was measured using a phytase kit, with 1 nmol of inorganic phosphorus released per gram of soil per hour from a 5 mmol / L sodium phytate solution as one unit of activity. The kit was purchased from Bioesn.

[0036] 4. Plant Sample Analysis

[0037] The experimental incubation period was 35 days. After the incubation period, the Shanghai green plants were collected, washed with distilled water, and the surface moisture was removed by filter paper. The fresh weight of the plants was then measured. The dry weight of the plants was then fixed at 105°C for 30 minutes and then dried at 80°C to constant weight. The dried plant samples were ground in a mortar and sealed for further analysis. The carbon and nitrogen contents of the plants were determined using an elemental analyzer (Elementarvario MACRO cube, Germany). The total phosphorus content of the plants was determined using nitric acid digestion-molybdenum blue colorimetry. The total potassium content of the plants was determined using nitric acid digestion-flame photometry. The nutrient absorption of the plants was calculated based on the dry weight and nutrient content of the plants.

[0038] 5. Data Processing

[0039] The data were organized using Excel, and the Duncan method in SPSS24.00 software was used to test the significance of the mean values of various indicators in different treatments (P < 0.05). GraphPadPrism 9.5 software was used to draw bar graphs, and R language was used to draw correlation analysis graphs.

[0040] 6. Experimental Results

[0041] 6.1 Changes in soil available phosphorus content and pH

[0042] As attached Figure 1 As shown, compared with the control, the LPY treatment significantly increased the resin-Pi content in the soil, while the LP, LPY, HP, and HPY treatments significantly increased the NaHCO3-Pi content in the soil. The NaHCO3-Pi content in the HP and HPY treatments was significantly higher than that in the H treatment. The resin-Pi, NaHCO3-Pi, and available phosphorus contents in the LPY treatment were significantly higher than those in the L and LY treatments. The LPY and HP treatments significantly increased the available phosphorus (resin-Pi + NaHCO3-Pi) content in the soil. The available phosphorus content in the HP treatment was significantly higher than that in the H treatment.

[0043] like Figure 2 As shown in the figure, compared with the control, under low Ti concentration conditions, the LPY treatment significantly reduced the soil pH, while the other treatments had no significant differences from the control. Under high Ti concentration conditions, compared with the control, the HP and HPY treatments significantly reduced the soil pH, while the HY treatment had no significant change in soil pH.

[0044] 6.2 Response of soil phosphatase activity

[0045] like Figure 3 As shown in Figure 2, low concentration Ti treatment had no significant effect on soil AcP activity, while HP and HY treatments significantly reduced soil AcP activity in high concentration Ti treatments ( Figure 3), the AcP activity in the soil treated with HP was significantly lower than that in the soil treated with H ( Figure 3 A). Compared with the control, under low concentration conditions, soil AlP activity in the LP treatment was significantly reduced; under high concentration treatments, all treatments significantly reduced soil AlP activity. Soil AlP activity in the HP treatment was significantly lower than that in the HY treatment ( Figure 3 B) In the low concentration group, except for LY, the soil PD activity of the other treatments was significantly lower than that of the control. The soil PD activity of L and LPY treatments was significantly lower than that of LY treatment. In the high concentration group, all four treatments significantly reduced the soil PD activity. The activity from low to high is: HPY <HY<HP<H( Figure 3 C). In the four low Ti concentration treatments, soil PhA activity was not significantly different from the control; in the high Ti concentration treatments, soil PhA activity in the HP treatment was significantly higher than that in the H and HPY treatments ( Figure 3 D).

[0046] 6.3 Phosphorus Uptake by Aboveground Plants

[0047] like Figure 4 As shown in the data, under low concentration conditions, the L, LP, and LPY treatments significantly increased the phosphorus absorption of green vegetables, and the phosphorus absorption of green vegetables in the LPY treatment was significantly higher than that in the L treatment; under the three high concentration nano-titanium dioxide treatments, the phosphorus absorption of green vegetables was significantly increased compared with the control, and there was no significant difference between the H treatment and the control.

[0048] 6.4 Growth Response of Shanghai Green

[0049] like Figure 5 As shown in the figure, compared with the control, except for the LY and H treatments, the other treatments significantly increased the fresh weight of green vegetables, among which the LP, PY, HP, and HPY treatments had particularly significant effects. In the low concentration group, the fresh weight of green vegetables treated with LP and LPY was significantly higher than that treated with L; in the high concentration group, the fresh weight of green vegetables treated with HP and HPY was significantly higher than that treated with H ( Figure 5 A). Compared with the control, only LPY, HP and HPY treatments significantly increased the dry weight of green vegetables. In the high concentration group, HPY treatment significantly increased the dry weight of green vegetables by 41.36% compared with H treatment ( Figure 5 B).

[0050] 6.5 Correlation between soil properties and plant growth

[0051] like Figure 6As shown in the results, under the experimental conditions, there was a significant positive correlation between soil AcP activity and resin-Pi content (P≤0.05). AlP activity was extremely significantly negatively correlated with soil NaHCO3-Pi content (P≤0.01) and with soil available phosphorus content (P≤0.05). Soil PD activity was extremely significantly positively correlated with soil PhA activity (P≤0.01) and with soil pH (P≤0.001). Soil resin-Pi content was significantly negatively correlated with soil pH (P≤0.05). Soil NaHCO3-Pi was extremely significantly negatively correlated with soil pH (P≤0.001). Soil AP content was extremely significantly negatively correlated with soil pH (P≤0.001).

[0052] The fresh weight, dry weight, and phosphorus uptake of green vegetables were all significantly negatively correlated with soil PD activity. The fresh weight, dry weight, and phosphorus uptake of green vegetables were also significantly negatively correlated with soil pH. Green vegetable fresh weight was significantly negatively correlated with soil AlP activity (P ≤ 0.01) and significantly positively correlated with resin-Pi (P ≤ 0.05). Soil NaHCO3-Pi was significantly positively correlated with green vegetable fresh weight and N, P, and K uptake (P ≤ 0.01). Green vegetable dry weight was significantly positively correlated with soil NaHCO3-Pi (P ≤ 0.05). Soil available phosphorus content was significantly positively correlated with plant N, P, and K uptake, as well as with plant fresh weight (P ≤ 0.001). Plant NPK nutrient uptake was also significantly positively correlated with both plant dry and fresh weight (P ≤ 0.001).

[0053] In summary, nano-TiO2 particles have a stimulating effect on phosphate-solubilizing bacteria, increasing their activity and metabolism, thereby promoting the dissolution of soil phosphorus. Phosphate-solubilizing bacteria can promote the dissolution of inorganic phosphorus in the soil by releasing organic acids. The application of yeast peptides can, on the one hand, input nutrients such as N and P into the soil, increasing the content of soil phosphorus; on the other hand, it can reduce the pH of the soil, alleviating the Ca content in calcareous soils to a certain extent. 2+ Phosphorus fixation. Specifically, the present invention utilizes nano-TiO2, phosphate-solubilizing bacteria, and yeast peptides to lower soil pH, thereby activating inorganic phosphorus in the soil. Increasing the available phosphorus content in the soil can enhance plant phosphorus absorption, thereby promoting crop growth.

[0054] The various embodiments in this specification are described in a progressive manner, and each embodiment focuses on the differences from other embodiments. The same and similar parts between the various embodiments can be referred to each other. The above description of the disclosed embodiments enables professionals and technicians in this field to implement or use the present invention. Various modifications to these embodiments will be apparent to professionals and technicians in this field, and the general principles defined herein can be implemented in other embodiments without departing from the spirit or scope of the present invention. Therefore, the present invention will not be limited to the embodiments shown herein, but will conform to the widest scope consistent with the principles and novel features disclosed herein.

Claims

1. A highly efficient synergistic calcareous soil phosphorus activation preparation, characterized in that: include: Nano-TiO2, phosphate-solubilizing bacteria and yeast peptide. The phosphate-solubilizing bacteria is Pseudomonas protegens LSOJM27, which is deposited in China Center for Type Culture Collection with a deposit number of CCTCC No: M20222101.

2. The highly efficient synergistic calcareous soil phosphorus activation preparation according to claim 1, characterized in that: Based on the weight of dry soil, the application amount of the nano-TiO2 is 25μg / kg-25mg / kg.

3. The highly efficient synergistic calcareous soil phosphorus activation preparation according to claim 1, characterized in that: Based on the weight of dry soil, the amount of phosphate-solubilizing bacteria applied is 6×10 7 CFU / g.

4. The highly efficient synergistic calcareous soil phosphorus activation preparation according to claim 1, characterized in that: The yeast peptide is applied in an amount of 0-10 mg / kg based on dry soil weight.

5. Use of the high-efficiency synergistic calcareous soil phosphorus activation preparation according to any one of claims 1 to 4 in the cultivation of crops in calcareous soil.

6. The use according to claim 5, characterized in that The crop is bok choy.

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