Efficient synergistic calcareous soil phosphorus activation preparation and application thereof

Abstract

The application discloses a kind of high-efficiency synergic calcareous soil phosphorus activation preparation and application thereof, comprising: nano TiO2, phosphorus-solubilizing bacteria and yeast peptide.It is applied to calcareous soil crop planting, and the absorption of plant to phosphorus is improved.Nano TiO2 is applied with phosphorus-solubilizing bacteria and yeast peptide, and the pH of calcareous soil can be reduced, the fixed state inorganic phosphorus in soil is activated, the content of effective phosphorus in soil is improved, and the supply of inorganic phosphorus in soil-plant system is promoted.Nano TiO2 is applied with phosphorus-solubilizing bacteria and yeast peptide, and it can help plant to obtain phosphorus nutrient in calcareous soil, and promote the growth of crop.

Description

TECHNICAL FIELD

[0001] The application relates to the technical field of soil improvers, in particular to a high-efficiency synergistic calcium soil phosphorus activation preparation and application thereof. BACKGROUND

[0002] In agricultural production, phosphorus fertilizers are mainly relied on to ensure the supply of phosphorus to crops and maintain the stability of crop yield. However, 80% of the phosphorus in the phosphorus fertilizer applied to calcareous soil is easily fixed, and only 10%-25% can be used for the growth of crops in the current season, which reduces the availability of phosphorus. This is due to the high pH and calcium carbonate level in the soil. Calcareous soil is widely distributed in arid and semiarid regions, has a high CaCO3 content and pH value, but a low effective phosphorus and organic matter content, and is an important resource for agricultural development. Long-term application of phosphorus fertilizer causes the gradual accumulation of Ca-P and O-P in calcareous soil. Related existing researches show that if the availability and supply capacity of the accumulated phosphorus in the soil can be improved in some way, it can support the use of plants for 100 years. Therefore, how to activate the fixed phosphorus in calcareous soil for biological utilization has important theoretical and practical significance.

[0003] Phosphate-solubilizing bacteria have the ability to convert insoluble phosphorus in the soil into phosphorus that can be directly absorbed and utilized by plants. Phosphate-solubilizing bacteria can reduce the pH value of the soil by producing organic acids such as 2-ketogluconic acid, so as to dissolve the insoluble inorganic phosphorus in the soil into soluble phosphorus and increase the availability of phosphorus in the soil. After applying the phosphorus-dissolving bacterial agent Burkholderia XQP35, the content of resin-Pi in the soil is significantly increased by 67%; after applying Raoultella SQP80, the content of NaHCO3-Pi in the soil is significantly increased. In addition, phosphate-solubilizing bacteria can secrete phosphatase to improve the hydrolysis capacity of soil organic phosphorus and increase the content of effective phosphorus in the soil. It is found that the application of phosphate-solubilizing bacteria can improve the activity of soil acid phosphatase, significantly increase the content of effective phosphorus in the rhizosphere soil of corn, and increase the phosphorus absorption amount of corn. Under the condition of reduced phosphorus, the application of Pseudomonas aeruginosa (MK 764942.1) bacterial agent can effectively dissolve the phosphorus in the soil and increase the phosphorus content and yield of peanuts. However, there are a large number of indigenous microorganisms in the soil, and there may be antagonism between phosphate-solubilizing bacteria and other microorganisms, thereby forming an obvious inhibitory effect on the growth and reproduction of phosphate-solubilizing bacteria and interfering with the effective phosphorus-dissolving effect of phosphate-solubilizing bacteria in the soil.

[0004] The by-product of the production process of beer yeast, yeast peptides, can be used as a nutrient source for rhizosphere microorganisms, enriching plant-beneficial microorganisms, and activating soil-insoluble phosphorus through the action of dissolving / hydrolyzing phosphorus. Yeast peptides can improve plant nutrient utilization, increase crop yield and quality. However, the synergistic effect of yeast peptides with other soil phosphorus activation measures (such as microbial inoculants) has not been fully studied, and 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 transformation / effectiveness, soil microbial population, and plant physiological characteristics. Studies have found that 20 mg·kg -1 of nano-TiO2 particles can significantly increase soil phosphatase activity and microbial biomass, 80 mg·kg -1 of nano-TiO2 particles can significantly change the community structure of soil microorganisms, and 100 mg·kg -1 of nano-TiO2 particles can significantly reduce soil acid and alkaline phosphatase activity and microbial biomass. Nano-TiO2 has a significant inhibitory effect when the concentration is higher than 250 mg·kg -1 , and with the increase of the concentration of nano-TiO2 particles, the pH value of the rhizosphere soil shows a downward trend, and the availability of phosphorus in the rhizosphere soil is significantly improved. TiO2 nanoparticles not only stimulate soil enzyme activity, but also increase plant phosphorus uptake and utilization, resulting in improved crop yield and quality. For example, nano-TiO2 particles help rhizobium adhere and grow in the root system, and promote the biomass of wheat seedlings. The application of nano-TiO2 and phosphorus-solubilizing bacteria can increase the number of root hairs and improve the activity of soil phosphatase and dehydrogenase, regulate the low mobility of phosphorus, and promote the absorption of phosphorus by plants.

[0006] However, excessive dosage of nanoparticles can have negative effects. For example, studies have found that 1000 mg·kg -1 of nano-TiO2 particles can inhibit the growth of wheat and inhibit the development of tobacco seedling leaves and roots. At the same time, the combined application of nano-TiO2 and other phosphorus efficiency technologies should also reduce the dosage.

[0007] Previously, most studies tended to focus on the effects of single nanoparticles, phosphorus-solubilizing bacteria or peptide treatment or the combination of the two on soil enzyme activity or pH, and lacked systematic studies on the combination effect of the three on soil phosphorus form transformation, plant phosphorus nutrient absorption and utilization, etc. Therefore, how to provide a reasonable nano-TiO2 combined with phosphorus-solubilizing bacteria and yeast peptide modifier is a problem to be solved by those skilled in the art. SUMMARY

[0008] In view of this, the present invention provides a highly efficient synergistic phosphorus activation agent for calcareous soil and its application, which uses nano-TiO2 combined with phosphate-solubilizing bacteria and yeast peptides as the main components, and optimizes the composition ratio to achieve the best effect on phosphorus activation in calcareous soil and phosphorus nutrient absorption and growth of green vegetables.

[0009] To achieve the above objectives, the present invention adopts the following technical solution:

[0010] First, this invention provides a highly efficient synergistic phosphorus activation agent for calcareous soils, comprising: nano-TiO2, phosphate-solubilizing bacteria, and yeast peptides. The phosphate-solubilizing bacteria is *Pseudomonas protegens* LSOJM27, deposited at the China Center for Type Culture Collection (CCTCC) with accession number CCTCC No.: M20222101, and deposited on December 29, 2022. Both the nano-TiO2 and the yeast peptides are commercially available, and the yeast peptides are produced by Angel Yeast Co., Ltd.

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

[0012] Furthermore, the nano-TiO2 content is >99.8%, the crystal form is anatase, the average particle size is 30 nm, and the specific surface area is 78 m². 2 / g.

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

[0014] Preferably, the yeast peptide is applied at a dosage of 0-10 mg / kg.

[0015] This invention also provides the application of the highly efficient synergistic phosphorus activator for calcareous soils described above in the cultivation of crops in calcareous soils.

[0016] Furthermore, the crop in question is Shanghai bok choy.

[0017] As can be seen from the above technical solution, compared with the prior art, the present invention discloses a highly efficient synergistic phosphorus activator for calcareous soils and its application, which has the following beneficial effects:

[0018] The combined application of nano-TiO2 with phosphate-solubilizing bacteria and yeast peptides can lower the pH of calcareous soils, activate fixed inorganic phosphorus in the soil, increase the content of available phosphorus in the soil, and promote the supply of inorganic phosphorus in the soil-plant system. The combined application of nano-TiO2 with phosphate-solubilizing bacteria and yeast peptides can help plants obtain phosphorus nutrients in calcareous soils and promote crop growth. Attached Figure Description

[0019] To more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the drawings used in the description of the embodiments or the prior art will be briefly introduced below. Obviously, the drawings described below are only embodiments of the present invention. For those skilled in the art, other drawings can be obtained based on the provided drawings without creative effort.

[0020] Figure 1 The available phosphorus content in the soil in the experimental case (different lowercase letters indicate significant differences between 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 The activities of enzymes related to soil P transformation in the experimental example include: A: acid phosphatase activity; B: alkaline phosphatase activity; C: phosphodiesterase activity; D: phytase activity.

[0023] Figure 4 For the absorption of Shanghai bok choy P in the experimental example;

[0024] Figure 5 The fresh weight and dry weight of Shanghai bok choy in the experimental example, where A: fresh weight of Shanghai bok choy and B: dry weight of Shanghai bok choy.

[0025] Figure 6 The Pearson correlation analysis between plant and soil indicators in the experimental case is shown. Among them, 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, and DW: plant dry weight. Detailed Implementation

[0026] The technical solution of the present invention will be clearly and completely described below with reference to embodiments and experimental examples. Obviously, the described embodiments are only some embodiments of the present invention, and not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of the present invention.

[0027] Experimental Example

[0028] I. Overview of the tested soil

[0029] The test soil was collected from Taohuatu (41°43'N, 120°36'E) in Shuangta District, Liaoning Province, which is located in low hilly area, belongs to temperate semi-arid and semi-humid continental monsoon climate, the average annual temperature is 8.4℃, the average annual precipitation is 483.1mm, the frost-free period is about 160d. The soil type is calcareous brown soil. The mixed sample was prepared by collecting 0-20cm soil from multiple points. The soil organic matter is 14.1g / kg, total nitrogen is 0.86g / kg, total phosphorus is 0.63g / kg, inorganic phosphorus is 0.38g / kg, organic phosphorus is 0.25g / kg, NaHCO3 extractable phosphorus is 5.98mg / kg, and pH is 7.38.

[0030] II. Test design

[0031] Before the start of the culture, the soil was passed through a 2mm sieve to remove roots, buds and other materials, and then the soil moisture content was adjusted to about 40% of the field water holding capacity, and incubated at 25℃ in the dark for two weeks to restore the activity of soil microorganisms. After the pre-incubation, 300g of dry soil was arranged for each treatment as follows: Control (no phosphorus activation treatment), low concentration of nanoparticle group: L (25μg / kg nano-TiO2), LP (25μg / kg nano-TiO2 and phosphate solubilizing bacteria), LY (25μg / kg nano-TiO2 and yeast extract) and LPY (25μg / kg TiO2, phosphate solubilizing bacteria and yeast extract), high concentration of nanoparticle group: H (25mg / kg TiO2), HP (25mg / kg TiO2 and phosphate solubilizing bacteria), HY (25mg / kg TiO2 and yeast extract) and HPY (25mg / kg TiO2, phosphate solubilizing bacteria and yeast extract). All treatments were applied with equivalent 200kg N / hm 2 urea and 73kg K / hm 2 . The amount of phosphate solubilizing bacteria added was 6×10 7 CFU / g dry soil, and the amount of yeast extract added was 10mg / kg dry soil. The soil moisture content was adjusted to 60% of the field water holding capacity, and the soil thickness was about 15cm. The test was carried out according to the complete random design, with 3 replicates for each treatment. The test crop was Shanghai green, which was sown with 2 seeds per hole in 5 holes per pot, with a sowing depth of about 1-1.5cm. During the incubation period, water was poured once every three to five days according to the growth needs of Shanghai green.

[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 Material Co., Ltd. The cabbage seeds were purchased from Hebei Cangzhou Heshuo Agricultural Technology Co., Ltd. The phosphate-solubilizing bacterial strain Pseudomonas protegens LSOJM27 was isolated and identified by the laboratory and preserved in the China Center for Type Culture Collection, Wuhan University, with the accession number CCTCC No: M20222101. The yeast peptide was provided by Angel Yeast Co., Ltd.

[0033] III. Soil sample analysis

[0034] The soil around the cabbage roots was collected after the end of the culture, and after passing through a 2 mm sieve, the soil was divided into two parts, one of which was stored in a 4°C refrigerator for the determination of soil enzyme activity; the other was air-dried under natural conditions for the determination of soil pH and available phosphorus content.

[0035] The soil pH was determined by a pH meter with a soil-to-water ratio of 1:2.5. The soil available phosphorus (resin-Pi and NaHCO3-Pi) was determined by the modified Hedley sequential extraction method, as follows: resin-Pi was extracted with anion exchange resin and deionized water, shaken for 16 h, then the resin was shaken with 0.25 M sulfuric acid for 1 h, and the content was determined by molybdenum blue colorimetry; the soil sample was centrifuged at 10000g for 10 min, and 30 mL of 0.5M NaHCO3(pH 8.5) was used to extract NaHCO3-Pi, shaken for 16 h, then centrifuged, and the supernatant was taken to determine its content by malachite green method. In this study, the sum of resin phosphorus and NaHCO3-extracted inorganic phosphorus content was defined as soil available phosphorus. The activities of soil acid phosphatase (AcP), alkaline phosphatase (AlP) and phosphodiesterase (PD) were determined using p-nitrophenyl phosphate (ρNPP, Sigma-Aldrich, USA) as the substrate, and the activity of phosphodiesterase (PD) was determined using bis-p-nitrophenyl phosphate (Sigma-Aldrich, USA) as the substrate. The activities of these three enzymes were characterized by the content of p-nitrophenol generated by incubating 1.00 g of fresh soil with different pH universal buffer (6.5, 11.0, 8.0) at 37°C for 1 h. The activity of phytase (PhA) was determined 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 a unit of enzyme activity. The kit was purchased from bioesn company.

[0036] IV. Plant sample analysis

[0037] The culture period for the experiment was 35 days. After the culture period, Shanghai bok choy plants were collected, washed with distilled water, blotted dry with filter paper, and weighed to determine the fresh weight of the plants. The dry weight of the plants was determined by blanching at 105℃ for 30 minutes, drying at 80℃ to constant weight, and then weighing. The dried plant samples were ground in a mortar and sealed for further analysis. The C and N contents of the plants were determined using an elemental analyzer (Elementarvario MACRO cube, Germany), the total phosphorus content was determined using the nitric acid digestion-molybdenum blue colorimetric method, and the total potassium content was determined using the nitric acid digestion-flame photometry method. The nutrient uptake of the plants was calculated based on the dry weight and nutrient content of the plants.

[0038] V. Data Processing

[0039] The data were organized using Excel. The Duncan method in SPSS 24.00 was used to test the significance of differences in the average values ​​of each indicator under different treatments (P < 0.05). GraphPadPrism 9.5 was used to create bar charts, and R language was used to create correlation analysis plots.

[0040] VI. Experimental Results

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

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

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

[0044] 6.2 Response of Soil Phosphatase Activity

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

[0046] 6.3 Phosphorus uptake of plant aboveground

[0047] As shown in Figure 4 , in low concentration conditions, L, LP, LPY treatment significantly improved the phosphorus uptake of Chinese cabbage, and the phosphorus uptake of Chinese cabbage in LPY treatment was significantly higher than that in L treatment; in high concentration of nano-titanium dioxide, the phosphorus uptake of Chinese cabbage was significantly improved compared with the control, and there was no significant difference between H treatment and the control.

[0048] 6.4 Growth response of Shanghai cabbage

[0049] As shown in Figure 5 , compared with Control, except LY and H treatment, the rest of the treatment significantly improved the fresh weight of Chinese cabbage, among which, LP, PY, HP, HPY treatment was particularly effective. In low concentration group, the fresh weight of Chinese cabbage in LP and LPY treatment was significantly higher than that in L treatment; in high concentration group, the fresh weight of Chinese cabbage in HP and HPY treatment was significantly higher than that in H treatment ( Figure 5 A). Compared with the control, only LPY, HP and HPY treatment significantly increased the dry weight of Chinese cabbage. In high concentration group, compared with H treatment, HPY treatment significantly increased the dry weight of Chinese cabbage by 41.36% ( Figure 5 B).

[0050] 6.5 Correlation between soil properties and plant growth

[0051] As shown in Figure 6The results showed that, under the experimental conditions, the AcP activity of the soil was significantly positively correlated with the resin-Pi content (P≤0.05). The AlP activity was significantly negatively correlated with the NaHCO3-Pi content in the soil (P≤0.01) and the available phosphorus content in the soil (P≤0.05). The PD activity of the soil was significantly positively correlated with the PhA activity (P≤0.01) and the soil pH (P≤0.001). The resin-Pi content in the soil was significantly negatively correlated with the soil pH (P≤0.05). The NaHCO3-Pi content in the soil was significantly negatively correlated with the soil pH (P≤0.001). The AP content in the soil was significantly negatively correlated with the soil pH (P≤0.001).

[0052] The fresh weight, dry weight and phosphorus absorption of the green vegetables were significantly negatively correlated with the PD activity of the soil. The fresh weight, dry weight and phosphorus absorption of the green vegetables were significantly negatively correlated with the soil pH. The fresh weight of the green vegetables was significantly negatively correlated with the AlP activity of the soil (P≤0.01) and significantly positively correlated with the resin-Pi content (P≤0.05). The NaHCO3-Pi content in the soil was significantly positively correlated with the fresh weight of the green vegetables and the N, P and K absorption of the green vegetables (P≤0.01). The dry weight of the green vegetables was significantly positively correlated with the NaHCO3-Pi content in the soil (P≤0.05). The available phosphorus content in the soil was significantly positively correlated with the N, P and K absorption of the green vegetables and the fresh weight of the green vegetables (P≤0.001). The NPK absorption of the green vegetables was significantly positively correlated with the dry weight and fresh weight of the green vegetables (P≤0.001).

[0053] In summary, the nano-TiO2 particles have a stimulating effect on the phosphorus-solubilizing bacteria, increase the activity and metabolism of the phosphorus-solubilizing bacteria and thus promote the dissolution of the soil phosphorus. The phosphorus-solubilizing bacteria can promote the dissolution of the soil inorganic phosphorus by releasing organic acids and the like. The application of the yeast peptides can input the N and P nutrients into the soil, increase the phosphorus content in the soil and reduce the soil pH to a certain extent, thereby alleviating the Ca 2+ phosphorus fixation. That is, the technical scheme of the present application can reduce the soil pH by applying the nano-TiO2, the phosphorus-solubilizing bacteria and the yeast peptides, so as to activate the inorganic phosphorus in the soil. The increase of the available phosphorus content in the soil can improve the phosphorus absorption of the plants and thus promote the growth of the crops.

[0054] The various embodiments described in this specification are intended to be illustrative of the invention and do not limit the scope of the invention. Although specific embodiments have been described herein, they are not to be taken as the only embodiments of the invention. Various modifications can be made to the embodiments described and other embodiments can be used without departing from the spirit or scope of the invention. Accordingly, the scope of the invention is to be limited only by the claims.

Claims

1. A highly synergistic calcareous soil phosphorus activation formulation characterized in that, Comprise: Nano-TiO2, phosphate-solubilizing bacteria and yeast peptides, wherein the phosphate-solubilizing bacteria is Pseudomonas protegens LSOJM27, which is preserved in China Center for Type Culture Collection, and the preservation number is CCTCC No: M20222101; The nano-TiO2 is applied in an amount of 25 μg / kg to 25 mg / kg, the phosphate-solubilizing bacteria is applied in an amount of 6 x 10 7 CFU / g, and the yeast peptide is applied in an amount of 10 mg / kg, based on the weight of dry soil.

2. The application of the high-efficiency synergistic calcium soil phosphorus activation preparation in claim 1 in the planting of crops in calcium soil.

3. Use according to claim 2, characterized in that, The crop is Shanghai green.

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