A method for increasing the available phosphorus content of soil by adding a plurality of organic phosphorus

Abstract

This invention discloses a method for increasing the available phosphorus content in soil by adding multiple organophosphorus compounds. The method, belonging to the field of soil improvement technology, provides a method for activating phosphorus-solubilizing microorganisms and increasing the available phosphorus content in soil by adding lecithin to multiple organophosphorus compounds. The method includes the following steps: using lecithin as a basic substrate, adding a combination of multiple organophosphorus compounds to the soil. The combination of organophosphorus compounds includes 1 to 7 of the following: DNA, calcium phytate, AMP, ATP, GTP, ADP, and glucose-6-phosphate. The amount of the added organophosphorus compounds is 0.5% to 1.5% of the mass of the top 0-20 cm soil layer. This invention employs a multi-phosphorus source combination technical solution, which, by encompassing organophosphorus compounds of different chemical types, forms a complex micro-ecological network in the soil, thereby stimulating the synergistic metabolic activity of diverse microbial groups and significantly improving the efficiency of organophosphorus mineralization and conversion. This multi-phosphorus source complementary mechanism not only improves the immediate supply capacity of available phosphorus in the soil but also maintains long-term fertility through continuous substrate supply.

Description

Technical Field

[0001] This invention belongs to the field of soil improvement technology, and in particular relates to a method for increasing the available phosphorus content in soil by adding various organic phosphorus compounds. Background Technology

[0002] Phosphorus, an essential macronutrient for plant growth and development, plays an irreplaceable role in core physiological processes such as photosynthesis, energy metabolism, and genetic material synthesis. However, agricultural production commonly faces the bottleneck of scarce available phosphorus in the soil—most soils contain extremely low levels of available phosphorus that can be directly absorbed and utilized by plants, making it difficult to meet the normal growth needs of crops. The core issue behind this phenomenon lies in the strong fixation characteristics of soil phosphorus: free phosphorus readily combines with metal ions such as calcium, iron, and aluminum in the soil or exists in the form of organic phosphorus, forming insoluble phosphorus compounds, leading to a significant decrease in the level of available phosphorus that plants can utilize.

[0003] From the perspective of soil phosphorus pool composition, organic phosphorus cannot be directly absorbed and utilized by plants, but it can serve as an important reserve of potential available phosphorus, accounting for 20% to 80% of total phosphorus. Its main forms include phytic acid, phospholipids, nucleotides, nucleic acids, phosphoric acid sugars, and phosphoproteins. Organic phosphorus plays a key role in maintaining the long-term availability of soil phosphorus. Studies have confirmed that during the conversion of subtropical forests to plantations, the decline in soil phosphorus availability is directly related to the decrease in organic phosphorus content (Yang L, Yang Z, Zhong X, et al. Decreases in soil P availability areas associated with soil organic P declines following forest conversion in subtropical China[J]. Catena, 2021, 205: 105459.).

[0004] Microorganisms are a core driving force in regulating soil phosphorus transformation. They mediate the mineralization process of organic phosphorus by expressing phosphorus transformation functional genes, releasing available phosphorus and thus influencing the soil's available phosphorus supply capacity. Notably, microorganisms have limited mineralization efficiency for single organic phosphorus groups, and different types of organic phosphorus exhibit varying microbial preferences. Furthermore, their phosphorus-solubilizing ability is significantly inhibited in acidified black soils. Therefore, we infer that higher diversity of organic phosphorus components leads to a richer variety of substrates available to phosphorus-solubilizing microorganisms, stronger co-metabolic activity, and potentially a corresponding increase in the amount of available phosphorus released.

[0005] However, existing research focuses on the application of single phosphorus sources. Such solutions can only meet the metabolic needs of specific microbial groups, resulting in limited organic phosphorus mineralization and difficulty in achieving efficient activation of soil phosphorus. Summary of the Invention

[0006] In view of this, the purpose of this invention is to provide a method for activating phosphorus-solubilizing microorganisms and increasing the available phosphorus content in soil by combining lecithin with multiple organophosphates. This invention employs a technical solution of combining lecithin with multiple phosphorus sources, forming a complex micro-ecological interaction network in the soil by encompassing different chemical types of organophosphate compounds. This stimulates the synergistic metabolic activity of diverse microbial communities, significantly improving the mineralization and conversion efficiency of organophosphates. This multi-phosphorus source complementary mechanism not only enhances the immediate supply capacity of available phosphorus in the soil but also maintains long-term fertility through continuous substrate supply.

[0007] This invention provides a method for activating phosphorus-solubilizing microorganisms and increasing the available phosphorus content in soil by adding a variety of organic phosphorus compounds to lecithin, comprising the following steps: adding an organic phosphorus compound to the soil, wherein the organic phosphorus compound comprises 2 to 8 of the following: lecithin, DNA, calcium phytate, AMP, ATP, GTP, ADP and glucose-6-phosphate; The amount of the organic phosphorus compound added is 0.5% to 1.5% based on the mass of the top 0-20 cm soil layer.

[0008] Preferably, when the organophosphorus combination includes lecithin, DNA, calcium phytate and AMP, the mass ratio of lecithin, DNA, calcium phytate and AMP is (0.7~1.3):(0.7~1.3):(0.7~1.3):(0.7~1.3).

[0009] Preferably, when the organophosphate combination includes lecithin, DNA, calcium phytate, AMP, ATP, GTP, ADP and glucose-6-phosphate, the mass ratio of lecithin, DNA, calcium phytate, AMP, ATP, GTP, ADP and glucose-6-phosphate is (0.7~1.3):(0.7~1.3):(0.7~1.3):(0.7~1.3):(0.7~1.3):(0.7~1.3):(0.7~1.3):(0.7~1.3).

[0010] Preferably, the amount of the organophosphorus compound added is 0.9% to 1.1%.

[0011] Preferably, the soil is cultured in a dark environment at 23-27°C for 6-8 days, and then an organophosphorus compound is added.

[0012] Preferably, increasing the available phosphorus content in soil includes increasing the content of soil acid phosphatase and phytase, increasing the abundance of soil phosphorus-solubilizing genes, and increasing the abundance of soil microorganisms with phosphorus-solubilizing functions.

[0013] Compared with the prior art, the present invention has the following beneficial effects: This invention systematically studies the impact of phosphorus source diversity gradients on changes in available phosphorus and mineralizing enzyme content in acidic black soil. Using lecithin as the basic substrate, four gradients of phosphorus source diversity were established in black soil to explore changes in the abundance of phosphate-solubilizing genes and the microbial community. Existing research largely focuses on the forms, transformations, and bioavailability of inorganic phosphorus in soil. This invention, however, focuses on organic phosphorus in soil, designing four gradients of phosphorus source diversity treatments to simulate the soil phosphorus cycle under different organic phosphorus source diversity conditions, comprehensively exploring the dynamic impact of phosphorus source diversity on available phosphorus in the soil. By comparing changes in available phosphorus content, phosphate-solubilizing enzyme activity, abundance of phosphate-solubilizing functional genes, and microbial community under different treatments, this invention aims to provide a scientific basis for soil phosphorus management and optimized fertilization strategies, and to lay a theoretical foundation for improving agricultural productivity and ecosystem service functions. Attached Figure Description

[0014] Figure 1 The changes in available phosphorus content in black soil after the addition of no organic phosphorus (CK), the addition of 2 types of organic phosphorus (P-2), the addition of 4 types of organic phosphorus (P-4), and the addition of 8 types of organic phosphorus (P-8) were investigated.

[0015] Figure 2 The changes in soil acid phosphatase and phytase activity in black soil after the addition of no organic phosphorus (CK), 2 types of organic phosphorus (P-2), 4 types of organic phosphorus (P-4), and 8 types of organic phosphorus (P-8) were investigated.

[0016] Figure 3 The changes in the abundance of phosphorus-solubilizing genes in black soil after the addition of no organic phosphorus (CK), 2 types of organic phosphorus (P-2), 4 types of organic phosphorus (P-4), and 8 types of organic phosphorus (P-8) were investigated.

[0017] Figure 4 The results for *Bacillus phosphate-solubilizing* spp. were: no organophosphates (CK), 2 organophosphates (P-2), 4 organophosphates (P-4), and 8 organophosphates (P-8). Bacillus The relative abundance plot of ). Detailed Implementation

[0018] This invention provides a method for increasing the available phosphorus content in soil by adding a variety of organophosphorus compounds, comprising the following steps: adding a combination of various organophosphorus compounds to the soil, with lecithin as the basic substrate, wherein the combination of organophosphorus compounds includes 2 to 8 of the following: lecithin, DNA, calcium phytate, AMP, ATP, GTP, ADP, and glucose-6-phosphate; the amount of the combination of organophosphorus compounds added is 0.5% to 1.5% based on the mass of the top 0 to 20 cm soil layer.

[0019] In this invention, the organophosphorus combination preferably includes lecithin, DNA, calcium phytate, and AMP; the mass ratio of lecithin, DNA, calcium phytate, and AMP is preferably (0.7~1.3):(0.7~1.3):(0.7~1.3):(0.7~1.3), more preferably (0.9~1.1):(0.9~1.1):(0.9~1.1):(0.9~1.1), and even more preferably 1:1:1:1.

[0020] In this invention, the organophosphorus combination more preferably includes lecithin, DNA, calcium phytate, AMP, ATP, GTP, ADP, and glucose-6-phosphate; the mass ratio of lecithin, DNA, calcium phytate, AMP, ATP, GTP, ADP, and glucose-6-phosphate is preferably (0.7~1.3):(0.7~1.3):(0.7~1.3):(0.7~1.3):(0.7~1.3):(0.7~1.3):(0.7~1.3):(0.7~1.3), further preferably (0.9~1.1):(0.9~1.1):(0.9~1.1):(0.9~1.1):(0.9~1.1):(0.9~1.1):(0.9~1.1):(0.9~1.1):(0.9~1.1), and even more preferably 1:1:1:1:1:1:1:1:1.

[0021] In this invention, different types of phosphorus sources are selected, representing different forms of organic phosphorus, covering a variety of types from monophosphates to polyphosphates. In the experimental design for phosphorus source selection and combination in this invention, each treatment is based on lecithin as the basic phosphorus source, gradually increasing the types of phosphorus sources while maintaining a constant total amount. In this invention, the total amount of the organic phosphorus combination, based on the mass of the 0-20 cm topsoil layer, is preferably 0.9% to 1.1%, more preferably 1.0%. This invention does not specifically limit the source of the organic phosphorus; commercially available products are acceptable.

[0022] In this invention, the soil is preferably pretreated before the addition of the organophosphorus compound. The pretreatment involves treating the soil at 24-26°C in the dark for 6-8 days, preferably at 25°C in the dark for 7 days. In this invention, the soil moisture content is preferably maintained at 20%-30% of its soil water holding capacity, more preferably 22%-28%, and most preferably 25%.

[0023] In this invention, after adding the organic phosphorus combination to the soil, it is kept in the dark for 25 to 35 days, preferably 27 to 33 days, and more preferably 30 days.

[0024] In this invention, increasing the available phosphorus content in soil preferably includes increasing the content of soil acid phosphatase and phytase, increasing the abundance of soil phosphorus-solubilizing genes, and increasing the abundance of soil microorganisms with phosphorus-solubilizing functions. This invention uses the above indicators to determine the influence and pathway of organic phosphorus combinations on the available phosphorus content in soil. This invention does not specifically limit the methods for determining the above indicators; methods known in the art can be used.

[0025] The technical solutions provided by the present invention will be described in detail below with reference to the embodiments, but they should not be construed as limiting the scope of protection of the present invention.

[0026] Example 1

[0027] 1) Phosphorus Source Selection and Combination. Different types of phosphorus sources are selected, using lecithin as the basic substrate, and combined with DNA, calcium phytate, AMP, ATP, GTP, ADP, and glucose-6-phosphate to form various phosphorus source combinations. These phosphorus sources represent different forms of organic phosphorus, covering a variety of types from monophosphates to polyphosphates. In the experimental design of phosphorus source selection and combination in this invention, each treatment is based on the phosphorus source of the previous treatment, gradually increasing the types of phosphorus sources, but the total amount added remains unchanged.

[0028] 2) Soil incubation. Soil samples used in this experiment were collected from Changchun City, Jilin Province (125°27′46″E, 43°47′12″N). Topsoil (0–20 cm) was collected, and 10 g of soil was used for incubation. The total amount of organic phosphorus source added to each soil sample was 1%. Three treatments were set up, with four replicates for each treatment: (1) Two phosphorus sources (P-2): 0.5 g lecithin and 0.5 g DNA (2) Four phosphorus sources (P-4): lecithin 0.025 g, DNA 0.025 g, calcium phytate 0.025 g, AMP 0.025 g; (3) Eight phosphorus sources (P-8): lecithin 0.0125 g, AMP 0.0125 g, DNA 0.0125 g, calcium phytate 0.0125 g, ATP 0.0125 g, GTP 0.0125 g, ADP 0.0125 g, glucose-6-phosphate 0.0125 g.

[0029] Soil without added organophosphorus sources served as the control group.

[0030] Soil samples were placed in plastic bags and incubated in the dark at 25 °C with moisture maintained at 25% of capacities. After one week of pre-incubation, a phosphorus source was added according to the established treatment plan, and incubation continued for one month. After the incubation period, samples were collected to determine the available phosphorus content, acid phosphatase, and phytase levels in the soil.

[0031] 3) Index Determination. Available phosphorus (AP) in the soil was extracted with 0.5 mol / L NaHCO3 at pH 8.5. After filtering the extract through phosphorus-free quantitative filter paper, the solution was developed using a molybdenum antimony solution, and then the available phosphorus content was determined using a microplate reader at a wavelength of 880 nm. Acid phosphatase activity was determined using a soil acid phosphatase (S-ACP) activity assay kit, purchased from Beijing Box Biotechnology Co., Ltd. Phytase activity was determined using a soil phytase activity assay kit, purchased from Solarbio. Acid phosphatase activity was determined using a microplate reader at a wavelength of 660 nm, and phytase activity was determined using a microplate reader at a wavelength of 700 nm.

[0032] 4) Determination of Phosphorus Transformation Functional Gene Abundance: 0.5 g of fresh soil was collected, and soil DNA was extracted using the Fast DNA SPIN Kit (MP Biomedical, France). The mass and concentration of the DNA sample were measured using a NanoDrop 2000 spectrophotometer. The extracted DNA was aliquoted; one portion was stored at -80℃ for later use, while the other portion was used for functional gene quantification analysis. To accurately quantify the abundance of functional genes closely related to phosphorus cycling in the soil sample, q-PCR technology was used for quantitative analysis. First, the extracted soil DNA was pre-amplified using conventional PCR with specific primers to enrich the target gene fragments. Subsequently, the PCR products were used as templates for quantification using the TB Green fluorescent dye method on a LightCycler® 96 real-time quantitative PCR system (Roche, Germany). The reaction mixture consisted of 20 μL of 10 μL Green Premix Ex Taq (Tli RNaseH Plus) (2X), 0.8 μL of forward primer (10 μM), 0.8 μL of reverse primer (10 μM), 1 μL of DNA template (10-100 ng), and 7.4 μL of nuclease-free water. Three technical replicates were performed for each sample, and a negative control without template was included. Absolute quantification was performed using a standard curve method with serial dilutions of plasmid DNA containing the target gene fragment (10²-10⁻¹⁰ ng). 8 A standard curve was constructed using copies / μL. Data acquisition and analysis were performed using LightCycler® 96 software. The copy number of the target gene was calculated by comparing the threshold cycle number (Ct value), and the abundance of functional genes was expressed as the number of gene copies per gram of dry soil. The amplification conditions and primers for phosphorus-converted functional genes are listed in Table 1.

[0033] Table 1 Primers and amplification conditions for phosphorus conversion functional genes

[0034] 5) High-throughput sequencing: Soil DNA samples were collected, and the universal primer pairs 515F (SEQ ID NO: 3, 5'-GTGYCAGCMGCCGCGGTAA-3') and 806R (SEQ ID NO: 4, 5'-GGACTACHVGGTWTCTAAT-3'), validated by NCBI Primer-BLAST, were selected for targeting the hypervariable region of the bacterial 16S rRNA gene V3-V4. The primers were purified by PAGE and prepared into a 10 μmol / L stock solution. PCR amplification was performed using a 25 μL optimized system containing 2 μL DNA template (final concentration 0.8 ng / μL), 1 μL forward / reverse primers (final concentration 0.4 μmol / L), 12.5 μL 2×Hieff Robust PCR Master Mix (containing HiFi Taq enzyme, dNTPs, and optimized buffer), and 8.5 μL sterile water. Three technical replicates and a template-free control were included. The thermal cycling parameters were set as follows: 95℃ pre-denaturation for 3 min to activate the hot-start enzyme; 30 cycles of 95℃ denaturation for 30 s, 55℃ annealing for 30 s, and 72℃ extension for 30 s; final extension at 72℃ for 5 min to ensure complete fragment synthesis; storage at 4℃. The product was separated by 2% agarose gel electrophoresis (1×TAE buffer, 100V constant voltage for 30 min). After confirming the target band of approximately 460 bp using a GeneRuler 100 bp DNA ladder, DNA was adsorbed using a GeneJET PCR Purification Kit with binding buffer added at a 1:5 ratio. After washing twice with 70% ethanol, the DNA was eluted with 50 μL LTE buffer for later use. The purified product was sequenced and analyzed using an Illumina MiSeq paired-end sequencing system. Using the DADA2 package (Version 1.18.0) in R software (Version 4.4.3), the data quality was first checked using the `plotQualityProfile` command, primers and high-error-rate leading / ending bases were removed, and low-quality sequences were filtered using parameters such as `maxN = 0`, `trunQ = 2`, and `rm.phix = TRUE`. Then, the `learnErrors` function was used to learn the errors in the sequencing data and build a model to remove sequencing errors and obtain "true" sequences. Next, the `mergePairs` command was used to merge paired-end data and build an ASV table, and chimeras were removed using the `removeBimeraDenovo` command (method = consensus). Finally, referring to the `silva_nr99_v138_train_set.fa.gz` database, the `assignTaxonomy` and `addSpecies` functions were used to complete species annotation.

[0035] Experimental results: 1) Changes in available phosphorus content in acidified black soil under different organic phosphorus source diversity gradients, such as... Figure 1 As shown, with the increase in phosphorus source diversity, the available phosphorus content in the soil gradually increases (no added organic phosphorus: 43.3 mg / kg, added with 2 kinds of organic phosphorus: 203 mg / kg, added with 4 kinds of organic phosphorus: 342 mg / kg, added with 8 kinds of organic phosphorus: 411 mg / kg). Lecithin combined with 7 kinds of organic phosphorus has the best effect on increasing the available phosphorus in the soil.

[0036] 2) Changes in acid phosphatase and phytase activities in acidified black soil under different organic phosphorus source diversity gradients, such as... Figure 2 As shown, the acid phosphatase content was highest under the treatments with the addition of 8 types of organophosphates (no organophosphates: 20625 mg / kg, 2 types of organophosphates: 21083 mg / kg, 4 types of organophosphates: 15709 mg / kg, 8 types of organophosphates: 42871 mg / kg). The phytase content increased significantly (no organophosphates: 1.66 U / g, 2 types of organophosphates: 10.8 U / g, 4 types of organophosphates: 10.2 U / g, 8 types of organophosphates: 13.4 U / g). This indicates that increasing phosphorus source diversity can effectively improve microbial phosphorus solubilization activity, improve soil phosphorus supply, and enhance phosphorus bioavailability.

[0037] 3) Changes in soil phosphorus solubility gene abundance in acidified black soil under different organic phosphorus source diversity gradients, such as... Figure 3 As shown: Compared with the control group and other treatments, the treatment with the addition of 8 organophosphates significantly increased the abundance of phosphate-solubilizing genes (control group: 5.34 × 10⁻⁶). 7 Copies / g, treatment with the addition of two organophosphates: 1.14 × 10⁻⁶ 8 Copies / g, Treatment with the addition of 4 organophosphates: 1.08 × 10 8 Copies / g, treatment with the addition of 8 organophosphates: 3.78 × 10⁻⁶ 8 (copies / g). Lecithin combined with 7 kinds of organic phosphorus has the best effect on enhancing the phosphorus-solubilizing genes in microorganisms.

[0038] 4) *Bacillus* spp. under different phosphorus diversity gradients ( Bacillus Changes in relative abundance, such as Figure 4 As shown: In the control group, the treatments with 2 types of organophosphates, 4 types of organophosphates, and 8 types of organophosphates, the relative abundance of Bacillus spp. gradually increased, reaching 2.71%, 1.69%, 3.12%, and 7.46%, respectively. Bacillus spp. is a typical phosphate-solubilizing bacterium, and the results indicate that lecithin combined with 7 types of organophosphates had the best effect on increasing the abundance of phosphorus-solubilizing species in the soil.

[0039] In summary, this invention takes acidified black soil as the research object, sets up a phosphorus source diversity gradient, and compares the changes in available phosphorus content, two phosphorus-solubilizing enzyme activities, phosphorus-solubilizing functional gene abundance, and phosphorus-solubilizing microbial abundance under different treatments. This provides a scientific basis for soil phosphorus management and optimized fertilization strategies, and lays a theoretical foundation for improving agricultural production efficiency and ecosystem service functions.

[0040] The above description is only a preferred embodiment of the present invention. It should be noted that for those skilled in the art, several improvements and modifications can be made without departing from the principle of the present invention, and these improvements and modifications should also be considered within the scope of protection of the present invention.

Claims

1. A method for increasing the available phosphorus content of soil by adding lecithin to a plurality of organic phosphorus-activating phosphorus-releasing microorganisms, characterized by, Includes the following steps: Using lecithin as a basic substrate, a variety of organic phosphorus compounds are added to the soil, wherein the organic phosphorus compounds include 1 to 7 of DNA, calcium phytate, AMP, ATP, GTP, ADP and glucose-6-phosphate; The amount of the organic phosphorus compound added is 0.5% to 1.5% based on the mass of the top 0-20 cm soil layer.

2. The method of claim 1, wherein, When the organophosphorus combination includes lecithin, DNA, calcium phytate and AMP, the mass ratio of lecithin, DNA, calcium phytate and AMP is (0.7~1.3):(0.7~1.3):(0.7~1.3):(0.7~1.3).

3. The method of claim 2, wherein, When the organic phosphorus combination includes lecithin, DNA, calcium phytate, AMP, ATP, GTP, ADP and glucose-6-phosphate; the mass ratio of lecithin, DNA, calcium phytate, AMP, ATP, GTP, ADP and glucose-6-phosphate is (0.7~1.3):(0.7~1.3):(0.7~1.3):(0.7~1.3):(0.7~1.3):(0.7~1.3):(0.7~1.3):(0.7~1.3).

4. The method according to claim 2 or 3, characterized in that, The amount of the organophosphorus compound added is 0.9% to 1.1%.

5. The method of claim 1, wherein, After the soil was cultured in a dark environment at 23-27℃ for 6-8 days, an organic phosphorus compound was added.

6. The method of claim 1, wherein, Increasing the available phosphorus content in soil includes increasing the content of soil acid phosphatase and phytase, increasing the abundance of soil phosphorus-solubilizing genes, and increasing the abundance of soil microorganisms with phosphorus-solubilizing functions.

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