Soil phosphorus solubilizing bacteria and carbon-based assembly material and application thereof in promoting rice growth
By screening the highly efficient phosphorus-solubilizing strain NJAU56 and preparing carbon-based assembly materials with biochar, the problems of poor compatibility of phosphorus-solubilizing bacteria and insufficient phosphorus supply in acidic red soil were solved, thus promoting rice growth and increasing yield.
Patent Information
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- ZHEJIANG FORESTRY UNIVERSITY
- Filing Date
- 2026-05-08
- Publication Date
- 2026-06-09
Smart Images
Figure CN122168485A_ABST
Abstract
Description
Technical Field
[0001] This invention relates to the field of biotechnology, specifically to a soil phosphate-solubilizing bacterium and a carbon-based assembly material, and their application in promoting rice growth. Background Technology
[0002] The typical double-cropping rice planting area in the middle reaches of the Yangtze River is an important grain production base in my country. However, the red soil widely distributed in this area has core problems such as high acidity (pH 4.5–6.5), lack of available phosphorus, and weak fertilizer retention capacity, which seriously restricts the absorption of phosphorus by rice and the increase in yield.
[0003] Phosphorus, an essential nutrient for plant growth and development, directly participates in photosynthesis, energy metabolism, and cell structure construction. However, in acidic red soils, available phosphorus readily combines with iron and aluminum ions to form insoluble iron phosphate and aluminum phosphate, resulting in extremely low bioavailability of phosphorus in the soil. At the same time, the nutrient-poor conditions of red soils and the complex structure of the native microbial community, along with significant competitive and predatory pressures, further exacerbate the phosphorus shortage.
[0004] To address the phosphorus deficiency problem in acidic soils, current technologies mainly involve applying chemical phosphate fertilizers or inoculating with phosphate-solubilizing bacteria. phosphate-solubilizing bacteria Phosphorus replenishment and activation can be achieved through the application of chemical phosphate fertilizers (PSB). However, the application of chemical phosphate fertilizers can easily lead to soil compaction and phosphorus leaching pollution, and the utilization rate is only 10%–25%. Although direct inoculation with phosphate-solubilizing bacteria can convert insoluble phosphorus by secreting organic acids and phosphatases, it faces serious challenges: on the one hand, the acidic environment will damage the cell membrane structure of phosphate-solubilizing bacteria and inhibit enzyme activity, leading to a decline in the activity of the strain; on the other hand, fierce competition from native microorganisms and predation by protozoa will cause the population of exogenous phosphate-solubilizing bacteria to decline rapidly, resulting in weak colonization ability and difficulty in continuously exerting phosphorus-solubilizing function.
[0005] In existing technologies, phosphate-solubilizing bacteria are mostly general-purpose strains (such as ATCC14581), which have poor adaptability to the acidic, anaerobic paddy field microenvironment, limited phosphate-solubilizing efficiency, and low strain survival rate, making it difficult to form a stable functional system. Furthermore, existing technologies have not clarified the phosphorus activation patterns of the biochar-phosphate-solubilizing bacteria composite system in different growth stages of rice and in rhizosphere and non-rhizosphere microdomains, making it impossible to achieve precise phosphorus supply. This results in inconsistent application effects in acidic red soil double-cropping rice areas. Whether biochar-based phosphate-solubilizing bacteria agents prepared by loading PSB with biochar can promote rhizosphere soil phosphorus activation and whether they can promote phosphorus absorption by plants remains unclear. Summary of the Invention
[0006] Objective of the invention: To provide a soil phosphate-solubilizing bacterium, and further to provide a carbon-based assembly material based on the above-mentioned soil phosphate-solubilizing bacterium and its application in promoting rice growth, so as to solve the above-mentioned problems existing in the prior art.
[0007] The inventors screened out a facultative anaerobic bacterium, NJAU56, with highly efficient phosphate-solubilizing ability. After two days of cultivation in phosphate-solubilizing medium, strain NJAU56 achieved a colony diameter (d) of 0.54 cm, a clear zone diameter (D) of 0.81 cm, and an SI value (SI = D / d) of 1.5. Morphological observation and molecular identification confirmed that NJAU56 is similar to *Serratia marcescens* (…). Serratia marcescens It has the highest homology, with a homology greater than 99%. The accession number is CGMCC No. 32793.
[0008] Specifically, the present invention is achieved through the following scheme: In a first aspect, the present invention provides a soil phosphate-solubilizing bacterium NJAU56 with accession number CGMCCNo.32793.
[0009] Soil phosphate-solubilizing bacteria NJAU56 and currently widely used phosphate-solubilizing bacteria ( Bacillus megaterium, Compared to ATCC14581, it exhibits superior phosphorus-solubilizing effects on both inorganic and organic phosphorus media.
[0010] In addition, the two phosphate-solubilizing bacteria were inoculated into inorganic phosphate liquid culture medium to monitor the phosphate solubilization dynamics. The phosphate solubilization capacity of the soil phosphate-solubilizing bacterium NJAU56 involved in this application tended to stabilize after 1 day of culture, and the phosphate solubilization capacity reached its maximum value on the fifth day. Its phosphate solubilization capacity was better than that of the phosphate-solubilizing bacterium ATCC14581.
[0011] In a second aspect, the present invention provides the application of the soil phosphate-solubilizing bacterium NJAU56 proposed in the first aspect above in the preparation of soil phosphate-solubilizing agents.
[0012] In a third aspect, the present invention provides a carbon-based assembly material comprising the soil phosphate-solubilizing bacterium NJAU56 proposed in the first aspect and biochar.
[0013] Preferably, the biochar is corn stalk biochar prepared by anaerobic pyrolysis at 450–500℃ for 2 hours. Specifically, the biochar is prepared by pyrolysis of corn stalks in a carbonization furnace under anaerobic conditions at 450–500℃ for 2 hours, and then passed through a 60-mesh sieve for later use. The basic physicochemical properties of the biochar are: pH 9.3, total carbon 42.4%, total nitrogen 1.0%, total phosphorus 0.2%, total potassium 0.3%, carbon-to-nitrogen ratio 41.74, and soluble organic carbon 612.84 mg / kg. -1 Ash content 15.7%, specific surface area 8.53 m² 2 g -1 .
[0014] Preferably, the mixing ratio of the biochar and the soil phosphate-solubilizing bacteria NJAU56 is 1:20 w / v (g mL). -1 ).
[0015] Specifically, the selected soil phosphate-solubilizing bacteria NJAU56 was cultured on LB medium at 28℃ and 150 r / min for 24 h until the end of the logarithmic growth phase, and the OD was measured. 600 =1.0; The corn straw biochar prepared above was incubated with soil phosphate-solubilizing bacteria NJAU56 at a ratio of 1:20 w / v in a shaker at 37°C for 24 hours, washed three times with physiological saline until neutral, and centrifuged (5000 rpm, 8 min) to obtain carbon-based assembly material.
[0016] In a fourth aspect, the present invention provides the application of the carbon-based assembly material proposed in the third aspect above in promoting rice growth.
[0017] The above applications include the use of carbon-based assembly materials in promoting phosphorus absorption during the rice growth period; The above applications also include the use of carbon-based assembly materials in increasing the available phosphorus content and phosphorus activation rate in soil; wherein the soil is moderately acidic soil (pH 4.5-5.5) or weakly acidic soil (pH 5.5-6.5).
[0018] Beneficial Effects: This invention relates to a soil phosphate-solubilizing bacterium and a carbon-based assembly material, and their applications. The soil phosphate-solubilizing bacterium NJAU56 is an anaerobic bacterium with highly efficient phosphate-solubilizing ability under acidic conditions. The phosphate-solubilizing ability of soil phosphate-solubilizing bacterium NJAU56 is superior to that of the widely used phosphate-solubilizing bacterium ATCC14581; it is naturally adapted to acidic red soil (pH 4.5-6.5) and the anaerobic microenvironment of flooded paddy fields, solving the problems of poor adaptability and rapid activity decay of existing general-purpose phosphate-solubilizing bacteria in target application scenarios.
[0019] For soils characterized by high acidity, scarce available phosphorus, and weak fertilizer retention in typical double-cropping rice growing areas of the middle reaches of the Yangtze River, a stable carbon-based assembly material was constructed. The survival rate of the phosphate-solubilizing bacterium NJAU56 reached 85.7%, avoiding the rapid decline in population caused by competition and predation by native microorganisms, and ensuring the continuous functioning of phosphorus-solubilizing properties. The application of carbon-based assembly materials can significantly improve the availability of soil phosphorus. It is specifically developed for the acidic red soil characteristics of double-cropping rice areas in the middle reaches of the Yangtze River. In the field, it can be applied by hole application or side application, which is simple to operate and solves the problem of phosphorus supply in acidic and low-phosphorus soils.
[0020] Information on the preservation of biological materials: The soil phosphate-solubilizing bacteria is *Serratia marcescens* (…). serratia marcescens NJAU56, deposited at the China General Microbiological Culture Collection Center (CGMCC) with accession number CGMCC No. 32793, deposited on November 25, 2024, at the Institute of Microbiology, Chinese Academy of Sciences, No. 3, Beichen West Road, Chaoyang District, Beijing, is in viable condition. Attached Figure Description
[0021] Figure 1 The phosphorus-solubilizing effect of the two strains in Example 2 on inorganic phosphorus solid culture medium; (left image ATCC14581, right image soil phosphorus-solubilizing bacteria NJAU56).
[0022] Figure 2 The phosphorus-solubilizing effect of the two strains in Example 2 on the organic phosphorus culture medium; (left image ATCC14581, right image soil phosphorus-solubilizing bacteria NJAU56).
[0023] Figure 3 This is a graph showing the dynamic changes in phosphorus solubility of the two strains in inorganic phosphorus liquid culture medium in Example 2.
[0024] Figure 4 The images show the surface morphology of biochar and phosphate-solubilizing bacteria in Example 3 under scanning electron microscopy (SEM) and laser confocal scanning microscopy (CLSM); where BC is biochar and BC & PBS are carbon-based assembly materials.
[0025] Figure 5 The biomass changes of different groups of rice during the critical production and development period in Example 4; (lowercase letters indicate significant differences between different treatments (P<0.05)).
[0026] Figure 6 The rhizosphere architecture changes of different groups of rice during key growth and development stages in Example 4; (lowercase letters indicate significant differences between different treatments (P<0.05)).
[0027] Figure 7 The changes in phosphorus absorption during the critical growth and development stages of rice in different groups in Example 4; (lowercase letters indicate significant differences between different treatments (P<0.05)).
[0028] Figure 8 The changes in available phosphorus content and phosphorus activation rate in soil of different groups in Example 4; (lowercase letters indicate significant differences between different treatments (P<0.05)). Detailed Implementation
[0029] This invention discloses a soil phosphate-solubilizing bacterium and a carbon-based assembly material, as well as their applications. Those skilled in the art can refer to the content of this document and appropriately modify the process parameters to achieve the desired results. It should be particularly noted that all similar substitutions and modifications are obvious to those skilled in the art and are considered to be included in this invention. The methods and applications of this invention have been described through preferred embodiments. Those skilled in the art can clearly modify or appropriately change and combine the methods and applications described herein without departing from the content, spirit, and scope of this invention to realize and apply the technology of this invention.
[0030] Example 1: Screening and isolation of functional strains from environmental soil in a typical double-cropping rice growing area in the middle reaches of the Yangtze River The red soil widely distributed in the double-cropping rice planting area in the middle reaches of the Yangtze River is characterized by high acidity, lack of available phosphorus, and weak fertilizer retention capacity. Under acidic conditions, available phosphorus is fixed, resulting in insufficient phosphorus absorption by rice and limited yield. Conventional fertilization methods are difficult to solve this problem.
[0031] Based on the above situation, the applicant's laboratory extensively collected environmental soil samples from typical double-cropping rice planting areas in the middle reaches of the Yangtze River, obtained 115 strains of facultative anaerobic, highly efficient lignocellulose-degrading bacteria, and determined the phosphorus solubilization ability of these 115 strains under acidic conditions, screening out a facultative anaerobic bacterium NJAU56 with highly efficient phosphorus solubilization ability.
[0032] After two days of cultivation in phosphate-solubilizing medium, strain NJAU56 achieved a colony diameter (d) of 0.54 cm, a clear zone diameter (D) of 0.81 cm, and an SI value (SI = D / d) of 1.5. Morphological observation and molecular identification confirmed that NJAU56 is similar to *Serratia marcescens* (…). Serratia marcescens It has the highest homology, with a homology of >99%.
[0033] Example 2: Evaluation of phosphorus solubilization effect and phosphorus solubilization capacity of strain NJAU56 The phosphorus-solubilizing effect and ability of the screened strain NJAU56 were compared with those of the widely used phosphorus-solubilizing bacterium ATCC14581. Comparison of phosphorus solubilization effects on inorganic phosphorus solid medium: Strain NJAU56 and phosphorus-solubilizing bacterium ATCC14581 were inoculated into inorganic phosphorus fixation medium and cultured for 48 hours. The phosphorus solubilization effects of the two strains were compared as follows: Figure 1 As shown, the size of the phosphate-solubilizing zone indicates that strain NJAU56 has a better phosphate-solubilizing effect than phosphate-solubilizing bacterium ATCC14581. Comparison of phosphorus solubilization effects on organophosphate media: Strain NJAU56 and phosphorus-solubilizing bacterium ATCC14581 were inoculated into organophosphate immobilization media and cultured for 48 hours. The phosphorus solubilization effects of the two strains were as follows: Figure 2 As shown, strain NJAU56 has a better potential for mineralizing organic phosphorus than phosphate-solubilizing bacteria ATCC14581. Phosphorus solubilization dynamics of two bacterial strains in inorganic phosphorus liquid medium: Two bacterial strains were inoculated separately into inorganic phosphorus liquid medium and cultured in shake flasks for 7 days, during which phosphorus solubilization dynamics were monitored. Figure 3 It can be seen that strain NJAU56 has a better phosphorus solubilizing ability than phosphorus solubilizing bacterium ATCC14581; among them, the phosphorus solubilizing ability of strain NJAU56 tends to stabilize after 1 day of culture, and the phosphorus solubilizing ability reaches its maximum value on the fifth day.
[0034] In this embodiment, the inorganic phosphorus culture medium consists of: 10.0 g glucose, 0.5 g (NH4)2SO4, 0.5 g yeast extract, 0.3 g NaCl, 0.3 g KCl, 0.3 g MgSO4·7H2O, 0.03 g FeSO4·7H2O, 0.03 g MnSO4·4H2O, 5.0 g Ca3(PO4)2, 1000 mL distilled water, and pH 7.0–7.5.
[0035] The organic phosphorus culture composition was as follows: glucose 10.0 g, (NH4)2SO4 0.5 g, NaCl 0.3 g, FeSO4·7H2O 0.03 g, MgSO4·7H2O 0.3 g, KCl 0.3 g, MnSO4·4H2O 0.03 g, lecithin 0.2 g / L, calcium carbonate 1.0 g / L, H2O 1000 mL, and pH 7.20–7.40.
[0036] Example 3: Preparation of carbon-based assembly materials The red soil widely distributed in the double-cropping rice planting area in the middle reaches of the Yangtze River has a low pH value and fierce competition from native microorganisms. When phosphate-solubilizing bacteria are directly inoculated, the strains are easily subjected to acid stress, competition from or predation by native microorganisms, resulting in a rapid decline in population size, weak colonization ability, and ineffective phosphate solubilization function, making it difficult to meet the crop's phosphorus absorption needs.
[0037] Based on this, the inventors prepared a mixture of biochar and soil phosphate-solubilizing bacteria NJAU56 at a ratio of 1:20 w / v (g / mL). -1 Carbon-based assembly materials; Culture of phosphate-solubilizing bacteria: The screened phosphate-solubilizing bacterium NJAU56 was inoculated into LB liquid medium and cultured in a constant temperature shaker at 28℃ and 150 r / min for 24 h until the strain reached the late logarithmic growth phase. The OD of the bacterial culture was measured by ultraviolet spectrophotometer. 600 The value is 1.0 (at this point, the bacterial concentration is approximately 1 × 10⁻⁶). 8 (cfu / mL), for later use; Preparation of corn stalk biochar: Dry corn stalks were crushed and placed in a carbonization furnace for anaerobic pyrolysis at 450-500℃ for 2 hours. After cooling, the biochar was crushed and passed through a 60-mesh standard sieve (0.25 mm aperture) to obtain corn stalk biochar. The physicochemical properties of the biochar were as follows: pH 9.3, total carbon content 42.4%, total nitrogen 1.0%, total phosphorus 0.2%, total potassium 0.3%, carbon-to-nitrogen ratio (C / N) 41.74, and soluble organic carbon 612.84 mg / kg. -1 Ash content 15.7%, specific surface area 8.53 m² 2 g -1It has the functions of neutralizing acidic soil, adsorbing and fixing microorganisms, and slowly releasing nutrients.
[0038] The corn straw biochar (BC) prepared above was mixed with phosphate-solubilizing bacteria NJAU56 (PSB) culture (OD). 600 =1.0) at 1:20 w / v (g mL) -1 The mixture was prepared in a ratio of 1:1 and incubated in a constant temperature shaker at 37℃ and 150 r / min for 24 h to allow PSB to be fully adsorbed onto the surface of biochar. After incubation, the mixture was washed three times with sterile physiological saline until the pH of the washing solution was neutral (pH 6.8-7.2). Then, it was centrifuged at 5000 rpm for 8 min and the precipitate was collected, which is the carbon-based assembly material (BC&PSB).
[0039] The surface morphology of BC & PSB was characterized by scanning electron microscopy (SEM-EDS), which showed that... Figure 4 In (c), PSB was successfully loaded onto BC; and the distribution of live PSB cells on BC was observed by laser confocal scanning microscopy (CLSM). Figure 4 In (d), quantitative analysis showed that the survival rate of PSB load on BC was 85.7%.
[0040] Example 4: Investigating the actual effects of carbon-based assembly materials on rice growth and soil phosphorus activation. To clarify the actual effects of carbon-based assembly materials (BC&PSB) on rice growth and soil phosphorus activation, the inventors used low-phosphorus soil in Fuyang District, Hangzhou City (30°04′N, 120°06′E) that had not been fertilized and had an available phosphorus content of 8.8 mg / kg as the test soil (pH 5.2±0.1, medium-acidic soil). The soil in this area is mainly red soil, which naturally has the characteristics of acidity and easy fixation of available phosphorus, consistent with the core physicochemical characteristics of red soil in the double-cropping rice area of the middle reaches of the Yangtze River.
[0041] Monitoring indicators: (Three key developmental stages of rice growth: jointing stage, heading stage, and maturity stage) 1. Plant indicators: plant height, fresh weight, root length, and phosphorus uptake during the three key developmental stages of rice. Second, soil indicators: effective phosphorus and phosphorus activation rate in rhizosphere and non-rhizosphere soils during the three key developmental stages of rice.
[0042] Experimental groups (see Table 1 for reference): Five treatments were set up: blank control (CK), biochar alone (BC), phosphate-solubilizing bacteria alone (PBS), simple mixture of biochar and PBS (BC+PSB), and carbon-based assembly material (BC&PSB). Germinated rice seeds (Oryza sativa L., variety: Yongyou 7850) were added. No phosphorus fertilizer was applied (nitrogen fertilizer: urea (N=46%), 300 mg / kg; potassium fertilizer: potassium chloride (K2O=62%), 175 mg / kg).
[0043] Based on the field water management model for rice paddies, the soil water level was maintained at 3-5 cm throughout the growth cycle. The experiment was set up with 4 replicates, and destructive sampling was carried out at 3 key developmental stages of rice (jointing stage, heading stage, and maturity stage), totaling 60 pots, which collected 120 samples of rhizosphere soil and non-rhizosphere soil.
[0044] Pot experiments were conducted to simulate paddy field production conditions. Plant growth indicators and soil phosphorus indicators during key developmental stages of rice were systematically monitored, and the effects of different treatments were compared.
[0045] Regarding the impact on phosphorus absorption in rice plants, such as Figure 5 , Figure 6 and Figure 7 During the jointing and maturity stages of rice, the carbon-based assembly material significantly increased the plant height of rice compared to other groups (P < 0.05).
[0046] Throughout the entire growth cycle of rice, the carbon-based assembly material significantly increased plant fresh weight and root length compared to other groups, and was particularly effective in promoting phosphorus absorption. Especially during the heading stage, phosphorus absorption was even better compared to the jointing and maturity stages.
[0047] Therefore, the growth-promoting effect of carbon-based assembly materials on rice lasts throughout the entire growth period. During the jointing and maturity stages, the rice plant height was significantly higher than that treated with biochar (BC) and phosphorus-solubilizing bacteria (PSB) alone. Throughout the entire growth period, it significantly increased the plant fresh weight and rice root length, and the developed root system further expanded the phosphorus absorption area.
[0048] like Figure 8 The effects on soil phosphorus content and phosphorus activation rate: In non-rhizosphere soil of rice, carbon-based assembly materials can significantly increase the available phosphorus content in non-rhizosphere soil during the jointing stage of rice (P<0.05). The soil phosphorus activation efficiency under this treatment is the best, and the phosphorus activation rate is increased. The non-rhizosphere is a phosphorus reserve, which can store nutrients for the early growth of rice. In rice rhizosphere soil, carbon-based assembly materials showed the best performance in increasing soil available phosphorus content and soil phosphorus activation efficiency during the rice heading stage, matching the peak phosphorus demand during the rice heading stage.
[0049] The higher the AP (AP content, or available phosphorus content) content in the rhizosphere soil, the greater the concentration gradient of absorbable soluble phosphorus around the roots, the more phosphorus enters the roots through active transport (requiring phosphorus transport proteins) and passive diffusion, and the higher the plant's phosphorus uptake. Figure 7 and 8 As shown, under the treatment of carbon-based assembly materials, the rhizosphere AP content reached its peak during the heading stage, and the phosphorus uptake of plants was also significantly higher during the same period.
[0050] A higher rhizosphere PAC (PAC is soil phosphorus activation rate, available phosphorus / total phosphorus × 100%) indicates a higher proportion of insoluble phosphorus in the rhizosphere soil (such as Fe-P and Al-P in red soil) converted into available phosphorus. Even if rhizosphere AP temporarily decreases due to absorption, PAC can maintain the continuous generation of available phosphorus, preventing root "supply shortage". Under the carbon-based assembly material treatment, the rhizosphere PAC at the heading stage was significantly better than other treatment groups. At this time, although a large amount of rhizosphere AP was absorbed, the high conversion efficiency of PAC ensured the continuous replenishment of AP, maintaining rhizosphere AP, which was significantly better than other treatment groups at the same time. Therefore, the plant's phosphorus uptake remained at a high level, ensuring the accumulation of photosynthetic products and grain development in rice, laying a solid foundation for yield improvement.
[0051] Table 1: Experimental groups and operating procedures ; Note ①: In the BC&PSB (Carbon-based Assembly Material) processing procedure, 7.5 g indicates the amount of BC used in preparing the carbon-based assembly material.
[0052] As described above, although the invention has been shown and described with reference to specific preferred embodiments, it should not be construed as limiting the invention itself. Various changes in form and detail may be made without departing from the spirit and scope of the invention as defined in the appended claims.
Claims
1. A soil phosphate-solubilizing bacterium, characterized in that, The phosphate-solubilizing bacteria is *Serratia marcescens* (… serratia marcescens NJAU56 was deposited on November 25, 2024, at the China General Microbiological Culture Collection Center (CGMCC) with accession number CGMCC No. 32793.
2. The application of the soil phosphate-solubilizing bacteria according to claim 1 in the preparation of soil phosphate-solubilizing bacterial agents.
3. A carbon-based assembly material, characterized in that: This includes soil phosphate-solubilizing bacteria NJAU56 and biochar.
4. The carbon-based assembly material according to claim 3, characterized in that: The biochar was mixed with soil phosphate-solubilizing bacteria NJAU56 at a ratio of 1:20 w / v (g / mL).
5. The carbon-based assembly material according to claim 3, characterized in that: The biochar is corn straw biochar prepared by anaerobic pyrolysis at 450-500℃ for 2 hours.
6. The application of the carbon-based assembly material according to any one of claims 3-5 in promoting rice growth.
7. The application according to claim 6, characterized in that, The applications include the use of carbon-based assembly materials in promoting phosphorus absorption during the rice growth period.
8. The application according to claim 6, characterized in that, The applications include the use of carbon-based assembly materials in increasing the available phosphorus content and activation rate of soil phosphorus.
9. The application according to claim 8, characterized in that, The soil is moderately acidic and / or weakly acidic.