Phosphorus-solubilizing bacteria with phosphorus-solubilizing and passivating dual functions, microbial inoculum prepared from phosphate-solubilizing bacteria and application of microbial inoculum

Abstract

The invention discloses a phosphate-solubilizing bacterium with dual functions of phosphate solubilizing and passivation, a microbial agent prepared from the phosphate-solubilizing bacterium and application of the phosphate-solubilizing bacterium, the phosphate-solubilizing bacterium is Bacillus sp and is preserved in Guangdong Microbial Culture Collection Center, and the preservation number of the phosphate-solubilizing bacterium is GDMCC NO: 66609. Through multiple rounds of screening, the strain capable of dissolving phosphorus, producing urease, IAA and siderophore, promoting corn growth and improving cadmium and lead tolerance of plants is obtained, the strain can greatly improve underground biomass of corn, meanwhile, urease, IAA and siderophore are produced, effectiveness of soil cadmium is reduced, and soil fertility is improved. The fertilizer can increase the yield of corn, improve the soil fertility and improve the market competitiveness of agricultural products. The phosphate-solubilizing bacterium disclosed by the invention is easy to culture and apply on a large scale, simple and convenient to operate, easy to accept and apply by agricultural producers, and relatively high in practicability. The strain and the preparation have relatively high agricultural application prospects and economic values.

Description

TECHNICAL FIELD

[0001] The present application relates to the field of microbial technology, in particular to a phosphorus-solubilizing bacteria with dual functions of phosphorus-solubilizing and passivation, a bacterial agent prepared therefrom and application thereof. BACKGROUND

[0002] The application of phosphorus fertilizer in cultivated land cannot be absorbed by crops because it combines with calcium, iron, aluminum and other ions to form insoluble compounds, and the agricultural land is at risk of heavy metal pollution such as cadmium and lead due to industrial activities. Although traditional chemical phosphorus-solubilizing agents can temporarily improve the availability of phosphorus, long-term use can lead to soil acidification and compaction, and may activate heavy metal ions in the soil to form bioavailable forms, thereby increasing the risk of agricultural product safety. Under this background, microbial remediation technology with the functions of phosphorus activation and heavy metal passivation has gradually become a research hotspot.

[0003] Phosphorus-solubilizing bacteria, as key regulators of the plant-soil-heavy metal interaction system, have important significance for the restoration of soil ecosystems due to their functional diversity. Existing studies have shown that excellent strains can convert insoluble phosphorus by secreting organic acids and phosphatases, and promote root development by producing plant hormones such as indole acetic acid (IAA). In addition, they can inhibit pathogenic bacteria by enhancing iron competition using siderophores, and regulate nitrogen metabolism cycles through urease. More importantly, some strains can reduce the bioavailability of heavy metals through mechanisms such as cell wall adsorption, ion transport and biomineralization. However, the currently reported phosphorus-solubilizing bacteria generally have single function and poor environmental adaptability, and often face challenges such as low survival rate and unstable function expression in field applications, especially in moderately to severely contaminated soils, where the activity of the strains is easily inhibited by heavy metal toxicity, leading to a sharp decrease in remediation efficiency.

[0004] Although some Bacillus and Pseudomonas phosphorus-solubilizing strains have been included in the microbial fertilizer registration directory, complex functional strains with high-efficiency phosphorus-solubilizing, multi-pathway growth-promoting and heavy metal passivation functions are still scarce resources. Existing bacterial agents generally have low heavy metal removal efficiency and large environmental factor interference on functional gene expression, which cannot meet the practical needs of remediation and production in contaminated farmland. Therefore, developing phosphorus-solubilizing strains with multiple mechanisms and establishing their adaptation to crop-soil systems have become key research directions to break through the current bottleneck of biological remediation technology. SUMMARY

[0005] The present application relates to the field of microbial technology, in particular to a phosphorus-solubilizing bacteria with dual functions of phosphorus-solubilizing and passivation, a bacterial agent prepared therefrom and application thereof.

[0006] The above technical purposes of the present application are achieved by the following technical solutions: The phosphorus solubilizing bacteria with the dual functions of phosphorus solubilization and passivation is Bacillus sp, which is preserved in the Guangdong Microbial Culture Collection Center with the strain preservation number of GDMCC NO: 66609.

[0007] The application further provides a microbial agent made of the phosphorus solubilizing bacteria with the dual functions of phosphorus solubilization and passivation, wherein the microbial agent is a liquid microbial agent, and the concentration of the phosphorus solubilizing bacteria in the microbial agent is 6*108 CFU / mL.

[0008] The production method is operated as follows: The phosphorus solubilizing bacteria are activated, the streak method is used to culture the bacteria on beef extract peptone solid culture medium at 28 DEG C for 24 hours, then the single colony is picked up by using a sterile inoculation loop and inoculated into liquid culture medium, and the bacteria are cultured by oscillation, and the strain is prepared into a phosphorus solubilizing bacteria liquid with a concentration of 6*108 CFU / mL by using sterile water.

[0009] The application further provides an application of the microbial agent made of the phosphorus solubilizing bacteria with the dual functions of phosphorus solubilization and passivation in promoting the growth of corn.

[0010] The application further provides an application of the microbial agent made of the phosphorus solubilizing bacteria with the dual functions of phosphorus solubilization and passivation in promoting growth and cadmium and lead tolerance.

[0011] To sum up, the application has the following beneficial effects: through multiple rounds of screening, a strain with the functions of phosphorus solubilization, urease production, IAA production, iron carrier production, corn growth promotion and plant cadmium and lead tolerance is obtained, the strain can greatly increase the underground biomass of corn, simultaneously produce urease, IAA and iron carrier, reduce the availability of soil cadmium, improve soil fertility, according to the description of examples, the application can improve the yield of corn and soil fertility, and improve the market competitiveness of agricultural products. The phosphorus solubilizing bacteria are easy to be cultured and applied on a large scale, the operation is simple, and the bacteria are easy to be accepted and applied by agricultural producers, and have high practicability. The strain and the preparation have high agricultural application prospect and economic value. BRIEF DESCRIPTION OF DRAWINGS

[0012] Figure 1 The figure is the colony morphology of the phosphorus solubilizing bacteria in the application; Figure 2 The figure is the phylogenetic tree between the phosphorus solubilizing bacteria and the reference strain in the application; Figure 3 The figure is the phosphorus solubilization effect of the phosphorus solubilizing bacteria in the application; Figure 4 The figure is the urease production coloration of the phosphorus solubilizing bacteria in the application; Figure 5 The figure is the IAA production coloration of the phosphorus solubilizing bacteria in the application; Figure 6The effect diagram of the iron carrier production of the phosphorus solubilizing bacteria in the application; Figure 7 The growth promoting effect diagram of the phosphorus solubilizing bacteria in the application. DETAILED DESCRIPTION

[0013] The application will be further described in detail below with reference to the accompanying drawings.

[0014] Example 1, a phosphorus solubilizing bacteria with the functions of phosphorus solubilizing and passivation, the classification and naming of the phosphorus solubilizing bacteria are Bacillus sp. , which is preserved in the Guangdong Provincial Microbial Culture Collection Center on June 30, 2025, and the strain preservation number is GDMCC NO: 66609.

[0015] Screening of the phosphorus solubilizing bacteria: 1. Collect corn rhizosphere soil in Pingyuan Town, Wenshan City, a high geological background area in Dian East, remove obvious stones and roots in the soil, and then take 5 g of the soil sample and add 50 ml of sterilized water into a constant temperature shaking incubator, shake for 4 hours to fully break and mix the bacteria contained in the soil sample, which is 10 -1 soil dilution liquid, take 1 mL of the above soil dilution liquid, and sequentially gradient dilute it with sterilized water to 10 -2 , 10 -3 , 10 -4 , 10 -5 , 10 -6 , 10 -7 , adopt the dilution plate method, sequentially streak on the inorganic phosphorus solid culture medium (glucose 10 g, sodium chloride 0.3 g, MgSO4·7H2O 0.3 g, MnSO4·4H2O 0.03 g, agar 15-18 g, (NH4)2SO4 0.5 g, KCl 0.3 g, FeSO4·7H2O 0.03 g, Ca3(PO4)2 10 g, pH 7.0-7.5, distilled water 1000 ml. 121℃ sterilization for 30 min) added with 10 mg / L Cd and 50 mg / L Pb, and place in a constant temperature incubator at 28℃ to culture until the colonies are produced, use a loop to pick typical clear single colonies on the culture medium, inoculate in beef extract peptone culture medium (peptone 10 g, sodium chloride 5 g, beef extract 3 g, agar 18 g, use a pH meter to adjust pH to 7.0-7.2, dissolve in 800 mL of distilled water, make up to 1000 mL, and use a high-pressure sterilization pot to sterilize at 121℃ for 30 min), repeatedly purify several times by the plate streaking method, preliminarily obtain cadmium and lead tolerant phosphorus solubilizing strains, and store the purified strains in a 4℃ refrigerator by the agar slant preservation method.

[0016] Preparation of bacterial suspension: The bacterial strain preserved on slant agar was cultured on beef extract peptone medium for 24 hours. A bacterial suspension was prepared using the turbidity counting method, with a McFarland cloud meter used as the standard for turbidity comparison. The suspension was prepared to contain approximately 6 × 10⁶ bacteria per milliliter. 8 One bacterial suspension was inoculated into the culture medium (molybdenum blue colorimetric method). 50 mL of inorganic phosphorus liquid culture medium (10 g glucose, 0.3 g sodium chloride, 0.3 g MgSO4·7H2O, 0.03 g MnSO4·4H2O, 0.5 g (NH4)2SO4, 0.3 g KCl, 0.03 g FeSO4·7H2O, 10 g Ca3(PO4)2, pH 7.0-7.5, 1000 mL distilled water. Sterilized at 121℃ for 30 min) was added to a 150 mL Erlenmeyer flask. 1 mL of bacterial suspension was inoculated into the medium and cultured at 28℃ with shaking at 180 rpm. An uninoculated inorganic phosphorus culture medium was used as a blank control. The culture was incubated for 72 h, and the OD600 value was measured after standing for 15 minutes. Then, the culture was centrifuged at 5000 rpm for 10 min, and the soluble phosphorus concentration in the supernatant was determined.

[0017] 2. Morphological and physiological-biochemical characteristics of phosphate-solubilizing bacteria The strain was streaked onto beef extract peptone medium (formula as above) and incubated at 28°C for 24 h. Colony characteristics such as shape, transparency, and color were observed, and the results are shown in the figure. The colony characteristics of the strain are as follows: Figure 1 The colonies were Gram-positive, with milky white, round, smooth, and opaque cells with regular edges.

[0018] 3. Molecular biological identification of phosphate-solubilizing bacteria Single colonies of bacteria were picked and cultured in beef extract peptone medium at 28°C for 24 hours. Bacteria in the logarithmic growth phase were preserved on dry ice and their 16S rDNA sequences were identified by Sangon Biotech (Shanghai) Co., Ltd. Genomic DNA was extracted from phosphate-solubilizing bacteria using the SK8255 Ezup column-based genomic DNA extraction kit. The 16S rDNA sequence of the strain was amplified by PCR using universal primers 27F and 1492R: (upstream primer, sequence 5'-AGAGTTTGATCMTGGCTCAG-3' SEQ ID NO. 2) and (downstream primer, sequence 5'-GGTTACCTTGTTACGACTT-3' SEQ ID NO. 3)). The sequencing results were compared with known sequences in the NCBI database to determine the species relationship of the strain and construct a phylogenetic tree. Figure 2 The strain shown was identified as Bacillus sp. based on its morphological, physiological and biochemical characteristics and molecular sequence. The Bacillus sp. was deposited in Guangdong Provincial Microbial Culture Collection Center in 2025 with accession number GDMCC 66609.

[0019] Example two, phosphorus bacteria dissolving phosphorus, producing urease, producing IAA and iron carrier ability effect analysis The screened phosphorus bacteria were inoculated into inorganic phosphorus medium, urea medium, King'B medium and CAS medium respectively to qualitatively and quantitatively analyze the function of the strains in dissolving phosphorus, producing urea, producing IAA and producing iron carrier. The specific operation steps are as follows: 1. Determination of phosphorus bacteria dissolving phosphorus: 50 mL of inorganic phosphorus liquid medium was added to a 150 mL conical flask, 1 mL of bacterial suspension was inoculated into the medium, and it was cultured at 28°C and 180 rpm. The inorganic phosphorus culture solution without bacteria (glucose 10 g, sodium chloride 0.3 g, MgSO4·7H2O 0.3 g, MnSO4·4H2O 0.03 g, agar 15-18 g, (NH4)2SO4 0.5 g, KCl 0.3 g, FeSO4·7H2O 0.03 g, Ca3(PO4)2 10 g, pH 7.0-7.5, distilled water 1000 ml. 121°C sterilization for 30 min) was used as a blank control, and the culture was incubated for 72 h. After 15 minutes of standing, the OD value was measured, then centrifuged at 5000 rpm for 10 min, and the supernatant was measured for soluble phosphorus concentration after centrifugation. 600

[0020] 2. Urea agar medium: NaCl 5 g, KH2PO4 2 g, glucose 1 g, phenol red 0.012 g, proteose peptone 0.2 g, agar 20 g, distilled water 950 mL, pH 6.8, 121°C sterilization for 30 min, after cooling to 50°C, add 50 mL of filtered and sterilized 40% urea solution, mix well, then plate, for colorimetric analysis of urease production ability.

[0021] 3. King'B medium: proteose peptone 10 g, glucose 10 g, Na2HPO4 4 g, NaH2PO4 2 g, MgSO4 0.2 g, CaCl2 0.2 g, distilled water 1 L, pH 7.0, 121°C high pressure sterilization for 30 min, for IAA production ability determination. Salksowski colorimetric solution II: FeCl3 4.5 g, distilled water 300 mL, 98% concentrated sulfuric acid 587.7 mL, cool to 1 L, for IAA colorimetric reaction. Preparation of standard curve: accurately weigh the IAA analysis reagent, prepare standard solutions with concentrations of 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 μg / mL, and mix with Salksowski colorimetric solution II at a volume ratio of 1:1, room temperature, avoid light for 30 min. With distilled water and Salksowski colorimetric solution II mixed solution as control, the OD 530 nm of each concentration was determined, and finally the IAA concentration was taken as the abscissa, and the OD 530 ​nm is plotted as the ordinate, that is, the IAA standard curve. The bacterial suspension is inoculated into 20 mL of King'B liquid medium at an inoculum of 2%, and cultured at 28°C and 180 r / min for 48 h. The bacterial liquid is centrifuged at 10,000 r / min for 10 min, and then 4 mL of the supernatant is mixed with Salksowski colorimetric solution II in an equal volume, shaken well, and placed in the dark at room temperature for 30 min. The OD 530 of the mixture is measured, wherein the mixture of the un-inoculated liquid medium and Salksowski colorimetric solution II in an equal volume is used as a control group. Finally, the corresponding IAA content is calculated according to the standard curve.

[0022] 4. CAS medium: chromium azure S 60.5 mg, cetyltrimethylammonium bromide 72.9 mg, FeCl3·6H2O 2.645 mg, NaH2PO4·2H2O 295.25 mg, Na2HPO4·12H2O 1213.5 mg, ammonium chloride 125 mg, KH2PO4 37.5 mg, sodium chloride 62.5 mg, agar 9 g, pH value 6.8±0.1, distilled water 1 L, high-pressure sterilization at 121°C for 30 min, used for screening of iron carrier production ability.

[0023] Results and analysis Figure 3 The phosphorus-dissolving effect of the phosphorus-dissolving bacteria, the color development of urease production, IAA production and iron carrier production are shown in Figs. Figure 4 , 5 and 6.

[0024] Table 1: Phosphorus-dissolving amount, phosphorus-dissolving rate of inorganic phosphorus and IAA production amount of phosphorus-dissolving bacteria The results in Table 1 show that the phosphorus-dissolving amount of the strain is 95.26 mg / L, which is obviously increased compared with the control, the phosphorus-dissolving rate is % and the IAA production amount is 27.90 ug / ml.

[0025] Example 3: Analysis of cadmium and lead removal rate effect of phosphorus-dissolving bacteria The phosphorus-dissolving bacteria bacterial suspension is inoculated into inorganic phosphorus liquid medium (formula same as in Example 2) containing Cd (10, 20, 40, 80 mg / L) and Pb (50, 100, 200, 400 mg / L) at an inoculum of 2%, and three groups of parallel are set. The cadmium and lead removal rates are determined. The heavy metal ion removal rate calculation formula is as follows: In the formula, C0is the initial mass concentration of Cd 2+ , Pb 2+ in the solution, mg / L; C s is the mass concentration of Cd 2+Pb 2 + Mass concentration, mg / L.

[0026] Results and analysis The results show that the bacteria suspension is inoculated in the inorganic phosphorus liquid medium containing cadmium at an inoculation amount of 2%, and the removal rate is measured under the stress of 10, 20, 40, 80 mg / L Cd concentration, and the removal rate of 50, 100, 200, 400 mg / L lead concentration is shown in Table 2: Table 2 Cadmium and lead removal rates of phosphorus solubilizing bacteria at different concentration gradients Example Four, Analysis of Corn Growth Promotion Effect of Phosphorus Solubilizing Bacteria 1. Test site: Experimental greenhouse of East Campus of Yunnan Agricultural University. The test soil is red soil with medium uniform soil fertility. Test variety: Yunrui 319. Collect the basic soil sample of the plough layer before planting, and analyze the soil routine 5 items.

[0027] 2. Data collection Collection of soil and plant samples Collect corn rhizosphere soil mixture, collect about 1 kg of soil sample, and measure soil pH, organic matter, available potassium, available phosphorus, and alkali-hydrolyzed nitrogen after natural air drying and removing impurities. Measure the SPAD value after 30 days of corn planting and collect the plants, wash them clean, then measure the corn plant height and stem thickness, and divide the corn plants into aboveground and underground parts, kill them at 105°C for 30 min, and then dry them in a 65°C oven. Use the thousandth scale to measure the dry matter quality. The preparation method of the microbial inoculant is the same as that in Example 1.

[0028] The phosphorus solubilizing bacteria are Bacillus sp selected and identified. The preparation of the inoculant: the slant-preserved strain is cultured on beef extract protein peptone medium for 24 h, and the bacterial suspension is prepared by turbidity counting method, with McFarland turbidity as the comparison standard of turbidity. About 6×108 bacteria per milliliter are contained (molybdenum blue colorimetric method).

[0029] 3. Test treatment design The corn variety is Yunrui 319, a Yunnan highland planting variety. After surface sterilization of corn seeds with 10% sodium hypochlorite (stirring with a glass rod for 1 min) and 75% alcohol (stirring for 1 min), the seeds are placed in a 25°C constant temperature incubator for 5 days of germination. After the seeds are 2-3 cm white, they are transplanted into pots. The pots are 23.5 cm in outer diameter and 14 cm in height, and are sterilized with 95% alcohol before the experiment.

[0030] CK: control (i.e. without inoculation of phosphorus solubilizing bacteria) PSB: inoculation of phosphate solubilizing bacteria (Bacillus sp.) Each treatment was repeated 5 times, a total of 10 pots, each pot filled with 5 kg of soil, and two corn plants were retained in each pot. The pot position was changed every seven days, and the soil was watered with deionized water every two days, with the soil humidity maintained at 60-70% of the absolute humidity of the soil. 10 mL of bacterial suspension was inoculated at the time of corn seeding. After 30 days of corn growth, corn plants and rhizosphere soil were collected for relevant index determination. The indexes included plant height, stem diameter, and biomass, which were determined after 30 days of corn planting, as shown in Table 3. Figure 7

[0031] Results and analysis Table 3: Corn plant height, stem diameter, aboveground and underground biomass under different treatments Note: In the table, the results of multiple comparison variance analysis are indicated by lowercase letters, which represent significant levels at p<0.05. The same applies to the following table.

[0032] The results of Table 3 show that the phosphate-solubilizing bacterial agent can significantly increase the plant height, stem diameter, and aboveground biomass of corn.

[0033] Example Five: Analysis of the Cadmium Tolerance Effect of Phosphate-Solubilizing Bacteria on Corn 1. Experimental site The experimental site was located in the experimental greenhouse of the East Campus of Yunnan Agricultural University. The soil used in the experiment was collected from the vicinity of an abandoned mining area in Huize, Qujing. The soil type was red soil. The basic physicochemical properties of the soil (pH 5.73, organic matter 27.83 mg / kg, alkali-hydrolyzable nitrogen 82.70 mg / kg, available phosphorus 4.80 mg / kg, available potassium 109.00 mg / kg, Pb 343 mg / kg, Cd 8.99 mg / kg) were as follows: the soil was dried and sieved through a 2 mm sieve. After high-temperature wet heat sterilization at 121°C for 2 hours, the soil was left at room temperature for 3-4 days for standby use. 2. Data collection as in Example 4 3. Test strain The phosphate-solubilizing bacteria used were laboratory-isolated and preserved strains, which were identified as Bacillus. The bacterial suspension was obtained by activating the preserved strains on beef extract peptone medium for 48 hours, and then counting the turbidity to prepare the bacterial suspension, with OD 600 =1.

[0034] 4. Test treatment design CK: control PSB: inoculation of PSB bacterial suspension ​Each treatment was repeated 5 times, a total of 10 pots, each flower pot filled with 5 kg of soil, each flower pot retained two corn plants, the flower pot position was changed every seven days, and the soil humidity was kept at 60-70% of the absolute humidity of the soil. 10 mL of bacterial suspension was inoculated at the same time as the corn was sown. After the corn grew for 45 days, the corn plants and rhizosphere soil were collected for determination of relevant indicators. The indicators determined include the cadmium and lead contents of the aboveground and underground parts of corn, the effective state of cadmium and lead in soil, and the five-step extraction state of cadmium and lead in soil.

[0035] Table 4 Cadmium and lead contents, accumulation, transport coefficient and enrichment coefficient of corn aboveground and underground parts Table 5 Effective state of cadmium and lead in soil Table 6 Five-step extraction state of cadmium and lead in soil Tessier The results in Table 4 show that compared with the control, the Cd content, Cd accumulation and transport coefficient of the aboveground and underground parts of corn are significantly reduced, and the Cd enrichment coefficient of the underground part is significantly reduced, indicating that the phosphorus-solubilizing bacterial agent can promote the transport of Cd in plants to the aboveground part. The Pb content, accumulation, transport coefficient of the aboveground part and the Pb accumulation of the underground part are significantly reduced compared with CK. The results in Table 5 show that the effective state of lead content is significantly increased, which is consistent with the results in Table 6, that is, the residual state of lead content in soil is reduced.

[0036] The specific embodiments are only an explanation of the present application, which is not a limitation of the present application. Those skilled in the art can make modifications to the embodiments without creative contribution after reading the specification, as long as the modifications are within the scope of the claims of the present application.

Claims

1. A phosphate-solubilizing bacteria having both phosphate-solubilizing and phosphate- immobilizing functions, characterized by: The phosphorus solubilizing bacteria are Bacillus sp. and are preserved in Guangdong Microbial Culture Collection Center with a strain preservation number of GDMCC NO: 66609.

2. The bacterial agent prepared from the phosphate-solubilizing bacteria with dual functions of phosphate solubilization and passivation according to claim 1, characterized in that: The microbial agent is a liquid microbial agent, and the concentration of the phosphorus solubilizing bacteria in the microbial agent is 6×108 CFU / mL.

3. The bacterial agent prepared from the phosphate-solubilizing and passivation dual functional phosphate-solubilizing bacteria according to claim 2, characterized in that, The preparation method is operated as follows: The phosphorus solubilizing bacteria are activated, a single colony is picked up by using a sterile inoculation ring and inoculated into a liquid culture medium after being cultured on beef extract peptone solid culture medium at 28°C for 24 h, and then the strain is prepared into a phosphorus solubilizing bacteria liquid with a concentration of 6×108 CFU / mL by using sterile water.

4. Application of the microbial agent prepared from the phosphorus solubilizing bacteria with dual functions of phosphorus solubilization and passivation to promoting corn growth according to claim 3.

5. Application of the microbial agent prepared from the phosphorus solubilizing bacteria with dual functions of phosphorus solubilization and passivation to promoting growth and cadmium and lead tolerance according to claim 3.

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