Phosphorus-solubilizing bacteria P6 and application thereof in promoting corn growth

Abstract

The invention discloses a phosphate-solubilizing bacterium P6 and application thereof in promotion of corn growth, the taxonomic name of the phosphate-solubilizing bacterium P6 is Pristia megaterium, the phosphate-solubilizing bacterium P6 is preserved in the China General Microbiological Culture Collection Center on April 16, 2024, and the preservation number is CGMCC No.30361. The phosphate-solubilizing bacterium P6 has the advantages that the phosphate-solubilizing bacterium P6 can be used for promoting corn growth; the phosphate-solubilizing bacteria P6 take a PKO inorganic phosphorus culture medium as a basic culture medium, is directly separated and purified from corn root soil, has the function of dissolving inorganic phosphorus, effectively promotes the growth of corn roots, stems and leaves, effectively improves the phosphorus content of corn seedling roots, stems and leaves, improves the enzymatic activity of corn rhizosphere soil and the content of available phosphorus, and improves the yield of corn seedlings. And the strain has great potential of being developed into a microbial agent.

Description

Technical Field

[0001] The invention belongs to the technical field of agricultural microorganisms and relates to a phosphate-dissolving bacterium P6 and an application thereof in promoting corn growth. Background Art

[0002] Corn is the common name for plants of the genus Zea mays in the family Poaceae. Corn is an annual tall herb. It is native to Central and South America. It is widely grown in tropical and temperate regions around the world. It has high nutritional value and is an excellent food crop. It is also one of the indispensable raw materials for food, medical and health care, light industry, chemical industry, etc.

[0003] Currently, most growers use a large amount of chemical fertilizers when growing corn, and the application of large amounts of chemical fertilizers will lead to soil and water pollution and deterioration of the ecological environment.

[0004] There are many microorganisms with phosphorus-solubilizing properties in the soil. They can convert ineffective phosphorus that is difficult for plants to absorb and utilize into an absorbable form, improve plant phosphorus nutrition, and promote the metabolic activities of beneficial microorganisms in the soil. Priococcus giganteus is a Gram-negative bacillus. Gram-negative bacillus is one of the common bacteria in the intestinal flora and one of the symbiotic bacteria in the intestines of humans and animals. It is usually found in soil, water, food, feces and other environments. It has the value of degrading organic phosphorus, producing polyhydroxyalkanoates, and is valuable in the field of fermented food and medicine.

[0005] At present, the patented strains reported to have a growth-promoting effect on corn are Penicillium purpurogenum (CN112375691A) and CR50 strain (CN112375717A). However, there is no report on the use of Priesteria gigantea to promote the growth of corn. The strain isolated in this application has the function of dissolving phosphorus and can promote the growth of corn roots, stems and leaves. Summary of the invention

[0006] The purpose of the present invention is to provide a phosphate-dissolving bacteria P6 and its application in promoting corn growth. The phosphate-dissolving bacteria P6 of the present invention has the function of dissolving phosphorus, so as to promote the absorption of phosphorus by corn and the growth of roots, stems and leaves of corn.

[0007] The technical solution of the present invention:

[0008] A phosphate-dissolving bacterium P6, the phosphate-dissolving bacterium P6 has a taxonomic name of Priestia megaterium, and was deposited in the China General Microbiological Culture Collection Center on April 16, 2024, with a deposit number of CGMCC No. 30361.

[0009] The aforementioned phosphate-solubilizing bacteria P6 was isolated from the rhizosphere soil of corn plants.

[0010] The above-mentioned method for separating and purifying the phosphate-solubilizing bacteria P6 is carried out according to the following steps:

[0011] (1) Weigh 1.0 g of fresh soil sample from the rhizosphere of corn plants into a 10 mL conical flask, add 9 mL of sterile water, mix well, and obtain product A for later use;

[0012] (2) Take product A and shake it in a shaker at 180 rpm and 30°C for 30 min to obtain 10 -1 After the soil suspension was diluted to 10, it was left to stand for 30 minutes. -3 ~10 -6 100 μL of soil suspension was taken with a sterile pipette and dropped onto the PKO inorganic phosphorus medium, spread evenly, and incubated upside down in a 28°C incubator for 5 days to obtain product B;

[0013] (3) Take out product B, use an inoculation loop to pick up the colonies with good growth and obvious transparent circles on the plate, and streak culture on beef extract protein solid culture medium. Separate and purify 3 to 5 times, inoculate the isolated and purified strain into PKO inorganic phosphorus culture medium, and culture at a constant temperature of 28°C to obtain phosphate-solubilizing bacteria P6.

[0014] In the above step (2), the PKO inorganic phosphorus medium is prepared by 25.0 g of Zn3(PO4), 10.0 g of sucrose, 0.5 g of NaCl, 0.2 g of KCl, 0.1 g of (NH4)2S04, 0.1 g of MgSO4·7H2O, 0.004 g of MnSO4, 0.5 g of yeast extract, 0.004 g of FeSO4, 1000 mL of distilled water, and 20.0 g of agar.

[0015] A corn growth promoting agent comprising the aforementioned phosphate-solubilizing bacteria P6.

[0016] For example, the aforementioned application of phosphate-solubilizing bacteria P6 in promoting corn growth, and the application of the phosphate-solubilizing bacteria P6 in preparing a preparation for promoting the growth of corn roots, stems and leaves.

[0017] A corn growth-promoting bacteria product, the active ingredients of which include the aforementioned phosphate-solubilizing bacteria P6.

[0018] A corn growth-promoting bacteria product, wherein the active ingredient of the product is the aforementioned phosphate-solubilizing bacteria P6.

[0019] A method for preparing a corn growth-promoting bacteria product, using the aforementioned phosphate-solubilizing bacteria P6 as an active ingredient or one of the active ingredients for preparing the product.

[0020] Compared with the prior art, the present invention has the following beneficial effects:

[0021] 1. The present invention discloses a phosphate-dissolving bacterium P6 and its application in promoting corn growth. The phosphate-dissolving bacterium P6 in the soil of corn roots is isolated and cultured by a tissue separation method, and the phosphate-dissolving bacterium P6 is back-inoculated into the corn pot seedlings for colonization, which can promote the growth of the corn seedlings.

[0022] 2. The results of the phosphate-dissolving effect test of phosphate-dissolving bacteria P6 showed that the strain P6 had a high ability to dissolve phosphorus. Using PKO liquid medium as the basic culture medium, the maximum value of 372.90 μg / ml was reached on the 4th day.

[0023] 3. Under the action of phosphate-dissolving bacteria P6, after 30 days of growth of corn seedlings, the total nitrogen, total phosphorus and total potassium contents in the soil decreased, and their contents ranged from 0.69-0.77g / kg, 1.27-1.75g / kg, and 22.77-27.82g / kg, respectively. However, the effective phosphorus content was significantly higher than that of CK, reaching a maximum of 95.51m / kg. The decrease in the total nitrogen, total phosphorus and total potassium contents in the soil and the increase in the effective phosphorus content promoted the absorption and utilization of nutrients by corn plants, which was beneficial to the growth of corn seedlings.

[0024] 4. Under the action of phosphate-solubilizing bacteria P6, the acid phosphatase activity (27.87±5.74U / g), alkaline phosphatase activity (15.39±3.83U / g) and catalase activity (18.36±0.48U / g) in the soil of corn seedlings increased significantly after 30 days of growth.

[0025] 5. Under the action of phosphate-dissolving bacteria P6, the phosphorus content in the roots, stems and leaves of corn increased significantly after 30 days of growth, reaching 7.77±0.95 mg / g, 2.37±0.31 mg / g and 5.44±0.21 mg / g respectively. Inoculation of phosphate-dissolving bacteria P6 promoted the growth of roots, stems and leaves of corn.

[0026] In summary, the phosphate-dissolving bacteria P6 of the present invention has the function of efficiently dissolving phosphorus, effectively promotes the growth of corn roots, stems and leaves, effectively increases the phosphorus content of corn seedling roots, stems and leaves, increases the enzyme activity and the content of available phosphorus in the corn rhizosphere soil, and has great potential for development into a microbial agent. BRIEF DESCRIPTION OF THE DRAWINGS

[0027] Figure 1 :The phosphate-dissolving effect of phosphate-dissolving bacteria in maize rhizosphere soil on inorganic phosphate medium (A: CK front; B: P6 front; C: P6 back);

[0028] Figure 2 : evolutionary tree;

[0029] Figure 3 :Changes in available phosphorus content of phosphate-solubilizing bacteria P6 during 7 days;

[0030] Figure 4: Comparison of plant height and stem girth of corn seedlings in CK group and P6 group after 30 days of growth;

[0031] Figure 5 : Comparison of above-ground and underground fresh weight of corn seedlings in CK group and P6 group after 30 days of growth;

[0032] Figure 6 : Comparison of above-ground and underground stems of corn seedlings in CK and P6 groups after 30 days of growth;

[0033] Figure 7 : Comparison of phosphorus content in roots / stems / leaves of corn seedlings in CK group and P6 group after 30 days of growth;

[0034] Figure 8 : Comparison of catalase activity / acid phosphatase activity / alkaline phosphatase activity in rhizosphere soil of corn seedlings in CK group and P6 group after 30 days of growth;

[0035] Fig. 9 : Comparison of soil total nitrogen between CK group and P6 group after 30 days of corn seedling growth;

[0036] Fig.10 : Comparison of soil total phosphorus between CK group and P6 group after 30 days of corn seedling growth;

[0037] Fig.11 : Comparison of soil total potassium between CK group and P6 group of corn seedlings after 30 days of growth;

[0038] Fig.12 : Comparison of soil available phosphorus between CK group and P6 group after 30 days of corn seedling growth; DETAILED DESCRIPTION

[0039] The present invention is further described below in conjunction with the accompanying drawings and embodiments, but they are not intended to limit the present invention.

[0040] Example 1: Isolation and purification of phosphate-solubilizing bacteria P6

[0041] (1) Weigh 1.0 g of fresh soil sample from the rhizosphere of corn plants into a 10 mL conical flask, add 9 mL of sterile water, mix well, and obtain product A for later use;

[0042] (2) Take product A and shake it in a shaker at 180 rpm and 30°C for 30 min to obtain 10 -1 After the soil suspension was diluted to 10, it was left to stand for 30 minutes. -3 100 μL of soil suspension was taken with a sterile pipette and dropped onto the PKO inorganic phosphorus medium, spread evenly, and incubated upside down in a 28°C incubator for 5 days to obtain product B;

[0043] (3) Take out product B, use an inoculation loop to pick up the colonies with good growth and obvious transparent circles on the plate, and streak culture on beef extract peptone solid culture medium. Separate and purify 3 to 5 times, inoculate the isolated and purified strain into PKO inorganic phosphorus culture medium, and culture at a constant temperature of 28°C to obtain phosphate-solubilizing bacteria P6.

[0044] Example 2: Isolation and purification of phosphate-solubilizing bacteria P6

[0045] (1) Weigh 1.0 g of fresh soil sample from the rhizosphere of corn plants into a 10 mL conical flask, add 9 mL of sterile water, mix well, and obtain product A for later use;

[0046] (2) Take product A and shake it in a shaker at 180 rpm and 30°C for 30 min to obtain 10 -1 After the soil suspension was diluted to 10, it was left to stand for 30 minutes. -4 100 μL of soil suspension was taken with a sterile pipette and dropped onto the PKO inorganic phosphorus medium, spread evenly, and incubated upside down in a 28°C incubator for 5 days to obtain product B;

[0047] (3) Take out product B, use an inoculation loop to pick up the colonies with good growth and obvious transparent circles on the plate, and streak culture on beef extract peptone solid culture medium. Separate and purify 3 to 5 times, inoculate the isolated and purified strain into PKO inorganic phosphorus culture medium, and culture at a constant temperature of 28°C to obtain phosphate-solubilizing bacteria P6.

[0048] Example 3: Isolation and purification of phosphate-solubilizing bacteria P6

[0049] (1) Weigh 1.0 g of fresh soil sample from the rhizosphere of corn plants into a 10 mL conical flask, add 9 mL of sterile water, mix well, and obtain product A for later use;

[0050] (2) Take product A and shake it in a shaker at 180 rpm and 30°C for 30 min to obtain 10 -1 After the soil suspension was diluted to 10, it was left to stand for 30 minutes. -5 100 μL of soil suspension was taken with a sterile pipette and dropped onto the PKO inorganic phosphorus medium, spread evenly, and incubated upside down in a 28°C incubator for 5 days to obtain product B;

[0051] (3) Take out product B, use an inoculation loop to pick up the colonies with good growth and obvious transparent circles on the plate, and streak culture on beef extract peptone solid culture medium. Separate and purify 3 to 5 times, inoculate the isolated and purified strain into PKO inorganic phosphorus culture medium, and culture at a constant temperature of 28°C to obtain phosphate-solubilizing bacteria P6.

[0052] Example 4: Isolation and purification of phosphate-solubilizing bacteria P6

[0053] (1) Weigh 1.0 g of fresh soil sample from the rhizosphere of corn plants into a 10 mL conical flask, add 9 mL of sterile water, mix well, and obtain product A for later use;

[0054] (2) Take product A and shake it in a shaker at 180 rpm and 30°C for 30 min to obtain 10 -1 After the soil suspension was diluted to 10, it was left to stand for 30 minutes. -6 100 μL of soil suspension was taken with a sterile pipette and dropped onto the PKO inorganic phosphorus medium, spread evenly, and incubated upside down in a 28°C incubator for 5 days to obtain product B;

[0055] (3) Take out product B, use an inoculation loop to pick up the colonies with good growth and obvious transparent circles on the plate, and streak culture on beef extract peptone solid culture medium. Separate and purify 3 to 5 times, inoculate the isolated and purified strain into PKO inorganic phosphorus culture medium, and culture at a constant temperature of 28°C to obtain phosphate-solubilizing bacteria P6.

[0056] Example 5: Method for promoting corn seedling growth by phosphate-dissolving bacteria P6

[0057] (1) Preparation of beef extract peptone solid culture medium: 3 g beef extract, 10 g peptone, 5 g NaCl, 15 g-20 g agar, 1000 ml water, Ph = 7.

[0058] (2) Preparation of beef extract peptone liquid culture medium: Beef extract peptone solid culture medium Agar was used as liquid culture medium.

[0059] (3) Preparation of P6 suspension: Prepare beef extract peptone liquid culture medium, then put 100 ml of the culture medium into 250 ml, sterilize at 121°C for 30 minutes, and after cooling, take 5 activated phosphate-solubilizing bacteria P6 bacterial cakes (diameter = 5 mm) with an inoculation loop under sterile conditions, and culture them on a shaker at 160 rpm for 16 hours to prepare P6 bacterial culture liquid. Then, adjust the suspension concentration to 1×10 8 CFU / mL, and obtain P6 suspension for later use.

[0060] (4) Inoculation: Before inoculation, the soil used for potted plants was sterilized; the potted experiments were conducted in a greenhouse; healthy, disease-free, and uniformly growing corn seedlings were selected and inoculated using the root irrigation method: the P6 suspension group was irrigated with 100 mL of suspension per corn plant.

[0061] Example 6: Method for promoting corn seedling growth by phosphate-dissolving bacteria P6

[0062] (1) Preparation of beef extract peptone liquid culture medium: Beef extract solid culture medium Agar is used as liquid culture medium.

[0063] (2) Preparation of P6 suspension: Prepare beef extract peptone liquid culture medium, then put 100 ml of the culture medium into 250 ml, sterilize at 121°C for 30 minutes, and after cooling, take 5 activated phosphate-solubilizing bacteria P6 bacterial cakes (diameter = 5 mm) with an inoculation loop under sterile conditions, and culture them on a shaker at 160 rpm for 16 hours to prepare P6 bacterial culture liquid. Then, adjust the suspension concentration to 1×10 8 CFU / mL, and obtain P6 suspension for later use.

[0064] (3) Inoculation: Before inoculation, the soil used for potted plants was sterilized; the potted experiments were conducted in a greenhouse; healthy, disease-free, and uniformly growing corn seedlings were selected and inoculated using the root irrigation method: the P6 suspension group was irrigated with 100 mL of suspension per corn plant.

[0065] The present invention has done a lot of analysis and verification experiments, and the following are the results of the experimental research of the present invention:

[0066] 1. Isolation and purification of phosphate-solubilizing bacteria P6

[0067] 1.1 Culture medium preparation

[0068] PKO inorganic phosphorus medium: (Zn3(PO4)25.0g, sucrose 10.0g, NaCl 0.5g, KCl 0.2g, (NH4)2S040.1g, MgSO4·7H2O 0.1g, MnSO4 0.004g, yeast extract 0.5g, FeSO4

[0069] 0.004 g, distilled water 1000 mL, agar 20.0 g, pH natural).

[0070] PKO inorganic phosphorus liquid culture: PKO inorganic phosphorus medium without Agar is used as liquid culture medium.

[0071] Preparation of beef extract peptone solid culture medium: 3g beef extract, 10g peptone, 5g Nacl, 15g-20g agar, 1000ml water, Ph=7.

[0072] Preparation of beef extract peptone liquid culture medium: Beef extract peptone solid culture medium Agar is used as liquid culture medium.

[0073] 1.2 Separation and purification process

[0074] Phosphate-solubilizing bacteria were isolated from the rhizosphere soil of corn plants in Huaxi District, Guiyang City, Guizhou Province, China (26°19′17″N, 106°39′18″E). First, 1.0 g of fresh soil sample was weighed into a 10 mL conical flask, and then 9 mL of sterile water was added and mixed. The phosphate-solubilizing bacteria were shaken at 180 rpm and 30°C for 30 min to obtain 10-1 After the soil suspension was left to stand for 30 min, the soil sample was diluted to 10 -2 , 10 -3 , 10 -4 , 10 -5 , 10 -6 Different dilution gradient soil suspension. Use a sterile pipette to pipette different dilution multiples (10 -3 , 10 -4 , 10 -5 , 10 -6 ) of the soil suspension, each aspirate 100 μL, drop it on the PKO inorganic phosphorus culture medium, spread it evenly, set three replicates for each dilution multiple, culture it upside down in a 28°C incubator for 5 days, take it out, use an inoculation loop to pick up the colonies on the plate that have good growth and obvious transparent circles around them, and streak culture them on beef extract peptone solid culture medium, separate and purify them 3 to 5 times, classify and mark them, culture them to the logarithmic growth phase, mix them with glycerol in a certain proportion in a bacteria preservation tube, and store them in a -80°C refrigerator for later use.

[0075] 2. Screening of phosphate-solubilizing bacteria P6

[0076] Initial screening: Under sterile conditions, the isolated strains to be tested were inoculated into beef extract solid medium for activation, and then inoculated into PKO inorganic phosphorus medium. Each treatment was repeated 3 times, and after incubation at 28°C for 7 days, the diameter of the phosphorus solubilization circle (D) and colony diameter (d) produced by each strain were measured, and the phosphorus solubilization index was calculated. The phosphorus solubilization ability of the strain was preliminarily judged based on the phosphorus solubilization index.

[0077] Solubilization index (SI) SI = D / d

[0078] Rescreening: Take 100 mL of PKO inorganic phosphorus liquid medium into a 250 mL Erlenmeyer flask and sterilize it at 121°C for 25 min. After cooling, use a pipette to take 2 mL of each bacterial suspension (10 8 CFU / mL) was inoculated into each culture medium, and the culture medium inoculated with an equal amount of high-temperature inactivated bacterial suspension was used as a control, and each treatment was repeated 3 times. After all treatments were placed in a shaker at 30°C and 180rpm for 7 days, each fermentation broth was centrifuged at 4°C and 8000rpm for 10 minutes, and the supernatant was taken to determine the effective phosphorus content and pH value of the bacterial broth. A standard curve was prepared using KH2PO4 as the standard sample, and the standard curve was paralleled 3 times, and the sample was repeated 3 times. The pH value was determined using a pH meter; the effective phosphorus content was determined using a spectrophotometer at 700nm by the molybdenum antimony colorimetric method, and the value minus the control was the amount of dissolved effective phosphorus. Finally, the phosphate-dissolving bacteria P6 with the best phosphate-dissolving effect was screened out.

[0079] 3. Identification of phosphate-solubilizing bacteria P6

[0080] The isolated and purified phosphate-dissolving bacteria P6 strain was inoculated into PKO inorganic phosphorus medium and cultured at 28°C. The colony characteristics (colony color, colony shape and edge characteristics) were observed and photographed with a camera. The characteristics of the colony are: the colony is round, the white edge is irregular, the surface is mucous, moist and smooth, and Gram-negative ( Figure 1 ).

[0081] Molecular biological identification of strains: PCR amplification of strains was performed using universal bacterial primers. The activated strains were stored in cryopreservation tubes and sent to Chongqing Qingke Xingye Biotechnology Co., Ltd. for sequencing. At the same time, the sequences were subjected to Blast similarity comparison in Genbank to obtain sequences with high similarity. Multiple sequence alignment was performed using ClustalW in MEGA7.0 software, and then the neighbor joining method (NJ) in MEGA7.0 was used to construct a phylogenetic tree ( Figure 2 ). The forward primer for PCR amplification was 27F (SEQ ID NO.4: 5'-AGAGTTTGATCCTGGCTCAG-3'), and the reverse primer was 1492R (SEQ ID NO.5: 5'-GGTTACCTTGTTACGACTT-3'). The results showed that the phosphate-soluble bacteria P6 strain had the highest homology with Priestia megaterium and the closest relationship.

[0082] 4 Determination of phosphorus capacity of phosphate-solubilizing bacteria P6

[0083] 4.1 Quantitative determination

[0084] The activated bacterial block (phosphate-soluble bacteria P6) was punched into a bacterial cake with a diameter of 5 mm using a hole punch, and inoculated into PKO inorganic phosphorus culture medium (Zn3(PO4) 25.0 g, sucrose 10.0 g, NaCl 0.5 g, KCl 0.2 g, (NH4)2S04 0.1 g, MgSO4·7H2O 0.1 g, MnSO4 0.004 g, yeast extract 0.5 g, FeSO4 0.004 g, distilled water 1000 mL, agar 20.0 g, pH natural) on a clean bench. After it grew stably, its periphery was observed to see whether a transparent phosphate-soluble ring appeared. The results showed that a transparent phosphate-soluble ring appeared. The phosphate-soluble index is shown in Table 1.

[0085] Table 1: D / d values ​​of phosphate solubilizing zone diameter and colony diameter of phosphate solubilizing bacteria P6 in solid PKO medium

[0086]

[0087] Note: Data are mean ± standard error

[0088] 4.2 Qualitative determination

[0089] Use PKO inorganic phosphorus medium without Agar as liquid culture medium, add 150 mL to each conical flask, and under sterile conditions, take 5 pieces of bacterial cakes with a diameter of 8.0 mm and put them into conical flasks with fermentation liquid respectively, move them to a shaker, and culture them at 28°C and 150 r / min for 7 days. After culture, take samples every day to determine the effective phosphorus content in the fermentation liquid.

[0090] (1) Determination of effective phosphorus content: Take 5 mL of supernatant, centrifuge at 12000 r / min for 5 min, take 3 ml of supernatant as the test solution and place it in a 50 ml conical flask, add 1 drop of 2,4-dinitrophenol indicator to each flask, the solution gradually develops color, then add 4 mol / L NaOH solution to make the fermentation liquid in the conical flask turn yellow, then add 1 mol / L sulfuric acid solution to adjust the pH so that the liquid is just slightly yellow. Add 5 ml of molybdenum antimony anti-colorimetric agent, distilled water to 50 ml, shake appropriately to make the liquid uniform, let it stand on a horizontal table for 30 min, and use a UV-visible spectrophotometer with a wavelength of 700 nm for colorimetry. Use the uninoculated PKO liquid fermentation liquid as the reference liquid, set the absorption value to zero, measure the color values ​​of different fermentation liquids respectively, and calculate their phosphorus concentrations on the phosphorus standard curve.

[0091] (2) Drawing of phosphorus standard curve: dilute 100 mg / L phosphorus standard solution to 5 mg / L, take 0, 2, 4, 6, 8, 10 mL of 5 mg / L phosphorus standard solution into a 50 mL conical flask, add distilled water to 30 mL, then add 2 drops of 2,4-dinitrophenol indicator, then add 4 mol / L NaOH dropwise to the conical flask until the liquid turns slightly yellow, add 1 mol / L H2SO4 until the yellow color fades, add 5 mL of molybdenum antimony anti-color developer, shake quickly, add distilled water to the 50 mL mark, color at room temperature for 2 h, and measure the absorbance value with a spectrophotometer at a wavelength of 700 nm. Draw the phosphorus standard curve, with the horizontal axis being the phosphorus concentration and the vertical axis being the absorbance value.

[0092] (3) Calculation formula: Effective phosphorus content P (μg / mL) = (ρ×V×Ts) / Vo

[0093] ρ: mass concentration of available phosphorus (p) obtained from the working curve (μg / mL);

[0094] V: the fixed volume of the color developing solution (mL), (here 50mL);

[0095] Ts: the multiple (here is 17 times);

[0096] Vo: volume of fermentation broth (mL) (here 3 mL).

[0097] Test results: Using PKO inorganic phosphorus liquid medium as the basic medium, the transparent zone was observed on the 4th day after inoculation. Figure 3 It can be seen that the overall trend of phosphate-solubilizing bacteria P6 was first rising and then falling. The effective phosphorus content gradually increased from the 1st day to the 4th day, reaching a maximum value of 372.90μg / ml on the 4th day, and dropped to 132.60μg / ml on the seventh day.

[0098] Effects of phosphate-solubilizing bacteria P6 on the growth and biomass of corn seedlings

[0099] 5.1 Preparation of P6 suspension

[0100] First, prepare beef extract peptone liquid culture medium, then put 100 ml of the culture medium into 250 ml, sterilize at 121°C for 20 minutes, and after cooling, take 5 activated phosphate-soluble bacteria P6 bacterial cakes (diameter = 5 mm) with an inoculation loop under sterile conditions, and culture them on a shaker at 160 rpm for 16 hours to prepare P6 bacterial culture liquid. Then, adjust the suspension concentration to 1×10 8 CFU / mL, and obtain P6 suspension for later use.

[0101] Beef extract peptone liquid medium: Beef extract peptone solid medium Agar is used as liquid culture medium.

[0102] 5.2 Grouping Scheme

[0103] The inoculation experiment was conducted in a potted plant experiment, with two treatment groups, namely the control group (CK) and the P6 suspension group, with one plant in each pot, 5 pots in each group, and a total of 10 experimental pots. Before inoculation, the soil used for potted plants was sterilized.

[0104] 5.3 Inoculation test

[0105] The pot experiment was conducted in a greenhouse. Healthy, disease-free, and uniformly growing corn seedlings were selected and inoculated using the root irrigation method: (1) the P6 suspension group was irrigated with 100 mL of suspension per corn plant; (2) the seedlings inoculated with 100 mL of sterile distilled water were used as the control group (CK); water was applied every three days, and the seedlings were harvested after 30 days of uniform cultivation management (e.g. Figure 4-Figure 6 ).

[0106] Table 2. Effects of phosphate-solubilizing bacteria in the rhizosphere of maize on the growth and biomass of maize seedlings under different treatments

[0107] Grouping Plant height (cm) Stem circumference (mm) Fresh weight above ground (g) Underground fresh weight (g) Above ground dry weight (g) Underground dry weight (g) CK 30.6±3.78 6.58±0.55 24.18±3.02 4.43±0.51 1.22±0.24 0.11±0.03 P6 33.4±3.78 7.33±0.79 25.37±3.05 4.64±0.55 1.42±0.21 0.13±0.03

[0108] Results: According to the results in Table 2, the plant height and stem girth of the P6 group grew better than those of the CK group. The aboveground fresh weight, underground fresh weight, aboveground dry weight and underground dry weight were all higher than those of the CK group, proving that the phosphate-solubilizing bacteria P6 was beneficial to the growth of corn seedlings.

[0109] 5.4 Effects of phosphate-solubilizing bacteria P6 on phosphorus content in roots, stems and leaves of corn seedlings

[0110] The roots, stems and leaves of the potted seedlings from the two groups were taken for determination of phosphorus content after 30 days of treatment. The results are shown in Tables 3 and Figure 7 The P6 group had the highest phosphorus content, with the root phosphorus content reaching 7.77±0.95 mg / g, the stem phosphorus content reaching 2.37±0.31 mg / g, and the leaf phosphorus content reaching 5.448±0.21 mg / g.

[0111] Table 3 Effects of phosphate-dissolving bacteria on phosphorus content in roots, stems and leaves of corn seedlings

[0112]

[0113] 5.5 Effects of phosphate-solubilizing bacteria P6 on enzyme activities in the rhizosphere soil of corn seedlings

[0114] The rhizosphere soil of the potted seedlings from the two groups after 30 days of treatment was taken for rhizosphere soil enzyme activity detection. The results are shown in Table 4 and Figure 8 As shown, the activities of acid phosphatase, alkaline phosphatase and catalase in group P6 increased significantly, which was beneficial to the absorption of soil nutrients by roots.

[0115] Table 4 Effects of phosphate-solubilizing bacteria strains on enzyme activities in the rhizosphere soil of corn seedlings

[0116]

[0117] 5.6 Effects of phosphate-solubilizing bacteria P6 on rhizosphere soil nutrients of corn seedlings

[0118] The two groups of potted corn seedlings were treated with bacterial solution applied to the root position, and the bacterial solution was applied once every 5 days. The amount of bacterial solution applied each time was 100 ml. Before applying the bacterial solution, part of the rhizosphere soil was taken to determine the total nitrogen, total phosphorus, total potassium and available phosphorus in the soil. The corn seedling stage was 30 days. The results are shown in Table 5. Figure 9-12 As shown in the figure, after 30 days of nutrient absorption, compared with CK, the P6 group reduced the total nitrogen, total phosphorus and total potassium content in the soil of corn seedlings, with total nitrogen content between 0.69-0.77g / kg, total phosphorus content between 1.27-1.75g / kg, and total potassium content between 22.77-27.82g / kg, but the available phosphorus content was significantly higher than CK, reaching a maximum of 95.51±26.39mg / kg. The reduction of total nitrogen, total phosphorus and total potassium content in the soil and the increase of available phosphorus content in the soil promoted the growth of corn seedlings in the later stage.

[0119] Table 5 Effects of phosphate-solubilizing bacteria on nutrients in the rhizosphere soil of corn seedlings

[0120]

[0121] P6(Priestia megaterium)

[0122] CGMCC NO.30361

[0123] DNA sequence:

[0124] >6_TSS20221014-028-01531

[0125]

Claims

1. A phosphate-dissolving bacterium P6, characterized in that: The phosphate-dissolving bacteria P6 has a taxonomic name of Priestia megaterium, and was deposited in the China General Microbiological Culture Collection Center on April 16, 2024, with a deposit number of CGMCC No.30361.

2. The phosphate-dissolving bacteria P6 according to claim 1, characterized in that: The phosphate-solubilizing bacteria P6 is isolated from the rhizosphere soil of corn plants.

3. The phosphate-dissolving bacteria P6 according to claim 1, characterized in that: The separation and purification method of the phosphate-solubilizing bacteria P6 is carried out according to the following steps: (1) Weigh 1.0 g of fresh soil sample from the rhizosphere of corn plants into a 10 mL conical flask, add 9 mL of sterile water, mix well, and obtain product A for later use; (2) Take product A and shake it in a shaker at 180 rpm and 30°C for 30 min to obtain 10 -1 After the soil suspension was diluted to 10, it was left to stand for 30 minutes. -3 ~10 -6 100 μL of soil suspension was taken with a sterile pipette and dropped onto the PKO inorganic phosphorus medium, spread evenly, and incubated upside down in a 28°C incubator for 5 days to obtain product B; (3) Take out product B, use an inoculation loop to pick up the colonies with good growth and obvious transparent circles on the plate, and streak culture on beef extract protein solid culture medium. Separate and purify 3 to 5 times, inoculate the isolated and purified strain into PKO inorganic phosphorus culture medium, and culture at a constant temperature of 28°C to obtain phosphate-solubilizing bacteria P6.

4. The phosphate-dissolving bacteria P6 according to claim 3, characterized in that: In the step (2), the PKO inorganic phosphorus culture medium is prepared by 25.0 g of Zn3(PO4), 10.0 g of sucrose, 0.5 g of NaCl, 0.2 g of KCl, 0.1 g of (NH4)2S04, 0.1 g of MgSO4·7H2O, 0.004 g of MnSO4, 0.5 g of yeast extract, 0.004 g of FeSO4, 1000 mL of distilled water, and 20.0 g of agar.

5. A corn growth promoting agent comprising the phosphate-solubilizing bacteria P6 according to claim 1.

6. The use of the phosphate-dissolving bacteria P6 in promoting corn growth as claimed in claim 1, characterized in that: The phosphate-dissolving bacteria P6 is used in preparing a preparation for promoting the growth of corn roots, stems and leaves.

7. A corn growth-promoting bacteria product, characterized in that: The active ingredient of the preparation includes the phosphate-solubilizing bacteria P6 described in claim 1.

8. A corn growth-promoting bacteria product, characterized in that: The active ingredient of the preparation is the phosphate-solubilizing bacteria P6 described in claim 1.

9. A method for preparing a corn growth-promoting bacteria product, characterized in that: The phosphate-dissolving bacteria P6 described in claim 1 is used as the active ingredient or one of the active ingredients for preparing the product.

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