P.megatherium as well as biological agent and application of P.megatherium

Abstract

The invention relates to P.megatherium as well as a biological agent and application thereof, and belongs to the technical field of microorganisms. The preservation number of the strain is CCTCC (China Center for Type Culture Collection) NO: M20251011, and the preservation number of the strain is CCTCC NO: M20251011. The P.megatherium SY39 can improve the soil agglomeration structure, has the growth promoting functions of dissolving organic and inorganic phosphorus, generating exopolysaccharides, indoleacetic acid, siderophores and the like, and can also generate antibacterial active substances to inhibit the growth of pathogenic bacteria.

Description

Technical Field

[0001] The present invention relates to a Priestia megaterium, a composite biological bacterial agent containing the same, and applications thereof, belonging to the field of microbial technology. Background Art

[0002] Phosphorus is one of the important elements for plant growth and development, and plays an important role in intracellular energy metabolism, carbon metabolism, nitrogen metabolism, and material transport processes. The available inorganic phosphorus that can be absorbed and utilized by plants in the soil is very low, generally only accounting for 2% - 3% of the total phosphorus content. When soluble phosphate fertilizers are applied to the soil, part of them can be absorbed and utilized by plants, while the other part reacts with soil components and then moves out of the soil phase, becoming insoluble phosphate forms that are difficult for plants to utilize.

[0003] Agricultural activities are the primary source of phosphorus pollution. Phosphorus in chemical fertilizers and livestock manure enters water bodies through surface runoff and soil leaching. Research shows that the total phosphorus emissions from agricultural sources in the Yangtze River Basin account for 67.62% of the total basin, among which livestock and poultry breeding and planting contribute 34.44% and 29.18% respectively. In addition, urban surface runoff, industrial wastewater, and domestic sewage are also important pollution sources. For example, the contribution rate of agricultural non-point sources to total phosphorus in the Taihu Lake Basin exceeds 80%.

[0004] Phosphate-solubilizing microorganisms can well decompose phosphorus in the soil, increase the content of available phosphorus, and can also improve the biological nitrogen fixation efficiency, improve other trace elements, and produce plant growth-promoting substances such as auxin (IAA), gibberellin (GA), cytokinin (CK), siderophore, HCN, etc. In view of the current situation of lack of available phosphorus and rich potential phosphorus sources in the soil, how to utilize phosphate-solubilizing microorganisms to activate soil-insoluble phosphorus and improve the utilization rate of chemical phosphate fertilizers is an urgent problem to be solved in current agricultural production in China, and is also one of the research hotspots in the fields of soil fertilizer and plant nutrition at home and abroad.

[0005] Bacillus has endospores, forms dormant endospores under extreme environments, and can tolerate harsh conditions such as high temperature, acid-base, and drought, and is easy to store and formulate. It has broad application potential in promoting plant growth, inhibiting pathogenic bacteria, decomposing environmental pollutants, etc. Currently, the Bacillus strains widely used for phosphorus pollution control mainly include: Bacillus subtilis ( Bacillus subtilis ), Bacillus velezensis ( Bacillus velezensis ), Bacillus amyloliquefaciens ( Bacliius amyloliquefaciens ), Bacillus megaterium ( Bacillus megaterium ), and Bacillus laterosporus ( Brevibacillus laterosporus), etc. It is reported that Wen Shaofu et al. used pot experiments to study the immobilization, transfer of lead in the rhizosphere soil of corn by Acinetobacter calcoaceticus and the effects and interrelationships on the microbial community structure. The results showed that the bacterial liquid and fermentation broth of Acinetobacter calcoaceticus significantly increased the activity of acid phosphatase (ACP) in the rhizosphere soil of corn. Song Gen et al. screened active strains using sweet potato stem nematodes as the target. The results showed that the screened Bacillus velezensis HM-3 not only had a strong contact-killing ability against sweet potato stem nematodes, but also had the functions of phosphorus solubilization, potassium solubilization and nitrogen fixation. It could convert insoluble or poorly soluble phosphorus and potassium elements in the soil into forms that could be absorbed by crops and fix nitrogen in the air for crop use, especially its potassium solubilization ability was the most prominent. However, there has been no report on Priestia megaterium having the characteristics of salt and alkali tolerance, biocontrol, phosphorus solubilization, potassium solubilization and improving soil structure, which is of great significance. Summary of the Invention

[0006] The purpose of the present invention is to provide a Priestia megaterium, its biological bactericide and application. The Priestia megaterium SY39 of the present invention can improve the soil aggregate structure, and at the same time has growth-promoting functions such as dissolving organic and inorganic phosphorus, producing extracellular polysaccharides, indole acetic acid and siderophores, and can also produce antibacterial active substances to inhibit the growth of pathogenic bacteria.

[0007] To achieve the above purpose, the present invention provides the following technical solutions: A strain of Priestia megaterium ( Priestia megaterium ) SY39, isolated from Dali in 2024, deposited in the China Center for Type Culture Collection, with the deposit number CCTCC NO: M 20251011. The deposit date is May 12, 2025, and the address of the China Center for Type Culture Collection is: Wuhan University, China, Wuhan.

[0008] The present invention also provides a biological bactericide, which contains the above-mentioned strain SY39 of Priestia megaterium or the metabolite of the strain.

[0009] The present invention also provides a biological preparation prepared from the above bactericide.

[0010] The present invention also provides any one of the following applications of the above bactericide and biological preparation: 1) For dissolving organic phosphorus or / and inorganic phosphorus; 2) For producing siderophores; 3) For producing extracellular polysaccharides; 4) For improving the soil aggregate structure; 5) For producing indole acetic acid; 6) For promoting plant growth; 7) For inhibiting pathogenic fungi; 8) For inhibiting pathogenic bacteria.

[0011] Pathogenic fungi include: Phytophthora parasitica var. nicotianae, Phytophthora parasitica Monilinia fructicola, Monilinia laxa Botrytis cinerea, Botrytis cinerea Rhizoctonia cerealis Rhizoctonia cerealis ).

[0012] Pathogenic bacteria include Pseudomonas syringae pv. lachrymans. Pseudomonas syringae ).

[0013] The beneficial effects produced after adopting the above technical solution are:

[0014] The Priestia megaterium SY39 of the present invention can improve soil aggregate structure, and at the same time has growth-promoting functions such as dissolving organic and inorganic phosphorus, producing exopolysaccharides, indole acetic acid and siderophores, and can also produce antibacterial active substances to inhibit the growth of pathogenic bacteria.

[0015] The Priestia megaterium SY39 of the present invention can still grow normally without inhibition under the stress of 8% salt concentration, and can tolerate a certain salt concentration; and still has the ability to dissolve phosphorus under high salt stress.

[0016] The average equivalent diameter of soil aggregates treated with the Priestia megaterium SY39 of the present invention increased by 32.52% compared with the clear water control group, and increased by 8.67% compared with the culture medium control group; the total falling time of the 500 mL soil leachate of the strain SY39 decreased by 6.82% and 26.37% respectively compared with the culture medium control and the clear water control.

[0017] The amount of IAA produced by the SY39 strain in the DF medium can reach 2.28 ± 0.11 mg / L, and the amount of IAA produced in the DF+ medium can reach 7.95 ± 0.19 mg / L.

[0018] The strain SY39 of the present invention has a good growth-promoting effect, which can increase the stem height of corn seedlings by 32.91%, the root length by 37.42%, the fresh weight of the stem by 50.30%, the fresh weight of the root by 94.24%, the dry weight of the stem by 19.21%, and the dry weight of the root by 35.94% (according to the method for detecting the growth-promoting effect of plant rhizosphere growth-promoting bacteria disclosed in CN101984067A).

[0019] The strain SY39 of the present invention has an antibacterial rate of 35.68% against Phytophthora parasitica var. nicotianae, 47.92% against Monilinia fructicola, 60.64% against Botrytis cinerea, and 57.12% against Rhizoctonia cerealis, and has great application potential in the prevention and control of plant diseases.

[0020] The strain SY39 of the present invention has a good inhibitory effect on Pseudomonas syringae pv. lachrymans, and the diameter of the inhibition zone is 31.05 ± 0.92 mm.

[0021] The strain SY39 of the present invention has a very good effect of dissolving phosphorus and promoting growth. Compared with the control group, it can increase the stem height of corn seedlings by 13.36%, the fresh weight of stems by 31.95%, the fresh weight of roots by 50.08%, the dry weight of stems by 28.67%, and the dry weight of roots by 51.00% (pot experiment). Description of the Drawings

[0022] Figure 1 Phosphorus-dissolving circle of SY39.

[0023] Figure 2 Growth of strain SY39 under different salt concentrations.

[0024] Figure 3 Ability of strain SY39 to produce siderophores.

[0025] Figure 4 Influence of strain SY39 on the mean weight diameter (MWD) of soil.

[0026] Figure 5 Influence of strain SY39 on the falling time of the first drop of leachate from the soil.

[0027] Figure 6 Influence of strain SY39 on the total falling time of 500 mL of leachate from the soil.

[0028] Figure 7 Growth-promoting effect of strain SY39 on corn seedlings.

[0029] Figure 8 Inhibitory effect of SY39 on pathogenic fungi.

[0030] Figure 9 Inhibitory effect of SY39 on Pseudomonas syringae pv. lachrymans.

[0031] Figure 10 Growth-promoting effect of strain SY39 on potted corn seedlings. Detailed Embodiments

[0032] Next, the technical solutions in the embodiments of the present invention will be clearly and completely described in conjunction with the accompanying drawings in the embodiments of the present invention. Obviously, the described embodiments are only a part of the embodiments of the present invention, rather than all the embodiments. All other embodiments obtained by those of ordinary skill in the art based on the embodiments of the present invention without creative efforts shall fall within the protection scope of the present invention.

[0033] Example 1. Isolation of the strain SY39 of Priestia megaterium ( Priestia megaterium ) In 2024, soil was collected from Dali. 10 g of the soil was added to an Erlenmeyer flask containing 100 ml of sterile normal saline. After standing for 20 min, it was shaken on a shaker at 28 °C and 200 rpm for 30 min. Then, 1 ml was taken and added to 9 ml of sterile normal saline, and further diluted successively by 10 2 , 10 3 , 10 4 times. 100 μl of the above soil suspensions were respectively taken and evenly spread on plates of phosphate growth medium (glucose 10.0 g / L, ammonium sulfate 0.5 g / L, sodium chloride 0.3 g / L, potassium chloride 0.3 g / L, magnesium sulfate heptahydrate 0.3 g / L, ferrous sulfate heptahydrate 0.03 g / L, manganese sulfate tetrahydrate 0.03 g / L, agar 15.0 g / L, pH 7.0) (calcium phytate 2 g / L for organic phosphorus selection and tricalcium phosphate 10.0 g / L for inorganic phosphorus selection). Colonies with a phosphorus-dissolving circle were selected, further streaked and purified to obtain single colonies, which were transferred to an LB slant for culture and stored at 4 °C for standby. The strain SY39 that produced a phosphorus-dissolving circle was isolated. The diameter (D) of the organic phosphorus-dissolving circle reached 17.53 ± 0.28 mm, and the ratio (D / d) of the diameter (D) of the phosphorus-dissolving circle to the diameter (d) of the colony was 1.45; the diameter (D) of the inorganic phosphorus-dissolving circle reached 9.06 ± 0.35 mm, and the ratio (D / d) of the diameter (D) of the phosphorus-dissolving circle to the diameter (d) of the colony was 1.38 (attached Figure 1 ).

[0034] After determining that the strain has the function of dissolving phosphorus, the phosphorus-dissolving ability of the strain is quantitatively determined, and the available phosphorus content in the fermentation supernatant is determined by the molybdenum-antimony anti-colorimetric method. After activating the strain on the LB liquid medium, inoculate it onto the liquid phosphorus-dissolving medium and shake culture at 28 °C for 7 days. Centrifuge the bacterial liquid at 12,000 rpm and 4 °C for 5 min, and keep the supernatant. The soluble phosphorus content in the supernatant is determined by the molybdenum-antimony anti-colorimetric method (Zhang Xiangsheng, 2008). Preparation of the standard curve: Weigh 0.2195 g of KH2PO4 dried at 105 °C for 2 h, dissolve it in 400 ml of water, add 5 ml of concentrated H2SO4 (analytical pure), transfer it to a 1 L volumetric flask, and make up the volume with water. This stock solution can be stored for a long time. Accurately pipette 25.0 ml of the phosphorus standard stock solution with ρ(P) = 50 mg / L, and accurately dilute it 10 times with water to obtain a standard working solution with ρ(P) = 5 mg / L, which is prepared and used immediately. Accurately pipette 0, 0.50, 1.00, 2.00, 4.00, 6.00, 8.00 ml of the phosphorus standard working solution (ρ(P) = 5 mg / L) (the corresponding phosphorus concentrations in this standard series solution are 0, 0.05, 0.10, 0.20, 0.40, 0.60, 0.80 mg / L P) into 50 ml volumetric flasks respectively, add water to about 30 ml, adjust the pH of the solution and develop the color (the method is the same as above), measure the absorbance of the series of solutions (882 nm), draw the calibration curve, and obtain the phosphorus concentration values in each solution according to the drawn standard curve.

[0035] The results show that the standard curve equation is y = 2.8109x - 0.1526 (R 2 = 0.9996), and it has a good linearity within the detection concentration range. After calculation, the inorganic phosphorus content of the strain SY39 is 115.03 ± 1.12 mg / L, and the organic phosphorus content is 46.65 ± 0.87 mg / L.

[0036] Example 2. Identification of Strain SY39

[0037] The strain SY39 is identified by molecular biology. Its 16S rDNA and gyrA sequences are amplified respectively, and the multi-locus sequence typing technology is used for identification. The specific identification results are as follows: First, amplify the 16S rDNA gene sequence of the strain. For the 16S rDNA, primers 27F: 5′-AGAGTTTGATCCTGGTCAGAACGAACGCT-3′ (SEQ ID NO.1) and 1492R: 5′-TACGGCTACCTTGTTACGACTTCACCCC-3′ (SEQ ID NO.2) are used. The 25-µl reaction system consists of 12.5 µl of 2×taqMix, 1 µl of each primer, 1.0 µl of primers, and 9.5 µl of ddH2O. The PCR reaction conditions are as follows: pre-denaturation at 94°C for 5 min, denaturation at 94°C for 30 s, annealing at 56°C for 30 s, extension at 72°C for 1 min. After 30 cycles, full extension is carried out at 72°C for 5 min.

[0038] For amplifying the gyrA sequence, primers 42F: 5'-CAGTCAGGAAATGCGTACGTCCTT-3' (SEQ ID NO.3) and 1066R: 5'-CAAGGTAATGCTCCAGGCATTGCT-3' (SEQ ID NO.4) are used. The 25-µl reaction system consists of 12.5 µl of 2×taqMix, 1 µl of each primer, 1.0 µl of primers, and 9.5 µl of ddH2O. The PCR reaction conditions are as follows: pre-denaturation at 94°C for 5 min, denaturation at 94°C for 30 s, annealing at 61.7°C for 30 s, extension at 72°C for 80 s. After 35 cycles, full extension is carried out at 72°C for 10 min.

[0039] After detecting the target band by agarose gel electrophoresis of the amplified product, it is sent to Beijing Tianyi Huiyuan Biotechnology Co., Ltd. for sequencing. The 16S rDNA sequence (SEQ ID NO.5) is shown in Table 1 below, and the gyrA sequence (SEQ ID NO.6) is shown in Table 2 below. According to the above analysis results and combined with the morphological characteristics of the strain, SY39 is identified as Priestia megaterium.

[0040] Table 1 Partial 16S rDNA sequence of strain SY39

[0041]

[0042] Table 2 gyrA gene sequence of strain SY39

[0043]

[0044] Example 3: Determination of the salt tolerance of strain SY39

[0045] First, activate the strain SY39 in LB liquid medium. Then, inoculate the activated strain into LB liquid media containing 2%, 5%, 8%, 10%, and 13% NaCl (w / v) at an inoculation amount of 1%, and culture it with shaking at 28 °C and 150 rpm for 48 h, and measure its OD 600 value. Use the blank medium as the negative control (CK1) and the LB medium as the positive control (CK2), with 3 replicates for each treatment. The results are as shown in the appendix Figure 2 After removing the OD 600 value of the negative control, the strain SY39 can still grow normally in the medium with a salt concentration of 8% without being inhibited, is inhibited in the 10% salt solution, and can hardly grow in the 13% salt solution. This indicates that the strain SY39 can tolerate a certain salt concentration.

[0046] Table 3 OD 600 value of strain SY39 under different salt concentrations

[0047] Note: Different letters indicate significant differences at the P < 0.05 level.

[0048] Further measure the phosphate-solubilizing ability of the strain SY39 under high salt stress. After streaking the strain SY39 on the LB medium, inoculate it into the liquid LB medium with a salt concentration of 8%, and then inoculate it onto the liquid phosphate-solubilizing medium with an 8% salt concentration, and culture it with shaking at 28 °C for 7 days. Centrifuge the bacterial solution at 12,000 rpm and 4 °C for 5 min, and keep the supernatant. Use the molybdenum antimony anti-colorimetric method to measure the content of soluble phosphorus in the supernatant (Zhang Xiangsheng, 2008), and the measurement method is the same as above. The results show that under salt stress, the inorganic phosphorus content of the strain SY39 is 95.01 ± 0.67 mg / L, and the organic phosphorus content is 26.14 ± 1.07 mg / L. This shows that the strain SY39 still has the phosphate-solubilizing ability.

[0049] Example 4. Determination of the ability to produce siderophores

[0050] Inoculate the strain SY39 activated on the LB medium onto the CAS plate and culture it at 30 °C for 7 d. Observe whether there is a yellow halo.

[0051] The preparation method of the CAS plate is as follows: Solution 1: CAS / HDTMA solution.

[0052] 1) CAS solution: 60.5 mg of CAS (Chrome Azurol S) is dissolved in 50 mL of water; 2) Iron solution: 1 mM of FeCl3·6H2O is dissolved in 10 mM of HCl, with a pH of 2.0; 3) HDTMA solution: 72.9 mg of cetyltrimethylammonium bromide is dissolved in 40 mL of water. Mix solution 1) with 10 mL of solution 2) and then add it to solution 3) and stir well. Autoclave the resulting blue-black liquid at 121 °C for 30 min. This liquid is the CAS / HDTMA solution.

[0053] Solution 2: Salts / Buffer solution.

[0054] 1) Salts (10×100 mL): 0.3 g of KH2PO4, 0.5 g of NaCl, 1.0 g of NH4Cl; 2) Pipes: 30.24 g is dissolved in Salts, the pH is adjusted to 6.8 with 50% (W / V) KOH, 15.0 g of agar is added, and the volume is made up to 800 mL with distilled water. Autoclave at 121 °C for 30 min and cool to 50 °C.

[0055] Solution 3: 2.00 g of glucose, 2.00 g of mannitol, 493 mg of MgSO4·7H2O, 11 mg of CaCl2, 1.4 mg of H3BO3, 1.2 mg of ZnSO4·7H2O, 1.17 mg of MnSO4·2H2O, 1 mg of Na2Mo4·2H2O, 40 μg of CuSO4, and the volume is made up to 750 mL with distilled water. Autoclave at 121 °C for 30 min.

[0056] Solution 4: 10.00 g of casamimoacid (Fluka, CAS: 65072 - 00 - 6) is dissolved in 100 mL of distilled water and filter sterilized.

[0057] After solution 3 is cooled to 50 °C, add solution 2 and mix it with 30 mL of filter-sterilized 10% (W / V) casamimoacid (Fluka, CAS: 65072 - 00 - 6), then add solution 1, and stir slowly (to avoid generating bubbles), and pour the plate.

[0058] As shown in the Figure 3 attachment, strain SY39 has an obvious yellow halo, indicating that SY39 has the ability to produce siderophores.

[0059] Example 5. Determination of the ability of SY39 to produce extracellular polysaccharides and improve soil structure

[0060] Ability to produce exopolysaccharide: The strain SY39 was inoculated into a 2 mL centrifuge tube containing 800 μL of LB liquid medium and cultured at 28 °C with continuous shaking at a speed of 160 rmp / min for 12 hours to obtain a seed solution. The fermented seed solution was inoculated into a sterile test tube containing 5 mL of polysaccharide fermentation medium (20.0 g of glucose, 20.0 g of peptone, 0.05 g of MgSO4, 2.0 g of Na2HPO4·12H2O, 1.0 g of NaH2PO4·2H2O, pH 7.0, volume made up to 1 L, autoclaved at 121 °C for 30 min) at a concentration of 1% (50 μL) and cultured for 48 h. The cells were removed by centrifugation at 10000 rmp / min for 10 min. Then, 2 mL of the supernatant was added to 3 volumes of 95% ethanol and shaken vigorously until a flocculent precipitate formed. After overnight precipitation at 4 °C, it was centrifuged at 10000 rmp / min for 10 min and the supernatant was removed. The obtained precipitate was the crude polysaccharide, which was dissolved by adding a quantitative amount of deionized water. The polysaccharide content was determined using the phenol-sulfuric acid method. The results showed that the exopolysaccharide yield of SY39 was 0.31 ± 0.01 mg / mL, indicating its high ability to produce exopolysaccharide.

[0061] Effect on soil aggregates: The strain SY39 was inoculated into a 150 mL Erlenmeyer flask containing 50 mL of LB liquid medium and the medium was continuously shaken at 28 °C at a speed of 160 rmp / min until the cell concentration reached OD 600 = 1 to obtain a seed solution. The fermented seed solution was inoculated into a 150 mL Erlenmeyer flask containing 50 mL of polysaccharide fermentation medium at an inoculation amount of 5% (2.5 mL) and cultured for 48 h. Then, the cell concentration was set to 1×10 8 (cfu / mL) for standby. The amount of the bacterial solution added was 5% of the soil amount, with 5 replicates set each time. 150 g of soil was used for each treatment. The soil culture container was a wide-mouth soil culture bottle with a volume of 200 mL and was cultured in the dark at 28 °C with a humidity of 70% in the incubator. Before the formal start of the experiment, the soil samples were completely mixed and the saturated water content was detected. The pre-experiment weighing method was used to obtain the amount of water required for the experimental soil samples to reach a field water holding capacity of 80%.

[0062] Spread the sample soil evenly on a kraft paper that is not easily wetted by water, and spray it evenly. After evenly sprinkling the bacterial liquid on each layer on average, gently shake it well, and try to avoid disturbing the soil during this period until all the bacterial liquid is added. Put 150 g of soil sample into a wide-mouth soil culture bottle with a height of 10 cm, a diameter of 12 cm, and a capacity of 200 mL. Place the soil culture bottle in a constant-temperature culture room for dark culture for 30 d, set the temperature at 28 °C, and the humidity of the incubator at 70%. Collect the soil sample on the 30th day of culture, and then use the collected undisturbed soil sample to measure soil aggregates.

[0063] Determination was carried out by the wet-sieving method. Accurately weigh 30 g of air-dried undisturbed soil and evenly spread it on a 2-mm sieve. Place sieves with apertures of 250 μm, 53 μm, and 20 μm in sequence below. Put the nested sieves into an iron bucket equipped with an aggregate analyzer, add deionized water to just cover the soil sample in the sieve and soak for 2 min; then vibrate vertically at a frequency of 40 times / min for 2 min. After sieving, take out the nested sieves from the water, and at the same time, wash the soil particles in each sieve into an aluminum box that has been pre-dried and weighed with a wash bottle. Put the aluminum box into an oven and dry it to a constant weight at 105 °C. Take it out, wait for it to cool naturally, and immediately weigh it with an analytical balance and record the weight to calculate the content of aggregates of each particle size. Aggregates can be classified into the following categories according to particle size: larger macro-aggregates (2 mm - 8 mm), smaller macro-aggregates (0.25 mm - 2 mm), micro-aggregates (0.053 mm - 0.25 mm), and silt plus clay fractions (<0.053 mm). For the aggregates of each level separated by the wet-sieving method (rapid wetting method), the mean weight diameter (MWD) was used as an evaluation index for soil structure stability. The results showed that the MWD of the SY39 treatment increased by 32.52% compared with the clear water control group and increased by 8.67% compared with the culture medium control group. There were significant differences in MWD between the SY39 treatment group, the culture medium control group, and the clear water control group (p < 0.05), as shown in Figure 4 shown.

[0064] Effect on soil water-stable structure: The addition amount of the bacterial liquid was 5% of the soil amount. The soil culture container was a self-made PVC soil column, and it was cultured at 28 °C with the humidity of the incubator at 70%. Before the experiment, first take the soil sample, mix it completely, and then detect the saturated water content. The pre-experiment weighing method was used to obtain the water amount required for the experimental soil sample to reach the field water holding capacity of 80%.

[0065] Lay the sample soil flat on a kraft paper that is not easily wetted by water, and spray it evenly. After evenly sprinkling the bacterial liquid on each layer on average, gently shake it, and try to avoid disturbing the soil during this period until all the bacterial liquid is added. Put 250 g of soil sample into a PVC tube, and note that the lower part of the PVC tube should be wrapped and sealed with plastic wrap first. Place the soil column in a constant temperature incubator for 30 d, and cultivate it at a temperature of 28 °C and a humidity of 70%. Collect the soil sample on the 30th day of cultivation. Before detecting the relevant indicators, first slowly remove the plastic wrap at the lower part of the soil column without damaging the soil structure, then place a qualitative filter paper of appropriate size, fix the filter paper with tape, and evenly punch 5 holes in the tape with a toothpick; fix the soil column vertically on the shelf with tape to ensure that the leaching solution falls vertically. Then leach the soil sample, and detect indicators such as the infiltration rate during this period.

[0066] Detection of the infiltration rate of the leaching solution: Regularly record the time when the leaching solution finishes infiltrating, including three indicators: the duration of the first drop of leaching solution falling from the soil column into the Erlenmeyer flask, the duration when all the leaching solution is added to the soil column, and the time when the soil surface in the soil column emerges from the water, and successively judge the change situation of the cultivated soil structure. The results show that the falling time of the first drop of leaching solution in the treatment group of strain SY39 decreased by 43.81% compared with the clear water control group and decreased by 12.35% compared with the culture medium control group (attached Figure 5 ). The total falling duration of 500 mL of leaching solution in the soil by strain SY39 decreased by 6.82% and 26.37% respectively compared with the culture medium control and the clear water control (attached Figure 6 ).

[0067] Example 6, Determination of IAA production ability

[0068] First, activate the strain in LB liquid medium, and inoculate the activated strain into DF (5.00 g of peptone, 1.50 g of yeast extract, 1.50 g of beef extract, 5.00 g of NaCl, 1000 mL of distilled water, pH 7.0, autoclaved at 121 °C for 30 min) and DF+ (add 0.50 g / L of tryptophan to the DF medium) media at a ratio of 1%. Shake culture at 28 °C for 7 days. After 7 days, take out the fermentation broth, centrifuge it at 12000 rpm for 5 min, and measure the content of IAA in the fermentation broth by the Salkowkin colorimetric method. The results show that the amount of IAA produced by SY39 in the DF medium is 2.08 ± 0.22 mg / L, and the amount of IAA produced by SY39 in the DF+ medium is 7.84 ± 0.15 mg / L.

[0069] Furthermore, the IAA synthesized by the strain was confirmed by HPLC analysis: after culturing the strain for 7 days, it was centrifuged at 12,000 rpm for 5 min, 30 mL of the supernatant was taken, and extracted 3 times with twice the volume of ethyl acetate in a constant temperature oscillator. The extracted solutions were combined, distilled under reduced pressure, then dissolved and made up to volume with 5 mL of methanol, and filtered through a 0.22 μm filter membrane.

[0070] Detection instrument: waters2998 high performance liquid chromatography; chromatographic column: AgilertZorbaxSB-C18 250 mm×4.6mm, 5 µm; mobile phase, methanol: acetonitrile: 0.6% acetic acid aqueous solution (50:5:45, v / v / v); injection volume: 20 µL; flow rate 0.8 mL / min; column temperature: room temperature; detection wavelength: 255 nm.

[0071] Detection results: The amount of IAA produced by SY39 in DF medium was 2.28±0.11 mg / L, and the amount of IAA produced in DF+ medium was 7.95±0.19 mg / L. Slightly higher than the detection results by colorimetry.

[0072] Example 7. Determination of growth promotion ability

[0073] Single colonies of strain SY39 were picked from the plate and activated in LB liquid medium, cultured at 28 °C and 150 rpm for 72 h. Corn seeds soaked with SY39 bacterial suspension (10 8 cfu / mL) were planted in a hydroponic container (10 cm×10 cm×9.7 cm), and seeds soaked with sterile normal saline were used as a control. There were 12 corn seedlings in each group, and 3 replicates were set for each treatment. All treatments were placed in a light incubator (25 °C, 16 h light and 18 °C, 8 h darkness) for culture, and the light intensity was 20000 Lx. According to the method for detecting the growth promotion effect of plant rhizosphere growth-promoting bacteria disclosed in CN101984067A, the growth promotion ability of SY39 fermentation broth on corn seedlings was detected, and the stem height, root length, stem fresh weight, root fresh weight, stem dry weight, and root dry weight of the corn seedlings were measured. The experimental results showed that strain SY39 had a good growth promotion effect (attached Figure 7 ) and could increase the stem height of corn seedlings by 32.91%, root length by 37.42%, stem fresh weight by 50.30%, root fresh weight by 94.24%, stem dry weight by 19.21%, and root dry weight by 35.94% (Table 4).

[0074] Table 4 Growth promotion effect of strain SY39 on corn seedlings

[0075] Note: Different letters indicate significant differences at the P<0.05 level Example 8. Determination of antibacterial ability

[0076] The confrontation culture method was used to detect the antifungal ability of SY39, and its inhibitory effects on Phytophthora parasitica var. nicotianae, Monilinia laxa , Botrytis cinerea , Rhizoctonia cerealis were determined respectively. First, the pathogenic bacteria were activated on PDA plates, and pathogen discs were made with a 5 mm punch and inoculated in the center of PDA plates. SY39 was point-inoculated with a toothpick at a distance of 2.5 cm from the disc and cultured at 25 °C for 4 - 7 days. The control diameter and inhibitory diameter were measured respectively, and the inhibition rate was calculated.

[0077]

[0078] The results are shown in Figure 8 and Table 5. The inhibition rates of strain SY39 against

[0079] var. nicotianae,

[0080] were 35.68%, 47.92%, 60.64% and 57.12% respectively, indicating that strain SY39 has great application potential in the control of plant diseases. Pseudomonas syringae pv.lachrymans were determined by the double-layer culture method: Strain SY39 was inoculated in the center of an LB plate and cultured at 28 °C for 24 h; the plate was placed upside down in a fume hood, and 5 mL of chloroform was placed in the lid of each plate and left overnight in the fume hood; the concentration of the pathogenic bacteria was adjusted to 10 8 cfu / mL with sterile normal saline. 100 μL was taken and added to 5 mL of water agar cooled to 50 °C after sterilization and poured onto the plate fumigated with chloroform, and cultured in an incubator at 28 °C for 12 h. The diameter of the inhibition zone was measured by the cross method. The results showed that strain SY39 had a good inhibitory effect on Figure 9 pv.lachrymans, and the diameter of the inhibition zone was 31.05 ± 0.92 mm (

[0081] Example 9. Pot experiment

[0082] A single colony of strain SY39 was picked into an LB liquid medium and cultured with shaking at 28 °C and 200 r / min for 24 h, then centrifuged at 6000 r / min for 15 min to collect the cells, and the cells were washed 3 times with sterile water. The concentration of the bacterial suspension was adjusted to 1×10 8CFU / mL. Uniform-sized corn seeds were sown in flower pots, with 3 seeds per pot, and 15 replicates were set for each treatment; 900.0 g of soil was filled in each pot. For the treatment group, 50.0 mL of the bacterial suspension of strain SY39 was poured, and the same amount of sterile water was poured for the blank control. The treatments were CK (low-phosphorus soil + calcium phytate, without applying bacteria) and SY39 (low-phosphorus soil + calcium phytate + bacteria). The indexes of the potted soil are shown in Table 4. During the potted period, other management measures for each group were the same. Water was appropriately irrigated. After 30 days, each index of each corn seedling was measured, and the measurement method was the same as above. The results showed that strain SY39 had a good effect on dissolving phosphorus and promoting growth (see Figure 10 , Table 6). Compared with the control group, it could increase the stem height of corn seedlings by 13.36%, the fresh weight of stems by 31.95%, the fresh weight of roots by 50.08%, the dry weight of stems by 28.67%, and the dry weight of roots by 51.00%.

[0083] Table 6 Physical and chemical properties of the soil

[0084] Table 7 Growth-promoting effect of strain SY39 on potted corn seedlings

[0085] Although the embodiments of the present invention have been shown and described, it will be understood by those of ordinary skill in the art that various changes, modifications, substitutions, and variations can be made to these embodiments without departing from the principles and spirit of the present invention. The scope of the present invention is defined by the appended claims and their equivalents.

Claims

1. A strain of Priestia megaterium ( Priestia megaterium ) SY39, with the deposit number of CCTCC NO: M20251011.

2. A biological bacterium agent, characterized in that: The microbial agent contains the SY39 strain of Priestia megaterium of claim 1 or the metabolite of said strain.

3. A biological agent prepared from the microbial agent of claim 2.

4. Any one of the following applications of the biological agent of claim 3: 1) For dissolving organic phosphorus and / or inorganic phosphorus; 2) For producing siderophores; 3) For producing exopolysaccharides; 4) For improving soil aggregate structure; 5) For producing indoleacetic acid; 6) For promoting plant growth; 7) For inhibiting pathogenic fungi; 8) For inhibiting pathogenic bacteria.

5. The application according to claim 4, wherein The pathogenic fungi include: Phytophthora parasitica var. nicotianae, Monilinia fructicola, Botrytis cinerea, Rhizoctonia solani.

6. The application according to claim 4, wherein, The pathogenic bacteria include Pseudomonas syringae pv. lachrymans.

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