Priesteria gigantea, its biological agent and application

Abstract

The present invention relates to Priesteria gigantea, its biological agent and application, and belongs to the field of microbial technology. Priestia megaterium The SY39 strain of the present invention is CCTCC No. M20251011. Priesteria gigantea SY39 can improve soil aggregate structure, dissolve organic and inorganic phosphorus, and promote growth by producing exopolysaccharides, indoleacetic acid, and siderophores. It also produces antimicrobial active substances to inhibit the growth of pathogens.

Description

Technical Field

[0001] The invention relates to Priesteria gigantea, a composite biological agent containing the bacterium and application thereof, and belongs to the technical field of microorganisms. Background Art

[0002] Phosphorus is an essential element for plant growth and development, playing a crucial role in intracellular energy metabolism, carbon metabolism, nitrogen metabolism, and material transport. The amount of inorganic phosphorus available for plant absorption and utilization in the soil is very low, generally accounting for only 2% to 3% of the total phosphorus content. When soluble phosphorus fertilizer is applied to the soil, some of it is absorbed and utilized by plants, while the remaining amount reacts with soil components and migrates out of the soil phase, becoming insoluble phosphates that are difficult for plants to utilize.

[0003] Agricultural activities are the primary source of phosphorus pollution. Phosphorus from fertilizers and livestock manure enters water bodies through surface runoff and soil leaching. Research shows that agricultural sources account for 67.62% of total phosphorus emissions in the Yangtze River Basin, with livestock and poultry farming and crop farming contributing 34.44% and 29.18%, respectively. Urban surface runoff, industrial wastewater, and domestic sewage are also significant sources of pollution. For example, in the Taihu Lake Basin, agricultural non-point source sources contribute over 80% of total phosphorus emissions.

[0004] Phosphate-solubilizing microorganisms effectively decompose soil phosphorus, increasing available phosphorus content. They also enhance the efficiency of biological nitrogen fixation, improve other trace elements, and produce substances that promote plant growth, such as auxin (IAA), gibberellins (GA), cytokinins (CK), siderophores, and HCN. Given the current situation of available phosphorus deficiency and abundant potential phosphorus sources in the soil, how to utilize phosphate-solubilizing microorganisms to activate insoluble phosphorus in the soil and improve the utilization rate of chemical phosphorus fertilizers is a pressing issue in agricultural production in my country and a hot topic of research in soil, fertilizer, and plant nutrition both domestically and internationally.

[0005] Bacillus has endogenous spores that form dormant spores in extreme environments. It can withstand harsh conditions such as high temperature, acidity, alkali and drought, and is easy to store and formulate. It has wide application potential in promoting plant growth, inhibiting pathogens, and decomposing environmental pollutants. Currently, the main Bacillus species widely used in phosphorus pollution control are: Bacillus subtilis ( Bacillus subtilis ), Bacillus velezinoffii ( Bacillus velezensis )、Bacillus amyloliquefaciens( Bacliius amyloliquefaciens ), Bacillus megaterium ( Bacillus megaterium ) and Bacillus laterosporus ( Brevibacillus laterosporus). Wen Shaofu et al. reportedly used pot experiments to investigate the effects of Acinetobacter calcoaceticus on lead fixation and translocation in maize rhizosphere soil, as well as the microbial community structure and interrelationships. The results showed that the bacterial culture and fermentation broth of Acinetobacter calcoaceticus significantly increased phosphatase (ACP) activity in maize rhizosphere soil. Song Gen et al. screened for active strains targeting the sweet potato nematode Ditylenchus spp. The resulting strain, Bacillus velezensis HM-3, not only demonstrated potent contact killing activity against the sweet potato nematode but also possessed phosphorus and potassium solubilization and nitrogen fixation capabilities. It converted insoluble or poorly soluble phosphorus and potassium in the soil into forms that could be absorbed by crops and fixed nitrogen from the air for crop use. Its potassium solubilization capacity was particularly prominent. However, there have been no reports of Priesteria gigantea exhibiting the combined properties of salt and alkali tolerance, biocontrol, phosphorus solubilization, potassium solubilization, and soil structure improvement, which is of great significance. Summary of the Invention

[0006] The present invention aims to provide Priesteria gigantea, a biological agent thereof and its application. The Priesteria gigantea SY39 of the present invention can improve soil aggregate structure, and has growth-promoting functions such as dissolving organic and inorganic phosphorus, producing exopolysaccharides, indoleacetic acid and siderogen, and can also produce antibacterial active substances to inhibit the growth of pathogens.

[0007] To achieve the above object, the present invention provides the following technical solutions:

[0008] A Priesterol bacterium ( Priestia megaterium SY39 strain, isolated from Dali in 2024, was deposited with the China Center for Type Culture Collection under the accession number CCTCC NO: M 20251011. The deposit date was May 12, 2025. The address of the China Center for Type Culture Collection is Wuhan University, Wuhan, China.

[0009] The present invention also provides a biological bacterial agent, which comprises the Priesteria gigantea SY39 strain or a metabolite of the strain.

[0010] The present invention also provides a biological preparation prepared from the above-mentioned bacterial agent.

[0011] The present invention also provides any of the following applications of the above-mentioned bacterial agents and biological preparations:

[0012] 1) Used to dissolve organic phosphorus and / or inorganic phosphorus;

[0013] 2) Used to produce siderophores;

[0014] 3) Used to produce extracellular polysaccharides;

[0015] 4) Used to improve soil aggregate structure;

[0016] 5) Used to produce indoleacetic acid;

[0017] 6) Used to promote plant growth;

[0018] 7) Used to inhibit pathogenic fungi;

[0019] 8) Used to inhibit pathogenic bacteria.

[0020] Pathogenic fungi include: tobacco black shank fungus ( Phytophthora parasitica var. nicotianae), peach brown rot fungus ( Monilinia laxa )、Botrytis cinerea( Botrytis cinerea ), wheat sheath blight pathogen ( Rhizoctonia cerealis ).

[0021] Pathogenic bacteria include Xanthomonas cucumeris ( Pseudomonas syringae pv. lachrymans).

[0022] The beneficial effects produced by adopting the above technical solution are:

[0023] The Priesteria gigantea SY39 of the present invention can improve the soil aggregate structure, and has growth-promoting functions such as dissolving organic and inorganic phosphorus, producing extracellular polysaccharides, indoleacetic acid and iron carriers, and can also produce antibacterial active substances to inhibit the growth of pathogenic bacteria.

[0024] The Priesteria gigantea SY39 of the present invention can still grow normally without being inhibited under the stress of 8% salt concentration, can tolerate a certain salt concentration, and still has the ability to solubilize phosphorus under high salt stress.

[0025] The average equivalent diameter of soil aggregates treated with Priesteria gigantea 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 500 mL of soil elution by strain SY39 was reduced by 6.82% and 26.37% compared with the culture medium control and the clear water control, respectively.

[0026] The SY39 strain could produce 2.28±0.11 mg / L of IAA in DF medium and 7.95±0.19 mg / L in DF+ medium.

[0027] 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 stem fresh weight by 50.30%, the root fresh weight by 94.24%, the stem dry weight by 19.21%, and the root dry weight by 35.94% (according to the method for detecting the growth-promoting effect of plant rhizosphere growth-promoting bacteria disclosed in CN101984067A).

[0028] The strain SY39 of the present invention has an inhibition rate of 35.68% against tobacco black shank pathogen, 47.92% against peach brown rot pathogen, 60.64% against gray mold pathogen, and 57.12% against wheat sheath blight pathogen, and has great application potential in the prevention and control of plant diseases.

[0029] The strain SY39 of the present invention has a good inhibitory effect on cucumber angular leaf spot pathogen, and the inhibition zone diameter is 31.05±0.92 mm.

[0030] The strain SY39 of the present invention has a good phosphorus tolerance and growth-promoting effect. Compared with the control group, it can increase the stem height of corn seedlings by 13.36%, the stem fresh weight by 31.95%, the root fresh weight by 50.08%, the stem dry weight by 28.67%, and the root dry weight by 51.00% (pot experiment). BRIEF DESCRIPTION OF THE DRAWINGS

[0031] Figure 1 SY39 phosphate dissolution circle.

[0032] Figure 2 Growth of strain SY39 under different salt concentrations.

[0033] Figure 3 Siderophore production ability of strain SY39.

[0034] Figure 4 Effect of strain SY39 on soil mean equivalent diameter (MWD).

[0035] Figure 5 Effect of strain SY39 on the falling time of the first drop of soil eluent.

[0036] Figure 6 Effect of strain SY39 on the total falling time of 500 mL soil eluent.

[0037] Figure 7 Growth-promoting effect of strain SY39 on corn seedlings.

[0038] Figure 8 Inhibitory effect of SY39 on pathogenic fungi.

[0039] Figure 9 Inhibitory effect of SY39 on Angular leaf spot pathogen of cucumber.

[0040] Figure 10 Growth-promoting effect of strain SY39 on potted corn seedlings. DETAILED DESCRIPTION

[0041] The following will clearly and completely describe the technical solutions in the embodiments of the present invention in conjunction with the accompanying drawings. Obviously, the described embodiments are only part of the embodiments of the present invention, not all of the embodiments. All other embodiments obtained by ordinary technicians in this field based on the embodiments of the present invention without making any creative efforts shall fall within the scope of protection of the present invention.

[0042] Example 1, Priesteria gigantea ( Priestia megaterium ) Isolation of SY39 strain

[0043] Soil was collected from Dali in 2024. 10 g of soil was added to a flask containing 100 ml of sterile saline. After standing for 20 min, it was shaken at 28°C and 200 rpm for 30 min. 1 ml of soil was added to 9 ml of sterile saline and diluted 10 2 , 10 3 , 10 4 Take 100 µl of each soil suspension and spread it evenly on phosphate growth medium plates (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 and tricalcium phosphate 10.0 g / L for inorganic phosphorus). Colonies with phosphate-soluble rings were selected and further streaked to purify. Single colonies were then transferred to LB slants and stored at 4°C for later use. The strain SY39 that produced a phosphate solubility zone was isolated. The diameter of the organic phosphorus solubility zone (D) reached 17.53±0.28 mm, and the ratio of the diameter of the phosphate solubility zone (D) to the diameter of the colony (d) (D / d) was 1.45. The diameter of the inorganic phosphorus solubility zone (D) reached 9.06±0.35 mm, and the ratio of the diameter of the phosphate solubility zone (D) to the diameter of the colony (d) (D / d) was 1.38 (see attached). Figure 1 ).

[0044] After confirming the strain's phosphate-solubilizing ability, its phosphate-solubilizing capacity was quantitatively determined. The available phosphorus content in the fermentation supernatant was determined using the molybdenum antimony colorimetric method. After activation in LB liquid medium, the strain was inoculated onto liquid phosphate-solubilizing medium and incubated with shaking at 28°C for 7 days. The culture was centrifuged at 12,000 rpm at 4°C for 5 minutes, and the supernatant was retained. The soluble phosphorus content in the supernatant was determined using the molybdenum antimony colorimetric method (Zhang Xiangsheng, 2008). To prepare a standard curve, weigh 0.2195 g of KH2PO4, oven-dried at 105°C for 2 hours, and dissolve it in 400 ml of water. Add 5 ml of concentrated H2SO4 (analytical grade), transfer the solution to a 1 L volumetric flask, and make up to volume with water. This stock solution was stored long-term. Accurately pipette 25.0 ml of the phosphorus standard stock solution with ρ(P) = 50 mg / L and dilute it 10-fold with water to obtain a standard working solution with ρ(P) = 5 mg / L. This solution was prepared for immediate use. Accurately pipette 0, 0.50, 1.00, 2.00, 4.00, 6.00, and 8.00 ml of the phosphorus standard working solution (ρ(P) = 5 mg / L) (the corresponding phosphorus concentrations in this standard series of solutions are 0, 0.05, 0.10, 0.20, 0.40, 0.60, and 0.80 mg / LP, respectively) into 50 ml volumetric flasks. Add water to approximately 30 ml. Adjust the pH and color development of the solution (as above). Measure the absorbance (882 nm) of the series of solutions, draw a calibration curve, and determine the phosphorus concentration in each solution based on the drawn standard curve.

[0045] The results showed that the standard curve equation was y=2.8109x-0.1526 (R 2 =0.9996), with good linearity within the assay concentration range. Calculated inorganic phosphorus content of strain SY39 was 115.03±1.12 mg / L, and organic phosphorus content was 46.65±0.87 mg / L.

[0046] Example 2, identification of strain SY39

[0047] Strain SY39 was identified by molecular biology, with its 16S rDNA and gyrA sequences amplified and multi-locus sequence typing used for identification. The specific identification results are as follows:

[0048] First, the 16S rDNA gene sequence of the strain was amplified using primers 27F: 5′-AGAGTTTGATCCTGGTCAGAACGAACGCT-3′ (SEQ ID NO. 1) and 1492R: 5′-TACGGCTACCTTGTTACGACTTCACCCC-3′ (SEQ ID NO. 2). A 25µl reaction system consisted of 12.5µl of 2× TaqMix, 1µl of each primer, 1.0% primer, and 9.5% ddH2O. PCR reaction conditions were: 94°C for 5 min, denaturation at 94°C for 30 s, annealing at 56°C for 30 s, and extension at 72°C for 1 min. Finally, 30 cycles of full extension at 72°C for 5 min were performed.

[0049] The gyrA sequence was amplified using primers 42F: 5'-CAGTCAGGAAATGCGTACGTCCTT-3' (SEQ ID NO. 3) and 1066R: 5'-CAAGGTAATGCTCCAGGCATTGCT-3' (SEQ ID NO. 4). A 25 µl reaction system contained 12.5 µl of 2× TaqMix, 1 µl of each primer, 1.0% primer, and 9.5% ddH2O. PCR reaction conditions were: 94°C for 5 min, denaturation at 94°C for 30 s, annealing at 61.7°C for 30 s, and extension at 72°C for 80 s. Finally, 35 cycles of full extension at 72°C for 10 min were performed.

[0050] The amplified product was analyzed by agarose gel electrophoresis to detect the target band and then 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. Based on the above analysis results and combined with the strain's morphological characteristics, SY39 was identified as Priesteria gigantea.

[0051] Table 1 Partial 16S rDNA sequence of strain SY39

[0052]

[0053] Table 2 gyrA gene sequence of strain SY39

[0054]

[0055] Example 3: Determination of salt tolerance of strain SY39

[0056] First, the strain SY39 was activated in LB liquid medium. The activated strain was inoculated into LB liquid medium containing 2%, 5%, 8%, 10%, and 13% NaCl (w / v) at a 1% inoculum size and cultured at 28°C and 150 rpm for 48 h. The OD 600 The blank culture medium was used as negative control (CK1), and LB culture medium was used as positive control (CK2). Each treatment was repeated 3 times. Figure 2 As shown, the negative control OD 600 After the values ​​were adjusted, strain SY39 could still grow normally in a medium with an 8% salt concentration without inhibition, but was inhibited in a 10% salt solution and could hardly grow in a 13% salt solution, indicating that strain SY39 can tolerate a certain salt concentration.

[0057] Table 3 OD of strain SY39 at different salt concentrations 600 value

[0058]

[0059] Note: Different letters indicate significant differences at the P<0.05 level.

[0060] To further test the phosphate solubilization ability of strain SY39 under high salt stress, strain SY39 was streaked onto LB medium and then inoculated into liquid LB medium with an 8% salt concentration. This was then inoculated into liquid phosphate solubilization medium containing an 8% salt concentration. The culture was shaken at 28°C for 7 days. The culture was centrifuged at 12,000 rpm at 4°C for 5 minutes, and the supernatant was retained. The soluble phosphorus content in the supernatant was determined using the molybdenum antimony colorimetric method (Zhang Xiangsheng, 2008), using the same method as above. The results showed that under salt stress, the inorganic phosphorus content of strain SY39 was 95.01 ± 0.67 mg / L, and the organic phosphorus content was 26.14 ± 1.07 mg / L, indicating that strain SY39 still had phosphate solubilization ability.

[0061] Example 4: Determination of siderophore production ability

[0062] After activation on LB medium, strain SY39 was inoculated onto CAS plates and cultured at 30°C for 7 days. The plates were then observed for the presence of yellow halos.

[0063] The CAS plate preparation method is as follows:

[0064] Solution 1: CAS / HDTMA solution.

[0065] 1) CAS solution: Dissolve 60.5 mg of CAS (chrome azurol) in 50 mL of water; 2) Iron solution: Dissolve 1 mM FeCl₃·6H₂O in 10 mM HCl, pH 2.0; 3) HDTMA solution: Dissolve 72.9 mg of cetyltrimethylammonium bromide in 40 mL of water. Mix solution 1) with 10 mL of solution 2) and add to solution 3) with stirring. Stir thoroughly. Autoclave the resulting blue-black liquid at 121°C for 30 minutes. This is the CAS / HDTMA solution.

[0066] Solution 2: Salts / Buffer solution.

[0067] 1) Salts (10 × 100 mL): KH2PO4 0.3 g, NaCl 0.5 g, NH4Cl 1.0 g; 2) Pipes: Dissolve 30.24 g of pipes in salts, adjust the pH to 6.8 with 50% (w / v) KOH, add 15.0 g agar, and dilute to 800 mL with distilled water. Autoclave at 121°C for 30 min and cool to 50°C.

[0068] Solution 3: glucose 2.00 g, mannitol 2.00 g, MgSO4·7H2O 493 mg, CaCl2 11 mg, H3BO3 1.4 mg, ZnSO4·7H2O 1.2 mg, MnSO4·2H2O 1.17 mg, Na2Mo4·2H2O 1 mg, CuSO4 40 μg, dilute to 750 mL with distilled water, and autoclave at 121°C for 30 min.

[0069] Solution 4: Dissolve 10.00 g of casamimoacid (Fluka, CAS: 65072-00-6) in 100 mL of distilled water and filter sterilize.

[0070] After solution 3 is cooled to 50°C, solution 2 and 30 mL of filter-sterilized 10% (W / V) casamimoacid (Fluka, CAS: 65072-00-6) are added and mixed. Then solution 1 is added, stirred slowly (to avoid bubbles), and plated.

[0071] As attached Figure 3 As shown, strain SY39 has an obvious yellow halo, indicating that SY39 has the ability to produce siderophore.

[0072] Example 5: Determination of the ability of SY39 to produce exopolysaccharides and improve soil structure

[0073] Exopolysaccharide production capacity: strain SY39 was inoculated into a 2 mL centrifuge tube containing 800 μL LB liquid medium and cultured at 28°C with continuous shaking at 160 rpm for 12 h to obtain seed solution. The fermented seed liquid was inoculated at a concentration of 1% (50 μL) into a sterile test tube containing 5 mL of polysaccharide fermentation medium (20.0 g glucose, 20.0 g peptone, 0.05 g MgSO4, 2.0 g Na2HPO4·12H2O, 1.0 g NaH2PO4·2H2O, pH 7.0, fixed to 1 L, and sterilized by moist heat at 121°C for 30 min). The culture was incubated for 48 h, and the cells were removed by centrifugation at 10,000 rpm for 10 min. 2 mL of the supernatant was then added to three volumes of 95% ethanol and shaken vigorously until a flocculent precipitate formed. After overnight precipitation at 4°C, the precipitate was centrifuged at 10,000 rpm for 10 min, and the supernatant was removed. The resulting precipitate, representing the crude polysaccharide, was dissolved by adding a predetermined amount of deionized water. Polysaccharide content was determined using the sulfuric acid-phenol method. The results showed that the extracellular polysaccharide production of SY39 was 0.31±0.01 mg / mL, which showed the ability to produce high extracellular polysaccharides.

[0074] Effect on soil aggregates: strain SY39 was inoculated into a 150 mL Erlenmeyer flask containing 50 mL LB liquid medium, and the medium was continuously shaken at 160 rpm at 28°C until the bacterial concentration reached OD 600 =1, and the seed solution was obtained. The fermented seed solution was inoculated into a 150 mL Erlenmeyer flask containing 50 mL of polysaccharide fermentation medium at a 5% inoculum volume (2.5 mL) and cultured for 48 h. The bacterial solution concentration was then set to 1×10 8 The bacterial solution was added at a rate of 5% of the soil volume. Five replicates were set up for each treatment, with 150 g of soil used for each treatment. The soil was cultured in 200 mL wide-mouthed flasks at 28°C in the dark, with an incubator humidity of 70%. Before the experiment began, soil samples were thoroughly mixed and tested for saturated water content. Pre-experimental weighing was used to determine the amount of water required for the experimental soil sample to reach a field water holding capacity of 80%.

[0075] Spread the sample soil flat on a piece of water-resistant kraft paper and evenly spray it with the bacterial solution. After each layer is evenly sprinkled with the bacterial solution, gently shake the soil to mix thoroughly. Minimize disturbance during the process until all the bacterial solution has been added. Place 150 g of soil sample into a 200 mL wide-mouthed soil culture bottle (10 cm high, 12 cm in diameter). Incubate the bottle in a constant temperature incubator in the dark for 30 days at 28°C and 70% humidity. Collect a soil sample on the 30th day of incubation. The undisturbed soil sample will be used to determine soil aggregates.

[0076] The wet sieving method was used. 30 g of air-dried, undisturbed soil was accurately weighed and evenly spread on a 2 mm sieve, followed by 250 μm, 53 μm, and 20 μm sieves. The sieves were placed in the iron bucket provided with the agglomerometer. Deionized water was added to just cover the soil sample and allowed to soak for 2 minutes. The sieves were then vertically shaken at a frequency of 40 times / min for 2 minutes. After sieving, the sieves were removed from the water and the soil particles from each sieve were rinsed with a washing bottle into a pre-dried and weighed aluminum box. The box was then placed in an oven at 105°C to a constant weight. After cooling, the box was immediately weighed using an analytical balance and the weight recorded. The aggregate content of each particle size was calculated. Aggregates can be divided into the following categories based on particle size: larger aggregates (2mm-8mm), smaller aggregates (0.25mm-2mm), microaggregates (0.053mm-0.25mm), and silt and clay fractions (<0.053mm). The soil aggregates of various levels separated by wet sieving (rapid wetting method) were evaluated using the mean equivalent diameter (MWD) as an indicator of soil structural stability. The results showed that the MWD of the SY39 treatment increased by 32.52% compared to the water control group and increased by 8.67% compared to the culture medium control group. There was a significant difference in MWD between the SY39 treatment group and the culture medium and water controls (p < 0.05). Figure 4 shown.

[0077] Effects on Soil Water Stability: The bacterial solution was added at a rate of 5% of the soil volume. The soil culture containers used self-made PVC soil columns. The soil was incubated at 28°C and 70% humidity. Before the experiment, soil samples were thoroughly mixed and tested for saturation water content. Pre-experimental weighing was used to determine the amount of water required to achieve a field water holding capacity of 80%.

[0078] Spread the sample soil flat on a piece of water-resistant kraft paper and evenly spray it with the bacterial solution. After evenly applying the bacterial solution to each layer, gently shake the soil to mix thoroughly, minimizing disturbance until the bacterial solution has been completely applied. Place a 250 g soil sample into a PVC tube, making sure to wrap the bottom of the tube tightly with plastic wrap. Place the soil column in a constant temperature incubator at 28°C and 70% humidity for 30 days. Collect a soil sample on the 30th day of incubation. Before testing relevant parameters, first slowly remove the plastic wrap from under the soil column without disrupting the soil structure. Place a qualitative filter paper of appropriate size on the column, secure it with tape, and use a toothpick to evenly punch five holes in the tape. Secure the soil column vertically to a rack using tape to ensure the eluent falls vertically. Leach the soil sample, monitoring infiltration rate and other parameters.

[0079] Eluent infiltration rate detection: Regularly record the time it takes for the eluent to infiltrate, including the time it takes for the first drop of eluent to fall from the soil column to the triangular flask, the time it takes for all the eluent to be added to the soil column, and the time it takes for the soil surface in the soil column to emerge from the water. These three indicators are used to determine the changes in the culture soil structure. The results showed that the time it took for the first drop of eluent to fall in the SY39 treatment group was 43.81% shorter than that in the water control group and 12.35% shorter than that in the culture medium control group (see Appendix). Figure 5 The total time for the 500mL soil eluent to fall by strain SY39 was reduced by 6.82% and 26.37% compared with the culture medium control and water control, respectively (Appendix Figure 6 ).

[0080] Example 6: IAA production capacity determination

[0081] The strain was activated in LB liquid medium. A 1% inoculum of the activated strain was then inoculated into DF medium (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, sterilized by autoclaving at 121°C for 30 min) and DF+ medium (DF medium supplemented with 0.50 g / L tryptophan). The culture was shaken at 28°C for 7 days. After 7 days, the fermentation broth was removed and centrifuged at 12,000 rpm for 5 minutes. The IAA content in the broth was determined by the Salkowkin colorimetric method. The results showed that SY39 produced 2.08 ± 0.22 mg / L of IAA in DF medium and 7.84 ± 0.15 mg / L in DF+ medium.

[0082] Furthermore, HPLC analysis was used to confirm the IAA synthesized by the strain: after culturing the strain for 7 days, the strain was centrifuged at 12,000 rpm for 5 minutes, 30 mL of the supernatant was taken, and it was fully extracted three times with twice the volume of ethyl acetate in a constant temperature oscillator. The extracts were combined and distilled under reduced pressure, then dissolved with 5 mL of methanol, fixed to volume, and filtered through a 0.22 μm filter membrane.

[0083] Detection instrument: Waters 2998 high-performance liquid chromatography; chromatographic column: Agilert Zorbax SB-C18 250 mm × 4.6 mm, 5 µm; mobile phase, methanol: acetonitrile: 0.6% glacial 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.

[0084] Test results: SY39 produced 2.28±0.11 mg / L of IAA in DF medium and 7.95±0.19 mg / L in DF+ medium, which were slightly higher than the results of colorimetric detection.

[0085] Example 7: Determination of growth-promoting ability

[0086] Single colonies of strain SY39 were picked from the plate and activated in LB liquid medium. The culture was shaken at 28°C and 150 rpm for 72 h. The SY39 bacterial suspension (10 8 cfu / mL) were planted in hydroponic containers (10 cm×10 cm×9.7 cm), and seeds soaked in sterile physiological saline were used as controls. Each group had 12 corn seedlings, and each treatment had 3 replicates. All treatments were cultured in a light incubator (25°C, 16 h light and 18°C, 8 h dark) with a light intensity of 20,000 Lx. According to the method for detecting the growth-promoting effect of plant rhizosphere growth-promoting bacteria disclosed in CN101984067A, the growth-promoting ability of SY39 fermentation liquid 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 the strain SY39 had a good growth-promoting effect (see Appendix). Figure 7 ), which can 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).

[0087] Table 4 Growth-promoting effect of strain SY39 on corn seedlings

[0088]

[0089] Note: Different letters indicate significant differences at the P<0.05 level

[0090] Example 8, determination of antibacterial ability

[0091] The antifungal activity of SY39 was tested by confrontation culture method, and its antifungal activity against tobacco black shank pathogen ( Phytophthora parasitica var. nicotianae), peach brown rot fungus ( Monilinia laxa )、Botrytis cinerea( Botrytis cinerea ), wheat sheath blight pathogen ( Rhizoctonia cerealis ), the pathogens were first activated on a PDA plate, a pathogen cake was made with a 5 mm hole puncher, and inoculated in the center of the PDA plate. SY39 was spotted with a toothpick 2.5 cm away from the cake, and cultured at 25°C for 4-7 days. The control diameter and inhibition diameter were measured, and the inhibition rate was calculated.

[0092]

[0093] The results are as attached Figure 8 As shown in Table 5, the inhibition rate of strain SY39 against tobacco black shank pathogen was 35.68%, peach brown rot pathogen was 47.92%, gray mold pathogen was 60.64%, and wheat sheath blight pathogen was 57.12%, indicating that strain SY39 has great application potential in the prevention and control of plant diseases.

[0094] Table 5 Inhibition rate of SY39 against pathogenic fungi

[0095]

[0096] The double-layer culture method was used to determine the strain SY39's ability to resist cucumber leaf spot pathogen ( Pseudomonas syringae pv. lachrymans) were inoculated in the center of an LB plate and cultured at 28°C for 24 h. The plates were placed upside down in a fume hood, and 5 mL of chloroform was placed in the lid of each plate. The plates were then placed in a fume hood overnight. The concentration of the pathogen was adjusted to 10 with sterile saline. 8 cfu / mL, 100 μL was added to 5 mL of sterilized water agar cooled to 50°C, poured onto a chloroform-fumigated plate, and incubated in a 28°C incubator for 12 h. The diameter of the inhibition zone was determined using the cross-hatch method. The results showed that strain SY39 had a good inhibitory effect on cucumber angular leaf spot pathogen, with an inhibition zone diameter of 31.05 ± 0.92 mm (see attached). Figure 9 ).

[0097] Example 9, potted plant experiment

[0098] A single colony of strain SY39 was picked and placed in LB liquid medium. After shaking culture at 28°C and 200 rpm for 24 h, the bacteria were collected by centrifugation at 6000 rpm for 15 min. The bacteria were washed three times with sterile water and the concentration of the bacterial suspension was adjusted to 1×10 8 CFU / mL. Corn seeds of uniform size were sown in pots, with 3 seeds per pot and 15 replicates per treatment. Each pot was filled with 900.0 g of soil. 50.0 mL of bacterial suspension of strain SY39 was added to the treatment group, while the blank control was poured with the same amount of sterile water. The treatments were CK (low-phosphorus soil + calcium phytate, no bacteria) and SY39 (low-phosphorus soil + calcium phytate + bacteria). Potted soil parameters are shown in Table 4. Other management measures were consistent across the treatment groups during potting, with appropriate irrigation. After 30 days, various parameters of each corn seedling were measured using the same method as above. The results showed that strain SY39 has a good phosphorus tolerance and growth-promoting effect (see Appendix). Figure 10 , Table 6), compared with the control group, the stem height of corn seedlings increased by 13.36%, the stem fresh weight increased by 31.95%, the root fresh weight increased by 50.08%, the stem dry weight increased by 28.67%, and the root dry weight increased by 51.00%.

[0099] Table 6 Soil physical and chemical properties

[0100]

[0101] Table 7 Growth-promoting effect of strain SY39 on corn potted seedlings

[0102]

[0103] While embodiments of the present invention have been shown and described, it will be appreciated by those skilled in the art that various changes, modifications, substitutions, and variations may be made to these embodiments without departing from the principles and spirit of the invention, and that the scope of the invention is defined by the appended claims and their equivalents.

Claims

1. A Priesterol bacterium ( Priestia megaterium ) SY39 strain, whose deposit number is CCTCC NO:M20251011.

2. A biological agent, characterized in that: The bacterial agent comprises the Priesteria gigantea SY39 strain according to claim 1.

3. A biological preparation prepared from the bacterial agent according to claim 2.

4. Any of the following uses of the biological agent according to claim 3: 1) Used to dissolve organic phosphorus and / or inorganic phosphorus; 2) Used to produce siderophores; 3) Used to produce extracellular polysaccharides; 4) Used to improve soil aggregate structure; 5) Used to produce indoleacetic acid; 6) Used to promote plant growth; 7) Used to inhibit tobacco black shank pathogen Phytophthora parasitica var. nicotianae, peach brown rot pathogen Monilinia laxa , Botrytis cinerea Botrytis cinerea Rhizoctonia solani Rhizoctonia cerealis ; 8) Used to inhibit cucumber angular leaf spot fungus Pseudomonas syringae pv.lachrymans.

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