P.megatherium and application of P.megatherium in plant growth promotion and N2O emission reduction

Abstract

The invention belongs to the field of microbial agents and plant growth promotion, and particularly relates to a P.megatherium YB15 strain and application thereof in plant growth promotion and N2O emission reduction. The preservation number of the P.megaterium YB15 is CCTCC (China Center for Type Culture Collection) NO: M 20251693, the P.megaterium YB15 is preserved in the China Center for Type Culture Collection on July 24, 2025, and the P.megaterium YB15 is classified and named as Pristia megaterium YB15. According to the present invention, the screened Priestia megaterium YB15 can significantly promote the plant growth and increase the yield through a variety of mechanisms such as secretion of plant growth regulating substances, dissolution of phosphorus, potassium and other nutrients, improvement of the soil environment and the like, and can reduce the dependence on chemical fertilizers and pesticides. Compared with a traditional chemical yield increasing mode, the method has the advantages that the pollution of agricultural production to the environment can be reduced, the soil health can be improved, and the agricultural sustainable development is promoted.

Description

TECHNICAL FIELD

[0001] The application belongs to the field of microbial agents and plant growth promotion, and particularly relates to a strain of Pristinamycetin YB15 and application of the strain in plant growth promotion and N2O emission reduction. BACKGROUND

[0002] Corn is one of the important food crops in China and plays a significant role in the agricultural economy. It is not only an important guarantee for food security but also widely used in feed, industrial raw materials, and bioenergy fields, making it a multi-functional crop. In recent years, with the rapid development of the livestock industry and the food processing industry, the demand for corn has continued to increase, further highlighting its importance. However, the use efficiency of corn planting resources and environmental problems have also attracted attention. Excessive use of fertilizers, irrigation, and other methods may lead to soil degradation and water waste. Therefore, plant growth-promoting bacteria (PGPB) as a green and environmentally friendly biological yield-increasing method have gradually gained attention.

[0003] Although PGPB has significant advantages in green agriculture, the complex natural soil environment, fluctuating conditions such as pH, temperature, and humidity, can directly affect the survival, colonization, and metabolic activity of PGPB. Moreover, the competition and antagonism of the original microbial community in the soil can inhibit the growth and reproduction of PGPB, leading to their inability to stably exert their growth-promoting functions. At the same time, there is a lack of adaptability to different growth stages of crops, and they cannot provide stable growth support throughout the entire process. SUMMARY

[0004] To solve the problems in the prior art, the application aims to provide a strain of Pristinamycetin YB15, which has a high ability to synthesize IAA, can increase the height, root length, root fresh weight, and root dry weight of corn, and has good application prospects.

[0005] The application further provides application of the above-mentioned Pristinamycetin YB15 in promoting crop growth and N2O emission reduction.

[0006] To achieve the above-mentioned purpose, the application adopts the following technical solutions: The application provides a strain of Pristinamycetin YB15. Priestia megaterium The preservation number of the Pristinamycetin YB15 is CCTCC NO: M 20251693, which was preserved in the China Center for Type Culture Collection on July 24, 2025, and is classified and named as YB15. Priestia megaterium

[0007] ​Preferably, the P. megaspora YB15 can grow in a culture medium with a salt content of 0-5%, and the culture temperature is 28-37℃.

[0008] The present application also provides a microbial inoculant containing the P. megaspora YB15, characterized in that the cell content of the P. megaspora YB15 is ≥1×10 9 CFU / mL.

[0009] The present application also provides a preparation method of the microbial inoculant, comprising the following steps: culturing the P. megaspora YB15 in an LB liquid culture medium to the logarithmic phase, collecting bacterial cells, washing with sterile water, resuspending in sterile water, and making the cell number ≥1×10 9 CFU / mL.

[0010] The P. megaspora YB15 provided by the present application has the effects of promoting crop growth and reducing N2O emission.

[0011] Most preferably, the crop is corn.

[0012] The strain YB15 is isolated from a red soil sample in Yunnan, and is prepared into a suspension and heated at 80℃ for 5 min, and then coated on an LB solid culture medium. The white colonies of the screened strain YB15 are formed after the LB solid culture medium is cultured at 37℃ for 1-2 d, and the colonies are round, smooth, convex, and have neat edges. The strain YB15 is further sequenced by 16S rRNA gene and a phylogenetic tree is constructed to determine its evolutionary status. It is found that the strain YB15 has the highest similarity (99.65%) with B8W22, and forms an independent branch with the strain B8W22 in the phylogenetic tree. Therefore, the strain YB15 is named as P. megaspora YB15. Priestia megaterium Priestia megaterium B8W22. Priestia megaterium

[0013] In the second aspect, the present application provides a culture condition of the P. megaspora YB15.

[0014] Preferably, the culture medium used is an LB culture medium, the salt content is 3%, and the culture temperature is 37℃.

[0015] In the third aspect, the present application provides a microbial inoculant containing the P. megaspora YB15. The microbial inoculant is a liquid inoculant, and the P. megaspora YB15 is cultured in an LB liquid culture medium to the logarithmic phase, bacterial cells are collected, washed with sterile water for 3 times, resuspended in sterile water, and made the cell number ≥1×10 9 CFU / mL.

[0016] ​​In a fourth aspect, the present application provides a plant growth promoting bacterial agent capable of promoting the growth of corn.

[0017] In a fifth aspect, the present application provides the use of the Pasteuria penetrans YB15 agent in promoting the plant height, root length, root fresh weight and root dry weight of corn.

[0018] In a sixth aspect, the present application provides the use of the Pasteuria penetrans YB15 and the agent in synthesizing IAA.

[0019] In a seventh aspect, the present application provides the use of the Pasteuria penetrans YB15 and the agent in relieving the N2O emission of corn plants.

[0020] The Pasteuria penetrans (P. penetrans) YB15 screened by the present application can significantly promote the growth of plants and increase the yield by secreting plant growth regulators, dissolving nutrients such as phosphorus and potassium, and improving the soil environment, while reducing the dependence on chemical fertilizers and pesticides. Priestia megaterium Compared with the traditional chemical yield-increasing method, the Pasteuria penetrans can not only reduce the pollution of agricultural production to the environment, but also improve the soil health and promote the sustainable development of agriculture.

[0021] The present application has the beneficial effects that the Pasteuria penetrans (P. penetrans) YB15 is isolated for the first time, the strain YB15 has the ability to secrete IAA, and can significantly promote the plant height, root length, root fresh weight and root dry weight of corn. Priestia megaterium Therefore, it has good application prospect.

[0022] Preservation information Preservation time: July 24, 2025; Preservation unit: China Center for Type Culture Collection; Preservation number: CCTCC NO: M 20251693; Address of the preservation unit: No. 299, Bayi Road, Wuchang District, Wuhan, Hubei Province; Classification and naming: Priestia megaterium YB15. Brief description of drawings

[0023] Figure 1 The figure is the colony of the strain YB15; Figure 2 The figure is the phylogenetic tree of the strain YB15 constructed based on the 16S rRNA gene sequence; Figure 3 The figure is the growth level of the strain YB15 under different salinity; Figure 4 The figure is the growth level of the strain YB15 under different temperature; Figure 5 The effect of strain YB15 on maize plant height; Figure 6 The effect of strain YB15 on maize root length; Figure 7 The effect of strain YB15 on the fresh weight of maize roots; Figure 8 The effect of strain YB15 on the dry weight of maize roots; Figure 9 A comparison of N2O emissions from maize plants by strain YB15; Figure 10 The graph shows the phosphate solubility of strain YB15. Figure 11 Fluorescence microscopy observation results of strain YB15 colonizing maize tissue. Detailed Implementation

[0024] The specific embodiments of the present invention will be further described in detail below with reference to the accompanying drawings and examples. The following examples are used to illustrate the present invention, but are not intended to limit the scope of the present invention. The parameters, proportions, etc. of the examples can be selected according to local conditions without substantially affecting the results. Unless otherwise specified, the methods described in the examples are all conventional methods, and the reagents used are all conventional reagents or reagents prepared in a conventional manner.

[0025] Example 1: Isolation, screening, and identification of *Priscilla megaterium* YB15 (1) Take 1g of Yunnan red soil sample (25°18′N, 103°21′E) and resuspend it in 10 mL of sterile physiological saline. Shake well to obtain a soil suspension. After standing, take the supernatant and perform 10 1000- ... -4 ~10 -6 Serial dilutions were performed, and the different dilutions were heated at 80°C for 5 min and then spread onto LB solid medium. The medium was incubated at 37°C for 48 h. Single colonies were picked and streaked onto LB solid medium for purification. This process was repeated 2-3 times to obtain pure strains. The pure strains were then stored in glycerol at -80°C.

[0026] The composition of the LB solid culture medium is as follows: take 0.5g of yeast extact, 1g of tryptone, 1g of NaCl, 1.5g of agar, and distilled water to a final volume of 100mL.

[0027] (2) Genomic DNA was extracted from the selected strains as a template for PCR amplification of their 16S rRNA gene. The primers used were: 27F (F: 5′-AGAGTTGATCCTGGCTCAG-3′) and 1492R (R: 5′-GGTTACCTTGTTACGACT-3′). The PCR amplification conditions were: 94℃ pre-denaturation for 2 min, 94℃ denaturation for 30 s, 54℃ annealing for 30 s, 72℃ extension for 45 s, 30 cycles, and 72℃ extension for 2 min. After purification, the PCR product was sequenced. The sequencing results were compared with BLAST data from NCBI, and species or genera with high similarity to the sequencing results were selected. A phylogenetic tree was constructed using MEGA 5 biological software, such as... Figure 2 As shown. The results showed that this strain had the highest homology with *Priscilla megaterium*, and was named... Priestia megaterium YB15.

[0028] (3) The selected strains were inoculated onto LB agar plates and incubated at 37°C for 24 h until colonies formed. Colony morphology was then observed. After 24 h of growth on LB agar, the strains were observed to form white, round colonies with smooth, raised surfaces and regular edges. Figure 1 As shown.

[0029] Strain strain YB15 was inoculated into LB medium with different salt contents (0, 1, 2, 3, 4, 5, 10%). After incubation for a period of time, the OD values ​​of strain YB15 in different treatment groups were measured. 600 The absorbance value at nm was measured. Additionally, after inoculating strain YB15 into LB liquid medium and culturing at 4℃, 28℃, and 37℃ for 12 h, the absorbance of strain YB15 in different treatment groups was measured. 600 Absorbance value at nm.

[0030] like Figure 3 As shown in the figure: This figure illustrates the inhibitory effect of different salt concentrations on the growth of YB15. When the salt concentration is below 10%, there is no significant difference in OD600 values ​​among the treatment groups. However, the OD600 value of the 10% salt concentration treatment group is significantly lower than that of the other treatment groups, indicating that it has a significant inhibitory effect on bacteria. Therefore, YB15 exhibits a certain degree of salt tolerance.

[0031] like Figure 4 As shown in the figure: This figure illustrates the effect of different temperatures on the growth of YB15. As can be seen from the figure, the OD600 value at 37℃ is significantly higher than that of the other two groups, indicating that the optimal growth temperature for YB15 is 37℃.

[0032] Example 2: Growth-promoting ability of *Priscilla megaterium* YB15 The YB15 strain, stored at -80 ℃, was activated. 200 μL of the bacterial culture was inoculated into 10 mL LB broth in a test tube and then incubated overnight in a shaker at 37 ℃. The next day, after the culture became turbid, it was transferred to an Erlenmeyer flask with an inoculation volume of 1%, and incubated in a shaker at 37 ℃. The fermentation broth of strain YB15 was centrifuged at 12000 rpm for 10 min, and the cells were collected, washed three times with sterile water, and resuspended in sterile water until the cell count reached 1 × 10⁻⁶. 9 CFU / mL.

[0033] The above-mentioned bacterial suspension was inoculated into the corn on day 1 and day 6 of growth, respectively. The negative control group was treated with an equal volume of sterile water as the bacterial suspension. During the experiment, sterile tap water was used daily to keep the soil moist and maintain the water required for growth. The culture period was 12 days. The plant height of the corn was measured during the same period. After the growth was completed, the root length, fresh weight of the roots, and dry weight of the roots were measured.

[0034] The composition of the LB liquid culture medium is as follows: take 0.5g of yeast extact, 1g of tryptone, 1g of NaCl, and distilled water to a final volume of 100mL.

[0035] like Figures 5-8 As shown, it can significantly promote corn plant height, root length, root fresh weight, and root dry weight.

[0036] This strain also exhibits certain resistance to pathogens. Plate confrontation experiments were conducted to investigate the resistance of strain YB15 to Fusarium graminearum (…). F. graminearum ), Fusarium graminearum ( F. pseudograminearum In terms of antagonistic effects, strain YB15 exhibited strong antagonistic ability against all strains. Among them, strain YB15 showed the highest inhibitory effect against *Fusarium graminearum*, with an inhibition rate approaching 70%. Example 3: Determination of IAA production capacity of *Priscilla megaterium* YB15 The fermentation broth of strain YB15 was transferred to TSB medium containing 5 mmol / L tryptophan and cultured at 30°C for 48 h. The fermentation broth was then centrifuged at 10,000 rpm for 5 min, and the supernatant was collected. One mL of the supernatant was taken, and 2 mL of Salkawski chromogenic reagent was added. The mixture was incubated in the dark for 30 min to induce an IAA color reaction. A positive reaction indicating IAA production by the strain resulted in a pink color change of the solution. The IAA yield of the strain was calculated using a standard curve.

[0037] Example 4: The effect of *Priestella megaterium* YB15 in reducing N2O emissions from maize plants. Three treatments were set in the pot experiment, which were inactivated strain, active strain and blank control treatment. 3 mL of bacterial agent (bacterial suspension) was added around the plants for inactivated strain and active strain treatments, 3 mL for each area, and the same amount of sterile water was added for blank control treatment. Periodic greenhouse gas determination was carried out.

[0038] The results are shown in Figure 9 The N2O emission of the blank control group was 0.052 mg-1kg-1d, which was slightly lower than that of the inactivated bacterial agent treatment group, but there was no significant difference between the two. The N2O emission of the active bacterial agent treatment group was significantly lower than that of the other two groups, which showed N2O absorption. It was proved that the selected P. megaspora could significantly reduce the N2O emission of corn plants, and the emission reduction rate was 157.69%.

[0039] Example 5 Determination of the phosphorus solubilizing ability of P. megaspora YB15 The test strain was inoculated on Pikovskaya medium, and the medium without inoculation was used as the control group. Each strain had 3 groups in parallel, and the strain growth and changes around the colonies were observed after 5 days of culture at 30°C. If the strain grew normally on the solid medium and a transparent circle appeared around the colony, as shown in Figure 10 , it indicated that the strain YB15 had the ability of dissolving phosphorus.

[0040] Example 6 Corn root colonization experiment (1) Seed disinfection: corn seeds (Zhongdan 909) were soaked in 75% anhydrous ethanol for 40 s, washed with sterile water for 5 times, then soaked in 0.2% sodium hypochlorite for 5 min, and washed with sterile water for 6 times; (2) The surface-disinfected seeds were evenly spread on sterile filter paper wetted with sterile water; the specific operation was as follows: the filter paper was laid flat in a sterile flat dish (2-3 mL of sterile water was added); the corn was placed on the filter paper with tweezers, and the transparent tape was sealed, and then placed in a 28°C incubator for germination; after about 2-3 days, the buds and roots grew out; then transferred to sterile moistened vermiculite, 25°C, light-dark ratio 16:8 (h / h); generally after one week (grew faster, 3-4 days, 2 leaves); the corn seedlings were transferred to Hoagland medium and cultured to 4 leaves.

[0041] (3) Strain preparation: after activation, the strain was transferred to a conical flask containing LB medium, cultured at 37°C, 200 rpm, to OD≈1.0; the bacterial cells were collected by low-speed centrifugation; after washing with sterile water, resuspended in 10 mL of sterile water. Take 2 mL and add to 200 mL of 1 / 4 Hoagland medium; the corn seedlings were moved to new Hoagland medium, 25°C, 16:8 (h / h), and cultured for 3 days; (4) Treatment: After cutting off the corn roots and washing them three times in sterile water, place the roots in 1 mL of sterile water and shake for 5 min. Then, dilute with PBS and spread 100 μL of the diluted solution on LB solid medium and incubate overnight at 37°C. Calculate the number of bacterial cells colonized per gram of corn root based on the fresh weight of the corn root system.

[0042] like Figure 11 As shown in the fluorescence micrographs, the strain labeled with FITC fluorescent dye was colonized in maize roots. Green fluorescence (strain YB15) is mainly distributed in the intercellular / textured regions of the maize tissue. The linear structures in the bright-field image correspond to the intercellular spaces and vascular structures of the plant tissue; the fluorescence signal intensity is high and the distribution is dense. The fluorescent region overlaps with the structure of the maize tissue (overlay image), indicating that strain YB15 successfully colonized the intercellular spaces / specific structures of the maize tissue. Therefore, this invention demonstrates that the strain provided by this invention can survive, reproduce, and occupy tissue space in maize, possessing colonization ability.

[0043] The above embodiments describe the basic principles, main features, and advantages of the present invention. Those skilled in the art should understand that the present invention is not limited to the above embodiments. The embodiments and descriptions in the specification are only illustrative of the principles of the present invention. Various changes and modifications can be made to the present invention without departing from the scope of the principles of the present invention, and all such changes and modifications fall within the protection scope of the present invention.

Claims

1. A strain of Priestella megaterium ( Priestia megaterium YB15, characterized in that, The *Priestella megaterium* YB15, with accession number CCTCC NO: M 20251693, was deposited at the China Center for Type Culture Collection on July 24, 2025, and classified as follows: Priestia megaterium YB15.

2. The *Priestella giantiflora* YB15 according to claim 1, characterized in that, The *Priestella megaterium* YB15 can grow in a medium with a salt content of 0%-5% at a temperature of 28℃-37℃.

3. A microbial inoculant containing *Priestella giantiflora* YB15 as described in claim 1 or 2, characterized in that, The microbial inoculant contains ≥1×10⁻⁶ cells of *Priscilla megaterium* YB15. 9 CFU / mL.

4. A method for preparing a microbial inoculant as described in claim 3, characterized in that, Includes the following steps: After culturing *Priscilla megaterium* YB15 in LB broth to the logarithmic growth phase, bacterial cells were collected, washed with sterile water, and resuspended in sterile water to ensure a cell count ≥1×10⁻⁶. 9 CFU / mL.

5. The application of *Priestella giantiflora* YB15 as described in claim 1 or 2 in promoting crop growth and reducing N2O emissions.

6. The application according to claim 4, characterized in that, The crop in question is corn.

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