Bacillus megaterium and application of biological fertilizer thereof in rice planting

Abstract

The invention discloses application of bacillus megaterium and a biological fertilizer thereof in rice planting, and relates to the technical field of microorganisms. The bacillus megaterium is preserved in the China General Microbiological Culture Collection Center on July 10, 2025, and the preservation number of the bacillus megaterium is CGMCC (China General Microbiological Culture Collection Center) NO.35179. The bacillus megaterium has the advantages that the bacillus megaterium is preserved in the China General Microbiological Culture Collection Center on July 10, 2025; the strain is a plant growth-promoting bacterium and has a good rice growth-promoting effect, and when the prepared biological fertilizer is applied to the rice planting process, compared with a control group, the tiller number of rice is increased by 64.44%, the plant height is increased by 2.77%, and the leaf chlorophyll content is increased by 8.38%. The bacillus megatherium provided by the invention and the biological fertilizer prepared by strain fermentation have the advantages of greenness, safety, high efficiency and the like, and have a better application prospect in the aspect of agricultural production.

Description

TECHNICAL FIELD

[0001] The present application relates to the field of microbial technology, and relates to a bacillus megaterium, a bio-fertilizer and application of the bacillus megaterium in rice planting. BACKGROUND

[0002] Rice is the staple food of more than half of the world's population, and its stable high yield is crucial to food security. Traditional rice farming relies heavily on chemical fertilizers, especially nitrogen fertilizers, to maintain high yields. However, long-term overuse of chemical fertilizers not only leads to ecological degradation problems such as soil compaction, acidification, and organic matter decline, but also causes serious non-point source pollution, such as water eutrophication. At the same time, the low utilization rate of chemical fertilizers (such as nitrogen fertilizer utilization rate is usually only 30%-40%) also means huge economic investment and resource waste. Therefore, developing environmentally friendly, resource-efficient new fertilizer technologies to achieve sustainable agricultural development has become a global consensus.

[0003] Under this background, microbial fertilizers are increasingly valued for their green, environmentally friendly, soil improvement, and improved fertilizer utilization. The core functional component of microbial fertilizers is live microorganisms, which help crops absorb nutrients, resist diseases, and promote growth through their life activities. Among them, bacillus megaterium, as a common plant growth-promoting rhizobacteria (PGPR), has shown great application potential. Making microorganisms into bio-fertilizers and applying them to rice planting is a promising technology to replace chemical fertilizers. However, the conversion of potential strains in the laboratory into stable effects in the field still faces challenges, mainly due to low in-situ activity, which makes it difficult to play a role and is easily competed by other microorganisms. Currently, there is a lack of systematic application research on bacillus megaterium bio-fertilizers in rice, an important food crop, and the technical system still needs to be further deepened and improved to promote the large-scale and industrialized application of this green technology. SUMMARY

[0004] In view of the lack of plant growth-promoting bacteria strains for rice and low in-situ activity, the present application provides a bacillus megaterium and a microbial inoculant, which can be applied to rice planting. The strain has a significant growth-promoting effect on rice planting.

[0005] The technical solutions adopted by the present application are as follows:

[0006] In a first aspect, the present application provides a bacillus megaterium, which was deposited with the China General Microbiological Culture Collection Center on July 10, 2025, and has a deposit number of CGMCC NO. 35179.

[0007] As a preferred embodiment of the first aspect, the nucleotide sequence of the 16S rRNA gene is shown in SEQ ID NO. 1.

[0008] In a second aspect, the present application provides a plant growth promoting microbial biofertilizer, wherein the effective component is the Bacillus megaterium according to the first aspect.

[0009] As a preferred embodiment of the second aspect, the minimum effective concentration of the Bacillus megaterium in the plant growth promoting microbial biofertilizer is 10 11 CFU / g.

[0010] In a third aspect, the present application provides a preparation method of a plant growth promoting microbial biofertilizer, wherein the Bacillus megaterium according to the first aspect is expanded by fermentation and then added to straw materials as a microbial inoculum, and then composted to obtain a plant growth promoting microbial biofertilizer product.

[0011] As a preferred embodiment of the third aspect, the Bacillus megaterium is expanded by fermentation, and then the obtained liquid microbial inoculum is mixed with a microbial inoculum carrier to prepare a solid microbial inoculum, which is then added to straw materials.

[0012] In a fourth aspect, the present application provides a plant growth promoting microbial biofertilizer prepared by the preparation method according to the third aspect.

[0013] In a fifth aspect, the present application provides an application of the Bacillus megaterium according to the first aspect in promoting rice growth.

[0014] In a sixth aspect, the present application provides an application of the plant growth promoting microbial biofertilizer according to the second aspect or the fourth aspect in promoting rice growth.

[0015] As a preferred embodiment of the sixth aspect, the plant growth promoting microbial biofertilizer is directly applied to rice soil, and the Bacillus megaterium in the biofertilizer is used to promote rice growth.

[0016] Compared with the prior art, the present application has the following beneficial effects:

[0017] The present application provides a Bacillus megaterium and a biofertilizer thereof, and the biofertilizer containing the Bacillus megaterium is applied to the process of rice planting. Compared with the control group, the number of tillers of rice is increased by 64.44%, the plant height is increased by 2.77%, and the chlorophyll content of leaves is increased by 8.38%. Therefore, the Bacillus megaterium and the biofertilizer thereof of the present application can be applied to rice planting to promote rice growth, and have the advantages of being green, safe, and efficient, and have a good application prospect in agricultural production. BRIEF DESCRIPTION OF DRAWINGS

[0018] Figure 1The colony morphology scanning electron microscope photograph of Bacillus megaterium in the embodiment of the present application.

[0019] Figure 2 The indole acetic acid production qualitative result of Bacillus megaterium in the embodiment of the present application, wherein A), B) and C) are IAA positive control group, IAA negative control group and Bacillus megaterium treatment group respectively.

[0020] Figure 3 The rice appearance comparison result of the biological fertilizer treatment group and the control group in the embodiment of the present application.

[0021] Figure 4 The rice tiller number, plant height and chlorophyll result comparison of the biological fertilizer treatment group and the control group in the embodiment of the present application, wherein the characterization parameters are chlorophyll plant SPAD value, tiller number and plant height respectively.

[0022] Figure 5 The in-situ activity characteristics of Bacillus megaterium in the biological fertilizer in the soil, the lower spectrum is the Raman spectrum of Bacillus megaterium in the soil without heavy water incubation, as a negative control; the upper spectrum is the Raman spectrum of Bacillus megaterium in the soil after 7 days of heavy water incubation, as a treatment group, wherein the activity of Bacillus megaterium is evaluated by its ability to intake heavy water in the soil, i.e. 2040-2300 cm -1 The C-D peak indicates the activity of Bacillus megaterium in the soil.

[0023] Biological preservation

[0024] Bacillus megaterium was preserved in China General Microbiological Culture Collection Center (CGMCC) on July 10, 2025, the preservation address is No. 1, Beichen West Road, Chaoyang District, Beijing, China, the postcode is 100101, and the preservation number is CGMCC NO. 35179. DETAILED DESCRIPTION

[0025] The present application will be further described in conjunction with the accompanying drawings, which are an explanation rather than a limitation of the present application. The methods used in the following examples are conventional methods, and the reagents used are commercially available products unless otherwise specified.

[0026] The application provides a bacillus megaterium which is preserved in the China General Microbiological Culture Collection Center on July 10, 2025, and has a preservation number of CGMCC NO. 35179, and a nucleotide sequence of a 16S rRNA gene of the bacillus megaterium is shown as SEQ ID NO. 1. The strain is a plant growth promoting bacteria, can be made into a biofertilizer, has a good rice growth promoting effect, and the biofertilizer prepared by the strain is applied to a rice planting process, and compared with a control group, the rice tillering number, plant height and leaf chlorophyll content are all improved.

[0027] The above-mentioned bacillus megaterium and the specific preparation process and application effect of the biofertilizer thereof are demonstrated through the following examples.

[0028] Example 1

[0029] In the embodiment, the separation and identification process of the bacillus megaterium strain involved in the application is mainly demonstrated, and the bacillus megaterium strain is as follows:

[0030] (1) In-situ high-activity single cell separation culture

[0031] In-situ high-activity single cell identification: under sterile conditions, 10 g of rice root soil is taken, 1 mL of heavy water is added and incubated for 7 days, 90 mL of water is added to the soil and mixed gently, impurities and suspended solids are removed by a cell screen with a pore size of 40 μm, and cells with C-D peaks are sorted by a PRECES CS-R300 Raman single cell sorter. The sorted cells are added to a sterile 96-well plate, and the 96-well plate is placed in a microplate shaker, incubated at 30°C and 180 r / min. The medium is selected as LB medium, the pH is adjusted to 7.0, and the medium is sterilized at 121°C for 20 min before use. The OD value at 600 nm of each microhole in the microhole plate is measured by a full-wavelength enzyme marker instrument to obtain the growth of the bacteria in each microhole.

[0032] It should be noted that the formula of the LB medium used in the embodiment is as follows: 10.0 g of tryptone, 5.0 g of yeast extract and 10.0 g of sodium chloride are dissolved in 1000 mL of distilled water, the pH is adjusted to 7, and the medium is sterilized at high temperature and high pressure. The formula of the LB medium used in the subsequent examples is the same, and is not described in detail.

[0033] (2) Separation and purification

[0034] The OD 600The highest and >0.5 bacterial suspension, 50 μL bacterial suspension was used for separation and purification. The bacterial suspension was spread on LB plate and incubated at 30 ℃ to grow colonies; single colonies on the plate were picked and purified by multiple "Z" line on LB plate, and no impurities were observed. The single colony was picked to obtain the purified strain.

[0035] (3) Strain identification

[0036] The physiological and biochemical characteristics of the bacteria and the 16S rRNA sequence were combined to identify the strain. The colony PCR primers were as follows: the bacterial universal primers 27F (5'-AGAGTTTGATCCTGGCTCAG-3') and 1492R (5'-GGTTACCTTGTTACGACTT-3') were used for colony PCR, and the strain species were preliminarily identified by BLAST sequence comparison of NCBI. According to the colony sequencing results of Bacillus megaterium, the nucleotide sequence of its 16S rRNA gene is shown in SEQ ID NO. 1.

[0037] In addition, the strain morphology of the above-mentioned Bacillus megaterium was characterized by scanning electron microscopy, and the specific method was as follows: the bacterial suspension was centrifuged (8000 g, 10 min), the bacterial body was collected, and was poured into 2.5% (v / v) glutaraldehyde solution, and was fixed at 4 ℃ overnight. After fixation, the bacterial body was rinsed with phosphate buffer (0.1 M, pH=7.0) for 15 min, repeated three times, and then fixed with 1% (v / v) osmium acid solution for 1-2 h. After fixation, the osmium acid waste liquid was removed by centrifugation, and the bacterial body was rinsed with phosphate buffer (0.1 M, pH=7.0) for 15 min, repeated three times. Sequentially dehydrated with gradient concentrations of 30%, 50%, 70%, 80%, 90%, and 95% ethanol for 15 min each time, and then treated with 100% ethanol for 20 min. After critical point drying and film plating, the morphology of the bacteria was observed by scanning electron microscopy. The morphological characteristics of the Bacillus megaterium under electron microscopy are shown in SEQ ID NO. 1. Figure 1

[0038] Finally, the strain identified in this embodiment is Bacillus megaterium, which was preserved in the China General Microbiological Culture Collection Center on July 10, 2025, with the preservation number of CGMCC NO. 35179.

[0039] In addition, the IAA production ability of the above-mentioned Bacillus megaterium was determined by Salkowski colorimetric method, and the presence of IAA was qualitatively and quantitatively determined by observing the color change. The specific method of the Bacillus megaterium treatment group was as follows:​

[0040] a. Standard curve drawing: a series of different concentrations of solutions were prepared with IAA standard, respectively added Salkowski reagent, after a period of time at room temperature, the absorbance was measured at 530 nm wavelength, the standard curve was drawn with IAA concentration as the abscissa and the absorbance as the ordinate.

[0041] b. Sample preparation: the strain to be tested was inoculated into the appropriate culture medium, cultured for a certain time, and then a certain amount of bacterial liquid was centrifuged, and the supernatant was collected as the sample to be tested.

[0042] c. Determination: a certain amount of supernatant was taken, Salkowski reagent was added, and the absorbance at 530 nm was measured after reaction. The content of IAA in the sample was calculated according to the standard curve.

[0043] At the same time, in addition to the Bacillus megaterium treatment group, IAA positive control group and IAA negative control group were also set up, and the respective IAA production qualitative results were as shown in Table 1. Figure 2 The results showed that the IAA production of the Bacillus megaterium strain was 19.45 μg / mL.

[0044] Example 2

[0045] In this embodiment, a plant growth promoting microbial biofertilizer made of Bacillus megaterium is provided, and its specific preparation method and application effect are shown below.

[0046] Based on the Bacillus megaterium (Bacillus megaterium) isolated in Example 1 (i.e. the aforementioned Bacillus megaterium with preservation number CGMCC NO. 35179), the fermentation experiment was gradually completed through the volume gradient of 5 L-50 L-500 L (i.e. first fermented to 5 L, then fermented to 50 L, and finally fermented to 500 L), and the microbial amount of Bacillus megaterium was gradually expanded. The OD600 of the liquid inoculum after 48 h of fermentation was 15. The parameters during the fermentation process were controlled as follows: sodium chloride 10 g / L, peptone 10 g / L, and yeast extract 5 g / L. The temperature was 32℃, the pressure was 0.05 MPa, the stirring speed was 200 rpm, and the air volume was 30 m 3 / h. In order to facilitate the use of the liquid inoculum after fermentation expansion, corn cob was selected as the inoculum carrier, and the liquid inoculum and the inoculum carrier were mixed and loaded according to the weight ratio of 1:8 to prepare the solid inoculum.

[0047] Then based on the above solid inoculant preparation of bio-fertilizer, the preparation method is as follows: taking rice straw as the main raw material, compounding sawdust and furfural residue as fermentation accessories, so that the C / N ratio of the final mixed fermentation material is 25:1, and the solid inoculant prepared above is added, the moisture content of the material is adjusted to 60%, the material is mechanically turned over, the frequency is 2 times per day, the aeration amount is 25m 3 / h for composting and maturation, and a plant growth promoting microbial bio-fertilizer product is obtained. After detection, the bacterial amount in the final composted bio-fertilizer is 2.6x10 11 CFU / g.

[0048] It should be noted that the fermentation method used in the preparation of the microbial inoculant in this embodiment is relatively mature, and the fermentation culture is mainly for amplifying the biomass of the microorganism to meet the subsequent experimental requirements. However, other fermentation methods in the prior art can also be used to prepare Bacillus megaterium microbial inoculants in the present application. In the final prepared bio-fertilizer, the effective component is Bacillus megaterium, but the viable bacterial content can be different, and the microbial inoculant can also contain other additional components produced during the fermentation process, which will not inhibit the growth and metabolism of Bacillus megaterium.

[0049] Example 3

[0050] In this embodiment, based on the plant growth promoting microbial bio-fertilizer prepared in Example 2, a method for application in rice planting is further provided, and the activity characteristics of Bacillus megaterium in actual soil and the promoting effect on rice growth are demonstrated through rice plot planting experiments.

[0051] In this embodiment, the bio-fertilizer obtained by final composting in Example 2 is used in rice soil, and the application time is the base fertilizer period of rice planting, and the addition amount of bio-fertilizer is 300 kg / acre. This experimental group is recorded as the bio-fertilizer treatment group. At the same time, a control group treatment group without applying bio-fertilizer is set in this embodiment.

[0052] The results of the rice plot experiment of the final bio-fertilizer treatment group and the control group treatment group are shown in Figure 3 and Figure 4 It can be found from Figure 3 and Figure 4 that the rice tillering number of the bio-fertilizer treatment group increased by 64.44%, the plant height increased by 2.77%, and the leaf chlorophyll content increased by 8.38% compared with the control group treatment group.

[0053] In addition, Figure 5The in-situ activity characteristics of Bacillus megaterium in the bio-fertilizer in the bio-fertilizer treatment group in the rice soil are shown. Here, the Bacillus megaterium incubated without heavy water is used as a negative control, and the sample 7 days after the Bacillus megaterium is added to the soil is used as a treatment group. The results show that the Bacillus megaterium still has a C-D peak, indicating that it has metabolic activity, wherein the Raman shift 2800-3100 cm -1 represents a C-H peak, and the Raman shift 2040-2300 cm -1 represents a C-D peak.

[0054] In addition, it should be noted that the soil to which the bio-fertilizer is applied in the present application is not limited to rice field soil, but can also be other farmland soil.

[0055] The above-described embodiments are only a preferred scheme of the present application, and are not intended to limit the present application. Those skilled in the art can make various changes and modifications without departing from the spirit and scope of the present application. Therefore, any technical scheme obtained by equivalent replacement or equivalent transformation falls within the protection scope of the present application.

Claims

1. A Bacillus megaterium strain, characterized in that, The Bacillus megaterium was preserved in China General Microbiological Culture Collection Center on July 10, 2025, and the preservation number is CGMCC NO. 35179.

2. The Bacillus megaterium according to claim 1, characterized in that, The nucleotide sequence of the 16S rRNA gene thereof is shown as SEQ ID NO.

1.

3. A plant growth promoting microbial biofertilizer characterized in that, The effective component is the Bacillus megaterium according to claim 1.

4. The plant growth promoting microbial biofertilizer according to claim 3, characterized in that, The minimum effective concentration of Bacillus megaterium in the plant growth promoting microbial biofertilizer is 10 11 CFU / g.

5. A method for preparing a plant growth promoting microbial biofertilizer, characterized by, The Bacillus megaterium according to claim 1 is expanded by fermentation and then added into straw materials as a microbial inoculum, and then composting and humification are performed to obtain a plant growth promoting microbial biofertilizer product.

6. The production method according to claim 5, wherein The liquid microbial inoculum obtained by expanding the Bacillus megaterium by fermentation is mixed with a microbial inoculum carrier to prepare a solid microbial inoculum, and then added into straw materials.

7. A plant growth promoting microbial biofertilizer prepared by the preparation method according to claim 5.

8. Application of the Bacillus megaterium according to claim 1 in promoting rice growth.

9. Application of the plant growth promoting microbial biofertilizer according to claim 3 or 7 in promoting rice growth.

10. Use according to claim 9, wherein The plant growth promoting microbial biofertilizer is directly applied to rice soil, and the Bacillus megaterium in the biofertilizer is used to promote rice growth.

Images