A strain of paenibacillus mojavensis d99 for mineralizing organic phosphorus in soil and its use

Abstract

The application provides a strain of mineralized soil organic phosphorus, Domibacillus indicus D99, and application of the strain in soil organic phosphorus mineralization, growth promotion of phosphorus solubilizing bacteria and plant growth promotion. The application first shows the function and role of Domibacillus indicus in soil organic phosphorus mineralization and plant growth promotion, and has important value for development of phosphorus solubilizing bacteria fertilizer and other microbial fertilizers. The strain of Domibacillus indicus D99 provided by the application has the ability of dissolving soil organic phosphorus, the acid phosphatase activity is 17.32, the alkaline phosphatase activity is 1.3, can promote plant growth, and improves the aboveground biomass by 50-115.

Description

Technical Field

[0001] The present invention belongs to the field of microbial technology. Specifically, the present application provides an Indian cleanroom Bacillus D99 that mineralizes soil organophosphorus and its application. Background Art

[0002] Phosphorus is a macronutrient essential for crop growth and development. However, the utilization efficiency of phosphate fertilizer applied to farmland is low, with the majority accumulating in the soil in various forms, including available phosphorus, insoluble inorganic phosphorus, and organic phosphorus. Specifically, 30%-65% of total phosphorus in soil exists as organic phosphorus, and in soils high in organic matter, this proportion can reach as high as 90%. Therefore, improving the efficiency of crop utilization of soil organic phosphorus is crucial for the sustainable development of modern agriculture.

[0003] Soil microorganisms are the primary drivers of organic phosphorus mineralization, and most phosphatases are produced by microorganisms. Therefore, microorganisms play a crucial role in the conversion of organic phosphorus into available phosphorus for plant uptake and utilization. Microorganisms capable of dissolving phosphorus are called phosphate-solubilizing microorganisms, and bacterial groups within this group are known as phosphate-solubilizing bacteria or phosphate-solubilizing bacteria. Utilizing these phosphate-solubilizing bacteria can effectively improve crop utilization of organic phosphorus in farmland, thereby promoting crop growth and possessing important agricultural applications.

[0004] Studies have shown that phosphate-solubilizing bacteria such as Bacillus spp., Pseudomonas spp., and Massetia spp. can promote plant growth. However, to date, there are no patents or research reports on the Indian cleanroom bacterium Domibacillus indicus improving soil organic phosphorus mineralization, promoting plant growth, or enhancing plant phosphorus uptake. Summary of the Invention

[0005] On the one hand, the present application provides an Indian cleanroom Bacillus (Domibacillus indicus) D99 that mineralizes soil organophosphate. The Indian cleanroom Bacillus D99 is deposited in the Guangdong Provincial Microbial Culture Collection Center with a deposit number of GDMCC No. 65675.

[0006] On the other hand, the present application provides a bacterial agent, which comprises the above-mentioned Indian cleanroom Bacillus D99.

[0007] The bacterial agent can be in various solid or liquid forms, including but not limited to freeze-dried bacterial powder, liquid bacterial solution, glycerol tube, sand tube, etc. Those skilled in the art can prepare these bacterial agents according to conventional preparation methods of microbial preparations. In addition to bacteria, the bacterial agent can also include culture medium, carrier and other components.

[0008] On the other hand, the present application provides the use of the above-mentioned bacteria or bacterial agents in soil organic phosphorus mineralization.

[0009] On the other hand, the present application provides the use of the above-mentioned bacteria or bacterial agents in promoting the growth of phosphate-solubilizing bacteria.

[0010] Furthermore, the phosphate-solubilizing bacteria are Bacillus and / or Pseudomonas.

[0011] On the other hand, the present application provides the use of the above-mentioned bacteria or bacterial agents in promoting plant growth.

[0012] Furthermore, the plant is corn.

[0013] On the other hand, the present application provides the use of the above-mentioned bacteria or bacterial agents in the preparation of microbial fertilizers.

[0014] The functions of the above-mentioned microbial fertilizer include one or more of the above-mentioned soil organic phosphorus mineralization, promotion of the growth of phosphate-solubilizing bacteria, and promotion of plant growth.

[0015] On the other hand, the present application provides a method for preparing the above-mentioned bacterial agent, the method comprising:

[0016] 1) Seed solution preparation: Indian cleanroom Bacillus D99 was inoculated into CASO liquid medium, cultured, and prepared into a seed solution;

[0017] 2) Fermentation: The seed liquid obtained in step 1) is inoculated into a fermentation medium, and fermented to obtain a fermentation liquid.

[0018] Furthermore, the culture conditions in step 1) are a culture temperature of 30°C, a rotation speed of 200 r / min, and a culture time of 8 hours; the culture conditions in step 2) are: the fermentation medium is LB medium, the inoculation amount is 1% of the fermentation volume, the initial pH value of the fermentation medium is 7.0-7.2, the culture temperature is 30°C, the rotation speed is 200 r / min, and the fermentation culture time is 1-2 days.

[0019] This application demonstrates for the first time the function and role of the Indian cleanroom Bacillus strain Domibacillus indicus in mineralizing soil organic phosphorus and promoting plant growth. This provides a reference for future research on this strain and is of great value for the development of microbial fertilizers such as phosphate-solubilizing fertilizers. The Indian cleanroom Bacillus strain D99 provided by this invention has the ability to dissolve soil organic phosphorus, with an acid phosphatase activity of 17.32 and an alkaline phosphatase activity of 1.3. It can promote plant growth, increase aboveground biomass by 50-115%, significantly reduce available organic phosphorus in black soil by 14.75 mg / kg, and promote the growth of indigenous phosphate-solubilizing bacteria in the soil. BRIEF DESCRIPTION OF THE DRAWINGS

[0020] Figure 1 This is the colony and bacterial morphology of Indian clean room Bacillus (Domibacillus indicus) D99 after culture on LB solid medium.

[0021] Figure 2 Schematic diagram of the test device for detecting the colonization ability of Indian cleanroom Bacillus D99 in field soil.

[0022] Figure 3 To demonstrate the survival of Indian cleanroom Bacillus D99 in field soil and its effect on the soil's organic phosphorus mineralization capacity.

[0023] Figure 4 Demonstrate the growth-promoting effect of Indian cleanroom Bacillus D99 on indigenous phosphate-solubilizing bacteria.

[0024] Figure 5 Demonstrate the growth-promoting effect of Indian cleanroom Bacillus D99 on indigenous phosphate-solubilizing bacteria.

[0025] Figure 6 To demonstrate the growth promoting effect of Indian cleanroom Bacillus D99 on maize in fluvo-aquic soil.

[0026] Figure 7 Demonstrating the growth promoting effect of Indian cleanroom Bacillus D99 on corn in brown soil. DETAILED DESCRIPTION

[0027] The following examples are provided to facilitate a better understanding of the present invention, but are not intended to be limiting. These examples are for illustrative purposes only and are not intended to limit the scope of protection of the present invention.

[0028] Example 1 Isolation and Identification of Indian Cleanroom Bacillus D99

[0029] (1) Strain isolation and screening

[0030] In 2022, a potted experiment was conducted in the greenhouse of the College of Resources and Environment at China Agricultural University to collect corn rhizosphere soil. 1 g of fresh rhizosphere soil temporarily stored at 4°C was weighed and placed in a centrifuge tube containing 9 mL of sterile water. The tube was shaken at 200 rpm for 30 minutes at room temperature. After shaking, the tube was placed in an 80°C water bath for 30 minutes to obtain a higher rate of Gram-positive bacteria. The soil mixture was diluted and spread using the dilution coating method, and three consecutive dilutions (10 -3 , 10 -4 , 10 -5) was spread onto the surface of CASO solid medium (CASO solid medium consists of 15g trypticase peptone, 5g soy peptone, 5g sodium chloride, 15g agar, 1000mL water, pH 7.0-7.2). The inoculated plates were incubated in a 30°C incubator for approximately 2 days. After colonies emerged, the dominant colonies were screened based on their growth and size. Single colonies were streaked twice to purify the strains. A total of 360 bacterial strains were obtained and stored in a -80°C freezer with 50% glycerol.

[0031] The obtained strain D99 was inoculated into LB solid medium and cultured at 30°C for 1 day. The colony morphology was observed: the colony was round in shape, milky white in color, smooth, with clear edges, and not sticky when picked up (such as Figure 1 shown).

[0032] (2) 16S rRNA gene sequencing and identification

[0033] Bacteria were inoculated into LB medium and shake-cultured overnight. PCR amplification of the 16S rRNA gene sequence was performed using the forward primer 27F (5'-AGAGTTTGATCCTGGCTCAG-3', SEQ ID NO. 1) and the reverse primer 1492R (5'-TACGACTTAACCCCAATCGC-3', SEQ ID NO. 2). Amplification conditions were: 95°C for 5 min, followed by 30 cycles of 95°C for 15 s, 60°C for 15 s, and 72°C for 15 s, followed by a final extension at 72°C for 5 min.

[0034] After the PCR amplification product was tested and qualified, it was sent to Anhui General Biotechnology Co., Ltd. for sequencing. The double-end sequencing sequence was spliced ​​using DNAMAN software, and the 16S rRNA gene sequence of strain D99 was subjected to BLAST comparison on the NCBI website. The results showed that the strain had the highest similarity with Domibacillus sp.DTU_2020, and the sequence was: CAGCGGCGGACGGGTGAGTAACACGTGGGTAACCTGCCTGTAAGACTGGGATAACTCCGGGAAACCGGGGCTAATACCGGATAACATCAAGAGCTGCATGGCTCTTGATTGAAAGGCGGCTTCGGCTGTCACTTACAGATGGACCCGCGGCGCATTAGCTAGTTGGTGAGGTAACGGCTCACCAAGGCGACGATGCGTAGCCGACCTGAGAGGGTGATCGGCCACACTG GGACTGAGACACGGCCCAGACTCCTACGGGAGGCAGCAGTAGGGAATCTTCCGCAATGGACGAAAGTCTGACGGAGCAACGCCGCGTGAGTGAAGAAGGTTTTCGGATCGTAAAACTCTGTTGTC AGGGAAGAACAAGTACGGGAGTAACTGCCCGTACCTTGACGGTACCTGGCCAGAAAGCCACGGCTAACTACGTGCCAGCAGCCGCGGTAATACGTAGGTGGCAAGCGTTGTCCGGAATTATTGGGCGTAAAGCGCGCGCAGGCGGCCTTTTAAGTCTGATGTGAAAGCCCACGGCTCAACCGTGGAGGGTCATTGGAAACTGGAAGGCTTGAGTGCAGAAGAGAAGAGCGGAATTCCACGTGTAGCGGTGAAATGCGTAGAGATGTGGAGGAACACCAGTGGCGAAGGCGGCTCTTTGGTCTGTAACTGACGCTGAGGCGCGAAAGCGTGGGGAGCGAACAGGATTAGATACCCTGGTAGTCCACGCCGTAAACGATGAGTGCTAAGTGTTGGGGGGTTTCCGCCCCTCAGTGCTGCAGCTAACGCATTAAGCACTCCGCCTGGGGAGTACGGCCGCAAGGCTGAAACTCAAAGGAATTGACGGGGGCCCGCACAAGCGGTGGAGCATGTGGTTTAATTCGAAGCAACGCGAAGAACCTTACCAGGTCTTGACATCCCGCTGACCGGCCTGGAGACAGATCTTTCCCTTCGGGGGCAGCGGTGACAGGTGGTGCATGGTTGTCGTCAGCTCGTGTCGTGAGATGTTGGGTTAAGTCCCGCAACGAGCGCAACCCTTGATCTTAGTTGCCAGCATTCAGTTGGGCACTCTAAGGTGACTGCCGGTGACAAACCGGAGGAAGGTGGGGATGACGTCAAATCATCATGCCCCTTATGACCTGGGCTACACACGTGCTACAATGGATGGTACAAAGGGCTGCAAGACCGCGAGGTTTAGCCAATCCCATAAAACCATTCTCAGTTCGGATTGCAGGCTGCAACTCGCCTGCATGAAGCTGGAATCGCTAGTAATCGCGGATCAGCATGCCGCGGTGAATACGTTCCCGGGCCTTGTACACACCGCCCGTCACACCACGAGAGTTTGCAACACCCGAAGTCGGTGGGGTAACCCTTACGGAGCCAGCCGCC(SEQ ID NO.3).

[0035] (3) Whole genome sequencing and identification

[0036] In order to more accurately identify the bacteria, a single colony of the D99 strain was picked and inoculated into 5 mL of LB liquid medium and cultured overnight at 30°C and 200 rpm. The fermentation broth was placed in a 1.5 mL centrifuge tube and centrifuged at 12,000 rpm for 1 min. The supernatant was removed and the retained bacteria were collected and sent to Nanjing Paisonno Gene Technology Co., Ltd. for whole genome sequencing.

[0037] The whole genome sequencing method uses the whole genome shotgun (WGS) sequencing strategy to construct libraries with different insert fragments. The libraries are sequenced using second-generation sequencing (NGS) and third-generation single-molecule sequencing technologies.

[0038] Sequencing results showed that D99 contained two circular chromosomes with sequence lengths of 3,578,255bp and 1,357,883bp, respectively. The whole genome data of D99 was compared with species classification in the comprehensive, high-quality microbial genome classification and annotation database GTDB (Genome Taxonomy Database). It had the highest similarity with GCF023715895.1 in the database, and D99 was identified as Domibacillus indicus, which is called Indian clean room Bacillus according to the Chinese name of CCTCC. It is deposited in Guangdong Provincial Microbial Culture Collection Center; the deposit number is GDMCC No.65675; the deposit date is December 25, 2024; the address is the Institute of Microbiology, Guangdong Academy of Sciences, 5th Floor, Building 59, Compound 100 Xianlie Middle Road, Guangzhou.

[0039] Example 2 Determination of Phosphatase Secretion Ability of Indian Cleanroom Bacillus D99

[0040] A single colony of Indian cleanroom Bacillus D99 was selected from the plate and inoculated into a centrifuge tube containing 5 mL of LB medium. Ferment at 30°C, 200 rpm, and 18 h. Pipette 0.5 mL of the fermentation broth into a 1.5 mL / 2 mL centrifuge tube, fill four tubes, and incubate at 8000 rpm for 3-5 min. Discard the supernatant and resuspend two tubes in 0.48 mL of Tris buffer (pH 5.4), followed by another 0.48 mL of Tris buffer (pH 9.4).

[0041] Treatment group: Directly add 20 μL of 100 mM pNPP to a final concentration of 4 mM. Incubate at 30°C for 1 hour. Then add 0.5 mL of 0.5 M NaOH for 10 minutes to terminate the enzyme reaction. Centrifuge at 8000 rpm for 3 minutes, and measure the absorbance of the supernatant at 405 nm.

[0042] Control group: First add 0.5mL NaOH (0.5M) to stop the enzyme activity reaction, then add 20μL 100mM pNPP, incubate at 30℃ for 1h, centrifuge at 8000rpm for 3min, take the supernatant and measure the absorbance at 405nm. The remaining fermentation liquid is used to measure OD 600 The phosphatase activity was calculated as follows: (PNP concentration in the supernatant of the treatment group - PNP concentration in the supernatant of the control group) / (reaction time × OD 600 reading)

[0043] The results showed that the acid phosphatase activity of Indian cleanroom Bacillus D99 was 17.32 and the alkaline phosphatase activity was 1.38.

[0044] Example 3: Colonization Ability Detection of Indian Cleanroom Bacillus D99 in Field Soil

[0045] The prerequisite for bacterial agents to work is that they survive stably in the soil. The number and relative abundance of the bacteria that survive represent the potential for action of the bacteria in farmland soil.

[0046] (1) Preparation of microbial agents

[0047] The Indian clean room Bacillus D99 was inoculated into LB liquid medium and cultured on a shaker (30°C, 200 rpm) for 48 hours to obtain a liquid bacterial agent. The bacterial agent was centrifuged at 5000 rpm for 5 minutes, the supernatant was removed, and the bacteria in the centrifuge tube were resuspended with sterile water and centrifuged again. After repeating twice, the mixture was adjusted to 0D. 600 =10, the concentration is about 2×10 7 CFU / mL, forming bacterial agents.

[0048] (2) Experimental materials and methods

[0049] Fresh soil was collected from the field at the Quzhou Experimental Station in Handan City, Hebei Province and stored at 4°C. In the experimental group, 10 mL of liquid microbial agent was added to 200 g of fresh soil and placed in a special soil incubation device (such as Figure 2 (as shown); the control group received only fresh soil, with four replicates per treatment group. The soil incubation device was buried in a corn field at the Quzhou Experimental Station in Handan City, Hebei Province, on July 21, 2024. The incubation device was harvested after three months of incubation, and 16S rRNA gene sequencing was used to determine the colonization of the strain in the field soil.

[0050] (3) Test results

[0051] The results of the bacterial colonization test on farmland soil are as follows Figure 3The results showed that, compared with the control group, the inoculated Indian Bacillus D99 strain stably colonized and survived in farmland soil, with its relative abundance increasing by 3.71-fold. Indian Bacillus D99 also significantly enhanced the soil's ability to mineralize organic phosphorus, with alkaline phosphatase activity increasing by 28.76% compared to the control group. These field trial results demonstrate that Indian Bacillus D99 can stably colonize farmland soil and significantly promote the activation of organic phosphorus in the soil, making it a promising resource for developing a stable and efficient microbial fertilizer strain.

[0052] Example 4 Effect of Indian Cleanroom Bacillus D99 on Other Phosphate-Solubilizing Bacteria in Soil

[0053] (1) Test method

[0054] After the inoculum was added to the soil, not only did Indian cleanroom Bacillus D99 exert its phosphate-solubilizing function, but native soil phosphate-solubilizing bacteria also played a role. This experiment was designed to test the growth-promoting effect of Indian cleanroom Bacillus D99 on native phosphate-solubilizing bacteria. Bacteria were cultured, isolated, and purified from the soil according to the method of Example 1. The purified bacteria were sequenced with 16S rDNA and identified, and their ability to mineralize organophosphate was also tested. The experiment yielded the native soil phosphate-solubilizing bacteria Bacillus sp. C67, Bacillus sp. A113, and Fictibacillus sp. A103.

[0055] Indian cleanroom Bacillus D99 was inoculated into M9 medium containing 1% glucose and cultured on a shaker (30°C, 200 rpm) for 48 hours to obtain a fermentation broth. The D99 fermentation broth was centrifuged at 5000 rpm for 5 minutes, and the supernatant was sterilized using a sterile 0.45 μm filter to obtain a D99 sterile fermentation broth.

[0056] The treatment groups consisted of three strains of indigenous phosphate-solubilizing bacteria, Bacillus sp. C67, Bacillus sp. A113, and Fictibacillus sp. A103, inoculated into sterile fermentation broth of Indian cleanroom Bacillus D99. The control group consisted of three strains of indigenous phosphate-solubilizing bacteria, Bacillus sp. C67, Bacillus sp. A113, and Fictibacillus sp. A103, inoculated into standard M9 medium supplemented with 1% glucose. The culture apparatus used a 100-well honeycomb plate, and the instrument and detection device used a Bioscreen C automated microbial growth instrument. The detection wavelength was 600 nm, the detection interval was 30 minutes, and the cumulative incubation time was 48 hours.

[0057] (2) Test results

[0058] Three strains of indigenous phosphate-solubilizing bacteria showed faster growth rates in the fermentation broth of Indian cleanroom Bacillus D99 ( Figure 4 and Figure 5 The D99 fermentation broth significantly increased the growth rate of the soil phosphate-solubilizing bacterium Bacillus sp. C67, with the plateau phase occurring 14 hours earlier than the control and the maximum OD value increasing by 0.43. The D99 fermentation broth also increased the growth rate of the soil phosphate-solubilizing bacterium Bacillus sp. A113, with the plateau phase occurring 6 hours earlier than the control. The D99 fermentation broth also increased the growth rate of the soil phosphate-solubilizing bacterium Fictibacillus sp. A103, with the plateau phase occurring 8 hours earlier than the control and the maximum OD value increasing by 0.23.

[0059] Example 5: Mineralization test of organic phosphorus in black soil by Indian clean room Bacillus D99

[0060] (1) Test method

[0061] The Indian cleanroom Bacillus D99 inoculum was prepared according to the method of the embodiment. The test plant was corn, variety Zhengdan 958. 2 kg of black soil from Gusheng Village, Dali City, Yunnan Province was potted for use. Selected corn seeds were soaked in a 2.5% sodium hypochlorite solution for 10 minutes, then surface-sterilized with 75% alcohol for 1 minute. After rinsing with sterile water 6-7 times, the seeds were placed on a petri dish and cultured for 24 hours. After germination, the seeds were sown in flower pots, with 5 seeds sown in each flower pot and covered with soil. The experimental group was irrigated with 10 ml of the inoculum, and the control group was irrigated with an equal volume of deionized water. Each group was repeated 4 times. After 40 days of growth, the corn plant height, dry weight, aboveground phosphorus content, and rhizosphere soil phosphatase activity were measured to analyze whether the Indian cleanroom Bacillus D99 had a mineralization effect on organic phosphorus in the black soil and whether it promoted phosphorus absorption by corn.

[0062] (2) Test results

[0063] The results of the pot experiment are shown in Table 1. They showed that, compared with the control group, inoculation with Indian Bacillus spp. D99 significantly reduced the content of available organic phosphorus in black soil, with degradation reaching 14.75 mg / kg. Phosphatase activity in the rhizosphere soil, reflecting the ability to mineralize organic phosphorus, was significantly reduced by the inoculation compared with the control group. However, alkaline phosphatase activity increased by 2.24-fold. Furthermore, aboveground phosphorus concentration in corn plants in the treated group increased significantly, by 16% compared with the control group.

[0064] Table 1 Effects of Indian cleanroom Bacillus D99 on the mineralization of organophosphate in black soil

[0065]

[0066] Note: The indicators marked with * show significant differences between the control group and the treatment group.

[0067] Example 6: Growth promotion test of corn by Indian cleanroom Bacillus D99 in damp soil

[0068] (1) Test method

[0069] The Indian cleanroom Bacillus D99 inoculant was prepared according to the method of Example 2. The test plant was corn, and the variety was Zhengdan 958. 2 kg of damp soil sampled from the field at Quzhou Experimental Station, Quzhou County, Handan City, Hebei Province was potted for standby use. Selected corn seeds were soaked in 2.5% sodium hypochlorite solution for 10 minutes, then surface disinfected with 75% alcohol for 1 minute, rinsed with sterile water 6-7 times, and then placed on a culture dish for 24 hours. After germination, the seeds were sown in flower pots. 5 seeds were sown in each flower pot and the surface was covered with soil. The experimental group was irrigated with 10 ml of the inoculant, and the control group was irrigated with an equal volume of deionized water. Each group was repeated 4 times. After 40 days of growth, the plant height, dry weight, aboveground phosphorus content, and rhizosphere soil phosphatase activity of the corn were measured to analyze whether the Indian cleanroom Bacillus D99 had a growth-promoting effect on corn in black soil.

[0070] (2) Test results

[0071] The results of the pot experiment are as follows Figure 6 Results showed that inoculation with Indian Bacillus D99 increased plant biomass by 115% compared to the control group. Phosphatase activity in the rhizosphere soil, reflecting the ability to mineralize organic phosphorus, significantly increased acid phosphatase activity in maize roots by 49% and alkaline phosphatase activity by 115% compared to the control group.

[0072] Table 2 Effects of Indian cleanroom Bacillus D99 on the mineralization of organophosphorus in black soil

[0073]

[0074]

[0075] Note: The indicators marked with * show significant differences between the control group and the treatment group.

[0076] Example 7: Growth promotion test of corn by Indian cleanroom Bacillus D99 in brown soil

[0077] (1) Test method

[0078] The Indian cleanroom Bacillus D99 bacterial agent was prepared according to the method of Example 2. The test plant was corn, and the variety was Zhengdan 958. 2 kg of brown soil from Tai'an City, Shandong Province was potted for use, and the selected corn seeds were soaked in 2.5% sodium hypochlorite solution for 10 minutes, and then surface disinfected with 75% alcohol for 1 minute. After rinsing with sterile water 6-7 times, the seeds were placed on a culture dish and cultured for 24 hours. After germination, the seeds were sown in flower pots, and 5 seeds were sown in each flower pot and the surface was covered with soil. The experimental group was irrigated with 10 ml of the bacterial agent, and the control group was irrigated with an equal volume of deionized water. Each group was repeated 4 times. After 40 days of growth, the plant height, dry weight, aboveground phosphorus content and rhizosphere soil phosphatase activity of the corn were measured to analyze whether the Indian cleanroom Bacillus D99 had a growth-promoting effect on corn in black soil.

[0079] (2) Test results

[0080] The results of the pot experiment are as follows Figure 7 Results showed that inoculation with Indian Bacillus D99 increased plant biomass by 50% compared to the control group. Phosphatase activity in rhizosphere soil, reflecting the ability to mineralize organic phosphorus, significantly increased acid phosphatase activity in maize roots by 20% and alkaline phosphatase activity by 8% compared to the control group.

[0081] Table 2 Effects of Indian cleanroom Bacillus D99 on the mineralization of organophosphorus in black soil

[0082]

[0083] Note: The indicators marked with * are significantly different between the control group and the treatment group.

Claims

1. An Indian cleanroom Bacillus that mineralizes soil organophosphates ( Domibacillus indicus )D99, characterized in that The Indian cleanroom Bacillus D99 is deposited in Guangdong Provincial Microbial Culture Collection Center with the deposit number GDMCC No. 65675.

2. A bacterial agent, characterized in that The bacterial agent comprises the Indian cleanroom Bacillus D99 according to claim 1.

3. Use of the Indian cleanroom Bacillus D99 according to claim 1 or the bacterial agent according to claim 2 in soil organic phosphorus mineralization.

4. Use of the Indian cleanroom Bacillus D99 according to claim 1 or the bacterial agent according to claim 2 in promoting the growth of phosphate-solubilizing bacteria; the phosphate-solubilizing bacteria are Bacillus and / or Pseudomonas.

5. Use of the Indian cleanroom Bacillus D99 according to claim 1 or the bacterial agent according to claim 2 in promoting corn growth.

6. Use of the Indian cleanroom Bacillus D99 according to claim 1 or the bacterial agent according to claim 2 in the preparation of microbial fertilizer.

7. The method for preparing the microbial agent according to claim 2, characterized in that: The method comprises: 1) preparing seed liquid: inoculating Indian clean room Bacillus D99 into CASO liquid culture medium, culturing and preparing seed liquid; 2) fermenting: inoculating the seed liquid obtained in step 1) into fermentation medium, fermenting and culturing to obtain fermentation liquid.

8. The method according to claim 7, wherein the culture conditions in step 1) are: culture temperature 30°C, rotation speed 200 r / min, and culture for 8 hours; and the fermentation culture conditions in step 2) are: fermentation medium is LB medium, the inoculum size is 1% of the volume of the fermentation medium, the initial pH value of the fermentation medium is 7.0-7.2, the culture temperature is 30°C, the rotation speed is 200 r / min, and the fermentation culture time is 1-2 days.

Images