India clean room bacillus D99 for mineralizing organophosphorus in soil and application of India clean room bacillus D99

Abstract

The invention provides an India clean room bacillus strain D99 for mineralizing organophosphorus in soil and application of the India clean room bacillus strain D99 in mineralizing the organophosphorus in the soil, promoting the growth of phosphate-solubilizing bacteria and promoting the growth of plants. According to the application, the functions and effects of the India clean room bacillus in the aspects of mineralizing organic phosphorus in soil and promoting plant growth are shown for the first time, and the India clean room bacillus has important value in development of microbial fertilizers such as phosphate-solubilizing bacterial fertilizers and the like. The strain India clean room bacillus D99 provided by the invention has the capability of dissolving organophosphorus in soil, the activity of acid phosphatase is 17.32, and the activity of alkaline phosphatase is 1.3; the plant growth can be promoted, and the aboveground biomass can be increased by 50-115.

Description

Technical Field

[0001] The present invention belongs to the field of microbial technology. Specifically, the present application provides a strain of Domibacillus indicus D99 that mineralizes soil organic phosphorus and its applications. Background Art

[0002] Phosphorus is one of the macronutrients essential for crop growth and development. However, the utilization rate of phosphate fertilizers applied to farmland is relatively low, and most of them accumulate in the soil in different forms such as available phosphorus, insoluble inorganic phosphorus, and organic phosphorus. Specifically, 30%-65% of the total phosphorus in the soil exists in the form of organic phosphorus, and in soils with high organic matter, this proportion can even be as high as 90%. Therefore, improving the utilization efficiency of soil organic phosphorus by crops is crucial for the sustainable development of modern agriculture.

[0003] Soil microorganisms are the main driving force for organic phosphorus mineralization, and most phosphatases are also produced by microorganisms. Therefore, microorganisms play an important role in the conversion of organic phosphorus into available phosphorus that can be absorbed and utilized by plants. Such phosphorus-solubilizing microorganisms are called phosphorus-solubilizing microorganisms, and the bacterial group among them is called phosphorus-solubilizing bacteria or phosphate-solubilizing bacteria. Using these phosphate-solubilizing bacteria can effectively improve the utilization rate of farmland organic phosphorus by crops, thereby promoting crop growth and having important agricultural application values.

[0004] Currently, it has been found that some phosphorus-solubilizing bacteria such as Bacillus spp., Pseudomonas spp., and Massetia spp. have plant growth-promoting functions. However, so far, there have been no relevant patents or research reports on Domibacillus indicus in terms of improving soil organic phosphorus mineralization, promoting plant growth, and enhancing plant phosphorus uptake. Summary of the Invention

[0005] On the one hand, the present application provides a strain of Domibacillus indicus D99 that mineralizes soil organic phosphorus. The Domibacillus indicus D99 is deposited in the Guangdong Provincial Microbial Culture Collection Center with the deposit number GDMCC No. 65675.

[0006] On the other hand, the present application provides a microbial agent, and the microbial agent contains the above-mentioned Domibacillus indicus D99.

[0007] The microbial 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 microbial agents according to the conventional preparation methods of microbial preparations. In addition to bacteria, the microbial agent can also include components such as culture media and carriers.

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

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

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

[0011] On the other hand, the present application provides the use of the above-mentioned bacterium or bacterial agent 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 bacterium or bacterial agent in the preparation of microbial fertilizers.

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

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

[0016] 1) Preparation of seed liquid: Inoculate Domibacillus indicus D99 into CASO liquid medium, culture and prepare a seed liquid;

[0017] 2) Fermentation: Inoculate the seed liquid obtained in step 1) into a fermentation medium, and perform fermentation culture to obtain a fermentation broth.

[0018] Furthermore, the culture conditions in step 1) are: culture temperature 30°C, rotation speed 200 r / min, culture for 8 h; 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] The present application first demonstrates the functions and effects of Domibacillus indicus in soil organic phosphorus mineralization and promoting plant growth, provides certain reference for the later research of this type of strain, and has important value for the development of microbial fertilizers such as phosphate-solubilizing bacterial fertilizers. The strain Domibacillus indicus D99 provided by the present 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 the above-ground biomass by 50 - 115%, significantly reduce the available organic phosphorus in black soil by 14.75 mg / kg, and has a growth-promoting effect on indigenous phosphate-solubilizing bacteria in the soil. Description of the Drawings

[0020] Figure 1 Colony and cell morphology diagrams of Domibacillus indicus D99 after cultivation on LB solid medium.

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

[0022] Figure 3 Showing the survival of Domibacillus indicus D99 in field soil and its effect on the mineralization ability of soil organic phosphorus.

[0023] Figure 4 Showing the growth promotion effect of Domibacillus indicus D99 on indigenous phosphorus-solubilizing bacteria.

[0024] Figure 5 Showing the growth promotion effect of Domibacillus indicus D99 on indigenous phosphorus-solubilizing bacteria.

[0025] Figure 6 Showing the growth promotion effect of Domibacillus indicus D99 on maize in fluvo-aquic soil.

[0026] Figure 7 Showing the growth promotion effect of Domibacillus indicus D99 on maize in brown soil. Specific implementation mode

[0027] The following examples facilitate a better understanding of the present invention, but are not limited thereto. These examples are for illustrative purposes only and do not limit the protection scope of the present invention.

[0028] Example 1 Isolation and identification of Domibacillus indicus D99

[0029] (1) Strain isolation and screening

[0030] In 2022, a pot experiment was carried out in the greenhouse of the College of Resources and Environment, China Agricultural University to collect maize rhizosphere soil. Weigh 1 g of fresh rhizosphere soil temporarily stored at 4 °C and place it in a centrifuge tube containing 9 mL of sterile water. Shake it at 200 rpm at room temperature for 30 min. After shaking, place it in a water bath at 80 °C for 30 min to obtain a higher acquisition rate of Gram-positive bacteria. The soil mixed liquid was used by the dilution coating method, and three consecutive dilution degrees (10 -3 , 10 -4 , 10 -5)Coated on the surface of CASO solid medium (CASO solid medium consists of 15 g of tryptone, 5 g of soy peptone, 5 g of sodium chloride, 15 g of agar, 1000 mL of water, pH 7.0 - 7.2). The inoculated plate was cultured in an incubator at 30 °C for about 2 days. After the colonies grew out, the dominant colonies were further screened according to the growth situation and the size of the colonies. Single colonies were picked and streaked continuously twice to purify the strains. A total of 360 bacterial strains were obtained, and the obtained strains were stored in a -80 °C ultra-low temperature refrigerator with 50% glycerol.

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

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

[0033] The bacteria were inoculated into LB medium and cultured overnight with shaking, and the 16S rRNA gene sequence was amplified by PCR. The forward primer used was 27F (5’-AGAGTTTGATCCTGGCTCAG-3’, SEQ ID NO.1), and the reverse primer was 1492R (5’-TACGACTTAACCCCAATCGC-3’, SEQ ID NO.2). The amplification conditions were: 95 °C for 5 min; 95 °C for 15 s, 60 °C for 15 s, 72 °C for 15 s, for 30 cycles; finally, extension at 72 °C for 5 min.

[0034] After the PCR amplification products passed the test, they were sent to Anhui General Biotech Co., Ltd. for sequencing. The paired-end sequencing sequences were assembled using DNAMAN software. The obtained 16S rRNA gene sequence of strain D99 was BLAST-aligned on the NCBI website. The results showed that the strain had the highest similarity with Domibacillus sp. DTU_2020, and the sequence was: CAGCGGCGGACGGGTGAGTAACACGTGGGTAACCTGCCTGTAAGACTGGGATAACTCCGGGAAACCGGGGCTAATACCGGATAACATCAAGAGCTGCATGGCTCTTGATTGAAAGGCGGCTTCGGCTGTCACTTACAGATGGACCCGCGGCGCATTAGCTAGTTGGTGAGGTAACGGCTCACCAAGGCGACGATGCGTAGCCGACCTGAGAGGGTGATCGGCCACACTGGGACTGAGACACGGCCCAGACTCCTACGGGAGGCAGCAGTAGGGAATCTTCCGCAATGGACGAAAGTCTGACGGAGCAACGCCGCGTGAGTGAAGAAGGTTTTCGGATCGTAAAACTCTGTTGTCAGGGAAGAACAAGTACGGGAGTAACTGCCCGTACCTTGACGGTACCTGGCCAGAAAGCCACGGCTAACTACGTGCCAGCAGCCGCGGTAATACGTAGGTGGCAAGCGTTGTCCGGAATTATTGGGCGTAAAGCGCGCGCAGGCGGCCTTTTAAGTCTGATGTGAAAGCCCACGGCTCAACCGTGGAGGGTCATTGGAAACTGGAAGGCTTGAGTGCAGAAGAGAAGAGCGGAATTCCACGTGTAGCGGTGAAATGCGTAGAGATGTGGAGGAACACCAGTGGCGAAGGCGGCTCTTTGGTCTGTAACTGACGCTGAGGCGCGAAAGCGTGGGGAGCGAACAGGATTAGATACCCTGGTAGTCCACGCCGTAAACGATGAGTGCTAAGTGTTGGGGGGTTTCCGCCCCTCAGTGCTGCAGCTAACGCATTAAGCACTCCGCCTGGGGAGTACGGCCGCAAGGCTGAAACTCAAAGGAATTGACGGGGGCCCGCACAAGCGGTGGAGCATGTGGTTTAATTCGAAGCAACGCGAAGAACCTTACCAGGTCTTGACATCCCGCTGACCGGCCTGGAGACAGATCTTTCCCTTCGGGGGCAGCGGTGACAGGTGGTGCATGGTTGTCGTCAGCTCGTGTCGTGAGATGTTGGGTTAAGTCCCGCAACGAGCGCAACCCTTGATCTTAGTTGCCAGCATTCAGTTGGGCACTCTAAGGTGACTGCCGGTGACAAACCGGAGGAAGGTGGGGATGACGTCAAATCATCATGCCCCTTATGACCTGGGCTACACACGTGCTACAATGGATGGTACAAAGGGCTGCAAGACCGCGAGGTTTAGCCAATCCCATAAAACCATTCTCAGTTCGGATTGCAGGCTGCAACTCGCCTGCATGAAGCTGGAATCGCTAGTAATCGCGGATCAGCATGCCGCGGTGAATACGTTCCCGGGCCTTGTACACACCGCCCGTCACACCACGAGAGTTTGCAACACCCGAAGTCGGTGGGGTAACCCTTACGGAGCCAGCCGCC(SEQ ID NO.3).

[0035] (3) Whole genome sequencing and identification

[0036] To more accurately identify the bacterium, a single colony of the D99 strain to be tested was picked and inoculated into 5 mL of LB liquid medium, and cultured overnight at 30 °C with a rotation speed of 200 rpm. The fermentation broth was taken and placed in a 1.5 mL centrifuge tube, centrifuged at 12,000 rpm for 1 min to remove the supernatant, and the remaining bacteria were collected and sent to Nanjing Personal Biotechnology Co., Ltd. for whole-genome sequencing.

[0037] The whole-genome sequencing method adopted the whole-genome shotgun (WGS) sequencing strategy to construct libraries with different insert fragments. The libraries were sequenced using the second-generation sequencing technology (Next-Generation Sequencing, NGS) and the third-generation single-molecule sequencing technology.

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

[0039] Example 2 Determination of the phosphatase secretion ability of Bacillus amyloliquefaciens subsp. plantarum D99

[0040] A single colony of Bacillus amyloliquefaciens subsp. plantarum D99 was picked from the plate and inoculated into a centrifuge tube containing 5 mL of LB medium, and fermented at 30 °C with a rotation speed of 200 rpm for 18 h. 0.5 mL of the bacterial fermentation broth was pipetted into 1.5 mL / 2 mL centrifuge tubes, with 4 tubes filled. The supernatant was discarded by centrifugation at 8000 rpm for 3 - 5 min. The bacteria in 2 centrifuge tubes were resuspended with 0.48 mL of Tris buffer (pH 5.4), and the bacteria in the other 2 centrifuge tubes were resuspended with 0.48 mL of Tris buffer (pH 9.4).

[0041] Treatment group: 20 μL of 100 mM pNPP was directly added to make the final concentration in the system 4 mM, and cultured in an incubator at 30 °C for 1 h. Then, 0.5 mL of NaOH (0.5 M) was added to react for 10 min to terminate the enzyme activity reaction. The supernatant was taken by centrifugation at 8000 rpm for 3 min and the absorbance was measured at 405 nm.

[0042] Control group: First, add 0.5 mL of NaOH (0.5 M) to terminate the enzymatic reaction. Then, add 20 μL of 100 mM pNPP and incubate in an incubator at 30 °C for 1 h. Centrifuge at 8000 rpm for 3 min and take the supernatant to measure the absorbance at 405 nm. The remaining fermentation broth is used to measure OD. 600 . The phosphatase activity calculation formula is = (concentration of PNP in the supernatant of the treatment group - concentration of PNP in the supernatant of the control group) / (reaction time × OD 600 reading)

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

[0044] Example 3 Detection of the colonization ability of Bacillus sporothermodurans D99 in field soil

[0045] The prerequisite for the fungicide to play a role is to survive stably in the soil. The number and relative abundance of its survival represent the action potential of the bacterium in farmland soil.

[0046] (1) Preparation of the fungicide

[0047] Inoculate Bacillus sporothermodurans D99 into LB liquid medium and shake culture (30 °C, 200 rpm) for 48 h to obtain a liquid fungicide. Centrifuge the fungicide at 5000 rpm for 5 min, remove the supernatant, resuspend the cells in the centrifuge tube with sterile water and centrifuge again. Repeat this process 2 times, and then adjust to 0D 600 = 10, with a concentration of approximately 2 × 10 7 CFU / mL to form the fungicide.

[0048] (2) Experimental materials and methods

[0049] Collect fresh soil from the field at the Quzhou Experimental Station in Handan City, Hebei Province and store it at 4 °C. In the experimental group, add 10 mL of liquid fungicide to 200 g of fresh soil and place it in a special soil incubation device (as Figure 2 shown); while in the control group, only add fresh soil, and set 4 replicates for each treatment group. The soil incubation device was buried in the corn field at the Quzhou Experimental Station in Handan City, Hebei Province on July 21, 2024, harvested after 3 months of cultivation, and the colonization of the strain in farmland soil was detected by 16S rRNA gene sequencing.

[0050] (3) Test results

[0051] The results of the bacterial colonization test in farmland soil are as Figure 3As shown, the results indicate that compared with the control group, inoculation with Bacillus amyloliquefaciens D99 can stably colonize and survive in farmland soil, and the relative abundance increases by 3.71 times. Bacillus amyloliquefaciens D99 significantly improves the ability of soil organic phosphorus mineralization, and the alkaline phosphatase activity increases by 28.76% compared with the control group. The above field test results show that Bacillus amyloliquefaciens D99 can stably colonize in farmland soil and significantly promote the activation of organic phosphorus in the soil, and can be used as a strain resource for developing stable and efficient microbial fertilizers.

[0052] Example 4 Effect of Bacillus amyloliquefaciens D99 on Other Phosphorus-Solubilizing Bacteria in Soil

[0053] (1) Test Method

[0054] After the inoculant is added to the soil, not only does Bacillus amyloliquefaciens D99 play a phosphorus-solubilizing function, but the native phosphorus-solubilizing bacteria in the soil can also play a certain role. In order to detect the growth-promoting effect of Bacillus amyloliquefaciens D99 on native phosphorus-solubilizing bacteria, this experiment was designed. Bacteria were cultured, isolated, and purified from the soil according to the method of Example 1. The purified bacteria were subjected to 16S rDNA sequencing and strain identification, and their ability to mineralize organic phosphorus was also detected. The native soil phosphorus-solubilizing bacteria Bacillus.sp.C67, Bacillus.sp.A113, and Fictibacillus.sp.A103 were obtained from the experiment.

[0055] Bacillus amyloliquefaciens D99 was inoculated into M9 medium containing 1% glucose and cultured in a shaker (30 °C, 200 rpm) for 48 hours to obtain a fermentation broth. The fermentation broth inoculant of D99 was centrifuged at 5000 rpm for 5 min, and the supernatant was filtered through a sterile 0.45 μm filter head to obtain the sterile fermentation broth of D99.

[0056] The treatment group was set as three native phosphorus-solubilizing bacteria, Bacillus.sp.C67, Bacillus.sp.A113, and Fictibacillus.sp.A103, which were respectively inoculated into the sterile fermentation broth of Bacillus amyloliquefaciens D99, while the control group was set as three native phosphorus-solubilizing bacteria, Bacillus.sp.C67, Bacillus.sp.A113, and Fictibacillus.sp.A103, which were respectively inoculated into ordinary M9 medium containing 1% glucose. The culture device used a 100-well honeycomb plate, and the culture instrument and detection device used a Bioscreen C microbial automatic growth instrument. The detection wavelength was 600 nm, the detection interval was 30 min, and the total culture time was 48 h.

[0057] (2) Test Results

[0058] Three indigenous phosphate-solubilizing bacteria will exhibit a faster growth rate in the fermentation broth of Bacillus amyloliquefaciens D99 from India( Figure 4 and Figure 5 as shown). The fermentation broth of D99 significantly increased the growth rate of the soil phosphate-solubilizing bacterium Bacillus.sp.C67. The stationary phase was 14 h faster than the control, and the maximum OD value increased by 0.43. The fermentation broth of D99 increased the growth rate of the soil phosphate-solubilizing bacterium Bacillus.sp.A113. The stationary phase was 6 h faster than the control. The fermentation broth of D99 increased the growth rate of the soil phosphate-solubilizing bacterium Fictibacillus.sp.A103. The stationary phase was 8 h faster than the control, and the maximum OD value increased by 0.23.

[0059] Example 5 Mineralization test of Bacillus amyloliquefaciens D99 from India on organic phosphorus in black soil

[0060] (1) Test method

[0061] Prepare the Bacillus amyloliquefaciens D99 inoculant according to the method of the example. The test plant is corn, and the variety is Zhengdan 958. Put 2 kg of black soil from Gusheng Village, Dali City, Yunnan Province into flower pots for use. Select corn seeds, soak them in 2.5% sodium hypochlorite solution for 10 min, then surface disinfect them with 75% alcohol for 1 min, rinse them 6 - 7 times with sterile water, place the seeds on a culture dish and cultivate them for 24 h, and then sow the seeds into the flower pots after germination. Sow 5 seeds in each flower pot and cover the surface with soil. Pour 10 ml of the inoculant into the test group and pour the same volume of deionized water into the control group. Repeat each group 4 times. After growing for 40 days, measure the plant height, dry weight, phosphorus content in the above-ground part, and phosphatase activity in the rhizosphere soil of the corn, etc., to analyze whether Bacillus amyloliquefaciens D99 has a mineralization effect on organic phosphorus in black soil and whether it promotes the phosphorus absorption of corn.

[0062] (2) Test results

[0063] The results of the pot experiment are shown in Table 1. The results show that compared with the control group, inoculating Bacillus amyloliquefaciens D99 significantly reduced the available organic phosphorus content in the black soil, and the degradation range of available organic phosphorus was 14.75 mg / kg. The phosphatase activity in the rhizosphere soil reflects the ability of organic phosphorus mineralization. Compared with the control group, inoculating the inoculant had little significant effect on the soil acid phosphatase activity; but the alkaline phosphatase activity increased by 2.24 times. In addition, the phosphorus concentration in the above-ground part of the corn in the treatment group increased significantly, by 16% compared with the control group.

[0064] Table 1 Effects of Bacillus amyloliquefaciens D99 on the mineralization of organic phosphorus in black soil

[0065]

[0066] Note: For the surface marked with *, there are significant differences in this index between the control group and the treatment group.

[0067] Example 6: Growth-promoting test of Bacillus amyloliquefaciens D99 on maize in fluvo-aquic soil

[0068] (1) Test method

[0069] Prepare the Bacillus amyloliquefaciens D99 bacterial agent according to the method of Example 2. The test plant is maize, and the variety is Zhengdan 958. Put 2 kg of fluvo-aquic soil sampled from the field of Quzhou Experimental Station, Quzhou County, Handan City, Hebei Province into pots for use. Select maize seeds, soak them in 2.5% sodium hypochlorite solution for 10 min, then surface disinfect them with 75% alcohol for 1 min, rinse them 6 - 7 times with sterile water, place the seeds on a culture dish and cultivate for 24 h, and then sow the germinated seeds into flower pots. After sowing 5 seeds in each flower pot, cover the surface with soil. Pour 10 ml of the bacterial agent into the test group, and pour the same volume of deionized water into the control group. Each group is repeated 4 times. After growing for 40 days, measure the plant height, dry weight, aboveground phosphorus content, and rhizosphere soil phosphatase activity of maize, etc., and analyze whether Bacillus amyloliquefaciens D99 has a growth-promoting effect on maize in fluvo-aquic soil.

[0070] (2) Test results

[0071] The results of the pot experiment are as Figure 6 shown. The results show that compared with the control group, inoculating Bacillus amyloliquefaciens D99 increases the plant biomass by 115%. The rhizosphere soil phosphatase activity reflects the ability of organic phosphorus mineralization. Compared with the control group, inoculating the bacterial agent significantly increases the acid phosphatase activity of maize roots by 49%; the alkaline phosphatase activity increases by 115%.

[0072] Table 2 Effects of Bacillus amyloliquefaciens D99 on the mineralization of organic phosphorus in fluvo-aquic soil

[0073]

[0074]

[0075] Note: For the surface marked with *, there are significant differences in this index between the control group and the treatment group.

[0076] Example 7: Growth-promoting test of Bacillus amyloliquefaciens D99 on maize in brown soil

[0077] (1) Test method

[0078] Prepare the Bacillus amyloliquefaciens D99 agent according to the method of Example 2. The test plant is corn, and the variety is Zhengdan 958. Put 2 kg of cinnamon soil from Tai'an City, Shandong Province into a pot for use. Select corn seeds, soak them in a 2.5% sodium hypochlorite solution for 10 min, then surface disinfect them with 75% alcohol for 1 min. After rinsing them 6 - 7 times with sterile water, place the seeds on a petri dish and cultivate them for 24 h. After germination, sow the seeds into the flower pots, and cover the surface with soil after sowing 5 seeds in each flower pot. Irrigate 10 ml of the agent to the experimental group, and irrigate the same volume of deionized water to the control group. Repeat each group 4 times. After growing for 40 days, measure the plant height, dry weight, phosphorus content in the above-ground part, and phosphatase activity in the rhizosphere soil of the corn, etc., and analyze whether there is a growth-promoting effect of Bacillus amyloliquefaciens D99 on corn in the black soil.

[0079] (2) Test results

[0080] The results of the pot experiment are as Figure 7 shown. The results show that compared with the control group, inoculating Bacillus amyloliquefaciens D99 increases the biomass of the plant, and the biomass increases by 50%. The phosphatase activity in the rhizosphere soil reflects the ability of organic phosphorus mineralization. Compared with the control group, inoculating the agent significantly increases the acid phosphatase activity of the corn roots by 20%; the alkaline phosphatase activity increases by 8%.

[0081] Table 2 Effects of Bacillus amyloliquefaciens D99 on the mineralization of organic phosphorus in black soil

[0082]

[0083] Note: Those with * indicate that this index is significant between the control group and the treatment group.

Claims

1. A strain of Domibacillus indicus D99 that mineralizes soil organophosphate, characterized in that: The Indian clean room Bacillus D99 is deposited in Guangdong Microbiological Culture Collection Center with the deposit number of GDMCC No.65675.

2. A bacterial agent, characterized in that: The bacterial agent comprises the above-mentioned Indian clean room Bacillus D99.

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

4. Use of the Indian clean room Bacillus D99 according to claim 1 or the bacterial agent according to claim 2 in promoting the growth of phosphate-solubilizing bacteria.

5. The use according to claim 4, wherein the phosphate-solubilizing bacteria are Bacillus and / or Pseudomonas.

6. Use of the Indian clean room Bacillus D99 according to claim 1 or the bacterial agent according to claim 2 in promoting plant growth.

7. The use according to claim 6, wherein the plant is corn.

8. Use of the Indian clean room Bacillus D99 according to claim 1 or the bacterial agent according to claim 2 in the preparation of microbial fertilizer.

9. The method for preparing the bacterial agent according to claim 2, characterized in that: The method comprises: 1) Preparation of seed solution: Indian clean room Bacillus D99 was inoculated into CASO liquid medium, cultured and prepared into seed solution; 2) Fermentation: The seed liquid obtained in step 1) is inoculated into a fermentation medium, and fermented to obtain a fermentation liquid.

10. The method according to claim 9, wherein 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 h; and 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.

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