Bacillus amyloliquefaciens and application thereof

Abstract

The present application belongs to the field of microorganisms, and provides a Bacillus amyloliquefaciens DS11 which is preserved in China Agricultural Microorganism Culture Collection Center with a preservation number of CCTCC NO: M 2022685. The inorganic phosphorus dissolving activity of the DS11 strain can reach 46.67±6.52 mg / L, the organic phosphorus dissolving activity can reach 107.99±4.29 mg / L, the organic phosphorus decomposition rate can reach 61.71%, the NH3-N production in the fermentation liquid can be determined to be 0.84±0.03 mg / mL when the strain is fermented in a nitrogen-free culture medium, the beta-glucanase activity can be produced to be 555.89±12.78 U / L when the strain is fermented in LB, and the potassium dissolving capacity of the strain is 4.03±0.25 mg / L. The strain has a significant crop growth promoting effect.

Description

Technical Field

[0001] This invention belongs to the field of microbiology, specifically relating to a type of Bacillus amyloliquefaciens and its applications. Background Technology

[0002] In agricultural production, the widespread use of chemical fertilizers has played a significant role in increasing grain yields. However, due to their inherent characteristics and improper application, they have also brought about a series of serious ecological problems. Firstly, the average utilization rate of chemical fertilizers by crops is not high. Statistics show that the utilization rates of nitrogen, phosphorus, and potassium fertilizers are 40%-50%, 10%-20%, and 30%, respectively. This not only increases the cost of agricultural production but also contributes to soil compaction and water pollution.

[0003] Microorganisms possess various functions, including nitrogen fixation, increasing the content of insoluble mineral elements in the soil (such as phosphorus and potassium), decomposing harmful substances in the soil (such as heavy metals), improving the soil environment, enhancing plant disease resistance, improving plant resistance to pests and diseases, and forming humic acid to improve soil fertility. These excellent properties make microorganisms a promising candidate for eliminating chemical fertilizers, making them a hot research topic for scientists. Microorganisms are diverse and widely distributed, representing a vast untapped resource. In recent years, *Bacillus amyloliquefaciens*, a novel plant biocontrol microorganism, has attracted considerable attention for its role in promoting plant growth. However, current germplasm resources of *Bacillus amyloliquefaciens* are insufficient, its phosphorus and potassium solubilization and nitrogen fixation capabilities need further improvement, and its effects on crop growth promotion are not yet significant. Summary of the Invention

[0004] To address the aforementioned technical problems, this invention provides a Bacillus amyloliquefaciens DS11, which is deposited at the China Agricultural Microbial Culture Collection Center, accession number CCTCC NO: M2022685.

[0005] The *Bacillus amyloliquefaciens* strain described in this invention was isolated from the rhizome of *Dendrobium huoshanense* and belongs to the plant endophytic bacteria category. Testing showed that the DS11 strain of this invention exhibits an inorganic phosphorus dissolving activity of 46.67±6.52 mg / L and an organic phosphorus dissolving activity of 107.99±4.29 mg / L, with an organic phosphorus decomposition rate of 61.71%. Fermentation of this strain on nitrogen-free medium yielded 0.84±0.03 mg / mL of NH3-N. Fermentation on LB medium produced 555.89±12.78 U / L of β-glucanase activity, and the strain's potassium-solubilizing capacity was 4.03±0.25 mg / L. This strain can produce 148.81±4.83 μg / ml of IAA. It has a significant effect on promoting crop growth. Simultaneously, this strain can also improve the plant's salt and alkali resistance.

[0006] The Bacillus amyloliquefaciens DS11 of this invention has the following applications:

[0007] In terms of the application of degrading organophosphorus compounds, the method is to dilute the LB fermentation broth of DS11 20-100 times and irrigate soil containing insoluble organophosphorus compounds.

[0008] For the application of dissolving insoluble inorganic potassium salts, the method is as follows: dilute 20-100 times the LB fermentation broth of DS11 and irrigate soil containing potassium feldspar.

[0009] In nitrogen fixation, the method is to dilute DS11 LB fermentation broth 20-100 times and irrigate the plants to increase the total nitrogen content of the plants.

[0010] In the application of β-glucanase production, the method is as follows: Bacillus amyloliquefaciens DS11 is cultured overnight in LB liquid medium at 37°C on a shaker, and the supernatant is collected.

[0011] In the application of indoleacetic acid production, the method is as follows: LB-activated strain DS11 is inoculated into King's culture medium and cultured in a shaker at 30°C and 100 rpm, and the supernatant is collected.

[0012] In its application to promote plant growth, the method is as follows: dilute the fermentation broth of Bacillus amyloliquefaciens DS11 by 20-100 times, soak the seeds in the fermentation broth for 0.5-1.5 hours, and then plant them.

[0013] In terms of improving the salt and alkali resistance of plants, the method is as follows: ferment the Bacillus amyloliquefaciens DS11 to obtain a fermentation broth, and then dilute the DS11 fermentation broth with irrigation water at a ratio of 1:8000 to irrigate the plants.

[0014] This invention provides a bacterial agent containing Bacillus amyloliquefaciens DS11 as described in this invention.

[0015] Compared with previously disclosed plant growth-promoting bacteria, the Bacillus amyloliquefaciens described in this invention has a more comprehensive and higher level of nitrogen-fixing, phosphorus-solubilizing, and potassium-solubilizing ability, especially its ability to decompose organic phosphorus, and it has excellent ability to secrete IAA, produce a high level of β-glucanase, and has a fairly stable passage stability. Attached Figure Description

[0016] Figure 1 Photograph of Bacillus amyloliquefaciens DS11 colonies

[0017] Figure 2 Cluster analysis of Bacillus amyloliquefaciens DS11;

[0018] Figure 3Example 7: Photographs of rapeseed seedlings promoted by Bacillus amyloliquefaciens DS11 from the first to the fifth generation;

[0019] Figure 4 The graph shows the growth-promoting effect of Bacillus amyloliquefaciens DS11 at different passage numbers on rapeseed seedlings in Example 7.

[0020] Preservation certificate: Culture name: Bacillus amyloliquefaciens DS11, this culture is deposited at the China Center for Type Culture Collection, the center completed the testing on May 27, 2022, and the result was viable, accession number CCTCCNO: M 2022685. Detailed Implementation

[0021] The present invention will be further described in detail below with reference to specific embodiments.

[0022] Example 1: Obtaining the DS11 strain

[0023] (I) Screening of bacterial strains

[0024] Root, stem, and leaf tissues from healthy *Dendrobium huoshanense* plants were disinfected, homogenized, and effectively diluted before being spread onto LB agar for culture. A total of 80 colonies with different morphologies were obtained. Colonies exhibiting hydrolysis zones were screened in phosphate-solubilized medium, while colonies producing clear zones were screened using nitrogen-free medium (Table 1), yielding 28 colonies. The β-glucanase activity of the bacterial cells in the liquid LB supernatant was determined using the NY / T911 spectrophotometric method. Strains with β-glucanase activity above 200 U / L were retained, and a seedling-promoting method was used to obtain the DS11 strain with significant viability.

[0025] Table 1. Culture media used for screening strains*

[0026]

[0027] *The table lists the components of liquid culture medium. If you need to make solid culture medium, please add 15-20 g / L agar.

[0028] (II) Identification of strains

[0029] 1. Morphological characteristics and basic physiological and biochemical properties

[0030] The bacterium was inoculated onto LB medium and incubated at 30°C. Single colony morphology was observed after 3 days. Figure 1 As shown, the colonies formed are cloudy white, round, with irregular edges, initially moist, later becoming drier, and have wrinkled surfaces.

[0031] Biochemical identification of the bacterium revealed that it is a Gram-positive bacterium that produces elliptical spores and has rod-shaped cells. The bacterium can grow in NaCl concentrations of 0-9%, with an optimal salt concentration of 3%. The bacterium can grow at temperatures ranging from 15-50℃, with an optimal growth temperature of 30℃ and a pH range of 6-8.

[0032] 2.16S rRNA sequence homology analysis

[0033] The conserved 16S rDNA sequence of this bacterium is as follows:

[0034] TGGGAGCTTGCTCCCTGATGTTAGCGGCGGACGGGTGAGTAACACGTGGGTAAC

[0035] CTGCCTGTAAGACTGGGATAACTCCGGGAAACCGGGGCTAATACCGGATGGTTGTCTGA

[0036] ACCGCATGGTTCAGACATAAAAGGTGGCTTCGGCTACCACTTACAGATGGACCCGCGGC

[0037] GCATTAGCTAGTTGGTGAGGTAACGGCTCACCAAGGCGACGATGCGTAGCCGACCTGAG

[0038] AGGGTGATCGGCCACACTGGGACTGAGACACGGCCCAGACTCCTACGGGAGGCAGCAG

[0039] TAGGGAATCTTCCGCAATGGACGAAAGTCTGACGGAGCAACGCCGCGTGAGTGATGAA

[0040] GGTTTTCGGATCGTAAAGCTCTGTTGTTAGGGAAGAACAAGTGCCGTTCAAATAGGGCG

[0041] GCACCTTGACGGTACCTAACCAGAAAGCCACGGCTAACTACGTGCCAGCAGCCGCGGT

[0042] AATACGTAGGTGGCAAGCGTTGTCCGGAATTATTGGGCGTAAAGGGCTCGCAGGCGGTT

[0043] TCTTAAGTCTGATGTGAAAGCCCCCGGCTCAACCGGGGAGGGTCATTGGAAACTGGGG

[0044] AACTTGAGTGCAGAAGAGGAGAGTGGAATTCCACGTGTAGCGGTGAAATGCGTAGAGA

[0045] TGTGGAGGAACACCAGTGGCGAAGGCGACTCTCTGGTCTGTAACTGACGCTGAGGAGC

[0046] GAAAGCGTGGGGAGCGAACAGGATTAGATACCCTGGTAGTCCACGCCGTAAACGATGA

[0047] GTGCTAAGTGTTAGGGGGTTTCCGCCCCTTAGTGCTGCAGCTAACGCATTAAGCACTCCG

[0048] CCTGGGGAGTACGGTCGCAAGACTGAAACTCAAAGGAATTGACGGGGGCCCGCACAAG

[0049] CGGTGGAGCATGTGGTTTAATTCGAAGCAACGCGAAGAACCTTACCAGGTCTTGACATC

[0050] CTCTGACAATCCTAGAGATAGGACGTCCCCTTCGGGGGCAGAGTGACAGGTGGTGCATG

[0051] GTTGTCGTCAGCTCGTGTCGTGAGATGTTGGGTTAAGTCCCGCAACGAGCGCAACCCTT

[0052] GATCTTAGTTGCCAGCATTCAGTTGGGCACTCTAAGGTGACTGCCGGTGACAAACCGGA

[0053] GGAAGGTGGGGATGACGTCAAATCATCATGCCCCTTATGACCTGGGCTACACACGTGCT

[0054] ACAATGGACAGAACAAAGGGCAGCGAAACCGCGAGGTTAAGCCAATCCCACAAATCTG

[0055] TTCTCAGTTCGGATCGCAGTCTGCAACTCGACTGCGTGAAGCTGGAATCGCTAGTAATC

[0056] GCGGATCAGCATGCCGCGGTGAATACGTTCCCGGGCCTTGTACACACCGCCCGTCACAC

[0057] CACGAGAGTTTGTAACACCCGAAGTCGGTGAG

[0058] DNA extracted from this strain was analyzed using 16S rDNA sequencing. A clear band of approximately 1400 bp was amplified using universal bacterial primers 16S rRNA27F and 1492R. Sequencing and alignment with GenBank showed a similarity of over 99.71% to *Bacillus amyloliquefaciens* strain INA01271. The housekeeping gene rpoB identified *Bacillus amyloliquefaciens* at 98.22%. Therefore, bacteria DS11 was identified as *Bacillus amyloliquefaciens*.

[0059] (III) Preservation

[0060] The isolated DS11 strain was deposited at the China Center for Type Culture Collection (CCTCC, Wuhan University, Wuhan, China) on May 20, 2022, with accession number CCTCC NO: M 2022685.

[0061] Example 2: Determination of the ability of bacterial strains to dissolve inorganic phosphorus (Ca3(PO4)2) and organic phosphorus (lecithin)

[0062] After activation, the frozen glycerol-preserved DS11 bacteria were inoculated at a ratio of 2% into lysed phosphorus liquid medium (inorganic phosphorus Ca3(PO4)2, organic phosphorus (lecithin)) (Table 1) and cultured on a shaker at 30°C and 100 rpm for 5 days. Commercially available Bacillus subtilis JCM 1465 and Bacillus megaterium ATCC 14581 were used as controls. Centrifuge the bacterial culture at 12000 r / min for 5 minutes, and take 1 mL of the supernatant into a test tube. Dilute with distilled water (dilute the supernatant for dissolving inorganic phosphorus by 2 times, and the supernatant for dissolving organic phosphorus by 10 times). Digest with nitric acid-perchloric acid (GB11893). Establish a standard curve with the concentrated sulfuric acid solution of KH2PO4 (Experimental Methods Lu Rukun, Soil Agricultural Chemical Analysis Methods [M], Beijing: China Soil Science Society: China Agricultural Science and Technology Press, 2000: 179-183). Use the molybdenum antimony resistance method to measure the color at a wavelength of 680 nm on a multi-parameter spectrophotometer (5B-3B V8, Lianhua Technology).

[0063] The following formula is used to calculate the phosphorus solubility rate: Phosphorus solubility rate = Phosphorus content in supernatant / Total phosphorus content

[0064] The ability to dissolve inorganic phosphorus reached 51.67±6.52 mg / L, and the phosphorus solubilization rate was between that of the two control bacteria (Table 2); the activity of DS11 strain in dissolving organic phosphorus was measured to reach 107.99±4.29 mg / L, and the organic phosphorus solubilization rate was 61.71%, which was much greater than that of the control Bacillus subtilis JCM1465 and Bacillus megaterium ATCC 14581 (Table 3).

[0065] Table 2. Inorganic phosphorus solubilization capacity of DS11 and control strains

[0066]

[0067] Table 3. Organophosphate solubilization capacity of DS11 and control strains

[0068]

[0069] *The two control strains showed highly significant differences (P<0.01).

[0070] Example 3: Determination of NH3-N production capacity of DS11

[0071] The LB-activated strain was inoculated at a ratio of 2% into nitrogen-free liquid medium and cultured in a shaker at 30℃ and 100 rpm for 7 days. The NH3-N content in the supernatant of the nitrogen-free liquid medium fermented by the strain was measured using Nessler's reagent (experimental method referred to Yue Yinling, Fan Rongtao, Zhang Lan, et al. Experimental study on test conditions for determination of ammonia nitrogen in water by Nessler's reagent spectrophotometry [J]. Hygiene Research, 2010, v.39(001):94-96.). Under the same conditions, the NH3 production capacity of DS11 was 0.84±0.03 mg / mL, which is much greater than that of Bacillus subtilis and Bacillus megaterium, which are known to have nitrogen-fixing capabilities (Table 4).

[0072] Table 4. NH3 production capacity of DS11 and reference strains

[0073] strain <![CDATA[NH3 production capacity (mg / mL)]]> Bacillus subtilis JCM 1465 0.49±0.02 Bacillus megaterium ATCC 14581 0.54±0.04 DS11 0.84±0.03*

[0074] *The two control strains showed highly significant differences (P<0.01).

[0075] Example 4: Determination of the potassium solubilizing ability of DS11

[0076] Single colonies of DS11 were inoculated into potassium-solubilizing culture medium using toothpicks. The medium was then shaken at 28°C and 180 rpm for 72 hours. The supernatant was collected and its potassium content was determined using a flame spectrophotometer. + The concentration was determined using a KCl standard curve and the method of determination was based on the findings of Wang Xunjue et al. (Wang Xunjue, Huang Qiaoyun, Cai Peng, et al. Comparison of methods for detecting potassium solubilization efficiency of potassium-solubilizing bacteria [J]. Journal of Huazhong Agricultural University, 2016(1):81-85.). The potassium solubilization capacity of DS11 was determined to be 4.03±0.25 mg / L.

[0077] Example 5: β-glucanase activity assay

[0078] The bacterial culture of LB liquid medium, which was cultured overnight in a shaker at 37°C, was used to determine the β-glucanase activity of the bacterial cells in the supernatant of LB liquid medium using the NY / T911 spectrophotometric method. The enzyme activity of DS11 was 555.89±12.78 U / L, which was significantly different from that of the reference strain (P<0.01).

[0079] Table 5. β-glucanase activities of DS11 and reference strains

[0080]

[0081] *The two control strains showed highly significant differences (P<0.01).

[0082] Example 6: Determination of IAA production capacity of strain

[0083] The LB-activated strain was inoculated into King's culture medium at a ratio of 1% (Table 1) and cultured in a shaker at 30℃ and 100 rpm for 3 days. The supernatant was taken, appropriately diluted, and Salkowski colorimetric reagent was added to detect the strain's ability to produce IAA (experimental method see: Liu Yuzhen, Deng Zhenshan, Gao Fei, et al. Identification of an endophytic bacterium of Giant Napier Grass and its preliminary analysis of its growth-promoting characteristics. Guangdong Agricultural Sciences, 2018, 45(03):88-93+173). The IAA produced by DS11 in the King fermentation supernatant was measured to be 148.81±4.83 mg / L. The difference was extremely significant compared with the reference strain and also extremely significant compared with the values ​​in published literature.

[0084] Table 6. IAA production capacity of DS11 and reference strains

[0085]

[0086] * Significantly different from the two control strains (P<0.01)

[0087] Example 7: The effect of DS11 on plant growth promotion

[0088] Take 10 cabbage seeds and 1 mL of DS11 LB fermentation broth (containing 10 cells) from different passage numbers. 8 -10 9 Mix the CFU / mL solution thoroughly, soak for 1 hour, and then plant in 9 cm diameter flowerpots. Use seeds soaked in an equal amount of pure water as a control. Water with an equal amount of water. Harvest the above-ground parts 60 days after planting and calculate the fresh weight. Results are shown in […]. Figure 3 As can be seen from the chart, DS11 has a significant growth-promoting effect on rapeseed seedlings from the first to the fifth generation.

[0089] Implementing 8 DS11 to enhance plant salt and alkali resistance

[0090] In an apple orchard in Aksu, Xinjiang (soil saturation mud EC value 3.86 mS / cm), with a pH of around 8.5, DS11 fermentation solution was diluted with irrigation water from the Tianshan Mountains every half month, starting from the spring budding period. In summer, due to increased temperature and excessive transpiration, soil salinization intensified. At this time, the leaves of orchard plants in the same irrigation water volume without DS11 fermentation solution turned yellow, while the leaves of orchards with DS11 fermentation solution added to the irrigation water showed a lower degree of yellowing. At this time, leaves were collected from the control and treated orchards to detect the total nitrogen, sodium, total phosphorus, total potassium, iron, and zinc contents of the plants. (Samples were prepared using the nitric acid-perchloric acid digestion method, and iron and zinc contents were detected using atomic absorption spectrophotometry. Total nitrogen content was detected using the Kjeldahl method according to NY / T2419-2013. Sodium and potassium contents were detected using a flame photometer according to NY / T 2420-2013. Total phosphorus was detected using the molybdenum-antimony method according to NY / T 2421-2013.) The results are shown in Table 7.

[0091] The results show that DS11 treatment significantly increased the total nitrogen and iron content of the plants. Although the sodium ion concentration was still high compared with the GSB-11 citrus leaf standard, the total nitrogen content was basically close to that of the standard plants. Iron content was slightly lower than the standard, but significantly higher than the control. Phosphorus and potassium levels were higher than the GSB-11 (citrus leaf) standard, which may be due to the high content of insoluble phosphorus and potassium in the Aksu soil of Xinjiang. DS11's phosphorus and potassium solubilizing effect allowed the plants to absorb large amounts of phosphorus and potassium.

[0092] Table 7 shows the effects of DS11 on mineral elements in apple plants grown in saline-alkali soil.

[0093]

[0094] *The difference was highly significant compared to the control plants (P<0.01).

[0095] The embodiments described above are merely preferred embodiments of the present invention and are not intended to limit the scope of the present invention. Various modifications and improvements made by those skilled in the art to the technical solutions of the present invention without departing from the spirit of the present invention should fall within the protection scope defined by the claims of the present invention.

Claims

1. Bacillus amyloliquefaciens ( Bacillus amyloliquefaciens The application of DS11 in promoting plant growth and improving plant salt and alkali resistance is characterized by, The specimen, with accession number CCTCC NO: M 2022685, is deposited at the China Center for Type Culture Collection. The described *Bacillus amyloliquefaciens* exhibits the following NH3 production capacity: 0.84±0.03 mg / mL; organic phosphorus degradation activity: 107.99±4.29 mg / L; inorganic phosphorus dissolution activity: 51.67±6.52 mg / L; sparingly soluble inorganic potassium salt dissolution activity: 4.03±0.25 mg / L; β-glucanase activity: 555.89±12.78 U / L; and IAA production capacity: 148.81±4.83 mg / L.

2. A microbial agent, characterized in that, It contains Bacillus amyloliquefaciens DS11, with accession number CCTCC NO: M2022685, and is deposited at the China Center for Type Culture Collection.

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