Lysinibacillus and application thereof

Abstract

The invention belongs to the technical field of microorganisms, and discloses lysine bacillus and application thereof. A strain of lysinibacillus is obtained from soil of a lysinibacillus iri planting plot, the classification name of the lysinibacillus iri is Lysinibacillus iri, the 16S rRNA sequence of the lysinibacillus iri is shown as SEQ ID NO.1, and the lysinibacillus iri has nitrogen fixation capacity, inorganic phosphorus dissolving capacity and IAA secreting capacity, has a remarkable promoting effect on growth of liquorice seedlings and is expected to serve as a plant growth-promoting bacterium to be used for planting of various crops.

Description

Technical Field

[0001] The invention belongs to the technical field of microorganisms and relates to biological control, and in particular to a lysine bacillus strain and application thereof. Background Art

[0002] The Chinese herbal medicine planting industry has been booming in recent years. However, many years of continuous cropping and excessive use of chemical fertilizers have led to soil degradation and a decline in the yield and quality of Chinese herbal medicines. These problems have seriously restricted the development of the Chinese herbal medicine industry. In addition, the large-scale use of chemical fertilizers has had a negative impact on the agricultural ecological environment and is not conducive to the sustainable development of agriculture.

[0003] Licorice is a perennial herb with important medicinal value. It is also widely used in cosmetics, food, light industry and other fields. Therefore, the demand for licorice has been increasing year by year. However, the growth cycle of licorice is relatively long, and it is generally harvested 2 to 4 years after planting. In addition, licorice may be attacked by a variety of pests and diseases such as powdery mildew and rust during its growth, which undoubtedly increases the management cost of licorice during its growth.

[0004] The inoculants prepared by plant growth-promoting bacteria (PGPB) have attracted widespread attention from researchers due to their green, environmentally friendly, plant growth-promoting, medicinal material quality-improving, and rhizosphere soil microorganism-regulating characteristics. They have great advantages and potential in meeting the needs of weight loss and efficiency improvement, agricultural sustainable development, and the construction of traditional Chinese medicine ecological agriculture.

[0005] Nitrogen (N) and phosphorus (P) are essential nutrients for plant growth and development, but 80% of nitrogen in the air cannot be directly used by plants, and the P element in the soil mostly exists in the form of Al-P and Fe-P, which is also difficult to be directly used by plants. Although PGPB has many advantages, the existing PGPB strain resources are still very scarce, and they are rarely used in the cultivation of Chinese medicine raw materials. Therefore, vigorously exploring PGPB strains that can utilize elements such as N and P, improving the ability of plants, especially slow-growing plants with long cultivation cycles, to utilize nutrients, promoting rapid plant growth and shortening the cultivation cycle are problems that need to be urgently solved in existing technologies. Summary of the invention

[0006] In order to promote the rapid growth of licorice plants and improve the ability of plants to utilize nutrients such as N and P, the present invention isolated a lysine Bacillus with the ability to fix nitrogen, dissolve inorganic phosphorus and secrete IAA from a wolfberry planting plot. The preservation information of the lysine Bacillus is as follows:

[0007] Taxonomic nomenclature: Lysinibacillus irui.

[0008] Strain number: XQY-2.

[0009] Depository: General Microbiology Center, China National Microbiological Culture Collection Administration.

[0010] The abbreviation of the collection agency is: CGMCC.

[0011] Address of the depository: Institute of Microbiology, Chinese Academy of Sciences, No. 3, Yard 1, Beichen West Road, Chaoyang District, Beijing.

[0012] Date of preservation: June 3, 2024.

[0013] Deposit number: CGMCC No.30838.

[0014] Furthermore, the 16S rRNA sequence of the lysinic Bacillus is shown as SEQ ID NO.1.

[0015] Furthermore, the lysinic Bacillus has the ability to fix nitrogen.

[0016] Furthermore, the lysinibacillus has the ability to dissolve inorganic phosphorus.

[0017] Furthermore, the lysinic Bacillus has the ability to secrete IAA.

[0018] On the other hand, the present invention further provides the use of the above-mentioned lysinibacillus in promoting plant growth, wherein the lysinibacillus is used as an active ingredient to prepare a bacterial agent, and the bacterial agent promotes plant growth.

[0019] Furthermore, in the above application, the lysinic Bacillus has nitrogen fixation ability.

[0020] Furthermore, in the above application, the lysinic Bacillus has the ability to dissolve inorganic phosphorus.

[0021] Furthermore, in the above application, the lysinic Bacillus has the ability to secrete IAA.

[0022] Furthermore, in the above application, the plant includes liquorice.

[0023] Compared with the prior art, the present invention "a strain of lysinic Bacillus and its application" has the following beneficial effects:

[0024] The lysinibacillus of the present invention is separated from soil and is a natural PGPB strain, which has no pollution to the environment, does not produce harmful chemical residues, and can help improve the quality of licorice.

[0025] The lysinibacillus of the present invention has significant effects of fixing nitrogen and dissolving inorganic phosphorus, which can help improve the ability of licorice to utilize nutrients in the soil and reduce the use of chemical fertilizers. In addition, the lysinibacillus has the ability to secrete IAA, which can effectively promote plant growth, help shorten the plant growth cycle, and reduce field management costs. BRIEF DESCRIPTION OF THE DRAWINGS

[0026] Figure 1 This is the phylogenetic tree of Lysinibacillus irui XQY-2 strain.

[0027] Figure 2 This is the verification result of the nitrogen fixation ability of Lysinibacillus irui XQY-2 strain.

[0028] Figure 3 These are the results of the verification of the ability of Lysinibacillus irui XQY-2 strain to dissolve inorganic phosphorus.

[0029] Figure 4 These are the results of the IAA secretion capability test of Lysinibacillus irui XQY-2 strain. Figure 4 CK+ indicates positive control, CK- indicates negative control, and XQY-2 indicates treatment with the fermentation broth of Lysinibacillus iruiXQY-2 strain.

[0030] Figure 5 is the IAA standard curve.

[0031] Figure 6 The growth-promoting effect of the bacterial agent dilution on licorice seedlings. The seedlings in the upper row are the control group treated with sterile water, and the seedlings in the lower row are the test group treated with the bacterial agent dilution. DETAILED DESCRIPTION

[0032] The technical solution of the present invention is clearly and completely described below in conjunction with the embodiments. Obviously, the described embodiments are only part of the embodiments of the present invention, not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without creative work are within the scope of protection of the present invention.

[0033] Example 1

[0034] This example describes the isolation and identification of Lysinibacillus irui XQY-2 strain.

[0035] 1. Separation

[0036] Use a sterile centrifuge tube to collect 1g of soil from the Ningxia wolfberry planting plot, add 9mL of sterile water, put it in a shaker at 28℃ and 180rpm for 30min, mix well and let it stand, then take 1mL of the supernatant and add it to a test tube containing 9mL of sterile water. Repeat several times to make a soil suspension of 10 -1 , 10 -2 , 10 -3 , 10 -4 100 μL of the supernatant was spread on beef extract peptone medium in turn to culture and isolate the dominant strains. The dominant strains were numbered and stored in -80℃ glycerol tubes for later use. After separation, a strain numbered XQY-2 was obtained.

[0037] 2. Identification

[0038] 1) The preserved XQY-2 strain was inoculated into beef extract peptone medium and cultured in a shaking incubator at 28°C and 180 rpm for 24 to 48 h.

[0039] 2) After centrifugation, a small amount of bacteria was taken and genomic DNA was extracted using a bacterial genomic DNA extraction kit (Shenzhen Segeno Biotechnology Co., Ltd., catalog number: R3040-50). The extraction steps were referred to the instructions of the bacterial genomic DNA extraction kit.

[0040] 3) Using genomic DNA as a template, PCR amplification was performed using universal primers 27F and 1492R. The primer sequences are as follows:

[0041] 27F: 5'-AGAGTTTGATCCTGGTCAGAACGAACGCT-3';

[0042] 1492R: 5'-TACGGCTACCTTGTTACGACTTCACCC-3'.

[0043] The PCR amplification reaction system (50 μL) includes: 5× buffer 10 μL; dNTP 4 μL; upstream primer 27F (10 μM) 1 μL; downstream primer 1492R (10 μM) 1 μL; Prime STAR 0.5 μL; template (1 μM) 1 μL; ddH2O 32.5 μL.

[0044] The PCR reaction conditions were as follows: pre-denaturation at 95°C for 5 min; denaturation at 94°C for 30 s, annealing at 55°C for 30 s, and extension at 72°C for 2 min, for 30 cycles; and extension at 72°C for 5 min.

[0045] 4) The amplified product was sequenced (Beijing Qingke Biotechnology Co., Ltd.), and the 16SrRNA sequence of the XQY-2 strain was obtained as shown in SEQ ID NO.1.

[0046] 5) SEQ ID NO.1 was compared with the NCBI nucleic acid database using BLAST, and some strains with high similarity were selected for phylogenetic analysis with the XQY-2 strain. The phylogenetic tree was constructed using the neighbor-joining method in MEGAX software ( Figure 1 ). According to the homology and phylogenetic tree, the XQY-2 strain was determined to be a bacterium of the genus Lysinibacillus, classified and named Lysinibacillus irui, and is currently deposited in the General Microbiology Center of the China Microbiological Culture Collection Administration, with a deposit number of CGMCC No.30838.

[0047] Example 2

[0048] This example describes the detection of nitrogen fixation ability of Lysinibacillus irui XQY-2 strain.

[0049] 1. Culture medium

[0050] Ashby nitrogen-free medium: mannitol 10.0 g, potassium dihydrogen phosphate 0.2 g, magnesium sulfate 0.2 g, sodium chloride 0.2 g, calcium sulfate dihydrate 0.1 g, calcium carbonate 5.0 g, agar 18-20 g, 0.5% Congo red solution 10 mL, distilled water 1000 mL, pH adjusted to 7.0, and sterilized by high-pressure steam at 121°C for 20 min.

[0051] 2. Test methods

[0052] The agar block confrontation method was used to detect whether Lysinibacillus irui XQY-2 had nitrogen fixation. The bacterial lawn of Lysinibacillus irui XQY-2 was scraped with a sterilized bamboo stick and inoculated into the sterilized Ashby nitrogen-free medium. Then it was inverted and placed in a 28°C constant temperature incubator for 3 to 7 days to observe whether a nitrogen-fixing transparent circle was formed at the inoculation site.

[0053] After the culture, it was found that the Lysinibacillus iruiXQY-2 strain could form a nitrogen-fixing transparent zone on Ashby nitrogen-free medium, indicating that it has a nitrogen-fixing effect.

[0054] The nitrogen fixation ability was further tested by the agar block confrontation method. The end of a sterilized 1mL pipette tip was used to punch holes in the solid culture medium where the bacteria grew, and agar truffle cakes of equal diameter were made. Then, the agar truffle cakes were inoculated in Ashby nitrogen-free medium, and 4 replicates were set. The culture was cultured in a 28℃ constant temperature incubator for 3 to 7 days. The diameter D of the nitrogen-fixing transparent circle and the diameter d of the agar truffle cake were measured and recorded using the cross method, and D / d was calculated. The D / d value of the Lysinibacillus irui XQY-2 strain was 2.50±0.13, indicating that the nitrogen fixation ability was strong. Figure 2 shown.

[0055] Example 3

[0056] This example describes the detection of the ability of Lysinibacillus irui XQY-2 strain to solubilize inorganic phosphorus.

[0057] 1. Culture medium

[0058] NBRIP inorganic phosphorus medium: sucrose (glucose) 10.0 g, calcium phosphate 5.0 g, magnesium chloride 5.0 g, magnesium sulfate 0.25 g, potassium chloride 0.2 g, ammonium sulfate 0.1 g, agar 18-20 g, distilled water 1000 mL, pH adjusted to 7.0, and sterilized by high-pressure steam at 115°C for 30 min.

[0059] 2. Test methods

[0060] The agar block confrontation method was used to detect whether the Lysinibacillus irui XQY-2 strain has the function of dissolving inorganic phosphorus. The bacterial lawn of the Lysinibacillus iruiXQY-2 strain was scraped with a sterilized bamboo stick and inoculated into the sterilized NBRIP inorganic phosphorus medium. Then it was inverted and placed in a 28℃ constant temperature incubator for 3 to 7 days to observe whether a phosphorus dissolving ring was formed at the inoculation site. After the incubation, it was found that the Lysinibacillus iruiXQY-2 strain could form a phosphorus dissolving ring on the NBRIP inorganic phosphorus medium, indicating that it has the function of dissolving inorganic phosphorus.

[0061] The inorganic phosphorus solubility ability was further tested by the agar block confrontation method. The end of the sterilized 1mL pipette tip was used to punch holes on the solid culture medium where the bacteria grew, and agar truffle cakes of equal diameter were made. Then, the agar truffle cakes were inoculated in the NBRIP inorganic phosphorus culture medium, and 4 replicates were set. The culture was cultured in a 28℃ constant temperature incubator for 3 to 7 days, and the diameter D of the phosphorus solubilization circle and the diameter d of the agar truffle cake were measured and recorded, and D / d was calculated. The measurement method is the same as above.

[0062] like Figure 3As shown, Lysinibacillus irui XQY-2 strain can form a phosphate-dissolving circle on the NBRIP inorganic phosphorus medium, indicating that it has the ability to dissolve inorganic phosphorus, and the D / d value is 1.41±0.05.

[0063] Example 4

[0064] This example describes the detection of the IAA secretion ability of Lysinibacillus irui XQY-2 strain.

[0065] 1. Preparation of Salkowski reagent colorimetric solution and culture medium

[0066] Salkowski reagent colorimetric solution: 150mL H2SO4, 250mL ddH2O, 7.5mL FeCl3 (0.5M).

[0067] Kings medium: 20.0 g of tryptone, 1.50 g of dipotassium hydrogen phosphate, 1.50 g of magnesium sulfate, 1000 mL of distilled water, pH adjusted to 7.2, and sterilized by high-pressure steam at 121°C for 20 min.

[0068] 2. Qualitative determination

[0069] 1) Take the frozen Lysinibacillus irui XQY-2 strain and inoculate it into 20 mL LB liquid culture medium at a 1% inoculation amount, and culture it in a shaking incubator at 180 rpm and 28° C. until the OD600 of the bacterial liquid is about 0.8.

[0070] 2) Take the activated bacterial solution and inoculate it into Kings liquid medium containing tryptophan with a final concentration of 0.2 g / L at a 1% inoculation rate. Repeat three times and culture in a shaker at 180 rpm and 28° C. for 3 days.

[0071] 3) After the culture is completed, the fermentation liquid is centrifuged at 12000rpm for 10 minutes, 800μL of the supernatant after centrifugation is placed on a white porcelain plate, an equal volume of Salkowski reagent colorimetric solution is added, and the plate is covered with tin foil to avoid light for 30 minutes. The positive control is 800μL of 100mg / L IAA standard solution and an equal volume of Salkowski reagent colorimetric solution. The negative control is 800μL Kings culture medium and an equal volume of Salkowski reagent colorimetric solution.

[0072] 4) Observe the results after the reaction. If the reaction solution is pink, it means that the Lysinibacillus iruiXQY-2 strain can secrete IAA. The darker the color, the stronger the ability to secrete IAA. If the color of the reaction solution does not change, it means that it does not have the ability to secrete IAA or indole compounds. Figure 4 , Figure 4 CK+ indicates positive control, CK- indicates negative control, and XQY-2 indicates treatment with the fermentation broth of Lysinibacillus iruiXQY-2 strain. Figure 4 It can be seen that after the reaction, the color of the reaction solution is between the negative control group and the positive control group, indicating that the Lysinibacillus irui XQY-2 strain has a certain ability to secrete IAA.

[0073] 3. Quantitative determination

[0074] 1) Drawing of IAA standard curve: Accurately weigh 0.005g of IAA, first dissolve it with a small amount of ethanol, and then dilute it to 50mL with deionized water to obtain an IAA standard stock solution with a concentration of 100mg / L. Accurately measure the standard stock solution, dilute it with deionized water, and obtain IAA standard solutions with standard concentrations of 0, 5, 10, 20, 30, and 40mg / L, respectively, and store them in the dark. Take 2mL of standard solution of each concentration, add an equal volume of Salkowski reagent colorimetric solution, and treat in the dark for 30min. Use a UV-visible spectrophotometer to detect the absorbance of the solution after treatment at 530nm, adjust it to zero with the standard solution of 0mg / L, and use the IAA standard concentration as the horizontal axis and the absorbance value as the vertical axis to draw the IAA standard curve, as shown below: Figure 5 shown.

[0075] 2) The Lysinibacillus irui XQY-2 strain was inoculated into Kings liquid medium containing tryptophan and cultured in triplicate in a shaking incubator at 180 rpm and 28°C for 3 days.

[0076] 3) After the culture is completed, the fermentation liquid is centrifuged at 12000 rpm for 10 min, 2 mL of the supernatant after centrifugation is placed in a glass test tube, 2 mL of Salkowski reagent colorimetric solution is added, and the tube is wrapped with tin foil and reacted for 30 min in the dark.

[0077] 4) After the reaction, the absorbance at 530 nm was measured by a UV spectrophotometer, and the IAA concentration in the supernatant was calculated based on the IAA standard curve. The quantitative determination result of IAA produced by Lysinibacillus irui XQY-2 strain was 10.85±0.37 mg / L.

[0078] Example 5

[0079] This example describes the preparation of a bacterial agent using the Lysinibacillus irui XQY-2 strain.

[0080] 1. Seed activation: Inoculate the frozen Lysinibacillus irui XQY-2 strain into LB solid culture medium and culture at 28-32°C for 24 hours to obtain activated seeds.

[0081] 2. Liquid seed culture: a single colony is selected from the activated seeds and inoculated into LB liquid culture medium, cultured at 180 rpm, 28-32° C. for 24 h, with a pH value of 7.0-7.2, and a seed solution is obtained after the culture is completed.

[0082] 3. Fermentation culture: The seed liquid was inoculated into a triangular flask filled with beef extract peptone liquid culture medium at an inoculum volume of 1%, and cultured at a temperature of 28-32°C, a pH value of 7.0-7.2, and 180-220 rpm for 24-72 hours until the OD600 reached about 1.0, i.e., the bacterial concentration reached 2×10 8 CFU / mL, and the bacterial agent is obtained.

[0083] Example 6

[0084] This example describes the growth-promoting effect of the bacterial agent prepared in Example 5 on licorice seedlings. When used, the bacterial agent was diluted to 1×10 7 CFU / mL.

[0085] 1. Seed pretreatment: First, select licorice seeds with uniform color, smooth seeds, plump and healthy. Soak the selected seeds in 85% concentrated sulfuric acid for 30-45 minutes, stirring every 3-5 minutes. After soaking, rinse with distilled water for 3 times, add 0.1% hydrogen peroxide for disinfection for 10 minutes, and then rinse with distilled water for 2 times. After washing, place in a beaker and soak in distilled water for 8 hours, and use sterilized filter paper to absorb the moisture on the surface of the seeds for later use.

[0086] 2. Culture method: This test adopts the culture dish filter paper method. The following operations are performed in a sterile operating table: two layers of suitable sterile filter paper are placed in a culture dish with a diameter of 90 mm, and diluted to 1×10 7 CFU / mL of the bacterial agent dilution (treatment group T), 5mL of sterile water (control group CK), 30 licorice seeds were evenly placed in each dish, and each treatment was repeated 3 times. Sterile water was added to a constant weight every day to maintain the concentration of the treatment solution. Samples were collected on the 9th day of cultivation to measure the length and thickness of the radicle and plumule of the seedlings.

[0087] 3. Growth index measurement: On the 9th day, 15 seedlings were randomly selected from each group to measure the length and thickness of the radicle and plumule. The plumule length and radicle length were measured with a ruler, and the plumule thickness and radicle thickness were measured with a vernier caliper with an accuracy of 0.01 mm. The measurement results and seedling growth conditions of the experimental group and the control group are shown in Tables 1 and Figure 6 shown. Figure 6 The growth-promoting effect of the bacterial agent dilution on licorice seedlings. The seedlings in the upper row are the control group treated with sterile water, and the seedlings in the lower row are the test group treated with the bacterial agent dilution.

[0088] Table 1. Growth-promoting effect of bacterial solution on licorice seedlings

[0089]

[0090] From Table 1 and Figure 6 It can be seen that the inoculant prepared by the Lysinibacillus iruiXQY-2 strain has a significant growth-promoting effect on licorice seedlings, which is manifested in that the radicle length, plumule length, radicle thickness and plumule thickness of the seedlings are significantly improved compared with the control group CK.

[0091] In summary, the Lysinibacillus irui XQY-2 strain provided by the present invention has the ability to fix nitrogen, dissolve inorganic phosphorus and secrete IAA, can significantly promote the growth of licorice seedlings, and has good application potential in improving plant nutrient supply, improving soil environment, enhancing the plant's ability to utilize nutrients in the soil, helping to reduce the use of chemical fertilizers, and promoting good plant growth.

[0092] The embodiments described above are only some embodiments of the present invention, not all embodiments. The detailed description of the embodiments of the present invention is not intended to limit the scope of the invention claimed for protection, but only represents selected embodiments of the present invention. All other embodiments obtained without creative work and related deductions and substitutions made by ordinary technicians in the field under the conditions of the concept of the present invention belong to the scope of protection of the present invention.

Claims

1. A lysinibacillus strain, characterized in that: The lysinibacillus is classified as Lysinibacillus irui and is deposited in the General Microbiological Center of China Microbiological Culture Collection Administration with a deposit number of CGMCC No.30838.

2. The lysinibacillus according to claim 1, characterized in that The 16SrRNA sequence of the lysinic Bacillus is shown as SEQ ID NO.

1.

3. The lysinibacillus according to claim 1, characterized in that The lysinic Bacillus has the ability to fix nitrogen.

4. The lysinibacillus according to claim 1, characterized in that The lysinic Bacillus has the ability to dissolve inorganic phosphorus.

5. The lysinibacillus according to claim 1, characterized in that The lysinic Bacillus has the ability to secrete IAA.

6. The use of the lysinibacillus according to claim 1 in promoting plant growth, characterized in that: The lysine bacillus is used as an active ingredient to prepare a bacterial agent, which has a promoting effect on plant growth.

7. The use according to claim 6, characterized in that: The lysinic Bacillus has the ability to fix nitrogen.

8. The use according to claim 6, characterized in that: The lysinic Bacillus has the ability to dissolve inorganic phosphorus.

9. The use according to claim 6, characterized in that: The lysinic Bacillus has the ability to secrete IAA.

10. The use according to claim 6, characterized in that: The plants include liquorice.

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