An endophytic Bacillus sp AN8 from bamboo, its use and isolation method

Abstract

The present invention discloses an endophytic Bacillus sp AN8 from bamboo shoots, its uses, and an isolation method. The endophytic Bacillus sp AN8 from bamboo shoots was deposited in the General Microbiology Center of the China Culture Collection Administration Committee on November 20, 2023, with a deposit number of CCTCC NO: 29065. The endophytic Bacillus sp AN8 from bamboo shoots has the ability to fix nitrogen and decompose inorganic phosphorus, and also has the function of producing proteases and amylases. It can be used as a soil remediation agent or for preparing a soil remediation agent to increase the content of organic matter, total nitrogen, hydrolyzable nitrogen, total phosphorus, available phosphorus, and available potassium in the soil; it is easy to cultivate and grows fast; and the isolation method is simple and easy to operate.

Description

Technical Field

[0001] The invention relates to an endophytic bacillus of bamboo, Bacillus sp AN8, a use thereof and an isolation method thereof, and belongs to the fields of microbial technology and ecological restoration. Background Art

[0002] Bamboo, belonging to the subfamily Bambusoideae of the Poaceae family, is an evergreen, perennial, facultative clonal plant that combines economic, ecological, and social benefits, primarily propagating asexually. China, known as the "Bamboo Kingdom," boasts over 500 species of bamboo across 39 genera. The bamboo forest area is 6.4116 million hectares. 2 , accounting for about 2.94% of the national forest area. Phyllostachys praecox is a high-quality bamboo species widely distributed in southern my country. It has many characteristics such as short cultivation cycle, easy cultivation, high yield, early shoots, delicious taste, and high economic benefits.

[0003] The application of chemical fertilizers can increase plant yield and quality, but it will reduce soil water holding capacity, reduce soil fertility, increase soil acidity, reduce microbial populations, and ultimately lead to soil nutrient imbalance. Microbial fertilizers, as an effective alternative to chemical fertilizers, are one of the important ways to increase agricultural product yield and quality and promote sustainable agricultural development. Microorganisms are widely distributed and are major participants in material circulation and energy flow, playing an important role in the growth and health of plants and animals. With the continuous exploration and utilization of microbial resources, the application of microorganisms in the fields of agriculture and forestry is becoming more and more extensive. The present invention isolated an endophytic Bacillus sp AN8 from Phyllostachys praecox, which has the functions of nitrogen fixation, inorganic phosphorus decomposition, and production of protease and amylase. It can be used as a soil remediation agent or for the preparation of soil remediation agents to increase the content of organic matter, total nitrogen, hydrolyzable nitrogen, total phosphorus, available phosphorus and available potassium in the soil. Summary of the Invention

[0004] The first object of the present invention is to provide an endophytic Bacillus sp AN8 from Phyllostachys praecox.

[0005] The second object of the present invention is to provide an application of an endophytic Bacillus sp AN8 of Phyllostachys praecox, which is used for soil remediation and promoting plant growth.

[0006] The third object of the present invention is to provide a method for isolating an endophytic Bacillus sp AN8 from Phyllostachys praecox.

[0007] In order to solve the above technical problems, the technical solutions adopted by the present invention are as follows:

[0008] An endophytic Bacillus sp AN8 from Phyllostachys thunbergii was deposited in the General Microbiology Center of China Culture Collection Administration, with a deposit date of November 20, 2023, and a deposit number of CGMCC NO: 29065.

[0009] The endophytic Bacillus sp AN8 of bamboo shoots of the present invention is obtained by screening by the inventors themselves.

[0010] The diameter of the colony of the endophytic Bacillus sp AN8 of the present invention on the nutrient agar plate is 1-2 mm. The bacteria are round, rough, opaque and white. The number of the strain can reach 1×10 9 cfu / ml or above.

[0011] The endophytic Bacillus sp AN8 of bamboo provided by the invention has the functions of fixing nitrogen, decomposing inorganic phosphorus and producing protease and amylase.

[0012] The 16S rDNA nucleotide sequence of the above-mentioned endophytic Bacillus sp AN8 of bamboo is:

[0013]

[0014] The endophytic Bacillus sp AN8 of Phyllostachys praecox can be used as a soil remediation agent, or used for preparing a soil remediation agent.

[0015] The endophytic Bacillus sp AN8 of Phyllostachys praecox can be used to increase the contents of organic matter, total nitrogen, hydrolyzable nitrogen, total phosphorus, available phosphorus and available potassium in the soil.

[0016] The above-mentioned endophytic Bacillus sp AN8 can be used to produce protease and amylase.

[0017] The above-mentioned endophytic Bacillus sp AN8 of Phyllostachys thunbergii can be used for nitrogen fixation.

[0018] The above-mentioned endophytic Bacillus sp AN8 of Phyllostachys thunbergii can be used to decompose inorganic phosphorus.

[0019] The above-mentioned method for isolating the endophytic Bacillus sp AN8 from Phyllostachys praecox comprises the following steps:

[0020] 1) Rinse the roots of Phyllostachys praecox with sterile water and blot dry with sterile filter paper. Soak in 70% alcohol for 1–2 minutes, then soak in sodium hypochlorite solution for 3–5 minutes. Rinse with sterile water until sterile after plate smear testing. Blot dry the surface of the sample with sterile absorbent paper.

[0021] 2) Cut the roots of Phyllostachys praecox obtained in step 1) into small pieces of 1-2 cm, grind them, and then dilute them with sterile saline for 10-10 min. 4 times;

[0022] 3) Take the dilutions of each concentration and spread them on LB medium plates. After drying, invert and incubate in a constant temperature box at 28-30°C for 48 hours until colonies appear.

[0023] 4) Pick a single colony with an inoculating needle based on its morphology, size, or color, and streak it continuously on the surface of the agar medium. Repeat 5-6 times to form a single isolated colony, thus obtaining a pure culture strain.

[0024] The technologies not mentioned in this invention are all referred to the prior art.

[0025] The endophytic Bacillus sp AN8 of the bamboo of the present invention has the ability to fix nitrogen and decompose inorganic phosphorus, and also has the function of producing protease and amylase, etc., and can be used as a soil remediation agent or for preparing a soil remediation agent, and improves the content of organic matter, total nitrogen, hydrolyzable nitrogen, total phosphorus, available phosphorus and available potassium in the soil; the culture is convenient, the growth rate is fast, and the isolation method is simple and easy to operate. BRIEF DESCRIPTION OF THE DRAWINGS

[0026] Figure 1 The growth of the endophytic Bacillus sp AN8 of the present invention on the Axubei nitrogen-fixing medium plate;

[0027] Figure 2 The growth of the endophytic Bacillus sp AN8 of the present invention on an inorganic phosphorus culture medium plate;

[0028] Figure 3 The growth of the endophytic Bacillus sp AN8 of the present invention on a skim milk powder medium plate;

[0029] Figure 4 The growth of the endophytic Bacillus sp AN8 of the present invention on a soluble starch culture medium plate; DETAILED DESCRIPTION

[0030] In order to better understand the present invention, the content of the present invention is further illustrated below in conjunction with the examples, but the content of the present invention is not limited to the following examples.

[0031] Example 1

[0032] Obtaining Bacillus sp AN8:

[0033] 1. Surface disinfection of plant tissues

[0034] The roots of two-year-old Phyllostachys praecox were rinsed with sterile water until no obvious turbidity was observed and then blotted dry with sterile filter paper. The roots were excised and surface disinfected (Beckers et al., 2017): soaked in 70% alcohol (70% alcohol by volume) for 2 min and then in sodium hypochlorite solution (2.5% active Cl - ) for 5 minutes, then rinse five times with sterile water. Finally, blot the sample surface with sterile absorbent paper. After the final sterile water wash is tested for sterility by plate smear, it is used for subsequent endophyte isolation and screening.

[0035] 2. Prepare dilution solution: Cut the surface-sterilized roots of Phyllostachys praecox into small pieces of 1-2 cm with sterile scissors, grind 5 g of sample with a sterile mortar, transfer it to a conical flask, add 45 ml of sterile saline, shake well and mix, then dilute 10-10 ml with sterile saline. 4 times.

[0036] 3. Culture: Take 200 μl of each dilution and spread it on LB medium plates (10 g / L peptone, 10 g / L NaCl, 5 g / L yeast extract, 16 g / L agar, the rest is water, pH 7.0, sterilize at 121°C for 20 minutes), cool and dry, then invert and culture in a constant temperature box at 30°C for 48 hours until colonies appear.

[0037] 4. Isolation and purification of strains: Pick up a single colony based on morphology, size or color with an inoculation needle, and streak it continuously on the surface of the agar medium for culture. After repeating this process five times, a single isolated colony will be formed, thus obtaining a pure culture strain.

[0038] 5. Glycerol preservation method: Select a single colony obtained in step 4 and inoculate it into 1 mL of LB liquid medium (10 g / L peptone, 10 g / L NaCl, 5 g / L yeast extract, the remainder water, pH 7.0, sterilized at 121°C for 20 minutes). Incubate in a shaking incubator at 37°C, 180 rpm for 12 hours to obtain the strain. Inoculate the strain into new LB liquid medium at a 1% (volume %) inoculum and shake incubate at 37°C, 180 rpm for 24 hours to obtain the strain culture. Add 0.7-0.8 ml of the strain culture and 0.2-0.3 ml of glycerol to a sterile culture tube, mix thoroughly, and store in a -80°C refrigerator.

[0039] 6. Bacterial species identification

[0040] (1) Morphological characteristics

[0041] In liquid culture medium, it is uniformly turbid with a trace amount of precipitation, which will disperse with a gentle shake. On nutrient agar plates, the colony diameter is 1-2 mm, the bacteria are round, rough, opaque, and white. When cultured in LB medium, the strain number can reach 1×10 9 cfu / ml or above.

[0042] (2) 16S rRNA sequence analysis

[0043] The isolated strain AN8 was further analyzed and compared with the 16S rRNA sequence. A pair of primers were designed based on the conserved sequence of bacterial 16S rDNA.

[0044] Upstream primer (27F): 5'-AAGAGTTTGATCMTGGCTCAG-3'

[0045] Downstream primer (1492R): 5'-GGTTACCTTGTTACGACTT-3'

[0046] PCR amplification was performed using the selected strains as templates. Reaction conditions included 30 cycles of pre-denaturation at 95°C for 5 minutes, denaturation at 94°C for 50 seconds, annealing at 57°C for 30 seconds, and extension at 72°C for 1 minute 30 seconds. The target fragments were cloned and sequenced according to conventional methods (Sambrook et al., 2001). The sequence of strain AN8 is as follows:

[0047] Bacillus sp AN8

[0048]

[0049] Sequencing of the strain AN8 was compared with 16S rRNA sequences registered in GenBank (accession numbers: LC769467.1, LC191186.1, and KC250199.1), revealing a gene identity exceeding 99%. Based on the "Bergey's Manual of Determinative Bacteriology" (Holt, JG, Gibbons, NE, 1994) and the "Handbook of Systematic Identification of Common Bacteria" (Dong Xiuzhu and Cai Miaoying, 2001), strain AN8 was identified as Bacillus through morphological characteristics and 16S rRNA sequence analysis.

[0050] Example 2

[0051] Determination of nitrogen fixation ability of strains:

[0052] The strain stored on the test tube slant was activated on a solid LB plate (10 g / L peptone, 10 g / L NaCl, 5 g / L yeast extract, 16 g / L agar, the remainder water, pH 7.0, sterilized at 121°C for 20 minutes). A single colony was selected and inoculated into 1 mL of LB liquid medium (10 g / L peptone, 10 g / L NaCl, 5 g / L yeast extract, the remainder water, pH 7.0, sterilized at 121°C for 20 minutes). The culture was then incubated in a shaking incubator at 37°C, 180 rpm for 12 hours to obtain the strain seed. The strain was then inoculated into a new LB liquid medium at a 1% (volume %) inoculum and incubated at 37°C, 180 rpm for 24 hours to obtain the strain seed solution. 10 μl of seed solution was spotted onto an Axubei nitrogen-fixing medium plate (0.2 g / L potassium dihydrogen phosphate, 0.2 g / L sodium chloride, 0.2 g / L magnesium sulfate, 5.0 g / L calcium carbonate, 0.1 g / L calcium sulfate, 10 g / L mannitol, 15.0 g / L agar, pH 7.0-7.5, autoclaved at 115°C for 30 min), with two spots per plate. The above operation was repeated three times. Incubate at 37°C for 5 days. Figure 1 As shown in the figure, the bacteria grew well, indicating that the strain has the ability to fix nitrogen.

[0053] Example 3

[0054] Determination of the ability of strains to decompose inorganic phosphorus:

[0055] The strain stored on the test tube slant was activated on a solid LB plate (10 g / L peptone, 10 g / L NaCl, 5 g / L yeast extract, 16 g / L agar, the remainder water, pH 7.0, sterilized at 121°C for 20 minutes). A single colony was selected and inoculated into 1 mL of LB liquid medium (10 g / L peptone, 10 g / L NaCl, 5 g / L yeast extract, the remainder water, pH 7.0, sterilized at 121°C for 20 minutes). The culture was then incubated in a shaking incubator at 37°C, 180 rpm for 12 hours to obtain the strain seed. The strain was then inoculated into a new LB liquid medium at a 1% (volume %) inoculum and incubated at 37°C, 180 rpm for 24 hours to obtain the strain seed solution. 10 μl of seed solution was spotted onto an inorganic phosphate medium plate (glucose 10.0 g / L, ammonium sulfate 0.5 g / L, sodium chloride 0.3 g / L, magnesium sulfate 0.3 g / L, manganese sulfate 0.03 g / L, potassium sulfate 0.3 g / L, ferrous sulfate 0.03 g / L, calcium phosphate 5.0 g / L, agar 15.0 g / L, pH 7.0-7.5, autoclaved at 115°C for 30 min), with two spots per plate. The above operation was repeated three times. Incubate at 37°C for 5 days. Figure 2 As shown in the figure, a transparent circle was observed around the strain, indicating that the strain had the ability to degrade organic phosphorus.

[0056] Example 4

[0057] Determination of protease production ability of strains:

[0058] The strain stored on the test tube slant was activated on a solid LB plate (10 g / L peptone, 10 g / L NaCl, 5 g / L yeast extract, 16 g / L agar, the remainder water, pH 7.0, sterilized at 121°C for 20 minutes). A single colony was selected and inoculated into 1 mL of LB liquid medium (10 g / L peptone, 10 g / L NaCl, 5 g / L yeast extract, the remainder water, pH 7.0, sterilized at 121°C for 20 minutes). The culture was then incubated in a shaking incubator at 37°C, 180 rpm for 12 hours to obtain the strain seed. The strain was then inoculated into a new LB liquid medium at a 1% (volume %) inoculum and incubated at 37°C, 180 rpm for 24 hours to obtain the strain seed solution. 10 μl of seed solution was spotted on a skim milk powder medium plate (15 g skim milk powder, 16 g agar powder, 1000 mL water, pH 7.0-7.2, sterilized at 108°C for 15 min), with 2 spots per plate. The above operation was repeated three times. Incubate at 37°C for 5 days. Figure 3 As shown, a transparent zone was observed around the strain, indicating that the strain could produce protease.

[0059] Example 5

[0060] Functional identification of amylase production by strains:

[0061] The strain stored on the test tube slant was activated on a solid LB plate (10 g / L peptone, 10 g / L NaCl, 5 g / L yeast extract, 16 g / L agar, the remainder water, pH 7.0, sterilized at 121°C for 20 minutes). A single colony was selected and inoculated into 1 mL of LB liquid medium (10 g / L peptone, 10 g / L NaCl, 5 g / L yeast extract, the remainder water, pH 7.0, sterilized at 121°C for 20 minutes). The culture was then incubated in a shaking incubator at 37°C, 180 rpm for 12 hours to obtain the strain seed. The strain was then inoculated into a new LB liquid medium at a 1% (volume %) inoculum and incubated at 37°C, 180 rpm for 24 hours to obtain the strain seed solution. Pipette 5 μL of seed solution and spot it on a soluble starch culture medium plate (soluble starch 10.0 g / L, potassium dihydrogen phosphate 1.0 g / L, magnesium sulfate 1.0 g / L, sodium chloride 1.0 g / L, ammonium sulfate 2.0 g / L, ferrous sulfate 0.001 g / L, manganese chloride 0.001 g / L, zinc sulfate 0.001 g / L, agar 10.0 g / L, pH 7.0-7.4, sterilized at 115°C for 20 min). Inoculate two spots on each culture dish and repeat the above operation three times. Place it upside down in a 28°C constant temperature box and culture for 2 days. Under sterile conditions, use 1% iodine solution to stain and continue constant temperature culture. Figure 4 As shown, the edge of the colony of the amylase-producing series has an obvious decolorization halo, which means that the strain has the function of producing amylase.

[0062] Example 6

[0063] Application of strains in bamboo forests:

[0064] The strain stored on the test tube slant was activated on a solid LB plate (10 g / L peptone, 10 g / L NaCl, 5 g / L yeast extract, 16 g / L agar, the remainder water, pH 7.0, sterilized at 121°C for 20 minutes). A single colony was selected and inoculated into 1 mL of LB liquid medium (10 g / L peptone, 10 g / L NaCl, 5 g / L yeast extract, the remainder water, pH 7.0, sterilized at 121°C for 20 minutes). The culture was then incubated in a shaking incubator at 37°C, 180 rpm for 12 hours to obtain the strain seed. The strain was then inoculated into a new LB liquid medium at a 1% (volume %) inoculum and incubated at 37°C, 180 rpm for 24 hours to obtain the strain seed solution. 5 ml of seed liquid was inoculated into 500 ml of LB liquid medium (10 g / L peptone, 10 g / L NaCl, 5 g / L yeast extract, the rest was water, pH 7.0, sterilized at 121°C for 20 minutes), and cultured at 37°C for 24 hours to obtain the fermentation liquid of the strain with a bacterial count of 2 × 10 9cfu / ml. The fermentation broth was diluted 10-fold with tap water and sprayed on the bamboo forest at a rate of 30 L / mu. Spraying was repeated 10 days later, for a total of two sprayings. A control group was treated without bacterial spraying. Testing after 120 days revealed that spraying with strain AN8 significantly increased soil organic matter, total nitrogen, hydrolyzable nitrogen, total phosphorus, available phosphorus, and available potassium compared to the control (Table 1).

[0065] Table 1 Effects of strain AN8 on the physical and chemical properties of bamboo forest soil

[0066] control group AN8 pH 4.70±0.16a 4.55±0.06b Organic matter (g / kg) 22.72±1.43b 41.46±0.63a Total nitrogen (g / kg) 1.42±0.05b 2.27±0.04a Hydrolyzable nitrogen (mg / kg) 134.38±22.47b 238.23±8.67a Total phosphorus (g / kg) 1.41±0.14b 2.08±0.06a Available phosphorus (mg / kg) 605.64±88.95b 832.66±109.06a Fast-acting potassium (mg / kg) 729.14±42.53b 850.22±15.42a

[0067] The above content is a further detailed description of the present invention in conjunction with specific preferred embodiments, and the specific implementation of the present invention should not be considered to be limited to these descriptions. For those skilled in the art to which the present invention belongs, they can make several simple deductions without departing from the concept of the present invention, which should be considered to fall within the scope of protection of the present invention.

Claims

1. A strain of Bacillus sp AN8 from Phyllostachys praecox, characterized by: It was deposited in the General Microbiology Center of China Culture Collection Administration, with the deposit date being November 20, 2023, and the deposit number being CGMCC NO:29065.

2. The use of the endophytic Bacillus sp AN8 from Phyllostachys praecox according to claim 1, characterized in that: It is used as a bamboo forest soil remediation agent, or for preparing a bamboo forest soil remediation agent; it is used to increase the content of organic matter, total nitrogen, hydrolyzable nitrogen, total phosphorus, available phosphorus and fast-acting potassium in bamboo forest soil.

3. The use of the endophytic Bacillus sp AN8 of bamboo shoots according to claim 1, characterized in that: Used for nitrogen fixation.

4. The use of the endophytic Bacillus sp AN8 from Phyllostachys praecox according to claim 1, characterized in that: Used to dissolve inorganic phosphorus.

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