Burkholderia sp D8 and application thereof

Abstract

The invention discloses a Burkholderia sp D8 strain and application thereof, the Burkholderia sp D8 strain is preserved in the China General Microbiological Culture Collection Center (CGMCC), the preservation date is November 15, 2024, and the preservation number is CGMCC NO: 32674. The Burkholderia sp D8 strain has the advantages that the Burkholderia sp D8 strain can be used for preparing the Burkholderia sp D8 strain, and the Burkholderia sp D8 strain can be used for preparing the Burkholderia sp D8 strain; the Burkholderia sp D8 disclosed by the invention is convenient to culture and high in growth speed; the capacity of rapidly and efficiently fixing nitrogen and dissolving inorganic phosphorus and organic phosphorus is achieved; the function of rapidly and efficiently generating protease and siderophore is realized; the salt-tolerant range is wide, and the adaptability is high; the compound can be used as a soil remediation agent, and can effectively improve the contents of organic matters, total nitrogen, alkali-hydrolyzable nitrogen, total phosphorus, available phosphorus and the like in soil.

Description

Technical Field

[0001] The invention relates to a bamboo forest Burkholderia sp D8 and application thereof, belonging to the technical fields of microorganisms and ecological restoration. Background Art

[0002] Bamboo is the most Chinese-specific non-wood forest resource and an important component of my country's forest resources. my country's bamboo resource area, species and reserves are among the highest in the world. As of 2021, my country's bamboo forest area is 7.5627 million hm2. 2 , accounting for 3.31% of the forest area, of which bamboo forest area is 5.2776 million hm2 2 , accounting for 69.78% of the total bamboo forest area. Moso bamboo has the characteristics of explosive and renewable growth. After a single afforestation, it can be harvested continuously for decades, and new bamboo shoots and new whips sprout every year for self-renewal. It has an amazing growth rate and contains a strong carbon sequestration capacity (Kuehl et al., 2013; Zhou et al., 2011). It has great potential in mitigating global climate change and has attracted the attention of more and more scientists (Cheng et al., 2022; Song et al., 2020). However, in recent years, due to the continuous decline in bamboo product prices and the continuous increase in labor costs, the economic benefits of bamboo forests have continued to decline, which has seriously affected the enthusiasm of bamboo farmers to operate, and has led to serious abandonment and mismanagement of bamboo forests. On the other hand, due to long-term overfertilization and predatory management, the ecological functions of moso bamboo forests have been seriously degraded, soil carbon emissions have increased, and the ecological fragility has become more and more serious (Chen et al., 2019; Qin et al., 2017).

[0003] Microbial fertilizer is one of the main ways to replace chemical fertilizers to achieve environmental friendliness. Microbial fertilizer is a kind of fertilizer with microbial life activities as its main component, which improves the target crops to obtain beneficial benefits through the activities of microorganisms. As a new type of low-cost and environmentally friendly fertilizer, microbial fertilizer can effectively improve soil, increase fertility, increase crop yields, and reduce crop losses caused by pathogens. It plays an important role in the national agricultural green development strategy.

[0004] The present invention separates a Burkholderia sp D8 from a bamboo forest. The Burkholderia sp D8 has the functions of fixing nitrogen, decomposing inorganic phosphorus, decomposing organic phosphorus, producing protease and iron carrier, and can be used as a soil remediation agent or for preparing a soil remediation agent to increase the contents of organic matter, total nitrogen, alkaline-hydrolyzed nitrogen, total phosphorus and available phosphorus in the soil. Summary of the invention

[0005] The first object of the present invention is to provide a bamboo forest Burkholderia sp D8.

[0006] The second object of the present invention is to provide an application of a bamboo forest Burkholderia sp D8, which is used for soil remediation and promoting plant growth.

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

[0008] A strain of Burkholderia sp D8 from moso bamboo forest was deposited in the General Microbiology Center of China Microorganism Culture Collection (CGMCC), with a deposit date of November 15, 2024 and a deposit number of CGMCC NO:32674.

[0009]

[0010] The bamboo forest Burkholderia sp D8 of the present invention is obtained by screening by the inventor himself.

[0011] The above-mentioned Burkholderia sp D8 from bamboo forest can be used as a soil remediation agent or used for preparing a soil remediation agent, and has a salt tolerance concentration of more than 7%.

[0012] Unless otherwise specified, % in this application are all mass percentages.

[0013] The above-mentioned Burkholderia sp D8 in bamboo forest can be used for nitrogen fixation, inorganic phosphorus decomposition and / or organic phosphorus decomposition.

[0014] The above-mentioned Burkholderia sp D8 from the bamboo forest can be used to produce protease.

[0015] The above-mentioned Burkholderia sp D8 from the bamboo forest can be used to produce siderophores.

[0016] The above-mentioned bamboo forest Burkholderia sp D8 has obvious inorganic phosphorus decomposition, organic phosphorus decomposition, protease production and iron carrier production functions after 18 hours of cultivation, and has obvious nitrogen fixation function after 42 hours of cultivation. However, the existing Burkholderia needs to be cultivated for 5 days or even longer to fix nitrogen and decompose organic phosphorus, and 5-7 days or even longer to decompose inorganic phosphorus.

[0017] The above-mentioned Burkholderia sp D8 in bamboo forest can be used to increase the contents of organic matter, total nitrogen, alkaline-hydrolyzed nitrogen, total phosphorus and available phosphorus in soil.

[0018] The above-mentioned Burkholderia sp D8 from bamboo forests can be used for various crops, and is preferably used for bamboo forests.

[0019] The extraction method of Burkholderia sp D8 in bamboo forest is as follows: the soil of bamboo forest is diluted with sterile physiological saline, and then coated on TSB solid culture medium (tryptone 17.0g, soy peptone 3.0g, sodium chloride 5.0g, potassium hydrogen phosphate 2.5g, glucose 2.5g, agar 18g, the rest is water, pH 7.0±0.5, sterilized at 115°C for 20 minutes) plate, and then dried and inverted in a constant temperature box at 30°C for 24-48h, and single colonies with different shapes, sizes and colors are picked with an inoculation needle, and they are streaked on the surface of TSB solid culture medium for 18-24h, and this is repeated 5-6 times to form separate isolated colonies, and multiple purified strains are obtained; the purified strains are inoculated into TSB liquid culture medium for 18-24h, and then spot-seeded on the organophosphorus culture medium plate, and the strain with the first transparent circle is strain D8.

[0020] The colony diameter of the bamboo forest Burkholderia sp D8 on the TSB solid culture medium plate is 0.5-0.8 mm, the colony is round, the edges are neat, opaque, the front is light yellow, the surface is smooth, moist, convex, the texture is soft, and it is easy to pick up. The strain number can reach 1×10 when cultured in TSB culture medium (tryptone 17.0 g, soy peptone 3.0 g, sodium chloride 5.0 g, dipotassium hydrogen phosphate 2.5 g, glucose 2.5 g, the rest is water, pH 7.0±0.5, sterilized at 115°C for 20 minutes). 8 cfu / ml.

[0021] The technologies not mentioned in the present invention are all referred to the prior art.

[0022] The bamboo forest Burkholderia sp D8 of the present invention has the following beneficial effects: 1. It is easy to cultivate and has a fast growth rate; 2. The strain has the ability to quickly and efficiently fix nitrogen, decompose inorganic phosphorus and decompose organic phosphorus; 3. The bacterium has the function of quickly and efficiently producing protease and iron carrier; 4. It has a wide salt tolerance range and strong adaptability; 5. It can be used as a soil repair agent and can effectively increase the content of organic matter, total nitrogen, alkaline nitrogen, total phosphorus and available phosphorus in the soil. BRIEF DESCRIPTION OF THE DRAWINGS

[0023] Figure 1 The growth of the Burkholderia sp D8 in the bamboo forest of the present invention on the inorganic phosphorus culture medium plate;

[0024] Figure 2 The growth of the Burkholderia sp D8 in the bamboo forest of the present invention on the organic phosphorus culture medium plate;

[0025] Figure 3The growth of the Burkholderia sp D8 in the bamboo forest of the present invention on a skimmed milk powder medium plate;

[0026] Figure 4 The figure shows the growth of Burkholderia sp D8 from the bamboo forest of the present invention on a CAS agar medium plate.

[0027] Figure 5 The growth of the bamboo forest Burkholderia sp D8 of the present invention on the Axubei nitrogen-fixing medium plate; DETAILED DESCRIPTION

[0028] In order to better understand the present invention, the content of the present invention is further explained below in conjunction with the embodiments, but the content of the present invention is not limited to the following embodiments.

[0029] Embodiment 1

[0030] Obtaining Burkholderia sp D8:

[0031] 1 Preparation of dilution: Take 10g of soil from bamboo forest in Anji, Zhejiang Province, add 90ml of sterile saline (mass concentration 0.9%), shake thoroughly and mix. Then dilute with sterile saline to 10 -1 , 10 -2 , 10 -3 , 10 -4 , 10 -5 , 10 -6 , 10 -7 , 10 -8 , 10 -9 There are nine concentration gradients in total.

[0032] 2 Cultivation: Select 10 -7 , 10 -8 , 10 -9 200ul of each of the three dilution gradients was taken and spread on TSB solid culture medium plates (17.0g tryptone, 3.0g soy peptone, 5.0g sodium chloride, 2.5g potassium dihydrogen phosphate, 2.5g glucose, 18g agar, the rest water, pH 7.0±0.5, sterilized at 115℃ for 20 minutes), cooled and dried, and then inverted in an incubator for culture at 30℃.

[0033] 3. Isolation and purification of strains: Cultivate on TSB solid culture medium at 30℃ for 48 hours until colonies appear. Use an inoculation needle to pick up individual colonies of different shapes, sizes, and colors, and streak them on the surface of TSB solid culture medium for 24 hours. Repeat this 6 times to form separate isolated colonies and obtain multiple purified strains.

[0034] The purified strains were inoculated into 5 mL of TSB liquid medium (17.0 g tryptone, 3.0 g soy peptone, 5.0 g sodium chloride, 2.5 g potassium dihydrogen phosphate, 2.5 g glucose, and the rest water, pH 7.0). 7.0±0.5, sterilized at 115℃ for 20 minutes), placed at 30℃, 180r / min, and cultured for 24h as seed solution; 10ul seed solution was spotted on an organophosphorus culture medium plate (glucose 10.0g / L, ammonium sulfate 0.5g / L, sodium chloride 0.3g / L, magnesium sulfate 0.3g / L, manganese sulfate 0.03g / L, potassium sulfate 0.3g / L, ferrous sulfate 0.03g / L, lecithin 5.0g / L, agar 15.0g / L, pH 7.0-7.5, high pressure sterilization at 115℃ for 30min), and it was observed that a transparent circle appeared around strain D8 at 18h (indicating that the strain has the ability to decompose organophosphorus), while the other strains began to show transparent circles after 48h, that is, the strain D8 showed the first transparent circle.

[0035] 4. Glycerol preservation method: Select the D8 seed liquid obtained in step 3, inoculate it into a new TSB liquid medium at 1% (volume %), and culture it at 30°C and 180r / min for 24 hours to obtain the strain culture liquid. Add 0.8ml of strain culture liquid and 0.2ml of glycerol to a sterile strain preservation tube, mix well, and store in a -80°C refrigerator.

[0036] 5. Bacterial species identification

[0037] (1) Morphological characteristics

[0038] In TSB liquid medium, it is uniformly turbid with a trace of precipitation, which will disperse with a slight shake. On TSB solid medium plates, the colony diameter is 0.5-0.8 mm, the colony is round, with neat edges, opaque, light yellow on the front, smooth, moist, convex surface, soft texture, and easy to pick up.

[0039] (2) 16S rRNA sequence analysis

[0040] The isolated strain D8 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.

[0041] Upstream primer (27F): 5'-A AGAGTTTGATCMTGGCTCAG-3'

[0042] Downstream primer (1492R): 5'-GGTTACCTTGTTACGACTT-3'

[0043] The strains screened were used as templates for PCR amplification. Reaction conditions: 95°C pre-denaturation for 5 min, 94°C denaturation for 50 s, 57°C annealing for 30 s, 72°C extension for 1 min 30 s, and 30 cycles. The target fragments were cloned and sequenced according to conventional methods (Sambrook, et al., 2001). The sequencing sequence of strain D8 is as follows:

[0044] Burkholderia sp D8

[0045]

[0046] The strain sequence determination results were compared with the 16S rRNA sequences registered in GenBank (accession numbers: NZ_CP011505.1, NZ_LR025744.1, NZ_CP156687.1 and NZ_CP009800.1, etc.), and the gene homology reached more than 99.50%. According to "Bergey's Manual of Determinative Bacteriology" (Holt, JG, Gibbons, NE, 1994) and "Common Bacterial System Identification Manual" (Dong Xiuzhu and Cai Miaoying, etc., 2001), strain D8 was determined to be Burkholderia through sexual characteristics and 16S rRNA sequence analysis.

[0047] Embodiment 2

[0048] Determination of the ability of strain D8 to decompose inorganic phosphorus

[0049] After activating the strain stored in the test tube slant, inoculate it on TSB liquid medium for activation. After culturing for 24 hours, use it as seed solution. Spot 10ul of seed solution on inorganic phosphorus medium plate (glucose 10.0g / L, ammonium sulfate 0.5g / L, sodium chloride 0.3g / L, magnesium sulfate 0.3g / L, manganese sulfate 0.03g / L, potassium sulfate 0.3g / L, ferrous sulfate 0.03g / L, calcium phosphate 5.0g / L, agar 15.0g / L, pH 7.0-7.5, autoclave at 115℃ for 30min), with 2 spots per dish, and repeat three times. Figure 2 As shown, after constant temperature culture at 30℃ for 18h, a transparent circle was observed around the strain, indicating that the strain has the ability to degrade organic phosphorus.

[0050] Embodiment 3

[0051] Determination of the efficiency of degrading inorganic phosphorus by strain D8

[0052] After the strain stored in the test tube slant was activated, it was inoculated on TSB liquid medium for activation. After 24 hours of culture, it was used as seed liquid. 100ul of seed liquid was inoculated into inorganic phosphorus liquid medium (glucose 10.0g / L, ammonium sulfate 0.5g / L, sodium chloride 0.3g / L, magnesium sulfate 0.3g / L, manganese sulfate 0.03g / L, potassium sulfate 0.3g / L, ferrous sulfate 0.03g / L, calcium phosphate 5.0g / L, pH 7.0-7.5, 115℃ high pressure sterilization for 30min), and 100ul of sterile water was added to the control group. Each group was repeated three times. Subsequently, it was placed on an oscillator at 180r / min, 30℃, and cultured for 18h; after the oscillation was completed, the culture solution was centrifuged at a speed of 10000r / min for 5min, and the supernatant was taken to determine its effective phosphorus content using the molybdenum antimony colorimetric method. Compared with the control group, the phosphorus solubility rate of D8 was 169.17% to 232.39%.

[0053] Embodiment 4

[0054] Determination of the ability of strain D8 to degrade organic phosphorus

[0055] After activating the strain stored in the test tube slant, inoculate it on TSB liquid medium for activation. After culturing for 48 hours, use it as seed solution. Spread 10ul of seed solution on an organic phosphorus medium plate (glucose 10.0g / L, ammonium sulfate 0.5g / L, sodium chloride 0.3g / L, magnesium sulfate 0.3g / L, manganese sulfate 0.03g / L, potassium sulfate 0.3g / L, ferrous sulfate 0.03g / L, lecithin 5.0g / L, agar 15.0g / L, pH 7.0-7.5, autoclave at 115℃ for 30min), with 2 planting points per dish, and repeat three times. Figure 3 As shown, after constant temperature culture at 30°C for 18 hours, a transparent circle was observed around the strain, indicating that the strain has the ability to degrade organic phosphorus.

[0056] Embodiment 5

[0057] Salt tolerance assay of strain D8

[0058] After the strain stored in the test tube slant was activated, it was inoculated on TSB liquid medium for activation. After culturing for 24 hours, it was used as seed liquid. The seed liquid was transferred to LB liquid medium (peptone 10g / L, NaCl 10g / L, yeast extract 5g / L, the rest was water, pH 7.0, sterilized at 121℃ for 20min) containing 0%, 1%, 3%, 4%, 5%, 7%, and 20% NaCl at an inoculation rate of 1%, respectively. The culture was carried out at 180r / min and 30℃ for 24 hours, and the absorbance OD was measured. 600 From Table 1, it can be seen that strain D8 has a wide range of salt tolerance (0-7%) in NaCl medium.

[0059]

[0060] Embodiment 6

[0061] Determination of protease production ability of strains

[0062] After activating the strain stored in the test tube slant, inoculate it on TSB liquid medium for activation. After culturing for 48 hours, use it as seed liquid. Spread 10ul of seed liquid on skim milk powder medium plate (skim milk powder 15g, agar powder 16g, water 1000mL, pH 7.0-7.2, sterilize at 115℃ for 15min), with 2 planting points per dish, and repeat three times. Figure 4 As shown, after constant temperature culture at 30°C for 18 hours, a transparent circle was observed around the strain, indicating that the strain can produce protease.

[0063] Embodiment 7

[0064] Functional identification of siderophore production by strains

[0065] After activating the strain stored in the test tube slant, inoculate it on TSB liquid medium for activation. After culturing for 24 hours, use it as seed liquid. Take 5μL of seed liquid and inoculate it on CAS solid medium (glucose 4.0g, peptone 5.0g / L, KCl0.5g / L, MgSO4·7H2O 0.5g / L, pH 6.8, water 800mL; add 100mL phosphate buffer and 100mL 0.06g / L CAS detection solution, mix well, sterilize at 115℃ for 20min), inoculate two points on each culture dish, repeat three times. Invert and place in a 30℃ constant temperature box and culture for 18h. Figure 5 As shown, a yellow halo appeared in the blue CAS medium, and the color change was very deep, indicating a strong ability to produce siderophores.

[0066] Embodiment 8

[0067] Determination of nitrogen fixation ability of strain D8

[0068] After activating the strain stored in the test tube slant, inoculate it in TSB liquid culture medium (17.0g tryptone, 3.0g soy peptone, 5.0g sodium chloride, 2.5g potassium dihydrogen phosphate, 2.5g glucose, the rest is water, pH 7.0±0.5, sterilize at 115℃ for 20 minutes) for activation. After culturing for 48 hours, use it as seed liquid and spot 10ul of seed liquid on the Axubei nitrogen-fixing medium plate (0.2g / L potassium dihydrogen phosphate, 0.2g / L sodium chloride, 0.2g / L magnesium sulfate, 5.0g / L calcium carbonate, 0.1g / L calcium sulfate, 10g / L mannitol, 15.0g / L agar, pH 7.0-7.5, autoclave at 115℃ for 30min), with 2 spots per dish, and repeat three times. Figure 1As shown, after constant temperature cultivation at 30°C for 42 hours, it was observed that the bacteria grew well, indicating that the strain has the ability to fix nitrogen.

[0069] Embodiment 9

[0070] Effects of strain D8 on physical and chemical properties of bamboo forest soil

[0071] After the strain stored in the test tube slant was activated, it was inoculated on TSB liquid medium for activation and cultured at 30℃ for 24h as seed liquid. 5ml of seed liquid was inoculated into 1000ml of LB liquid medium and cultured at 30℃ for 24h. The culture liquid after fermentation was diluted to 50L in equal amounts and sprayed on the bamboo forest, with no spraying of bacterial liquid as the control. After 120d, it was found that compared with the control, spraying strain D8 and others significantly increased the content of organic carbon, total nitrogen, total phosphorus, alkaline nitrogen and available phosphorus in the soil (Table 2).

[0072] Table 2 Effects of strain AN8 on the physical and chemical properties of bamboo forest soil

[0073]

[0074] The above contents are further detailed descriptions of the present invention in combination with specific preferred embodiments, and it cannot be determined that the specific implementation of the present invention is limited to these descriptions. For ordinary technicians in the technical field to which the present invention belongs, several simple deductions can be made without departing from the concept of the present invention, which should be regarded as falling within the protection scope of the present invention.

Claims

1. A plant of Burkholderia sp D8, Its characteristics are: It is deposited at the General Microbial Center of the China Microbial Sperm Preservation and Management Committee. The deposit date is November 15, 2024, and the deposit number is CGMCCNO:32674.

2. The Burkholderia sp D8 of claim 1, Its characteristics are: Its 16SrDNA nucleotide sequence is: ACTTCTGGTAAAACCCACTCCCATGGTGTGACGGCGGTGTGTACAAGACCCGGGAACGTATTCACCGCGGCATGCTGATCCGCGATTACTAGCGATTCCAGCTTCATGCACTCGAGTTGCAGAGTGCAATCCGGACTACGATCGGTTTCTGGGA TTAGCTCCCCCCTCGCGGGTTGGCAACCCCTCTCTGTTCCGACCATTGTATGACGTGTGAAGCCCTACCCATAAGGGCCATGAGGACTTGACGTCATCCCCACCTTCCTCTCCCGGTTTGTCACCGCAGTCTCCTTAGAGTGCTCTTGCGTAGCAACTAAGG ACAAGGGTTGCGCTCGTTGCGGGACTTAACCCCAACATCTCACGACGAGCTGACGACAGCCATGCAGCCATGCAGCACCTGTGTATCGGTTCTCTTTCGAGCACTCCCCACCTCTCAGCAGGATTCCGACCATGTCAAGGGTAGGTAAGGTTTTCGCGTTGCA TCGAATTAATCCACATCATCCACCACCGCTTGTGCGGGTCCCCGTCAATTCCTTTGAGTTTAATCTTGCGACCGTACTCCCCCAGGCGGTCAACTTCACGCGTTAGCTACTAAGGAAATGAATCCCCCAACAACTAGTTGACATCGTTTAGGGCGT GGACTACCAGGGTATCTAATCCTGTTTGCTCCCCACGCTTTCGTGCATGAGCGTCAGTATTGGCCCAGGGGCTGCCTTCGCCATCGGTATTCCTCCACATCTCTACGCATTTCACTGCTACACGTGGAATTCTAG CCTGCCAGTCACCAATGCAGGTCCCAGGTTGAGCCCGGGATTTCACATCGGTCTTAGCAAACCGCCTGCGCACGCTTTACGCCCAGTAATTCCGATTAACGCTCGCACCCTACGTATTACCGCGGCTGCTGGCACGTAGTTAGCCGGTGCTTATTC TTCCGGTACCGTCATCCCCGGCTATATTAGAACCAAGGATTTCTTTCCGGACAAAAGTGCTTTACAACCCGAAGGCCTTCTTCACACACGCGGCATTGCTGGATCAGGCTTTCGCCATTGTCCAAAATTCCCCACTGCTGCCTCCCGTAGGAGT CTGGGCCGTGTCTCAGTCCCAGTGTGGCTGTCGTCGTCCTCTCAGACCAGCTACTGATCGTCGCCTTGGTAGCCTTTACCCCACCAACTAGCTAATCAGCCATCGGCCAACCCTATAGCGCGAGGCCCCGAAGGTCCCCCGCTTTCATCCGTAGATCG.

3. The use of Burkholderia sp D8 as claimed in claim 1 or 2, Its characteristics are: Used as a soil repair agent, or used to prepare soil repair agents.

4. The use of Burkholderia sp D8 according to claim 3, Its characteristics are: The salt tolerance concentration is above 7%.

5. The use of Burkholderia sp D8 as claimed in claim 1 or 2, Its characteristics are: Used for nitrogen fixation, deorganic phosphorus and / or deorganic phosphorus.

6. The use of Burkholderia sp D8 as claimed in claim 1 or 2, Its characteristics are: Used for protease production.

7. The use of Burkholderia sp D8 of claim 1 or 2, Its characteristics are: Used for iron-producing carriers.

8. Use of Burkholderia sp D8 as claimed in claim 1 or 2, Its characteristics are: Used to increase the content of organic matter, total nitrogen, alkaline nitrogen, total phosphorus and effective phosphorus in soil.

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