Pseudomonas sp. YP-2023-11 strain and application thereof

Abstract

The invention discloses a pseudomonas strain YP-2023-11 (Pseudomonas sp. YP-2023-11) and an application of the pseudomonas strain YP-2023-11 (Pseudomonas sp. YP-2023-11). The strain is preserved in the China Center for Type Culture Collection (CCTCC), the preservation number is CCTCC No.M 20241193, and the preservation date is June 11, 2024. The pseudomonas sp. YP-2023-11 strain is applied to the application of the pseudomonas sp. YP-2023-11 strain as a lignin degrading bacterium and the application of the pseudomonas sp. YP-2023-11 strain in promoting degradation of tobacco stems in a low-concentration nicotine environment.

Description

Technical Field

[0001] The invention relates to a Pseudomonas sp. YP-2023-11 strain and application thereof, and belongs to the technical field of microorganisms and applications. Background Art

[0002] Tobacco stalks are the main part of the tobacco plant residues left after the tobacco leaves are picked for cigarette production. Due to their high moisture content and hard texture, they are found in the daily processing process to be neither suitable as fuel nor easy to return to the fields as fertilizer. For a long time, how to deal with tobacco plant residues such as tobacco stalks has been a puzzling problem.

[0003] In the actual production process, a large amount of tobacco waste is generated in the flue-cured tobacco production process. If it is discarded or burned at will, it will not only pollute the environment, but also cause the spread of tobacco diseases and pests. The existing visible technology is to crush the tobacco stems to make fertilizer or activated carbon, but in the process of making fertilizer, because the tobacco straw structure is denser than other straws, with high lignin content and nicotine content, it is difficult to decompose under natural conditions. The straw composting agents currently on the market mostly work on corn, rice and other straws, but tobacco stems contain nicotine, which many microorganisms cannot tolerate and are difficult to play a role in the degradation of tobacco stems, which leads to slow formation of fertilizer made from tobacco stems.

[0004] Therefore, the invention aims to solve the technical problem that how to reasonably deal with tobacco waste has become the current resource utilization of agricultural waste. Summary of the invention

[0005] The technical problem to be solved by the present invention is to provide a Pseudomonas sp. YP-2023-11 strain and application thereof, wherein the strain is tolerant to low concentration of nicotine, can degrade tobacco stems well, and can overcome the shortcomings of the prior art.

[0006] The technical solution of the present invention: Pseudomonas sp. YP-2023-11 strain, characterized in that: the strain is preserved in the China Center for Type Culture Collection (CCTCC), the preservation number is CCTCC No. M20241193, and the preservation date is June 11, 2024.

[0007] The Pseudomonas sp. YP-2023-11 strain is used as a lignin-degrading bacterium.

[0008] The Pseudomonas sp. YP-2023-11 strain is used as a tobacco stem degrading bacterium in a low-concentration nicotine environment.

[0009] The Pseudomonas sp. YP-2023-11 strain is used to promote the degradation of tobacco stems by composting organic fertilizers with tobacco stems and other waste as the main raw materials or by directly returning tobacco stems to the fields.

[0010] Compared with the prior art, the present invention isolates and identifies the Pseudomonas sp. YP-2023-11 strain, which is deposited in the China Center for Type Culture Collection (CCTCC) with a deposit number of CCTCC No. M 20241193 and a deposit date of June 11, 2024.

[0011] The obtained Pseudomonas sp. YP-2023-11 strain is derived from tobacco stem waste in a tobacco-growing environment, has a strong adaptability to field conditions, and has tolerance to low concentrations of nicotine through experiments, and can degrade tobacco stems well. Therefore, it can not only be used in organic fertilizer composting with tobacco stems and other waste as the main raw materials, but also can be used in direct return of tobacco stems to the field, and can still play an effective role in the field and in the soil. BRIEF DESCRIPTION OF THE DRAWINGS

[0012] Figure 1 It is the colony morphology of Pseudomonas sp. YP-2023-11 strain;

[0013] Figure 2 Aniline blue fading reaction results of Pseudomonas sp. YP-2023-11 strain;

[0014] Figure 3 Growth of Pseudomonas sp. YP-2023-11 strain on a culture medium plate with tobacco stems as the sole carbon source;

[0015] Figure 4 Changes in the appearance of tobacco stalks 15 days after application of Pseudomonas sp. YP-2023-11 strain.

[0016] The isolated strain Pseudomonas sp. YP-2023-11 of the present invention is deposited in the China Center for Type Culture Collection (CCTCC) with a deposit number of CCTCC No. M 20241193, a deposit date of June 11, 2024, and a deposit address of Wuhan, China. DETAILED DESCRIPTION

[0017] 1. Screening of lignin-degrading bacteria

[0018] 1.1 Source and collection of strain screening materials

[0019] Materials for screening of lignin-decomposing bacteria and sampling locations: tobacco-growing soil in Xixiu District, Anshun.

[0020] 1.2 Preliminary screening of lignin decomposing bacteria

[0021] 1.2.1 Isolation of decomposing bacteria

[0022] Weigh 10 g of soil sample into 90 mL of sterile water containing glass beads and shake it on a shaker at 150 r / min for 1 h, then let it stand for 5 min. Then take 2 mL of the supernatant into the sterilized and cooled enrichment culture medium and culture it at 20°C and 180 r / min for 3 days.

[0023] According to the requirements of sterile operation, the enriched culture fluid was diluted gradiently, and 0.1 mL of each of the dilutions 10-6, 10-7, and 10-8 was applied to different culture media for bacterial isolation; NA medium was used as the bacterial isolation medium; Martin's medium was used as the fungal isolation medium; and modified Gao's medium No. 1 was used as the actinomycete culture medium.

[0024] 1.2.2 Purification of decomposing bacteria

[0025] After colonies grow on the plate, select representative colonies according to the number, morphology, color and surface condition of the colonies, pick out single colonies with a sterile inoculating loop, and streak culture the bacteria and fungi on the corresponding solid culture medium according to the sterile operation requirements until a single colony is purified. For actinomycetes, pick a single colony into different culture media for liquid culture, and then use an inoculating loop to take a small amount of bacterial liquid and streak culture on the actinomycete culture medium until a single colony is obtained. The obtained single colony is stored in a test tube and stored at 4°C for later use after it grows plump.

[0026] 1.2.3 Screening of decomposing bacteria

[0027] Pick out the isolated and purified bacteria, fungi, and actinomycetes and inoculate them on aniline blue medium and guaiacol medium, and do three parallel tests for each colony. Incubate them upside down in a 30°C constant temperature incubator for 3-5 days, observe the growth of the strains, and obtain the strains that produce peroxidase, Mn peroxidase, and laccase based on whether there are transparent circles.

[0028] 2 Strain identification

[0029] 2.1 Morphological identification

[0030] The strains were purified and the colony morphology was described after the strains grew stably. The lignin decomposing bacteria screened were subjected to Gram staining test, and the morphological characteristics were observed under a microscope. The different colors of the staining reaction were used to determine whether they belonged to Gram-positive bacteria (G+) or Gram-negative bacteria (G-).

[0031] 2.2 Molecular Biological Identification

[0032] The bacterial genomic DNA extraction kit (spin column type) from Tiangen Biochemical Technology (Beijing) Co., Ltd. was used to extract the strain DNA. The PCR amplification primers used were bacterial 16SrDNA universal primers, the forward primer was 27F (5'-AGAGTTTGATCMTGGCTCAG-3'), and the reverse primer was 1492R

[0033] (5'-TACGGYTACCTTGTTACGACTT-3'). PCR reaction system 25μL: 1.1×T3 Super PCR Mix 22μL, primer 27F (10μM) 1.0μL, primer 1492R (10μM) 1.0μL, Template DNA 1μL. PCR thermal cycle conditions: 98℃ pre-denaturation 2min, 98℃ denaturation 10min, 55℃ annealing 10s, 72℃ extension 20s, 72℃ repair extension 2min, 4℃ termination reaction∞, 32 cycles. Take 2μL of PCR product for electrophoresis detection on 2% agarose gel.

[0034] The PCR amplification product was subjected to 1% agarose gel electrophoresis to determine the band brightness. The PCR amplification product was sent to the Chongqing Branch of Beijing Qingke Biotechnology Co., Ltd. for gene sequence determination.

[0035] >YP-2023-11

[0036]

[0037] The sequencing results were searched for sequence similarity using the National Center for Biotechnology Information (NCBI) BLAST program.

[0038] Nicotine tolerance test of 3 strains

[0039] The bacteria were inoculated on a medium with tobacco stems (lignin content 13.83%, nicotine content 0.60%) as the only carbon source. After 5 days of culture, large colonies ( Figure 2 ), which proved that the strain was tolerant to nicotine in tobacco stems and could be used as a subsequent test strain.

[0040] Study on the Degradation Ability of 4 Bacteria to Flue-cured Tobacco Straw

[0041] 4.1 Test materials

[0042] Flue-cured tobacco straw: Process the tobacco straw into powder or 2 cm size.

[0043] 4.2 Tobacco stem degradation test

[0044] Take several 500mL triangular flasks, put 50g of processed tobacco straw and 250g of soil in each flask, mix well and sterilize.

[0045] The cultured bacterial solution (strain number ≥ 107) was inoculated into a triangular flask containing 50g of sterilized flue-cured tobacco straw and 250g of soil, and then 100-200g of tobacco straw was added for continuous cultivation. Electron microscopy scanning was performed on the 15th day. Each treatment was repeated 3 times. The tobacco straw degradation rate was determined at the same time. Clean water was used instead of the cultured bacterial solution as a control (CK).

[0046] Degradation rate % = (original straw weight - straw weight after 15 days of degradation) / original straw weight × 100%

[0047] 4.3 Quantitative detection of ligninase activity

[0048] The bacterial liquid cultured for 24 hours was inoculated into the enzyme production medium as the seed liquid and cultured at 30°C and 180r / min. After continuous culture for 5 days, samples were taken and centrifuged at 4°C and 12000r / min for 10 minutes. The supernatant was the crude enzyme liquid. The enzyme activity was determined using the lignin peroxidase (Lip) activity detection kit, manganese peroxidase (Mnp) activity detection kit and laccase (Lac) activity detection kit of Beijing Solarbio Technology Co., Ltd. (Solarbio).

[0049] Test results:

[0050] Pseudomonas sp. YP-2023-11 (Pseudomonas sp. YP-2023-11) Figure 1), the lignin degradation screening and nicotine tolerance test showed that the strain had a strong decolorization ability for aniline blue, with a transparent zone D / d = 3.80 ( Figure 2 ), which has a strong ability to decompose lignin. It was then sent to the China Center for Type Culture Collection (CCTCC), with the deposit number CCTCC No.M 20241193, the deposit date is June 11, 2024, and the deposit address is Wuhan, China.

[0051] The Lip activity of Pseudomonas YP-2023-11 was the highest, which was 137.63 U / mL, and the Mnp activity was 17.08 U / mL.

[0052] The bacteria were inoculated on a medium with tobacco stems (lignin content 13.83%, nicotine content 0.60%) as the only carbon source. After 5 days of culture, large colonies ( Figure 3 ), proving that the strain was tolerant to nicotine in tobacco stems.

[0053] According to the experimental procedure of "Study on the Degradation Ability of 4 Strains on Flue-cured Tobacco Straw", after culturing for 15 days, the electron microscope scanning results showed that ( Figure 4 ), the tobacco stems became loose and porous, showing a more significant degradation state. The degradation rate of the tobacco stems was measured to be 39.76%.

[0054] Finally, it should be noted that the above embodiments are only used to illustrate the technical solution of the present invention rather than to limit it. Although the present invention has been described in detail with reference to the preferred embodiments, those skilled in the art should understand that the technical solution of the present invention can be modified or replaced by equivalents without departing from the purpose and scope of the technical solution, which should be included in the scope of the claims of the present invention.

Claims

1. A Pseudomonas sp. YP-2023-11 strain, characterized in that: The strain is deposited in the China Center for Type Culture Collection (CCTCC) with the deposit number CCTCC No.M 20241193 and the deposit date of June 11, 2024.

2. Use of the Pseudomonas sp. YP-2023-11 strain as claimed in claim 1 as a lignin-degrading bacterium.

3. Use of the Pseudomonas sp. YP-2023-11 strain as claimed in claim 1 as a tobacco stem degrading bacterium in a low nicotine concentration environment.

4. The Pseudomonas sp. YP-2023-11 strain according to claim 3, characterized in that: Its application in promoting the degradation of tobacco stems is to use tobacco stems and other wastes as the main raw materials in organic fertilizer composting or to directly return tobacco stems to the fields.

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