A salt-tolerant actinomycete in saline-alkali land and its application

Abstract

The present invention provides a salt-tolerant actinomycete in saline-alkali land and its application, named as strain NHKJ0003, which has been deposited in the General Microbiological Center of China Microbiological Culture Collection Committee on April 26, 2024, with a deposit number of CGMCC No.30450, and a deposit address of No. 3, Yard No. 1, Beichen West Road, Chaoyang District, Beijing, Institute of Microbiology, Chinese Academy of Sciences, and taxonomic name Streptomyces rochei. The strain can inhibit Fusarium oxysporum, Pseudomonas aeruginosa and Alternaria alternata. Compared with traditional chemical pesticides for preventing and controlling plant diseases, it has significant prevention and control effects, no pollution to the soil, green and environmentally friendly, and has broad application prospects.

Description

Technical Field

[0001] The invention belongs to the technical field of environmental microorganisms and relates to a salt-tolerant actinomycete in saline-alkali land and application thereof. Background Art

[0002] Salt-alkali land is a widespread type of problematic land distributed over a large area in northern my country. The so-called salinization is caused by the excessive salt content in the soil, so that the salt deposits and the corrosiveness of chemical substances will make the land infertile and lack of materials suitable for crop growth.

[0003] Microorganisms are an important component of soil, especially some microorganisms that must use plant residues as energy, such as nitrifying bacteria and humic bacteria. Soil microorganisms can affect vegetation in many ways, such as participating in biotransformation processes, providing nutrients, and positively / negatively regulating the loss of nutrients in the soil, pathogens, beneficial organisms, etc. on the health of the soil. Therefore, studying the diversity and abundance of soil microorganisms in saline-alkali land is of great value for the management of saline-alkali land and the improvement of feed crops. Summary of the invention

[0004] The invention aims at a salt-tolerant actinomycete in saline-alkali land and application thereof, which is named as strain NHKJ0003. The actinomycete can inhibit Fusarium oxysporum, Pseudomonas aeruginosa and Alternaria alternata.

[0005] To achieve the above object, the technical solution adopted by the present invention is:

[0006] The present invention provides a salt-tolerant actinomycete in saline-alkali land, named as strain NHKJ0003, which was deposited in the General Microbiology Center of China Microorganism Culture Collection Committee on April 26, 2024, with a deposit number of CGMCC No.30450. The deposit address is Institute of Microbiology, Chinese Academy of Sciences, No. 3, Yard 1, Beichen West Road, Chaoyang District, Beijing, and the taxonomic name is Streptomyces rochei.

[0007] Furthermore, the 16S rDNA sequence of strain NHKJ0003 is shown in SEQ ID NO:3.

[0008] The present invention also provides the use of the salt-tolerant actinomycetes in saline-alkali land in inhibiting pathogenic bacteria, wherein the pathogenic bacteria include Fusarium oxysporum, Staphylococcus aureus and Alternaria alternata.

[0009] The present invention further provides a plant bacteriostatic agent, comprising the above-mentioned saline-alkali land salt-tolerant actinomycetes.

[0010] The beneficial effects of the present invention are:

[0011] The present invention screened a salt-tolerant actinomycete from a saline-alkali soil sample, named strain NHKJ0003, which can inhibit Fusarium oxysporum, Pseudomonas aeruginosa and Alternaria alternata. Compared with traditional chemical pesticides for preventing and controlling plant diseases, it has significant prevention and control effects, no pollution to the soil, is green and environmentally friendly, and has broad application prospects. BRIEF DESCRIPTION OF THE DRAWINGS

[0012] Figure 1 Shows some of the actinomycetes obtained in the initial screening.

[0013] Figure 2 Some actinomycetes obtained by purification and culture after rescreening.

[0014] Figure 3 A graph showing the inhibitory effect of strain No. 2 (i.e., the deposited strain NHKJ0003) on Fusarium oxysporum LYG 9-1.

[0015] Figure 4 A graph showing the inhibitory effect of strain No. 2 (i.e., the preserved strain NHKJ0003) on Alternaria alternata sy-1.

[0016] Figure 5 A graph showing the inhibitory effect of strain No. 2 (i.e., the deposited strain NHKJ0003) on Pseudomonas aeruginosa JX-8.

[0017] Figure 6 The phylogenetic tree analysis of strain No. 2 (i.e., deposited strain NHKJ0003) is shown. DETAILED DESCRIPTION

[0018] In order to explain the present invention more clearly, the present invention is further described in detail below in conjunction with embodiments and with reference to the accompanying drawings. It should be understood by those skilled in the art that the content described below is illustrative rather than restrictive, and should not be used to limit the scope of protection of the present invention.

[0019] Example

[0020] The present invention provides a salt-tolerant actinomycete in saline-alkali land, named as strain NHKJ0003, which was deposited in the General Microbiology Center of China Microorganism Culture Collection Committee on April 26, 2024, with a deposit number of CGMCC No.30450. The deposit address is Institute of Microbiology, Chinese Academy of Sciences, No. 3, Yard 1, Beichen West Road, Chaoyang District, Beijing, and the taxonomic name is Streptomyces rochei.

[0021] This embodiment mainly includes the following steps:

[0022] 1. Soil sample separation

[0023] Prepare 0.85% saline solution, select 250mL flasks, put 50mL in each flask, sterilize by high pressure at 121℃ for 20min, cool and set aside. Label the bags containing saline-alkali soil samples and take photos for preservation. Weigh 5g of each soil sample and add it to the flask containing 0.85% saline solution, shake it on a shaker at 28℃ and 180rpm / min for 30min, let it stand for 10min, draw the supernatant, and dilute it to 10 with sterile water. -9 ,spare.

[0024] 2. Screening of salt-tolerant strains

[0025] (1) Initial screening

[0026] Prepare the above 10 -1 -10 -8 10 -3 -10 -7 Spread 50 μL of the dilution on the plate, and repeat 3 times for each concentration. If the colonies are too dense or too few, spread other concentration gradients of soil dilution. Place the coated G1 plate in a 28℃ incubator and observe the growth of the colonies on the plate every day.

[0027] (2) Rescreening

[0028] During the initial screening, the colonies grown on the G1 plate ( Figure 1 ), and then streak pure culture on high-salt G1 medium (NaCl content is 15%), observe the growth of actinomycetes on G1 medium, and select the strains whose growth rate is not obviously inhibited.

[0029] 3. Antibacterial test

[0030] (1) Activation of plant pathogens

[0031] The tomato-specific strain of Fusarium oxysporum f.sp.lycopersici LYG9-1 (isolated from tomato plants with Fusarium wilt in Lianyungang, see the master's thesis of Huaiyin Institute of Technology "Evaluation and Preliminary Exploration of the Mechanism of Resistance and Preservation Effect Induced by Dihydrochlorin Iron in Fruits and Vegetables", Li Siyu), Stemphylium eturmiunum JX-8 (isolated from garlic leaves, identified by ITS sequence sequencing, the ITS sequence is shown in SEQ ID NO: 4), and Alternaria sp. sy-1 (isolated from garlic leaves, identified by ITS sequence sequencing, the ITS sequence is shown in SEQ ID NO: 5) were selected and activated and cultured on PDA medium.

[0032] (2) Flat plate confrontation method

[0033] Use a sterilized puncher to punch holes along the edge of the plant pathogen colony to obtain a round bacterial cake with a diameter of 5 mm, and inoculate it in the center of the PDA plate with antibiotics. Actinomycetes grow slowly, so do not use the point inoculation method, just draw a 20 mm long line. Take the plate inoculated with pathogens only as the control group (CK), and set up 3 parallel tests. Place the above PDA plate at 28°C for static culture and observe it every 24 hours. Repeat the antibacterial test many times to screen out the strain with the best effect.

[0034] (3) Calculation of inhibition rate

[0035] The diameter of the pathogen was measured by the cross method, and the inhibition rate was calculated using the formula.

[0036] Inhibition rate (%) = (control pathogen colony diameter - treated pathogen colony diameter) / control diameter × 100%

[0037] Through the antibacterial test, it was found that there were 4 strains of actinomycetes with good inhibitory effects. The present application selected strain No. 2 (i.e., the deposited strain NHKJ0003), and its antibacterial effect is shown in Table 1. The inhibition rate of strain No. 2 on the tomato-specific strain LYG9-1 of Fusarium oxysporum was 27.5%, the inhibition effect on the pathogenic Alternaria sy-1 was 50%, and the inhibition rate on the pathogenic Portobellomyces JX-8 was 73.80%. The antibacterial test plate was shown in Table 1. Figure 3 , Figure 4 and Figure 5 .

[0038] Table 1 Statistical table of the inhibition rate of strain No. 2 on pathogens

[0039]

[0040]

[0041] 4. Strain identification

[0042] (1) Morphological identification

[0043] Activate strain No. 2 (i.e., deposited strain NHKJ0003) on a plate and culture in a constant temperature incubator for 48-72 hours, and observe and record the colony color, growth rate, shape, edge, degree of protrusion, surface condition, etc. (Table 2). Note: If the colony grows slowly, the culture time can be extended to 4 days.

[0044] Table 2 Identification of colony morphology of strain No. 2 on G1 medium

[0045]

[0046] (2) Molecular identification

[0047] The selected strains were identified by molecular biology using 16S rDNA universal primers 27F and 1492R (see Table 3).

[0048] Table 3 Primer sequences

[0049]

[0050] PCR amplification reaction system (25 μL): 2×Taq Mix 12.5 μL, primers F and R 1 μL each, template DNA 1 μL, ddH2O 9.5 μL.

[0051] PCR amplification reaction conditions: 94℃ pre-denaturation for 5min; 94℃ denaturation for 30s; 54℃ annealing for 30s; 72℃ extension for 1min, 34 cycles; 72℃ extension for 10min. PCR products were detected by 1% agarose gel electrophoresis at 130V, and the products were sent to Bio-Tech for sequencing. The 16S rDNA sequence of strain NHKJ0003 is shown in SEQ ID NO: 3.

[0052] The obtained sequences were uploaded to NCBI (https: / / www.ncbi.nlm.nih.gov) for BLAST sequence homology comparison, the top 10 sequences with the highest similarity to the target sequence were downloaded, and the phylogenetic tree was constructed using MEGA software, such as Figure 6 shown.

[0053] As shown in Table 4, the 16S sequence of strain No. 2 has the highest consistency with the sequence of Streptomyces rocheri 173831, which can reach 95.69%.

[0054] Table 4 Summary of 16S rDNA homology comparison results of strain 2

[0055]

[0056]

[0057] Obviously, the above embodiments of the present invention are merely examples to more clearly illustrate the present invention, and are not limitations on the implementation methods of the present invention. For ordinary technicians in the relevant field, other different forms of changes or modifications can be made on the basis of the above description. It is impossible to list all the implementation methods here. All obvious changes or modifications derived from the technical solution of the present invention are still within the protection scope of the present invention.

Claims

1. A salt-tolerant actinomycete in saline-alkali land, named strain NHKJ0003, was deposited in the General Microbiology Center of China Microorganism Culture Collection Administration on April 26, 2024, with the deposit number CGMCC No.30450. The deposit address is Institute of Microbiology, Chinese Academy of Sciences, No. 3, Yard 1, Beichen West Road, Chaoyang District, Beijing, and the taxonomic name is Streptomyces rochei.

2. The salt-tolerant actinomycetes for saline-alkali land according to claim 1, characterized in that: The 16S rDNA sequence of strain NHKJ0003 is shown in SEQ ID NO:

3.

3. Use of the salt-tolerant actinomycetes in saline-alkali land according to claim 1 or 2 in inhibiting pathogenic bacteria, wherein the pathogenic bacteria include Fusarium oxysporum, Staphylococcus aureus and Alternaria alternata.

4. A plant antibacterial agent comprising the saline-alkali land salt-tolerant actinomycete according to claim 1 or 2.

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