Streptomyces virginiae and application thereof in promoting tobacco growth

Abstract

The invention discloses streptomyces virginiae and application thereof in promoting tobacco growth, streptomyces virginiae A15 is preserved in China General Microbiological Culture Collection Center (CGMCC), and the preservation number is CGMCC No.30461. The streptomyces virginiae A15 has the advantages that the streptomyces virginiae A15 is preserved in the China General Microbiological Culture Collection Center (CGMCC); the rhizosphere strain which can promote tobacco seed germination and has IAA production capacity, siderophore production capacity and protease production capacity is screened from rhizosphere soil, and the rhizosphere strain has important significance in the aspects of promoting plant growth, regulating and controlling soil micro-ecological balance, reducing the dosage of chemical fertilizer, improving the quality of agricultural products and increasing the yield.

Description

Technical Field

[0001] The invention relates to microorganisms and applications thereof, in particular to a strain of Streptomyces virginiana and applications thereof in promoting tobacco growth. Background Art

[0002] Soil health is related to the green and sustainable development of agriculture. However, due to excessive use of fertilizers, pesticides and improper planting, my country is facing a series of soil degradation problems such as soil compaction, continuous cropping obstacles, salinization, and heavy metal pollution. Not only is the fertilizer utilization rate low and the amount used large, but it also seriously affects crop yields and food security. Plant growth-promoting rhizobacteria (PGPR) are rhizosphere microorganisms that are beneficial to plant growth. They have the functions of promoting crop nutrient absorption, inhibiting pathogenic microorganisms, improving abiotic stress environments, and secreting plant hormones. They are of great significance to promoting plant growth and maintaining soil ecological health.

[0003] Rhizosphere secretions are key factors in the activity of rhizosphere microbial communities and also affect the diversity of rhizosphere microbial communities. Plant rhizospheres secrete sugars, organic acids, enzymes, amino acids, phenolic acids, flavonoids, fatty acids, growth factors and other substances. These secretions can provide carbon sources, nitrogen sources and other nutrients for microorganisms to attract microorganisms. The mechanism by which plant rhizosphere growth-promoting bacteria assist plant growth promotion mainly includes five aspects: nitrogen transformation, phosphorus solubilization, potassium solubilization, iron carrier production, and plant hormone secretion. In addition to promoting plant growth, the bacterial agents and fertilizers developed using plant rhizosphere growth-promoting bacteria can also help plants resist soil-borne diseases, improve plant stress resistance and improve the soil environment. With the continuous increase in environmental pressure, the separation and screening of suitable plant rhizosphere growth-promoting bacteria and the development of microbial agents will become an important research direction for sustainable agricultural development and have great prospects. Summary of the invention

[0004] In view of the deficiencies of the prior art, the present invention provides a strain A15 and an application thereof in promoting tobacco growth.

[0005] The technical solution of the present invention is as follows:

[0006] A strain of Streptomyces virginiana, wherein the Streptomyces virginiana A15 is deposited in the General Microbiological Center of China Microorganism Culture Collection Administration, and the deposit number is CGMCC No.30461.

[0007] The application of the Streptomyces virginia can promote tobacco growth, has the ability to produce IAA, siderophore, and extracellular protease, and can promote tobacco seed germination.

[0008] The bacterial cells, sterile fermentation liquid, or a mixture of the bacterial cells and the sterile fermentation liquid of the Streptomyces virginiana are used to promote tobacco growth.

[0009] A biocontrol agent, the components of which include the bacterial body of Streptomyces virginia according to claim 1, or the fermentation liquid of Streptomyces virginia according to claim 1, or a mixture of the bacterial body of Streptomyces virginia according to claim 1 and the fermentation liquid.

[0010] The biocontrol agent is applied after being processed into an agriculturally acceptable preparation.

[0011] The biocontrol agent is used to promote tobacco growth. The Virginia Streptomyces has the ability to produce IAA, siderophore and extracellular protease, and can promote tobacco seed germination.

[0012] The present invention screens out rhizosphere strains that can promote tobacco seed germination and have the ability to produce IAA, iron carriers and proteases from rhizosphere soil, which is of great significance in promoting plant growth, regulating soil microecological balance, reducing the use of chemical fertilizers, improving the quality of agricultural products and increasing yield. BRIEF DESCRIPTION OF THE DRAWINGS

[0013] Figure 1 Colony morphology of strain A15;

[0014] Figure 2 Phylogenetic tree of strain A15;

[0015] Figure 3 Detection of IAA production ability of strain A15;

[0016] Figure 4 Detection of the ability of strain A15 to produce siderophore;

[0017] Figure 5 Detection of the ability of strain A15 to produce extracellular protease;

[0018] Figure 6 Seed germination experiment results: The 5 plants on the left are the control group, and the 5 plants on the right are the A15 treatment group;

[0019] Figure 7 The results of the potted plant experiment show the control on the left and the A15 treatment on the right; DETAILED DESCRIPTION

[0020] The present invention is described in detail below in conjunction with specific embodiments.

[0021] (1) Strain Isolation Steps

[0022] The samples were collected from rhizosphere soil of tobacco fields in Ankang City, Shaanxi Province.

[0023] The target biocontrol bacteria in soil samples were isolated by dilution plate spreading method. Weigh 10 g of soil sample and add it to 90 mL of sterile water and shake until the large soil particles are completely broken up. Let it stand for more than 30 minutes to make it clear. Under sterile conditions, use a pipette to take 1 mL of supernatant and add it to a test tube containing 9 mL of sterile water and shake it to dilute it to 10 -1 Use different pipettes to take liquid from the diluted bacterial solution in turn, and dilute the bacterial solution to 10 times the original solution. -1 , 10 -2 , 10 -3 , 10 -4 , 10 -5 Take appropriate amount of soil suspension with different dilution concentrations and spread them evenly on solid LB, PDA and Gao's No. 1 medium, respectively, with 3 replicates for each medium. Place LB plate, PDA plate and Gao's No. 1 plate in a 28℃ constant temperature incubator for 48h.

[0024] Select colonies with different morphologies and streak them separately, and then streak them continuously to obtain purified strains. The culture morphology of the selected A15 strain on the LB plate is shown in Figure 1 The A15 strain appeared as regular round, yellow, opaque, smooth, and raised colonies on the LB plate.

[0025] (2) Identification steps of strain A15

[0026] The selected target strains were amplified by colony PCR using the universal primers 27F and 1492R for 16s rRNA amplification. The primer sequences were 27F: 5'-AGAGTTTGATCAATGGCTCAG-3, 1492R: 5'-TACGGYTACCTTGTTACGACTT-3'. 30μL PCR reaction system: 15μL 2×Taq PCR Master Mix; 1.5μL of upstream and downstream primers, 12μL H2O; a small amount of bacteria was taken with a sterilized toothpick as a DNA template. Reaction procedure: 95℃ pre-denaturation for 4min; 95℃ denaturation for 15s; 57℃ annealing for 15s; 72℃ extension for 1min; 30 cycles in total; 72℃ supplementary extension for 10min; PCR products were stored at 4℃. 1% agarose gel was used for PCR product detection, and the PCR product was sent to Shanghai Biotechnology Co., Ltd. for bidirectional sequencing to obtain a 16srRNA sequence with a fragment size of 1425bp. The sequencing results were compared with the Genbank nucleic acid database by Blast sequence homology. The target strain had a 16S rRNA sequence homology greater than 99% with multiple strains of Streptomyces virginiae, and was located in the same branch as Streptomyces virginiae with accession number CBS314.55; the phylogenetic tree was constructed using MEGA software. A strain of Streptomyces virginiae was obtained and named A15 (for the evolutionary tree, see Figure 2 ).

[0027] The 16s rRNA gene sequence of strain A15 is shown in SEQ ID NO:3.

[0028] The preservation information is as follows:

[0029] Classification name: Streptomyces virginiae

[0030] Storage time: April 26, 2024

[0031] Depository: China National Microbiological Culture Collection Administration General Microbiology Center

[0032] Address of the depository: Institute of Microbiology, Chinese Academy of Sciences, No. 3, Yard 1, Beichen West Road, Chaoyang District, Beijing

[0033] Deposit number: CGMCC No.30461

[0034] (3) Determination of IAA production capacity of strains

[0035] Inoculate a single A15 colony with liquid LB culture medium, culture at 28°C, 220rpm shaking for 2 days, transfer to 50mL LB liquid culture medium supplemented with 100mg / L L-tryptophan at a ratio of 1:100, culture at 28°C, 220rpm shaking for 5 days, then centrifuge the bacterial solution at 12000rpm for 5min, add an equal volume of Salkowskis colorimetric solution to the supernatant after centrifugation, develop color at room temperature in the dark for 30min, and measure the absorbance at 530nm. Prepare standard solutions with IAA concentrations of 0mg / L, 5mg / L, 10mg / L, 15mg / L, 20mg / L, and 25mg / L, add an equal volume of Salkowskis colorimetric solution, develop color at room temperature in the dark for 30min, measure the absorbance at 530nm, and draw a standard curve of IAA concentration content. The standard curve is as shown below. Figure 3 The IAA concentration produced by strain A15 was measured to be 7.41 mg / L.

[0036] (4) Determination of the ability of strains to produce siderophores

[0037] Inoculate a single colony of A15 with liquid LB medium and culture on a shaker at 28°C and 220 rpm for 2 days. Add 20 μL of the culture solution prepared under the above conditions to the center of the CAS plate and culture at 28°C for 7 days to observe the yellow transparent circle ( Figure 4 ). The yellow transparent circle is more obvious, which shows that strain A15 has a good ability to produce siderophores.

[0038] (5) Determination of the ability of strains to produce extracellular proteases

[0039] Inoculate a single colony of A15 with liquid LB medium and culture on a shaker at 28°C and 220 rpm for 2 days. Add 20 μL of the culture solution prepared under the above conditions to the center of the skim milk plate and culture at 28°C for 7 days to observe the transparent zone ( Figure 5 ). The transparent zone is more obvious, from which it can be concluded that the A15 strain has a good ability to produce extracellular protease.

[0040] (6) Effect of fermentation liquid on tobacco seed germination

[0041] A single colony of A15 was inoculated with liquid LB medium, cultured in a shaking incubator at 28°C and 220rpm for 2 days, and then transferred to a 150mL conical flask containing 50mL LB liquid medium at a ratio of 1:100, and cultured in a shaking incubator at 28°C and 220rpm for 5 days; the tobacco seeds were soaked and disinfected in 75% alcohol for 30 seconds, washed with sterile water 3 times, and the sterilized seeds were added with 1mL of A15 fermentation liquid cultured under the above conditions and soaked for 6 hours. A layer of sterile filter paper was laid in the culture dish, the filter paper was moistened with sterile water, and 50 seeds soaked in A15 fermentation liquid were evenly sown in each culture dish. The sterile water treatment was used as the control group, and repeated 3 times. The seeds were cultured in a 25°C incubator. After 10 days, observation was carried out. 30 seedlings were randomly selected from each culture dish, and the seedling length was measured with a ruler and the average value was calculated. 100 seedlings were selected and weighed for fresh weight. The results showed that the fresh weight of the treated seedlings increased and was significantly higher than that of the control group. The experimental results are shown in Table 1. Figure 6 .

[0042] Table 1 Seed germination experimental results

[0043]

[0044] (7) Potted plant growth promotion experiment

[0045] A single colony of A15 was inoculated with liquid LB culture medium, cultured in a shaking incubator at 28°C and 220rpm for 2 days, and then transferred to a 150mL conical flask containing 50mL LB liquid culture medium at a ratio of 1:100. Cultured in a shaking incubator at 28°C and 220rpm for 5 days to obtain the A15 fermentation liquid. The seedling medium and organic fertilizer (PINDSTRUP 0-6) were mixed with water at a ratio of 3:1 until the soil could be gathered into blocks and could be dispersed by light shaking. The soil was then divided into flower pots and tobacco seeds were planted. The tobacco seedlings were transplanted after germination. After transplanting, 10% of the A15 fermentation liquid (diluted with sterile water) was poured into the rhizosphere soil of the tobacco plants. The amount used for each tobacco seedling was 10mL. The control was treated with an equal amount of sterile water every 7 days. 30 days after the tobacco was transplanted, the growth of the plants was observed, and the plant height, fresh weight of the aboveground part, and dry weight of the aboveground part of the tobacco seedlings in the control and treatment groups were measured. Results (Table 2, Figure 7 ) showed that the tobacco seedlings treated with A15 fermentation liquid grew better than those of the control, and the plant height, aboveground fresh weight and aboveground dry weight increased, indicating that A15 fermentation liquid has a good growth-promoting function on tobacco.

[0046] Table 2 Pot test results

[0047]

[0048] It should be understood that those skilled in the art can make improvements or changes based on the above description, and all these improvements and changes should fall within the scope of protection of the appended claims of the present invention.

Claims

1. A strain of Streptomyces virginiana, characterized in that The Streptomyces virginiana A15 is deposited in the General Microbiology Center of China Microorganism Culture Collection Administration, with the deposit number being CGMCC No.30461.

2. The use of the Streptomyces virginiana according to claim 1, wherein the Streptomyces virginiana can promote tobacco growth, has the ability to produce IAA, siderophore, and extracellular protease, and can promote tobacco seed germination.

3. Use of the bacterial cells, sterile fermentation liquid, or a mixture of the bacterial cells and the sterile fermentation liquid of the Streptomyces virginiana according to claim 1 in promoting tobacco growth.

4. A biocontrol agent, characterized in that: The components of the biocontrol agent include the bacterial body of Streptomyces virginia according to claim 1, or the fermentation liquid of Streptomyces virginia according to claim 1, or a mixture of the bacterial body of Streptomyces virginia according to claim 1 and the fermentation liquid.

5. The biocontrol agent according to claim 4, characterized in that The biocontrol agent is processed into an agriculturally acceptable formulation and then applied.

6. The biocontrol agent according to claim 5, characterized in that The biocontrol agent is used for promoting tobacco growth; the Virginia Streptomyces has the ability to produce IAA, iron carriers and extracellular proteases, and can promote tobacco seed germination.

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