Streptomyces griseolus SR09 and application thereof in prevention and treatment of tobacco root rot

Abstract

The invention provides a streptomyces ruberogriseus SR09 and application thereof, the streptomyces ruberogriseus SR09 is named as Streptomyces ruberogriseus SR09 in taxonomy, and the preservation number of the streptomyces ruberogriseus SR09 is CCTCC (China Center for Type Culture Collection) NO.2024783. The streptomyces griseogriseus has a good disease prevention effect on tobacco root rot and has a remarkable growth promoting effect on tobacco plants. The streptomyces griseogriseus has a good disease prevention effect on tobacco root rot and has a remarkable growth promoting effect on tobacco plants.

Description

Technical Field

[0001] The invention relates to the technical field of microbiology, and in particular to a Streptomyces erythrorhizium SR09 and application thereof in preventing and controlling tobacco root rot. Background Art

[0002] Tobacco occupies an extremely important economic position in China. It is not only one of the main sources of tax revenue, but also promotes the economic development of many regions. China's tobacco planting area and cigarette production are both ranked first in the world, fully demonstrating its important contribution to the national economy. However, in recent years, the widespread root rot in tobacco planting has become a key issue restricting production. The disease is common in my country's tobacco-growing areas, seriously affecting the quality and yield of tobacco leaves, and causing heavy economic losses to tobacco farmers and tobacco companies. How to effectively prevent and control root rot has become an important issue to ensure the healthy development of the tobacco industry.

[0003] Tobacco root rot is caused by Fusarium spp., which is a common fungal disease affecting tobacco production in my country. It can break out in both the seedbed stage and the field stage, and the incidence rate is stable at 3% to 5%. Due to the extension of continuous cropping time and changes in soil ecological environment and cultivation patterns, Fusarium root rot has gradually become the main disease in tobacco production. The current means of preventing and controlling root rot are mainly disease-resistant varieties and chemical control, but the resources of disease-resistant varieties are limited and the cultivation cycle is long, and chemical control is prone to cause environmental pollution. In contrast, biological control has become a research hotspot because of its non-residue and environmentally friendly characteristics. In particular, actinomycetes in plant rhizosphere soil, as a natural disease prevention resource, can show great potential in pest and disease control, promoting plant growth and enhancing plant disease resistance.

[0004] Therefore, it is necessary to screen and identify a strain that can prevent and treat root rot from soil or plant materials. Summary of the invention

[0005] The present invention aims to provide a strain of Streptomyces erythronicus SR09 and an application thereof. The strain of Streptomyces erythronicus has a good disease prevention effect on tobacco root rot and has a significant growth-promoting effect on tobacco plants.

[0006] In order to achieve the above object, the present invention adopts the following technical solution:

[0007] In a first aspect of the present invention, a strain of Streptomyces rubrogriseus SR09 is provided, and the deposit number of the Streptomyces rubrogriseus SR09 is: CCTCCNO.2024783.

[0008] In a second aspect of the present invention, a fermentation agent is provided, the fermentation agent comprising:

[0009] Fermenting and culturing the Streptomyces erythrorhizon SR09 to obtain a fermentation liquid or bacterial suspension;

[0010] Or the fermentation liquid is spray-dried to obtain a dry powder bacterial agent.

[0011] Furthermore, the method for preparing the fermentation broth comprises:

[0012] The Streptomyces erythrorhizon SR09 is inoculated into a liquid culture medium for fermentation to obtain a fermentation liquid.

[0013] Furthermore, the fermentation culture conditions include: temperature of 26-30°C and pH of 5-9.

[0014] In the third aspect of the present invention, the use of the Streptomyces erythrocereus SR09 in preventing and controlling tobacco root rot is provided.

[0015] In the fourth aspect of the present invention, the use of the fermentation bacteria agent in preventing and treating tobacco root rot is provided.

[0016] In the fifth aspect of the present invention, the application of the Streptomyces erythrocereus SR09 in promoting tobacco growth is provided.

[0017] In the sixth aspect of the present invention, there is provided the use of the fermentation bacteria agent in promoting tobacco growth.

[0018] One or more technical solutions in the embodiments of the present invention have at least the following technical effects or advantages:

[0019] The embodiment of the present invention provides a strain of Streptomyces SR09 and its application, which has a good disease prevention effect on tobacco root rot and a significant growth-promoting effect on tobacco plants. It can be seen that the strain has good theoretical research value and application prospects. It can not only be used as a good experimental material for studying the interaction between soil actinomycetes and plants and their mechanism of action, but also can be used for the research and development of soil biofertilizers and biofungicides. At the same time, it may also become a good carrier for multifunctional engineering bacteria such as disease prevention and growth promotion, providing a new bacterial strain resource for the biological control of tobacco root rot.

[0020] The deposit date of Streptomyces rubrogriseus SR09 of the present invention is April 25, 2024, and the deposit number is CCTCCNO.2024783. Its classification name is Streptomyces rubrogriseus SR09, and the depository name is China Center for Type Culture Collection, and the address is Wuhan University, Wuhan City, Hubei Province, China, Postal Code: 430072. BRIEF DESCRIPTION OF THE DRAWINGS

[0021] In order to more clearly illustrate the technical solutions in the embodiments of the present invention, the following briefly introduces the drawings required for use in the description of the embodiments. Obviously, the drawings described below are some embodiments of the present invention. For ordinary technicians in this field, other drawings can be obtained based on these drawings without creative work.

[0022] Figure 1 This is a graph showing the antibacterial effect of Streptomyces erythrorhizium SR09 on Fusarium oxysporum;

[0023] Figure 2 The disease prevention effect of the strain on tobacco root rot; Figure A shows the growth of tobacco seedlings, and Figure B is a comparison of the seedlings and roots (rhizomes);

[0024] Figure 3 Phylogenetic tree of strains. DETAILED DESCRIPTION

[0025] The present invention will be described in detail below in conjunction with specific implementations and examples, and the advantages and various effects of the present invention will be more clearly presented. It should be understood by those skilled in the art that these specific implementations and examples are used to illustrate the present invention, rather than to limit the present invention.

[0026] Throughout the specification, unless otherwise specifically stated, the terms used herein should be understood as meanings commonly used in the art. Therefore, unless otherwise defined, all technical and scientific terms used herein have the same meanings as those generally understood by those skilled in the art to which the present invention belongs. In the event of a conflict, the present specification takes precedence.

[0027] Unless otherwise specified, various raw materials, reagents, instruments and equipment used in the present invention can be purchased from the market or obtained by existing methods.

[0028] The technical solution of the embodiment of the present application is to solve the above technical problems, and the overall idea is as follows:

[0029] The inventors of the present application obtained an actinomycete from tobacco-growing soil collected from disease-free fields in Nanzhang, Zaoyang and other places through separation, purification and screening. It was found that the strain had an antagonistic effect on tobacco oxysporum Fusarium. The actinomycete was analyzed by colony morphology, biochemistry and 16S rRNA sequencing. The homology of the strain with multiple strains of Streptomyces rubrogriseus was more than 96%. Combined with physiological and biochemical characteristics, the strain was preliminarily determined to be of the genus (Streptomyces rubrogriseus) and named Streptomyces rubrogriseus.

[0030] The Streptomyces rubrum of the invention has a good promoting effect on the growth of tobacco plants and a good disease prevention effect on tobacco root rot, thus providing a theoretical basis for the subsequent development of biological microbial agents.

[0031] The following will describe in detail a strain of Streptomyces erythrorhizium SR09 of the present application and its application in preventing and controlling tobacco root rot in combination with examples and experimental data.

[0032] Example 1: Isolation, purification and identification of Streptomyces erythrocereus SR09

[0033] 1. Isolation and purification of tobacco field soil microorganisms

[0034] Test medium: ISP2 medium: 4g yeast extract powder, 4g glucose, 10g malt extract powder, distilled water to 1000mL, pH7.2, sterilized at 115℃ for 20 minutes. Gao's medium No. 1: KNO31.0 g, K2HPO40.5 g, MgSO4·7H2O0.5g, NaCl 0.5g, FeSO4·7H2O 0.01g, soluble starch 20.0g, agar 15.0-20.0g, distilled water 1000ml, pH 7.2-7.4, sterilized at 115℃ for 20 minutes. Potato dextrose medium (PDA): 200g potato, 20g glucose, 15-20g agar, 1000mL distilled water, pH7.0-7.4, sterilized at 115℃ for 20 minutes.

[0035] LB solid medium: 10 g sodium chloride, 10 g peptone, 5 g yeast powder, 1000 mL water, 15 g agar, sterilized. Potato dextrose medium (PDA): 200 g potato, 20 g glucose, 15-20 g agar, 1000 mL distilled water, pH 7.0-7.4, sterilized.

[0036] Weigh 10 g of soil and place it in a 250 mL conical flask. Then add 100 mL of sterile water and place the conical flask on an oscillator at 180 rpm and 28°C for 2 h. -1 -10 -5 Dilution of rhizosphere soil suspension; absorb 10 -2 -10 -5 100 μL of soil suspension with different dilutions were evenly spread on the culture medium plate, and 3 parallels were set for each dilution, and then placed in a constant temperature incubator at 28°C for 5 days. After the colonies grew, they were streaked and purified 2-3 times according to various characteristics such as color, morphology, structure, and glossiness. The purified strains were numbered and stored in a 4°C refrigerator for future use.

[0037] 2. Isolation and screening of strains

[0038] (1) Primary screening: Fusarium oxysporum, a tobacco root rot pathogen, was used as an indicator bacteria, and the plate confrontation method was used to determine the inhibitory activity of the test strain against Fusarium oxysporum. Fusarium oxysporum was pre-activated on a PAD plate, and a sterile punch with a diameter of 5 mm was used to punch a bacterial cake at the edge of the colony. It was inoculated in the center of a new PDA plate, and different actinomycetes isolated and purified were inoculated in parallel lines 2.5 cm from the center of the plate. The blank control was inoculated with only pathogens. Each treatment was repeated 3 times, and the plates were cultured in a constant temperature box at 28°C. When the control colony grew to 2 / 3 of the culture dish, it was observed whether there was an obvious antagonistic effect, and the strains that had an inhibitory effect on the growth of Fusarium oxysporum were selected for re-screening.

[0039] 64 actinomycetes were isolated from the soil, and after preliminary screening, 10 strains were found to have antagonistic effects against tobacco Fusarium.

[0040] (2) The 10 strains obtained from the initial screening were rescreened using the plate confrontation method. The screening method is the same as above, but after each plate is inoculated with fungi, the same strain is inoculated on both sides. The control is not inoculated with fungi and the inhibition zone of the strain is recorded after the plate is full of growth. The above initial screening plate is taken out, the size of the inhibition zone is measured and the inhibition rate is calculated. The calculation formula is as follows:

[0041]

[0042] After repeated screening, a strain with good antagonistic effect on tobacco Fusarium was finally screened out and named SR09. Figure 1 The results of the antibacterial test of SR09 of the present invention on tobacco root rot are shown in Table 1.

[0043] Table 1 Antibacterial assay of SR09 against tobacco Fusarium

[0044]

[0045] It can be seen from Table 1 that the width of the inhibition zone of strain SR09 against tobacco Fusarium solani is 31 mm, which is 53.67 mm less than the diameter of the indicator bacteria. The inhibition rate can reach 63.39%, and the antagonistic effect can be clearly observed on the plate. Therefore, it was selected as the target strain for screening.

[0046] 3. Identification of strains

[0047] The strain was picked up and added to LB liquid medium for overnight culture, and then the sediment was centrifuged to extract the genomic DNA of the bacteria. The strain gene DNA was extracted by the bacterial suspension boiling water bath method, and the universal primers for actinomycetes were used.

[0048] 27F (5'-AGAGTTTGATCCTGGCTCAG-3') and 1541R (5'-AAGGAGGTGATCCAGCCGCA-3') were PCR amplified. The PCR reaction system included 1 μL DNA template, 1 μL upstream and downstream primers, 12.5 μL Taq polymerase, and ddH2O to 25 μL. The PCR amplification program was: 98°C pre-denaturation for 5 min, 98°C denaturation for 30 s, 58°C annealing for 30 s, 72°C extension for 1 min 30 s, 35 cycles, and finally 72°C extension for 5 min. The PCR reaction products were detected by 1% agarose gel electrophoresis, and the PCR amplification products of 16S rRNA were recovered and purified and sequenced by Wuhan Qingke Biotechnology Co., Ltd. The sequencing results (shown in SEQ ID NO.1) were submitted to the NCBI database for BLAST homology analysis, and then the sequence alignment and phylogenetic tree construction were performed using MEGA 11.0 software. Data processing was analyzed using Excel and SPSS27 software.

[0049] The 16S rRNA sequence of the strain to be tested obtained by PCR (shown in SEQ ID NO.1) was compared and analyzed by BLAST in NCBI, and it was found that the sequence homology between the strain and Streptomyces rubrogriseus was 90%. Combined with the morphology and physiological and biochemical characteristics of the strain, the strain was determined to be Streptomyces rubrogriseus. The strain has been deposited in the China Center for Type Culture Collection with the deposit number of CCTCC NO.2024783.

[0050] Example 2, Preparation of strain fermentation broth

[0051] The SR09 strain of Streptomyces rubrae was inoculated into 5 mL of ISP2 liquid culture medium and cultured at 28°C and 180 r / min for 5 days to prepare seed solution. Then, it was inoculated into ISP2 culture medium at a 1% inoculum amount and cultured for 7 days at 28°C and 180 r / min to obtain the fermentation liquid of the strain. Before the pot test, the concentration of live bacteria in the fermentation liquid was adjusted to 1×10 8 cfu / mL.

[0052] Example 3: Determination of the growth-promoting effect on tobacco

[0053] The fermented liquid of SR09 strain was used to irrigate the roots and spray the leaves of tobacco seedlings with uniform growth at the 4-5 leaf stage, once every 5 days, for a total of 3 times. The control group CK1 was irrigated with water, and CK2 was irrigated with ISP2 liquid culture medium. There were 3 plants in each treatment, and 3 replicates were set. According to the tobacco industry standard: Tobacco agronomic traits survey and measurement method (YC / T142-2010), the agronomic traits of tobacco plants were measured. After 15 days, the differences in plant height, stem girth, leaf length, leaf width, fresh weight, and dry weight between the treatment group and the control group were measured and compared. The results are shown in Table 2.

[0054] Table 2 Effect of SR09 on tobacco growth

[0055]

[0056] Note: Different lowercase letters in the vertical row represent significant differences among different treatment groups (P<0.05).

[0057] From the results in Table 2, it can be seen that the experimental group irrigated with the fermentation liquid of the SR09 strain had a significant increase in both fresh weight and dry weight compared with the control group. The fresh weight of the aboveground part of the experimental group reached 24.09g, while the aboveground fresh weights of CK1 and CK2 were 11.73g and 15.91g, respectively. The tobacco plants in the treatment group grew significantly better, and the growth of tobacco with ISP2 medium was slightly better than that of the water group. In terms of dry weight, the aboveground weight of tobacco plants irrigated with SR09 was 0.78g, which was higher than 0.45g and 0.56g of CK1 and CK2. The plant height, stem girth, maximum leaf length and maximum leaf width of tobacco plants in the treatment group were significantly higher than those in the water treatment group, which were 32.50cm, 1.78cm, 20.33cm and 10.29cm, respectively, indicating that it has a good promoting effect on tobacco growth.

[0058] Example 4: Experiment on the effectiveness of strains in preventing tobacco root rot in indoor potted plants

[0059] The experiment was designed as blank group CK1, control group T2, and treatment group T1. The unpreserved Streptomyces durocortorensis (Streptomyces durocortorensis) obtained from the same batch of screening in Example 1 was used to treat CK2. Pre-select tobacco seedlings with consistent growth, use sterilized scissors to cut off 1 cm from the root end of the seedlings, cut 5 roots for each plant, and pierce the rhizomes with a sterilized needle. Each rhizome is pierced with 5 wounds, and then moved to a sterilized nutrient pot (with nutrient matrix inside). Each treatment has 5 plants, 3 repetitions, and a total of 15 plants. The plants are irrigated with root irrigation 2 days after transplanting. Each plant in the blank group and the control group was irrigated with 15 mL of sterile water; the treatment group was irrigated with the biocontrol bacteria fermentation liquid prepared in Example 2 at a rate of 15 mL per plant, and the control group and the treatment group were irrigated with a suspension of Fusarium oxysporum at a rate of 15 mL per plant after 48 hours. Among them, the concentration of fungal spores in the bacterial suspension is 5×106 / mL, the concentration of the biocontrol bacteria fermentation liquid was 1×10 8 CFU / mL. When the control group became ill after 15 days of growth, the incidence was counted. The grading standard of tobacco root rot was based on the tobacco industry standard of the People's Republic of China (GB / T23222-2008).

[0060] Incidence rate = (number of diseased plants / total number of plants surveyed) × 100%

[0061] Disease index = [∑ (number of diseased plants or leaves at each level × value of the disease level) / (total number of plants or leaves surveyed × highest level value)] × 100

[0062] Relative control effect = [(control disease index - treatment disease index) / control disease index] × 100%

[0063] The results are as follows Figure 2 As shown in A, the tobacco seedlings in the control group wilted and turned yellow. Figure 2 B shows that the root and stem of the seedlings of the control group T2 inoculated with pathogens rotted and the plants died, producing a pathological phenotype similar to that in the field. This proves that indoor inoculation with pathogens can cause tobacco to become sick and die, but inoculation with the potential biocontrol bacteria SR09 can effectively prevent the plants from becoming sick and dying. The specific disease statistics are as follows:

[0064] Table 3 Inhibition of tobacco root rot by biocontrol bacteria SR09

[0065]

[0066] As shown in Table 3, in the control group T2, 15 tobacco seedlings were wilted, yellowed and died due to Fusarium oxysporum, and the incidence of root rot reached 100.00%, while the incidence of seedlings inoculated with SR09 in the treatment group T1 was 13.33%, and the disease index of the diseased plants was reduced. The relative control effect was 81.08%, which was increased by 56.53% compared with the black-skinned Streptomyces screened from the same batch.

[0067] In summary, the Streptomyces erythroxysporum SR09 provided by the present invention has a good disease prevention effect on tobacco root rot and has a significant growth-promoting effect on tobacco plants.

[0068] The above embodiments are preferred implementation modes of the present invention, but the implementation modes of the present invention are not limited to the above embodiments. Any other changes, modifications, substitutions, combinations, and simplifications that do not deviate from the spirit and principles of the present invention should be equivalent replacement methods and are included in the protection scope of the present invention.

[0069] Finally, it should be noted that the terms "comprises," "includes," or any other variations thereof are intended to cover non-exclusive inclusion, such that a process, method, article, or apparatus that includes a series of elements includes not only those elements, but also other elements not explicitly listed, or also includes elements that are inherent to such process, method, article, or apparatus.

[0070] Although the embodiments of the present invention have been described, those skilled in the art may make additional changes and modifications to these embodiments once they have learned the basic creative concept. Therefore, the appended claims are intended to be interpreted as including the embodiments and all changes and modifications falling within the scope of the present invention.

[0071] Obviously, those skilled in the art can make various changes and modifications to the present invention without departing from the spirit and scope of the present invention. Thus, if these modifications and variations of the present invention fall within the scope of the claims of the present invention and their equivalents, the present invention is also intended to include these modifications and variations.

Claims

1. A strain of Streptomyces erythrocereus SR09, characterized in that: The taxonomic name of the Streptomyces rubrogriseus is Streptomyces rubrogriseus SR09, and its preservation number is: CCTCC NO.2024783.

2. Use of the Streptomyces erythroxysporum SR09 according to claim 1 in preventing and treating tobacco root rot.

3. Use of the Streptomyces erythroxysporum SR09 according to claim 1 in promoting the growth of tobacco plants.

4. A fermentation agent, characterized in that: The fermentation agent comprises: Fermentation culture of the Streptomyces erythrocereus SR09 as claimed in claim 1 to obtain a fermentation liquid or a bacterial suspension; Or the fermentation liquid is spray-dried to obtain a dry powder bacterial agent.

5. The fermentation agent according to claim 4, characterized in that: The preparation method of the bacterial suspension comprises: The Streptomyces erythrorhizon SR09 is inoculated into a liquid culture medium for fermentation to obtain a fermentation liquid.

6. The fermentation agent according to claim 4, characterized in that: The fermentation culture conditions include: temperature of 26-30° C. and pH of 5-9.

7. Use of the fermentation agent according to any one of claims 2 to 4 in preventing and treating tobacco root rot.

8. Use of the fermentation agent according to any one of claims 2 to 4 in promoting the growth of tobacco plants.

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