Streptomyces purpureus SP33 and application thereof in prevention and treatment of tobacco root rot

Abstract

The invention provides streptomyces purpureus SP33 and application thereof, the streptomyces purpureus SP33 is named as Streptomyces purpureus SP33 in taxonomy, and the preservation number of the streptomyces purpureus SP33 is CCTCC (China Center for Type Culture Collection) NO.2024784. The streptomyces purpureus strain has a good disease prevention effect on tobacco root rot and has a remarkable growth promoting effect on tobacco plants. The streptomyces purpureus strain 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 strain of Streptomyces rhodochrous SP33 and application thereof in preventing and controlling tobacco root rot. Background Art

[0002] China's tobacco industry occupies a pivotal position in the global market. Its planting area and cigarette production are among the highest in the world, becoming an important economic pillar of the country. Tobacco planting provides economic support and employment opportunities for many regions, and plays an irreplaceable role in local economic development. However, the frequent outbreaks of tobacco root rot have seriously affected the growth and yield of tobacco, becoming one of the main problems troubling tobacco farmers. The spread of root rot not only causes huge economic losses to the tobacco industry, but also threatens the stability of the tobacco supply chain. Prevention and control work needs to be strengthened urgently.

[0003] Tobacco root rot is caused by Fusarium spp. and is a common fungal disease in tobacco production in my country. It can occur in both the seedbed stage and the field stage, with an annual incidence rate of 3% to 5%. With the extension of continuous cropping years and changes in soil microecology and cultivation systems, Fusarium root rot has gradually become a major disease. Currently, commonly used prevention and control measures include the selection of disease-resistant varieties and chemical pesticide control. However, the resources of disease-resistant varieties are limited and the breeding cycle is long, while chemical pesticides may produce residual pollution and threaten human health. In contrast, biological control has the advantages of no residue and environmental friendliness, and has become an effective means of preventing and controlling tobacco root rot. In particular, actinomycetes in the rhizosphere soil of plants, as natural biological control resource bacteria, can not only prevent and control diseases, but also play a long-term protective role through growth promotion and biological nitrogen fixation.

[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 purpurogenus SP33 and application thereof, wherein the strain of Streptomyces purpurogenus 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 puniceus SP33 is provided, and the deposit number of the Streptomyces puniceus SP33 is: CCTCCNO.2024784.

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

[0009] Fermenting and culturing the Streptomyces rhodochrous SP33 to obtain a fermentation liquid or a 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 rhodochrous SP33 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 rhodochrous SP33 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 rhodochrous SP33 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 purpurogenus SP33 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 species resource for the biological control of tobacco root rot.

[0020] The preservation date of Streptomyces puniceus SP33 of the present invention is April 25, 2024, and the preservation number is CCTCCNO.2024784. Its classification name is Streptomyces puniceus SP33, and the preservation unit 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 diagram showing the antibacterial effect of Streptomyces purpurogenum SP33 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 puniceus was more than 96%. Combined with physiological and biochemical characteristics, the strain was preliminarily determined to be of the genus (Streptomyces puniceus) and named Streptomyces puniceus.

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

[0031] The following will describe in detail a strain of Streptomyces purpurogenum SP33 of the present application and its application in preventing and controlling tobacco root rot in combination with embodiments and experimental data.

[0032] Example 1: Isolation, purification and identification of Streptomyces rhodochrous SP33

[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 SP33. Figure 1 The results of the antibacterial test of SP33 of the present invention on tobacco root rot are shown in Table 1.

[0043] Table 1 Antibacterial assay of SP33 against tobacco Fusarium

[0044]

[0045] It can be seen from Table 1 that the width of the inhibition zone of strain SP33 against tobacco Fusarium is 32 mm, which is 52.67 mm less than the diameter of the indicator bacteria. The inhibition rate can reach 62.20%, 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. The genomic DNA of the bacteria was extracted, and the strain gene DNA was extracted by the bacterial suspension boiling water bath method. The universal primers 27F (5'-AGAGTTTGATCCTGGCTCAG-3') and 1541R (5'-AAGGAGGTGATCCAGCCGCA-3') of actinomycetes were used for PCR amplification. The PCR reaction system was 1 μL of DNA template, 1 μL of upstream and downstream primers, 12.5 μL of Taq polymerase, and ddH2O was added to 25 μL. The PCR amplification program was: 98℃ pre-denaturation for 5min, 98℃ denaturation for 30s, 58℃ annealing for 30s, 72℃ extension for 1min30s, 35 cycles, and finally 72℃ extension for 5min. 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 were submitted to the NCBI database for BLAST homology analysis, and then the MEGA 11.0 software was used for sequence alignment and phylogenetic tree construction. Data processing was performed using Excel and SPSS27 software.

[0048] The 16S rRNA sequence of the strain to be tested obtained by PCR (shown in SEQ ID NO.1) was compared by BLAST (https: / / blast.ncbi.nlm.nih.gov / Blast.cgi) in the public database NCBI, and the strain SP33 was finally determined to be Streptomyces puniceus through homology detection. The strain has been deposited in the China Center for Type Culture Collection with the deposit number of CCTCC NO.2024784.

[0049] Example 2, Preparation of strain fermentation broth

[0050] The Streptomyces purpurogenus SP33 strain was inoculated into 5 mL of ISP2 liquid culture medium and cultured at 28°C, 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, 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.

[0051] Example 3: Determination of the growth-promoting effect on tobacco

[0052] SP33 strain fermentation liquid was used to irrigate roots and spray 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. Each treatment had 3 plants and 3 replicates. 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.

[0053] Table 2 Effect of SP33 on tobacco plant growth

[0054]

[0055] Note: Different lowercase letters in the vertical row represent significant differences among different treatment groups (P<0.05).

[0056] From the results in Table 2, it can be seen that the experimental group irrigated with the fermentation liquid of the SP33 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 20.40g, 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 SP33 was 0.75g, 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 31.50cm, 1.82cm, 20.17cm and 10.17cm, respectively, indicating that it has a good promoting effect on tobacco growth.

[0057] Example 4: Experiment on the effectiveness of strains in preventing tobacco root rot in indoor potted plants

[0058] 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).

[0059] Incidence rate = (number of diseased plants / total number of plants surveyed) × 100%

[0060] 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

[0061] Relative control effect = [(control disease index - treatment disease index) / control disease index] × 100%

[0062] 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 in the control group 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 potential biocontrol bacteria SP33 can effectively prevent the plants from becoming sick and dying. The specific disease statistics are as follows:

[0063] Table 3 Inhibition of tobacco root rot by biocontrol bacteria SP33

[0064]

[0065] 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 SP33 in the treatment group T1 was 13.33%, and the disease index of the diseased plants was reduced, with a relative control effect of 79.34%. Compared with the black-skinned Streptomyces screened from the same batch, the relative control effect increased by 54.79%.

[0066] In summary, the Streptomyces purpurogenum SP33 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.

[0067] 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.

[0068] 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.

[0069] 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.

[0070] 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 rhodochrous SP33, characterized in that: The taxonomic name of the Streptomyces puniceus SP33 is Streptomyces puniceus SP33, and its preservation number is: CCTCC NO.2024784.

2. Use of the Streptomyces rhodochrous SP33 according to claim 1 in preventing and treating tobacco root rot.

3. Use of the Streptomyces rhodochrous SP33 according to claim 1 in promoting the growth of tobacco plants.

4. A fermentation agent, characterized in that: The fermentation agent comprises: Fermentation broth or bacterial suspension obtained by fermenting the Streptomyces rhodochrous SP33 as claimed in claim 1; 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 rhodochrous SP33 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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