Microbial agent for preventing and treating tobacco target leaf spot as well as preparation method and application of microbial agent

Abstract

The invention relates to the technical field of microorganisms, and discloses a microbial agent for preventing and treating tobacco target leaf spot as well as a preparation method and application of the microbial agent. Streptomyces marxianus is bacillus amyloliquefaciens SE4-2, and is named as Streptomyces sp.SE4-2 in taxonomy; the strain is applied to prevention and treatment of tobacco target leaf spot, diseases are well prevented and treated, and pathogenic bacteria are prevented from generating drug resistance.

Description

Technical Field

[0001] The present invention relates to the technical field of microorganisms, and particularly relates to a microbial agent for controlling tobacco target spot pathogen and its preparation method and application. Background Art

[0002] Tobacco target spot disease caused by Rhizoctonia solani is an important foliar disease on tobacco. The lesions are light brown, often accompanied by concentric rings, and the necrotic part in the center of the lesions is easy to break and form perforations. This disease spreads rapidly and can achieve multiple reinfections within a growing season. It is widespread in many tobacco-growing areas such as Liaoning, Yunnan, Guizhou, Hunan, and Sichuan, causing significant economic losses to tobacco leaf production.

[0003] Currently, the control of tobacco target spot disease mainly relies on chemical control. However, problems such as environmental pollution caused by long-term use of chemical agents, enhanced pathogen resistance, and deterioration of tobacco leaf quality are becoming increasingly prominent. Biological control has the characteristics of environmental friendliness and harmlessness to humans and livestock, and is an ideal alternative to chemical control. The main biocontrol microorganisms used for biological control include Bacillus, Pseudomonas, Streptomyces, and Trichoderma, etc. Among them, Streptomyces can produce a variety of secondary metabolites with antibacterial activity and can promote plant growth, which is one of the research hotspots in biological control in recent years. Streptomyces has good application prospects in the control of tobacco target spot disease.

[0004] Therefore, obtaining Streptomyces that antagonizes tobacco target spot pathogen is of great significance for controlling tobacco target spot disease. Summary of the Invention

[0005] The purpose of the present invention is to provide a microbial agent for controlling tobacco target spot pathogen and its preparation method and application to solve the above problems.

[0006] The solution of the present invention is

[0007] A strain of Streptomyces, Streptomyces sp. SE4-2 with the preservation number of CCTCC NO: M 20242638, was preserved in the China Center for Type Culture Collection on November 26, 2024, and its taxonomic name is Streptomyces sp. SE4-2.

[0008] As a preferred technical solution, the 16S rDNA sequence of the Streptomyces sp. SE4-2 is shown as SEQ ID NO.1.

[0009] The present invention also discloses a microbial agent for controlling tobacco target spot pathogen, which is characterized in that it contains Streptomyces sp. SE4-2 with the preservation number of CCTCC NO: M 20242638.

[0010] The present invention also discloses a method for preparing a microbial agent for controlling tobacco target spot pathogen, comprising the following steps:

[0011] 1) Strain activation: The Streptomyces malaysiensis SE4-2 described in claim 1 is subjected to plate culture through a culture medium to obtain activated Streptomyces malaysiensis SE4-2;

[0012] 2) Preparation of seed liquid: An inoculation loop is used to pick up the activated Streptomyces malaysiensis SE4-2 and inoculate it into a seed culture medium, and then shake-cultured to obtain a seed liquid;

[0013] 3) Preparation of the microbial agent: The seed liquid is inoculated into a fermentation culture medium, and the volume ratio of the seed liquid to the fermentation culture medium is 1:10 - 20. Then it is placed in a constant temperature incubator for fermentation culture, and after that, the cultured fermentation product is dried at 25 - 35 °C and pulverized into powder to obtain a biocontrol microbial agent.

[0014] As a preferred technical solution, the culture medium in 1) is an LB solid culture medium; in 2), it is shake-cultured at 28 °C and 180 r / min for 48 h.

[0015] As a preferred technical solution, in 3), it is fermented and cultured in a constant temperature incubator for 7 - 14 d, and the temperature of the constant temperature incubator is 28 °C.

[0016] The present invention also discloses an application of Streptomyces in the preparation of a microbial agent for controlling tobacco target spot pathogen.

[0017] The present invention also discloses an application of a microbial agent for controlling tobacco target spot pathogen in antagonizing tobacco target spot pathogen.

[0018] Advantages of the present invention:

[0019] The present invention discloses and provides a Streptomyces malaysiensis for antagonizing tobacco target spot pathogen, which has a relatively high inhibition rate against tobacco target spot pathogen, is green and environmentally friendly, and will not cause pollution to the environment. Description of the Drawings

[0020] In order to more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the following will briefly introduce the drawings required for use in the description of the embodiments or the prior art. Obviously, the drawings in the following description are only the embodiments of the present invention. For those of ordinary skill in the art, other drawings can be obtained according to the provided drawings without creative efforts.

[0021] Figure 1 It is a confrontation culture result diagram of Streptomyces malaysiensis SE4-2 and tobacco target spot in Example 1 of the present invention;

[0022] Figure 2Colony morphology diagram of Streptomyces malaysiensis SE4-2 provided by the present invention on Gause's No. 1 medium;

[0023] Figure 3 Gel electrophoresis diagram of 16S rDNA PCR product of Streptomyces malaysiensis SE4-2 in Example 2 of the present invention;

[0024] Figure 4 Phylogenetic tree result diagram of Streptomyces malaysiensis SE4-2 of the present invention;

[0025] Figure 5 Antagonistic effect of Streptomyces malaysiensis SE4-2 against test pathogenic bacteria in Example 3 of the present invention, where a is Rhizoctonia solani of rice sheath blight, b is Fusarium graminearum of wheat head blight, c is Pyricularia oryzae of rice blast, and d is Phytophthora fragariae of strawberry blight. Detailed implementation manners

[0026] In order to make the technical means, creative features, achieved purposes and effects of the present invention easy to understand, the present invention will be further described below in conjunction with specific embodiments.

[0027] Materials, reagents and instruments used in the examples:

[0028] Tobacco rhizosphere soil: Rhizosphere soil of healthy tobacco plants collected from severely diseased tobacco fields of tobacco target spot disease in Hunan tobacco-growing area.

[0029] Tobacco variety: Yunyan 87

[0030] Test strains: Rhizoctonia solani of tobacco target spot disease, Phytophthora fragariae of strawberry blight, Rhizoctonia solani of rice sheath blight, Fusarium graminearum of wheat head blight, and Pyricularia oryzae of rice blast are preserved in the Plant Pathology Laboratory of Hunan Agricultural University.

[0031] Culture media and medicaments

[0032] Potato dextrose agar medium: 200 g of potatoes are washed, peeled, boiled in 1 L of distilled water, filtered through a gauze, 20 g of glucose and 15 - 20 g of agar powder are added, stirred with a glass rod until the drugs are dissolved, and then made up to 1 L with distilled water. 1 L of the medium is dispensed into 500 mL conical flasks, 200 mL is added to each flask, sterilized by autoclaving at 121 °C for 30 min, and cooled for standby.

[0033] LB liquid medium: Weigh 10 g of NaCl, 10 g of tryptone, and 5 g of yeast powder, and add 500 mL of deionized water. Stir with a glass rod until all the drugs are dissolved, then add deionized water to make up to 1 L. The sterilization and sub-packaging methods are the same as above.

[0034] LB solid medium: Add 15 - 20 g / L of agar powder to the LB liquid medium, stir until the drugs are dissolved, and the sterilization and sub-packaging methods are the same as above.

[0035] Reagents: Glucose, agar powder, tryptone, NaCl, yeast powder, L-tryptophan (100 mg / L), indole-3-acetic acid (IAA) solution, purchased from Sinopharm Chemical Reagent Co., Ltd.; Salkowskis colorimetric solution, 150 mL of concentrated sulfuric acid is dissolved in 250 mL of pure water, and 7.5 mL of 0.5 mol / L ferric chloride [FeCl 3 ·6H 2 O] is added.

[0036] Experimental instruments

[0037] 2 L graduated cylinder, glass rod, 2 mL centrifuge tube, 15 mL centrifuge tube, pipette, spreader, disposable petri dish, autoclave, laminar flow hood, shaker, etc.

[0038] Example 1: Strain screening

[0039] 1) Sampling

[0040] Dig out the rhizosphere soil of healthy tobacco plants from tobacco target spot disease-affected fields in Hunan tobacco-growing areas and put it into a sterilized paper bag for storage. After bringing it back to the laboratory, sieve the coarse soil to leave the fine soil.

[0041] 2) Isolation and purification of soil bacteria

[0042] Take 10 g of soil sample and put it into a triangular flask containing 90 mL of sterile water. Incubate it at 30 °C and 180 r / min for 30 min. After standing for 5 min, take the soil suspension and dilute it with sterile water to concentration gradients of 10 -1 、10 -2 、10 -3 、10 -4 、10 -5 、10 -6 。Use a pipette to suck 0.1 mL of the soil dilution of each gradient, spread it evenly on the LB solid medium plate, and culture it in an incubator at 28 °C. After 2 days, pick different morphological single colonies for multiple purifications. For the selected fungi, pick the hyphae or punch a bacterial cake at the edge of the single colony for purification. Number the isolated strains and store them at 4 °C for the next antagonistic experiment identification. A total of 27 strains were isolated, including 21 strains of bacteria, 4 strains of fungi, and 2 strains of streptomyces.

[0043] 3) Screening of biocontrol bacteria

[0044] Using the tobacco target spot pathogen (Rhizoctonia solani) as the target, the strains isolated from tobacco rhizosphere soil were co-cultured with Rhizoctonia solani. The specific steps were as follows:

[0045] Use a 5-mm punch to cut fresh tobacco target spot pathogen discs at the edge of the plate and place them in the center of a fresh PDA plate. Then, spot inoculate single colonies of the isolated bacteria about 2.5 cm away from the pathogen discs. Use a PDA plate with only the pathogen discs as a control. Each treatment has 3 replicates. After culturing at 30 °C for 5 days, observe the presence of inhibition zones. Use the cross method with a vernier caliper to measure the hyphal diameters of the pathogen in the control group and the treatment group, and calculate the inhibition rate. The inhibition rate of antagonistic bacteria was calculated by the following formula.

[0046] Inhibition rate (%) = (control colony diameter - treatment colony diameter) / (control colony diameter - disc diameter) × 100.

[0047] A total of 21 strains of bacteria were isolated and purified. Among them, strains such as SE4-2, B2-1, B5-1, I10-1, and B10-1 had obvious antagonistic effects on the tobacco target spot pathogen. Their inhibition rates were 71.1%, 66.89%, 65.14%, 64.6%, and 53.72% respectively (Table 1). The antagonistic effect of strain SE4-2 was significantly higher than that of other strains, and the diameter of the inhibition zone reached 28.70 mm ( Figure 1 ).

[0048] Table 1 Antagonistic effects of antagonistic bacteria on the tobacco target spot pathogen

[0049]

[0050]

[0051] Note: The values are mean ± standard error. Different letters in the same column indicate significant differences (P < 0.05). The same below.

[0052] 4) Identification of biocontrol bacteria strains

[0053] Morphological observation

[0054] Strain SE4-2 was opaque white on Gause's No. 1 plate.

[0055] Physiological and biochemical identification of biocontrol bacteria

[0056] The physiological and biochemical identification of the strain was carried out with reference to the "Manual for the Systematic Identification of Common Bacteria", including methyl red test, V.P. test, catalase test, starch hydrolysis test, gelatin liquefaction reaction, oxidase test, indole reaction, and hydrogen sulfide production test, etc.

[0057] The determination of physiological and biochemical characteristics showed that the starch hydrolysis and gelatin liquefaction tests were positive, while the methyl red (M.R.) test, V.P. test, catalase test, oxidase test, indole reaction, and hydrogen sulfide production test were negative (Table 2).

[0058] Table 2 Physiological and Biochemical Characteristics of Strain SE4-2

[0059]

[0060] Note: In the table, "+" indicates a positive reaction and "-" indicates a negative reaction.

[0061] Example 2: Molecular Identification of Biocontrol Bacteria

[0062] Single colonies of SE4-2 were picked and PCR amplification was carried out using the universal primers for 16S rDNA amplification (27F: 5′-AGAGTTTGATCCTGGCTCAG-3′ and 1492R: 5′-TACGGTTACCTTGTTACGAC-3′). The reaction system was (50 μL): ddH2O (20.5 μL); 27F (1.5 μL); 1492R (1.5 μL); Mix (25 μL); DNA template (1.5 μL).

[0063] The reaction program was: pre-denaturation at 95°C for 5 min; denaturation at 94°C for 30 s; annealing at 59°C for 30 s; extension at 72°C for 1.5 min; 35 cycles; extension at 72°C for 10 min; storage at 4°C. After the reaction was completed, the PCR product was sent to Sangon Biotech (Shanghai) Co., Ltd. for sequencing. The obtained sequence was subjected to BLAST nucleic acid homology alignment on NCBI. A phylogenetic tree was established using MEGA 11 to determine the taxonomic status of the strain.

[0064] PCR amplification of the 16S rDNA sequence of strain SE4-2 showed that the size of the PCR product band was about 1500 bp ( Figure 3 ), and the obtained sequence was subjected to BLAST alignment on NCBI. The results showed that the sequence similarity between strain SE4-2 and Streptomyces sp. AM6-12 from Malaysia (GenBank: PQ202303.1) was 99%, and the taxonomic name was Streptomyces sp.

[0065] The obtained 16S rDNA sequence is shown as SEQ ID NO.1.

[0066] Example 3: Determination of the antibacterial spectrum of the biocontrol bacterial strain

[0067] The broad-spectrum antibacterial ability of strain SE4-2 was determined using another 4 kinds of pathogenic bacteria preserved in the laboratory, and the mycelial growth and antibacterial effect were observed. It was found that it had good inhibitory effects on 4 kinds of pathogenic bacteria such as the rice sheath blight pathogen and the wheat scab pathogen, and the inhibition rate was 51.85% - 63.68% (Table 3, Figure 5 ), indicating that strain SE4-2 has broad-spectrum antibacterial activity.

[0068] Table 3 Determination of the broad-spectrum antibacterial ability of strain SE4-2

[0069]

[0070] The above shows and describes the basic principles, main features and advantages of the present invention. Those skilled in the art should understand that the present invention is not limited by the above embodiments. The above embodiments and the descriptions in the specification only illustrate the principles of the present invention. Without departing from the spirit and scope of the present invention, the present invention will have various changes and improvements, and these changes and improvements all fall within the scope of the present invention claimed. The scope of protection claimed by the present invention is defined by the appended claims and their equivalents.

Claims

1. A strain of Streptomyces, characterized in that: Malaysian Streptomyces sp. SE4-2, with the deposit number CCTCC NO:M 20242638, was deposited in the China Center for Type Culture Collection on November 26, 2024, and the taxonomic name is Streptomyces sp. SE4-2.

2. The Streptomyces according to claim 1, characterized in that: The 16S rDNA sequence of the Malaysian Streptomyces SE4-2 is shown in SEQ ID NO.

1.

3. A microbial agent for controlling tobacco target spot disease, characterized in that: It comprises the Malaysian Streptomyces sp. SE4-2 with the deposit number of CCTCC NO: M 20242638 as claimed in claim 1.

4. A method for preparing the microbial agent for controlling tobacco target spot disease as claimed in claim 3, characterized in that: The following steps are involved: 1) Activating the strain, culturing the Malaysian Streptomyces SE4-2 according to claim 1 on a plate using a culture medium to obtain activated Malaysian Streptomyces SE4-2; 2) preparing seed solution, picking activated Malaysian Streptomyces SE4-2 with an inoculation loop and inoculating it into a seed culture medium, and culturing it on a shaking table to obtain seed solution; 3) preparing the microbial agent, inoculating the seed liquid into the fermentation medium, wherein the volume ratio of the seed liquid to the fermentation medium is 1:10-20, and then placing it in a constant temperature incubator for fermentation and culture, and then drying the cultured fermented product at 25-35° C. and crushing it into powder to obtain the biocontrol microbial agent.

5. A method for preparing a microbial agent for controlling tobacco target spot disease according to claim 4, characterized in that: The culture medium in 1) is LB solid culture medium; and the culture in 2) is carried out by shaking at 28° C. and 180 r / min for 48 h.

6. A method for preparing a microbial agent for controlling tobacco target spot disease according to claim 4, characterized in that: The fermentation is carried out in a constant temperature incubator for 7 to 14 days at a temperature of 28°C.

7. Use of the Streptomyces according to any one of claims 1 or 2 in the preparation of a microbial agent for controlling tobacco target spot disease.

8. Use of the microbial agent for controlling tobacco target spot disease as claimed in any one of claims 3 to 5 in antagonizing tobacco target spot disease pathogens.

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