A strain antagonizing tobacco wildfire pathogenic bacteria and its application

Abstract

The invention discloses a strain antagonizing tobacco wildfire pathogenic bacteria. The strain is Pseudomonas aeruginosa 565-1, which is deposited in China Center for Type Culture Collection on November 1, 2022. The deposit number is CCTCCNO: M20221707, the deposit address is Wuhan University, Wuhan, China, and the taxonomic name is Pseudomonas aeruginosa 565-1. The strain antagonizing tobacco wildfire pathogenic bacteria is used in the prevention and treatment of tobacco wildfire, which can prevent the pathogenic bacteria from developing drug resistance and achieve long-term prevention and treatment effects.

Description

Technical Field

[0001] The invention relates to the technical field of strain screening, and more specifically to a strain antagonizing tobacco wildfire pathogenic bacteria and application thereof. Background Art

[0002] Tobacco wildfire is a bacterial disease caused by Pseudomonas syringae pv. tobacco. Tobacco wildfire can occur in the seedling stage, cluster stage and vigorous growth stage of tobacco, mainly harming leaves, stems, fruits and leaves. In the early stage of the disease, small brown dots appear on the leaves, and then a clear yellow halo forms around them, which is caused by the toxins secreted by the wildfire pathogen. Under the suitable conditions of high temperature and high humidity, the lesions expand rapidly and connect together over a large area, like a fire, and the yellow halo also becomes larger. When there is continuous rainfall and high humidity, the lesions expand over a large area, and pus will be produced on the back of the leaves. When there is little rain and dryness on the day, the lesions on the leaves are easy to fall off, and high temperature is conducive to the occurrence of wildfire.

[0003] At present, the prevention and control measures for tobacco wildfire are mainly chemical pesticides. However, the use of chemical pesticides has brought about serious "3R" problems. The use of chemical pesticides for prevention and control will leave pesticide residues, seriously pollute the environment and harm humans and animals. In addition, long-term use will easily lead to drug resistance, which will greatly reduce the prevention and control effect. Biological control does not pollute the environment, will not harm the health of humans and animals, and will not cause pathogens to develop drug resistance.

[0004] Therefore, how to provide a strain that can antagonize tobacco wildfire pathogens is a technical problem that technical personnel in this field urgently need to solve. Summary of the invention

[0005] In view of this, the present invention provides a strain antagonistic to tobacco wildfire pathogens, and applies the strain to the prevention and treatment of wildfire.

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

[0007] A strain antagonistic to tobacco wildfire pathogens, the strain is Pseudomonas aeruginosa 565-1, deposited in the China Center for Type Culture Collection, the preservation date is November 1, 2022, the preservation number is CCTCC NO: M 20221707, the preservation address is Wuhan University, Wuhan, China, and the taxonomic name is Pseudomonas aeruginosa 565-1.

[0008] As an inventive concept identical to the above technical solution, the present invention also seeks to protect the use of the Pseudomonas aeruginosa 565-1 strain in the preparation of a microbial preparation for preventing and treating tobacco wildfire disease.

[0009] As an inventive concept identical to the above technical solution, the present invention also seeks to protect a microbial preparation for preventing and treating tobacco wildfire disease, characterized in that it comprises Pseudomonas aeruginosa 565-1 strain.

[0010] Through the above technical scheme, it can be known that the present invention screened out a Pseudomonas aeruginosa 565-1 strain for preventing and controlling tobacco wildfire from tobacco fields in various tobacco-growing areas in Hunan where tobacco wildfire occurs seriously. By spraying tobacco plants with the fermentation liquid of the strain, the incidence rate of tobacco wildfire can be reduced to 20.19%, the morbidity index can be reduced to 6.95, and the prevention effect is as high as 65.52%, which lays a theoretical foundation for the research and development of microbial preparations for preventing and controlling tobacco wildfire. BRIEF DESCRIPTION OF THE DRAWINGS

[0011] In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings required for use in the embodiments or the description of the prior art will be briefly introduced below. Obviously, the drawings described below are only embodiments of the present invention. For ordinary technicians in this field, other drawings can be obtained based on the provided drawings without paying creative work.

[0012] Figure 1 This is a diagram showing the antibacterial effect of the 565-1 strain provided by the present invention on tobacco wildfire pathogens;

[0013] Figure 2 This is a diagram showing the antibacterial effect of the 565-1 sterile fermentation supernatant provided by the present invention on tobacco wildfire pathogens;

[0014] Figure 3 This is a colony morphology diagram of the 565-1 strain provided by the present invention;

[0015] Figure 4 The 16S rDNA PCR product gel electrophoresis diagram of the 565-1 strain provided by the present invention;

[0016] Figure 5 This is a phylogenetic tree result diagram of the 565-1 strain provided by the present invention. DETAILED DESCRIPTION

[0017] The following will be combined with the drawings in the embodiments of the present invention to clearly and completely describe the technical solutions in the embodiments of the present invention. Obviously, the described embodiments are only part of the embodiments of the present invention, not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without creative work are within the scope of protection of the present invention.

[0018] Materials, reagents and instruments used in the examples:

[0019] Tobacco rhizosphere soil: Soil from the roots of tobacco seedlings collected from tobacco fields where tobacco wildfire disease was seriously incurred in five tobacco-producing areas including Guiyang County, Jiahe County, Yongxing County of Chenzhou City, Hunan Province, and Ningyuan County and Xintian County of Yongzhou City.

[0020] Culture medium and reagents

[0021] LB medium: Use an electronic balance to weigh 10g of peptone, 5g of yeast powder and 10g of NaCl, add 500ml of deionized water, stir with a glass rod to dissolve all the drugs, then add deionized water to make up to 1L, divide 1L of the medium into 500ml conical flasks, add 200ml to each bottle, then add 1.5g of agar to the conical flask, sterilize it in a high-pressure steam sterilizer at 121℃ for 25min, prepare it into LB solid medium, and cool it for use;

[0022] LB liquid culture medium: The preparation method is the same as above, but without adding agar. The culture medium is divided into 50 ml conical flasks, 20 ml per bottle, and sterilized in a high pressure steam sterilizer at 121°C for 25 minutes.

[0023] Prepare LB liquid culture medium;

[0024] Reagents: peptone, yeast powder, NaCl, agar powder, CTAB, anhydrous ethanol, phenol-chloroform-isoamyl alcohol (25:24:1), chloroform, isopropanol, sterile water, etc.;

[0025] Experimental Instruments

[0026] 2L measuring cylinder, glass rod, 2ml centrifuge tube, 15ml centrifuge tube, pipette, coating rod, disposable culture dish, high pressure steam sterilizer, clean bench, shaker, etc.

[0027] Example 1 Strain Screening

[0028] 1) Sampling

[0029] Healthy tobacco seedlings were dug up with their roots from tobacco fields in Hunan where tobacco wildfire disease was serious. The seedlings were stored in sterilized paper bags. After being brought back to the laboratory, the soil at the roots of the tobacco was shaken off and the coarse soil was sieved to leave the fine soil.

[0030] 2) Preparation of soil suspension

[0031] Weigh 0.1 g of soil sample into a 15 ml centrifuge tube, add 9.9 ml of sterile water to make 10 -2 g / ml soil suspension, using a 1ml pipette to take a 10 -2 g / ml soil suspension was added to 9ml of sterile water to make 10ml of 10 - 3g / ml soil suspension, continue to dilute the soil suspension in this way to obtain a concentration of 10 -6 g / ml, 10 -7 g / ml and 10 -8 g / ml soil suspension, take 200ul of each of the three concentrations of suspension, add them to PDA medium and LB medium for smearing and bacteria, apply three culture dishes with each concentration of suspension, put the coated PDA culture dish in a 28℃ incubator, and put the coated LB culture dish in a 37℃ incubator. The culture dishes of two different culture media are inverted for culture. After a period of time, observe the growth of strains at different dilution concentrations, select suitable single colonies for streaking purification of the selected bacteria, and purify the selected fungi by picking hyphae or making cakes at the edge of single colonies. The isolated strains are numbered and stored at 4℃ for the next antagonism experiment identification. A total of 31 strains were isolated, including 22 bacterial strains, 6 fungal strains and 3 actinomycetes.

[0032] 3) Screening of biocontrol bacteria

[0033] Taking tobacco wildfire pathogen (Pseudomonas syringan pv.tabaci (Wolf et Foster) Young & Dye Wiki) as the target, the strains isolated from the rhizosphere soil of tobacco were screened by the inhibition zone method. The specific steps are as follows: First, each isolated and purified strain was cultured in a shaking incubator at 30°C and 160r / min for 48h, 50μl of bacterial solution was spread on LB agar medium, and after the colony covered the entire plate, it was cut into 8mm diameter bacterial cakes with a puncher. The tobacco wildfire pathogen was inoculated on an LB liquid culture dish, cultured in a shaking incubator at 30°C and 160r / min for 48h, and the LB agar medium was heated to dissolve. When it cooled to about 50°C, 1ml of tobacco wildfire pathogen was added to every 150ml of culture medium, and the plate was poured after being fully shaken. Then, the selected bacterial and fungal cakes were inverted and inoculated on LB plates mixed with tobacco wildfire pathogens. At the same time, sterile PDA cakes and sterile water were inoculated as a control group. Each strain was inoculated three times. After culturing at 28℃ for 2 to 3 days, the inhibition zone of each strain on tobacco wildfire pathogens was observed. The biocontrol bacteria with obvious inhibitory effects on tobacco wildfire pathogens were screened out, numbered 565-1, and the results are shown in the figure. Figure 1 The average diameters of colonies and inhibition zones are shown in Table 1.

[0034] Table 1 Average diameter of colonies and average diameter of inhibition zones

[0035]

[0036] The selected biocontrol bacteria were inoculated into LB liquid culture medium and fermented at 30℃ and 160r / min for 48h. After the fermentation liquid was centrifuged, the supernatant was filtered through a 0.22μm filter membrane twice to obtain a sterile fermentation supernatant. The LB plate with the tobacco wildfire pathogen was fully mixed and punched with a diameter of 8mm. 300μl of sterile fermentation supernatant was injected into each hole. After culturing at 28℃ for 2-3d, the formation of the inhibition zone was observed. The results are shown in the figure. Figure 2 .

[0037] 4) Identification of biocontrol bacteria strains

[0038] Morphological observation

[0039] The colonies of strain 565-1 are round with smooth edges, gray-green in color, and have a metallic luster. Figure 3 ;

[0040] Molecular Biology Identification

[0041] Pick the 565-1 bacterial colony and use the universal primers for 16S rDNA amplification (27F: 5′-AGAGTTTGATCCTGGCTCAG-3′1492R: 5′-TACGGTTACCTTGTTAC GAC-3′) for PCR amplification. The reaction system is (50μL): dd H2O (20.5μL); 27F (1.5μL); 1492R (1.5μL); Mix (25μL); DNA template (1.5μL). The reaction procedure is: 95℃ pre-denaturation for 5min; 94℃ denaturation for 30s; 59℃ annealing for 30s; 72℃ extension for 1.5min; 35 cycles; 72℃ extension for 10min; 4℃ storage. After the reaction is completed, the PCR product is sent to Shanghai Sangon Biotechnology (Shanghai) Co., Ltd. for sequencing. The obtained sequence is compared by BLAST nucleic acid homology on NCBI. The phylogenetic tree was constructed using MEGA 10.0 to determine the taxonomic status of the strains. The bacterial sequences of the genus Pseudomonas were downloaded from NCBI, and the phylogenetic tree was constructed using the NJ method with Escherichia coli as the outgroup. Figure 5 .

[0042] 16SrDN:

[0043] AGTCGAACGGTAACAGGAAGCAAGCTTGCTNCTTNGCTGACGAGTGGC

[0044] GGACGGGTGAGTAATGTCTGGGAAACTGCCTGATGGAGGGGGATAACT

[0045] ACTGGAAACGGTAGCTAATACCGCATAACGTCGCAAGACCAAAGAGGG

[0046] GGACCTTCGGGCCTCTTGCCATCGGATGTGCCCAGATGGGATTAGCTNG

[0047] TNGGTGGGGTAACGGCTCACCTAGGCGACGATCCCTAGCTGGTCTGAG

[0048] AGGATGACCAGCCACACTGGAACTGAGACACGGTCCAGACTCCTACGG

[0049] GAGGCAGCAGTGGGGAATATTGCACAATGGGCGCAAGCCTGATGCAGC

[0050] CATGCCGCGTGTATGAAGAAGGCCTTCGGGTTGTAAAGTACTTTCAGCG

[0051] GGGAGGAAGGGAGTAAAGTTAATAACCTTTGCTCATTGACGTTACCCGC

[0052] AGAAGAAGCACCGGCTAACTCCGTGCCAGCAGCCGCGGTAATACGGAG

[0053] GGTGCAAGCGTTAATCGGAATTACTGGGCGTAAAGCGCACGCAGGCGG

[0054] TTTGTTAAGTCAGATGTGAAATCCCCGGGCTCAACCTGGGAACTGCATC

[0055] TGATACTGGCAAGCTTGAGTCTCGTAGAGGGGGTAGAATTCCAGGTG

[0056] TAGCGGTGAAATGCGTAGAGATCTGGAGGAATACCGGTGGCGAAGGCG

[0057] GCCCCCTGGACGAAGACTGACGCTCAGGTGCGAAAGCGTGGGGAGCAA

[0058] ACAGGATTAGATACCCTGGTAGTCCACGCCGTAAACGATGTCGACTTG

[0059] GAGGTTGTGCCCTTGAGGCGTGGCTTCCGGAGCTAACGCGTTAAGTCG

[0060] ACCGCCTGGGGAGTACGGCCGCAAGGTTAAAACTCAAATGAATTGACG

[0061] GGGGCCCGCACAAGCGGTGGAGCATGTGGTTTAATTCGATGCAACGCG

[0062] AAGAACCTTACCTGGTCTTGACATCCACGGAAGTTTTCAGAGATGAGA

[0063] ATGTGCCTTCGGGAACCGTGAGACAGGTGCTGCATGGCTGTCGTCAGCT

[0064] CGTGTTGTGAAATGTTGGGTTAAGTCCCGCAACGAGCGCAACCCTTATC

[0065] CTTTGTTGCCAGCGGTCCGGCCGGGAACTCAAAGGAGACTGCCAGTGA

[0066] TAAACTGGAGGAAGGTGGGGATGACGTCAAGTCATCATGGCCCTTACG

[0067] ACCAGGGCTACACACGTGCTACAATGGCGCATACAAAGAGAAGCGACC

[0068] TCGCGAGAGCAAGCGGACCTCATAAAGTGCGTCGTAGTCCGGATTGGA

[0069] GTCTGCAACTCGACTCCATGAAGTCGGAATCGCTAGTAATCGTGGATCA

[0070] GAATGCCACGGTGAATACGTTCCCGGGCCTTGTACACACCGCCCGTCAC

[0071] ACCATGGGAGTGGGTTGCAAAAGAAGTAGGTAGCTTAACCTTCGGGAGGGCGCTTACCACTTTGTGATTCATGACTGGGGT, as shown in SEQ ID NO.1.

[0072] The strain 565-1 is 1421bp in size, and has a sequence homology of up to 99% with the Pseudomonas aeruginosa with accession number MN636758.1. Based on phylogenetic tree analysis, it can be preliminarily determined that the biocontrol bacteria isolated from the tobacco rhizosphere soil is Pseudomonas aeruginosa (Pseudomonas aeruginosa), which is deposited in the China Center for Type Culture Collection on November 1, 2022. The deposit number is CCTCCNO: M 20221707, and the deposit address is Wuhan University, Wuhan, China. The taxonomic name is Pseudomonas aeruginosa 565-1.

[0073] Example 2 Field evaluation of strain 565-1 against tobacco wildfire

[0074] Two mu of tobacco fields with severe wildfire disease were selected in Guiyang County, Chenzhou City. Four treatments were set up in the experiment: 20% thiophanate-methyl wettable powder; 10% kasugamycin aqueous solution; 565-1 fermentation liquid; culture medium (control). Each treatment was set up with 4 replicates, a total of 16 plots, each plot was about 50m 2 . The dosage of each treatment is: 20% thiophanate-methyl wettable powder diluted 1000 times for application; 565-1 fermentation liquid is 200ml / mu, diluted with 50kg of water for application; 10% kasugamycin aqueous solution is diluted 600 times for application. Start applying the medicine when sporadic lesions are first seen, and continue to apply the medicine 3 times every 5-7 days. Method of application: spray application, spray on both sides of the leaves.

[0075] Surveys were conducted before each application and 10 days after the last application. All leaves of 15 tobacco plants were surveyed in each plot. According to the Grading and Survey Methods of Tobacco Pests and Diseases (GBT 23222-2008), the survey was divided into 9 levels with leaves as the unit. The incidence rate, disease index and relative control effect of each treatment were calculated: disease index = 100 × ∑ (number of diseased leaves at each level × representative value of each level) / (total number of leaves surveyed × highest level representative value), relative control effect (%) = (control disease index - treatment disease index) / control disease index × 100.

[0076] The results of the field trial investigation showed that with the extension of the application time, the incidence rate and disease index of the control group (CK), 20% thiophanate-methyl wettable powder, 10% kasugamycin aqueous solution, and 565-1 fermentation liquid treatment groups gradually increased, reaching the highest in the third investigation, with the incidence rates of 58.43%, 30.36%, 25.18% and 20.19%, respectively. The incidence rate and disease index of the 565-1 fermentation liquid treatment group were significantly lower, and the maximum prevention effect on tobacco wildfire disease could reach 65.52%.

[0077] Table 2 The control effect of 565-1 on tobacco wildfire

[0078]

[0079] The various embodiments in this specification are described in a progressive manner, and each embodiment focuses on the differences from other embodiments. The same or similar parts between the various embodiments can be referenced to each other.

[0080] The above description of the disclosed embodiments enables one skilled in the art to implement or use the present invention. Various modifications to these embodiments will be apparent to one skilled in the art, and the general principles defined herein may be implemented in other embodiments without departing from the spirit or scope of the present invention. Therefore, the present invention will not be limited to the embodiments shown herein, but rather to the widest scope consistent with the principles and novel features disclosed herein.

Claims

1. A strain antagonistic to tobacco wildfire pathogens, characterized in that: The strain is Pseudomonas aeruginosa ( Pseudomonas aeruginosa )565-1, deposited in China Center for Type Culture Collection, deposit date is November 1, 2022, deposit number is CCTCC NO: M 20221707, deposit address is Wuhan University, Wuhan, China, taxonomic name is Pseudomonas aeruginosa 565-1.

2. Use of the Pseudomonas aeruginosa 565-1 strain according to claim 1 in the preparation of a microbial preparation for preventing and treating tobacco wildfire.

3. A microbial preparation for preventing and treating tobacco wildfire, characterized in that: It includes the Pseudomonas aeruginosa 565-1 strain described in claim 1.

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