Aeromonas hydrophila ztyl3 for tobacco bacterial wilt prevention and application thereof

Abstract

The application discloses Aeromonas hydrophila ZTYL3 for tobacco bacterial wilt prevention and application thereof, and the strain ZTYL3 of Aeromonas hydrophila has a preservation certificate number of CCTCC No: M 2019922. The disease control effect of the obtained Aeromonas hydrophila ZTYL3 is evaluated directly by taking the disease control ability as an index instead of antagonistic effect, and it is found by plate antagonistic ability determination that the strain ZTYL3 has no antibacterial ability to Ralstonia, which indicates that the disease control mechanism of the strain ZTYL3 is different from that of traditional antagonistic bacteria, and the strain ZTYL3 plays a disease control role mainly by not producing antibiotics, and may prevent diseases by mechanisms such as ecological niche competition, plant disease resistance induction and rhizosphere microbial community regulation. The Aeromonas hydrophila ZTYL3 provides a new biocontrol resource for crop bacterial wilt prevention.

Description

Technical Field

[0001] This application relates to the field of microbial technology, and in particular to Aeromonas hydrophila ZTYL3 for the prevention and control of tobacco bacterial wilt and its applications. Background Technology

[0002] Bacterial wilt, caused by Ralstonia spp., is an important soil-borne disease of crops. Ralstonia has a wide host range, infecting more than 400 crop species, but is particularly damaging to bananas, tomatoes, potatoes, and tobacco. In my country, tobacco bacterial wilt is mainly caused by Ralstonia nicotianae, which is prevalent in tobacco-growing areas south of the Yangtze River (Liu Jun-Ying, Zhang Jian-Feng, Wu Han-Lian, et al. Proposal to classify Ralstonia solanacearum phylotype I strains as Ralstonianicotianae sp.nov., and a genomic comparison between members of the genus Ralstonia. Frontiers in Microbiology, 2023, 14:1135872. doi:10.3389 / fmicb.2023.1135872.). The paper "Genomic Analysis of Ralstonia syzygii LLRS-1" published by Lu Canhua, Li Junying, and Mi Mengge in 2021 ([C] / / Chinese Society for Plant Pathology. Technological Innovation and Green Control in Plant Pathology - Proceedings of the 2021 Annual Meeting of the Chinese Society for Plant Pathology. China Agricultural Science and Technology Press, 2021:1.DOI:10.26914 / c.cnkihy.2021.063949.) has identified that the disease occurs in 43 districts (counties) of 12 prefectures and cities in Yunnan Province, including Wenshan, Baoshan, Lincang, Honghe, Kunming, Yuxi, Qujing, Zhaotong, Dali, Lijiang, Chuxiong, and Dehong, with the Wenshan, Lincang, Honghe, and Pu'er tobacco-growing areas experiencing more severe outbreaks. Although tobacco bacterial wilt is a serious disease, the scarcity of disease-resistant resources and the lack of effective chemical control have made it a significant factor restricting the improvement of tobacco yield and quality.

[0003] Biological control has received widespread attention due to its environmental friendliness. Numerous biocontrol resources have been extensively studied as biological control methods for soil-borne diseases. For example, the reported biocontrol bacteria for bacterial wilt mainly include: Pseudomonas aeruginosa, Bacillus spp., Streptomyces spp., Acinetobacter spp., Burkholderia spp., and Paenibacillus spp.

[0004] my country has developed and registered 11 biocontrol bacteria for tobacco bacterial wilt, mainly fungicides such as *Pseudomonas fluorescens*, *Bacillus amyloliquefaciens*, *Bacillus polymyxa*, and *Bacillus subtilis*. Most of these agents exhibit antagonistic activity against *Ralstonia*. Domestically reported patents related to biocontrol bacteria for tobacco bacterial wilt mainly include: *Enterbacter tabaci* 65B7 (CN115678806A), *Stenotrophomonas indicatrix* strain 107E3 (CN115612651A), and *Ralstonia*. sp.) 56D2 (CN113502250A), *Pseudomonas parafulva* DW15 (CN114934001A), *Pseudomonas koreensis* CLP-23 (CN111705016A), *Bacillus amyloliquefaciens* TBA03 (CN111117936A), *Bacillus polymyxa* NX1-4 -4 (CN107365729A), Bacillus subtilis biocontrol strain Trb3 (CN102747013A); the compound microbial agent mainly consists of three strains of Pseudomonas lurida FGD5-2, P. koreensis HCH2-3 and P. rhodesiae MTD4-1 (CN112920965A), and Streptomyces flavus. The following fungi were identified: *Streptomyces microflavus* CGMCC 12841, *Streptomyces albidoflavus* CGMCC 12842, *Streptomyces pratensis* CGMCC 12843, and a combination of *Pseudomonas aeruginosa* (CN106754563A); *Streptomyces leucovorum*, *Streptomyces reciprocitatum*, *Bacillus saffron*, *Bacillus mesoprothiolane*, and *Trichoderma rubrum* (CN106465734A); *Trichoderma ressei* ACCC 30150 and *Streptomyces silaceus* ACCC 40021 (CN103315005A); and a combination of *Polymycinium* and *Bacillus subtilis* (PD20200380).

[0005] Currently, there are no reports of Aeromonas bacteria being used in crop disease control, and no reports of Aeromonas bacteria being able to control or inhibit bacterial wilt in crops.

[0006] The existing screening of bacterial wilt biocontrol bacteria is obtained through indoor plate antagonistic screening and greenhouse biotesting. The obtained bacteria have a significant growth inhibitory effect on the pathogen. However, long-term use of such biocontrol bacteria will enhance the drug resistance of the pathogen and affect the control effect on bacterial wilt.

[0007] The information disclosed in the background section is intended only to enhance the understanding of the overall background of this application and should not be construed as an admission or in any way implying that such information constitutes prior art known to those skilled in the art. Summary of the Invention

[0008] This application addresses the aforementioned technical problems by providing Aeromonas hydrophila ZTYL3 for the control of tobacco bacterial wilt and its application. The ZTYL3 strain exerts its disease control effect not primarily by producing antibiotics, but may prevent disease through mechanisms such as niche competition, inducing plant resistance, and regulating the rhizosphere microbial community, which is beneficial to improving the safety of biocontrol agents.

[0009] This application provides a *Aeromonas hydrophila* ZTYL3 for the prevention and control of tobacco bacterial wilt, with preservation certificate number CCTCC No: M2019922.

[0010] Preferably, the carbon sources that this bacterium can utilize include: N-acetyl-D-glucosamine, Tween 40, D-salicylic acid, gelatin, inosine, α-D-glucose, D-trehalose, glycerol, L-malic acid, D-mannitol, D-galactose, sucrose, L-arginine, β-formyl-D-glucosinolate, D-fructose-6-phosphate, L-lactic acid, D-mannose, D-fructose, L-serine, D-glucose-6-phosphate, 3-formylglucose, D-malonic acid, and acetyl-D-glucosinolate. Maltose, N-acetyl-D-galactosamine, dextrin, D-serine, D-gluconic acid, L-aspartic acid, L-alanine, L-histamine, L-glutamic acid, aminoacetyl-L-proline, citric acid, propionic acid, mucoic acid, pectin, bromo-succinic acid, methyl pyruvate, acetic acid, D-fructose, L-galacturonic acid lactone, D-methyl lactate, glucuronide, glycosaminoglycans, inositol, D-glucuronic acid, L-pyroglutamic acid, D-aspartic acid;

[0011] Preferably, the carbon sources that this bacterium cannot utilize include: formic acid, melitriose, acetoacetic acid, N-acetylneuraminic acid, D-sorbitol, quinic acid, p-hydroxyphenylacetic acid, stachyose, L-rhamnose, γ-aminobutyric acid, α-D-lactose, D-galacturonic acid, gentiobiose, N-acetyl-β-D-mannosamine, D-cellobiose, D-minobiose, D-malic acid, α-hydroxybutyric acid, D-arabinol, L-fructose, α-ketoglutarate, β-hydroxy-D,L-butyric acid, α-keto-butyric acid, and melitriose;

[0012] Preferably, the bacteria grow better under conditions containing 1% NaCl, 1% sodium lactate, D-serine, pH 5, pH 6, sodium butyrate, vancomycin, rifamycin SV, tetrazolium blue, lincomycin, guanidine hydrochloride, and sodium tetradecanoate sulfate.

[0013] Preferably, the bacteria grow slowly under conditions containing 4% NaCl, 8% NaCl, dimethylaminetetracycline, potassium tellurite, tetrazolium violet, clostridial acid, aztreonam, lithium chloride, sodium bromate, naridinone acid, and acetosine.

[0014] Aeromonas hydrophila strain ZTYL3 does not control bacterial wilt by antagonizing or inhibiting Ralstonia nicotianae, Ralstonia solanacearum, Ralstonia pseudosolanacearum, or Ralstonia syzygii.

[0015] Another aspect of this application provides a biocontrol agent for tobacco bacterial wilt, comprising: the strain described above.

[0016] This microbial agent can be formulated by those skilled in the art based on the teachings and inspiration of this application, and according to actual production needs, by combining appropriate excipients with commonly used technical means in the field of microbial processes, to produce various dosage forms of Aeromonas hydrophila strain ZTYL3 with accession number CCTCC No: M2019922 that meet the requirements of various process production, such as powders, tablets, liquids, etc.

[0017] The dosage form of the microbial agent in this application is not limited to powder. Based on the teachings and inspiration of this application, and for actual production needs, those skilled in the art can select appropriate excipients to formulate various other dosage forms of Aeromonas hydrophila strain ZTYL3, which has accession number CCTCC No: M 2019922, in accordance with the requirements of various production processes. These dosage forms include tablets, liquids, sprays, granules, etc.

[0018] Preferably, the biocontrol agent also includes: excipients.

[0019] Preferably, the excipients are selected from at least one of the following: solvents, propellants, solubilizers, cosolvents, emulsifiers, colorants, binders, disintegrants, fillers, lubricants, wetting agents, osmotic pressure regulators, stabilizers, flow aids, flavoring agents, preservatives, suspending agents, coating materials, fragrances, anti-adhesion agents, binding agents, penetration promoters, pH adjusters, buffers, plasticizers, surfactants, foaming agents, defoamers, thickeners, encapsulating agents, humectants, absorbents, diluents, flocculants, anti-flocculation agents, filter aids, and release inhibitors.

[0020] Preferably, the biocontrol agent is in powder form.

[0021] Preferably, the concentration of strain ZTYL3 in the biocontrol agent is greater than or equal to 10. 7 CFU / mL, more preferably, is 10. 7 ~10 9 CFU / mL.

[0022] Preferably, the preparation method of the biocontrol agent is as follows: centrifuge the fermentation broth of strain ZTYL3, collect the bacterial cells, mix the bacterial cells with diatomaceous earth at a mass ratio of 1:1, add sucrose accounting for 5‰ of the total mass of the mixture, dry and pulverize to obtain the powder of the biocontrol agent.

[0023] Preferably, the drying process involves air-drying in a cool, dry, and ventilated place until the moisture content of the material reaches 10±2%, after which it is pulverized.

[0024] Preferably, the method of applying the biocontrol agent is as follows: applying the biocontrol agent or its diluted form to the roots of the tobacco plants via root irrigation.

[0025] Preferably, the biocontrol agent is diluted in the form of a fermentation diluent or a bacterial powder suspension.

[0026] Preferably, the pathogen of tobacco bacterial wilt is at least one of Ralstonia nicotianae, Ralstonia solanacearum, Ralstonia pseudosolanacearum, or Ralstonia syzygii.

[0027] Another aspect of this application provides a fermentation method for the strain described above, comprising the following steps: inoculating the seed culture of strain ZTYL3 into a fermentation medium for aerobic fermentation.

[0028] Preferably, the fermentation medium consists of 5 g / L glucose, 20 g / L soybean meal, 2.5 g / L bone peptone, 15 g / L corn starch, 2.5 g / L yeast extract, 0.5 g / L magnesium sulfate, 1 g / L dipotassium hydrogen phosphate, and pH 7.3.

[0029] Preferably, the inoculation ratio of the seed liquid of strain ZTYL3 is 1:100 of the volume ratio of seed liquid to fermentation medium.

[0030] Preferably, the fermentation conditions are: stirring at 120 r / min, temperature fluctuating around 37℃ by 0.5℃, and aeration rate of 15 m³ / min. 3 / h, the tank pressure is maintained at 0.05Mpa during fermentation until fermentation is completed;

[0031] Preferably, the fermentation time is 40 hours.

[0032] Specifically, the seed culture of strain ZTYL3 was transferred at a ratio of 1:100 to fermentation medium containing 150L of medium (5g / L glucose, 20g / L soybean meal, 2.5g / L bone peptone, 15g / L corn starch, 2.5g / L yeast extract, 0.5g / L magnesium sulfate, 1g / L dipotassium hydrogen phosphate, pH 7.3).

[0033] The fermentation process parameters are as follows: (1) Sterilization: The air sterilization conditions are: 0.15MPa, 123℃, 1h; the actual sterilization conditions are: 0.15MPa, 123℃, 35min; (2) Inoculation: Inoculation begins when the culture medium temperature drops to 37℃ after sterilization, and the inoculation ratio is 1:120 for inoculum / culture medium; (3) Fermentation: Rotation speed: maintain 120r / min until fermentation ends; Aeration rate: maintain 15m 3 Maintain a pressure of 0.05 MPa throughout the fermentation process; allow for short-term fluctuations in aeration rate due to equipment limitations. Temperature: Maintain a temperature of 37°C throughout the fermentation process, with a fluctuation of 0.5°C. Fermentation time: 40 hours.

[0034] In this document, the biocontrol bacteria, Aeromonas hydrophila ZTYL3, ZTYL3, strain ZTYL3, strain ZTYL3, and Aeromonas hydrophila mentioned in the invention content and specific embodiments section all refer to Aeromonas hydrophila strain ZTYL3 with accession number CCTCC No: M 2019922 of this application.

[0035] The beneficial effects that this application can produce include:

[0036] 1) The Aeromonas hydrophila ZTYL3 strain and its application provided in this application for the control of bacterial wilt in tobacco are evaluated based on their disease control ability, not antagonistic effect. Plate antagonistic ability testing revealed that strain ZTYL3 showed no inhibitory effect against Rawlstonella, indicating that the disease control mechanism of strain ZTYL3 differs from that of traditional antagonistic bacteria. This suggests that strain ZTYL3's disease control effect is not primarily through antibiotic production, but may involve mechanisms such as niche competition, inducing plant resistance, and regulating the rhizosphere microbial community. Aeromonas hydrophila ZTYL3 provides a new biocontrol resource for the control of bacterial wilt in crops.

[0037] 2) The Aeromonas hydrophila ZTYL3 strain and its application provided in this application for the control of tobacco bacterial wilt do not induce resistance in Rawlstonella to strain ZTYL3 or its metabolites when applied in the field. Long-term use of this biocontrol can achieve the effect of controlling bacterial wilt while avoiding the development of drug resistance. The application of strain ZTYL3 has good safety. Attached Figure Description

[0038] Figure 1 These are colony morphology and strain cell morphology images of Aeromonas hydrophila ZTYL3 cultured for 48 hours in Example 1 of this application, where a) is a photograph of a culture dish; b) is magnified 3 times; and c) is a cell morphology image.

[0039] Figure 2 This is a phylogenetic tree diagram of Aeromonas hydrophila ZTYL3 constructed based on the whole genome in Example 3 of this application.

[0040] Figure 3 This is a diagram illustrating the effect of the ZTYL3 powder from Example 3 of this application on controlling tobacco bacterial wilt.

[0041] Figure 4 These are comparative photographs of the colony morphology of Aeromonas hydrophila ZTYL3 and Rawlstonella QBRS-1 in confrontation culture of Example 3 of this application.

[0042] The preservation information of Aeromonas hydrophila ZTYL3 provided in this application is as follows: Preservation number: CCTCC NO: M 2019922; Classification name: Aeromonas hydrophila ZTYL3; Preservation date: November 12, 2019; Preservation institution: China Center for Type Culture Collection; Preservation address: Wuhan University, Wuhan, China. Detailed Implementation

[0043] The present application will now be described in further detail with reference to the accompanying drawings and embodiments, but this does not limit the present application in any way. Any modifications or improvements made based on the teachings of the present application shall fall within the protection scope of the present application.

[0044] Example

[0045] Unless otherwise specified, all materials and instruments used in the following embodiments were obtained through commercial channels; and all detection methods used are existing methods unless otherwise specified.

[0046] 1. Sources of biological materials

[0047] I. The tobacco material used in the following embodiments is the well-known and widely used tobacco variety, Honghua Dajinyuan, which is kept in the applicant's laboratory and can also be purchased commercially.

[0048] II. Source of Rollstonella used in the following examples:

[0049] Ralstonia nicotianae RS T For "Liu Jun-Ying, Zhang Jian-Feng, Wu Han-Lian, et al. Proposal to classify Ralstonia solanacearum phylotypeI strains as Ralstonia nicotianae sp.nov., and a genomic comparison between members of the genus Ralstonia.Frontiers in Microbiology, 2023,14:1135872.doi:10.3389 / fmicb.2023.1135872." RS recorded in the article T The strain has completed genome sequencing, and the sequence has been submitted to the GenBank database. The Bioproject Number is PRJNA594457, and the GenBank assembly accession number is GCA_018243235.1. The strain was deposited at the Guangdong Provincial Microbial Culture Collection Center on July 14, 2022, with the accession number GDMCC 1.3533.

[0050] Ralstonia syzygii LLRS-1 is the strain described in the article "Can-Hua Lu, Jun-Ying Li, Meng-Ge Mi, et al. Complete Genome Sequence of Ralstonia syzygii subsp. indonesiensis Strain LLRS-1, Isolated from Wilted Tobacco in China. Phytopathology, 2021, 111:12:2392-2395".

[0051] Ralstonia solanacearum FQY_4 is the strain described in the article "Cao Yi, Tian Baoyu, Liu Yanxia, ​​et al. Genome Sequencing of Ralstonia solanacearum FQY_4, Isolated from a Bacterial Wilt Nursery Used for Breeding Crop Resistance. Genome Announcements, 2013, 1(3):e00125-13."

[0052] Ralstonia nicotianae BSRS-1, QBRS-1, and PEJG01 are all strains preserved in the applicant's laboratory. The applicant promises to distribute them to the public free of charge for 20 years from the date of this application to verify the effectiveness of this application.

[0053] 2. The culture media and seedling cultivation methods used in the following experimental examples:

[0054] I. Culture medium used

[0055] LB liquid medium contains 1% tryptone, 0.5% yeast extract, 1% sodium chloride and 0.5% sucrose;

[0056] CG medium contains 0.1% acid-hydrolyzed casein, 0.25% glucose, and 2% peptone;

[0057] CGA contains 0.1% acid-hydrolyzed casein, 0.25% glucose, 2% peptone, and 1.5% agar;

[0058] The oligotrophic medium CN contains 0.1% casein amino acids, 0.1% nutrient broth, and 1.5% agar;

[0059] TZC medium contains 1% peptone, 25% glucose, 0.1% casein hydrolysate, 1.5% agar, and 0.005% triphenyltetrazolium chloride (TTC).

[0060] II. Floating Seedling Raising

[0061] Using the diseased tobacco variety Honghua Dajinyuan as the test subject, tobacco seedlings were cultivated in a floating seedling stage until the 4-5 leaf stage.

[0062] III. Pathogen Culture

[0063] Activation of Rollston bacteria RS from a -80°C ultra-low temperature freezer T The sample was placed on TZC medium [1% peptone, 0.25% glucose, 0.1% casein hydrolysate, 1.5% agar and 0.005% triphenyltetrazolium chloride (TTC)] and incubated in a constant temperature incubator at 28℃ for 36–48 h.

[0064] Select typical colonies with wide white edges, strong fluidity, and a pink or light red liquid center, and inoculate them into Erlenmeyer flasks containing 100 mL of CG liquid medium (1% peptone, 0.25% glucose, 0.1% casein hydrolysate). Incubate at 28℃ and 225 r / min for 24 h with constant temperature shaking.

[0065] Take 100 μL and dilute to 10 -7 Take 100μL of 10 -5 10 -6 10 -7 The diluted solution was spread onto TZC plates, and the colony morphology and colony count were observed after 48 hours. The amount of bacteria in the culture solution was then calculated.

[0066] Example 1: Isolation of strain ZTYL3

[0067] I. Trapping, Separation and Culture

[0068] After removing impurities and large lumps from the soil sample (collected from tobacco soil in Hongta District, Yuxi City, Yunnan Province, by Lu Canhua, collected in May 2019), the soil was placed in a 120mm diameter glass petri dish with a soil layer thickness of about 1.5cm and moistened with distilled water using a titration bottle.

[0069] Preparation of the microbial trapping device: First, apply glue to the edge of a microporous filter membrane with a diameter of 50 mm and a pore size of 0.45 μm, and place a stainless steel flat-bottomed washer on the microporous filter membrane; then add 3 mL of solid culture medium (1.2% gellan gum and 1.0% vitamins) into the inner cavity of the washer; then apply glue to the upper surface of the metal washer, and cover it with another microporous filter membrane with a pore size of 0.45 μm;

[0070] Place the microbial trapping device on the moist soil sampled, gently compact the device to ensure that the microporous filter membrane is in full contact with the soil, then completely cover the device with the remaining soil, and moisten the soil again with a titration bottle containing distilled water.

[0071] Cover the petri dish, seal the culture device with sealing film, and place it in a 30℃ incubator for 7 days. During this period, observe the soil moisture. If the soil moisture is low, replenish it with sterile water.

[0072] Remove the culture device from the incubator after induction, crush the solid culture medium, add 3 mL of sterile water and let stand for 10 min, then serially dilute to 10. -4 ;

[0073] Take 10 -4 10 -5 The bacterial culture was spread on oligotrophic medium CN (containing 0.1% casein amino acids, 0.1% nutrient broth, and 1.5% agar), with 5 plates for each gradient. The plates were dried in a laminar flow hood and incubated in a 30°C incubator for 7 days.

[0074] Through the above experiments, pure strain ZTYL3 was obtained by streaking the culture from CN culture dishes. A single colony of ZTYL3 was then inoculated into a test tube containing 2.5 mL of LB liquid medium and incubated at 28℃ with shaking at 225 rpm for 48 h. The colony morphology of the obtained strain is shown below. Figure 1 As shown.

[0075] Example 2: Identification of strain ZTYL3

[0076] I. Culture characteristics and morphological features: Conventional bacterial identification was performed with reference to the literature "Handbook of Systematic Identification of Common Bacteria" (edited by Dong Xiuzhu et al., Science Press, 2001).

[0077] Cultivation characteristics and morphological features of strain ZTYL3: When cultured in NA medium at 28℃, round colonies form after 24 hours. Initially, the colonies are pale, later becoming pale grayish-white with a slight reddish tinge, smooth edges, and a raised center. Strain ZTYL3 can grow in LB, NB, and PDB media. In LB liquid medium, strain ZTYL3 can grow at temperatures ranging from 20 to 35℃, with 25℃ being the optimal temperature. Microscopic examination of the strain reveals rod-shaped bacteria with multiple tufts of flagella. The bacteria are Gram-negative.

[0078] II. The compound metabolic characteristics of strain ZTYL3 were analyzed using Biolog GEN III plates.

[0079] 1. Metabolic characteristics of compounds:

[0080] The growth of strain ZTYL3 under various carbon sources and growth inhibition conditions is as follows. Available carbon sources include: N-acetyl-D-glucosamine, Tween 40, D-salicylic acid, gelatin, inosine, α-D-glucose, D-trehalose, glycerol, L-malic acid, D-mannitol, D-galactose, sucrose, L-arginine, β-formyl-D-glucoside, D-fructose-6-phosphate, L-lactic acid, D-mannose, D-fructose, L-serine, and D-glucose-6-phosphate. 3-Formylglucose, D-maltose, N-acetyl-D-galactosamine, dextrin, D-serine, D-gluconic acid, L-aspartic acid, L-alanine, L-histamine, L-glutamic acid, aminoacetyl-L-proline, citric acid, propionic acid, mucoic acid, pectin, bromo-succinic acid, methyl pyruvate, acetic acid, D-fructose, L-galacturonic acid lactone, D-methyl lactate, glucuronide, glycolic acid, inositol, D-glucuronic acid, L-pyroglutamic acid, D-aspartic acid;

[0081] The following carbon sources cannot be utilized: formic acid, melitriose, acetoacetic acid, N-acetylneuraminic acid, D-sorbitol, quinic acid, p-hydroxyphenylacetic acid, stachyose, L-rhamnose, γ-aminobutyric acid, α-D-lactose, D-galacturonic acid, gentiobiose, N-acetyl-β-D-mannosamine, D-cellobiose, D-minobiose, D-malic acid, α-hydroxybutyric acid, D-arabinol, L-fructose, α-ketoglutarate, β-hydroxy-D,L-butyric acid, α-keto-butyric acid, and melitriose.

[0082] It grows better under the conditions of containing 1% NaCl, 1% sodium lactate, D-serine, pH 5, pH 6, sodium butyrate, vancomycin, rifamycin SV, tetrazolium blue, lincomycin, guanidine hydrochloride, and sodium tetradecanoate sulfate.

[0083] However, it grows slowly under conditions containing 4% NaCl, 8% NaCl, dimethylaminetetracycline, potassium tellurite, tetrazolium violet, fusoxanol, aztreonam, lithium chloride, sodium bromate, naphthidone acid, and acetosine.

[0084] 2. Strain identification based on the Gen III database

[0085] Gen III identification results showed that strain ZTYL3 had the highest similarity to Aeromonas hydrophila in the database (0.589), and other similar species included Vibrio cholerae O1 / O139 (0.184), Aeromonas jandaei (0.139), and Vibrio metschnikovii (0.088).

[0086] III. Identification of strain ZTYL3 using 16S rDNA and genome sequence

[0087] 1. Molecular identification methods are as follows: A bacterial genomic DNA extraction kit was used; the method is described in the kit instructions. The 16S rDNA sequence was amplified by PCR using universal primers F27 / R1492 under standard conditions. After gel recovery, the amplified product was ligated into the pEAZY-T5 Zero vector. The vector was then heat-shocked and transformed into competent *E. coli* cells DH5α. Colonies were picked and identified by colony PCR using primers M13F / M13R. Positive clones were sent to Shanghai Yingjun Biotechnology Co., Ltd. for sequencing. The Ezbiocloud database (https: / / www.ezbiocloud.net / ) was used to analyze the type species closely related to strain ZTYL3, preliminarily confirming the strain's primary taxonomic position.

[0088] 2. Obtain the complete genome of the strain using genome sequencing technology. Compare the DNA with the TYPE database to calculate the dDDH (dMolecular Hybridization Hierarchy) value between the patented strain and its closest related species, ultimately determining the strain's molecular taxonomic position. Figure 2 As shown in Table 2.

[0089] Table 2. Comparative analysis of the genome similarity between ZTYL3 and bacteria of the genus Zygomycetes

[0090]

[0091] 16S rDNA sequence analysis showed that strain ZTYL3 is related to Aeromonas hydrophila subsp. hydrophila ATCC 7966. T The highest similarity was 99.93%, with *Aeromonas hydrophila* subsp. *ranae* LMG19707. T A.media CECT 4232 T The sequence similarity was 99.86% and 99.73%, respectively, indicating that strain ZTYL3 belongs to the genus Aeromonas. After genome sequencing, analysis using the Type (Strain) Genome Server (https: / / tygs.dsmz.de / ) database showed that strain ZTYL3 is similar to Aeromonas hydrophila ATCC 7966. T The highest similarity was 73.6% (dDDH4), followed by Aeromonas hydrophila subsp.ranae CIP 107985. T(71.6%), exceeding the identification threshold of 70.0% for new species; strain ZTYL3 is similar to Aeromonas aquariorum CECT 7289. T , Aeromonas dhakensis CIP107500 T The DDH(d4) values ​​of strain ZTYL3 and other Aeromonas hydrophila were 50.4% and 50.2%, respectively; the dDDH values ​​of strain ZTYL3 and other Aeromonas hydrophila were less than 50%. Based on the genomes of strain ZTYL3 and its closely related species, a phylogenetic tree was constructed, showing that strain ZTYL3 and the standard strain ATCC 7966 of Aeromonas hydrophila... T and Aeromonas hydrophila subsp.ranae CIP107985 T The three are most closely related, and they come together as one branch. Figure 2 The above results indicate that strain ZTYL3 is Aeromonas hydrophila.

[0092] Based on the above identification results, strain ZTYL3 was confirmed to be a strain of Aeromonas hydrophila, named ZTYL3, and deposited for preservation. Its preservation information is as follows: Preservation number: CCTCC NO: M2019922; Preservation date: November 12, 2019; Preservation institution: China Center for Type Culture Collection; Preservation address: Wuhan University, Wuhan, China.

[0093] Application of strain ZTYL3 in the control of bacterial wilt

[0094] In the following embodiments, bacterial wilt refers to tobacco bacterial wilt, a disease caused by infection with at least one of the following pathogens: Ralstonia tobaccoicotianae, Ralstonia solanacearum, Ralstonia pseudosolanacearum, or Ralstonia syzygii.

[0095] 1. Evaluation of the efficacy of biocontrol bacterium ZTYL3 in controlling tobacco bacterial wilt:

[0096] The efficacy evaluation is divided into indoor bioassay for initial screening and secondary screening. The specific procedures are as follows:

[0097] Step S1, initial screening by bioassay

[0098] Tobacco seedling treatment: Tobacco seedlings were cultivated to the 4-5 leaf stage using the floating seedling method. One day before the seedlings were used, the seedlings were taken out of the seedling pond and dried in the floating trays. The next day, sterile blades were used to make incisions on both sides of the root of the seedlings 1.5 cm away from the center of the plant. The seedlings were divided into experimental group and control group, with 2 seedlings in each group.

[0099] Biocontrol bacteria pretreatment: 1 mL of the tested biocontrol bacteria was inoculated into the root substrate of the tobacco plants in the experimental group, while the tobacco seedlings inoculated only with LB culture medium were set as the control group;

[0100] Tobacco seedling suspension culture: Place a thick plastic sheet slightly larger than the floating plate for cultivating tobacco seedlings on the culture rack in a 28℃ constant temperature artificial climate chamber, and place 5 disposable culture dishes as supports at the 4 corners and center of the plastic sheet. Place the floating plate on it for 1 day of cultivation, and water it three times a day (morning, noon and evening) with a water sprayer. The amount of water should be such that the water in the holes of the floating plate does not drip.

[0101] Pathogen inoculation: One day after pretreatment with biocontrol bacteria, 0.5 mL of *R. tobaccolorius* RS was inoculated into both the experimental and control groups. T The 10-fold diluted solution was cultured in a constant temperature artificial climate chamber at 28℃ for 15-20 days, during which time it was watered and kept moist with a sprayer three times a day, morning, noon and evening.

[0102] Observation and Recording: Observe the disease incidence in the experimental group and the control group. When the disease incidence in the control group is >80%, record the disease incidence in the tobacco plants in the experimental group. Healthy tobacco plants are recorded as 1, tobacco plants with disease but not dead are recorded as 0.5, and tobacco plants that have died are recorded as 0. The value of each tobacco plant treated with the tested bacteria is the sum of the values ​​of the two tobacco plants. Select the strain with the highest value among the tested bacteria as the potential biocontrol bacteria for indoor rescreening.

[0103] Step S2, biological testing and rescreening

[0104] Step S2, biological test rescreening, is the same as step S1, biological test initial screening, except for the following different tests.

[0105] 1) The tested strains were potential biocontrol bacteria obtained from the initial screening in step S1;

[0106] 2) The number of tobacco plants treated in the bioassay was increased, with both the treatment group and the control group treated with 8 tobacco seedlings;

[0107] 3) The disease occurrence of tobacco plants in each treatment group was investigated 10 and 20 days after inoculation with Rollstonella, as shown in Table 1.

[0108] Table 1. Effect of indoor strain ZTYL3 on tobacco bacterial wilt control

[0109]

[0110]

[0111] Experimental results showed that in the initial screening, both tobacco plants treated with strain ZTYL3 were healthy, hence the value was assigned 2.0. In the greenhouse secondary screening, 8 and 5 tobacco plants were healthy at 10 and 20 dpi, respectively, while the control group had 5 and 0 healthy plants, respectively. These results indicate that ZTYL3 has a good control effect and can be used to evaluate its efficacy in greenhouse potted plants.

[0112] 2. Greenhouse pot experiment on the control of tobacco bacterial wilt by biocontrol bacterium ZTYL3

[0113] Experiment setup: The experiment was conducted in a greenhouse with a temperature control of 28-30℃. The experiment included a biocontrol treatment group and a control group (root irrigation with an equal volume of LB medium + tap water). Each treatment was replicated 3 times, with 10 tobacco plants per replicate.

[0114] Culture of the test strain: The biocontrol bacteria ZTYL3 obtained by indoor screening was cultured in LB liquid medium (1% tryptone, 0.5% yeast extract, 1% sodium chloride and 0.5% sucrose) and shaken at 225 r / min 30℃ for 48 h to obtain the bacterial agent ZTYL3;

[0115] Four strains of Rawlstonella from Yuxi City (LLRS-1), Lincang City (BSRS-1), Pu'er City (PEJG01) in Yunnan Province and Fujian Province (FQY_4) were selected as pathogens in the experiment. The Rawlstonella pathogens were cultured in CG liquid medium and shaken at 225 r / min and 30℃ for 24 h.

[0116] Tobacco seedling transplanting: The tobacco seedlings are cultivated using the floating seedling raising method. The seedlings are those with two leaf cuts and have been cultivated for about 50 days. When transplanting, the red soil and organic matter are mixed at a ratio of 3:1 before transplanting the seedlings.

[0117] Inoculation: After transplanting, dilute 250 mL of biocontrol bacteria strain ZTYL3 fermentation broth 25 times, and apply 200 mL of the diluted solution to the roots of each tobacco plant (the concentration of ZTYL3 bacteria in the diluted solution is 10). 7 -10 8 CFU / mL);

[0118] The control group included treatment with an equal volume of LB dilution;

[0119] Dilute the chemical agent thiabendazole copper (pesticide registration certificate number PD20086024, produced by Zhejiang Longwan Chemical Co., Ltd.) 500 times with tap water and drench the roots of each tobacco plant with 200 mL.

[0120] The following day, the *Rolstonia* culture medium, which had been shaken for 24 hours, was diluted 100-fold, and each tobacco plant was inoculated with 100 mL of the diluted *Rolstonia* solution (inoculation concentration approximately 10). 7 (CFU / mL)

[0121] Survey and statistics: Disease index was assessed every 7 days after vaccination, for a total of 3-6 times. The incidence, disease index, and control efficacy of tobacco bacterial wilt were calculated using the following formula:

[0122] Incidence rate = number of diseased plants / total number of plants surveyed × 100%; Disease index = [Σ(disease level × number of strains at this level) / (highest level × total number of plants)] × 100;

[0123] Prevention and control effect = (control disease index - treatment disease index) / control disease index × 100%.

[0124] The disease index was determined according to the Tobacco Industry Standard of the People's Republic of China (GB / T23222-2008) on the classification of diseases as follows: Level 0: No disease in the whole plant; Level 1: Less than half of the leaves on the diseased side wither; Level 3: Half to two-thirds of the leaves on the diseased side wither; Level 5: More than two-thirds of the leaves on the diseased side wither; Level 7: All the leaves of the diseased plant wither; Level 9: The diseased plant is basically dead.

[0125] The results of the experiment are shown in Table 3.

[0126] Table 3. Control efficacy of bacterial agent ZTYL3 against different types of Rawlstonella.

[0127]

[0128] The above results indicate that tobacco plants treated with fungal agent ZTYL3 showed lower disease indices than the water control group and the thiabendazole copper treatment group during the observation period. Furthermore, ZTYL3 demonstrated control efficacy against *Ralstonia solanacearum* strains from different geographical origins, with efficacy ranging from 35.66% to 78.95%. It showed particularly good control efficacy against bacterial wilt caused by *Ralstonia solanacearum* strains from Pu'er (PEGJ01) and Yuxi (LLRS-1), reaching 59.28% and 78.95%, respectively. The control efficacy of ZTYL3 against *Ralstonia solanacearum* strain FQY_4 from Fujian was similar to that of the control agent thiabendazole copper. These results demonstrate that ZTYL3 has good control efficacy against bacterial wilt caused by *Ralstonia solanacearum* strains from different geographical origins.

[0129] 3. Evaluation of the greenhouse control efficacy of biocontrol agent ZTYL3 against tobacco bacterial wilt.

[0130] The differences between this experiment and "2. Greenhouse pot experiment on the control of tobacco bacterial wilt by biocontrol bacteria ZTYL3" are as follows:

[0131] 1. Seed culture: Pick one loopful of biocontrol bacteria ZTYL3 and inoculate it into a 500mL centrifuge tube containing 500mL seed culture medium (10.0g / L peptone, 3.0g / L beef extract, 5.0g / L sodium chloride, 15.0g / L agar, pH 7.0), and incubate at 28℃ in a shaker for 15h.

[0132] 2. Fermentation: The seed culture of biocontrol bacteria ZTYL3 was transferred at a ratio of 1:100 to 150L of fermentation medium (5g / L glucose, 20g / L soybean meal, 2.5g / L bone peptone, 15g / L corn starch, 2.5g / L yeast extract, 0.5g / L magnesium sulfate, 1g / L dipotassium hydrogen phosphate, pH 7.3).

[0133] The fermentation process parameters are as follows: (1) Sterilization: The air sterilization conditions are: 0.15MPa, 123℃, 1h; the actual sterilization conditions are: 0.15MPa, 123℃, 35min; (2) Inoculation: Inoculation begins when the culture medium temperature drops to 37℃ after sterilization, and the inoculation ratio is 1:120 for inoculum / culture medium; (3) Fermentation: Rotation speed: maintain 120r / min until fermentation ends; Aeration rate: maintain 15m 3 Maintain a pressure of 0.05 MPa throughout the fermentation process; allow for short-term fluctuations in aeration rate due to equipment limitations. Temperature: Maintain a temperature of 37°C throughout the fermentation process, with a fluctuation of 0.5°C. Fermentation time: 40 hours.

[0134] 3. Powdering: Centrifuge the fermentation broth (approximately 16.743 billion CFU / mL) and collect the centrifuged cells as raw materials. Mix them with diatomaceous earth at a 1:1 mass ratio and add sucrose at 5‰ of the total mass of the mixture as nutrients. Place the mixture in a cool, dry and ventilated place to dry. When the moisture content reaches about 10%, pulverize it to obtain ZTYL3 powder (5.2 billion CFU / g).

[0135] 4. Prevention and control: Apply 2.5g of bacterial agent to each tobacco seedling, dilute with 50mL of water to obtain a bacterial powder suspension with a bacterial concentration of 260 million CFU / mL, and drench the roots. Set up 3 replicates for every 15 seedlings, and set up water as a control. Inoculate with Tobacco Rawstone's QBRS-1 the next day, 0.1OD per seedling. Investigate the disease level once a week and calculate the disease index.

[0136] The test results are as follows Figure 3 As shown, by Figure 3It can be seen that as the number of days after inoculation increased, the disease severity in the control group gradually increased, while the disease severity in the tobacco plants treated with fungal agent ZTYL3 was milder. At 24 days after inoculation, the area under the disease index curve was 352.6±124.6, while that in the control group was 1219±86.26. Based on the area under the disease index curve, the control efficacy of the ZTYL3 treatment group was 71.07%, indicating good control efficacy.

[0137] 4. ZTYL3 strain plate confrontation antagonism against Rawlstonella

[0138] The inhibition zone of the bacterial strain was measured using the plate confrontation culture method.

[0139] The operating steps are as follows: Roldstone bacteria QBRS-1 and RS, cultured on CG medium for 24 hours respectively... T LLRS-1 and BSRS-1 were serially diluted to 10 -4 Take 100 μL of the diluted solution and place it on the surface of CGA medium. Spread it evenly with a spreader and air dry in a cleanroom. Pick a single colony of strain ZTYL3 and the antagonistic bacterium Bacillus velezensis WY2 (depository location: Institute of Microbiology, Chinese Academy of Sciences, Datun Road, Chaoyang District, Beijing; depository institution: China General Microbiological Culture Collection Center; accession number: CGMCC No. 6662) and inoculate it on the surface of a medium containing Rawlstonella. After culturing for 2-3 days, observe the antibacterial effect of each strain. For strains with antibacterial effect, use the four-point confrontation method to culture and measure the inhibition zone and inhibition band of the strain. The results are as follows. Figure 4 As shown in Table 4.

[0140] Table 4. Antagonistic ability of strain ZTYL3 against different tobacco wilt pathogens

[0141] <![CDATA[RS T ]]> 0.00±0.00 0.00±0.00 LLRS-1 0.00±0.00 0.00±0.00 BSRS-1 0.00±0.00 0.00±0.00 QBRS-1 0.00±0.00 0.00±0.00

[0142] The experimental results showed that strain ZTYL3 was effective against Rawlstonella RS. T None of the LLRS-1, BSRS-1, and QBRS-1 showed antagonistic effects. These results indicate that the biocontrol bacterium ZTYL3 does not control bacterial wilt through antibacterial activity, but may exert its effects through non-antagonistic niche competition, inducing resistance, and regulating the rhizosphere microecological structure.

[0143] Although this application has been described in detail with reference to the foregoing embodiments, those skilled in the art can still modify the technical solutions described in the foregoing embodiments or make equivalent substitutions for some of the technical features. Any modifications, equivalent substitutions, improvements, etc., made within the spirit and principles of this application should be included within the protection scope of this application.

Claims

1. An Aeromonas hydrophila species used for the control of tobacco bacterial wilt ( Aeromonas hydrophila ZTYL3 is characterized by, Aeromonas hydrophila ( Aeromonas hydrophila ZTYL3 Collection Certificate No.: CCTCCNo: M 2019922.

2. A biocontrol agent for tobacco bacterial wilt, characterized in that, include: Aeromonas hydrophila ZTYL3 as described in claim 1.

3. The biocontrol agent according to claim 2, characterized in that, Biocontrol agents also include: excipients; excipients are selected from at least one of the following: solvents, propellants, solubilizers, co-solvents, emulsifiers, colorants, binders, disintegrants, fillers, lubricants, wetting agents, osmotic pressure regulators, stabilizers, flow aids, flavoring agents, preservatives, suspending agents, coating materials, fragrances, anti-adhesion agents, binding agents, penetration promoters, pH adjusters, buffers, plasticizers, surfactants, foaming agents, defoamers, thickeners, encapsulating agents, humectants, absorbents, diluents, flocculants, anti-flocculation agents, filter aids, and release inhibitors; the dosage form of biocontrol agents is powder.

4. The biocontrol agent according to claim 2, characterized in that, The concentration of strain ZTYL3 in the biocontrol agent is greater than or equal to 10. 7 CFU / mL.

5. The biocontrol agent according to claim 2, characterized in that, Preparation method of biocontrol agent: Centrifuge the fermentation broth of strain ZTYL3, collect the bacterial cells, mix the bacterial cells with diatomaceous earth at a mass ratio of 1:1, add sucrose accounting for 5‰ of the total mass of the mixture, dry and pulverize to obtain the powder of biocontrol agent; The drying process involves air-drying the material in a cool, dry, and well-ventilated place until the moisture content reaches 10±2%, at which point the material is then pulverized.

6. The biocontrol agent according to claim 2, characterized in that, The method of applying the biocontrol agent is as follows: apply the biocontrol agent or its diluted form to the roots of the tobacco plants for root irrigation; the biocontrol agent is diluted in the form of fermentation dilution or bacterial powder suspension.

7. The biocontrol agent according to claim 2, characterized in that, The pathogens causing tobacco bacterial wilt are at least one of Ralstonia nicotianae, Ralstonia solanacearum, or Ralstonia syzygii.

8. The fermentation method of Aeromonas hydrophila ZTYL3 according to claim 1, characterized in that, Includes the following steps: The seed culture of strain ZTYL3 was inoculated into a fermentation medium for aerobic fermentation.

9. The fermentation method according to claim 8, characterized in that, The fermentation medium consisted of 5 g / L glucose, 20 g / L soybean meal, 2.5 g / L bone peptone, 15 g / L corn starch, 2.5 g / L yeast extract, 0.5 g / L magnesium sulfate, and 1 g / L dipotassium hydrogen phosphate, with a pH of 7.

3. The inoculation ratio of the seed culture of strain ZTYL3 was 1:100 (seed culture to fermentation medium volume). The fermentation conditions were: stirring at 120 r / min, temperature fluctuating around 37℃ by 0.5℃, and aeration rate of 15 m³ / min. 3 / h, the tank pressure is maintained at 0.05Mpa during fermentation until the end of fermentation; the fermentation time is 40h.

Images