Acetobacter xylinum 79A6 for preventing tobacco bacterial wilt and application thereof

Abstract

The application discloses Achromobacter deleyi 79A6 for tobacco bacterial wilt prevention and application thereof, and the preservation number of the Achromobacter deleyi strain 79A6 is CCTCC No: M 20211382. The disease control mechanism of the strain 79A6 is different from that of existing bacterial wilt antagonistic bacteria, and the strain 79A6 can prevent diseases through mechanisms such as ecological niche competition, plant disease resistance induction and rhizosphere microbial community regulation, so that it is proved that the strain 79A6 plays a disease control role mainly by not producing antibiotics, and the strain 79A6 has good safety.

Description

Technical Field

[0001] This application relates to the field of microbial technology, and in particular to a strain of Achromobacter de Lisbeckia orientalis 79A6 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 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.). 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, and a large number of biological control resources have been studied in depth as biological control methods for soil-borne diseases. For example, the reported biological control 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] Most of the biocontrol bacteria mentioned above are screened through indoor plate antagonistic screening and greenhouse biocontrol screening, resulting in the selected strains having antibacterial effects. Long-term use of such biocontrol bacteria leads to increased drug resistance in pathogens, affecting the biocontrol effect.

[0006] Achromobacter species are important agricultural microbial resources. For example, Achromobacter panius strain JD417 (CN114292764A) can effectively reduce total nitrogen, nitrate nitrogen, and sulfonamide compounds in water bodies; Achromobacter JY-2-3R (CN116162564A) can control aconite white mold; and Achromobacter insuavis SL8, Enterobacter cancerogenus SL12, Alcaligenes faecalis SL7, and Serratia... The compound microbial agent of *Bacillus marcescens* SL11 has the ability to effectively remove cadmium and can also prevent and control plant blight and gray mold (CN115418326A); *Achromobacterium ningpoensis* NPDY20Z has the effect of antagonizing plant pathogenic fungi and preventing and controlling fungal diseases (CN115772479A); the microbial agent prepared from *Bacillus subtilis*, *Oligotrophic Monotrophus* and *Achromobacterium ningpoensis* has the ability to prevent and control tobacco red spot disease (CN115868505A) and target spot disease (CN115558621A).

[0007] Currently, there is no application of Achromobacterium bacteria in crop disease control, and there are no reports of Achromobacterium bacteria that can control or inhibit bacterial wilt in crops.

[0008] The information disclosed in the background section is intended only to enhance the understanding of the overall background of the invention 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

[0009] This application addresses the aforementioned technical problems by providing a strain of *Achromobacterium deltae* 79A6 for the control of tobacco bacterial wilt and its application.

[0010] This application provides a strain of Achromobacter deleyi 79A6 for the prevention and control of tobacco bacterial wilt. The preservation number of Achromobacter deleyi strain 79A6 is CCTCC No: M 20211382.

[0011] Preferably, bacterial wilt is tobacco bacterial wilt, caused by infection with Ralstonia nicotianae, Ralstonia solanacearum, Ralstonia pseudosolanacearum, or Ralstonia syzygii.

[0012] Preferably, Achromobacter deleyi strain 79A6 does not control bacterial wilt by antagonizing or inhibiting Ralstonia nicotianae, Ralstonia solanacearum, Ralstonia pseudosolanacearum, or Ralstonia syzygii.

[0013] Preferably, the carbon sources that strain 79A6 can utilize include: aminoacetyl-L-proline, L-alanine, L-aspartic acid, L-glutamic acid, L-pyroglutamic acid, D-gluconic acid, glycosylate, p-hydroxy-phenylacetic acid, methyl pyruvate, L-lactic acid, citric acid, D-malic acid, L-malic acid, bromo-succinic acid, β-hydroxy-D,L-butyric acid, and acetic acid;

[0014] Preferably, strain 79A6 grows slowly under conditions containing D-fructose, D-fructose, L-fructose, D-fructose-6-phosphate, L-histamine, L-serine, D-galacturonic acid, L-galacturonic acid lactone, D-glucuronic acid, glucuronide, α-ketoglutarate, γ-aminobutyric acid, α-hydroxybutyric acid, α-ketobutyric acid or propionic acid;

[0015] Preferably, the carbon sources that strain 79A6 cannot utilize include: dextrin, D-maltose, D-trehalose, D-cellobiose, gentiobiose, sucrose, D-minobiose, stachyose, metriose, raffinose, α-D-lactose, metriose, β-formyl-D-glucosinolate, D-salicylic acid, N-acetyl-D-glucosamine, N-acetyl-β-D-mannosamine, N-acetyl-D-galactosamine, N... - Acetylneuraminic acid, α-D-glucose, D-mannose, D-galactose, 3-formylglucose, L-rhamnose, inosine, D-sorbitol, D-mannitol, D-arabinol, inositol, glycerol, D-glucose-6-phosphate, D-aspartic acid, D-serine, gelatin, L-arginine, pectin, mucoacid / mucilage, quinic acid, D-methyl lactate, Tween 40, acetoacetic acid or formic acid;

[0016] Preferably, strain 79A6 grows better under conditions containing pH 6, 1% NaCl, 4% NaCl, 1% sodium lactate, clostridial acid, acetomycin, rifamycin SV, lincomycin / gentamicin, guanidine hydrochloride, vancomycin, tetrazolium violet, nalidixic acid, tetrazolium blue, aztreonam, sodium butyrate, or sodium tetradecanoate sulfate.

[0017] Preferably, strain 79A6 grows slowly in the presence of potassium tellurite, pH 5, D-serine or lithium chloride, and does not grow in the presence of 8% NaCl, dimethylaminetetracycline or sodium bromate.

[0018] Another aspect of this application provides a biocontrol agent comprising: the strain described above.

[0019] The dosage form of the microbial agent of the present invention is not limited to powder. Those skilled in the art can, based on the teachings and inspiration of the present invention and for actual production needs, combine commonly used technical means in the field of microbial processes (e.g., the Encyclopedia of Pharmaceutical Technology, Pharmaceutical Preparation Technology, etc.), select appropriate excipients to formulate various other dosage forms of Achromobacter deleyi strain 79A6 with accession number CCTCC No: M 20211382 of the present invention, such as tablets, liquids, sprays, granules, etc., that meet the requirements of various production processes.

[0020] Preferably, the biocontrol agent further includes: excipients;

[0021] Preferably, the 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.

[0022] Preferably, the biocontrol agent is in powder form.

[0023] Preferably, the concentration of strain ZTYL3 in the biocontrol agent is greater than or equal to 10. 7 CFU / mL.

[0024] Preferably, the preparation method of the biocontrol agent is as follows: the activated strain 79A6 is inoculated into LB liquid medium and shaken at 225 r / min and 30℃ for 48 h to obtain the agent;

[0025] 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.

[0026] 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.

[0027] 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.

[0028] Preferably, the biocontrol agent is diluted in the form of a fermentation diluent or a bacterial powder suspension.

[0029] This biocontrol agent is applicable to diseased plants that are tobacco plants infected with Ralstonia nicotianae, Ralstonia solanacearum, Ralstonia pseudosolanacearum, or Ralstonia syzygii, which cause bacterial wilt.

[0030] Another aspect of this application provides a fermentation method for strain 79A6 as described above, comprising the following steps: inoculating the activated strain into a fermentation medium and carrying out fermentation culture.

[0031] Preferably, the fermentation medium is LB liquid medium.

[0032] Preferably, the fermentation culture conditions are shaking culture at 225 r / min and 30℃ for 48 h.

[0033] In this article, the biocontrol bacteria, Achromobacter deleyi 79A6, 79A6, strain 79A6, and Achromobacter deleyi mentioned in the invention content and specific embodiments all refer to: Achromobacter deleyi strain 79A6 with accession number CCTCC No: M 20211382 of this invention.

[0034] The beneficial effects that this application can produce include:

[0035] 1) The *Achromobacter deleyi* 79A6 strain and its application provided in this application for the control of tobacco bacterial wilt, when screening *Achromobacter deleyi* 79A6, the efficacy evaluation did not use antagonistic effect as the evaluation index, but rather disease control ability. Plate antagonistic ability testing revealed that strain 79A6 had no inhibitory effect on *R. rockstone*, indicating that the disease control effect of strain 79A6 is not mainly through antibiotic production. The disease control mechanism of strain 79A6 is different from existing bacterial wilt antagonists, possibly through niche competition, inducing plant resistance, and regulating the rhizosphere microbial community. Therefore, when applying strain 79A6 in the field to control tobacco bacterial wilt, it will not induce *R. rockstone* tolerance to strain 79A6 or its metabolites, and the application of strain 79A6 has good safety.

[0036] 2) The Achromobacter deleyi 79A6 and its application provided in this application for the control of bacterial wilt in tobacco. The biocontrol bacteria for bacterial wilt in tobacco are mainly Bacillus, Pseudomonas, and Streptomyces. There are no reports on the use of Achromobacter spp. in the biocontrol of bacterial wilt in crops. The Achromobacter deleyi 79A6 involved in this invention provides a new biocontrol resource for the control of bacterial wilt in tobacco, enriching the biocontrol resources. Moreover, the bacterial agent 79A6 has a good control effect on bacterial wilt caused by Rawstone's bacteria from different geographical sources.

[0037] The preservation information for Achromobacter deleyi 79A6 is as follows: Accession number: CCTCCNO:M 20211382; Classification name: Achromobacter deleyi 79A6; Preservation date: November 8, 2021; Preservation institution: China Center for Type Culture Collection; Preservation address: Wuhan University, Wuhan, China. Attached Figure Description

[0038] Figure 1 This is a colony morphology image of Achromobacterium 79A6 cultured for 48 hours in Example 1 of this application, where a) is a photograph of a culture dish; b) is an optical micrograph of strain 79A6 cells;

[0039] Figure 2 This is a phylogenetic tree diagram of Achromobacterium 79A6 constructed based on the whole genome in Example 2 of this application.

[0040] Figure 3 This demonstrates the effect of powder 79A6 in Example 3 of this application on the prevention and control of tobacco bacterial wilt. Detailed Implementation

[0041] The present invention will now be described in further detail with reference to the accompanying drawings and embodiments, but this does not limit the present invention in any way. Any modifications or improvements made based on the teachings of the present invention shall fall within the protection scope of the present invention.

[0042] Example

[0043] 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.

[0044] Sources of biomaterials

[0045] I. The tobacco material used in the following examples 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.

[0046] II. The Rollstonella strain used in the following examples:

[0047] 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.

[0048] 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".

[0049] Ralstonia solanacearum CQPS-1 is the strain described in the article "Liu Y, Tang Y, Qin X, et al. Genome Sequencing of Ralstonia solanacearum CQPS-1, a Phylotype I Strain Collected from a Highland Area with Continuous Cropping of Tobacco. Front. Microbiology, 2017, 8:974."

[0050] Ralstonia nicotianae BSRS-1 and QBRS-1 are 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 the invention.

[0051] III. Culture media used in the following examples

[0052] LB liquid medium contains 1% tryptone, 0.5% yeast extract, 1% sodium chloride and 0.5% sucrose;

[0053] CG medium contains 0.1% acid-hydrolyzed casein, 0.25% glucose, and 2% peptone;

[0054] CGA contains 0.1% acid-hydrolyzed casein, 0.25% glucose, 2% peptone, and 1.5% agar;

[0055] The oligotrophic medium CN contains 0.1% casein amino acids, 0.1% nutrient broth, and 1.5% agar;

[0056] TZC medium contains 1% peptone, 25% glucose, 0.1% casein hydrolysate, 1.5% agar, and 0.005% triphenyltetrazolium chloride (TTC).

[0057] IV. Floating Seedling Raising

[0058] 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.

[0059] V. Pathogen Culture

[0060] 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.

[0061] 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), and incubate at 28℃ with constant temperature shaking at 225 r / min for 24 h.

[0062] 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.

[0063] Example 1: Collection, isolation and culture of strain 79A6

[0064] After removing impurities and large lumps, the soil samples (collected in May 2020, collected by Lu Canhua, collected from tobacco soil in Hongta District, Yuxi City, Yunnan Province) were placed in a 120mm diameter glass petri dish with a soil layer thickness of about 1.5cm. The soil was moistened with distilled water using a titration bottle.

[0065] 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;

[0066] Place the microbial trapping device on the moist soil in (1), 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 distilled water using a titration bottle;

[0067] 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.

[0068] 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 ;

[0069] Take 10 -4 10 -5The 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 clean bench and incubated at 30°C for 7 days.

[0070] Through the above experiments, pure bacteria 79A6 were obtained by streaking the culture from CN petri dishes onto CN medium again. A single colony of 79A6 was then inoculated into a test tube containing 2.5 mL of LB liquid medium and incubated at 28℃ with constant shaking at 225 rpm for 48 h. The colony morphology is as follows. Figure 1 As shown.

[0071] Preliminary bacterial identification: Routine bacterial identification was performed with reference to the literature "Handbook of Systematic Identification of Common Bacteria".

[0072] (Dong Xiuzhu et al., eds. Science Press, 2001).

[0073] Culture characteristics and morphological features of the strain:

[0074] Strain 79A6 can grow in LB, NB, and PDB media. In LB liquid medium, strain 79A6 can grow at temperatures ranging from 20 to 35°C, with 25°C being the optimal temperature. The bacterial cells are rod-shaped and Gram-negative.

[0075] Example 2: Identification of strain 79A6

[0076] 1. The compound metabolic characteristics and preliminary identification of strain 79A6 were analyzed using Biolog GEN III plates.

[0077] Gen III identification results showed that the growth of strain 79A6 under various carbon sources and growth inhibition conditions was as follows. The available carbon sources included: aminoacetyl-L-proline, L-alanine, L-aspartic acid, L-glutamic acid, L-pyroglutamic acid, D-gluconic acid, glycolic acid, p-hydroxy-phenylacetic acid, methyl pyruvate, L-lactic acid, citric acid, D-malic acid, L-malic acid, bromo-succinic acid, β-hydroxy-D,L-butyric acid, and acetic acid.

[0078] It grows slowly in the presence of D-fructose, D-fructose, L-fructose, D-fructose-6-phosphate, L-histamine, L-serine, D-galacturonic acid, L-galacturonic acid lactone, D-glucuronic acid, glucuronide, α-ketoglutarate, γ-aminobutyric acid, α-hydroxybutyric acid, α-ketobutyric acid or propionic acid.

[0079] The following carbon sources cannot be utilized: dextrin, D-maltose, D-trehalose, D-cellobiose, gentiobiose, sucrose, D-minobiose, stachyose, metriose, raffinose, α-D-lactose, metriose, β-formyl-D-glucosinolate, D-salicylic acid, N-acetyl-D-glucosamine, N-acetyl-β-D-mannosamine, N-acetyl-D-galactosamine, N-acetylneuraminic acid, α-D-glucose, D-mannose, D-galactose, 3-formylglucose, L-rhamnose, inosine, D-sorbitol, D-mannitol, D-arabinol, inositol, glycerol, D-glucose-6-phosphate, D-aspartic acid, D-serine, gelatin, L-arginine, pectin, mucoacid / mucolytic acid, quinic acid, D-methyl lactate, Tween 40, acetoacetic acid or formic acid;

[0080] It grows better in the presence of pH 6, 1% NaCl, 4% NaCl, 1% sodium lactate, clostridial acid, acetomycin, rifamycin SV, lincomycin / gentamicin, guanidine hydrochloride, vancomycin, tetrazolium violet, nalidixic acid, tetrazolium blue, aztreonam, sodium butyrate or sodium tetradecanoate.

[0081] It grows slowly in the presence of potassium tellurite, pH 5, D-serine, or lithium chloride, and does not grow in the presence of 8% NaCl, dimethylaminetetracycline, or sodium bromate.

[0082] Based on the growth of the strain under the aforementioned carbon source, nitrogen source, and stress conditions, and comparing it with the GEN_III_v2.8.0.I5G database, strain 79A6 was found to be most similar to *Achromobacter insolitus*, with a similarity of 0.772. Other species with high similarity included *Achromobacter ruhlandii* / *denitrificans* (0.110), *Bordetella hinzii* (0.068), and *Bordetella petrii* (0.049). These results indicate that strain 79A6 belongs to the genus *Achromobacter*.

[0083] 2. Molecular identification of the strain:

[0084] Molecular identification methods are as follows: A bacterial genomic DNA extraction kit was used (see 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, transformed into *E. coli* competent cells DH5α by heat shock, and colonies were picked for colony PCR identification 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 79A6, preliminarily confirming the primary taxonomic position of the strain.

[0085] 16S rDNA sequence analysis showed that strain 79A6 is related to Achromobacter deleyi subsp. ATCC 7966. T , Achromobacter kerstersii LMG 3441 T , Achromobacter spanius LMG5911 T The highest similarity was found in both, at 99.93%; and it was also similar to Achromobacter piechaudii NBRC 102461. T , Achromobacter insuavis LMG 26845 T The similarity was second only to that of other strains, both at 99.72%. Strain 79A6 also showed high 16S rDNA sequence similarity with other strains of the genus *Achromobacter*, all exceeding 98.70%. These results indicate that strain 79A6 belongs to the genus *Achromobacter*, and its species classification cannot be determined solely by its 16S rDNA sequence.

[0086] After genome draft sequencing, analysis using the Type (Strain) Genome Server (https: / / tygs.dsmz.de / ) database showed that strain 79A6 is related to Achromobacter deleyi LMG 3458. T The highest similarity was observed for dDDH0, dDDH4, and dDDH6, with values ​​of 78.1%, 54.1%, and 75.4%, respectively. The molecular hybridization values ​​of genomic DNA of strain 79A6 with other achromobacteria were less than 60% (Table 1).

[0087] Phylogenetic trees were constructed based on the genomes of strain 79A6 and its closely related species. The results showed that strain 79A6 is related to the model bacterium LMG 3458 of *Achromobacter deleyi*. TThey are closely related, but the two strains form separate clades. Figure 2 The above results indicate that strain 79A6 is an achromobacterium, most closely related to *Achromobacter deleyi*. Because the dDDH threshold for bacterial species identification is 70%, dDDH4 is used as the primary reference when the genome sequence is a draft or when there are differences in three dDDHs.

[0088] In this invention, 79A6 and Achromobacter deleyi LMG 3458 T With a prevalence of only 54.1%, significantly different from *Achromobacter de Lisbeckia*, strain 79A6 may be a new species within the *Achromobacter* genus. Because strain 79A6 is most closely related to *Achromobacter de Lisbeckia*, it is tentatively named *Achromobacter de Lisbeckia*.

[0089] Table 1. Comparative analysis of genomic similarity between 79A6 and Achromobacterium spp. (unit: %)

[0090]

[0091]

[0092] 3. Preservation of Achromobacter deleyi 79A6

[0093] Based on the above identification results, strain 79A6 was confirmed to be a strain of *Achromobacter deleyi*, named 79A6, and deposited for preservation. Its preservation information is as follows: Accession number: CCTCC NO: M20211382; Classification name: *Achromobacter deleyi* 79A6; Preservation date: November 23, 2021; Preservation institution: China Center for Type Culture Collection; Preservation address: Wuhan University, Wuhan, China.

[0094] Example 3: Evaluation of the efficacy of biocontrol bacterium 79A6 in controlling tobacco bacterial wilt:

[0095] 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.

[0096] I. The specific procedures for evaluating the effectiveness of prevention are as follows:

[0097] Step S1, initial screening for biological testing

[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 pretreatment: 1 mL of the tested biocontrol bacteria was inoculated into each tobacco plant in the experimental group, and the tobacco seedlings inoculated 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, 1 mL of Rawlstonella 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, rescreening of biological samples

[0104] Except for the following test operations, the remaining steps of step S2 bioassay screening are the same as those of step S1 bioassay screening.

[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 screening 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. The results are shown in Table 2.

[0108] Table 2. Effects of strain 79A6 in the growth chamber on the control of tobacco bacterial wilt.

[0109]

[0110] As shown in Table 2, in the initial screening, one of the two tobacco plants treated with strain 79A6 wilted while the other remained healthy, hence the value was assigned 1.5. In the greenhouse rescreening, 8 and 5 tobacco plants were healthy at 10 and 20 dpi, respectively, while the control group had 6 and 2 healthy plants, respectively. These results indicate that 79A6 has a certain control effect and can be used to evaluate its efficacy in greenhouse potted plants.

[0111] II. Application of strain 79A6 in the control of tobacco bacterial wilt

[0112] 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.

[0113] Culture of the test strain: The biocontrol bacteria 79A6 obtained by indoor screening was cultured in LB liquid medium and shaken at 225 r / min and 30℃ for 48 h to obtain the bacterial agent 79A6;

[0114] The experiment selected Rawlstonella QBRS-1 from Qiubei, Wenshan, which was preserved in the laboratory. The pathogen Rawlstonella was cultured in CG liquid medium and shaken at 225 r / min and 30℃ for 24 h.

[0115] 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.

[0116] Inoculation: After transplanting, dilute 250 mL of biocontrol bacteria fermentation solution 25 times, and apply 200 mL of the diluted solution to the roots of each tobacco plant (the concentration of 79A6 bacteria in the diluted solution is 10). 7 ~10 8 CFU / mL), the control group was treated with an equal volume of LB dilution; the next day, the *Rolstonia* cultured on CG medium for 24 hours was diluted 100-fold, and each tobacco plant was inoculated with 100 mL of the *Rolstonia* dilution (inoculation concentration approximately 10). 7 (CFU / mL)

[0117] Survey and statistics: Disease index was assessed every 7 days after vaccination, for a total of 5 surveys. The incidence, disease index, and control efficacy of tobacco bacterial wilt were calculated using the following formula:

[0118] 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;

[0119] Control effect = (Control disease index - Treatment disease index) / Control disease index × 100%. The disease index was determined according to the People's Republic of China Tobacco Industry Standard for Classification and Investigation of Tobacco Diseases (GB / T 23222-2008).

[0120] The disease is classified as follows: Level 0 is when the whole plant is disease-free; Level 1 is when less than half of the leaves on the diseased side wither; Level 3 is when one-half to two-thirds of the leaves on the diseased side wither; Level 5 is when more than two-thirds of the leaves on the diseased side wither; Level 7 is when all the leaves on the diseased plant wither; and Level 9 is when the diseased plant is basically dead.

[0121] The results are shown in Table 3:

[0122] Table 3. Control efficacy of inoculant 79A6 against different types of Rawlstonella.

[0123]

[0124] The above experimental results show that the disease index of tobacco plants treated with inoculant 79A6 was lower than that of the water control group during the observation period. When using unsterilized soil as the medium in a greenhouse, the relative control efficacy at 35 dpi was 51.79% (see Table 3 for results). Figure 3 The above results indicate that the fungal agent 79A6 has a good control effect on bacterial wilt caused by Rawstone's bacterium.

[0125] III. The antagonistic effect of strain 79A6 against Rawlstonella

[0126] The inhibition zone of the bacterial strain was measured using the plate confrontation culture method.

[0127] The operating steps are as follows: Roldstone bacteria QBRS-1 and RS, cultured on CG medium for 24 hours respectively... T LLRS-1, CQPS-1 and BSRS-1 were serially diluted to 10 -4 Take 100 μL of the dilution solution and place it on the surface of the CGA medium. Spread it evenly with the spreader and place it in a cleanroom to dry.

[0128] Single colonies of strain 79A6 were inoculated onto the surface of a culture medium containing Rawlstonella. After culturing for 2–3 days, the antibacterial activity of each strain was observed. Strains with antibacterial effects were cultured using the four-point method, and the inhibition zone and inhibition band of the strains were measured (see Table 4 for the results).

[0129] Table 4. Antagonistic ability of biocontrol bacterium 79A6 against different Rawstone bacteria

[0130]

[0131]

[0132] The experimental results showed that strain 79A6 was effective against Rawlstonella RS. T None of the four inhibitors, LLRS-1, CQPS-1, BSRS-1, and QBRS-1, showed antagonistic effects. These results indicate that biocontrol bacterium 79A6 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.

[0133] Example 4: Biocontrol agent containing strain ZTYL3

[0134] The fermentation broth of strain ZTYL3 was centrifuged (79A6 fermentation broth was obtained by inoculating activated biocontrol bacteria 79A6 into LB liquid medium and shaking at 225 r / min and 30℃ for 48 h). The bacterial cells were collected and mixed with diatomaceous earth at a mass ratio of 1:1. Sucrose at a mass ratio of 5‰ of the total mass of the mixture was added. The mixture was then dried in a cool, dry and ventilated place until the moisture content of the material reached 10±2%. The material was then pulverized to obtain the powder of the biocontrol agent.

[0135] Although the present invention 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 the present invention should be included within the protection scope of the present invention.

Claims

1. A type of Achromobacterium delieri used for the control of tobacco bacterial wilt ( Achromobacter deleyi 79A6, characterized in that, Achromobacter delyeli ( Achromobacter deleyi The accession number of 79A6 is CCTCC No: M20211382.

2. A biocontrol agent, characterized in that, include: The Achromobacterium delleoides 79A6 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 79A6 in the biocontrol agent is greater than or equal to 10. 7 CFU / mL; Preparation method of biocontrol agent: The activated strain 79A6 was inoculated into LB liquid medium and shaken at 225 r / min and 30℃ for 48 h to obtain the agent.

5. The biocontrol agent according to claim 2, characterized in that, Preparation method of biocontrol agent: Centrifuge the fermentation broth of strain 79A6, 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 operation is to air dry in a cool, dry and ventilated place, and after drying, the moisture content of the material reaches 10±2%, and then pulverize.

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. A fermentation method for *Achromobacterium deltae* 79A6 as described in claim 1, characterized in that, Includes the following steps: The activated bacterial strain was inoculated into the fermentation medium and fermented.

8. The fermentation method according to claim 7, characterized in that, The fermentation medium was LB liquid medium.

9. The fermentation method according to claim 8, characterized in that, The fermentation conditions were 225 r / min and 30℃ for 48 h of shaking culture.

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