Indicator stenotrophomonas 114E6 and application thereof

Abstract

The invention belongs to the technical field of microorganisms, and particularly relates to an indicator stenotrophomonas 114E6 and application thereof. The invention provides an indicator stenotrophomonas indicatrix, and the preservation number of the indicator stenotrophomonas indicatrix is CCTCC (China Center For Type Culture Collection) NO: M 20211383. The invention also provides an application of the indicator Stenotrophomonas indicatrix in the prevention and treatment of tobacco bacterial wilt and the promotion of tobacco growth. The invention further provides the application of the indicator Stenotrophomonas indicatrix in the prevention and treatment of tobacco bacterial wilt and the promotion of tobacco growth. Tests prove that when the indicator stenotrophomonas is independently applied or compounded with other strains, the disease index of tobacco plants suffering from bacterial wilt can be effectively maintained at a low level.

Description

Technical Field

[0001] The invention belongs to the technical field of microorganisms, and in particular relates to an indicator strain of oligotrophic monocytogenes 114E6 and an application thereof. Background Art

[0002] Bacterial wilt is caused by infection with Ralstonia spp. and is an important soil-borne disease of crops. Ralstonia has a wide host range and can infect more than 400 crops. It is more serious in bananas, tomatoes, potatoes, and tobacco. In my country, tobacco bacterial wilt is mainly caused by infection with Ralstonia nicotianae, which is common in the Yangtze River Basin and tobacco-growing areas to its south (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.). Lu Canhua, Li Junying, Mi Mengge et al. published an article titled "Genomic Analysis of Ralstonia syzygii LLRS-1" in 2021 ([C] / / Chinese Society of Plant Pathology. Plant Pathology Technological Innovation and Green Control-Proceedings of the 2021 Academic Annual Meeting of the Chinese Society of Plant Pathology. China Agricultural Science and Technology Press, 2021:1.DOI:10.26914 / c.cnkihy.2021.063949.) It has been found that the disease has occurred in 43 districts (counties) in 12 prefectures and cities in Yunnan Province, including Wenshan, Baoshan, Lincang, Honghe, Kunming, Yuxi, Qujing, Zhaotong, Dali, Lijiang, Chuxiong and Dehong. Among them, Wenshan, Lincang, Honghe and Pu'er tobacco areas have more serious diseases. Due to the lack of disease-resistant resources and the lack of specific chemical control drugs, the disease has always been an important factor restricting the improvement of tobacco production and quality.

[0003] Biological control has attracted wide attention due to its environmental friendliness. my country has developed and registered 11 biocontrol bacteria for tobacco bacterial wilt, mainly Pseudomonas fluorescens, Bacillus amyloliquefaciens, Paenibacillus polymyxa, Bacillus subtilis and other fungicides, most of which have the ability to antagonize Ralstonia.

[0004] Stenotrophomonas, as an important biocontrol resource, has been applied in soil improvement, organic fertilizer fermentation, and disease control. For example, Stenotrophomonas pavanii LC00168 controls Bursaphelenchus xylophilus (CN112877240A), Stenotrophomonas rhizophila Sneb 1777 induces peanut resistance to Meloidogyne incognita (CN106148221A), Stenotrophomonas rhizophila S11 controls rice blast (CN110643551A), Stenotrophomonas maltophilia controls cucumber green mottle mosaic virus disease (CN105219673A), Stenotrophomonas maltophilia ZL-2 controls wheat rust (CN112708584A), Stenotrophomonas maltophilia TDJN1 controls cucumber downy mildew (CN105002111A), and Stenotrophomonas acidaminiphila strain BJ1 controls apple tree canker (CN102851225A).There are also reports on the combination of Stenotrophomonas and bacteria of other genera to form a compound microbial inoculum. For example, Acinetobacter sp. NXH1, Klebsiella sp. NXH2, Stenotrophomonas sp. NXH3, and Pseudomonas sp. XKS are combined to form an ecological improvement substrate (CN111011159A). Stenotrophomonas maltophilia 37-1 is used to prepare microbial enzymolysis products of rapeseed meal for preventing and controlling tomato root-knot nematode disease (CN102154159A), pepper blight (CN101948780A), and melon fusarium wilt (CN101691549A). A microbial inoculum composed of Achromobacter xylosoxidans, Enterobacter sp., Ochrobactrum sp., Stenotrophomonas sp., and Bacillus subtilis is used to prevent and control tobacco brown spot disease (CN110402964A). A synthetic bacterial community composed of Stenotrophomonas rhizophila, Stenotrophomonas maltophilia, Variovorax boronicumulans, Pseudomonas putida, Chitinophaga niastensis, and Pseudomonas fluorescens has the functions of disease prevention, growth promotion, and quality improvement (CN115161406A).

[0005] There is no relevant report on Stenotrophomonas indicatrix that can both prevent and control bacterial wilt of crops and promote plant growth. Summary of the Invention

[0006] The object of the present invention is to provide a Stenotrophomonas indicatrix that can prevent and control tobacco bacterial wilt and promote the growth of tobacco, and its application.

[0007] The present invention provides a strain of Stenotrophomonas indicatrix named 114E6, and its deposit number is CCTCC NO: M 20211383.

[0008] The present invention also provides a bacterial agent, which comprises the above-mentioned Stenotrophomonas indicatrix.

[0009] The bacterial agent may be a compound bacterial agent, and may also comprise one or more strains selected from Ralstonia mannitolilytica strain 56D2, Enterobacter mori strain 65D6, Agrobacterium deltaense strain 18A6, and Stenotrophomonas indicatrix strain 107E3;

[0010] Among them, the preservation number of strain 56D2 is CCTCC NO: M 2019921; the preservation number of strain 65D6 is GDMCC NO: 64022; the preservation number of strain 18A6 is CCTCC NO: M 2020041; the preservation number of strain 107E3 is CCTCC NO: M 2019923.

[0011] The contents of the various strains in the compound bacterial agent may be the same.

[0012] The bacterial agent may be a liquid agent or a powder agent.

[0013] The application of the above-mentioned Stenotrophomonas indicatrix in the preparation of a product for preventing and treating tobacco bacterial wilt and / or promoting the growth of tobacco also belongs to the protection scope of the present invention.

[0014] The application of the above-mentioned Stenotrophomonas indicatrix or the above-mentioned bacterial agent in preventing and treating tobacco bacterial wilt and / or promoting the growth of tobacco also belongs to the protection scope of the present invention.

[0015] In the above application, the fermentation broth of the above-mentioned Stenotrophomonas indicatrix or the suspension prepared from the bacterial agent can be used for root irrigation, root dipping, mixing with substrate or organic fertilizer of tobacco seedlings.

[0016] In some embodiments, the bacterial concentration of the fermentation broth of the above-mentioned Stenotrophomonas indicatrix or the suspension prepared from the bacterial agent is greater than or equal to 10 7 CFU / mL.

[0017] In the above application, the tobacco bacterial wilt can be caused by Ralstonia nicotianae, Ralstonia syzygii, Ralstonia solanacearum or Ralstonia pseudosolanacearum.

[0018] The beneficial effects of the present invention are as follows:

[0019] (1) Different disease prevention mechanisms

[0020] For the screening of conventional biocontrol bacteria against bacterial wilt, primary screening is carried out by indoor plate antagonism and secondary screening is carried out by greenhouse bioassay. However, for the evaluation of the control effect of the Stenotrophomonas indicatrix 114E6 in the present invention, the control ability is directly used as the evaluation index instead of the antagonistic effect against pathogenic bacteria. Through the determination of the plate antagonistic ability, it is found that strain 114E6 has no antibacterial ability against Ralstonia, indicating that the disease control mechanism of strain 114E6 is different from that of traditional antagonistic bacteria. Research shows that strain 114E6 has a good control effect on tobacco bacterial wilt (Experimental Example 5), and the control effect is better when compounded with other biocontrol bacteria (Experimental Examples 6 and 7), even higher than that of the chemical agent chlorine urea·copper sulfate. It is very likely to prevent diseases through mechanisms such as the coordinated action between biocontrol bacteria or the regulation of rhizosphere microbial communities. In addition, strain 114E6 also has the effect of promoting the growth of tobacco seedlings (Experimental Example 9, Figure 4 ).

[0021] (2) No drug resistance

[0022] Strain 114E6 has no inhibitory ability on the growth of Ralstonia, indicating that strain 114E6 does not mainly exert its disease control effect by producing antibiotics. Therefore, when strain 114E6 is applied in the field to control tobacco bacterial wilt, the tolerance of Ralstonia to strain 114E6 or the metabolites of the strain will not be generated, and it has good safety.

[0023] (3) Enriched biocontrol resources

[0024] The biocontrol bacteria against tobacco bacterial wilt are mainly Bacillus, Pseudomonas and Streptomyces. The Stenotrophomonas indicatrix 114E6 of the present invention provides a new biocontrol resource for the control of tobacco bacterial wilt.

[0025] The patent preservation information of the Stenotrophomonas indicatrix 114E6 of the present invention is as follows:

[0026] Deposit number: CCTCC NO: M 20211383;

[0027] Taxonomic name: Stenotrophomonas indicatrix 114E6;

[0028] Deposit date: November 8, 2021;

[0029] Depositary institution: China Center for Type Culture Collection;

[0030] Deposit address: Wuhan University, Wuhan, China. Description of the drawings

[0031] Figure 1 This is the colony morphology diagram of Stenotrophomonas indicatrix 114E6 cultured for 48 h in Experimental Example 1 of the present invention.

[0032] Figure 2 This is the phylogenetic tree diagram of Stenotrophomonas indicatrix 114E6 constructed based on the whole genome in Experimental Example 3 of the present invention.

[0033] Figure 3 This is the comparison diagram of the colony morphology of the confrontation culture between Stenotrophomonas indicatrix 114E6 and Ralstonia nicotianae QBRS-1 in Experimental Example 8 of the present invention. The antagonistic bacterium is Bacillus amyloliquefaciens strain WY11.

[0034] Figure 4 This is the effect of Stenotrophomonas indicatrix 114E6 on the germination, fresh weight and root length of tobacco seeds in Experimental Example 9 of the present invention. Detailed implementation manners

[0035] The technical solutions of the present invention will be further described in detail below in combination with specific embodiments and drawings, but the present invention is not limited to the following technical solutions.

[0036] Tobacco material

[0037] The tobacco materials used in Experimental Example 2, Experimental Example 4, Experimental Example 5, Experimental Example 6 and Experimental Example 7 are the well-known and commonly used tobacco variety 'Honghuadajinyuan', which is stored in the applicant's laboratory and can also be obtained commercially. The tobacco material used in Experimental Example 9 is the well-known and commonly used tobacco variety 'Yunyan 87', which is stored in the applicant's laboratory and can also be obtained commercially.

[0038] Pathogen

[0039] The Ralstonia nicotianae RS used in Experimental Example 2 and 8 TRS described in the article "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." T This bacterium 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. This bacterium was deposited in the Guangdong Provincial Culture Collection Center of Microorganisms on July 14, 2022, with the deposit number GDMCC NO: 1.3533.

[0040] Ralstonia nicotianae QBRS-1 used in Experimental Examples 4, 5, 6, 7 and 8 is a strain preserved in the applicant's laboratory.

[0041] Ralstonia syzygii LLRS-1 used in Experimental Example 8 is the LLRS-1 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".

[0042] Ralstonia nicotianae BSRS-1 used in Experimental Example 8 is a strain preserved in the applicant's laboratory.

[0043] Regarding the above biological materials, the applicant undertakes to freely distribute them to the public within 20 years from the filing date for verifying the effects of the present invention.

[0044] Antagonistic bacterium

[0045] The Bacillus amyloliquefaciens WY11 used in Experimental Example 8 is described in the Chinese patent with the patent number ZL201210435610.5 and the title "A Bacillus and Its Bacterial Agent, Preparation Method and Application", and is preserved in the General Microbiological Center of the China Committee for Culture Collection of Microorganisms (CGMCC). Preservation date: October 11, 2012, Preservation number: CGMCC NO: 6663.

[0046] Culture medium

[0047] LB liquid medium contains 1% tryptone, 0.5% yeast extract, 1% sodium chloride and 0.5% sucrose;

[0048] CG medium contains 0.1% acid-hydrolyzed casein, 0.5% glucose and 2% peptone;

[0049] CGA contains 0.1% acid-hydrolyzed casein, 0.5% glucose, 2% peptone and 1.5% agar;

[0050] Oligotrophic medium CN contains 0.1% casein amino acids, 0.1% nutrient broth and 1.5% agar;

[0051] TZC medium contains 1% peptone, 1% glucose, 0.1% casein hydrolysate, 1.5% agar and 0.005% triphenyltetrazolium chloride (TTC).

[0052] Floating seedling raising

[0053] Using the susceptible tobacco variety 'Honghuadajinyuan' as the bioassay object, floating seedlings are cultivated until the 4-5 leaf stage.

[0054] Pathogen culture

[0055] The Ralstonia is taken out from the -80°C ultra-low temperature refrigerator, inoculated on the surface of the TZC medium, and placed in an incubator at 28°C for 36-48 h to activate the strain;

[0056] Pick the typical colonies with a relatively wide white edge, strong fluidity and a pink or light red dilute liquid in the middle, and inoculate them into a triangular flask containing 100 mL of CG liquid medium (1% peptone, 1% glucose, 0.1% casein hydrolysate), and place it in a constant temperature shaker at 28°C and 225 r / min for 24 h;

[0057] Take 100 μL of the bacterial liquid and dilute it to 10 -7 Take 100 μL of 10 -5, 10 -6 , 10 -7 Dilute the liquid and spread it on a TZC plate. Observe the colony morphology and count the number of colonies after 48 h, and calculate the amount of bacteria contained in the cultured bacterial solution.

[0058] Example Group 1: Strain 114E6 of the present invention

[0059] This group of examples provides a strain of Stenotrophomonas indicatrix named 114E6, and its preservation number is CCTCC NO: M 20211383.

[0060] Any act of using, selling, offering for sale, producing, preparing, culturing, propagating, or fermenting the Stenotrophomonas indicatrix strain 114E6 with the preservation number CCTCC NO: M20211383 falls within the protection scope of the present invention.

[0061] Any product containing the Stenotrophomonas indicatrix with the preservation number CCTCC NO: M 20211383 falls within the protection scope of the present invention.

[0062] 114E6, Stenotrophomonas indicatrix 114E6, biocontrol bacterium 114E6, strain 114E6, and 114E6 strain described herein all refer to the Stenotrophomonas indicatrix with the preservation number CCTCC NO: M 20211383.

[0063] Example Group 2: The bacterial agent of the present invention

[0064] This group of examples provides a bacterial agent, which contains the Stenotrophomonas indicatrix strain 114E6 with the preservation number CCTCC NO: M 20211383.

[0065] In some embodiments, the microbial agent is a compound microbial agent, further comprising one or more strains selected from Ralstonia mannitolilytica strain 56D2, Enterobacter mori strain 65D6, Agrobacterium deltaense strain 18A6, and Stenotrophomonas indicatrix strain 107E3; wherein, the preservation number of strain 56D2 is CCTCC NO: M2019921; the preservation number of strain 65D6 is GDMCC NO: 64022; the preservation number of strain 18A6 is CCTCC NO: M2020041; the preservation number of strain 107E3 is CCTCC NO: M 2019923.

[0066] In some embodiments, the contents of the strains in the compound microbial agent are the same.

[0067] In some embodiments, the dosage form of the microbial agent is a liquid agent or a powder agent.

[0068] The dosage form of the microbial agent of the present invention is not limited to a liquid agent or a powder agent. Those skilled in the art, according to the teachings and inspirations of the present invention and for actual production needs, combine common technical means in the field of microbial technology, select appropriate excipients for formulation, and prepare various other dosage form products that meet the requirements of process production from Stenotrophomonas indicatrix strain 114E6 with the preservation number of CCTCC NO: M 20211383, such as tablets, sprays, granules, etc.

[0069] Group 3 embodiments: Application of strain 114E6 of the present invention

[0070] This group of embodiments provides the application of Stenotrophomonas indicatrix strain 114E6 with the preservation number of CCTCC NO: M 20211383 in the preparation of products for preventing and treating tobacco bacterial wilt and / or promoting the growth of tobacco.

[0071] In some embodiments, the tobacco bacterial wilt is caused by Ralstonia nicotianae, Ralstonia syzygii, Ralstonia solanacearum, or Ralstonia pseudosolanacearum.

[0072] Group 4 of Examples: Application of Strain 114E6 of the Present Invention

[0073] This group of examples provides the application of Stenotrophomonas indicatrix strain 114E6 with the preservation number of CCTCC NO: M 20211383 in controlling tobacco bacterial wilt and / or promoting tobacco growth.

[0074] In some examples, the tobacco seedlings are irrigated with the root, dipped in the root, mixed with the matrix or organic fertilizer with the fermentation broth of the Stenotrophomonas indicatrix or the suspension prepared from the bacterial agent of the present invention.

[0075] In some examples, the bacterial concentration of the fermentation broth of the Stenotrophomonas indicatrix or the suspension prepared from the bacterial agent of the present invention is greater than or equal to 10 7 CFU / mL.

[0076] In some examples, the tobacco bacterial wilt is caused by Ralstonia nicotianae, Ralstonia syzygii, Ralstonia solanacearum or Ralstonia pseudosolanacearum.

[0077] Experimental Example 1 Obtaining of Strains

[0078] I. Trapping, Isolation and Cultivation

[0079] After removing impurities and large lumps from the soil sample, it is loaded into a glass petri dish with a diameter of 120 mm, and the soil layer thickness is about 1.5 cm. The soil is wetted with distilled water using a burette;

[0080] Prepare a microbial trapping device: First, apply glue to the edge of a microporous membrane with a diameter of 50 mm and a pore size of 0.45 μm, and place a stainless steel flat washer on the microporous membrane; then add 3 mL of solid medium (1.2% gellan gum and 1.0% vitamin) into the inner cavity of the washer; then apply glue to the upper surface of the metal washer and cover it with another microporous membrane with a pore size of 0.45 μm;

[0081] Place the microbial trapping device on the wetted soil above, gently press the device to ensure full contact between the microporous membrane and the soil, and then completely cover the device with the remaining soil, and wet the soil again with distilled water through a burette;

[0082] Cover the petri dish, seal the culture device with sealing film, and place it in an incubator at 30°C for 7 days. Observe the soil humidity during this period. If the soil humidity is low, supplement it with sterile water.

[0083] Take out the culture device that has been trapped from the incubator, crush the solid medium, add 3 mL of sterile water, let it stand for 10 minutes, and then perform gradient dilution.

[0084] Take 10 -4 、10 -5 Spread the bacterial liquid on the oligotrophic medium CN (containing 0.1% casein amino acid, 0.1% nutrient broth, 1.5% agar). Spread the bacterial liquid of each concentration gradient on 5 petri dishes, dry it in a laminar flow hood, and place it in an incubator at 30°C for 7 days.

[0085] II. Experimental Results

[0086] Through the above experiment, pick the colonies from the CN medium and streak them again on a new CN medium to obtain the pure bacterium 114E6; pick a single colony of 114E6 and inoculate it into a test tube containing 2.5 mL of LB liquid medium (1% tryptone, 0.5% yeast extract, 1% sodium chloride, and 0.5% sucrose), and place it in a constant temperature shaker at 28°C and 225 r / min for 48 h. Its colony morphology is as Figure 1 shown.

[0087] Experimental Example 2 Evaluation of the Control Effect of Strain 114E6 against Tobacco Bacterial Wilt

[0088] The control effect evaluation is divided into primary screening and re-screening in indoor bioassays, using the susceptible tobacco variety Honghuadajinyuan as the bioassay object.

[0089] Step S1, primary screening by bioassay

[0090] Treatment of tobacco seedlings: Cultivate tobacco seedlings by the floating seedling method until the 4-5 leaf stage. Take out the spare tobacco seedlings from the seedling nursery pond 1 day before seeding, dry the floating trays, and the next day, use a sterile blade to wound both sides of the tobacco seedling roots 1.5 cm away from the center of the tobacco plant. Divide the tobacco seedlings into the experimental group and the control group, with 2 tobacco seedlings in each group.

[0091] Pre-treatment of biocontrol bacteria: Inoculate 1 mL of the test bacteria (about 10 9 CFU / mL) into the tobacco seedlings in the experimental group, and inoculate the tobacco seedlings in the control group with LB liquid medium.

[0092] Suspension culture of tobacco seedlings: Place a thick plastic sheet slightly larger than the floating tray for cultivating tobacco seedlings on the culture rack in a constant temperature artificial climate chamber at 28°C, and place a total of 5 disposable petri dishes at the four corners and the center of the plastic sheet as supports. Place the floating tray on it and culture for 1 day. During this period, water with a watering device three times in the morning, middle, and evening, and the amount of water should be such that the water in the holes of the floating tray does not drip.

[0093] Pathogen inoculation: One day after the pretreatment with the biocontrol bacterium, 0.5 mL of a 10-fold diluted solution of Ralstonia solanacearum RS (about 10 T CFU / mL) was inoculated onto the tobacco seedlings in the experimental group and the control group, and the seedlings were further cultured in an artificial climate chamber at a constant temperature of 28°C for 15 - 20 days. During this period, the seedlings were watered three times a day (in the morning, at noon, and in the evening) with a watering device to keep the humidity. 8 Observation and recording: Observe the disease incidence in the experimental group and the control group. When the disease incidence in the control group > 80%, record the disease incidence of the tobacco plants in the experimental group. A healthy tobacco plant is recorded as 1, a diseased but not withered tobacco plant is assigned a value of 0.5, and a withered tobacco plant is assigned a value of 0. The value of each tobacco plant treated with the test bacterium is the sum of the values assigned to two tobacco plants. Select the strain with the highest value among the test bacteria as the potential biocontrol bacterium for indoor re-screening.

[0094]

[0095] Step S2, secondary screening by bioassay

[0096] Step S2: Biological assay for re-screening. Except for the following differences in the experiment, the remaining operation steps are the same as those in Step S1 for the primary biological assay.

[0097] 1) The test strains are the potential biocontrol bacteria obtained from the primary biological assay in Step S1;

[0098] 2) Increase the number of tobacco plants to be treated in the biological assay for re-screening. Both the experimental group and the control group treat 8 tobacco seedlings;

[0099] 3) Investigate the disease occurrence of the tobacco plants in the experimental group and the control group 10 days and 20 days after inoculation with Ralstonia solanacearum RS. The results are shown in Table 1. T

[0100]

[0101] Table 1. Control effect of strain 114E6 on tobacco bacterial wilt in the growth chamber

[0102]

[0103] The experimental results show that in the primary biological assay, both of the two tobacco plants treated with strain 114E6 were diseased but not withered, so the assigned value was 1.0. In the biological assay for re-screening, 6 and 5 tobacco plants in the experimental group were healthy 10 days and 20 days after inoculation with strain 114E6, respectively, while 4 and 2 tobacco plants in the control group survived. The above results indicate that strain 114E6 has a certain control effect on bacterial wilt and can be used to evaluate its control effect in greenhouse pot experiments for the control of bacterial wilt.

[0104] Experimental Example 3: Strain identification and preservation

[0105] I. Strain identification

[0106] 1. Cultural characteristics and morphological characteristics of the strain

[0106] The strain 114E6 was routinely identified as a bacterium with reference to the literature "Manual for Systematic Identification of Common Bacteria" (edited by Dong Xiuzhu et al., Science Press, 2001). The results showed that the strain 114E6 was a Gram-negative bacterium, strictly aerobic, without endospores, with flagella, motile, rod-shaped, 1.1 - 1.6 μm in width and 1.7 - 2.5 μm in length. The strain 114E6 grew under the conditions of 10 - 37 °C, pH 6.0 - 9.0, and 0 - 4% NaCl, and did not grow under 8% NaCl or pH 5 conditions.

[0107] 2. Compound metabolic characteristics of the strain

[0108] The compound metabolic characteristics of strain 114E6 were analyzed using the Biolog GEN III plate. The results showed that strain 114E6 had the highest similarity of 0.713 with Stenotrophomonas maltophilia in the database; the other relatively similar species included Stenotrophomonas rhizophila (0.019), Xanthomonas campestris pv.ti A (0.001), and Xanthomonas campestris pv.oryzihabitans (0.001). The above results indicated that strain 114E6 was a bacterium of the Lysobacteraceae family and had a relatively close genetic relationship with bacteria of the Stenotrophomonas genus. The carbon sources that strain 114E6 could utilize well included D-maltose, D-trehalose, D-cellobiose, gentiobiose, sucrose, D-isomaltose, α-D-lactose, melibiose, β-formyl-D-glucoside, D-salicin, N-acetyl-D-glucosamine, N-acetyl-D-galactosamine, D-mannose, gelatin, glycyl-L-proline, L-alanine, L-histidine, L-serine, methyl pyruvate, L-lactic acid, citric acid, α-ketoglutaric acid, L-malic acid, bromosuccinic acid, Tween 40, propionic acid, and acetic acid; it grew weakly under the condition of using dextrin, α-D-glucose, D-fructose, L-fructose, D-fructose-6-phosphate, L-aspartic acid, pectin, D-galacturonic acid, D-glucuronic acid, glucuronamide, or acetoacetic acid as the sole carbon source; the carbon sources that it could not utilize included stachyose, melezitose, raffinose, N-acetyl-β-D-mannosamine, N-acetylneuraminic acid, D-galactose, 3-formylglucose, D-fructose, L-rhamnose, inosine, D-sorbitol, D-mannitol, D-arabitol, inositol, glycerol, D-glucose-6-phosphate, D-aspartic acid, D-serine, L-arginine, L-glutamic acid, L-pyroglutamic acid, L-galactonolactone, D-gluconic acid, mucic acid, quinic acid, saccharic acid, p-hydroxy-phenylacetic acid, methyl D-lactate, D-malic acid, γ-aminobutyric acid, α-hydroxybutyric acid, β-hydroxy-D,L-butyric acid, α-ketobutyric acid, and formic acid; it could grow well under the condition containing pH 6, 1% NaCl, 1% sodium lactate, fusidic acid, oleandomycin, rifamycin SV, lincomycin / clindamycin, guanidine hydrochloride, vancomycin, tetrazolium violet, nalidixic acid, tetrazolium blue, lithium chloride, or aztreonam, but grew slightly weakly under the condition containing pH 5, 4% NaCl, sodium tetradecyl sulfate, potassium tellurite, and sodium butyrate, and could not grow under the condition containing 8% NaCl, D-serine, minocycline, or sodium bromate.

[0109] 3. Molecular identification of the strain

[0110] (1) 16S rDNA sequence analysis

[0111] The genomic DNA of strain 114E6 was extracted using a bacterial genomic DNA extraction kit according to the kit instructions. Using the extracted genomic DNA as a template, PCR was performed with universal primers F27 / R1492 to amplify the 16S rDNA sequence. After the amplified product was recovered by gel extraction, it was ligated into the pEASY-T5 Zero vector, and heat shock transformation was used to transform Escherichia coli DH5α competent cells. Colonies were picked and colony PCR identification was performed using M13F / M13R as primers. The positive clones were sent to Shanghai Invitrogen Biotechnology Co., Ltd. for sequence determination. Based on the 16S rDNA sequence, the Ezbiocloud database (https: / / www.ezbiocloud.net / ) was used to analyze the type species similar to strain 114E6, and the genus-level taxonomic status of the strain was initially confirmed. The results showed that strain 114E6 had a high homology with multiple species within the genus Stenotrophomonas, and the top 3 species with higher similarity were Stenotrophomonas cyclobalanopsidi TPQG1-4 T (99.58%), Stenotrophomonas maltophilia MTCC 434 T (99.52%), and Stenotrophomonas hibiscicola ATCC 19867 T (99.38%). Therefore, strain 114E6 belongs to Stenotrophomonas, but strain 114E6 cannot be effectively identified only by 16S rDNA sequence analysis.

[0112] (2) Whole-genome analysis

[0113] Furthermore, the whole genome of strain 114E6 was obtained using genome sequencing technology. The TYPE database was used for alignment, and the DNA molecular hybridization value (dDDH) between the patented strain and the species with the closest genetic relationship was calculated to finally determine the molecular taxonomic status of the strain. The results are as Figure 2 shown in Table 2. After analysis by the Type(Strain)Genome Server (https: / / tygs.dsmz.de / ) database, strain 114E6 had the highest similarity with Stenotrophomonas indicatrix WS40 T at 84.9% (dDDH4), which is greater than the identification threshold of 70.0% for new species; strain 114E6 had a similarity with Stenotrophomonas lactitubi M15 TThe dDDH4 value was 53.1%; the dDDH4 values of strain 114E6 and other Stenotrophomonas were less than 50%. A phylogenetic tree was constructed based on the genomes of strain 114E6 and its related species. The results showed that strain 114E6 was most closely related to the type strain WS40 of Stenotrophomonas indicatrix T and the two clustered together ( Figure 2 ). The above results indicate that strain 114E6 is Stenotrophomonas indicatrix.

[0114] Table 2. Comparative analysis of genomic similarity between strain 114E6 and Stenotrophomonas

[0115] Type strain dDDH0 / % dDDH4 / % dDDH6 / % G + C content difference / % <![CDATA[Stenotrophomonas indicatrix WS40 T > 90.6 84.9 92.3 0.1 <![CDATA[Stenotrophomonas lactitubi M15 T > 74.5 53.1 72.1 0.44 <![CDATA[Stenotrophomonas cyclobalanopsidis TPQG1-4 T > 57.2 33.1 50.8 0.83 <![CDATA[Stenotrophomonas beteli LMG 978 T > 60.2 32.7 52.8 0.51 <![CDATA[Stenotrophomonas geniculata JCM 13324 T > 58.6 32.6 51.6 0.14 <![CDATA[Stenotrophomonas africana LMG 22072 T > 62.2 32.6 54.2 0.01 <![CDATA[Stenotrophomonas pavanii DSM 25135 T > 65.4 32.5 56.4 1.07 <![CDATA[Stenotrophomonas maltophilia NBRC 14161 T > 57.9 32.4 51 0.14 <![CDATA[Stenotrophomonas sepilia SM16975 T > 59.2 32.2 51.8 0.13 <![CDATA[Stenotrophomonas hibiscicola ATCC 19867 T > 67 32.2 57.4 0.12 <![CDATA[Stenotrophomonas chelatiphaga DSM 21508 T > 36.6 25.7 32.9 0.51 <![CDATA[Stenotrophomonas pennii Sa5BUN4 T > 36.8 25.3 32.9 0.12

[0116] II. Strain preservation

[0117] Based on the above identification results, it was confirmed that strain 114E6 is a strain of Stenotrophomonas indicatrix. The patent preservation of strain 114E6 was carried out, and the preservation information is as follows:

[0118] Preservation number: CCTCC NO:M 20211383;

[0119] Taxonomic name: Stenotrophomonas indicatrix 114E6;

[0120] Preservation date: November 8, 2021;

[0121] Preservation unit: China Center for Type Culture Collection;

[0122] Preservation address: Wuhan University, Wuhan, China.

[0123] Experimental example 4 Evaluation of the greenhouse control effect of the fermentation broth of strain 114E6 against tobacco bacterial wilt

[0124] Test settings: The test was carried out in a greenhouse with a temperature control of 28 - 30 °C. The test set up a biocontrol bacteria treatment group and a control group (irrigation with an equal volume of LB liquid medium + tap water). Each treatment had 3 replicates, and each replicate had 10 tobacco plants. The susceptible tobacco variety Honghuadajinyuan was used as the bioassay object.

[0125] Cultivation of the tested strains: The biocontrol strain 114E6 obtained by indoor screening was cultured in LB liquid medium (1% tryptone, 0.5% yeast extract, 1% sodium chloride, and 0.5% sucrose), shaken at 225 r / min at 30 °C for 48 h to obtain the 114E6 fermentation broth. The tobacco Rhodococcus QBRS-1 isolated from Wenshan Prefecture, Yunnan Province and preserved in the laboratory was selected as the pathogen, and the pathogen was cultured in CG liquid medium (0.1% acid-hydrolyzed casein, 0.5% glucose, and 2% peptone), shaken at 225 r / min at 30 °C for 24 h.

[0126] Transplanting of tobacco seedlings: Tobacco seedlings were cultivated by the floating seedling raising method. The tobacco seedlings were those with secondary leaf pruning, cultivated for about 50 days. When transplanting, red soil and organic matter were mixed at a ratio of 3:1 and then the tobacco seedlings were transplanted.

[0127] Inoculation: After transplanting, 250 mL of the 114E6 fermentation broth of the biocontrol strain was diluted 25 times, and 200 mL of the diluted solution was irrigated to the root of each tobacco seedling in the biocontrol treatment group. The concentration of the 114E6 bacteria in the diluted solution was about 10 7 CFU / mL; the control group was treated with an equal volume of the diluted LB liquid medium; the next day, the tobacco Rhodococcus QBRS-1 shaken in the CG liquid medium for 24 h was diluted 100 times, and 100 mL of the diluted solution of the tobacco Rhodococcus QBRS-1 was inoculated to each tobacco seedling. The inoculation concentration was about 10 7 CFU / mL.

[0128] Investigation and statistics: The disease index was investigated once every 7 days after inoculation, for a total of 5 times. The incidence, disease index, and control effect of tobacco bacterial wilt were calculated according to the following formulas: Incidence = number of diseased plants / total number of investigated plants × 100%; Disease index = [Σ (disease level × number of strains at this level) / (highest level × total number of plants)] × 100; Control effect = (control disease index - treatment disease index) / control disease index × 100%. The disease index was investigated according to the tobacco disease grading and investigation method of the tobacco industry standard of the People's Republic of China (GB / T 23222-2008). The disease grading was as follows: 0 level for the whole plant without disease, 1 level for the leaves withering below half on the diseased side, 3 level for the leaves withering from half to two-thirds on the diseased side, 5 level for the leaves withering more than two-thirds on the diseased side, 7 level for all the leaves of the diseased plant withering, and 9 level for the diseased plant being basically dead.

[0129] Test results: As shown in Table 3, the disease index of the tobacco plants treated with the 114E6 fermentation broth was lower than that of the control group during the observation period, and the 114E6 fermentation broth had a certain control effect on the tobacco Rhodococcus QBRS-1. The control effect at 35 dpi after inoculating the pathogen was 31.62%.

[0130] Table 3. Control effect of the 114E6 fermentation broth of the strain on the tobacco Rhodococcus

[0131]

[0132] Evaluation of the Greenhouse Control Effect of the Powder of Strain 114E6 on Tobacco Bacterial Wilt

[0133] In Experimental Example 5, except for the following tests and results being different, the rest are the same as Experimental Example 4.

[0134] 1. Seed liquid culture: Pick a loop of biocontrol bacterium 114E6 and inoculate it into a 500 mL centrifuge tube containing 500 mL of seed culture medium (peptone 10.0 g / L, beef extract 3.0 g / L, sodium chloride 5.0 g / L, agar 15.0 g / L, pH 7.0), and place it in a constant temperature shaker at 28 °C for 15 h.

[0135] 2. Fermentation: Transfer the seed liquid of biocontrol bacterium 114E6 to 150 L of fermentation medium (glucose 20 g / L, soybean meal powder 25 g / L, yeast extract 4 g / L, dipotassium hydrogen phosphate 1.5 g / L, pH 7.3) at a ratio of 1:100 for fermentation. The fermentation process parameters are as follows: (1) Sterilization: The empty sterilization conditions are 0.15 MPa, 123 °C, 1 h; the actual sterilization conditions are 0.15 MPa, 123 °C, 35 min; (2) Inoculation: After sterilization is completed, start inoculating when the temperature of the culture medium drops to 37 °C, and the inoculation ratio is 114E6 seed liquid / culture medium 1:100; (3) Fermentation: Keep the rotation speed at 120 r / min until the end of fermentation; keep the ventilation volume at 15 m 3 / h until the end of fermentation; keep the tank pressure at 0.05 Mpa throughout the process; due to equipment reasons, the ventilation volume is allowed to fluctuate within a short period of time. Keep the temperature at 37 °C throughout the fermentation process, with a fluctuation range of ±0.5 °C. Run the fermentation for 42 h.

[0136] 3. Powder preparation: Centrifuge the fermentation broth (about 3.05 billion CFU / mL), use the collected thalli as raw materials, mix them with diatomaceous earth at a mass ratio of 1:1, and add 5‰ of sucrose based on the total mass of the mixed materials as nutrients. Place the mixed materials in a cool, dry, and ventilated place to dry. When the moisture content reaches about 10%, pulverize them to obtain the 114E6 powder (620 million CFU / g).

[0137] 4. Control: Apply 2.5 g of 114E6 powder to each tobacco seedling in the biocontrol bacterium treatment group, add 50 mL of water, and use the obtained bacterial powder suspension with a bacterial concentration of 310 million CFU / mL for root irrigation. Each replicate has 15 plants, and 3 replicates are set; the tobacco seedlings in the control group are irrigated with the same volume of clear water; inoculate Pseudomonas rolfsii QBRS-1 on the next day, and inoculate 0.1 OD for each tobacco seedling; investigate the disease grade once a week and calculate the disease index.

[0138] Test results: As shown in Table 4, with the increase of inoculation days, the disease condition of the control group gradually worsened, while the tobacco plants treated with 114E6 powder had a lighter disease incidence. At 35 days after inoculation, the area under the disease index curve of the 114E6 powder treatment group was 687±150.3, while that of the control group was 1219±86.26 at this time. Calculated by the area under the disease index curve, the control effect of the 114E6 powder treatment group was 43.64%. This indicates that the 114E6 powder has a good control effect on tobacco bacterial wilt.

[0139] Table 4. Evaluation of the control effect of strain 114E6 powder on tobacco bacterial wilt

[0140]

[0141] Experimental Example 6 Greenhouse control effect evaluation of compound microbial agents on tobacco bacterial wilt

[0142] Except for the following tests and results being different, the rest of Experimental Example 6 is the same as Experimental Example 4.

[0143] 1. The experiment was carried out in a temperature-controlled (28°C) greenhouse indoors, and the tobacco plants used tobacco floating seedling substrate (specially for tobacco, produced by Yunnan Mile Tobacco Supplies Co., Ltd.) as the culture medium.

[0144] 2. After the biocontrol bacteria 56D2, 18A6, and 114E6 were separately cultured, their fermentation broths were mixed in equal proportions to obtain compound microbial agent A. The tobacco seedlings in the biocontrol bacteria treatment group were treated with compound microbial agent A. The biocontrol bacteria 56D2 and 18A6 were also strains screened in this laboratory, and their patent preservation information is as follows:

[0145] 56D2 is Ralstonia mannitolilytica, preservation number: CCTCC NO: M2019921, taxonomic name: Ralstonia mannitolilytica 56D2, preservation date: November 12, 2019, preservation unit: China Center for Type Culture Collection, address: Wuhan University, Wuhan, China.

[0146] 18A6 is Agrobacterium deltaense, preservation number: CCTCC NO: M 2020041, taxonomic name: Agrobacterium deltaense 18A6, preservation date: January 13, 2020, preservation unit: China Center for Type Culture Collection, address: Wuhan University, Wuhan, China.

[0147] 3. The disease grade was investigated every 3 days, and the disease severity and the control effect of the treatment were calculated.

[0148] Test results: As shown in Table 5, after pre-treating tobacco plants by evenly mixing the fermentation broths of the three biocontrol strains 56D2, 18A6, and 114E6 in equal proportions and then inoculating with Ralstonia solanacearum QBRS-1, tobacco bacterial wilt can be effectively controlled. The control effect of the composite bactericide composed of the three strains was 48.13% at 22 days after inoculation. The above results indicate that the composite bactericide composed of the three strains 56D2, 18A6, and 114E6 can effectively control tobacco bacterial wilt.

[0149] Table 5. Evaluation of the effect of composite bactericide A on controlling tobacco bacterial wilt

[0150]

[0151] Experimental Example 7 Greenhouse control effect evaluation of composite bactericide on tobacco bacterial wilt

[0152] Experimental Example 7 is the same as Experimental Example 6 except for the following tests and results.

[0153] After separately culturing the biocontrol strains 56D2, 65D6, 18A6, 107E3, and 114E6, the fermentation broths were mixed in equal proportions to obtain composite bactericide B. Tobacco seedlings in the biocontrol strain treatment group were treated with composite bactericide B. The biocontrol strains 65D6 and 107E3 were also screened in this laboratory, and their patent preservation information is as follows:

[0154] 65D6 is Enterobacter mori, preservation number: GDMCC NO:64022, taxonomic name: Enterobacter mori 65D6, preservation date: November 14, 2023, preservation unit: Guangdong Provincial Microbial Culture Collection Center, address: 5th Floor, Building 59, No. 100 Compound, Xianlie Middle Road, Guangzhou, Guangdong Institute of Microbiology, Chinese Academy of Sciences.

[0155] 107E3 is Stenotrophomonas indicatrix, preservation number: CCTCCNO:M 2019923, taxonomic name: Stenotrophomonas indicatrix 107E3, preservation date: November 12, 2019, preservation unit: China Center for Type Culture Collection, address: Wuhan University, Wuhan, China.

[0156] Chemical agent group: Chlorothalonil·copper sulfate (pesticide registration number PD20160275, produced by Nanjing Nannong Pesticide Science and Technology Development Co., Ltd.) was diluted 1000 times with tap water according to the instructions, and 200 mL was irrigated into the root of each tobacco seedling.

[0157] Control group: Dilute LB liquid medium of equal volume, and 200 mL was irrigated into the root of each tobacco seedling.

[0158] Test results: As shown in Table 6, when the fermentation broths of the five biocontrol strains 56D2, 65D6, 18A6, 107E3, and 114E6 were pre-treated on tobacco plants after being mixed in equal proportions, tobacco bacterial wilt could be effectively controlled. When the composite microbial agent composed of the five strains was inoculated for 22 days, the control effect reached 58.21%, while the control effect of the chemical agent chlorothalonil·copper sulfate was 54.11%. The above results indicate that the composite microbial agent composed of the five strains 56D2, 65D6, 18A6, 107E3, and 114E6 can effectively control tobacco bacterial wilt.

[0159] Table 6. Effect of composite microbial agent B on controlling tobacco bacterial wilt

[0160]

[0161] Experimental Example 8 Antagonistic effect of strain 114E6 against Ralstonia

[0162] Using the plate confrontation culture method, the inhibition zone of the strain was measured. The operation steps are as follows: Ralstonia QBRS-1, RS T , LLRS-1, and BSRS-1 cultured in CG medium (0.1% acid-hydrolyzed casein, 0.5% glucose, and 2% peptone) for 24 h were serially diluted to 10 -4 ; 100 μL of the dilution was placed on the surface of CGA medium (0.1% acid-hydrolyzed casein, 0.5% glucose, 2% peptone, and 1.5% agar), spread evenly, and dried on a clean bench; a single colony of strain 114E6 was picked and inoculated on the surface of the medium containing Ralstonia. After culturing for 2-3 days, the antibacterial effect of 114E6 against various Ralstonia was observed. Strains with antibacterial effects were cultured by the four-point confrontation method and the inhibition zone and antibacterial band of the strain were measured. Bacillus amyloliquefaciens WY11 was used as the antagonistic bacteria control ( Figure 3 ).

[0163] Test results: As shown in Table 7, strain 114E6 had no antagonistic effect against Ralstonia RS T , LLRS-1, BSRS-1, and QBRS-1. This indicates that the biocontrol strain 114E6 does not control bacterial wilt by antibacterial action, and may play a disease-resistant role through non-antagonistic niche competition, induced resistance, and regulation of the rhizosphere microecological structure.

[0164] Table 7. Antagonistic ability of biocontrol strain 114E6 against different Ralstonia

[0165] Ralstonia Biocontrol bacterium Inhibition zone / cm Inhibitory band / cm <![CDATA[RS T > 114E6 0.00±0.00 0.00±0.00 LLRS-1 114E6 0.00±0.00 0.00±0.00 BSRS-1 114E6 0.00±0.00 0.00±0.00 QBRS-1 114E6 0.00±0.00 0.00±0.00

[0166] Experimental Example 9 Effect of strain 114E6 on the germination of tobacco seeds

[0167] Preparation of the supernatant of the biocontrol bacterium culture solution: Add 5 mL of LB + 0.5% sucrose culture solution to a 50 mL centrifuge tube, inoculate the biocontrol bacterium 114E6, ferment at 28 °C and 180 r / min for 48 h, centrifuge to obtain the supernatant and filter to remove bacteria. Dilute the supernatant 10-fold, 25-fold, 50-fold and 100-fold respectively, and add it to the germination bed. Finally, select the 100-fold supernatant dilution with better effect for the experiment.

[0168] Seed pretreatment: The tested flue-cured tobacco variety is the naked seed 'Yunyan 87'. Disinfect the naked seeds of 'Yunyan 87' with 75% ethanol and 0.5% sodium hypochlorite for 30 s in sequence. After rinsing 3 - 4 times with sterile water, soak the seeds in 1 mL of sterile water for 16 h. After sucking off a part of the sterile water, place the seeds on a paper towel for wiping hands and dry for later use.

[0169] Growth promotion experiment: Lay a layer of sponge in a petri dish, place two pieces of filter paper on the sponge, add 12 mL of the 100-fold dilution of the sterile supernatant of the biocontrol bacterium 114E6 prepared. Use sterile water as a control, keep the filter paper moist, sow the seeds on the filter paper, and germinate in an incubator at 25 °C with 12 h of light / dark each. Each repetition (each petri dish) has 25 seeds (5×5), and repeat 3 times. During this period, add the 100-fold dilution of the supernatant and sterile water once every two days, and exchange positions. Generally, a batch is completed within 14 d, and investigate the germination rate, fresh weight and radicle length of tobacco seeds, and analyze the significance of differences.

[0170] Test results: There was no significant difference in the germination rate of the seeds treated with the supernatant of the 114E6 biocontrol bacterium culture solution and the sterile water control group, and the germination rate could reach over 98% in both cases. However, there were extremely significant differences in the fresh weight and root length of the seeds treated with the supernatant of the 114E6 biocontrol bacterium culture solution and the sterile water control group ( Figure 4 ). This indicates that strain 114E6 can significantly promote the growth of tobacco seeds.

Claims

1. A strain of Stenotrophomonas indicatrix with a preservation number of CCTCC NO: M20211383.

2. A bacterial agent comprising the Stenotrophomonas indicatrix described in claim 1.

3. The bacterial agent according to claim 2, characterized in that the bacterial agent is a compound bacterial agent and further comprises one or more strains selected from Ralstonia mannitolilytica strain 56D2, Enterobacter mori strain 65D6, Agrobacterium deltaense strain 18A6, and Stenotrophomonas indicatrix strain 107E3; wherein, the preservation number of strain 56D2 is CCTCC NO: M 2019921; the preservation number of strain 65D6 is GDMCC NO: 64022; the preservation number of strain 18A6 is CCTCC NO: M 2020041; the preservation number of strain 107E3 is CCTCC NO: M2019923.

4. The bacterial agent according to claim 3, characterized in that the contents of the strains in the compound bacterial agent are the same.

5. The bacterial agent according to any one of claims 2 to 4, characterized in that the bacterial agent is a liquid agent or a powder agent.

6. Use of the Stenotrophomonas indicatrix described in claim 1 in the preparation of a product for preventing and treating tobacco bacterial wilt and / or promoting tobacco growth.

7. The use according to claim 6, characterized in that the tobacco bacterial wilt is caused by Ralstonia nicotianae, Ralstonia syzygii, Ralstonia solanacearum or Ralstonia pseudosolanacearum.

8. Use of the Stenotrophomonas indicatrix described in claim 1 or the bacterial agent according to any one of claims 2 to 5 in preventing and treating tobacco bacterial wilt and / or promoting tobacco growth.

9. The use according to claim 8, characterized in that the fermentation broth of the Stenotrophomonas indicatrix or the suspension prepared from the bacterial agent is used for root irrigation, root dipping, mixing with substrate or organic fertilizer of tobacco seedlings.

10. The use according to claim 8 or 9, characterized in that The tobacco bacterial wilt is caused by Ralstonia nicotianae, Ralstonia syzygii, Ralstonia solanacearum or Ralstonia pseudosolanacearum.

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