A Pseudomonas mollissima strain antagonistic to tobacco target spot

Abstract

The invention discloses a Pseudomonas mosselii antagonistic to tobacco target spot disease, belongs to the field of microbial technology, is preserved in China Center for Type Culture Collection, and has a preservation number of CCTCC NO: M2022859. It provides an important biological control resource for biological control of tobacco target spot disease.

Description

Technical Field

[0001] The invention relates to the technical field of microorganisms, and in particular to Pseudomonas mosselii antagonistic to tobacco target spot disease. Background Art

[0002] Due to the good compatibility of biological control with the environment, it provides new ideas and options for plant disease and insect pest control. There have been reports on the use of Pseudomonas to control rice sheath blight: For example, Zhang Qingxia reported that Pseudomonas chlororaphis 7-5 mainly controls rice sheath blight by producing antibiotics such as phenazine-1-carboxylic acid, siderophore, hydrocyanic acid, protease, pyocyanin and 2,4-diacetylphloroglucinol, with a control efficiency of 69.0%; Zhang Ya's research showed that Pseudomonas SU8 can inhibit the activity of rice sheath blight by producing phenazine substances; Wang Xiaoying analyzed the fermentation liquid of Pseudomonas through ultraviolet, infrared, mass spectrometry, nuclear magnetic resonance and other spectra, and identified a new compound as 2-heptyl-5-hexylfuran-3-carboxylic acid (HHCA), which has antagonistic ability to rice sheath blight. However, so far, there are still few reports on biological control resources for tobacco target spot pathogens.

[0003] Tobacco target spot is a fungal disease caused by the fungus Pseudomonas aeruginosa. Tobacco target spot can occur from the seedling stage to the maturity of tobacco leaves, infecting both leaves and stems. The infected leaves are initially circular water-soaked spots. If the temperature is high, the humidity is high, and the leaves are moist for a long time, the spots will expand rapidly to form irregular spots with a diameter of 2-20 cm, with concentric rings, forming chlorotic halo spots, and the necrotic part of the spots is fragile and perforated, resembling the holes left on the target after a bullet is shot, so it is called target spot. When the humidity is high, the mycelium of the fungus and the hymenium and basidiospores of the sexual generation often appear on the edge of the spots on the lower surface of the leaves.

[0004] At present, the prevention and control measures for tobacco target spot disease are mainly chemical pesticides. However, the use of chemical agents for prevention and control will leave pesticide residues, seriously pollute the environment and harm humans and animals. In addition, long-term use of the same chemical pesticide will make the plants develop certain resistance, and the prevention and control effect will be greatly reduced. Biological control will not pollute the environment, will not harm the health of humans and animals, and can effectively prevent the spread of the disease.

[0005] Therefore, how to provide a strain that can antagonize tobacco target spot pathogens is a technical problem that those skilled in the art urgently need to solve. Summary of the invention

[0006] In order to overcome the technical problems in the background technology, the present invention provides a Pseudomonas mosselii antagonistic to tobacco target spot pathogens.

[0007] The technical solution adopted by the present invention to solve its technical problem is:

[0008] A Pseudomonas mosselii strain antagonistic to tobacco target spot

[0009] CZ-56, which is deposited in China Center for Type Culture Collection with the deposit number: CCTCC NO: M2022859.

[0010] The present invention also provides a bacterial agent, which contains the Pseudomonas mosselii CZ-56.

[0011] The present invention also provides a composition, which comprises the Pseudomonas mosselii CZ-56.

[0012] The composition may be in solid form or in liquid form. The composition may also contain other substances that have a synergistic effect with the Pseudomonas morganii of the present invention, and the other substances may be antibacterial active substances of microbial origin or antibacterial compounds. The composition may further include adjuvants, thickeners and / or dispersants that match the Pseudomonas morganii of the present invention, for example, the adjuvant may be selected from cottonseed oil, castor oil, tung oil, liquid paraffin, soybean oil, A1, A2, dimethyl phthalate, dibutyl phthalate, etc.; thickeners include bentonite, aluminum stearate, QH gel, dragon glue powder, F1, xanthan gum, etc.; dispersants include pull open powder, NNO, LFS, B1, carbon black, etc.

[0013] Preferably, at least one of the Pseudomonas mosselii CZ-56, the bacterial agent or the composition is used in preventing and treating target spot disease.

[0014] Preferably, the target spot disease is tobacco target spot disease.

[0015] Beneficial effects of the present invention:

[0016] The experiment proved that the Pseudomonas mosselii CZ-56 provided by the present invention can reach an inhibition rate of 83.33% on tobacco target spot pathogens through plate confrontation, which is significantly higher than the current biological pesticide Jinggangmycin for preventing and treating tobacco target spot pathogens. The indoor detached leaf prevention effect determination and potted plant prevention effect determination were further carried out on tobacco target spot pathogens. The indoor detached leaf prevention effect determination showed that the fermentation liquid can prevent and treat tobacco leaves, and the potted plant prevention effect determination showed that the relative prevention effect of the fermentation liquid on tobacco potted plants was the highest. The growth parameters and resistance-related enzyme activities of tobacco seedlings treated with the CZ-56 strain were further statistically analyzed and determined, and it was found that the fermentation liquid root irrigation treatment of tobacco seedlings can effectively promote the growth of plants and the accumulation of nutrients, and can also effectively promote the enhancement of PPO, SOD, POD and PAL enzyme activities. In summary, a biocontrol bacterium with good biocontrol potential was screened, which provides an important biocontrol resource for the biological control of tobacco target spot disease. It also provides a reference for the use of Pseudomonas members in biological control and promoting plant disease resistance. Therefore, the promotion and application of CZ-56 is expected to replace chemical pesticides to control tobacco target spot disease, protect the environment, reduce pesticide residues, significantly improve the safety of tobacco leaves, and has good prospects for the development of biological pesticides. BRIEF DESCRIPTION OF THE DRAWINGS

[0017] Figure 1 This is a comparison chart of the antibacterial effect of CZ-56 of the present invention and its influence on mycelium;

[0018] Figure 2 The morphology and scanning electron microscope photos of CZ-56 bacteria of the present invention;

[0019] Figure 3 Comparative photos of physiological and biochemical characteristics of CZ-56 of the present invention;

[0020] Figure 4 This is the result diagram of the CZ-56 phylogenetic tree of the present invention;

[0021] Figure 5 The comparison photos are of the preventive and therapeutic effects of different treatments of the present invention on potted tobacco inoculated with tobacco target spot pathogen;

[0022] Figure 6 The photos are comparisons of the growth-promoting effects of different treatments of CZ-56 on tobacco;

[0023] Figure 7 This is a comparison chart of PPO enzyme activity determination of CZ-56 treated with different methods in the present invention;

[0024] Figure 8 This is a comparison chart of POD enzyme activity determination of CZ-56 under different treatments of the present invention;

[0025] Fig. 9This is a comparison chart of SOD enzyme activity determination of CZ-56 under different treatments of the present invention;

[0026] Fig.10 This is a comparative diagram of PAL enzyme activity determination of CZ-56 with different treatments of the present invention. DETAILED DESCRIPTION

[0027] The following will be combined with the drawings in the embodiments of the present invention to clearly and completely describe the technical solutions in the embodiments of the present invention. Obviously, the described embodiments are only part of the embodiments of the present invention, not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without creative work are within the scope of protection of the present invention.

[0028] Embodiment 1

[0029] The present invention provides a Pseudomonas mosselii CZ-56 for antagonizing tobacco target spot disease, which is deposited in the China Center for Type Culture Collection with a deposit number of: CCTCCNO: M2022859, address: Wuhan University, Wuhan, China, and a deposit date of June 10, 2022.

[0030] Materials, reagents and instruments used in the examples:

[0031] 1.1 Test strains

[0032] The antagonistic strain was isolated and screened from the rhizosphere soil of healthy tobacco in a tobacco field in Chenzhou with tobacco target spot disease, and was numbered CZ-56. The test strain, tobacco target spot pathogen, was isolated and identified by the research team from the target spot disease leaves of tobacco in the field in the early stage, and was numbered BB-11. Both strains are stored in the Hunan Provincial Key Laboratory of Plant Pest Biology and Control, Hunan Agricultural University.

[0033] 1.2 Test culture medium

[0034] Nutrient liquid medium (NB): weigh 10 g of peptone, 3 g of beef extract, and 5 g of sodium chloride, and dissolve them in 1000 mL of deionized water, pH 7.3 ± 0.1;

[0035] Nutrient agar medium (NA): Add 15 g of agar to the nutrient liquid medium;

[0036] Nitrate reduction medium: weigh 5 g of peptone, 3 g of beef extract, 10 g of glucose, and 1 g of potassium nitrate, and dissolve them in 1000 mL of distilled water, pH 7.3 ± 0.3;

[0037] Starch hydrolysis medium: weigh 10 g of peptone, 5 g of beef extract, 5 g of sodium chloride, 2 g of soluble starch, and 15-20 g of agar, and dissolve in 1000 mL of distilled water, pH 7.1±0.1;

[0038] Methyl red medium (MP): weigh 5 g of peptone, 5 g of glucose, and 5 g of dipotassium hydrogen phosphate and dissolve them in 1000 mL of distilled water;

[0039] Vapour medium (VP): weigh 5 g of peptone, 5 g of glucose, and 5 g of potassium dihydrogen phosphate and dissolve them in 1000 mL of distilled water;

[0040] Gelatin culture medium: weigh 5g of peptone, 3g of glucose, 3g of beef extract, and 120g of gelatin, and dissolve them in 1000mL of distilled water. Dissolve the above ingredients in a water bath, pH 7.3±0.1;

[0041] Hydrogen sulfide medium: weigh 20 g of peptone, 5 g of sodium chloride, 0.5 g of sodium thiosulfate, 0.5 g of ammonium ferric citrate, and 15-20 g of agar, and dissolve them in 1000 mL of distilled water;

[0042] All the above culture media were sterilized by high pressure at 121°C for 15 min.

[0043] 1.3 Main reagents and instruments

[0044] In this example, the flue-cured tobacco variety Yunyan 87 was selected as the test variety, and the fungicide used was 10% Jinggangmycin aqueous solution (Tonglu Huifeng Biotechnology Co., Ltd., Zhejiang Province).

[0045] Test reagents: peptone, beef extract, sodium chloride, agar, potassium nitrate, soluble starch, dipotassium hydrogen phosphate, gelatin, ammonium ferric citrate, sodium thiosulfate, creatine, sorbitol, glucose, D-fructose, sucrose, maltose, galactose, D-xylose, and mannitol were purchased from Shanghai Shenggong Company and Sinopharm Chemical Reagent Co., Ltd. Crystal violet, 95% ethanol, fuchsin stain, 3% H 2 O 2 , 0.03%H 2 O 2 , 40% NaOH, 0.1mol / L phosphate (PBS) buffer pH 6.8, 0.1mol / L phosphate (PBS) buffer pH 8.8, 0.05mol / L phosphate (PBS) buffer pH 7, 0.05mol / L phosphate (PBS) buffer pH 7.8, 1.3×10 -2 mol / L methionine, 7.5×10 -5 mol / L nitro blue tetrazolium (NBT), 2×10 -6 mol / L riboflavin, 12×10 -6 mol / L EDTA, 6mol / L hydrochloric acid, 0.02mol / L L-phenylalanine, 0.1mol / L catechol, 0.2% guaiacol, 1% PVP, DNA purification and recovery kit were purchased from OMEGA, and agarose was purchased from Thermo Fisher Scientific.

[0046] Iodine solution: iodine 1g, potassium iodide 2g, distilled water 300mL.

[0047] Gries reagent A solution: 0.5 g of p-aminobenzenesulfonic acid, 150 mL of dilute acetic acid.

[0048] Gries reagent B solution: α-naphthylamine 0.1 g, distilled water 20 mL, dilute acetic acid 150 mL.

[0049] Diphenylamine reagent: 0.5 g of diphenylamine is dissolved in 100 mL of concentrated sulfuric acid and diluted with 20 mL of distilled water. Methyl red indicator: 0.1 g of methyl red, 300 mL of 95% ethanol, and 200 mL of distilled water.

[0050] Main instruments: Biometra TAdvanced PCR instrument (Jena, Germany), UV-visible spectrophotometer (752E RS), -20℃ refrigerator, microwave oven, induction cooker (Midea), -80℃ refrigerator (Haier), clean bench (Antai Company of Sujing Group), electronic balance (Shanghai Guangzheng Medical Instrument Co., Ltd.), desktop high-speed refrigerated centrifuge, pipette (Eppendorf), constant temperature incubator (Shaoguan Taihong Medical Equipment Co., Ltd.), biological microscope (McOdy Industrial Group Co., Ltd.), constant temperature shaker (Thermo), Japan TOMYSX500 high pressure sterilizer, ultrapure water machine, CNC super low temperature constant temperature bath (Ningbo Tianheng Instrument Factory), American AlghaImager HP gel imaging system, Japanese SANYO ice machine, electrophoresis instrument, electrophoresis tank (Beijing Junyi Vertical Electrophoresis Tank Technology Co., Ltd.), ASD induction cooker (Zhejiang ASD Electric Co., Ltd.), Leici HY-2 vortex mixer (Shanghai Yidian Scientific Instrument Co., Ltd.).

[0051] Embodiment 2

[0052] 1.4 Isolation and screening of antagonistic bacteria

[0053] 1.4.1 Isolation of bacteria

[0054] The dilution and spreading separation method was used for separation. The soil sample was placed in a ventilated and dry place to air dry naturally, 5 g of soil was weighed and poured into 50 mL of sterile water with sterilized glass beads, and then placed on a shaker for 30 minutes. 1 mL of the suspension was drawn and diluted with sterile water, and then evenly spread on the NA agar plate, and then placed in a 28°C biochemical incubator for culture. Single colonies with different culture characteristics were selected and transferred to NA culture medium for purification culture. After purification for 3 times, they were placed in a 4°C refrigerator for storage.

[0055] 1.4.2 Determination of antibacterial effect of antagonists

[0056] Antagonistic bacteria were screened by plate confrontation method. A bacterial cake was taken from the plate with activated strain BB-11 and inoculated in the center of the PDA plate. After activating the isolated strain, a line was drawn at 2.5 cm on both sides of BB-11, and a cover glass was inserted obliquely into the culture medium. The plate was cultured in a constant temperature incubator at 28°C for 3 days, and the inhibition rate was calculated. The PDA plate with no antagonistic bacteria but only strain BB-11 was used as a blank control, and 3 replicates were set for each treatment.

[0057]

[0058] Embodiment 3

[0059] 1.5 Identification of the antagonistic strain CZ-56

[0060] 1.5.1 Morphological observation and physiological and biochemical identification

[0061] Purify and culture as described above, grow a single colony on the NA plate, and observe its morphology. The physiological and biochemical characteristics refer to the eighth edition of the Bergey's Manual of Bacterial Identification.

[0062] Gram staining: Use an inoculating needle to pick up a small amount of CZ-56 bacterial moss cultured on NA medium for 24 hours, evenly spread it on a drop of sterile water on a clean glass slide, and air-dry to fix; primary stain with crystal violet for 1 minute, wash with water; mordant with iodine solution for 1 minute, wash with water, and dry; decolorize with 95% ethanol for 30 seconds, and dry; restain with safranin for 1-2 minutes, wash with water, and air-dry; observe under an oil microscope, dark purple is Gram-positive bacteria, and red is Gram-negative bacteria.

[0063] Growth curve determination: The strain to be tested was activated twice in succession, and 24 hours after the second activation, it was transferred into NB culture medium, cultured at 180rpm / min, 28℃ for 24 hours, and then its OD was measured. 600 Then take 1mL and inoculate it into an equal amount of NB culture medium, culture it at 180rpm / min and 28℃, take samples every 2h, and measure it until 72h, and use the spread plate method to determine the number of live bacteria in the sample and draw a growth curve.

[0064] Growth temperature range determination: 1 mL of bacterial solution was inoculated into an equal amount of NA culture medium. Parallel experiments were set up and cultured in the dark at 4, 10, 15, 20, 25, 28, 30, 35, 37, 40 and 45 °C. Samples were taken at 24 h of culture and the OD was determined. 600 The effects of different temperatures on the growth of strains were compared and analyzed.

[0065] Effect of sodium chloride on growth: NaCl was added to the culture medium at a ratio of 0.5%, 1%, 2%, 5% and 10% (w / v), and the test bacteria were streaked and cultured in the culture medium containing NaCl. After culturing at 28°C for 7 days, the growth of the test bacteria was observed, and the culture medium without NaCl was used as a negative control.

[0066] Determination of growth pH range: Prepare NA culture medium with pH values ​​of 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, and 13, inoculate the strain to be tested in equal amounts into the culture medium with different pH values, culture at 28°C for 24 hours, and determine the OD value. 600 The values ​​and effective viable counts were compared and analyzed to analyze the effects of different pH on the growth of strains.

[0067] Carbon source utilization determination: The test bacteria were streaked and inoculated on NA medium (without adding any carbon source) containing 1.0% glucose, D-fructose, sucrose, maltose, galactose, sorbitol, mannitol, and D-xylose, and cultured in the dark at 28°C. The growth was observed after 7 and 14 days. If the test bacteria can grow on this medium, it indicates that the carbon source can be utilized. If it cannot grow, it means that it cannot be utilized. Each carbon source treatment was repeated 5 times.

[0068] Nitrate reduction test: Inoculate the bacteria to be tested in nitrate culture medium, place it in a constant temperature incubator at 28℃ and culture it for 1d, 3d, and 5d, and use the nitrate culture medium without bacteria as a blank control. Add 0.5mL of bacterial solution cultured for 1d, 3d, and 5d to sterilized empty test tubes, and then add 1 drop of Griess reagent A solution and B solution to each. Add 1 drop of Griess reagent A solution and B solution to the control tube, and repeat three times for each group. If the culture medium turns pink, rose red, orange, brown, etc., it indicates the presence of nitrite, which is a positive reaction for nitrate reduction; if no red appears, add 1 or 2 drops of diphenylamine reagent. If it is blue, it means that there is nitrate in the culture medium, and there is no nitrite reaction, indicating that there is no nitrate reduction effect, which is a negative reaction; if it is not blue, it means that the nitrate and the formed nitrite have become other substances, so it is still treated as a positive reaction for nitrate reduction.

[0069] Catalase test: Take the test bacteria grown for 18-24 hours on a clean slide and add 3% H 2 O 2, observe whether there are bubbles produced. If there are bubbles within half a minute, it is positive, if there are no bubbles, it is negative.

[0070] Starch hydrolysis test: Inoculate the bacteria to be tested on a plate of starch hydrolysis medium and culture it in a constant temperature incubator at 28℃ for 2d-5d. After obvious colonies are formed, add iodine solution on the plate and the plate will turn blue-black. If a transparent circle without discoloration appears around the colony, it is positive for starch hydrolysis; if no transparent circle appears around the colony, it is negative.

[0071] Methyl red test (MR): Inoculate the bacteria to be tested into the methyl red culture medium, and use the culture medium without bacteria as the blank control. Repeat three times for each group. Culture in a 37℃ constant temperature box, and add 1 drop of methyl red indicator to the culture medium at 2d and 6d (if negative, the culture time can be appropriately extended). If the culture medium turns red, it is positive; if it turns yellow, it is negative.

[0072] Gelatin liquefaction test: Inoculate the test bacteria in a test tube of gelatin culture medium, culture it in a constant temperature incubator at 20℃, and observe whether the culture medium is liquefied after 2-7 days. If part or all of the gelatin clot turns into a flowable liquid below 20℃, it is a positive reaction for gelatin hydrolysis; if the test bacteria have grown, there is no depression on the surface of the gelatin and it is a stable clot, it is a negative reaction. If the test bacteria have grown, the gelatin has not liquefied, but the gelatin shows a small depression under the bacterial moss (it must be compared with the blank control without the test bacteria inoculated, and the gelatin cultured for too long will appear slightly depressed due to water loss), it is also considered as mild hydrolysis and recorded as a positive reaction for gelatin liquefaction.

[0073] VP assay: Inoculate the bacteria to be tested into VP culture medium, and use the medium without bacteria as the blank control, repeat three times each time. Incubate at a constant temperature in a constant temperature incubator. After 7 days, take the culture medium and mix it with 40% NaOH solution in equal amounts, add a small amount of creatine, and observe after standing for 10 minutes (sometimes it needs to be left there for longer). If red appears, it is positive, and if no red appears, it is negative. 3.1.5.2 Multi-gene sequence determination

[0074] The genomic DNA of CZ-56 was extracted using a bacterial genomic DNA extraction kit as a template. The bacterial 16S rDNA gene universal primers 27F (5′-AGAGTTTGATCCTGGCTCAG-3′) and 1492R (5′-TACGGCTACCTTGTTACGACTT-3′) and gyrB gene primers F (5′-GACAAGCTGGTGTCTTCCGA-3′) and R (5′-CATCTCGCCCAGACCTTTGT-3′) were used for PCR amplification and sequencing. The PCR reaction system (25 μL) was as follows: 1 μL DNA template, 1 μL upstream and downstream primers (10 μmol / L) each, 1 μL, 2×EasyTaq PCR Super Mix 12.5 μL, and 9.5 μL ddH2O. The PCR conditions for 16S rDNA and gyrB genes were as follows: 94°C for 5 min; 94°C for 30 s, 56°C for 30 s, 72°C for 1.5 min, 36 cycles; 72°C for 10 min. The sequencing results were submitted to the NCBI database for BLAST comparison, and finally the multi-gene phylogenetic tree was constructed using the neighbor-joining method using MEGA 7.0 software.

[0075] 1.6 Indoor efficacy test of strain CZ-56 against tobacco target spot

[0076] 1.6.1 Detection of biocontrol activity of CZ-56 on detached tobacco leaves

[0077] The bacterial cake was used to inoculate the strain CZ-56 against tobacco target spot pathogens in detached leaves, and 10% Jinggangmycin aqueous solution was used as the control. After the strain CZ-56 was cultured in NB medium in a constant temperature incubator at 28°C for 2 days, it was centrifuged at 8000rpm for 10 minutes. The supernatant was the fermentation stock solution, and the precipitate was suspended with sterile water to obtain OD 600 The value was 0.8. The mycelium blocks with a diameter of 7 mm were inoculated on the surface of tobacco leaves. 24 hours before inoculation, the tobacco leaves were soaked in water, 10% Jinggangmycin dilution, 6.4×10 8 cfu / m bacterial suspension, fermentation stock solution, fermentation solution diluted 10 times (10×), and fermentation solution diluted 50 times (50×), soaked for 5 minutes in 6 treatments as a preventive test (or treated 24 hours after inoculation as a therapeutic test), with 6 tobacco leaves in each treatment, repeated 3 times, and the cross-cross method was used to measure the length of the lesions 4 days after inoculation.

[0078] 1.6.2 Detection of biocontrol activity of CZ-56 against tobacco

[0079] The bacterial cake inoculation method was used to determine the efficacy of strain CZ-56 against tobacco target spot pathogens in living tobacco plants, with clean water as the control. According to step 1.6.1, 6 treatments were prepared, and the target spot pathogens were sprayed on tobacco leaves 24 hours before inoculation as a preventive test (treatment 24 hours after inoculation was a therapeutic test). Each treatment had 8 pots of tobacco, with 3 replicates. Four days after inoculation, the disease index was calculated using the tobacco target spot disease grading standard to calculate the efficacy and analyze it.

[0080] 1.7 Determination of the growth-promoting ability of CZ-56

[0081] Plant a single tobacco plant in a pot, with 30 g of substrate in each pot. Prepare 6 treatments according to step 1.6.1, with 12 pots in each treatment replicated, irrigate each pot with 5 mL of water, once a week for a total of 3 times, and investigate the plant height, stem width, root length, maximum leaf length and width, fresh weight, and dry weight 7 days after the last root irrigation.

[0082] 1.8 Determination of disease resistance-related enzyme activities of CZ-56

[0083] Root irrigation was carried out according to the above treatment, and the largest leaf in each pot was taken for resistance-related enzyme activities (polyphenol oxidase (PPO), peroxidase (POD), superoxide dismutase (SOD), and phenylalanine ammonia-lyase (PAL)).

[0084] Extraction of enzyme solution: Select the largest leaf in each pot, take 1g of leaf tissue for each treatment and put it into a pre-cooled mortar, add 1.5mL of enzyme extract and an appropriate amount of sterilized quartz sand, grind in an ice bath and transfer to a sterile centrifuge tube, rinse the mortar with 1.5mL of enzyme extract, and add a total of 3mL of enzyme extract before and after. Centrifuge at 0℃ / 10000rpm for 20min, and the supernatant is the enzyme solution, which is stored in a -20℃ refrigerator for later use.

[0085] PPO: PPO activity was measured by the catechol method. Add 1.5 mL of 0.1 mol / L phosphate buffer with a pH of 6.8 to a test tube, then add 1.4 mL of 0.1 mol / L catechol solution and 0.05 mL of enzyme solution in sequence. Replace the enzyme solution with phosphate buffer for control. After shaking thoroughly, measure the absorbance at 410 nm with a UV spectrophotometer, and record the reading every minute for a total of 5 times. One enzyme activity unit (U) is calculated as the amount of enzyme per gram of tissue per minute when A410 changes by 0.01. All samples were repeated 3 times.

[0086] POD: The guaiacol method was used to determine the enzyme activity of POD. Take two test tubes as test and control, and add 1 mL of H 2 O 2 , 1mL 0.05mol / L pH 7.0 phosphate buffer, 0.95mL 0.2% guaiacol solution and 0.05mL enzyme solution. For the control, the enzyme solution was replaced with phosphate buffer. After thorough mixing, the OD value was measured at 470nm and recorded once every minute for a total of 5 times. One enzyme activity unit (U) is the amount of enzyme per gram of plant tissue when the A470 changes by 0.01 per minute. All samples were repeated 3 times.

[0087] SOD: The SOD enzyme activity was determined by the nitroblue tetrazolium photoreduction method. Two test tubes were used as the test and control tubes. 0.3 mL of 1.3 × 10 -2 mol / L methionine, 0.3mL concentration is 2×10 -6 mol / L riboflavin, 0.3mL concentration is 7.5×10 -5 mol / L nitroblue tetrazolium (NBT), 0.3mL concentration is 12×10 -6 mol / L EDTA, 1.5mL phosphate buffer with a concentration of 0.05mol / L and a pH value of 7.8, add 0.05mL enzyme solution to the test group, add the same volume of phosphate buffer to the control group, add sterile water to make up to 3mL, put the test tube under 4000LX fluorescent light for 30min, then immediately block the light and measure the OD value at 560nm, record once every minute for a total of 5 times. One enzyme activity unit (U) is the amount of enzyme per gram of plant tissue when the A560 changes by 0.01 per minute. All samples were repeated 3 times.

[0088] PAL: Take two test tubes as test and control, add 1mL 0.02mol / L phenylalanine and 2mL 0.1mol / L pH 8.8 phosphate buffer to each test tube, add 0.05mL enzyme solution to the test group, and add the same volume of phosphate buffer to the control group. After fully mixing, put it in a 30℃ water bath for 1h, and add 0.2mL 6mol / L hydrochloric acid to the test tube of the test group to terminate the reaction. Immediately measure the OD value at 290nm with a UV spectrophotometer, and record it once every minute for a total of 5 times. One enzyme activity unit (U) is the amount of enzyme per gram of plant tissue when the A290 changes by 0.01 per minute. All samples are repeated 3 times.

[0089] Enzyme activity (ΔOD λ ·g -1 ·min -1 )=(ΔOD λ ·VT) / (W·VS·0.01·t)

[0090] ΔOD: Optical density value during reaction time

[0091] VT: Total volume of sample extract (mL)

[0092] W: fresh weight of sample (g)

[0093] VS: Sample extraction volume during measurement (mL)

[0094] t: reaction time (min)

[0095] 1.9 Data Analysis

[0096] IBM SPSS Statistics 26 and Microsoft Excel 2010 were used to analyze the experimental data and plot them. Duncan's new differential repolarization method was used to test the significance of differences.

[0097] Embodiment 4

[0098] 2. Results and Analysis

[0099] 2.1 Screening of antagonistic bacteria and determination of antibacterial effects

[0100] 711 strains were isolated from the rhizosphere soil of healthy tobacco in tobacco fields in Chenzhou, Hunan, and antagonistic screening was performed using tobacco target spot pathogens as indicator bacteria through confrontation culture. It was found that 26 strains had good antagonistic effects on tobacco target spot pathogens as shown in Table 1. After treatment with the antagonistic bacteria CZ-56, the average diameter of tobacco target spot pathogen colonies was 1.5±0.05cm, and the inhibition rate was 83.33%±0.68%, with obvious inhibition effects. Figure 1 , A, C: control group; B, D: treatment group, Figure 1 The antibacterial effect of antagonistic bacteria CZ-56 and its influence on mycelium, and the external structure and internal cytoplasm of Rhizoctonia solani mycelium were greatly affected by confrontation culture. Figure 1 As shown in the figure, the outer part of the control treated mycelium is slender as a whole, the connection between mycelium is far away, and the internal cytoplasm is evenly distributed; the mycelium of Rhizoctonia solani cultured in confrontation with CZ-56 can be observed from the external mycelium that the connection between mycelium is closer, and some mycelium is swollen and has signs of breaking. From the internal observation, it can be seen that the white empty segments in the mycelium have increased significantly, and the internal cytoplasm has the characteristics of dissipation and lysis. The gaps produced cause the cell membrane to expand outward, and a small number of mycelium have overflowed their contents and the mycelium is hollow. Therefore, this study selected the relatively stable strain CZ-56 as the target bacteria for subsequent experiments.

[0101] Table 1 Inhibitory effect of biological control materials on tobacco target spot

[0102]

[0103]

[0104] 2.2 Identification of antagonistic strain CZ-56

[0105] 2.2.1 Morphological observation, physiological and biochemical identification and biological characteristics analysis

[0106] After culturing on NA medium for 24 hours, the colonies of CZ-56 were white with smooth, neat and slightly convex edges. Figure 2 As shown, a: CZ-56 colony image; b: CZ-56 bacterial morphology under scanning electron microscope magnification 20000 times. The results of the determination of physiological and biochemical indicators of strain CZ-56 showed that the strain was positive for contact enzyme, methyl red test, nitrate reduction, and hydrogen sulfide test, and negative for Gram staining, starch hydrolysis, acetylmethyl carbinol, and gelatin liquefaction test as shown in Table 2 and Figure 3 As shown, A: Gram staining; B: catalase reaction; C, D: starch hydrolysis assay; E: nitrate reduction reaction; F: MR assay; G: VP assay; H: hydrogen sulfide assay. These physiological and biochemical characteristics preliminarily speculated that the bacteria was Pseudomonas mosselii.

[0107] The strain CZ-56 was cultured for 56 h, and the OD 600 Measure the absorbance and colony count. CZ-56 enters the logarithmic growth phase at 6 hours, when the bacterial growth rate is the fastest and the metabolism is vigorous; it enters the stable growth phase at 12 hours, and the total colony count is the largest at 28 hours, with the most accumulated metabolites, and more important secondary metabolites are produced at this time; after growing for 46 hours, the turbidity of the bacteria begins to decrease, and cells undergo apoptosis at 52 hours. The growth temperature range test found that strain CZ-56 can grow at 4-45°C, and at 28°C and 37°C, the OD 600 The absorbance value reached the highest; the effect of sodium chloride on growth was determined and it was found that strain CZ-56 could survive when the NaCl concentration reached 5% and 10%, but the content was low; the pH range of strain CZ-56 growth showed that the strain could survive at pH 2, but not at pH 11.

[0108] Table 2 Physiological and biochemical characteristics of strain CZ-56

[0109]

[0110] 2.2.2 Molecular Biological Identification

[0111] like Figure 4As shown in the figure, the GenBank accession number is in brackets; the numbers on the branch points represent the bootstrap values; the scale 0.5 represents the nucleotide substitution rate. According to the sequencing results, the PCR product length of the 16S rDNA gene of strain CZ-56 is 1434bp. The BLASTn comparison results in the NCBI database show that the CZ-56 strain has 100% similarity with the 16S rDNA gene of P. mosselii strain yy161 (MN177227.1), and 99.86% similarity with the 16S rDNA gene of P. mosselii strain ODW 1.5.1 (MK503435.1); the PCR product length of part of the gyrB gene of strain CZ-56 is 579bp. The BLASTn comparison results in the NCBI database show that the CZ-56 strain has 97.14% similarity with the whole genome of P. mosselii strain PtA1 (CP024159.1), and has 99.86% similarity with the 16S rDNA gene of P. mosselii strain ODW 1.5.1 (MK503435.1). The whole genome of BS011 (CP023299.1) has a similarity of 97.50%. The sequences of CZ-56 and other strains were selected to construct a phylogenetic tree. The results showed that CZ-56 and Pseudomonas morganii were on the same branch. Figure 5 Based on the results of sequence alignment analysis, morphological and physiological and biochemical identification, strain CZ-56 was preliminarily identified as Pseudomonas mosselii (P. mosselii).

[0112] 2.3 Determination of indoor protection effect of CZ-56

[0113] 2.3.1 Detection of biocontrol activity of CZ-56 on detached tobacco leaves

[0114] The results of the control test of different treatments of strain CZ-56 on tobacco target spot pathogen inoculated on detached leaves are shown in Table 3. In the prevention test, the long diameter of the lesions in the fermentation liquid of the strain CZ-56 treatment group was 1.600±0.255cm, which was larger than the 1.175±0.035cm lesion size treated with 10% jinggangmycin solution, but still significantly smaller than the 3.033±0.255cm lesion size treated with water. In the treatment test, 6.4×10 8 The diameters of the lesions treated with the bacterial suspension with a concentration of cfu / mL, the original fermentation liquid and the fermentation liquid diluted 10 times were 0.324±0.231cm, 0.084±0.061cm and 0.145±0.201cm, which were significantly smaller than the diameter of the lesions treated with the clear water control (2.231±0.246cm), and the effect was better than that of the 10% Jinggangmycin aqueous solution. This shows that strain CZ-56 has biocontrol potential.

[0115] Table 3 The preventive and therapeutic effects of different treatments on tobacco target spot pathogens

[0116]

[0117] Note: The data in the table are mean ± standard error. Different lowercase letters after the data in the same column indicate significant differences (P<0.05).

[0118] 2.3.2 Determination of the efficacy of CZ-56 on indoor potted plants

[0119] The results of the indoor potted plant protection test are shown in Table 4 and Figure 5 As shown, the treatments from left to right are control group, 10% validamycin aqueous solution, 6.4×10 8 cfu / mL bacterial suspension, fermentation stock solution, fermentation diluted 10 times, and fermentation diluted 50 times. In the prevention test, the four treatment groups of antagonistic bacteria CZ-56 and the control group of Jinggangmycin were sprayed for 24 hours before inoculation of tobacco target spot disease. The disease index of the water treatment was 66.30±9.26, and the disease index of the fermentation stock solution treatment was 17.52±2.24, and the relative prevention efficiency reached 72.58±7.21%. Although it is lower than the prevention efficiency of 96.36±1.36% of Jinggangmycin, this treatment can have a good preventive effect on tobacco target spot disease. In the treatment test, tobacco target spot pathogens were first inoculated and then four treatments of CZ-56 were carried out. The results showed that the relative prevention efficiencies of the fermentation stock solution and the 10-fold diluted solution treatment were 83.06±3.06% and 78.12±3.12%, respectively, which were significantly higher than the prevention efficiency of 62.74±2.74% of Jinggangmycin. In addition, 6.4×10 8 The bacterial suspension treatment with a concentration of cfu / mL also had a protective effect of 67.92±2.92%. This experiment shows that CZ-56 has significant therapeutic and preventive effects on tobacco target spot pathogens, and the therapeutic effect is more significant than the preventive effect.

[0120] Table 4 The preventive and therapeutic effects of different treatments on target spot disease in living tobacco

[0121]

[0122] Note: The data in the table are mean ± standard error. Different lowercase letters after the data in the same column indicate significant differences (P<0.05)

[0123] 2.4 Determination of the growth-promoting ability of CZ-56 on tobacco seedlings

[0124] The results of the test on the growth promotion effect of CZ-56 on tobacco seedlings are shown in Table 5. The results showed that strain CZ-56 promoted the growth of tobacco seedlings. Compared with the water treatment control, the fermentation liquid and 6.4×10 8 cfu / mL bacterial suspension root irrigation treatment can promote the growth of tobacco seedlings, at the 5% confidence level there is a significant difference such as Figure 6And Table 5, A: clean water; B: 6.4×10 8 cfu / mL bacterial suspension; C: fermentation stock solution; D: fermentation solution diluted 10 times; E: fermentation solution diluted 50 times. 6.4×10 8 cfu / mL bacterial suspension treatment, tobacco stem diameter, plant height, maximum leaf length, maximum leaf width and fresh weight increased by 37.78%, 50.54%, 26.03%, 27.82% and 39.25%, respectively; after fermentation solution treatment, tobacco stem diameter, maximum leaf length, maximum leaf width, fresh weight and dry weight increased by 28.89%, 28.43%, 39.39%, 42.53% and 15.09%, respectively; fermentation solution diluted 10 times and root irrigation increased the dry weight of tobacco by 15.09%.

[0125] Table 5 Growth-promoting effect of antagonistic bacteria CZ-56 on tobacco

[0126]

[0127] Note: The data in the table are mean ± standard error. Different lowercase letters after the data in the same column indicate significant differences (P<0.05).

[0128] 2.5 Determination of disease resistance-related enzyme activities

[0129] After different treatments of CZ-56, the activities of PPO, SOD, PAL and POD resistance enzymes were tested in tobacco plants. The results are as follows: Figure 7-10 As shown in the figure, they are PPO, POD, SOD and PAL enzyme activity determination, T1 represents clean water, T2 represents 6.4×10 8 cfu / mL bacterial suspension, T3 represents CZ-56 fermentation stock solution, T4 represents CZ-56 fermentation liquid diluted 10 times, T5 represents CZ-56 fermentation liquid diluted 50 times, different lowercase letters after the same column of data indicate significant differences. The fermentation stock solution treatment of tobacco seedlings can make the PPO, SOD and PAL enzyme activities reach the highest level, which are 1239.33, 77.67 and 15.00 (ΔOD λ / g·min), which were 348.91%, 242.61% and 400.00% higher than those of the clean water treatment. 8 cfu / mL bacterial suspension treatment of tobacco seedlings can make the POD enzyme activity reach the highest level of 1214.00 (ΔOD λ / g·min), which was 240.16% higher than that of the clean water treatment.

[0130] In the detection of PPO enzyme activity, in addition to the highest enzyme activity after fermentation liquid treatment, the fermentation liquid diluted 10 times treatment also showed significant differences from the control group (P<0.05). In the detection of POD enzyme activity, all four treatments showed significant differences from the water treatment (P<0.05), 6.4×108 cfu / mL bacterial suspension treatment of tobacco seedlings can make the POD enzyme activity the highest. From the results of the fermentation solution to the 50-fold dilution of the fermentation solution, it can be seen that as the fermentation solution is diluted to treat tobacco seedlings, the POD enzyme activity of the treated plants gradually weakens. The POD enzyme activity of the treated plants reaches 1083 (ΔOD λ / g·min), which was 184.25% higher than that of the clean water treatment. In the SOD enzyme activity test, the enzyme activity of the four treatment groups was higher than that of the control group. Among them, the enzyme activity of tobacco seedlings treated with the fermentation liquid was the highest, followed by the fermentation liquid diluted 10 times. In the PAL enzyme activity test, except for the fermentation liquid treatment, 6.4×10 8 The PAL enzyme activity after treatment with cfu / mL bacterial suspension was also higher than that of the control group and had a significant difference (P<0.05). In summary, the treatment of tobacco seedlings with fermentation stock solution can cause a significant increase in the activities of PPO, POD, SOD and PAL resistance enzymes in leaves, among which the activities of PPO, SOD and PAL enzymes reached the highest level.

[0131] Through plate confrontation, it was found that the inhibition rate of the bacteria on tobacco target spot pathogens can reach 83.33%. Through the morphological characteristics, physiological and biochemical characteristics, and multi-gene sequence system evolution analysis of the bacteria, the strain was identified as Pseudomonas mosselii (P. mosselii). Further indoor detached leaf efficacy determination and potted efficacy determination were carried out on tobacco target spot pathogens. The indoor detached leaf efficacy determination showed that the fermentation liquid could have a preventive and therapeutic effect on tobacco leaves, and the potted efficacy determination showed that the fermentation liquid had the highest relative efficacy on tobacco potted plants. Further statistics and determination of growth parameters and resistance-related enzyme activities of tobacco seedlings treated with CZ-56 strains were conducted, and it was found that root irrigation of tobacco seedlings with fermentation liquid could effectively promote plant growth and the accumulation of nutrients, and could also effectively promote the enhancement of PPO, SOD, POD and PAL enzyme activities. In summary, this study screened a biocontrol bacterium with good biocontrol potential, providing an important biocontrol resource for the biological control of tobacco target spot disease. It also provides a reference for the use of Pseudomonas members in biological control and promoting plant disease resistance.

[0132] Pseudomonas is a kind of bacteria with great potential for biological control. They are widely distributed in rhizosphere soil and plants and can produce a variety of antimicrobial substances and a variety of secondary metabolites. Some bacteria of this genus can also promote the absorption of nutrients and the growth of plants by hydrolyzing phosphorus and potassium and secreting plant hormones and ferritin. Studies have reported that biocontrol bacteria can play a biocontrol role by direct antagonism or indirectly activating the induced systemic resistance (ISR) of plants. Plants can stimulate ISR by sensing elicitors secreted by microorganisms (or pathogens), that is, molecular patterns (MAMPs or PAMPs) similar to those associated with microorganisms (or pathogens). MAMPs or PAMPs are recognized by plant pattern recognition receptors associated with cell membranes, thereby triggering plant innate immunity. Pseudomonas can secrete a variety of elicitors to stimulate the production of plant ISR. It is reported that Pseudomonas can control pathogens by stimulating plant ISR through the production of secondary metabolites such as CLPs, Pyochelin (Pch), precursor salicylic acid (SA) and phenazine compound pyocyanin. At present, there are also studies reporting that Pseudomonas can promote the improvement of plant disease resistance: the HrpZ protein encoded by Pseudomonas Syringa can stimulate the allergic response of non-host plants, promote tobacco growth, and improve tobacco resistance to tobacco mosaic virus (TMV) and tobacco late blight. The above research background shows that some Pseudomonas have biocontrol potential, growth-promoting functions, and inducing plant disease resistance, and have important application value. The biocontrol strain CZ-56 discovered in this study has a good antagonistic effect and biocontrol potential against tobacco target spot pathogens, and has the function of promoting tobacco seedling growth and improving resistance enzyme activity.

[0133] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention, rather than to limit it. Although the present invention has been described in detail with reference to the aforementioned embodiments, those skilled in the art should understand that they can still modify the technical solutions described in the aforementioned embodiments, or make equivalent replacements for some of the technical features therein. However, these modifications or replacements do not deviate the essence of the corresponding technical solutions from the spirit and scope of the technical solutions of the embodiments of the present invention.

Claims

1. A Pseudomonas mosselii CZ-56 antagonistic to tobacco target spot disease, which is deposited in China Center for Type Culture Collection with a deposit number of CCTCC NO: M2022859.

2. A bacterial agent, characterized in that The bacterial agent comprises the Pseudomonas mosselii CZ-56 as described in claim 1.

3. A composition, characterized in that The composition comprises the Pseudomonas mosselii CZ-56 as claimed in claim 1.

4. Use of at least one of the Pseudomonas aeruginosa according to claim 1, the bacterial agent according to claim 2, and the composition according to claim 3 for preventing and controlling target spot disease.

5. The use according to claim 4, characterized in that: The target spot disease is tobacco target spot disease.

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