Pseudomonas wrinkles resistant to botrytis cinerea, leaf mould and early blight bacteria of tomatoes and application of pseudomonas wrinkles

Abstract

The invention relates to pseudomonas wrinkularis capable of resisting botrytis cinerea, leaf mould and early blight bacteria of tomatoes and application of the pseudomonas wrinkularis. The pseudomonas wrinkularis is pseudomonas wrinkularis YC-2305, and the preservation number of the pseudomonas wrinkularis YC-2305 is CGMCC (China General Microbiological Culture Collection Center) NO.29940; the thalli and the fermentation liquor of the Pseudomonas wrinkularis YC-2305 disclosed by the invention have relatively strong antibacterial activity on various fungal diseases of tomatoes, and the antibacterial effect is lasting and stable, so that the Pseudomonas wrinkularis YC-2305 has a huge potential of developing a novel, efficient and broad-spectrum biocontrol bactericide from the Pseudomonas wrinkularis YC-2305.

Description

Technical Field

[0001] The present invention relates to the technical field of microorganisms, in particular to a Pseudomonas rugosa resistant to tomato gray mold, leaf mold and early blight fungus and an application thereof. Background Art

[0002] Tomatoes are beloved for their nutritious properties and delicious taste, whether eaten raw or cooked. my country is a major tomato producer, with the cultivated area expanding annually. Currently, most tomatoes are grown in greenhouses, where high temperatures and humidity are common, making them susceptible to diseases. Botrytis cinerea, leaf mold, and early blight are three common diseases that damage the fruit, stems, and leaves, causing fruit rot and plant wilting, resulting in economic losses for tomato production. Currently, pesticides are the primary treatment for tomato diseases, but these can easily lead to pesticide resistance, leaving residual pesticides that harm human and animal health and pollute the environment.

[0003] Plant endophytes are widely present within healthy plant tissues and have co-evolved with them over a long period of time. They are ubiquitous in all studied terrestrial and aquatic plants. They are natural components of plant microecologies and maintain a complex microecological relationship with plants. Endophytes can produce a variety of biological effects within plants, participating in plant defenses, promoting rapid growth, and enhancing plant resistance to adversity, disease, and animal damage. Currently, researchers have screened strains capable of antagonizing a variety of tomato diseases through extensive testing, with significant success. Summary of the Invention

[0004] The purpose of the present invention is to provide a Pseudomonas rugosa resistant to tomato gray mold, leaf mold and early blight fungus and application thereof.

[0005] The technical solutions of the present invention are as follows.

[0006] A rugosa Pseudomonas spp. resistant to tomato gray mold, leaf mold, and early blight, wherein the rugosa Pseudomonas spp. is Pseudomonas spp. YC-2305, which is deposited in the General Microbiology Center of the China Culture Collection Administration of Microorganisms, with a deposit address of CGMCC NO. 29940 and a deposit date of March 5, 2024, and is classified as Pseudomonas rugosa Pseudomonas corrugata .

[0007] As a preferred technical solution, the Pseudomonas rugosa YC-2305 strain has a milky white colony when grown on an LB agar plate, which later turns yellow. It is a Gram-negative bacterium with a smooth and bright surface, moist texture, neat edges, opaque, round spots, and a colony diameter of 1 to 2 mm.

[0008] As a preferred technical solution, the 16S rDNA sequence of the Pseudomonas rugosa YC-2305 is shown as SEQ ID NO: 1.

[0009] The present invention also discloses an application of Pseudomonas rugosa that is resistant to tomato gray mold, leaf mold, and early blight. The bacteria and fermentation liquid of the Pseudomonas rugosa YC-2305 are used to inhibit or prevent tomato gray mold and the diseases caused by it; The bacterial bodies and fermentation liquid of the Pseudomonas rugosa YC-2305 are used to inhibit or prevent tomato leaf mold and the diseases caused by it; The bacterial body and fermentation liquid of the Pseudomonas rugosa YC-2305 are used for inhibiting or preventing tomato early blight fungus and diseases caused by it.

[0010] The appearance of a yellow halo around the colony of Pseudomonas rugosa YC-2305 indicates that Pseudomonas rugosa YC-2305 can produce siderophore, which promotes plant growth and has a preventive and therapeutic effect on diseases.

[0011] As a preferred technical solution, the bacterial culture conditions of the rugosa Pseudomonas rugosa YC-2305 are as follows: the activated strain is inoculated at a 1% inoculum into LB medium with an aeration volume of 50 mL to 75 mL / 250 mL, pH 5.5 to 8, 35°C, and constant temperature shaking culture at 150 rpm on a shaker.

[0012] As a preferred technical solution, the pH is 6 to 8; the antibacterial effect of the cultivated Pseudomonas rugosa is more significant.

[0013] Advantages of the present invention: The antibacterial activity of the bacteria and fermentation liquid of the present invention is determined by plate confrontation method and colony growth rate method. The bacteria YC-2305 has a good antagonistic effect on tested tomato gray mold, leaf mold and early blight.

[0014] The bacterial body and fermentation liquid of the rugosa Pseudomonas rugosa YC-2305 of the present invention both have strong antibacterial activity against various tomato fungal diseases, and the antibacterial effect is long-lasting and stable, and has great potential for developing new, efficient and broad-spectrum biocontrol fungicides therefrom. BRIEF DESCRIPTION OF THE DRAWINGS

[0015] Figure 1 This is a colony morphology diagram of Pseudomonas rugosa YC-2305 in Example 1 of the present invention; Figure 2 This is a graph showing the Gram test results of Pseudomonas rugosa YC-2305 in Example 1 of the present invention; Figure 3 This is a phylogenetic tree diagram of the Pseudomonas rugosa YC-2305 strain constructed based on the 16S rDNA gene sequence in Example 1 of the present invention; Figure 4This is a graph showing the results of the resistance of Pseudomonas rugosa YC-2305 to tomato leaf mold in Example 3 of the present invention; Figure 5 This is a graph showing the resistance of Pseudomonas rugosa YC-2305 to Botrytis cinerea in Example 3 of the present invention; Figure 6 This is a graph showing the results of the resistance of Pseudomonas rugosa YC-2305 to tomato early blight fungus in Example 3 of the present invention; Figure 7 This is a graph showing the siderophore production test results of Pseudomonas rugosa YC-2305 in Example 8 of the present invention. DETAILED DESCRIPTION

[0016] The present invention will be further described below with reference to specific embodiments.

[0017] Example 1: Isolation and identification of Pseudomonas rugosa YC-2305 The present invention's rugosa Pseudomonas YC-2305 resistant to various tomato fungal diseases was obtained from Chinese cabbage leaves at the vegetable base of Yantai Academy of Agricultural Sciences in Shandong Province by culture and identification in the laboratory under the following conditions.

[0018] Isolation and purification of YC-2305: A homogenate slurry coating method was used. Twenty pieces of 0.8 cm × 0.8 cm were cut from seedling-stage Chinese cabbage leaves. These leaves were soaked in 75% ethanol for 30 seconds in a laminar flow hood, then immersed in a 2.5% NaClO solution (10% available Cl₂) for 3 minutes. The leaves were then rinsed four times with sterile water. 1 mL of the sterile water from the final rinse was aspirated and smeared onto a plate as a control to verify the thoroughness of surface disinfection. The disinfected Chinese cabbage leaves were placed in a sterile mortar and pestle with a small amount of quartz sand to create a slurry. The resulting slurry was diluted to 1 mL, 2 mL, and 5 mL, respectively, and smeared onto NA solid medium plates. This was repeated three times. After incubation at 37°C for 1–2 days, individual colonies were selected and stored for future use.

[0019] Pseudomonas rugosa YC-2305 was identified based on colony morphology, individual morphological characteristics, physiological and biochemical characteristics, and phylogenetic tree of 16S rDNA gene sequences.

[0020] The individual morphology and colony characteristics of this bacterium are as follows: YC-2305 colonies are milky white, turning yellow later in life. They have a smooth, bright surface, a moist texture, neatly margined, and opaque, rounded spots with a diameter of 1-2 mm. The bacteria are straight or slightly curved, not spiral, lack a stipe or sheath, and do not produce spores. This Gram-negative bacterium requires oxygen and undergoes a strictly respiratory metabolism, using oxygen as the final electron acceptor. Figure 1 The colony morphology, Figure 2 Gram test results.

[0021] 16S rDNA Homology Analysis: PCR amplification of the 16S rDNA gene was performed using genomic DNA from this strain as a template using the universal bacterial primers 27-F / 1492-R. Primer sequences were: 27-F: 5'-AGAGTTTGATCCTGGCTCAG-3', 1492-R: 5'-GGTTACCTTGTTACGACTT-3'. The PCR product was a single band, which was recovered, purified, and sequenced. The 16S rDNA sequence of this strain was 1444 bp in length, and the nucleotide sequence is shown in SEQ ID NO: 1. The 16S rDNA sequence of this strain was submitted to the GenBank database and obtained the sequence number GenBank NO. SUB14112590. Figure 3 This is the phylogenetic tree of Pseudomonas rugosa YC-2305 strain constructed based on the 16S rDNA gene sequence.

[0022] Example 2: Optimal conditions for the cultivation and fermentation of Pseudomonas rugosa YC-2305 (1) Slant culture conditions: beef extract peptone solid medium: beef extract powder 3 g, peptone 10 g, sodium chloride 5 g, agar powder 15 g, water 1000 mL, pH 7.3±0.2. Autoclave at 121°C, 0.1 MPa for 20 min; culture temperature 37°C, culture time 2-4 days.

[0023] (2) Fermentation culture conditions: LB medium: 10 g tryptone, 5 g yeast extract, 10 g sodium chloride, 1000 mL water.

[0024] Adjust the pH of 50 mL of LB liquid culture medium using phosphate buffer to 5.5, 6.0, 6.5, 7.0, and 7.5. Sterilize the medium with steam at 121°C, 0.1 MPa for 20 minutes. After inoculation, incubate the medium in a shaker at 30°C, 150 rpm, and maintain a constant temperature. Count the viable cells using the LB medium plating method to determine the optimal pH for growth of different strains.

[0025] The activated strain was inoculated into 50 mL of LB liquid medium and cultured in a constant temperature incubator at 25°C, 28°C, 30°C, 35°C, and 40°C for 48 h. 1 mL of each treatment was diluted 10 times using the stepwise dilution method. 5 ~ 10 7 Each treatment was repeated three times, and 50 μL LB was taken to spread on the plate. The number of live colonies on the plate was counted after incubation at 30°C for 24 h to 36 h.

[0026] Inoculate the activated strain at a 1% inoculum concentration into 250 mL Erlenmeyer flasks containing 25 mL, 50 mL, 75 mL, 100 mL, and 150 mL of LB liquid medium. Incubate the cultures at 30°C with a shaker at 150 rpm. Count the viable cells using the LB plating method to determine the optimal aeration volume for the strain's growth.

[0027] The number of viable bacteria per milliliter of culture medium (cfu / mL) = the number of colonies on the plate * dilution factor (2000) ———————————— 0.05 mL The results are shown in Table 1.

[0028] Table 1

[0029] Experiments have shown that the optimal culture conditions for Pseudomonas rugosa YC-2305 are to inoculate the activated strain at a 1% inoculum into LB medium with an aeration volume of 50 mL ~ 75 mL / 250 mL, and culture at a pH of 5.5 ~ 6.0, 35°C, and a constant temperature of 150 rpm on a shaker.

[0030] Example 3: Analysis of the antibacterial activity of Pseudomonas rugosa YC-2305 against tomato pathogens Antibacterial validation test against tomato leaf mold: using the plate confrontation method, a 6 mm diameter cake of tomato leaf mold was placed in the middle of a PDA solid culture medium plate, and YC-2305 was inoculated 2.5 cm around it. One bacterial strain was inoculated on each plate, and three points were inoculated symmetrically. 70 μL of YC-2305 solution (concentration of 10 5 cfu / mL), repeated 3 times. Place in a 25°C incubator and culture with no bacteria as a control. When the control grows to the edge of the culture dish, measure the size of the inhibition zone, see Table 2 and Figure 4 .

[0031] Table 2 Size of the inhibition zone of Pseudomonas rugosa YC-2305 against tomato leaf mold

[0032] Antibacterial verification test on tomato early blight: Using the colony growth rate method, take the bacterial solution of Pseudomonas rugosa YC-2305 of the present invention, and add 200 μL of the solution with a concentration of 10 5The bacterial liquid with a concentration of 100 cfu / mL was evenly spread on the sterilized PDA solid culture medium. After the bacteria were slightly dry, a 6 mm diameter indicator bacteria (tomato early blight pathogen, tomato gray mold) cake was placed in the center of the plate. The PDA solid culture medium plate inoculated with only the indicator bacteria (tomato early blight pathogen, tomato gray mold) cake was used as the control. When the indicator bacteria (tomato early blight pathogen, tomato gray mold pathogen) covered the plate, the diameter of the treated colony was measured to determine the antibacterial effect of the antagonistic bacteria YC-2305 on the indicator bacteria (tomato early blight pathogen, tomato gray mold pathogen). The results are shown in Table 3. Figure 5 、 Figure 6 .

[0033] Table 3 Inhibitory effect of Pseudomonas rugosa YC-2305 on tomato early blight and tomato gray mold

[0034] From Table 2, Table 3 and Figure 4 、 Figure 5 、 Figure 6 It can be seen that the size of the inhibition zone of Pseudomonas rugosa YC-2305 against tomato leaf mold is 20.1 mm, and the inhibition rates against tomato early blight and tomato gray mold are 92.16% and 89.17% respectively, indicating that Pseudomonas rugosa YC-2305 has resistance or inhibitory effects on the above pathogens.

[0035] Example 4: Analysis on the adaptability of Pseudomonas rugosa YC-2305 bacterial solution to antibacterial conditions Thermal adaptability analysis: The bacterial suspension of Pseudomonas rugosa YC-2305 was placed at 20°C, 40°C, 60°C, 80°C, and 100°C for 30 min, and then at 121°C for 20 min. Tomato leaf mold, tomato early blight, and tomato gray mold were used as indicator bacteria. The plate confrontation method was used. A 6 mm diameter cake of tomato leaf mold was placed in the middle of a PDA solid culture medium plate. The treated YC-2305 bacterial suspension was inoculated 2.5 cm around it. Two points were symmetrically placed on each plate. 70 μL of the treatment solution (concentration of 10 5 cfu / mL) with three replicates. The samples were incubated in a 25°C incubator, and the antibacterial activity of the antimicrobial substances was determined using an indicator bacteria-only control. The results showed no significant changes in the inhibitory activity against tomato leaf mold, tomato early blight, and tomato gray mold at temperatures between 20°C and 40°C. However, at temperatures above 60°C, bacterial viability and inhibitory activity decreased significantly.

[0036] pH adaptability analysis: The initial pH value of the antibacterial substance extract of Pseudomonas rugosa YC-2305 was determined and used as a control group. The YC-2305 bacterial solution was adjusted to pH values ​​of 2, 4, 6, 8, 10 and 12 with 1 mol / L HCL and NaOH. After 2 hours, the pH value was adjusted back to the initial value. Tomato leaf mold, tomato early blight and tomato gray mold were used as indicator bacteria. The plate confrontation method was used. A 6 mm diameter cake of tomato leaf mold was placed in the middle of a PDA solid culture medium plate. The treated YC-2305 bacterial solution was inoculated 2.5 cm around it. Two points were symmetrically placed on each plate. 70 μL of the treatment solution (concentration of 10 5 cfu / mL) and replicated three times. The cells were incubated in a 25°C incubator, with indicator bacteria alone used as a control. The antibacterial activity of the antimicrobial substances was determined. The results showed that the antibacterial effect remained unchanged within the pH range of 4 to 10. However, bacterial viability decreased significantly under strong acid and alkaline conditions, which had a certain impact on the antibacterial effect.

[0037] Example 5: Experimental study on the control of tomato gray mold by using Pseudomonas rugosa YC-2305 1. Preparation of pathogen inoculum: Wash the collected diseased tomato leaves, cut the border between the diseased and healthy leaves into small pieces, and culture them on a culture medium. For gray mold, use PDA medium. After 2-3 days of culture, isolate and purify single spores. Culture in a constant temperature incubator at 28°C on PDA medium for 14 days. After sporulation, wash the spores with sterile water, filter through double-layer gauze, and obtain a conidia suspension. Dilute the suspension to 5×10 7 / mL for later use.

[0038] 2. Preparation of YC-2305 inoculum: Inoculate the activated strain at a 1% inoculum rate into 50 mL to 75 mL / 250 mL of LB medium with an aeration volume. Incubate at a pH of 5.5, 35°C, and 150 rpm on a shaker. The resulting fermentation broth is the YC-2305 inoculum.

[0039] 3. Experiment on the control of tomato gray mold with YC-2305 bacterial agent: The tomato variety was Yanfen 201. Seeds were sown in plug trays, with 10 seedlings per replicate for a total of three replicates. Treatment was performed when the tomatoes had 4 to 6 leaves.

[0040] Pathogen inoculation treatment: Spray the conidia suspension of Botrytis cinerea on the surface and back of the true leaves of tomatoes using the spray method. Each plant should have approximately 1 mL of conidia suspension, and the conidia concentration should be 5×10 7Moisturize the room for 12 hours before inoculation to ensure that the relative humidity of the air reaches 90% to 100%. After inoculation, the humidity should be maintained above 85% for 8 hours, and then normal management can be performed.

[0041] YC-2305 preventive treatment: spray YC-2305 on the surface and back of tomato leaves, about 1 mL of the agent per plant, with a spore concentration of 10 5 cfu / mL. After the inoculum dried, inoculate the conidia suspension of Botrytis cinerea with approximately 1 mL of conidia per strain, and the conidia concentration of the conidia was 5×10 7 / mL, the humidity was maintained above 85% for 8 h after inoculation, and then normal management was carried out.

[0042] YC-2305 treatment: spray the conidia suspension of tomato gray mold with about 1 mL of conidia suspension per plant, with a spore concentration of 5×10 7 After inoculation, the humidity was kept above 85% for 8 h, and then YC-2305 was sprayed on the surface and back of the true leaves of tomatoes, with about 1 mL of the agent per plant and a spore concentration of 10 5 cfu / mL, and then managed normally.

[0043] Control treatment: Use the spray method to spray LB culture medium on the surface and back of tomato true leaves, about 1 mL per plant. After spraying, the humidity is maintained above 85% for 8 hours, and then normal management is carried out.

[0044] The disease index was investigated 7 days after treatment. Level 0: no lesions; Level 1: the area of ​​lesions accounts for less than 5% of the total leaf area; Level 3: the area of ​​lesions accounts for 6% to 15% of the total leaf area; Level 5: the area of ​​lesions accounts for 16% to 25% of the total leaf area; Level 7: the area of ​​lesions accounts for 26% to 50% of the total leaf area; Level 9: the area of ​​lesions accounts for more than 50% of the total leaf area.

[0045] Disease index = [∑(number of diseased leaves at each level × relative level value) / (total number of leaves surveyed × 9)] × 100 Control effect (%) = (disease index of CK treatment - disease index of YC-2305 treatment) / disease index of CK treatment × 100%.

[0046] Experimental results: The disease investigation results showed that on the 7th day after inoculation with tomato gray mold, the disease occurred to varying degrees in each treatment. The prevention and treatment treatments with the Pseudomonas rugosa YC-2305 agent showed strong disease prevention effects. The disease index of the prevention treatment was 8.9, and the disease index of the treatment treatment was 12.22, both lower than that of the control treatment. The prevention efficiencies were 82.56% and 76.09%, respectively, indicating that prevention was better than treatment (Table 4) and had good application value.

[0047] Table 4 Control effect of Pseudomonas rugosa YC-2305 on tomato gray mold

[0048] Example 6: Experimental study on the control of tomato leaf mold by using Pseudomonas rugosa YC-2305 bacterial agent The tomato variety was Yanfen 201. Seeds were sown in plug trays, with 10 seedlings per replicate for a total of three replicates. Treatment was performed when the tomatoes had 3 to 4 leaves.

[0049] Pathogen inoculation treatment: Spray the tomato leaf mold conidia suspension on the surface and back of the tomato true leaves using the spray method. Each plant should have about 1 mL of conidia suspension, and the conidia concentration should be 5×10 7 Moisturize the room for 24 hours before inoculation to ensure that the relative humidity of the indoor air reaches 90% to 100%. After inoculation, the humidity should be maintained above 85% for 8 hours, and then normal management can be carried out.

[0050] YC-2305 preventive treatment: spray YC-2305 on the surface and back of tomato leaves, about 1 mL of the agent per plant, with a spore concentration of 10 5 cfu / mL. After the inoculum dried, the conidia suspension of tomato leaf mold was inoculated, with approximately 1 mL of bacterial solution per plant and a spore concentration of 5×10 7 / mL, the humidity was maintained above 85% for 8h after inoculation, and then normal management was carried out.

[0051] YC-2305 treatment: Spray the tomato leaf mold conidia suspension with about 1 mL of the suspension per plant. The spore concentration of the suspension is 5×10 7 After inoculation, the humidity was kept above 85% for 8 h, and then YC-2305 was sprayed on the surface and back of the true leaves of tomatoes, with about 1 mL of the agent per plant and a spore concentration of 10 5 cfu / mL, and then managed normally.

[0052] The disease index was investigated 14 days after treatment. Level 0: No symptoms; Level 1: Chlorotic to yellow spots appear on the inoculated leaves; Level 3: A thin and sparse layer of mold appears on the spots on the inoculated leaves; Level 5: An obvious layer of mold appears on the spots on the inoculated leaves; Level 7: A dense layer of mold appears on the spots on the inoculated leaves, and the upper leaves are also infected; Level 9: In addition to the dense layer of mold on the spots on the inoculated leaves, the mold layer on the upper leaves is also obvious or the diseased leaves begin to turn yellow.

[0053] Disease index = [∑(number of diseased leaves at each level × relative level value) / (total number of leaves surveyed × highest level value)] × 100 Control effect (%) = (disease index of CK treatment - disease index of YC-2305 treatment) / disease index of CK treatment × 100%.

[0054] Experimental results: The disease investigation results showed that on the 14th day after inoculation with tomato leaf mold, the disease occurred to varying degrees in each treatment. The prevention and treatment treatments of Pseudomonas rugosa YC-2305 showed strong disease prevention effects. The disease index of the prevention treatment was 7.5, and the disease index of the treatment treatment was 11.11, both lower than that of the control treatment. The prevention efficiencies were 86.5% and 80%, respectively, indicating that prevention was better than treatment (Table 5) and had good application value.

[0055] Table 5 Control effect of Pseudomonas rugosa YC-2305 on tomato leaf mold

[0056] Example 7: Experimental study on the control of tomato early blight by using Pseudomonas rugosa YC-2305 bacterial agent The tomato variety was Yanfen 201. Seeds were sown in plug trays, with 10 seedlings per replicate for a total of three replicates. Treatment was performed when the tomatoes reached the 4-leaf stage.

[0057] Pathogen inoculation treatment: Spray the conidia suspension of tomato early blight fungus on the surface and back of the true leaves of tomatoes using the spray method. Each plant should have about 1 mL of conidia suspension, and the conidia concentration should be 5×10 6 Moisturize the room air for 24 hours before inoculation to ensure that the relative humidity reaches 90% to 100%. After inoculation, the humidity should be maintained above 85% for 24 hours, and then normal management can be performed.

[0058] YC-2305 preventive treatment: spray YC-2305 on the surface and back of tomato leaves, about 1 mL of the agent per plant, with a spore concentration of 10 5 cfu / mL. After the inoculum dried, inoculate the conidia suspension of tomato early blight fungus with approximately 1 mL of conidia per plant. The conidia concentration of the conidia was 5×10 6 After inoculation, the humidity was maintained above 85% for 24 hours and then managed normally.

[0059] YC-2305 treatment: Spray the conidia suspension of tomato early blight fungus with 1 mL of conidia suspension per plant at a spore concentration of 5 × 10 6 After inoculation, the humidity was kept above 85% for 24 h, and then YC-2305 was sprayed on the surface and back of the true leaves of tomatoes, with about 1 mL of the agent per plant and a spore concentration of 10 5 cfu / mL, and then managed normally.

[0060] The disease index was investigated 7 days after treatment. Level 0: No lesions on leaves; Level 1: The area of ​​lesions occupies less than 1 / 4 of the pinnate leaves; Level 2: The area of ​​lesions occupies 1 / 4 to 1 / 2 of the pinnate leaves; Level 3: The area of ​​lesions occupies 1 / 2 to 3 / 4 of the pinnate leaves, and nearly half of the leaflets are dead; Level 4: The area of ​​lesions occupies more than 3 / 4 of the pinnate leaves, and more than half or all of the leaflets are dead.

[0061] Disease index = [∑(number of diseased leaves at each level × relative level value) / (total number of leaves surveyed × highest level value)] × 100 Control effect (%) = (disease index of CK treatment - disease index of YC-2305 treatment) / disease index of CK treatment × 100%.

[0062] Experimental results: The disease investigation results showed that on the 7th day after inoculation with tomato early blight, the disease occurred to varying degrees in all treatments. The prevention and treatment treatments with the Pseudomonas rugosa YC-2305 agent showed strong disease prevention effects. The disease index of the prevention treatment was 12.5, and the disease index of the treatment treatment was 18.75, both lower than that of the control treatment. The prevention efficiencies were 75% and 62.5%, respectively, indicating that prevention was more effective than treatment (Table 6) and had good application value.

[0063] Table 6 Control effect of Pseudomonas rugosa YC-2305 on tomato early blight

[0064] Example 8: Siderophore production ability of Pseudomonas rugosa YC-2305 Siderophore detection is performed using a culture medium (CAS medium). The main components of CAS medium are: chrome azurol S (CAS), hexadecyltrimethylammonium bromide (HDTMA), ferric chloride hexahydrate, sodium dihydrogen phosphate dihydrate, disodium hydrogen phosphate dodecahydrate, ammonium chloride, potassium dihydrogen phosphate, sodium chloride, and agar. The pH is 6.8 ± 0.1. Plates are used to form the siderophore detection plates. Activated Pseudomonas rugosa YC-2305 bacteria are inoculated onto the siderophore detection plates and incubated at 28°C for 72 hours. Colonies are observed for the formation of a yellow halo around them. The presence of a yellow halo around a colony indicates siderophore production due to the chelation of iron ions by EDTA in the siderophore competition medium.

[0065] The results showed that a yellow halo appeared around the colony of Pseudomonas rugosa YC-2305. Pseudomonas rugosa YC-2305 can produce siderophores, which can promote plant growth and prevent diseases ( Figure 7 ).

[0066] The above shows and describes the basic principles, main features, and advantages of the present invention. Those skilled in the art should understand that the present invention is not limited to the above embodiments. The above embodiments and descriptions are merely illustrative of the principles of the present invention. Various changes and modifications may be made to the present invention without departing from the spirit and scope of the present invention. Such changes and modifications are intended to fall within the scope of protection of the present invention. The scope of protection of the present invention is defined by the appended claims and their equivalents.

Claims

1. A Pseudomonas rugosa resistant to tomato gray mold, leaf mold, and early blight fungus, characterized in that: The rugosa Pseudomonas is rugosa Pseudomonas YC-2305, which is deposited in the General Microbiological Center of the China Microbiological Culture Collection Administration, with a deposit address of CGMCC NO.29940 in Beijing, China, a deposit date of March 5, 2024, and a classification name of Pseudomonas rugosa Pseudomonas corrugata .

2. The Pseudomonas rugosa resistant to tomato gray mold, leaf mold, and early blight fungus according to claim 1, characterized in that: When the strain of Pseudomonas rugosa YC-2305 grows on LB agar plates, the colonies are milky white and turn yellow later. It is a Gram-negative bacterium with a smooth and bright surface, moist texture, neat edges, opaque, round spots, and a colony diameter of 1 to 2 mm.

3. The Pseudomonas rugosa resistant to tomato gray mold, leaf mold, and early blight fungus according to claim 1, characterized in that: The 16S rDNA sequence of the Pseudomonas rugosa YC-2305 is shown in SEQ ID NO:

1.

4. A use of Pseudomonas rugosa against tomato gray mold, leaf mold and early blight as claimed in any one of claims 1 to 3, characterized in that: The bacterial body and fermentation liquid of the Pseudomonas rugosa YC-2305 are used to inhibit or prevent tomato gray mold and the diseases caused by it; The bacteria and fermentation liquid of Pseudomonas rugosa YC-2305 are used to inhibit or prevent tomato leaf mold and the diseases caused by it; The bacterial body and fermentation liquid of the rugosa Pseudomonas YC-2305 are used for inhibiting or preventing tomato early blight fungus and the diseases caused by it.

5. The use of Pseudomonas rugosa against tomato gray mold, leaf mold and early blight as claimed in claim 4, characterized in that: The bacterial culture conditions of the rugosa Pseudomonas YC-2305 are as follows: the activated strain is inoculated at a 1% inoculum into an LB medium with an aeration volume of 50 mL to 75 mL / 250 mL, pH 5.5 to 8, 35°C, and a constant temperature shaking culture on a shaker at 150 rpm.

6. The use of Pseudomonas rugosa against tomato gray mold, leaf mold and early blight as claimed in claim 5, characterized in that: The pH is 6-8.

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