Pseudomonas aeruginosa NEAU-L06 and application of pseudomonas aeruginosa NEAU-L06 in prevention and treatment of lonicera caerulea leaf spot disease

Abstract

The invention relates to the technical field of microorganisms, in particular to pseudomonas aeruginosa NEAU-L06 and application of the pseudomonas aeruginosa NEAU-L06 in prevention and treatment of lonicera caerulea leaf spot disease. The pseudomonas aeruginosa NEAU-L06 is preserved in China General Microbiological Culture Collection Center (CGMCC), the address is Institute of Microbiology, Chinese Academy of Sciences, No.3, No.1 Yard, Beichen West Road, Chaoyang District, Beijing, the preservation date is October 23, 2024, and the preservation number is CGMCC No.32310. The pseudomonas aeruginosa NEAU-L06 can be used for preparing the pseudomonas aeruginosa NEAU-L06. The strain disclosed by the invention is wide in antibacterial spectrum, has antibacterial activity on various pathogenic fungi harmful to the lonicera caerulea leaf spot disease in lonicera caerulea production, and has important significance on green prevention and control of lonicera caerulea diseases.

Description

Technical Field

[0001] The present invention relates to the field of microbial technology, and particularly to a Pseudomonas aeruginosa NEAU-L06 and its application in the prevention and control of blue honeysuckle leaf spot disease. Background Art

[0002] The occurrence of plant diseases will reduce the quality and yield of crops. Due to reasons such as climate change and the simplification of crop varieties, the probability of plant diseases is increasing, and the prevention and control difficulty is gradually increasing. Although chemical control is a commonly used method, its side effects such as soil pollution, water pollution, and killing of beneficial insects are becoming increasingly prominent.

[0003] In recent years, with the emphasis on green agriculture and sustainable development strategies in China, biological control has developed rapidly. Biological control has good environmental protection, has little impact on the environment and ecosystem, is conducive to maintaining ecological balance and biodiversity, specifically targets the target pathogen, has a continuous effect, and helps to control diseases in the long term. Due to its advantages such as safety, economy, and high efficiency, biological control has attracted more and more attention.

[0004] Blue honeysuckle, scientifically named Lonicera caerulea L. Lonicera caerulea L. ), is a deciduous shrub crop of the genus Lonicera in the family Caprifoliaceae, widely distributed in countries such as China, Japan, Russia, and Poland. Especially in Northeast China, blue honeysuckle has become an important cash crop, bringing significant economic benefits. However, as the main disease of blue honeysuckle, leaf spot disease causes leaf wilting and reduces the yield of blue honeysuckle, resulting in serious economic losses. No bactericide has been found in the prior art that can be used to prevent and control blue honeysuckle leaf spot disease. Therefore, a bactericide for preventing and controlling blue honeysuckle leaf spot disease is needed to meet the deficiencies of the prior art. Summary of the Invention

[0005] The purpose of the present invention is to provide a Pseudomonas aeruginosa NEAU-L06 and its application in the prevention and control of blue honeysuckle leaf spot disease.

[0006] To achieve the above-mentioned invention purpose, the present invention provides the following technical solutions: The present invention provides a strain of Pseudomonas aeruginosa ( Pseudomonas aeruginosa ), NEAU-L06, which is deposited in the China General Microbiological Culture Collection Center, located at No. 3, Institute of Microbiology, Chinese Academy of Sciences, No. 1 Beichen West Road, Chaoyang District, Beijing. The deposit date is October 23, 2024, and the deposit number is CGMCC No. 32310.

[0007] The present invention also provides the application of the above-mentioned Pseudomonas aeruginosa ( Pseudomonas aeruginosa ), NEAU-L06, in the preparation of a product for inhibiting plant pathogens.

[0008] Preferably, the plant pathogenic bacteria include Alternaria tenuissima( Alternaria tenuissima ), Fusarium asiaticum( Fusarium asiaticum ), Epicoccum sorghinum( Epicoccum sorghinum ), Pestalotiopsis rosae( Neopestalotiopsis rosae ) and Pleiochaeta cucumerina( Stagonosporopsis cucurbitacearum ), or one or more of them.

[0009] The present invention also provides the use of the Pseudomonas aeruginosa( Pseudomonas aeruginosa ) NEAU-L06 in the preparation of enzyme-producing products; The enzymes include one or more of amylase, cellulase and protease.

[0010] The present invention also provides a microbial inoculum, including the Pseudomonas aeruginosa( Pseudomonas aeruginosa ) NEAU-L06 and its bacterial suspension or fermentation broth.

[0011] The present invention also provides the use of the microbial inoculum in the preparation of products for inhibiting plant pathogenic bacteria; The plant pathogenic bacteria include Alternaria tenuissima( Alternaria tenuissima ), Fusarium asiaticum( Fusarium asiaticum ), Epicoccum sorghinum( Epicoccum sorghinum ), Pestalotiopsis rosae( Neopestalotiopsis rosae ) and Pleiochaeta cucumerina( Stagonosporopsis cucurbitacearum ), or one or more of them.

[0012] The present invention also provides the use of the microbial inoculum in the preparation of enzyme-producing products; The enzymes include one or more of amylase, cellulase and protease.

[0013] The present invention also provides a biocontrol inoculum for preventing and controlling Alternaria tenuissima leaf spot of Lonicera caerulea, including the Pseudomonas aeruginosa( Pseudomonas aeruginosa ) NEAU-L06 and its bacterial suspension or fermentation broth.

[0014] The present invention also provides a method for preventing and controlling Alternaria tenuissima leaf spot of Lonicera caerulea, including the following steps: Spraying and / or irrigating the roots of Lonicera caerulea with the biocontrol inoculum.

[0015] The present invention provides a strain of Pseudomonas aeruginosa NEAU-L06 and its application in the prevention and control of Alternaria tenuissima leaf spot of Lonicera caerulea. The present invention isolates a strain of Pseudomonas aeruginosa( Pseudomonas aeruginosa)Pseudomonas aeruginosa NEAU-L06 is a safe and non-toxic bacterium, friendly to the environment, with simple cultivation conditions, fast reproduction speed, easy to preserve and transport; and it has a wide antibacterial spectrum and antibacterial activity against various pathogenic fungi that endanger the leaf spot disease of Lonicera caerulea var. enulis during production. It is a broad-spectrum microbial bactericide and bacterial fertilizer with good development and application prospects and is of great significance for the green prevention and control of Lonicera caerulea var. enulis diseases.

[0016] Depositing Instructions

[0017] Pseudomonas aeruginosa ( Pseudomonas aeruginosa )NEAU-L06 was deposited in the General Microbiological Center of the China Committee for Culture Collection of Microorganisms at No. 3, Institute of Microbiology, Chinese Academy of Sciences, Beichen West Road, Chaoyang District, Beijing 100101, China on October 23, 2024, with the deposit number CGMCC No. 32310. Description of the Drawings

[0018] Figure 1 is the colony morphology diagram of Pseudomonas aeruginosa NEAU-L06; Figure 2 is the phylogenetic tree of Pseudomonas aeruginosa NEAU-L06; Figure 3 is the antibacterial effect of Pseudomonas aeruginosa NEAU-L06 on the pathogenic fungus Alternaria tenuissima on a Petri dish; Figure 4 is the control effect of Pseudomonas aeruginosa NEAU-L06 on the potted plant experiment of Lonicera caerulea var. enulis leaf spot disease, where A: healthy Lonicera caerulea var. enulis; B: treatment group; C: control group; D: control effect. Detailed Embodiments

[0019] The following describes the solutions provided by the present invention in detail with reference to the embodiments, but they should not be construed as limiting the protection scope of the present invention.

[0020] In the embodiments of the present invention, the LB solid medium uses deionized water as a solvent and includes components with the following concentrations: yeast extract powder 5 g / L, tryptone 10 g / L, sodium chloride 5 g / L, agar powder 20 g / L. This medium is used for the isolation and purification of strains; In the embodiments of the present invention, the LB liquid medium uses deionized water as a solvent and includes components with the following concentrations: yeast extract powder 5 g / L, tryptone 10 g / L, sodium chloride 5 g / L. This medium is used for the propagation of strains; In the embodiments of the present invention, the PDA solid medium uses deionized water as a solvent and includes components with the following concentrations: potato 200 g / L, glucose 20 g / L, agar powder 20 g / L. This medium is used for the activation culture of strains; In the embodiment of the present invention, the gelatin medium uses deionized water as a solvent and includes components with the following concentrations: sodium chloride 5 g / L, tryptone 10 g / L, beef extract 3 g / L, gelatin 120 g / L. This medium is used for the gelatin liquefaction test; In the embodiment of the present invention, the starch hydrolysis determination agar medium uses deionized water as a solvent and includes components with the following concentrations: soluble starch 10 g / L, potassium nitrate 1 g / L, dipotassium hydrogen phosphate 0.3 g / L, magnesium carbonate 1 g / L, sodium chloride 0.5 g / L, agar 20 g / L, pH 7.2. This medium is used for the starch hydrolysis test.

[0021] In the embodiment of the present invention, the skim milk powder plate medium uses deionized water as a solvent and includes components with the following concentrations: potassium chloride 2 g / L, magnesium sulfate heptahydrate 1 g / L, calcium chloride dihydrate 0.48 g / L, sodium bicarbonate 0.06 g / L, ferric chloride 0.001 g / L, sodium chloride 40 g / L, skim milk powder 10 g / L, agar 15 g / L, pH 7.5. This medium is used for the protease test.

[0022] In the embodiment of the present invention, the CMC medium uses deionized water as a solvent and includes components with the following concentrations: dipotassium hydrogen phosphate 2.5 g / L, disodium hydrogen phosphate 2.5 g / L, sodium carboxymethyl cellulose 20.0 g / L, peptone 2.0 g / L, yeast extract powder 0.5 g / L, agar 20 g / L, autoclaved at 121 °C for 15 min.

[0023] The above-mentioned media are all sterile media. The LB solid medium and LB liquid medium are the media obtained after sterilization at 121 °C for 20 min.

[0024] The PDA solid medium, gelatin medium and starch hydrolysis determination agar medium are the media obtained after sterilization at 121 °C for 20 min.

[0025] The skim milk powder plate medium is the medium obtained after sterilization at 115 °C for 20 min.

[0026] In the embodiment of the present invention, the Congo red solution is a 0.2 wt% Congo red solution. The specific preparation method is: 2 g of Congo red powder and 1 L of distilled water, and a small amount of alcohol can be added to help dissolve evenly.

[0027] In the embodiment of the present invention, the sodium chloride solution is a 1 M sodium chloride solution. The specific preparation method is: mix 58.5 g of sodium chloride with 1 L of distilled water and stir evenly to obtain the sodium chloride solution.

[0028] Example 1

[0029] Isolation, Identification and Preservation of Strain NEAU-L06

[0030] I. Isolation and Purification of Strain NEAU-L06 Collect the rhizosphere soil on the surface of 1 - 10 cm of healthy blue honeysuckle plants in the Xiangyang Base of Northeast Agricultural University and store it at 4°C for later use. Weigh 0.3 g of each soil sample, add it to 30 mL of sterile water, place it in a constant temperature shaker at 30°C and shake it at 180 rpm for 30 min. Then, pipette 30 μL and spread it on the LB solid medium, and culture it in an incubator at 30°C for 48 h. After single colonies grow on the spread plate, use a sterilized toothpick to pick single colonies with different morphologies and streak them on the LB solid medium plate for purification. Place it in a constant temperature incubator at 30°C until single colonies with consistent morphology grow, and store it in a refrigerator at 4°C for later use. Strain NEAU-L06 was isolated.

[0031] II. Identification of Strain NEAU-L06 1. Morphological Identification Observe the morphological characteristics of strain NEAU-L06. The results are as Figure 1 shown. The results show that the colonies are round, the surface and edge are rough, yellowish-green, and there is a metallic luster on the colony surface.

[0032] 2. Physiological and Biochemical Identification Refer to the methods in "Common Bacterial System Identification Booklet" and "Bergey's Manual of Determinative Bacteriology" to conduct the following physiological and biochemical tests on strain NEAU-L06: Gram test, growth temperature test, salt tolerance test, motility test, catalase test, starch hydrolysis test, gelatin liquefaction test, protease test, etc. The results are shown in Table 1. The results show that strain NEAU-L06 is positive for gelatin liquefaction, maltose, xylose, fructose, sucrose, glucose, lactose, mannitol, galactose, sorbitol, citrate, protease, organophosphorus decomposition, starch hydrolysis, and cellulose hydrolysis reactions. It is negative for the catalase reaction and cannot grow normally at a temperature of 4°C and a salt concentration of 5% - 7%.

[0033] Table 1 Physiological and Biochemical Identification Results of Strain NEAU-L06

[0034] In Table 1, "+" indicates positive and "-" indicates negative.

[0035] 3. Molecular Biology Identification of Strain NEAU-L06 Use a bacterial genomic DNA extraction kit to extract the genomic DNA of strain NEAU-L06, and use primers to PCR amplify the 16S rDNA gene sequence of strain NEAU-L06.

[0036] 16S rDNA gene amplification primers: 27F (SEQ ID NO.1): 5'-AGAGTTTGATCCTGGCTCAG-3'; 1492R (SEQ ID NO.2): 5'-AAGGAGGTGATCCAGCCGCA-3'.

[0037] PCR amplification system: 25 μL, including 1 μL of each upstream primer and downstream primer, 1 μL of template DNA; 12.5 μL of T-Taq Mix; 9.5 μL of sterile water. PCR reaction procedure: Pre-denaturation at 94°C for 5 min; denaturation at 94°C for 30 s, annealing at 55°C for 30 s, extension at 72°C for 90 s, 35 cycles; extension at 72°C for 10 min.

[0038] After amplification, 3 μL of the PCR product was taken for gel electrophoresis on a 1.5% (w / v) agarose gel. The PCR product was sent to Sangon Biotech (Shanghai) Co., Ltd. for sequencing and preliminarily aligned by BLAST in the NCBI database. Gene sequences related to constructing the phylogenetic tree were obtained through the GenBank database. The phylogenetic tree was constructed using the Neighbor-Joining Tree method in MEGA 7.0 software to determine the species relationship of strain NEAU-L06.

[0039] Based on the 16S rDNA gene sequence (SEQ ID NO.3) of strain NEAU-L06, a phylogenetic tree of the NEAU-L06 gene was constructed, and the results are as Figure 2 shown. The results indicate that strain NEAU-L06 clusters together with Pseudomonas aeruginosa ( Pseudomonas aeruginosa ) BP C1 with a bootstrap support rate of 100%.

[0040] Based on the comprehensive observation of the growth characteristics, morphological characteristics, physiological and biochemical tests, and molecular biological identification of strain NEAU-L06, it was determined to be Pseudomonas aeruginosa Pseudomonas aeruginosa.

[0041]

[0042] III. Preservation of Strain NEAU-L06 Pseudomonas aeruginosa ( Pseudomonas aeruginosa ) NEAU-L06 was deposited in the General Microbiology Center of the China Committee for Culture Collection of Microorganisms at the Institute of Microbiology, Chinese Academy of Sciences, No. 3, Yard 1, Beichen West Road, Chaoyang District, Beijing. The deposit date was October 23, 2024, and the deposit number was CGMCC No. 32310.

[0043] Example 2

[0044] Antibacterial Spectrum Identification of Strain NEAU-L06

[0045] Using Alternaria tenuissima Alternaria tenuissima LD-12 as the target (pathogenic bacterium), the antibacterial activity of bacterium NEAU-L06 was determined by the plate confrontation method.

[0046] A sterile punch with a diameter of 5 mm was used to punch holes in the pathogenic bacterium colony, and the bacterial cake was transferred to the center of the PDA solid plate medium. The single colony of NEAU-L06 was spot-inoculated on the plate at 4 points 3 cm away from the center of the plate by the cross method. The control group was not spot-inoculated. The experiment was set with 3 parallels and cultured at 28°C. The investigation was carried out when the plate without the NEAU-L06 antagonistic strain grew to the edge of the plate. The diameter of the control colony and the treated colony of the target bacterium were measured and expressed by the inhibition rate. Inhibition rate (%) = [(control colony diameter - treated colony diameter) / (control colony diameter - 5mm)]×100%.

[0047] The antibacterial effect of strain NEAU-L06 against Alternaria tenuissima Alternaria tenuissima LD-12 is as Figure 3 . The results showed that strain NEAU-L06 had a strong inhibitory effect on Alternaria tenuissima Alternaria tenuissima LD-12, and the inhibition rate was 69.32±1.01%. Strain NEAU-L06 could inhibit the mycelial growth of the target bacterium.

[0048] Five pathogenic fungi of blue honeysuckle leaf spot were selected, and the antibacterial spectrum of strain NEAU-L06 was determined according to the above-mentioned plate confrontation method. The five pathogenic fungi of blue honeysuckle leaf spot and the antibacterial results are shown in Table 2 specifically.

[0049] Table 2 Antibacterial Results of Strain NEAU-L06 against Five Pathogenic Bacteria of Blue Honeysuckle Leaf Spot

[0050] Data are mean ± standard error.

[0051] The results showed that the strain NEAU-L06 could effectively inhibit five fungi, namely Alternaria tenuissima, Fusarium asiaticum, Pestalotiopsis rosae, Septoria cucurbitacearum, and Epicoccum sorghinum, and the inhibition rates were all above 49%.

[0052] Example 3

[0053] Determination of biocontrol factors of strain NEAU-L06

[0054] 1. Detection of cellulase Pick a single colony of strain NEAU-L06 and spot-inoculate it at the center of the CMC medium. After culturing at 28 °C for 24 h, add 3 mL of 0.2 wt% congo red solution, stain for 30 min, pour out the dye, and then add 5 mL of 1 M sodium chloride solution for decolorization for 15 min. Observe the digestion zone. Each treatment was repeated 3 times. The results are shown in Table 1.

[0055] 2. Detection of protease Pick a single colony of strain NEAU-L06 and spot-inoculate it at the center of the skim milk powder medium. Observe the digestion zone after culturing at 28 °C for 24 h. Each treatment was repeated 3 times. The results are shown in Table 1.

[0056] Example 4

[0057] Determination of the control effect of strain NEAU-L06 on Alternaria tenuissima leaf spot of Lonicera caerulea

[0058] Place the strain NEAU-L06 in LB liquid medium and culture until OD 600 = 0.8 to obtain a bacterial suspension for standby.

[0059] Control group: Spray sterile water on the Lonicera caerulea plants until the leaves are covered with water droplets but not dripping.

[0060] Treatment group: Dilute the bacterial suspension 100 times with sterile water and spray it on the Lonicera caerulea plants until the leaves are covered with the liquid medicine but not dripping.

[0061] After 24 h of treatment, each plant in the treatment group was spray-inoculated with Alternaria tenuissima at a concentration of 1×10 8 cfu / mL ( Alternaria tenuissima)Suspension of LD-12. Keep it moist at 28°C, with 8 plants per treatment and 3 replicates. Investigate the disease incidence after 10 days of treatment. Grade the diseases according to the "Guidelines for Field Efficacy Trials of Pesticides" (Bioassay Laboratory, Institute for the Control of Agrochemicals, Ministry of Agriculture and Rural Affairs, 1993), and calculate the disease index and control effect. Disease index = ∑(number of diseased leaves at each level × disease level) / (total number of leaves investigated × highest disease level) × 100. Disease level classification standard: Grade 0: no disease spots; Grade 1: disease spot area accounts for less than 5% of the total leaf area; Grade 2: disease spot area accounts for 6% - 25% of the total leaf area; Grade 3: disease spot area accounts for 26% - 50% of the total leaf area; Grade 4: disease spot area accounts for 51% - 75% of the total leaf area; Grade 5: disease spot area accounts for more than 75% of the total leaf area.

[0062] Control effect (%) = (control disease index - treatment disease index) / control disease index × 100%.

[0063] The results are as Figure 4 shown.

[0064] The results show that the control effect of strain NEAU-L06 on blueberry leaf spot disease reaches 78.17%, which is significantly higher than that of the control group.

[0065] As can be seen from the above embodiments, the present invention provides a Pseudomonas aeruginosa NEAU-L06 and its application in the control of blueberry leaf spot disease. The present invention isolates a Pseudomonas aeruginosa ( Pseudomonas aeruginosa ) NEAU-L06 from the rhizosphere soil of blueberry plants. This bacterium is safe, non-toxic, environmentally friendly, has simple culture conditions, fast reproduction speed, and is easy to store and transport; moreover, it has a wide antibacterial spectrum and has antibacterial activity against a variety of pathogenic fungi that endanger blueberry leaf spot disease in blueberry production. It is a broad-spectrum microbial bactericide / bacterial fertilizer with good development and application prospects and is of great significance for the green prevention and control of blueberry diseases.

[0066] The above are only the preferred embodiments of the present invention. It should be noted that for those of ordinary skill in the art, without departing from the principle of the present invention, several improvements and refinements can be made, and these improvements and refinements should also be regarded as the protection scope of the present invention.

Claims

1. A strain of Pseudomonas aeruginosa ( Pseudomonas aeruginosa ) NEAU-L06, characterized in that, It is deposited in the General Microbiology Center of China Microbiological Culture Collection Administration, located at the Institute of Microbiology, Chinese Academy of Sciences, No. 3, Yard 1, Beichen West Road, Chaoyang District, Beijing. The deposit date is October 23, 2024, and the deposit number is CGMCC No.32310.

2. The Pseudomonas aeruginosa according to claim 1 ( Pseudomonas aeruginosa ) Application of NEAU-L06 in the preparation of products for inhibiting plant pathogens.

3. The use according to claim 2, characterized in that: The plant pathogenic bacteria include Alternaria tenuissima ( Alternaria tenuissima ), Fusarium asiatica ( Fusarium asiaticum ), Echinococcus sorghumus ( Epicoccum sorghinum ), Pseudodiscus roseus ( Neopestalotiopsis rosae ) and Pseudomonas cucurbitae ( Stagonosporopsis cucurbitacearum ) 4. The Pseudomonas aeruginosa according to claim 1 ( Pseudomonas aeruginosa ) Application of NEAU-L06 in the preparation of enzyme-producing products; The enzymes include one or more of amylase, cellulase and protease.

5. A microbial agent, characterized in that: comprising the Pseudomonas aeruginosa according to claim 1 ( Pseudomonas aeruginosa ) NEAU-L06 and its bacterial suspension or fermentation broth.

6. Use of the microbial agent according to claim 5 in the preparation of a product for inhibiting plant pathogens; The plant pathogenic bacteria include Alternaria tenuissima ( Alternaria tenuissima ), Fusarium asiatica ( Fusarium Asiaticum ), Echinococcus sorghumus ( Epicoccum sorghinum ), Pseudodiscus roseus ( Neopestalotiopsis rosae ) and Pseudomonas cucurbitae ( Stagonosporopsis cucurbitacearum ) 7. Use of the microbial agent according to claim 5 in the preparation of enzyme-producing products; The enzymes include one or more of amylase, cellulase and protease.

8. A biocontrol agent for preventing and treating Alternaria edulis leaf spot disease, characterized in that: comprising the Pseudomonas aeruginosa according to claim 1 ( Pseudomonas aeruginosa ) NEAU-L06 and its bacterial suspension or fermentation broth.

9. A method for preventing and treating Alternaria caerulea leaf spot disease of Lonicera edulis, characterized in that: The steps include: The biocontrol agent according to claim 8 is sprayed on blue honeysuckle and / or irrigated to its roots.

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