A strain of Pseudomonas aeruginosa NEAU-L06 and its application in the control of blueberry leaf spot disease

Abstract

The present invention relates to the field of microbial technology, and in particular to a strain of Pseudomonas aeruginosa NEAU-L06 and its application in the prevention and treatment of blue indigo leaf spot disease. Pseudomonas aeruginosa NEAU-L06 of the present invention is deposited in the General Microbiology Center of the China Culture Collection Administration Committee, with the address being the Institute of Microbiology, Chinese Academy of Sciences, No. 3, Yard 1, Beichen West Road, Chaoyang District, Beijing, with a deposit date of October 23, 2024, and a deposit number of CGMCC No. 32310. The strain of the present invention has a wide antibacterial spectrum and has antibacterial activity against a variety of pathogenic fungi that harm blue indigo leaf spot disease in blue indigo production, which is of great significance to the green prevention and control of blue indigo diseases.

Description

Technical Field

[0001] The present invention relates to the technical field of microorganisms, and in particular to a Pseudomonas aeruginosa NEAU-L06 strain and application thereof in preventing and treating blueberry leaf spot disease. Background Art

[0002] Plant diseases can reduce crop quality and yield. With climate change and the increasing homogenization of crop varieties, plant diseases are becoming increasingly common and more difficult to control. While chemical control is a common method, its side effects, such as soil and water pollution and the killing of beneficial insects, are becoming increasingly prominent.

[0003] In recent years, with my country's emphasis on green agriculture and sustainable development strategies, biological control has experienced rapid development. Biological control is environmentally friendly, with minimal impact on the environment and ecosystems, contributing to ecological balance and biodiversity maintenance. It specifically targets pathogens, exhibits sustained effects, and contributes to long-term disease control. Biological control is gaining increasing attention due to its safety, cost-effectiveness, and high efficiency.

[0004] Blue indigo fruit, scientific name blue fruit honeysuckle ( Honeysuckle blue L. Lonicera edulis is a deciduous shrub crop of the genus Lonicera in the family Caprifoliaceae, widely distributed in countries such as China, Japan, Russia, and Poland. In Northeast China, indigo berries have become an important cash crop, generating significant economic benefits. However, leaf spot disease, a major disease of indigo berries, causes leaf wilting, reduces berry yields, and results in significant economic losses. Existing technologies have not identified any bacterial agents that can control leaf spot disease in indigo berries. Therefore, a bacterial agent for controlling leaf spot disease in indigo berries is needed to address the shortcomings of existing technologies. Summary of the Invention

[0005] The present invention aims to provide a Pseudomonas aeruginosa NEAU-L06 and its application in preventing and treating Lonicera edulis leaf spot disease.

[0006] In order to achieve the above-mentioned object of the invention, the present invention provides the following technical solutions:

[0007] The present invention provides a strain of Pseudomonas aeruginosa ( Pseudomonas aeruginosa )NEAU-L06, deposited in the General Microbiology Center of China Culture Collection Administration, located at 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.

[0008] The present invention also provides the Pseudomonas aeruginosa ( Pseudomonas aeruginosa ) Application of NEAU-L06 in the preparation of products for inhibiting plant pathogens.

[0009] Preferably, the plant pathogenic bacteria include Alternaria tenuissima ( Alternaria tenuissima ), Fusarium asiatica ( Fusarium asiatica ), Escherichia coli ( Epicoccum sorghum ), Rose pseudodiscus hirsutus ( Neopestalotiopsis rosae ) and Pseudomonas cucurbitae ( Stagonosporopsis cucurbitaceae )

[0010] The present invention also provides the Pseudomonas aeruginosa ( Pseudomonas aeruginosa ) Use of NEAU-L06 in the preparation of enzyme-producing products;

[0011] The enzymes include one or more of amylase, cellulase and protease.

[0012] The present invention also provides a microbial agent, comprising the Pseudomonas aeruginosa ( Pseudomonas aeruginosa ) NEAU-L06 and its bacterial suspension or fermentation broth.

[0013] The present invention also provides the use of the microbial agent in preparing products that inhibit plant pathogens;

[0014] The plant pathogens include Alternaria tenuissima ( Alternaria tenuissima ), Fusarium asiatica ( Fusarium asiatica ), Escherichia coli ( Epicoccum sorghum ), Rose pseudodiscus hirsutus ( Neopestalotiopsis rosae ) and Pseudomonas cucurbitae ( Stagonosporopsis cucurbitaceae )

[0015] The present invention also provides the use of the microbial agent in the preparation of enzyme-producing products;

[0016] The enzymes include one or more of amylase, cellulase and protease.

[0017] The present invention also provides a biocontrol agent for preventing and treating Alternaria edulis leaf spot disease, comprising the Pseudomonas aeruginosa ( Pseudomonas aeruginosa ) NEAU-L06 and its bacterial suspension or fermentation broth.

[0018] The present invention also provides a method for preventing and treating Alternaria caerulea leaf spot disease of Lonicera edulis, comprising the following steps:

[0019] The biocontrol agent is sprayed on the blue loquat fruit and / or irrigated to its roots.

[0020] The present invention provides a strain of Pseudomonas aeruginosa NEAU-L06 and its application in preventing and treating leaf spot disease of blue honeysuckle. The present invention isolates a strain of Pseudomonas aeruginosa (NEAU-L06) from the rhizosphere soil of blue honeysuckle plants. Pseudomonas aeruginosa)NEAU-L06, this bacterium is safe and non-toxic, environmentally friendly, has simple culture conditions, reproduces quickly, and is easy to store and transport; it also has a broad antibacterial spectrum and has antibacterial activity against a variety of pathogenic fungi that harm blue indigo leaf spot in blue indigo production. It is a broad-spectrum microbial agent and fertilizer with good development and application prospects, and is of great significance to the green prevention and control of blue indigo diseases.

[0021] Preservation Instructions

[0022] Pseudomonas aeruginosa ( Pseudomonas aeruginosa )NEAU-L06, deposited in the General Microbiology Center of China Culture Collection Administration, located at 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. BRIEF DESCRIPTION OF THE DRAWINGS

[0023] Figure 1 is the colony morphology of Pseudomonas aeruginosa NEAU-L06;

[0024] Figure 2 is the phylogenetic tree of Pseudomonas aeruginosa NEAU-L06;

[0025] Figure 3 The plate inhibition effect of Pseudomonas aeruginosa NEAU-L06 on the pathogenic fungus Alternaria tenuis;

[0026] Figure 4 This is the control effect of Pseudomonas aeruginosa NEAU-L06 on blue indigo leaf spot in a potted experiment, where A: healthy blue indigo fruit; B: treatment group; C: control group; D: control effect. DETAILED DESCRIPTION

[0027] The solutions provided by the present invention are described in detail below with reference to the embodiments, but they should not be understood as limiting the scope of protection of the present invention.

[0028] The LB solid medium described in the embodiment of the present invention uses deionized water as a solvent and includes the following components at the following concentrations: yeast extract powder 5 g / L, tryptone 10 g / L, sodium chloride 5 g / L, and agar powder 20 g / L. This medium is used to isolate and purify strains;

[0029] The LB liquid culture medium described in the embodiment of the present invention uses deionized water as a solvent and includes the following components at the following concentrations: yeast extract 5 g / L, tryptone 10 g / L, and sodium chloride 5 g / L. This culture medium is used for the propagation of strains.

[0030] The PDA solid culture medium described in the embodiment of the present invention uses deionized water as a solvent and includes the following components at the following concentrations: 200 g / L potato, 20 g / L glucose, and 20 g / L agar powder. This culture medium is used for the activation culture of the strain.

[0031] The gelatin culture medium described in the embodiment of the present invention uses deionized water as a solvent and includes the following components at the following concentrations: 5 g / L sodium chloride, 10 g / L tryptone, 3 g / L beef extract, and 120 g / L gelatin. This culture medium is used for gelatin liquefaction experiments.

[0032] The starch hydrolysis assay agar medium described in the embodiment of the present invention uses deionized water as a solvent and includes the following components at 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 culture medium was used for starch hydrolysis test.

[0033] The skim milk powder plate medium described in the embodiment of the present invention uses deionized water as a solvent and includes the following components at the following concentrations: 2 g / L potassium chloride, 1 g / L magnesium sulfate heptahydrate, 0.48 g / L calcium chloride dihydrate, 0.06 g / L sodium bicarbonate, 0.001 g / L ferric chloride, 40 g / L sodium chloride, 10 g / L skim milk powder, and 15 g / L agar, with a pH of 7.5. This medium is used for protease assays.

[0034] The CMC medium in the embodiment of the present invention uses deionized water as a solvent and includes the following components at the following concentrations: 2.5 g / L dipotassium hydrogen phosphate, 2.5 g / L disodium hydrogen phosphate, 20.0 g / L sodium carboxymethyl cellulose, 2.0 g / L peptone, 0.5 g / L yeast extract powder, and 20 g / L agar, and is sterilized by autoclaving at 121°C for 15 min.

[0035] The above-mentioned culture media are all sterile culture media. LB solid culture medium and LB liquid culture medium are culture media obtained by sterilizing at 121°C for 20 minutes.

[0036] PDA solid medium, gelatin medium and starch hydrolysis assay agar medium were obtained by sterilizing at 121°C for 20 min.

[0037] The skim milk powder plate culture medium is a culture medium obtained by sterilizing at 115°C for 20 min.

[0038] The Congo red solution described in the embodiment of the present invention is a 0.2 wt% Congo red solution, and the specific preparation method is: 2 g of Congo red powder, 1 L of distilled water, and a small amount of alcohol can be added to help dissolve it evenly.

[0039] The sodium chloride solution described in the embodiment of the present invention is a 1 M sodium chloride solution, and the specific preparation method is: 58.5 g of sodium chloride is mixed with 1 L of distilled water, and stirred evenly to obtain a sodium chloride solution.

[0040] Example 1

[0041] Isolation, identification and preservation of strain NEAU-L06

[0042] 1. Isolation and purification of strain NEAU-L06

[0043] Rhizosphere soil from the 1–10 cm surface of healthy blue loquat plants was collected from the Xiangyang Base of Northeast Agricultural University and stored at 4°C until use. 0.3 g of each soil sample was weighed and added to 30 mL of sterile water. The soil was then incubated at 30°C in a shaker at 180 rpm for 30 min. 30 μL of the sample was then spread onto LB solid medium and incubated in a 30°C incubator for 48 h. After single colonies grew on the plate, individual colonies of varying morphology were selected with a sterile toothpick and streaked onto LB solid medium plates for purification. The samples were then incubated in a 30°C incubator until single colonies of uniform morphology emerged. The strains were then stored in a refrigerator at 4°C until use. The strain NEAU-L06 was isolated.

[0044] 2. Identification of strain NEAU-L06

[0045] 1. Morphological identification

[0046] The morphological characteristics of strain NEAU-L06 were observed. Figure 1 As shown, the results showed that the colonies were round, with rough surfaces and edges, yellow-green in color, and a metallic luster on the surface.

[0047] 2. Physiological and biochemical identification

[0048] With reference to the methods in the "Common Bacterial Systematic Identification Manual" and the "Bergey's Manual of Bacterial Identification," strain NEAU-L06 was subjected to the following physiological and biochemical tests: Gram test, growth temperature test, salt tolerance test, motility test, catalase test, starch hydrolysis test, gelatin liquefaction test, and protease test. The results are shown in Table 1. The strain NEAU-L06 tested positive for gelatin liquefaction, maltose, xylose, fructose, sucrose, glucose, lactose, mannitol, galactose, sorbitol, citrate, protease, organophosphate hydrolysis, starch hydrolysis, and cellulose hydrolysis. However, it tested negative for catalase and could not grow normally at 4°C and a salt concentration of 5% to 7%.

[0049] Table 1 Physiological and biochemical identification results of strain NEAU-L06

[0050]

[0051] In Table 1, “+” indicates positive, and “-” indicates negative.

[0052] 3. Molecular biological identification of strain NEAU-L06

[0053] The genomic DNA of strain NEAU-L06 was extracted using a bacterial genome extraction kit, and the 16S rDNA gene sequence of strain NEAU-L06 was amplified by PCR using primers.

[0054] 16S rDNA gene amplification primers: 27F (SEQ ID NO. 1): 5'-AGAGTTTGATCCTGGCTCAG-3'; 1492R (SEQ ID NO. 2): 5'-AAGGAGGTGATCCAGCCGCA-3'.

[0055] PCR amplification system: 25 μL, including 1 μL each of upstream and downstream primers, 1 μL template DNA, 12.5 μL T-Taq Mix, and 9.5 μL sterile water. PCR reaction program: 94°C initial denaturation for 5 min; 35 cycles of 94°C denaturation for 30 s, 55°C annealing for 30 s, and 72°C extension for 90 s; and 72°C extension for 10 min.

[0056] After amplification, 3 μL of the PCR product was aspirated and run on a 1.5% agarose gel for electrophoresis. The PCR product was sequenced at Sangon Biotech (Shanghai) Co., Ltd., and a preliminary BLAST comparison was performed against the NCBI database. Gene sequences relevant for constructing a phylogenetic tree were obtained from the GenBank database. A phylogenetic tree was constructed using the Neighbor-Joining Tree method in MEGA 7.0 software to determine the species relationships of strain NEAU-L06.

[0057] Based on the 16S rDNA gene sequence of strain NEAU-L06 (SEQ ID NO.3), the phylogenetic tree of NEAU-L06 was constructed. Figure 2 As shown, the results showed that strain NEAU-L06 and Pseudomonas aeruginosa ( Pseudomonas aeruginosa )BP C1 are clustered together with a support rate of 100%.

[0058] Based on 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.

[0059]

[0060] 3. Deposit of strain NEAU-L06

[0061] Pseudomonas aeruginosa ( Pseudomonas aeruginosa )NEAU-L06, deposited in the General Microbiology Center of China Culture Collection Administration, located at 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.

[0062] Example 2

[0063] Identification of the antibacterial spectrum of strain NEAU-L06

[0064] Alternaria tenuissima Alternaria tenuissima LD-12 was used as the target (pathogen), and the plate confrontation method was used to determine the antibacterial activity of bacteria NEAU-L06.

[0065] Puncture the pathogen colonies with a 5 mm diameter sterile punch and transfer the resulting bacterial cake to the center of a PDA solid plate. Using the cross-hatch method, single NEAU-L06 colonies were spot-inoculated onto the plate at four points 3 cm from the center. A control group was not spot-inoculated. Three replicates were set up and incubated at 28°C. Plates not inoculated with the NEAU-L06 antagonist strain were observed after growth to the edge of the plate. The diameters of the control and treated colonies of the target bacteria were measured and expressed as the inhibition rate. Inhibition rate (%) = [(control colony diameter - treated colony diameter) / (control colony diameter - 5 mm)] × 100%.

[0066] Alternaria tenuissima Alternaria tenuissima The antibacterial effect of LD-12 is as follows Figure 3 The results showed that strain NEAU-L06 was effective against Alternaria tenuissima Alternaria tenuissima The inhibitory effect was strong, with an inhibition rate of 69.32±1.01%. Strain NEAU-L06 was able to inhibit the mycelial growth of the target bacteria.

[0067] Five fungal pathogens of blue loquat leaf spot were selected, and the antifungal spectrum of strain NEAU-L06 was determined using the plate standoff method described above. The five fungal pathogens of blue loquat leaf spot and the antifungal results are shown in Table 2.

[0068] Table 2 Inhibition results of strain NEAU-L06 against five pathogens of blue loquat leaf spot

[0069]

[0070] Data are mean ± standard error.

[0071] The results showed that strain NEAU-L06 could effectively inhibit five fungi, including Alternaria tenuissima, Fusarium asiatica, Pseudomonas rosea, Pseudomonas cucurbitae, and Ephemerococcus sorghum, with inhibition rates of all of them being above 49%.

[0072] Example 3

[0073] Determination of biocontrol factors of strain NEAU-L06

[0074] 1. Detection of cellulase

[0075] A single colony of strain NEAU-L06 was picked and spot-inoculated at the center of CMC medium. After incubation at 28°C for 24 hours, 3 mL of 0.2 wt% Congo red solution was added and stained for 30 minutes. The dye was then discarded, and 5 mL of 1 M sodium chloride solution was added for decolorization for 15 minutes. The digestion zone was observed. Each treatment was repeated three times. The results are shown in Table 1.

[0076] 2. Detection of protease

[0077] A single colony of strain NEAU-L06 was spot-inoculated at the center of a skim milk powder culture medium. The digestion zone was observed after incubation at 28°C for 24 hours. Each treatment was repeated three times. The results are shown in Table 1.

[0078] Example 4

[0079] Determination of the Control Effect of Strain NEAU-L06 on Alternaria caerulea Leaf Spot Disease in Lonicera edulis

[0080] The strain NEAU-L06 was placed in LB liquid medium and cultured until OD 600 =0.8, and obtain bacterial suspension for later use.

[0081] Control group: Sterile water was sprayed on the blue indigo fruit plants until the leaves were covered with water droplets but no water was dripping.

[0082] Treatment group: Dilute the bacterial suspension 100 times with sterile water and spray it on the blue indigo fruit plants until the leaves are covered with the liquid without dripping.

[0083] After 24 hours of treatment, the plants in the treatment group were sprayed with an inoculation concentration of 1×10 8 cfu / mL of Alternaria tenuissima ( Alternaria tenuissima) LD-12 suspension. Moisturize at 28°C, treat eight plants per treatment, replicate three times, and investigate disease activity 10 days after treatment. Disease classification was performed according to the "Guidelines for Field Efficacy Tests of Pesticides" (Laboratory of the Pesticide Testing Institute, Ministry of Agriculture and Rural Affairs, 1993), and the disease index and control efficacy were calculated. Disease index = ∑(number of diseased leaves at each level × number of disease levels) / (total number of leaves surveyed × highest disease level) × 100. Disease classification criteria: Level 0: No lesions; Level 1: Lesion area less than 5% of the total leaf area; Level 2: Lesion area 6%-25% of the total leaf area; Level 3: Lesion area 26%-50% of the total leaf area; Level 4: Lesion area 51%-75% of the total leaf area; Level 5: Lesion area greater than 75% of the total leaf area.

[0084] Control effect (%) = (control disease index - treatment disease index) / control disease index × 100%.

[0085] The results are as follows Figure 4 shown.

[0086] The results showed that the control effect of strain NEAU-L06 on blue indigo leaf spot reached 78.17%, which was significantly higher than that of the control group.

[0087] As can be seen from the above examples, the present invention provides a strain of Pseudomonas aeruginosa NEAU-L06 and its application in the prevention and treatment of blue honeysuckle leaf spot disease. The present invention isolated a strain of Pseudomonas aeruginosa ( Pseudomonas aeruginosa ) NEAU-L06, this bacterium is safe and non-toxic, environmentally friendly, has simple culture conditions, reproduces quickly, and is easy to store and transport; it also has a broad antibacterial spectrum and has antibacterial activity against a variety of pathogenic fungi that cause blue indigo leaf spot disease in blue indigo fruit production. It is a broad-spectrum microbial agent / fertilizer with good development and application prospects, and is of great significance to the green prevention and control of blue indigo fruit diseases.

[0088] The above is only a preferred embodiment of the present invention. It should be pointed out that for ordinary technicians in this technical field, several improvements and modifications can be made without departing from the principles of the present invention. These improvements and modifications should also be regarded as within the scope of protection 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 Culture Collection Administration, located at 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 ) Use of NEAU-L06 in the preparation of products for inhibiting plant pathogens; The plant pathogens include Alternaria tenuissima ( Alternaria tenuissima ), Fusarium asiatica ( Fusarium asiaticum ), Escherichia coli ( Epicoccum sorghinum ), Rose pseudodiscus hirsutus ( Neopestalotiopsis rosae ) and Pseudomonas cucurbitae ( Stagonosporopsis cucurbitacearum ) 3. 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.

4. Use of the microbial agent according to claim 3 in the preparation of a product for inhibiting plant pathogens; The plant pathogens include Alternaria tenuissima ( Alternaria tenuissima ), Fusarium asiatica ( Fusarium asiaticum ), Escherichia coli ( Epicoccum sorghinum ), Rose pseudodiscus hirsutus ( Neopestalotiopsis rosae ) and Pseudomonas cucurbitae ( Stagonosporopsis cucurbitacearum ) 5. A biocontrol agent for preventing and treating Alternaria leaf spot of Lonicera caerulea, characterized in that: comprising the Pseudomonas aeruginosa according to claim 1 ( Pseudomonas aeruginosa ) NEAU-L06 and its bacterial suspension or fermentation broth.

6. A method for preventing and treating Alternaria caerulea leaf spot of Lonicera edulis, characterized in that: The steps include: The biocontrol agent according to claim 5 is sprayed on blue loquat fruit and / or irrigated to its roots.

Images