Arthrobacter pasenii and application thereof

Abstract

The invention relates to the technical field of prevention and control of microorganisms and nematodes, in particular to arthrobacter pastoris and application thereof. The arthrobacter pastoris disclosed by the invention is classified and named as Arthrobacter pascens, the strain number of the arthrobacter pastoris is YCH-T2, the arthrobacter pastoris is preserved in the China General Microbiological Culture Collection Center on March 19, 2024, and the preservation number of the arthrobacter pastoris is CGMCC No.30072. The strain has strong lethal activity on second-stage larvae of the root-knot nematode, has strong inhibition effect on incubation of eggs of the root-knot nematode, and has important application value in prevention and treatment of root-knot nematode diseases of crops.

Description

Technical Field

[0001] The present invention relates to the technical field of microorganism and nematode control, and in particular to Parsons' arthropod and application thereof. Background Art

[0002] Currently, chemical pesticides are the primary method for controlling root-knot nematode disease in agricultural production due to their rapid effectiveness and economic benefits. However, chemical pesticides are non-specific in their toxicity to nematodes, often harming non-target organisms. Long-term use can also lead to drug resistance in nematodes, causing serious pollution to soil microorganisms, plants, water sources, and the atmosphere, posing a threat to ecological safety and human health. Biological control technologies, which use living organisms or their metabolites to reduce the population density or adverse effects of specific pests, are considered the most promising control methods due to their environmental friendliness, safety, and effectiveness.

[0003] Therefore, exploring microbial resources for controlling nematodes and developing and utilizing biocontrol agents to effectively replace chemical pesticides are of great significance for ensuring the production safety, quality safety and environmental ecological safety of greenhouse vegetables. Summary of the Invention

[0004] In response to the above technical problems, the present invention provides a Parsons' Arthritis Bacterium and its application.

[0005] One of the objects of the present invention is to provide a Parsons' Arthritis Bacillus ( Arthrobacter pascens ), the Parsons' arthropod was deposited in the General Microbiology Center of the China Culture Collection Administration on March 19, 2024, with the deposit number CGMCC No.30072.

[0006] The present invention discovered a Parsons' arthropod YCH-T2, which, compared with other existing strains, has stronger lethal activity against second-instar larvae of root-knot nematodes and a stronger inhibitory effect on the hatching of root-knot nematode eggs, and has important application value for the prevention and control of root-knot nematode disease in crops.

[0007] The second object of the present invention is to provide a bacterial agent prepared from the Parsons' Arthritis Bacterium.

[0008] In some specific embodiments, the bacterial agent is a bacterial suspension or fermentation supernatant of the Parsons' Arthritis Bacterium.

[0009] In some specific embodiments, the bacterial suspension is prepared by: The seed liquid of Parsons' arthropod was inoculated into a fermentation medium, cultured at 36-37°C, with a rotation speed of 180-200 r / min for 24-26 hours, centrifuged, and the supernatant was removed. The solution was prepared with water to obtain a viable bacterial count of 1.0×10 8CFU / mL bacterial suspension.

[0010] In some specific embodiments, the method for preparing the fermentation broth supernatant is: The seed liquid of Parsons' arthropod is inoculated into a fermentation medium, cultured at 36-37° C., at a rotation speed of 180-200 r / min, for 24-26 hours, centrifuged, and the supernatant is collected and filtered for sterilization to obtain a sterile fermentation liquid supernatant.

[0011] The third object of the present invention is to provide the use of the Parsons' Arthritis Bacillus or the bacterial agent in the preparation of products for controlling root-knot nematodes.

[0012] In some embodiments, the controlling comprises killing root-knot nematodes and inhibiting the hatching of root-knot nematode eggs.

[0013] In some embodiments, the root-knot nematode comprises Meloidogyne incognita.

[0014] The present invention has the following beneficial effects: The fermentation broth of Parsons' Arthritis Bacillus YCH-T2 discovered by the present invention has a significant lethal effect on southern root-knot nematodes, with nematode mortality rates of 85.19% and 98.91% after 24 hours and 48 hours of treatment, respectively. The sterile fermentation broth supernatant of Parsons' Arthritis Bacillus YCH-T2 has an egg hatching inhibition rate of 91.11% after 4 days of treatment. The field control effect on southern root-knot nematodes can reach 57%. The application of Parsons' Arthritis Bacillus YCH-T2 in the preparation of products for controlling root-knot nematodes has broad application prospects.

[0015] Additional aspects and advantages of the present invention will be given in the detailed description, and in part will become obvious from the following description, or may be learned through practice of the present invention.

[0016] Preservation Instructions: Species name: Parsons' arthrocera; Latin name: Arthrobacter pascens ; Strain ID: YCH-T2; Depository: China General Microbiology Culture Collection Center (CGMCC); Address: Institute of Microbiology, Chinese Academy of Sciences, No. 3, Yard 1, Beichen West Road, Chaoyang District, Beijing; Deposit date: March 19, 2024; Deposit number: CGMCC No. 30072. BRIEF DESCRIPTION OF THE DRAWINGS

[0017] Figure 1 The colony morphology (A) and cell morphology (B) of Parsons' arthrocera YCH-T2.

[0018] Figure 2 Phylogenetic tree of Parsons' Arthritis Bacterium YCH-T2 constructed based on 16S rDNA sequences.

[0019] Figure 3 The lethal effect of Parsons' arthropod YCH-T2 on the southern root-knot nematode.

[0020] Figure 4 The results show that Parsons's arthropod YCH-T2 can inhibit the egg hatching of the southern root-knot nematode. DETAILED DESCRIPTION

[0021] The present invention will be described in detail below with reference to the accompanying drawings and specific examples, but they should not be construed as limiting the present invention. Unless otherwise specified, the technical means used in the following examples are conventional means well known to those skilled in the art, and the materials, reagents, etc. used in the following examples, unless otherwise specified, can be obtained from commercial sources.

[0022] Example 1: Screening and identification of strains with nematocidal effects.

[0023] 1 Materials and methods

[0024] 1.1 Test soil.

[0025] Soil was collected from the rhizosphere soil of rapeseed in Dali County, Weinan City, Shaanxi Province. The soil samples were air-dried and passed through a 5 mm sieve before use.

[0026] 1.2 Test culture medium.

[0027] Enrichment medium (g / L): 10 g tryptone, 5 g yeast extract, 10 g sodium chloride, and the balance distilled water.

[0028] Autoclave at 121°C for 20 min. If solid culture medium is required, add 1.5% agar powder.

[0029] 2 Test method.

[0030] (1) Isolation of bacterial strains.

[0031] Take 1 g of soil sample and inoculate it into 100 mL of enrichment medium (100 mL / 250 mL conical flask), shake and culture at 37°C and 160 r / min for 24 h, take 1 mL of the upper suspension and add it into a centrifuge tube containing 9 mL of sterile water, and mix it by pipetting (get 10 -1 dilution solution), and follow the same steps to dilute to 10 -7 , take 10 respectively -5 , 10 -6 , 10 -7Spread 0.2 mL of each dilution onto enrichment medium and incubate at 30°C for 24 h. Select strains with distinct colony characteristics and perform streak separation. Repeat this step three or more times for purification until a single pure strain is obtained.

[0032] (2) Screening of bacterial strains.

[0033] 100 isolated bacterial strains were selected and transferred to 10 mL of enrichment medium. The culture was shaken at 37°C and 180 rpm for 24 hours. The fermentation broth was centrifuged at 8000 rpm for 3 minutes to obtain the supernatant. The supernatant was sterilized by filtration through a 0.22 μm sterile filter to obtain sterile fermentation filtrates of different bacterial strains.

[0034] Add 500 μL of the sterile fermentation filtrate to be tested to each 24-well plate. Add 50 incognita root-knot nematodes to each well and incubate at 28°C for 48 hours, or 50 incognita root-knot nematodes eggs to each well and incubate at 28°C for 4 days. Count the number of nematodes and eggs at the beginning and end of the assay, calculate nematode mortality and egg hatching inhibition rates, and screen for strains with significant inhibitory effects and identify them.

[0035] (2) Identification of bacterial species.

[0036] Morphological identification: Inoculate a single bacterium into the enrichment medium and culture at 37°C for 24 hours before observing the colony morphology.

[0037] Gram staining: Take a clean glass slide and place a drop of sterile distilled water in the center. Use a sterile inoculating loop to pick a small amount of bacterial strain and apply it to the slide. Break up the colony, mix thoroughly with sterile water, and spread it into an even thin layer. After air-drying, quickly pass the slide over a flame three times to fix the bacteria on the slide. Add a drop of crystal violet stain and stain for 2 minutes, then rinse with water. Add a drop of iodine solution and stain for 2 minutes, then rinse with water. Then, add 95% ethanol and rinse with water after 30 seconds. Add safranin and stain for 2 minutes, rinse with water, and air-dry. After air-drying, the slide is ready for microscopic examination.

[0038] Physiological and biochemical characteristics were identified using conventional methods.

[0039] 16S rDNA sequence identification: PCR amplification using bacterial 16S DNA universal primers: 27F (SEQ ID NO. 1): 5′-AGAGTTTGATCMTGGCTCAG-3′.

[0040] 1492R (SEQ ID NO. 2): 5′-GGTTACCTTGTTACGACTT-3′.

[0041] The reaction system consisted of 5 µL of 10× Ex Taq Buffer, 5 µL of dNTP Mixture, 2 µL of upstream primer, 2 µL of downstream primer, 5 µL of template DNA, 1 µL of Ex Taq DNA polymerase, and 30 µL of sterile ddH2O. The PCR protocol was as follows: 94°C for 5 min, 35 cycles of 94°C for 30 s, 57°C for 1 min, and 72°C for 120 s, followed by 10 min at 72°C and storage at 4°C. After completion of the PCR reaction, the resulting products were spotted on a 0.8% agarose gel and electrophoresed at 120 V for 20 min. The gel was visualized using a UV-visible gel imaging system, and the products were then purified.

[0042] The purified samples were sequenced. A phylogenetic tree was constructed based on the 16S rRNA gene of the strains finally sequenced to determine their taxonomic placement. The specific steps for constructing the phylogenetic tree were as follows: 16S rRNA sequences obtained from sequencing were analyzed for homology using the BLAST function on the National Center for Biotechnology Information (NCBI) website. Gene sequences from model strains closely related to the sequenced gene were selected for analysis. The Clustal W function of MEGA7 was used to align the target gene sequence with related sequences. The aligned sequences were then used to construct a phylogenetic tree using the neighbor-joining method in MEGA7 software.

[0043] 3Test results.

[0044] A strain with nematocidal activity against southern root-knot nematodes was isolated from the rhizosphere soil of rapeseed flowers in Dali County, Weinan City, Shaanxi Province. The strain was identified as Parsons spp. ( Arthrobacter pascens ), strain number YCH-T2 ( Figure 1 and Figure 2 , Table 1), a Gram-positive bacterium. The strain was deposited with the General Microbiology Center of the China Culture Collection Administration on March 19, 2024, with the deposit number CGMCC No. 30072.

[0045] Table 1: Physiological and biochemical characteristics of Parsons' arthropod YCH-T2 Note: - indicates negative, + indicates positive.

[0046] 16S rDNA sequence results (1386 bp, as shown in SEQ ID NO. 3):

[0047] Example 2: Control effect of Parsonsella pneumoniae YCH-T2 on root-knot nematodes.

[0048] 1 Method.

[0049] (1) Prepare fermentation broth.

[0050] 1) Preparation of fermentation seed solution: A single colony of Parsons' Arthritis Bacterium YCH-T2 was inoculated into beef extract peptone liquid medium (beef extract 3 g / L, peptone 10 g / L, NaCl 5 g / L, the balance being distilled water, pH 7.2), cultured in a constant temperature shaker at 37°C and 200 r / min for 18 h, and used as fermentation seed liquid for later use.

[0051] 2) Preparation of fermentation medium: Sucrose 20 g / L, peptone 10 g / L, beef extract 3 g / L, NaCl 5 g / L, the balance is distilled water, pH 7.2.

[0052] 3) Fermentation: The fermentation seed liquid was inoculated into the fermentation medium with an inoculation rate of 1%, a fermentation temperature of 37°C, a rotation speed of 200 r / min, and a culture temperature of 37°C for 24 hours to obtain a fermentation liquid. The number of viable bacteria of Parsons' arthropod YCH-T2 in the fermentation liquid was 5.8×10 9 CFU / mL, in line with the implementation standard of agricultural microbial agents GB20287-2006.

[0053] (2) In vitro experiments.

[0054] Fermentation broth of Arthritis Parsons YCH-T2 was centrifuged at 8000 rpm for 3 minutes. The supernatant was collected and sterilized by filtration through a 0.22 μm cell filter to prepare a sterile fermentation supernatant. To a 24-well cell culture plate, 1.5 mL of the sterile fermentation supernatant of Arthritis Parsons YCH-T2 was added, along with 100 second-instar larvae of the southern root-knot nematode J2s. Uninoculated sterile fermentation broth served as a blank control (CK). Three replicates were used for each treatment. The culture was incubated at 28°C, and root-knot nematode mortality was measured after 24 and 48 hours of treatment.

[0055] The supernatant of sterile fermentation broth of Parsons' Arthritis Bacillus YCH-T2 was added to a 24-well cell culture plate, and 100 eggs of the southern root-knot nematode were added to each well. The effect on the egg hatching effect was statistically analyzed.

[0056] (3) Prevention efficacy experiment.

[0057] The field experiment was repeated for two years from August 2023 to November 2024 in a carrot field in Dali County, Weinan City, Shaanxi Province. The fermentation broth of Arthritis Parsons YCH-T2 was centrifuged at 8 rpm / min for 3 min, the supernatant was discarded, and the mixture was prepared with water to 1.0×10 8 A 0.5 CFU / mL bacterial suspension was applied to the roots of carrots in the field. Plain water served as the control (CK). Two rows (35 m x 0.7 m) were placed in each treatment. Application was repeated every 30 days from the time of sowing for three consecutive times. Disease status was investigated by randomly surveying 30 seedlings per row. Disease index and control efficacy were calculated.

[0058] 2 results.

[0059] like Figure 3 The results showed that the fermentation liquid of Parsons' arthropod YCH-T2 had a significant lethal effect on the southern root-knot nematode, with the nematode mortality rates of 85.19% and 98.91% after 24h and 48h treatment, respectively. Figure 3 ).

[0060] like Figure 4 The results showed that the egg hatching inhibition rate of southern root-knot nematode was 91.11% after 4 days of treatment.

[0061] The results of the prevention experiment showed that 35 days after the treatment, the prevention and control effect of the root-knot nematode could reach 57%.

[0062] Table 2: YCH-T2 field efficacy statistics It should be noted that when the claims of the present invention involve numerical ranges, it should be understood that the two endpoints of each numerical range and any numerical value between the two endpoints can be selected. In order to avoid redundancy, the present invention describes preferred embodiments.

[0063] Although the preferred embodiments of the present invention have been described, those skilled in the art may make additional changes and modifications to these embodiments once they have learned the basic creative concept. Therefore, the appended claims are intended to be interpreted as including the preferred embodiments and all changes and modifications that fall within the scope of the present invention.

Claims

1. A Parsons' Arthritis Bacillus for controlling root-knot nematodes ( Arthrobacter pascens ), characterized in that, The Parsons' arthropod was deposited in the General Microbiology Center of the China Culture Collection Administration on March 19, 2024, with the deposit number CGMCC No.30072.

2. A bacterial agent prepared from the Parsons' Arthritis Bacterium according to claim 1.

3. The microbial agent according to claim 2, characterized in that The bacterial agent is the bacterial suspension or fermentation supernatant of the Parsons' Arthritis Bacillus.

4. The microbial agent according to claim 3, characterized in that The preparation method of the bacterial suspension is: The seed liquid of Parsons' arthropod was inoculated into a fermentation medium, cultured at 36-37°C, with a rotation speed of 180-200 r / min for 24-26 hours, centrifuged, and the supernatant was removed. The solution was prepared with water to obtain a viable bacterial count of 1.0×10 8 ~3.0×10 8 CFU / mL bacterial suspension.

5. The microbial agent according to claim 3, characterized in that The preparation method of the bacterial suspension is: The seed liquid of Parsons' arthropod was inoculated into a fermentation medium, cultured at 36-37°C, with a rotation speed of 180-200 r / min for 24-26 hours, centrifuged, and the supernatant was removed. The solution was prepared with water to obtain a viable bacterial count of 1.0×10 8 ~3.0×10 8 CFU / mL bacterial suspension.

6. Use of the Arthrobacterium Parsonii according to claim 1 or the bacterial agent according to any one of claims 2 to 5 in the preparation of a product for controlling root-knot nematodes.

7. The use according to claim 6, characterized in that The control includes killing the root-knot nematodes and inhibiting the hatching of root-knot nematode eggs.

8. The use according to claim 7, characterized in that The root-knot nematodes include the incognita root-knot nematode.

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