A parsonomyces sp. and applications thereof
By using the bacterial agent prepared from the fermentation broth of Bacillus parsniformis YCH-T2, the problems of non-specific toxicity and environmental pollution caused by chemical pesticides for root-knot nematode disease have been solved, achieving highly efficient control of root-knot nematodes.
Patent Information
- Authority / Receiving Office
- CN · China
- Patent Type
- Patents(China)
- Current Assignee / Owner
- SHAANXI INST OF BIOLOGICAL AGRI
- Filing Date
- 2025-05-21
- Publication Date
- 2026-06-02
AI Technical Summary
Chemical pesticides have non-specific toxic effects in controlling root-knot nematode disease, harming non-target organisms, causing environmental pollution and pesticide resistance, and existing biological control methods have limited effectiveness.
A bacterial agent was prepared using Bacillus parsniformis YCH-T2 and its fermentation broth. This agent killed second-instar larvae of root-knot nematodes and inhibited the hatching of their eggs. The resulting bacterial suspension or fermentation broth supernatant was then used to control root-knot nematode disease.
The fermentation broth of Bacillus parsniformis YCH-T2 showed an 85.19% lethality rate and a 91.11% egg hatching inhibition rate against southern root-knot nematodes, with a field control effect of 57%.
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Figure CN120464533B_ABST
Abstract
Description
Technical Field
[0001] This invention relates to the field of microbial and nematode control technology, specifically to a type of *Pasculococcus parsenium* and its applications. Background Technology
[0002] Currently, chemical pesticides are the primary means of controlling root-knot nematode diseases in agricultural production due to their rapid effectiveness and cost-effectiveness. However, the toxic effects of chemical pesticides on nematodes are non-specific, often harming non-target organisms. Furthermore, long-term use can lead to nematode resistance, causing serious pollution to soil microorganisms, plants, water sources, and the atmosphere, which is detrimental to ecological security and human health. Biological control technologies, which rely on living organisms or their metabolites to reduce the population density or adverse effects of specific pests, are environmentally friendly, safe, and highly efficient, and are considered the most promising control methods.
[0003] Therefore, exploring and utilizing microbial resources for nematode control to develop and utilize biocontrol agents as effective alternatives to chemical pesticides is of great significance for ensuring the safety of greenhouse vegetable production, quality, and environmental ecology. Summary of the Invention
[0004] To address the aforementioned technical problems, this invention provides Parseniorhynchus and its applications.
[0005] One objective of this invention is to provide a Parsenella spp. strain for the control of root-knot nematodes. Arthrobacter pascens The *Parsenulobacillus* was deposited on March 19, 2024, at the China General Microbiological Culture Collection Center (CGMCC) with accession number CGMCC No. 30072.
[0006] This invention discovers a strain of *Plasmodium parsniformis* YCH-T2, which, compared to other existing strains, exhibits stronger lethality against second-instar larvae of root-knot nematodes and a stronger inhibitory effect on the hatching of root-knot nematode eggs, making it of significant application value for the prevention and control of root-knot nematode disease in crops.
[0007] A second objective of this invention is to provide a bacterial agent prepared from the aforementioned *Arthrobacter parsenii*.
[0008] In some specific embodiments, the bacterial agent is a bacterial suspension of the Parsenella arthritis or the supernatant of the fermentation broth.
[0009] In some specific embodiments, the method for preparing the bacterial suspension is as follows:
[0010] The seed culture of *Pasculococcus parsniensis* was inoculated into a fermentation medium and cultured at 36–37°C and 180–200 rpm for 24–26 h. After centrifugation, the supernatant was discarded, and the culture was diluted with water to a viable count of 1.0 × 10⁻⁶ cells / mL. 8 CFU / mL bacterial suspension.
[0011] In some specific embodiments, the method for preparing the fermentation broth supernatant is as follows:
[0012] The seed culture of *Pasculocytobacter parsnipae* was inoculated into a fermentation medium and cultured at 36-37°C and 180-200 r / min for 24-26 h. After centrifugation, the supernatant was collected, filtered, and sterilized to obtain a sterile fermentation broth supernatant.
[0013] A third objective of this invention is to provide the application of the *Pasculococcus parseniaceus* or the bacterial agent in the preparation of products for the prevention and control of root-knot nematodes.
[0014] In some specific embodiments, the control includes killing root-knot nematodes and inhibiting the hatching of root-knot nematode eggs.
[0015] In some specific embodiments, the root-knot nematode includes the southern root-knot nematode.
[0016] The present invention has the following beneficial effects:
[0017] The fermentation broth of *Arthrobacter parsniensis* YCH-T2 discovered in this invention has a significant lethal effect on *Strombus parsniensis* (southern root-knot nematode). After 24 hours and 48 hours of treatment, the nematode mortality rates were 85.19% and 98.91%, respectively. Treatment of the supernatant of the sterile fermentation broth of *Arthrobacter parsniensis* YCH-T2 for 4 days inhibited the hatching rate of *Strombus parsniensis* eggs by 91.11%, and the field control effect on *Strombus parsniensis* reached 57%. The application of *Arthrobacter parsniensis* YCH-T2 in the preparation of products for controlling root-knot nematodes has broad application prospects.
[0018] Additional aspects and advantages of the invention will be set forth in the detailed description, and in some respects will be obvious from the description which follows, or may be learned by practice of the invention.
[0019] Preservation instructions:
[0020] Bacterial strain name: Parseniorhynchus;
[0021] Latin name: Arthrobacter pascens ;
[0022] Strain number: YCH-T2;
[0023] Preservation institution: China General Microbiological Culture Collection Center (CGMCC);
[0024] Address: Institute of Microbiology, Chinese Academy of Sciences, No. 3, Courtyard 1, Beichen West Road, Chaoyang District, Beijing;
[0025] Deposit date: March 19, 2024;
[0026] Accession number: CGMCC No. 30072. Attached Figure Description
[0027] Figure 1 The colony morphology (A) and cell morphology (B) of Parseniorhynchus YCH-T2 are shown.
[0028] Figure 2 A phylogenetic tree of Parsenella arthritis YCH-T2 constructed based on the 16S rDNA sequence.
[0029] Figure 3 The lethal effect of Parsenella arthritis YCH-T2 on southern root-knot nematodes.
[0030] Figure 4 The inhibitory effect of Parseniobacterium arthritis YCH-T2 on the hatching of Southern root-knot nematodes eggs. Detailed Implementation
[0031] The present invention will now be described in detail with reference to the accompanying drawings and specific embodiments, but this should not be construed as limiting the invention. Unless otherwise specified, the technical means used in the following embodiments are conventional means well known to those skilled in the art, and the materials, reagents, etc. used in the following embodiments are commercially available unless otherwise specified.
[0032] Example 1: Screening and identification of strains with nematicidal activity.
[0033] 1. Materials and Methods
[0034] 1.1 Test soil.
[0035] Soil samples were collected from the rhizosphere soil of rapeseed flowers in Dali County, Weinan City, Shaanxi Province. The soil samples were air-dried and then passed through a 5 mm sieve for later use.
[0036] 1.2 Test culture medium.
[0037] Enrichment medium (g / L): 10g tryptone, 5g yeast extract, 10g sodium chloride, and the remainder is distilled water.
[0038] Autoclave at 121℃ for 20 minutes. If solid culture medium is required, add 1.5% agar powder.
[0039] 2. Experimental methods.
[0040] (1) Isolation of bacterial strains.
[0041] Take 1 g of soil sample and inoculate it into 100 mL of enrichment medium (100 mL / 250 mL Erlenmeyer flask). Incubate at 37℃ with shaking at 160 rpm for 24 h. Take 1 mL of the supernatant suspension and add it to a centrifuge tube containing 9 mL of sterile water. Mix well by pipetting (to obtain 10 g of soil sample). -1 (Diluent), serially diluted to 10 using the same steps. -7 Take 10 respectively -5 10 -6 10 -7 Spread 0.2 mL of each of the three dilutions onto enrichment medium and incubate upside down at 30°C for 24 h. Select strains with distinctly different colony characteristics for streak isolation. Repeat this step at least three times for purification until pure single bacteria are obtained.
[0042] (2) Screening of strains.
[0043] One hundred isolated bacterial colonies were picked and transferred to 10 mL of enrichment medium, and cultured on a shaker at 37°C and 180 rpm for 24 h. The fermentation broth was centrifuged at 8000 rpm for 3 min to obtain the supernatant, which was then filtered through a 0.22 μm sterile filter to obtain sterile fermentation filtrates for different bacterial strains.
[0044] Add 500 μL of the sterile fermentation filtrate to each well of a 24-well plate, then add 50 Southern Root-knot Nematodes to each well and incubate at 28°C for 48 h; or add 50 Southern Root-knot Nematode eggs to each well and incubate at 28°C for 4 days. Count the number of nematodes and eggs at the beginning and end, calculate the nematode mortality rate and egg hatching inhibition rate, and screen out strains with significant inhibitory effects for identification.
[0045] (2) Identification of strains.
[0046] Morphological identification: Single bacteria were inoculated into enrichment medium and incubated at 37°C for 24 hours before observing colony morphology.
[0047] Gram staining: Take a clean glass slide, place a drop of sterile distilled water in the center of the slide, use a sterile inoculation loop to pick up a small amount of bacterial inoculum and spread it on the slide. Break up the colonies, mix them thoroughly with sterile water, and spread them into a uniform thin layer. After air-drying, quickly pass the slide over a flame three times to fix the bacteria onto the slide. Add a drop of crystal violet staining solution and stain for 2 minutes, then wash with water. Add a drop of iodine solution and stain for 2 minutes, then wash with water. Next, add 95% ethanol and wash with water after 30 seconds. Finally, add safranin and stain for 2 minutes, then wash with water and air-dry. After air-drying, the slide preparation is complete and it is ready for microscopic examination.
[0048] Physiological and biochemical characteristics were identified using conventional methods.
[0049] 16S rDNA sequence identification: PCR amplification used universal primers for bacterial 16S DNA.
[0050] 27F (SEQ ID NO. 1): 5′-AGAGTTTGATCMTGGCTCAG-3′.
[0051] 1492R (SEQ ID NO. 2): 5′-GGTTACCTTGTTACGACTT-3′.
[0052] 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 dd H2O. The PCR reaction program was: 94℃ for 5 min; 94℃ for 30 s; 57℃ for 1 min; 72℃ for 120 s, 35 cycles; 72℃ for 10 min, and storage at 4℃. After the PCR reaction, all the products were spotted onto a 0.8% agarose gel and electrophoresed at a constant voltage of 120 V for 20 min. The electrophoresis results were observed using a UV gel imaging system, and the products were purified.
[0053] The purified samples were sequenced. A phylogenetic tree was constructed based on the 16S rRNA genes of the finally determined strains to determine their position in the taxonomic system. The specific steps for constructing the phylogenetic tree were as follows: Homology alignment analysis of the 16S rRNA sequences obtained from sequencing was performed using the BLAST function on the National Center for Biotechnology Information (NCBI) website. The corresponding gene sequences of type strains closely related to the determined gene sequences were selected, and these sequences were used as analysis objects. The Clustal W function of MEGA7 was used to align the target gene sequence with related sequences. Finally, the phylogenetic tree was constructed using the Neighbor-joining method with MEGA7 software.
[0054] 3. Experimental results.
[0055] A strain of bacteria with nematicidal activity against southern root-knot nematodes was isolated from the rhizosphere soil of rapeseed flowers in Dali County, Weinan City, Shaanxi Province. Identification results, based on bacterial cell morphology, physiological and biochemical tests, and 16S rDNA molecular detection, showed that this strain is *Arthrobacter parsiensis* (…). Arthrobacter pascens ), strain number YCH-T2 ( Figure 1 and Figure 2(Table 1) is a Gram-positive bacterium. This strain was deposited at the China General Microbiological Culture Collection Center (CGMCC) on March 19, 2024, with accession number CGMCC No. 30072.
[0056] Table 1: Physiological and biochemical characteristics of Parseniorhynchus YCH-T2
[0057]
[0058] Note: - indicates negative, + indicates positive.
[0059] 16S rDNA sequence result (1386bp, as shown in SEQ ID NO.3):
[0060]
[0061] Example 2: The control effect of Parseniobacterium arthritis YCH-T2 on root-knot nematodes.
[0062] 1. Method.
[0063] (1) Preparation of fermentation broth.
[0064] 1) Preparation of fermentation seed liquid:
[0065] A single colony of *Plasmodium parsnifolium* YCH-T2 was inoculated into beef extract peptone liquid medium (3 g / L beef extract, 10 g / L peptone, 5 g / L NaCl, the remainder being distilled water, pH 7.2), and cultured in a constant temperature shaker at 37°C and 200 r / min for 18 h to obtain a seed culture for fermentation.
[0066] 2) Preparation of fermentation medium:
[0067] Sucrose 20g / L, peptone 10g / L, beef extract 3g / L, NaCl 5g / L, balance distilled water, pH 7.2.
[0068] 3) Fermentation:
[0069] The fermentation seed culture was inoculated into the fermentation medium at an inoculum size of 1%, fermented at 37℃, and rotated at 200 r / min for 24 h to obtain the fermentation broth. The viable count of *Arthrobacter parsniformis* YCH-T2 in the fermentation broth was detected to be 5.8 × 10⁻⁶. 9 CFU / mL, which meets the implementation standard GB20287-2006 for agricultural microbial agents.
[0070] (2) In vitro experiments.
[0071] The fermentation broth of *Arthrobacter parsniformis* YCH-T2 was centrifuged at 8000 rpm for 3 min, and the supernatant was collected and filtered through a 0.22 μm cell filter to prepare sterile fermentation broth supernatant. 1.5 mL of the sterile fermentation broth supernatant of *Arthrobacter parsniformis* YCH-T2 was added to a 24-well cell culture plate, along with 100 second-instar larvae (J2s) of *Southern Root-knot Nematodes*. Uninoculated sterile fermentation broth served as a blank control (CK). Each treatment was performed in triplicate. The plates were incubated at 28℃, and the mortality rate of root-knot nematodes was recorded at 24 h and 48 h.
[0072] The supernatant of sterile fermentation broth of *Plasmodium parsniformis* YCH-T2 was added to a 24-well cell culture plate, and 100 eggs of *Stromboma septemlobus* were added to each well. The effect on egg hatching efficiency was statistically analyzed.
[0073] (3) Prevention efficacy test.
[0074] The field trial was conducted from August 2023 to November 2024 in a carrot field in Dali County, Weinan City, Shaanxi Province, with two years of replication. The *Pasculococcus parsnii* YCH-T2 fermentation broth was centrifuged at 8 rpm for 3 min, the supernatant was discarded, and the broth was diluted with water to a concentration of 1.0 × 10⁻⁶. 8 CFU / mL bacterial suspension was applied to the roots of carrot fields via root irrigation, with water serving as the control (CK). Two rows (35m × 0.7m) were used for each treatment. The solution was applied every 30 days from sowing time for a total of three applications. Disease incidence was assessed by randomly surveying 30 seedlings per row, and the disease index and control efficacy were calculated.
[0075] 2. Results.
[0076] like Figure 3 The results showed that the fermentation broth of *Pasculococcus parsnifolius* YCH-T2 had a significant lethal effect on southern root-knot nematodes, with mortality rates of 85.19% and 98.91% after 24 and 48 hours of treatment, respectively. Figure 3 ).
[0077] like Figure 4 The results showed that the hatching inhibition rate of southern root-knot nematodes eggs was 91.11% after 4 days of treatment.
[0078] The efficacy test results showed that 35 days after treatment with the fungicide, the control effect on root-knot nematodes could reach 57%.
[0079] Table 2: Field efficacy statistics of YCH-T2
[0080]
[0081] It should be noted that when numerical ranges are mentioned in the claims of this invention, it should be understood that the two endpoints of each numerical range and any value between the two endpoints can be selected. To avoid redundancy, the present invention describes preferred embodiments.
[0082] Although preferred embodiments of the invention have been described, those skilled in the art, upon learning the basic inventive concept, can make other changes and modifications to these embodiments. Therefore, the appended claims are intended to be interpreted as including the preferred embodiments as well as all changes and modifications falling within the scope of the invention.
Claims
1. A Parsenella arthritis strain used to control root-knot nematodes ( Arthrobacter pascens ), characterized in that, The *Parsenulobacillus* was deposited on March 19, 2024, at the China General Microbiological Culture Collection Center (CGMCC) with accession number CGMCC No. 30072.
2. The bacterial agent prepared from the *Arthrobacter parsnitis* as described in claim 1.
3. The microbial agent according to claim 2, characterized in that, The bacterial agent is a bacterial suspension or fermentation broth supernatant of the *Arthrobacter parsenii*.
4. The microbial agent according to claim 3, characterized in that, The method for preparing the bacterial suspension is as follows: The seed culture of *Pasculococcus parsniensis* was inoculated into a fermentation medium and cultured at 36–37°C and 180–200 rpm for 24–26 h. After centrifugation, the supernatant was discarded, and the culture was diluted with water to a viable count of 1.0 × 10⁻⁶ cells / mL. 8 ~3.0×10 8 CFU / mL bacterial suspension.
5. The use of the *Pasciolarioides parsnipae* of claim 1 or the bacterial agent of any one of claims 2 to 4 in the preparation of products for controlling southern root-knot nematodes.
6. The application according to claim 5, characterized in that, The control measures include killing the southern root-knot nematode and inhibiting the hatching of southern root-knot nematode eggs.