Corynebacterium glycinophilus T68, which produces nematicidal active compounds, and its applications

Abstract

This invention discloses a strain of Corynebacterium glycinophilus T68 that produces nematicidal active compounds and its applications. In a pot experiment for controlling root-knot nematodes, its control efficacy reached 62.03%. The LC50 activity of the compound phenylacetamide produced by this strain against southern root-knot nematodes was [not specified in the original text]. 50 The concentration was 122.58 μg / ml, demonstrating good control efficacy against root-knot nematodes. This actinomycete can be effectively used to control root-knot nematode disease. It has great application potential in solving root-knot nematode damage in vegetable production.

Description

Technical Field

[0001] This invention belongs to the field of agricultural microbiology and biological control technology, specifically relating to a strain of Corynebacterium glycinophilus T68 that produces nematicidal active compounds and its applications. Background Technology

[0002] Root-knot nematodes (RKNs: Meloidogyne Southern root-knot nematodes (S. spp.) are a group of soil-borne plant parasitic nematodes that cause serious damage, covering more than 5,000 crop species and causing crop yield losses of 10%-75%. Their damage is particularly prominent in greenhouse cultivation systems for cash crops such as cucumbers and tomatoes. In my country, the southern root-knot nematode (S. spp.) is a significant species. Meloidogyne incognita Nematodes are a major pathogenic species in greenhouse vegetable production areas. Their infection can cause root knots in crops, hindering nutrient absorption and transport, and significantly reducing agricultural yield and market quality. While traditional chemical control can quickly suppress nematode populations, long-term application can easily induce nematode resistance, and the residues of highly toxic pesticides can cause environmental risks such as soil ecological imbalance. Biological control, as a core technology of green pest control systems, has attracted widespread attention due to its high environmental compatibility and strong target specificity. Actinomycetes, with their rich secondary metabolic gene clusters, can synthesize structurally diverse active compounds, making them an important reserve for biocontrol resource development. Current research mainly focuses on soil-derived actinomycetes (such as...). Streptomyces griseus However, the study of symbiotic actinomycetes in the unique microecological environment of insect guts has been insufficient. In fact, the long-term co-evolutionary relationship between insect guts and parasitic nematodes may drive the evolution of unique metabolic pathways in symbiotic actinomycetes. The active substances produced by these symbiotic actinomycetes provide potential value for the development of novel nematicides, and have important research value and application prospects.

[0003] Potato tuber moth ( Phthorimaea operculella Lepidoptera, belonging to the order Lepidoptera, are widely distributed in over 100 tropical and subtropical countries. Their gut symbiotic systems, resulting from long-term co-evolution, may contain novel bioactive metabolites. Studies show that actinomycetes are the dominant flora in the gut of Lepidoptera, playing a crucial role in the host's immune defense. Summary of the Invention

[0004] The purpose of this invention is to provide a strain of Corynebacterium glycinophilum T68 that produces nematicidal active compounds and its applications.

[0005] A strain of glycine-loving Corynebacterium ( Corynebacterium glycinophilum The strain, with preservation number CGMCCNo.35590, is named T68.

[0006] The glycemicotrophobic bacteria ( Corynebacterium glycinophilum Application of ) in the prevention and control of root-knot nematode disease.

[0007] Corynebacterium glycinate ( Corynebacterium glycinophilum Inoculate in Erlenmeyer flasks with LB broth, incubate at 25-32℃ with shaking for 5-9 days, and then inoculate into the rhizosphere soil of cucumber seedlings.

[0008] The glycemicotrophobic bacteria ( Corynebacterium glycinophilum Application of nematicidal active compounds.

[0009] The nematicidal active compound is 2-phenylacetamide.

[0010] A microbial pesticide for controlling root-knot nematode disease, comprising the aforementioned *Corynebacterium glycinophilum* (… Corynebacterium glycinophilum ).

[0011] A type of glycine-loving Corynebacterium ( Corynebacterium glycinophilum The fermentation broth was prepared by the *Corynebacterium glycinophilum* (…). Corynebacterium glycinophilum Obtained through fermentation.

[0012] A microbial fungicide for controlling root-knot nematode disease, said fungicide containing the aforementioned *Corynebacterium glycinophilum* (…). Corynebacterium glycinophilum ) and / or its fermentation products.

[0013] The classification name of Corynebacterium glycinophilum T68 in this invention is: Corynebacterium glycinophilum ( Corynebacterium glycinophilum The strain was deposited on August 13, 2025, at the China General Microbiological Culture Collection Center (CGMCC), with proof of viability. The depository address is: Institute of Microbiology, Chinese Academy of Sciences, No. 1, Beichen West Road, Chaoyang District, Beijing, with accession number CGMCC No. 35590.

[0014] The beneficial effects of this invention: The *Corynebacterium glycinophilum* isolated from the intestine of the potato tuber moth (…) Corynebacterium glycinophilum Strain T68, the first strain of this bacterium to be used to control root-knot nematodes, exhibits a high lethality against these nematodes due to its nematicidal product, phenylacetamide. *Corynebacterium glycinophilus* strain T68 is safe and harmless to crops and has a controlling effect on root-knot nematodes, demonstrating safety in application and making it suitable for biological control of root-knot nematodes. The supernatant of the fermentation broth from *Corynebacterium glycinophilus* strain T68 achieved a 100% lethality against second-instar larvae of root-knot nematodes, and a control effect of 62.03% in pot experiments, demonstrating high and stable control efficacy, suitable for agricultural production needs. Attached Figure Description

[0015] Figure 1 Corynebacterium glycinophilum in the embodiments of the present invention ( Corynebacterium glycinophilum Morphological diagram of strain T68 on LB solid medium.

[0016] Figure 2 The image shows the hydrogen nuclear magnetic resonance spectrum of compound FR12-9 in this embodiment of the invention.

[0017] Figure 3 The image shows the carbon NMR spectrum of compound FR12-9 in this embodiment of the invention.

[0018] Figure 4 This is a mass spectrometry data diagram of the negative ion source of compound FR12-9 in the embodiments of the present invention. Detailed Implementation

[0019] To facilitate understanding of the present invention, a more comprehensive description will be given below. However, the present invention can be implemented in many different forms and is not limited to the embodiments described herein. Rather, these embodiments are provided to provide a thorough and complete understanding of the disclosure of the present invention.

[0020] Example 1: Isolation, purification and identification of Corynebacterium glycinophilus T68

[0021] (1) Isolation and purification of strain T68

[0022] After dissecting and obtaining the intestinal tissue of the potato tuber moth, it was ground and purified using the spread dilution method. Single colonies were picked and cultured on LB solid medium. Each liter of LB solid medium contained 10g of tryptone, 5g of yeast extract, 10g of sodium chloride, 15g of agar, and pH 7.2, in preparation for subsequent classification and identification of the strains.

[0023] (2) Identification of T68 strain

[0024] The 16S rRNA sequence of this strain was amplified using primers 27F and 1492R. The PCR amplification reaction volume was 50 μL, containing 25 μL ExTaq enzyme, 1.5 μL 27F, 1.5 μL 1492R, 2 μL DNA template, and 20 μL sterile water. The amplification conditions were: pre-denaturation at 94 °C for 5 min, followed by 30 cycles of denaturation at 94 °C for 30 s, annealing at 45 °C for 30 s, extension at 72 °C for 2.5 min, and a final extension at 72 °C for 10 min. The PCR products were separated and identified by agarose gel electrophoresis, and bidirectional sequencing was performed directly on the PCR products.

[0025] The primer sequence is as follows:

[0026] 27F: 5'-AGAGTTTGATCCTGGCTCAG-3' (SEQ ID NO: 1);

[0027] 1492R: 5'-GGTTACCTTGTTACGACTT-3' (SEQ ID NO: 2).

[0028] Test results: See Figure 1 The T68 strain was obtained on LB agar plates. After incubation at 28°C for 3 days, single colonies were milky white, smooth with regular edges, 0.5-2.0 mm in size, round, smooth, moist, soft in texture, and easily picked up. The bacteria were rod-shaped and Gram-positive. 16S rRNA sequence amplification was performed using primers 27F and 1492R. Sequencing analysis showed that the amplified sequence was 1487 bp long. BLAST analysis of the 16S rRNA sequence (as shown in SEQ ID NO: 3) in the NCBI database showed that the T68 strain was similar to *Corynebacterium glycinophilum* (…). Corynebacterium glycinophilum The similarity reached 100%. The identified strain was Corynebacterium glycinophilum (…). Corynebacterium glycinophilum ).

[0029] Example 2: Nematode-killing effect of the supernatant from the fermentation broth of Corynebacterium glycinophilus T68 strain

[0030] (1) Preparation of supernatant from fermentation broth of strain T68

[0031] The T68 strain was inoculated into a 500 mL Erlenmeyer flask containing 150 mL of LB liquid medium. Each liter of LB liquid medium contained 10 g of tryptone, 5 g of yeast extract, 10 g of sodium chloride, and a pH of 7.2. The flask was then incubated at 28°C with shaking at 180 rpm for 7 days. Subsequently, the bacterial culture was transferred to centrifuge tubes in a clean bench and centrifuged at 12,000 rpm for 5 min to collect the supernatant.

[0032] (2) Preparation of experimental nematodes

[0033] Southern root-knot nematodes were propagated using chili peppers as the primary material. After 35 days of inoculation, egg masses were collected using tweezers. The egg masses were sterilized with 0.5% NaClO and rinsed repeatedly with sterile water until no NaClO residue remained on the surface. The sterilized egg masses were then placed in 20 mL of sterile water in a sterile culture dish and incubated at 28°C in the dark. J2-stage nematodes were collected after 24 hours for subsequent experiments.

[0034] (3) Determination of nematicidal activity of fermentation broth supernatant of strain T68

[0035] The activity assay was performed using 24-well plates, with LB liquid medium as the negative control. Each well contained a 1 ml system (990 μl of the original fermentation broth supernatant + 10 μl of nematodes, or 100 μl of the 10-fold diluted fermentation broth supernatant + 10 μl of nematodes + 890 μl of water), and was performed in triplicate. The 24-well plates were incubated at 28°C in the dark. After 24 hours, the morphology of the nematodes was observed under a microscope. The nematodes were stimulated with 0.5M NaOH; if they remained rigid, they were considered dead. The corrected mortality rate was calculated. The formula for calculating the corrected mortality rate is as follows:

[0036] Corrected mortality rate = [(treatment mortality rate - control mortality rate) ÷ (1 - control mortality rate)] × 100%

[0037] Experimental Results: The results showed that different fermentation broth concentrations had different effects on the killing of root-knot nematodes after 24 hours of treatment. The results indicated that different fermentation broth concentrations had different effects on nematodes. The corrected mortality rate of root-knot nematodes was 100% with the original fermentation broth, and the nematode-killing effect was still 96.8% in the 10-fold dilution. The experiment shows that the fermentation broth of strain T68 has a good effect on the control of root-knot nematodes.

[0038] Example 3: Pot Experiment of Fermentation Broth of Corynebacterium glycinophilus T68

[0039] (1) Preparation for pot experiment

[0040] Zhongnong No. 6 cucumber seeds were germinated for three days in a sterile petri dish lined with sterile, moistened filter paper at a constant temperature of 28°C. After sowing, when the Zhongnong No. 6 cucumber seedlings reached the one-leaf stage, they were transplanted into flowerpots with a volume of 9 cm × 9 cm × 10 cm. After transplanting, three 1 ml blue syringe tips were evenly inserted into each flowerpot. The experiment was conducted when the cucumbers reached the two-leaf stage.

[0041] (2) Pot experiment treatment

[0042] 1 ml of *Corynebacterium glycinophilus* T68 was inoculated into a 500 mL Erlenmeyer flask containing 150 mL of LB liquid medium. After culturing at 28℃ and 180 rpm for 7 days, the bacterial solution was poured into the cucumber roots. In the treatment group, 10 ml of T68 bacterial solution was poured into each pot. The control group received water and LB medium. The positive control concentration was 10 ml of 0.18 mg / ml abamectin. Each treatment consisted of 10 pots, with three independent replicates. After 48 h of treatment, three blue nozzles were removed from each pot, and 200 μl of a nematode suspension containing 100 J2s nematodes was inoculated into each well, for a total of 300 J2s per pot. After inoculation, the small holes left after nozzle removal were covered with substrate soil. Cucumber growth data were recorded after 35 days. The control effect was calculated using the following formula.

[0043] Control efficacy (%) = [(Number of root knots per plant in control group - Number of root knots per plant in treatment group) ÷ Number of root knots per plant in control group] × 100%

[0044] Experimental results: The number of root knots on cucumber roots was significantly reduced after treatment with Corynebacterium glycinophilum T68 strain. The control effect of strain T68 on root-knot nematodes reached 62.03%, indicating that Corynebacterium glycinophilum T68 strain can effectively control cucumber root-knot nematodes and has important value in the prevention and control of root-knot nematode disease in agricultural production.

[0045] Table 1. Number of root knots and control effect of different treatments

[0046]

[0047] Example 3: Isolation, identification, and nematicidal activity of pure Corynebacterium glycinophilus T68 strain.

[0048] (1) Liquid fermentation of strain T68

[0049] The *Corynebacterium glycinophilus* T68 strain, stored at -80℃, was spread onto LB agar plates using the spreader method and incubated at 28℃ for 3 days. Single colonies were then picked and inoculated into LB liquid medium, and cultured on a shaker at 28℃ for 3 days to obtain the T68 strain.

[0050] 1 ml of Corynebacterium glycinophilus T68 was inoculated into a 500 mL Erlenmeyer flask containing 150 mL of LB liquid medium and cultured aseptically in a shaker at 28 °C for 7 days, for a total of 20 L of fermentation culture.

[0051] (2) Extraction of crude extract

[0052] The fermentation culture obtained in step (1) was soaked in an equal volume of ethyl acetate, sonicated for 30 min, and allowed to stand at room temperature to separate into layers. The organic phase was filtered using four layers of filter paper, and the ethyl acetate was recovered by rotary evaporation. The crude extract was dried to obtain 580.74 mg.

[0053] (3) Separation of crude extract and determination of nematicidal activity of components

[0054] Crude extract separation and nematicidal activity assay: The crude extract was separated using a dextran gel column [60cm x 1.8cm, purchased from Newwell Glass Instruments Co., Ltd.; the gel packing material was Sephadex LH-20 dextran gel (purchased from GEIlea thcare Bio Sciences AB, 30g of gel packed in the column], with methanol:dichloromethane (1:1, v / v) as the elution system. After gradient elution, 21 fractions, Fr1 to Fr21, were collected. The nematicidal activity of each fraction was screened using a 96-well plate assay. The experimental system was set as follows: each well had a total reaction volume of 100μl, containing 5μl of the test fraction (initial concentration 20mg / ml, final concentration 1mg / ml) and 95μl of a suspension of second-instar larvae (J2) of *Strombus haemolyticus*; a negative control (methanol) was also included. Each treatment was performed in triplicate. After incubating the 96-well plates at 28°C for 24 hours, the morphology of nematodes was observed under a microscope, and the mortality rate was recorded. The results showed that, compared to other components treated at a concentration of 1 mg / ml, component Fr12 exhibited the best nematicidal activity, with a corrected mortality rate of 100% after 24 hours of treatment. A total of 32.65 mg of the active component Fr12 was collected for compound isolation and preparation.

[0055] (4) Compound preparation

[0056] Fraction Fr12 was purified by semi-preparative HPLC using the following HPLC preparation conditions: phase B was acetonitrile, and phase C was water. Separation was performed using a Kromasil 100-5-C18 reversed-phase semi-preparative column (10 μm, 10 x 250 mm) at a flow rate of 2.0 mL / min with gradient elution: 0–20 min, 20% phase B; 20–21 min, 100% phase B; 21–30 min, 100% phase B. 9 mg of compound Fr12-9 was obtained (retention time tR = 15.88 min).

[0057] (5) Determination of the nematicidal activity of the pure compound and LC 50 Value determination

[0058] The isolated pure compound was prepared into five concentrations: 10 mg / ml, 7 mg / ml, 5 mg / ml, 2.5 mg / ml, and 1.25 mg / ml. 5 μl of sample and 95 μl of nematodes were added to each well of a 96-well plate, and the mixture was subjected to three independent replicates. Regression analysis was performed between the logarithmic values ​​of the compound concentrations and the corresponding corrected mortality rates. The LC50 of the compound was calculated based on the regression curve equation. 50 .

[0059] (6) Structural identification

[0060] The thoroughly dried compound was completely dissolved in 550 μL of deuterated acetone, then transferred to an NMR tube and sealed for testing. 1 H-NMR and 13 The C-NMR nuclear magnetic resonance spectroscopy was performed by the Institute of Microbiology, Chinese Academy of Sciences.

[0061] Experimental results: The compound is a white powdery solid, readily soluble in methanol and acetone. Based on the mass spectrometry, the molecular ion peak of [M-Na]- is 112.9856, suggesting the molecular formula is C8H9NO. Further analysis using 1H NMR (12-12-12-13-14-14-14-15-16 ... 1 H-NMR and carbon spectroscopy (H-NMR) 13 C-NMR analysis was compared with that of the known compound 2-phenylacetamide reported in the literature, and its structural formula was determined as shown in Formula I. Its chemical name is phenylacetamide (C-NMR). Figures 2 - 3 ).

[0062] Its structure is shown below, and detailed NMR data are as follows:

[0063]

[0064] Formula I

[0065] Carbon and proton NMR data of the compound (Methanol-d4):

[0066] 1 H-NMR (600MHz, Methanol-d4)

[0067] δ: 5.02 (5H, m, H-2~6), 2.00 (1H, s, H-7);

[0068] 13 C-NMR (125MHz, Methanol-d4)

[0069] δ: 172.92 (C-8), 136.05 (C-1), 130.31 (C-2, 6), 129.22 (C-3, 5), 127.60 (C-4), 41.43 (C-7).

[0070] The nematicidal activity of different concentrations of the compound after 24 h of treatment is shown in the table. The regression curve is Y = 0.0774x + 28.16, R0. 2 =0.98, LC50 of the compound 50 It is 122.58 μg / ml.

[0071] Table 2. Catechic activity of compounds at different concentrations

[0072]

[0073] In summary, from *Corynebacterium glycinophilum* ( Corynebacterium glycinophilum The natural compound FR12-9 was extracted and isolated. After further purification, the structural characteristics of the compound were identified by high performance liquid chromatography, mass spectrometry, and nuclear magnetic resonance (NMR) techniques, clarifying the LC-linked immunosorbent assay (LC-LC) assay of the compound. 50 The value was 122.58 μg / ml.

[0074] The embodiments described above are merely illustrative of several implementations of the present invention, and while the descriptions are relatively specific and detailed, they should not be construed as limiting the scope of the invention patent. It should be noted that those skilled in the art can make various modifications and improvements without departing from the concept of the present invention, and these all fall within the protection scope of the present invention. Therefore, the protection scope of this invention patent should be determined by the appended claims.

Claims

1. A Corynebacterium ammoniagenes (C. Corynebacterium glycinophilum ) strain, having a preservation number of CGMCC No. 35590 and named as T68.

2. Use of the Corynebacterium g1ycerolipophilum (Corynebacterium sp. ATCC 10145) according to claim 1 for the control of root-knot nematode disease. Corynebacterium glycinophilum ) in the control of root-knot nematode disease.

3. Use according to claim 2, characterized in that, Corynebacterium ammoniagenes Corynebacterium glycinophilum Corynebacterium ammoniagenes was inoculated into a flask of LB culture solution, and cultured at 25-32°C for 5-9 days, and then inoculated into the rhizosphere soil of cucumber seedlings.

4. The use of a Corynebacterium ammoniagenes strain according to claim 1 for the production of a nematicidal active compound, characterized in that Corynebacterium glycinophilum the strain is a Corynebacterium ammoniagenes strain according to claim 1. The nematicidal active compound is 2-phenylacetamide.

5. A microbial pesticide for controlling root-knot nematode disease, characterized by, The coryneform bacterium of claim 1, Corynebacterium glycinophilum ).

6. A microbial fungicide for controlling root-knot nematode disease, characterized by comprising the microorganism according to any one of claims 1 to 5. The bactericide contains the Gordonia amarae of claim 1 Corynebacterium glycinophilum ).

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