Use of dimethoxyphenol and its biological preparation in the control of plant pathogenic nematodes

Abstract

The application discloses application of dimethoxyphenol and a biological preparation thereof in plant pathogenic nematode control, and aims to solve the problems of single function, unknown active ingredient, unclear mechanism and unstable quality of existing microbial source preparations. The application accurately identifies 2,3-dimethoxyphenol as an important nematicidal active substance through metabolomics, and realizes stable co-accumulation of 2,3-dimethoxyphenol and a growth-promoting factor, isopentenyladenine, by combining with a specific strain (pink helical polysporal mold NF-06 and bacillus velezensis YB-1652) co-culture process, so that technical effects of clear composition, synergistic disease-preventing and growth-promoting function, controllable quality and environment friendliness are achieved.

Description

Technical Field

[0001] This invention relates to the field of biological control technology, specifically to the application of dimethoxyphenol and its biological agents in the control of plant pathogenic nematodes. Background Technology

[0002] Plant nematode diseases are a significant biological stressor affecting agricultural production, causing substantial yield losses annually. Chemical control is increasingly limited due to environmental residues, ecological risks, and nematode resistance. Meanwhile, modern agriculture demands ever-growing regulation of green and efficient crop growth. Developing novel, highly targeted, and environmentally compatible plant protection products utilizing microbial metabolites has become an important direction for sustainable agricultural development.

[0003] However, the research and development of microbial pesticides and plant growth promoters still face significant limitations. First, existing research largely focuses on the single-function development of individual strains; for example, a single strain might be used only for disease control or growth promotion, resulting in limited functionality that fails to meet the demands of multifunctional products in production. Second, the key material basis driving these biological activities remains unclear. Much research is limited to observing the phenotypic activity of strains or crude extracts, failing to clearly identify and link their core active ingredients. This leads to vague mechanisms of action, unstable effects, and an inability to optimize production processes for specific active substances. Third, the active ingredients in existing biological agents are often unknown and complex mixtures, making it difficult to guarantee standardized production, stable quality control, and consistent efficacy.

[0004] Therefore, there is an urgent need in this field to overcome existing limitations and develop novel biological agents with clearly defined active ingredients, well-defined mechanisms of action, stable and controllable preparation processes, and the ability to simultaneously control diseases and promote plant growth.

[0005] The information disclosed in this background section is intended only to enhance the understanding of the background technology of this disclosure and should not be construed as an admission or in any way implying that the information constitutes prior art known to those skilled in the art. Summary of the Invention

[0006] This invention discloses the application of dimethoxyphenol and its biological agents in the control of plant pathogenic nematodes, aiming to solve the technical problems of existing microbial pesticides and growth promoters, such as single function, unclear active substances, vague mechanism of action, uncontrollable process, and unstable product efficacy.

[0007] The inventor's related research has confirmed that *Polyspora pinkis* (Pink Spiral Polyspora) Clonostachys rosea NF-06 and Bacillus belye ( Bacillus velezensisThe combined application of YB-1652 showed superior synergistic effects compared to single-strain applications in controlling root-knot nematodes and promoting plant growth, demonstrating the potential of this combination in biocontrol applications. Metabolomics analysis revealed metabolic reprogramming in the co-culture system, accumulating various differentially expressed metabolites with potential functional significance. Based on this, this invention further utilized targeted liquid chromatography-tandem mass spectrometry (LC-MS / MS) to qualitatively and quantitatively analyze the metabolites of *Polyspora pulmonarius* and *Bacillus belesiensis*, revealing that the metabolites 2,3-dimethoxyphenol and isopentenyl adenine are the key material basis for nematode control and plant viability promotion, respectively, and elucidating their synthesis and accumulation characteristics in single-culture and co-culture systems.

[0008] The first aspect disclosed in this application relates to the use of 2,3-dimethoxyphenol in the preparation of plant pathogenic nematode control agents.

[0009] In some embodiments of this disclosure, the 2,3-dimethoxyphenol is isolated from the fermentation product of *Polyspora pinkis*, or from the co-culture product of *Polyspora pinkis* and *Bacillus belye*.

[0010] In some embodiments of this disclosure, the plant nematode is Southern Root-knot Nematode, Skribner Short-bodied Nematode, Oat Cyst Nematode, or Philippine Cyst Nematode.

[0011] The second aspect of this application discloses a biological agent for controlling plant nematodes, comprising 2,3-dimethoxyphenol as an active ingredient, wherein the 2,3-dimethoxyphenol is isolated from the fermentation product of *Polyspora pinkis*, or from the co-culture product of *Polyspora pinkis* and *Bacillus belye*.

[0012] The third aspect disclosed in this application provides a compound biological agent that combines nematicide and life-promoting properties, comprising the biological agent and isopentenyl adenine.

[0013] The fourth aspect disclosed in this application provides a method for preparing the aforementioned composite biological agent, wherein *Polyspora pinkis* and *Bacillus belye* are simultaneously inoculated into a culture medium for co-culture to obtain a fermentation product containing both 2,3-dimethoxyphenol and isopentenyl adenine.

[0014] In some embodiments of this disclosure, the *Polyspora pinkis* is *Polyspora pinkis* with accession number CGMCC No. 16262. Clonostachys rosea NF-06; The Bacillus belyssus mentioned is Bacillus belyssus with accession number CGMCC No. 36152 (NF-06); Bacillus velezensis YB-1652.

[0015] In some embodiments of this disclosure, the preparation method includes the following steps: (1) The activated *Polyspora pinkis* and *Bacillus belye* strains were inoculated into the fermentation medium at an inoculation rate of 1% to 5% and co-cultured. (2) The co-culture conditions are: temperature 25~30℃, rotation speed 150~200 rpm, and culture time 40~56 h; (3) After the culture is completed, solid-liquid separation is performed, and the supernatant is collected.

[0016] In some embodiments of this disclosure, the culture medium contains per liter: 12-22 g corn flour, 1-5 g sucrose, 0.2-0.8 g MgSO4, 0.02-0.08 g FeSO4∙7H2O, 0.02-0.08 g ZnSO4∙7H2O, 6-13 g soybean meal, 1-5 g wheat bran, and the remainder is water.

[0017] The fifth aspect of this application discloses a method for controlling plant pathogenic nematodes, which involves applying the biological agent or the compound biological agent through root irrigation.

[0018] One or more technical solutions provided in the embodiments of this application have at least one of the following technical effects or advantages: 1. Clear Mechanism and Definite Components: Through metabolomics and targeted analysis, 2,3-dimethoxyphenol and isopentenyl adenine were identified for the first time in a microbial interaction system as the key material basis for nematicidal action and life-promoting effects, respectively. Their synthesis and accumulation characteristics under co-culture conditions were also elucidated. This elevates product development from a "black box" operation relying on unknown complexes to a rational design stage based on clearly defined active ingredients, laying a solid foundation for elucidating the mechanism of action and quality control, resulting in significant comprehensive agronomic benefits.

[0019] 2. Synergistic Function and Significant Enhancement: The two active ingredients target two core aspects—pests and diseases (nematodes) and plant health (growth)—respectively, and their combined use exhibits a significant synergistic effect. Experiments have shown that this composition not only effectively kills nematodes but also simultaneously promotes plant growth, overcoming the drawbacks of some chemical agents that inhibit plant growth.

[0020] 3. Stable process and controllable quality: By limiting specific strain combinations (CGMCC No. 16262 and CGMCC No. 36152), synchronous inoculation methods, culture medium formulation, and culture parameters, stable co-accumulation of 2,3-dimethoxyphenol and isopentenyladenine in fermentation broth was achieved. Three batch replication experiments showed that the relative standard deviation (RSD) of the target metabolite content was <8%, significantly better than single-strain fermentation or non-optimized co-culture systems, providing a reliable process basis for industrial scale-up and batch consistency control.

[0021] 4. Environmentally friendly and highly safe: The active ingredient is a natural product derived from microorganisms, exhibiting good environmental compatibility, easy degradation, and safety for both the environment and non-target organisms. This invention provides a novel technological path and product prototype for developing a new generation of efficient, safe, high-quality, and multifunctional green agricultural inputs, with broad application prospects. Attached Figure Description

[0022] Figure 1 This is a targeted liquid chromatography-tandem mass spectrometry verification of 2,3-dimethoxyphenol in one embodiment of this application; wherein, A is the full scan mass spectrum (MS1) of 2,3-dimethoxyphenol standard; B is the tandem mass spectrum (MS / MS) of 2,3-dimethoxyphenol standard; and C is the tandem mass spectrum (MS / MS) of co-culture sample.

[0023] Figure 2 This is an ion current chromatogram (XIC) of a co-cultured sample at m / z 155.0 in one embodiment of this application.

[0024] Figure 3 External standard calibration curve for quantifying 2,3-dimethoxyphenol in one embodiment of this application ( R 2 >0.999).

[0025] Figure 4 The absolute concentrations of 2,3-dimethoxyphenol in monocultures of *Polyspora pinki*, *Bacillus belye*, and their co-cultures in one embodiment of this application are shown (different lowercase letters indicate statistically significant differences). p <0.05); where Cl: *Alternaria pinki* Clonostachys rosea Single culture; Ba: Bacillus belesiensis Bacillus velezensi s: single culture; Co: co-culture.

[0026] Figure 5This document provides a targeted liquid chromatography-tandem mass spectrometry (LC-MS / MS) validation of isopentenyl adenine in one embodiment of this application. A represents the full scan mass spectrum (MS1) of the isopentenyl adenine standard; B represents the tandem mass spectrum (MS / MS) of the isopentenyl adenine standard; and C represents the tandem mass spectrum (MS / MS) of the co-cultured sample.

[0027] Figure 6 This is an ion current chromatogram (XIC) of a co-cultured sample at m / z 204.1 in one embodiment of this application.

[0028] Figure 7 This is an external standard calibration curve for the quantification of isopentenyl adenine in one embodiment of this application. R 2 >0.999).

[0029] Figure 8 The absolute concentrations of isopentenyl adenine in monocultures of *Polyspora pinki*, *Bacillus belyssica*, and their co-cultures in one embodiment of this application are shown (different lowercase letters indicate statistically significant differences). p <0.05); where Cl: *Alternaria pinki* Clonostachys rosea Single culture; Ba: Bacillus belesiensis Bacillus velezensi s: single culture; Co: co-culture.

[0030] Figure 9 This invention relates to an embodiment of the effect of isopentenyl adenine on the growth of tomato seedlings; wherein, A: plant height; B: root length; C: fresh weight. Detailed Implementation

[0031] The following examples illustrate specific implementations of the present invention. However, these examples are merely for illustrative purposes and do not limit the scope of the invention in any way.

[0032] Unless otherwise specified, the instruments and equipment involved in the following embodiments are all conventional instruments and equipment; unless otherwise specified, the reagents and materials involved are all commercially available conventional products; unless otherwise specified, the test and detection methods involved are all conventional methods.

[0033] Example 1: Culture of the test strain and preparation of fermentation samples (1) Microbial strains

[0034] Pink Spiral Polyporus ( Clonostachys rosea , hereinafter referred to as C. rosea NF-06, originally named "Pink Broom Mold NF-06", has the accession number CGMCC No.16262 (for the source and isolation process, please refer to the applicant's earlier patent document CN109762743A).

[0035] Bacillus belesiensis ( Bacillus velezensis , hereinafter referred to as B. velezensis YB-1652, with accession number CGMCC No.36152 (for the source and separation process, please refer to the applicant's previous patent application CN202511673699.2).

[0036] (2) Preparation of seed culture

[0037] Seed cultures were prepared by separate fermentation: A single colony of *Polyspora pinkis* NF-06 was picked from a potato dextrose agar (PDA; containing 200 g / L potato extract, 20 g / L glucose, and 20 g / L agar) plate and inoculated into 100 mL of potato dextrose broth (PDB; 200 g / L potato extract and 20 g / L glucose). The broth was incubated at 25°C and 150 rpm for 48 h with shaking.

[0038] Single colonies of Bacillus belyssus YB-1652 were picked from LA plates (10 g / L tryptone, 5 g / L yeast extract, 10 g / L NaCl, 20 g / L agar) and inoculated into 100 mL LB broth (10 g / L tryptone, 5 g / L yeast extract, 10 g / L NaCl) and cultured at 30°C with shaking at 180 rpm for 48 h.

[0039] Fungal conidial concentrations were normalized to 1.0 × 10⁻⁶ using a hemocytometer. 7 Spores / mL, while bacterial cell density was normalized to 1.0 × 10⁻⁶ by serial dilution plate counting. 8 CFU / mL. These standardized cultures were used as inoculum for subsequent experiments.

[0040] (3) Preparation of cell-free fermentation filtrate

[0041] Culture medium composition: 18 g / L corn flour, 2 g / L sucrose, 0.5 g / L MgSO4, 0.05 g / L FeSO4·7H2O, 0.05 g / L ZnSO4·7H2O, 8 g / L soybean meal, 2 g / L wheat bran. PDB was used as a control for conventional culture medium.

[0042] For the above culture medium, four treatments were prepared in parallel: ① Co-culture (2% v / v) C. rosea + 2% v / v B. velezensis ); ② C. rosea Single culture (2%, v / v); ③ B. velezensis Single culture (2%, v / v).

[0043] All cultures were cultured in 250 mL Erlenmeyer flasks at 28°C with shaking at 180 rpm for 48 h, and autoclaved at 121°C for 20 min before inoculation. After culturing, the broth was centrifuged at 10,000 g at 4°C for 5 min. The supernatant was filtered and sterilized through a 0.22 μm filter membrane to obtain cell-free fermentation filtrate. The cell-free fermentation filtrate obtained from co-culture is the compound biological agent.

[0044] Example 2: Accumulation characteristics analysis of 2,3-dimethoxyphenol in single and co-culture systems

[0045] The co-culture system of *Polyspora pinkis* and *Bacillus belye* accumulated a variety of metabolites with potential functional significance. Based on the preliminary identification of 2,3-dimethoxyphenol as a key metabolite using non-targeted analysis, targeted liquid chromatography-tandem mass spectrometry (LC-MS / MS) based on real standards was further employed to confirm and quantify it.

[0046] (1) Sample preparation:

[0047] Fermentation filtrates from simultaneous co-cultures and corresponding single cultures of *C. rosea* and *B. velezensis* were used for targeted analysis. Frozen aliquots were thawed at room temperature and vortexed. For extraction, 200 μL of sample was mixed with 800 μL of ice-cold methanol, vortexed for 5 min, and centrifuged at 12,000 g for 5 min at 4 °C. 80 μL of the supernatant was filtered through a 0.22 μm PTFE syringe filter into glass autosampler vials for subsequent LC-MS / MS analysis.

[0048] (2) Qualitative verification of targeted LC-MS / MS of 2,3-dimethoxyphenol:

[0049] Analysis was performed using an ultra-high performance liquid chromatography (UHPLC) system coupled with an AB Sciex Triple Quad™ 4500 mass spectrometer (Flemingham, Massachusetts, USA). Chromatographic conditions: Separation was achieved on a Thermo Scientific™ HYPERSIL GOLD C18 column (3 μm, 2.1 × 100 mm) at a temperature maintained at 35 °C. The mobile phase was (A) 0.1% (v / v) formic acid aqueous solution and (B) acetonitrile, with a flow rate of 0.3 mL / min. The injection volume was 3 μL. The gradient program was: 0–3 min, 10% B; 3–6 min, 10–90% B; 6–6.5 min, 90% B; 6.5–6.6 min, 90–10% B; 6.6–10 min, 10% B. Detection was performed using a Turbo Spray® ionization source in positive electrospray ionization mode. The relevant parameters were set as follows: ion spray voltage, +5500 V; source temperature, 550 °C; curtain gas, 30 psi; collision gas, 9 psi. 2,3-Dimethoxyphenol was quantified in multiple reaction monitoring mode by monitoring the m / z transition from 155 to 95.

[0050] The protonated molecular ion [M+H]⁺ of the 2,3-dimethoxyphenol standard exhibits a high-response, sharp peak at a mass-to-charge ratio (m / z) of approximately 155.0. Figure 1 A). To confirm the structure, the tandem mass spectra (MS / MS) of the target ion (m / z 155.0) in the standard and the co-culture sample were further compared. The results showed that the two were completely identical in terms of fragment ion species, mass-to-charge ratio, and relative intensity, confirming the presence of 2,3-dimethoxyphenol in the sample. Figure 1 B, C).

[0051] (3) Validation of chromatographic separation and quantitative methods:

[0052] Calibration curves were constructed using a dilution series of 2,3-dimethoxyphenol standards (Sigma-Aldrich, USA), with each concentration analyzed three times. A linear regression of the average peak area against the standard concentration (μg / mL) yielded the calibration equation. The concentration of the sample extract was determined by interpolating the peak area into this curve. The final concentration in the original fermentation filtrate was calculated using the formula: Cfinal (μg / mL) = c × Vtotal / vsample, where c is the interpolated concentration, Vtotal is the total volume of the extraction mixture, and vsample is the volume of the original fermentation filtrate used for extraction. Method reliability was monitored by periodically analyzing the mixed QC sample and by interspersing low, medium, and high concentrations of calibration standards throughout the analytical sequence.

[0053] The detection of 2,3-dimethoxyphenol was further validated by extractive ion chromatography (XIC), demonstrating that it achieved effective chromatographic separation from other components in the matrix. Figure 2 Quantitative analysis employed the external standard calibration curve method. This calibration curve exhibited excellent linearity across the tested concentration range. R² >0.999), ensuring the accuracy and precision of the measurement. Figure 3 ).

[0054] (4) Characteristics of 2,3-dimethoxyphenol content in the co-culture system:

[0055] Consistent with the trends revealed by non-targeted metabolomics, absolute quantitative results confirm ( Figure 4 ),exist Bacillus velezensis Single culture (Ba) and Clonostachys rosea In the monoculture (Cl) system, the content of this metabolite was at extremely low levels (approximately 0.9 μg / L and 1.0 μg / L); however, in the co-culture (Co) system, the content of 2,3-dimethoxyphenol increased significantly to approximately 25 μg / L, which was significantly higher than in the two monoculture systems. The results indicate that... C. rosea and B. velezensis The co-culture specificity triggered the reprogramming of metabolic pathways, significantly inducing the biosynthesis and accumulation of 2,3-dimethoxyphenol.

[0056] (5) Nematicidal activity of 2,3-dimethoxyphenol:

[0057] By mixing 100 μL of filtrate with an equal volume of wheat cyst nematode pathogen (Philippine cyst nematode) Heteradera filipjevi Tomato root-knot nematode pathogen (Southern root-knot nematode) Meloidogyne incognita ) and the pathogen of maize root rot (Scribneria short-bodied nematode) Pratylenchus scribneri The nematicidal activity was evaluated by mixing a suspension of second-instar larvae (J2s) (2,000 J2s / mL) with the biological agent obtained in Example 1, cell-free fermentation filtrate, and 2,3-dimethoxyphenol standard in 96-well plates. Uninoculated culture medium served as a negative control. After exposure at 25°C for 24 h and 48 h, immobile nematodes were detected with 4% NaOH (w / v) to confirm death. The relative mortality rate was calculated as follows: Relative mortality rate (%) = [(T−C) / (100−C)]×100, where T is the mortality rate of the treatment group and C is the mortality rate of the control group.

[0058] The results (Table 1) showed that the 2,3-dimethoxyphenol standard and biological agents all exhibited good nematicidal activity against the three plant pathogenic nematodes, and the overall corrected mortality rate increased with the extension of treatment time (24 h → 48 h). At 24 h, the corrected mortality rates of the 2,3-dimethoxyphenol standard against *Symplocos spp.*, *Spora spp.*, and *Skeribneria spp.* (85.10%, 84.60%, 75.20%) were higher than those of the biological agent (80.20%, 76.80%, 70.50%), indicating that the standard had superior nematicidal activity under short-term action. However, at 48 h, the corrected mortality rates of the biological agent against *Spora spp.* and *Skeribneria spp.* (94.60%, 90.40%) exceeded those of the standard (86.50%, 87.90%), while the corrected mortality rate against *Symplocos spp.* (96.50%) was close to that of the standard (97.60%), demonstrating that the nematicidal effect of the biological agent increased more significantly over time. In terms of nematode sensitivity, *Strombus spp.* (southern root-knot nematode) was the most sensitive to both treatments, with a 48-hour corrected mortality rate approaching 97%. *Strombus sp. Phillips* showed a significantly greater increase in mortality under the biological agent treatment (76.80%→94.60%) than under the standard treatment (84.60%→86.50%). *Strombus skribnerii* (skeribnerii short-bodied nematode) exhibited a significantly enhanced nematode-killing effect over time under both treatments. These results indicate that 2,3-dimethoxyphenol has a good lethal effect on all three pathogenic nematodes.

[0059] Table 1. Lethal effects of 2,3-dimethoxyphenol standard and biological agents on plant pathogenic nematodes.

[0060] Example 3: Accumulation Characteristics Analysis of Isopentenyladenine in Single and Co-culture Systems

[0061] Based on the preliminary identification of isopentenyl adenine as a key metabolite using non-targeted analysis, further confirmation and quantification were performed. The method was the same as in Example 2.

[0062] (1) Qualitative verification of targeted LC-MS / MS of isopentenyl adenine:

[0063] The key metabolite isopentenyladenine in non-targeted screening was validated and quantified using liquid chromatography-tandem mass spectrometry (LC-MS / MS). The protonated molecular ion [M+H]⁺ of the isopentenyladenine standard exhibited a high-response, sharp peak at approximately 204.1 m / z. Figure 5To confirm the structure, the tandem mass spectra (MS / MS) of the target ion (m / z 204.1) in the standard and the co-culture sample were further compared. The results showed that the two were completely identical in terms of fragment ion species, mass-to-charge ratio, and relative intensity, confirming the presence of isopentenyl adenine in the sample.

[0064] (2) Validation of chromatographic separation and quantitative methods:

[0065] The detection of isopentenyl adenine was further validated by extractive ion chromatography (XIC), demonstrating its effective chromatographic separation from other components in the matrix. Figure 6 Quantitative analysis employed the external standard calibration curve method. This calibration curve exhibited excellent linearity (R0) across the tested concentration range. 2 >0.999), ensuring the accuracy and precision of the measurement. Figure 7 ).

[0066] (3) Characteristics of isopentenyl adenine content in different culture systems:

[0067] Based on the results of target metabolomics detection ( Figure 8 The content of isopentenyladenine (AAA) varied significantly across different culture systems: the lowest content (0.007 μg / L) was observed in the *C. rosea* monoculture (Cl) system; approximately 1.5 μg / L in the *B. velezensis* monoculture (Ba) system; and a significantly higher content (approximately 2.0 μg / L) in the co-culture (Co) system. These results indicate that co-culturing *C. rosea* and *B. velezensis* significantly induces the biosynthesis and accumulation of AAA. As a key cytokinin-like plant growth regulator, the synergistic accumulation of AAA further supports the core mechanism by which co-culture enhances plant growth by promoting the production of metabolites, thereby increasing crop growth.

[0068] Example 4: Effects of isopentenyl adenine on the growth of tomato seedlings

[0069] To investigate the physiological effects of isopentenyladenine on plant growth, this study analyzed its influence on the growth indicators of tomato seedlings. Tomato seeds (variety: Zhongza 9) were surface-sterilized with 75% (v / v) ethanol for 30 seconds, rinsed five times with sterile distilled water, and treated with isopentenyladenine standard at 2 μM / g. The seeds were then sown in a sterile substrate (peat:vermiculite:perlite = 3:1:1, v / v / v) and cultured in a 25℃ greenhouse for 20 days. The seedling height, root length, and fresh weight were then measured.

[0070] The results show that ( Figure 9 Treatment with isopentenyl adenine standard significantly promoted the overall growth of tomato seedlings. The plant height (approximately 25 cm), root length (approximately 17 cm), and fresh plant weight (approximately 1.8 g) in the treatment group were significantly higher than those in the control group (approximately 20 cm, 13 cm, and 1.0 g, respectively) (p<0.05).

[0071] Example 5: Verification of the effect of compound biological agents on the control of cucumber root-knot nematode disease in indoor potted plants.

[0072] Transplant cucumber seedlings into diseased soil infected with root-knot nematodes and perform root drenching treatments using the following methods: (1) Solitary culture of *Polyspora pinkis* (Cl) 10 mL / plant for root irrigation; (2) Bacillus belyi culture (Ba) 10 mL / plant root irrigation; (3) Co-culture (Co, compound biological agent) 10 mL / plant for root irrigation; (4) 2,3-Dimethoxyphenol standard, 2 μM / plant, for root irrigation; (5) Isoprene adenine standard 2 μM / plant for root irrigation; (6) 2,3-Dimethoxyphenol standard 2 μM / plant root irrigation + isopentenyl adenine standard 2 μM / plant root irrigation; (7) Comparison.

[0073] The microbial culture solutions of treatments (1), (2), and (3) were the cell-free fermentation filtrates of each treatment in Example 1. One cucumber seedling was planted in each pot, and each treatment group was biologically replicated 6 times. The treatment groups were cultured in an incubator at 25℃. The plants were harvested 40 days after transplanting, and the soil attached to the roots was gently rinsed. The severity of the disease was determined according to the percentage of root knots using a 0-5 grade grading standard, and the root knot index and control effect were calculated. At the same time, plant height, root length, and fresh weight were measured to comprehensively evaluate the growth-promoting effect of the tested fermentation products on cucumber plants.

[0074] The results (Table 2) showed that the root irrigation treatment with co-culture (Co) fermentation broth (compound biological agent) demonstrated good overall effects in this experiment. In terms of disease control, the root knot index was as low as 16.83, with a control effect of 71.60%. This was not significantly different from the combined treatment with 2,3-dimethoxyphenol + isopentenyl adenine standard (72.06%), and was significantly superior to the two single microbial treatments (Cl and Ba) and the treatment using isopentenyl adenine alone. Regarding growth promotion, the Co treatment achieved the highest levels of root length (18.01 cm) and fresh weight (2.20 g), and the plant height (26.17 cm) was also significantly higher than the control.

[0075] The results showed that the effect of Co treatment exceeded that of its single components (Cl and Ba), demonstrating the synergistic effect that co-cultivation may produce. Compared with chemical treatment, Co treatment (compound biological agent) achieved overall efficacy comparable to the standard "2,3-dimethoxyphenol + isopentenyl adenine" combined root irrigation, realizing the dual effect of highly efficient nematode control and plant growth promotion.

[0076] Table 2. Control effects of different treatments on cucumber root-knot nematodes in indoor potted plants and their plant growth-promoting effects.

[0077] Although some 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.

[0078] Obviously, those skilled in the art can make various modifications and variations to this invention without departing from its spirit and scope. Therefore, if these modifications and variations fall within the scope of the claims of this application and their equivalents, this invention is also intended to include these modifications and variations.

Claims

1. 2,3-Dimethoxyphenol in the preparation of plant pathogenic nematode control agents.

2. The application according to claim 1, characterized in that, The 2,3-dimethoxyphenol was isolated from the fermentation product of *Polyspora pinkis*, or from the co-culture product of *Polyspora pinkis* and *Bacillus belye*.

3. The application according to claim 1, characterized in that, The plant nematodes mentioned are Southern Root-knot Nematode, Skribner's Short-bodied Nematode, Philippine Cyst Nematode, or Oat Cyst Nematode.

4. A biological agent for controlling plant nematodes, characterized in that, It contains 2,3-dimethoxyphenol as an active ingredient, which is isolated from the fermentation product of *Polyspora pinkis* or from the co-culture product of *Polyspora pinkis* and *Bacillus belye*.

5. A compound biological agent possessing both nematicidal and life-promoting properties, characterized in that, It comprises the biological agent of claim 4 and isopentenyl adenine.

6. A method for preparing the composite biological agent according to claim 5, characterized in that, By simultaneously inoculating *Polyspora pinkis* and *Bacillus belye* into a culture medium for co-culture, a fermentation product containing both 2,3-dimethoxyphenol and isopentenyl adenine was obtained.

7. The preparation method according to claim 6, characterized in that, The *Polyspora pinkis* species is *Polyspora pinkis* with accession number CGMCC No. 16262. Clonostachys rosea NF-06; The Bacillus belyssus mentioned is Bacillus belyssus with accession number CGMCC No. 36152 (NF-06); Bacillus velezensis YB-1652.

8. The preparation method according to claim 6 or 7, characterized in that, Includes the following steps: (1) The activated *Polyspora pinkis* and *Bacillus belye* strains were inoculated into the fermentation medium at an inoculation rate of 1% to 5% and co-cultured. (2) The co-culture conditions are: temperature 25~30℃, rotation speed 150~200 rpm, and culture time 40~56 h; (3) After the culture is completed, solid-liquid separation is performed, and the supernatant is collected.

9. The preparation method according to claim 8, characterized in that, The culture medium contains per liter: 12-22 g corn flour, 1-5 g sucrose, 0.2-0.8 g MgSO4, 0.02-0.08 g FeSO4∙7H2O, 0.02-0.08 g ZnSO4∙7H2O, 6-13 g soybean meal, 1-5 g wheat bran, and the remainder is water.

10. A method for controlling plant pathogenic nematodes, characterized in that, The biological agent of claim 4 or the compound biological agent of claim 5 is used for root irrigation.

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