Composite microbial preparation for preventing and treating root-knot nematode and promoting plant growth and preparation and application thereof

Abstract

This invention discloses a compound microbial preparation for controlling root-knot nematodes and promoting plant growth, as well as its preparation and application. Addressing the technical problems of existing biocontrol agents, such as lack of target optimization based on root-knot nematodes, unclear metabolite profiles, poor synergistic effects, and unstable insecticidal and growth-promoting properties, this invention screens *Polyspora pinki* and *Bacillus belye* for co-culture, and through inter-bacterial interactions, directionally enriches synergistic metabolites such as daidzein, isopentenyl adenine, and melatonin. When applied to the rhizosphere, this preparation reduces the root-knot index of cucumbers by ≥40% and increases the fresh weight of aboveground parts by ≥25%, significantly superior to single-strain or physical mixture systems. Furthermore, the product is flexible in form, possessing both viable bacterial colonization ability and metabolite stability; its activity retention rate is >80% after 90 days of storage at 35℃, making it suitable for various application methods such as root irrigation and seed coating, providing a highly efficient and stable technical solution for the green control of root-knot nematodes.

Description

Technical Field

[0001] This invention relates to the field of agricultural biotechnology, specifically to a compound microbial preparation for controlling root-knot nematodes and promoting plant growth, and its preparation and application. Background Technology

[0002] Root-knot nematodes ( Meloidogyne spp The plant parasitic nematode (BNP) is one of the most damaging plant parasitic nematodes in global agricultural production, infecting over 3,000 crop species. It invades and induces the formation of nodular root knots, disrupting water and nutrient absorption, leading to stunted growth, reduced yields, and even crop failure. Simultaneously, the root wounds caused by nematodes easily induce secondary infections such as soil-borne fungi and bacteria, forming complex diseases and further exacerbating agricultural losses. With the gradual banning and restriction of highly toxic chemical nematicides such as aldicarb and carbofuran, the development of environmentally friendly and sustainable biological control technologies has become an urgent need for the industry.

[0003] Microbial preparations are considered an important direction for green pest control due to their good environmental compatibility and diverse mechanisms of action. Current technical approaches mainly include: ① Fermentation preparations using single biocontrol strains, which have a limited metabolic spectrum and cannot simultaneously achieve multiple functions such as nematode control and growth promotion; ② Physical mixing of fermentation broths from two strains, which can integrate the metabolites of different strains, but lacks inter-strain interactions, failing to induce unique synergistic metabolites or optimize metabolite ratios, resulting in unstable biological activity and poor batch-to-batch reproducibility; ③ Co-culturing of microorganisms, such as the co-culturing of *Polyspora pinki* and *Bacillus subtilis*, but its antagonistic specificity and synergistic potential against nematodes are unsatisfactory, and the spectrum of key active metabolites enriched is still unclear. This leads to unclear product active ingredients and a lack of precise chemical basis for quality stability control, limiting its upgrade to standardized and highly efficient preparations.

[0004] Therefore, there is an urgent need in this field to break through the existing technological framework and develop a compound microbial preparation and its preparation technology that can significantly stimulate synergistic interactions between microorganisms, efficiently enrich clearly defined active metabolites, and simultaneously achieve efficient and stable insecticidal and growth-promoting effects.

[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 addresses the technical problems of existing biocontrol agents, such as lack of optimization based on root-knot nematode targets, unclear metabolite profiles, poor synergistic effects, and unstable insecticidal and growth-promoting properties. It employs a key technical approach: co-culturing *Polyspora pinkis* and *Bacillus belye* to activate inter-bacterial interactions and directionally enrich synergistic metabolites such as daidzein, isopentenyl adenine, and melatonin. This achieves dual stable efficacy, reducing the root-knot index by ≥40% and increasing the aboveground fresh weight of plants by ≥25%, while also ensuring product storage stability and field applicability.

[0007] According to one aspect of this disclosure, a compound microbial preparation is provided, the active ingredient of which is composed of Polyspora pinkis (…). Clonostachys rosea ) and Bacillus belesi ( Bacillus velezensis Produced through co-culture and fermentation; At least one of the following was detected in the active ingredient: daidzein, isopentenyl adenine, and melatonin; the compound microbial preparation has at least one of the following functions: (a) Inhibits root-knot nematode infection; (b) Promotes root growth and / or aboveground biomass accumulation; (c) Improve plant health.

[0008] In some embodiments of this disclosure, the *Polyspora pinkis* is *Polyspora pinkis* NF-06 with accession number CGMCC No. 16262; and the *Bacillus belyssus* is *Bacillus belyssus* YB-1652 with accession number CGMCC No. 36152.

[0009] According to another aspect of this disclosure, a method for preparing the compound microbial preparation is provided, wherein *Polyspora pinkis* NF-06 and *Bacillus belye* YB-1652 are inoculated into a liquid culture medium and fermented with shaking at 25–30°C and 150–200 rpm for 40–50 h; then centrifuged at 8000–12000 g and 3–6°C, and the supernatant obtained by filtration through a filter membrane is sterilized to obtain the final product.

[0010] In some embodiments of this disclosure, the liquid culture medium contains, in g / L: 15-25g corn flour, 0.2-1.0g MgSO4, 0.02-0.1g FeSO4·7H2O, 0.02-0.1g ZnSO4·7H2O, 5-12g soybean meal, 5-12g wheat bran, and the remainder is water.

[0011] According to another aspect of this disclosure, the application of the compound microbial preparation in the prevention and control of plant root-knot nematode disease and / or the promotion of plant growth is provided.

[0012] According to another aspect of this disclosure, a method for preventing and controlling plant root-knot nematode disease is provided, wherein the compound microbial preparation is applied to the plant rhizosphere, seeds or seedling substrate, with an effective dose of 5-15 mL / plant, applied 1-3 times.

[0013] According to another aspect of this disclosure, a plant health-promoting composition is provided, comprising effective amounts of daidzein, isopentenyl adenine, and melatonin as active ingredients, wherein the daidzein, isopentenyl adenine, and melatonin are derived from the co-culture fermentation products of *Polyspora pinkis* and *Bacillus belye*.

[0014] According to another aspect of this disclosure, an agricultural formulation is provided, comprising the compound microbial preparation or the plant health promoting composition as described above, and an agriculturally acceptable carrier and / or adjuvant.

[0015] One or more technical solutions provided in the embodiments of this application have at least one of the following technical effects or advantages: 1. Achieving synergistic effects based on specific bacterial pairings: Through co-culturing *Polyspora pinkis* NF-06 and *Bacillus belyssus* YB-1652, experiments showed that the synergistic effect produced by this specific pairing was significantly better than its co-culture combinations with *Bacillus subtilis* or other reported Bacillus species in terms of lethal activity against second-instar larvae of root-knot nematodes.

[0016] 2. A clear understanding of the core active substances enhances product controllability: The specific co-culture system allows for the specific and efficient enrichment of key functional metabolites such as daidzein, isopentenyl adenine, and melatonin. This clearly defined chemical basis enables the quality control, activity evaluation, and efficacy stability of this formulation to leap from a "mixture of fermentation products" to a "composition of specific active ingredients," resolving the issues of unclear product composition and large batch-to-batch fluctuations inherent in existing co-culture technologies.

[0017] 3. Achieved superior overall effects of disease prevention and growth promotion: Thanks to the above-mentioned synergistic effects and the enrichment of specific metabolites, this formulation has consistently surpassed the effects of co-culture filtrate reported in existing technologies in pot and field trials in terms of control of root-knot nematodes and growth promotion indicators of crops (such as root length and biomass), thus achieving a dual enhancement of disease prevention and growth promotion functions.

[0018] 4. High product stability and convenient application: The main active ingredient of this invention is derived from fermentation metabolites and is a sterile product, completely avoiding the problems of unstable survival rate, harsh storage conditions, and short shelf life associated with live bacteria preparations. The activity retention rate is >80% after 90 days of storage at 35℃. The product has strong quality uniformity and good batch stability, making it easy to standardize production. Furthermore, this preparation can be flexibly applied to various agronomic operations such as seed treatment, root irrigation, foliar spraying, and hole application, demonstrating strong adaptability, ease of use, and promising prospects for industrial application. Attached Figure Description

[0019] Figure 1 The relative abundances of isopentenyl adenine, melatonin, and daidzein in the single-culture and co-culture systems of one embodiment of this application are shown; wherein, Cl: Polyspora pinkis single culture; Ba: Bacillus belyss single culture; Co: synchronous co-culture.

[0020] Figure 2 This is a comparison of the growth-promoting effects of different treatment groups on tomato seedlings in one embodiment of this application; where A: plant height; B: root length; C: fresh weight; D: representative phenotypic image of tomato seedlings. Data are mean ± standard error; bars marked with different lowercase letters indicate statistically significant differences between treatment groups (…). p <0.05).

[0021] Figure 3 The lethal effect of the compound microbial preparation on nematodes after being stored at 35°C for 120 days in one embodiment of this application is shown. Detailed Implementation

[0022] Unless otherwise specified, the instruments and equipment involved in the following embodiments are all conventional instruments and equipment; the industrial raw materials involved are all commercially available conventional industrial raw materials; and the processing and manufacturing methods involved are all conventional methods unless otherwise specified.

[0023] The preparation of the aforementioned biomaterials is involved. 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).

[0024] 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).

[0025] Preparation of inoculum for Southern Root-Knot Nematodes: Southern root-knot nematodes ( Meloidogyne incognita The colony was initially collected from Henan Province, China, and maintained on tomato plants in a controlled environment incubator (25°C, 70% relative humidity). Egg masses were manually collected from infected roots, surface-sterilized with 1% (v / v) H₂O₂ for 3 min, and thoroughly rinsed with sterile distilled water. The sterilized egg masses were incubated in sterile water at 25°C to allow second-instar larvae (J2s) to hatch. Newly hatched J2s were collected using a Baermann funnel and their viability was confirmed under an optical microscope. The larval suspension was adjusted to a density of 2000 J2s / mL with sterile tap water for subsequent bioassays.

[0026] Example 1: Preparation and Component Analysis of Compound Microbial Preparations Co-culture process of *Polyspora pinkis* NF-06 and *Bacillus belyssus* YB-1652: (1) Seed liquid preparation: A single colony of *Polyspora pinki* 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, 20 g / L glucose). The mixture was incubated at 25°C with shaking at 150 rpm for 48 h. The fungal conidial concentration was normalized to 1.0 × 10⁻⁶ using a hemocytometer. 7 Spores / mL.

[0027] Single colonies of *Bacillus belye* YB-1652 were picked from LB agar (LA; 10 g / L tryptone, 5 g / L yeast extract, 10 g / L NaCl, 20 g / L agar) plates 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. Bacterial cell density was normalized to 1.0 × 10⁻⁶ cells / mL using serial dilution plate counting. 8 CFU / mL.

[0028] (2) Co-culture to obtain cell-free fermentation filtrate: The seed culture of *Polyspora pinkis* NF-06 and the seed of *Bacillus belyssus* YB-1652 were inoculated into a special culture medium (calculated as g / L: corn flour 20, MgSO4 0.5, FeSO4·7H2O 0.05, ZnSO4·7H2O 0.05, soybean meal 10, wheat bran 10), and cultured for fermentation at 28℃ and 180 rpm for 48 h by shaking; the mixture was then centrifuged and filtered at 10,000 g and 4℃ to obtain sterile fermentation supernatant, which is the compound microbial preparation described in this example.

[0029] To verify the synergistic effect of the co-culture system, multiple treatment groups were set up: ① Co-culture: 2% v / v C. rosea + 2% v / v B. velezensis Simultaneous vaccination; ② C. rosea Single culture (Cl): 2% v / v C. rosea Vaccination; ③ B. velezensis Single culture (Ba): 2% v / v B. velezensis Vaccination.

[0030] Cell-free fermentation filtrate was obtained from the above treatment groups. For metabolite extraction, 50 μL of fermentation filtrate was mixed with 150 μL of pre-chilled extraction solvent (methanol:acetonitrile = 4:1, v / v) containing an internal standard mixture. The mixture was vigorously vortexed for 3 min, then centrifuged at 4°C and 12,000 ×g for 10 min. 150 μL of the supernatant was transferred to a new tube and incubated at -20°C for 30 min to precipitate residual proteins, then centrifuged again under the same conditions for 3 min. Finally, 120 μL of the supernatant was collected and transferred to a glass-lined tube for LC-MS analysis. The results are shown in Table 1 (each group was tested 6 times, and the average value was taken).

[0031] Table 1 Key differential abundance metabolites identified in the co-culture system of *Polyspora pinki* and *Bacillus belyss*.

[0032] Note: Metabolites were identified by matching precise mass, MS / MS spectra, and retention times to real standards or database records. Relative abundance was based on LC-MS peak area comparisons. Statistical significance between Cosim and individual cultures was assessed using independent samples t-tests.

[0033] From the relative abundance results of non-target metabolomics ( Figure 1 Compared with single-strain fermentation broth, the co-culture fermentation broth of *Polyspora pinki* NF-06 and *Bacillus belyssae* YB-1652 (Cosim) significantly increased the content of several key plant growth regulators. The relative abundances of isopentenyl adenine, daidzein, and melatonin in the co-culture system were significantly higher than in the single-strain system. This result indicates that the interaction between the two microorganisms can synergistically activate related metabolic pathways, promoting the production of metabolites with plant growth regulation (isopentenyl adenine, melatonin) and nematicidal bioactivity (daidzein), providing metabolic support for the co-culture system to enhance plant growth and stress resistance potential.

[0034] Furthermore, the overall efficacy of this metabolite composition is not a linear superposition of the metabolic profiles of a single strain, but rather a non-additive synergistic system formed through inter-strain interactions (such as precursor sharing, signal induction, or metabolic flux redirection) in a co-culture microenvironment. This specific metabolite ratio and composition endows it with dual functions of promoting growth and resisting nematodes, effectively solving the technical bottleneck of existing biocontrol agents that cannot simultaneously achieve "growth promotion" and "protection".

[0035] Example 2: Verification of the growth-promoting effect of compound microbial preparations This embodiment verifies the growth-promoting effect of the biological agent obtained in Example 1 on tomato seedlings.

[0036] Tomato seeds (variety: Zhongza 9) were surface-sterilized with 75% (v / v) ethanol for 30 seconds, rinsed five times with sterile distilled water, soaked in the corresponding cell-free fermentation filtrate described in Example 1 for 4 hours, and sown in a sterile substrate (peat:vermiculite:perlite = 3:1:1, v / v / v) and grown under controlled conditions. After 20 days, the seedling height, root length, and fresh weight were measured.

[0037] The processing groups are as follows: Cl (Pink Spiral Polyporus monoculture); Ba (Bacillus berberis single culture); CoSim (simultaneous inoculation and co-culture); CoSeq (Sequential Seeding Co-culture): First inoculate... C. rosea (2%, v / v) cultured for 24 hours, then added B. velezensis (2%, v / v) and incubate for another 24 hours; Cl+Ba (a 1:1 physical mixture of single-culture filtrate); Control (uninoculated culture medium).

[0038] The results are as follows Figure 2 Compared with other treatment groups, the CoSim treatment group showed the best overall effect in promoting the growth of tomato seedlings. Compared with the control group, the tomato seedlings in the CoSim treatment group showed an increase of 42.28% in plant height, 20.3% in root length, and 87.35% in fresh weight. Figure 2 These quantitative improvements correspond to visible phenotypic advantages, including stronger apical dominance and more extensive root branching.

[0039] Example 3: Pot experiment to verify the control effect on cucumber root-knot nematodes Under controlled environmental conditions, pot experiments were conducted to evaluate the control effects of various fermentation products on southern root-knot nematodes.

[0040] The experiment included six treatments: (1) Cl (root irrigation with fermentation broth from a single culture of *Polyspora pinkis*). (2) Ba (root irrigation with Bacillus belesiensis single culture fermentation broth); (3) CoSim (simultaneous inoculation with co-culture fermentation broth for root irrigation); (4) Cl+Ba (a 1:1 physical mixture of single culture filtrate for root irrigation); (5) Chemical control (0.5% abamectin granules applied in holes); (6) Untreated control (root irrigation with sterile water).

[0041] For each of the above treatments, 10 mL of the liquid was applied to the soil immediately after transplanting the corresponding cucumber seedlings, while the chemical control was applied by placing 2 g of granules into the planting hole. The experiment used a completely randomized design with 12 replicates for each treatment (one cucumber seedling per pot). The plants were maintained in an incubator at 25°C and watered regularly.

[0042] Two days after transplanting, each cucumber seedling was inoculated with southern root-knot nematodes ( Meloidogyne incognita The nematode inoculation was performed by injecting approximately 1,000 J²s into two 1 cm deep holes located 2 cm from the stem base, resulting in a total inoculation of approximately 2,000 J²s per plant. Plants were harvested 40 days after nematode inoculation. The roots were gently washed to remove soil. Disease severity was assessed using a standard 0-5 grading system based on the percentage of root nodules. The root knot index and control efficacy were then calculated as previously described. Plant growth was also evaluated by recording plant height, root length, and fresh weight. The experiment employed a completely randomized design with 12 replicates (plants) for each treatment.

[0043] Pot experiments confirmed that co-culture treatment with co-inoculation exhibited superior biocontrol efficacy against southern root-knot nematodes (Table 2). The root knot index of the co-culture group was 17.33, significantly lower than the untreated control group's 58.52 and all other microbial treatments, including single culture of *Polyspora pinkis* (22.96), single culture of *Bacillus belyssioides* (28.15), and a physical mixture of the fermentation broths of the two single cultures (25.18). Therefore, co-culture achieved the highest control efficacy (70.39%), a result that was not statistically significantly different from the control efficacy of the chemical agent avermectin (68.75%).

[0044] In addition to suppressing nematodes, co-culture treatment significantly promoted cucumber plant growth (Table 2). Plants treated with co-culture achieved the greatest plant height (48.56 cm), root length (18.99 cm), and fresh weight (19.79 g); all these values ​​were significantly higher than those of the control plants (27.21 cm, 12.47 cm, and 10.23 g, respectively). Notably, co-culture also outperformed individual cultures and their physical mixtures, indicating that co-culture induces synergistic interactions, thereby promoting plant growth.

[0045] Table 2. Control effects of different treatments on southern root-knot nematode disease in cucumber and their plant growth-promoting effects. .

[0046] Example 4: Study on the Changes in the Nematicidal Activity of Biological Agents Based on Thermal Stability Evaluation The nematicidal activity of this biological agent gradually decreased with prolonged storage time under storage conditions of 35°C. Figure 3 The relative mortality rate was highest at day 0 of storage (92.17%), decreasing to 89.56%, 87.78%, 83.64%, and 78.53% after 30, 60, 90, and 120 days of storage, respectively. These results indicate that although high-temperature storage leads to the gradual degradation of the active ingredient, the formulation still maintained a mortality rate of 78.53% after 120 days, demonstrating that its active ingredient possesses good thermal stability and has potential for practical application and storage.

[0047] In summary, the compound microbial preparation provided by this invention exhibits significant dual biological functions. Metabolite analysis shows that the preparation is rich in various active ingredients related to plant growth regulation and nematode inhibition; plant growth promotion experiments verified its significant promoting effect on cucumber seedling growth; in vivo efficacy tests further confirmed that the sterile fermentation broth can effectively inhibit the infection and damage of southern root-knot nematodes on live plants. This composition can simultaneously achieve the goals of promoting plant health and controlling root-knot nematodes without relying on live bacterial colonization, effectively solving the technical problems of existing biological agents having single functions, insufficient stability, and limited field application, and has good application prospects and industrialization potential.

[0048] 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.

[0049] 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. A compound microbial preparation, characterized in that, Its active ingredient is composed of *Polyspora pinkis* (… Clonostachys rosea ) and Bacillus belesi ( Bacillus velezensis Produced through co-culture and fermentation; At least one of the following was detected in the active ingredient: daidzein, isopentenyl adenine, and melatonin; the compound microbial preparation has at least one of the following functions: (a) Inhibits root-knot nematode infection; (b) Promotes root growth and / or aboveground biomass accumulation; (c) Improve plant health.

2. The compound microbial preparation according to claim 1, 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.

3. A method for preparing the composite microbial preparation according to claim 1 or 2, characterized in that, Inoculate *Polyspora pinkis* NF-06 and *Bacillus belye* YB-1652 into liquid culture medium and ferment at 25–30°C and 150–200 rpm for 40–50 h with shaking. Then centrifuge at 8000–12000 g and 3–6°C, filter the supernatant through a filter membrane, and sterilize to obtain the final product.

4. The preparation method according to claim 3, characterized in that, The liquid culture medium contains, in g / L: 15-25g corn flour, 0.2-1.0g MgSO4, 0.02-0.1g FeSO4·7H2O, 0.02-0.1g ZnSO4·7H2O, 5-12g soybean meal, 5-12g wheat bran, and the remainder is water.

5. The application of the compound microbial preparation according to claim 1 or 2 in the prevention and control of plant root-knot nematode disease and / or the promotion of plant growth.

6. A method for controlling plant root-knot nematode disease, characterized in that, The compound microbial preparation described in claim 1 or 2 is applied to the plant rhizosphere, seeds, or seedling substrate at an effective dose of 5-15 mL / plant, 1-3 times.

7. A plant health-promoting composition, characterized in that, It contains effective amounts of daidzein, isopentenyl adenine, and melatonin as active ingredients, and the daidzein, isopentenyl adenine, and melatonin are derived from the co-culture fermentation products of *Polyspora pinkis* and *Bacillus belye*.

8. An agricultural formulation, characterized in that, It contains the compound microbial preparation as described in claim 1 or the plant health promoting composition as described in claim 7, and an agriculturally acceptable carrier and / or adjuvant.

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