Compound microbial preparation for preventing and treating root-knot nematode and application of compound microbial preparation
Through the synergistic effect of compound microbial agents, the problem of root-knot nematode disease control has been solved, achieving efficient and environmentally friendly control, improving crop yield and quality, and maintaining soil ecological balance.
Patent Information
- Application Number
- CN202511182417.9
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- Filing Date
- 2025-08-22
- Publication Date
- 2025-11-21
AI Technical Summary
In existing technologies, root-knot nematode disease seriously affects the yield of greenhouse vegetables, and traditional pesticide control measures lead to environmental pollution. There is a lack of safe, efficient and environmentally friendly control methods.
A compound microbial agent, composed of Streptomyces rochei S36, Trichoderma sp. M2, and Bacillus velezensis Y10, is used to control root-knot nematodes by applying the mixed fermentation liquid to the soil or drenching the roots of plants.
It significantly reduces nematode hatching and survival rates, decreases nematode parasitism density, forms a long-lasting protective barrier, enhances crop root vitality, improves fruit quality and yield, and maintains soil microecological balance, with no residues or pollution.
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Figure CN120988906A_ABST
Abstract
Description
Technical Field
[0001] This invention relates to the field of microbial preparation technology, and in particular to a compound microbial preparation for the prevention and control of root-knot nematodes and its application. Background Technology
[0002] Root-knot nematode (Meloidogyne spp.) is one of the most serious root diseases affecting vegetable yields in greenhouse cultivation worldwide, characterized by its wide distribution, multiple host sites, strong infectivity, and insidious occurrence and damage. With the rapid development of greenhouse vegetable cultivation, the incidence of root-knot nematodes has been increasing year by year, with severe areas experiencing yield reductions of 30-40%, or even total crop failure. Currently, the most important control methods in agriculture are still pesticides and soil disinfection, resulting in large amounts of pesticide residues and environmental pollution. Therefore, improving vegetable quality and yield, and seeking safe, efficient, and environmentally friendly root-knot nematode control measures has become an urgent and important need for the vegetable industry. Summary of the Invention
[0003] The purpose of this invention is to provide a compound microbial preparation for the prevention and control of root-knot nematodes and its application, so as to solve the problems existing in the prior art.
[0004] To achieve the above objectives, the present invention provides the following solution:
[0005] One of the technical solutions of this invention is a composite microbial agent composed of Streptomyces rochei S36, Trichoderma sp. M2 and Bacillus velezensis Y10.
[0006] The second technical solution of the present invention, the preparation method of the compound microbial agent, includes the following steps: inoculating Actinomycetes S36, Trichoderma M2 and Bacillus belye Y10 into a culture medium for cultivation to obtain three fermentation broths, and then mixing the three fermentation broths.
[0007] The third technical solution of the present invention is the application of the compound microbial agent in the control of root-knot nematodes.
[0008] The fourth technical solution of the present invention is a method for controlling root-knot nematodes, which involves applying the compound microbial agent to the soil by fertigation or drenching the roots of plants.
[0009] Based on the above technical solution, the present invention has the following technical effects:
[0010] The compound microbial preparation disclosed in this invention has eco-friendliness as its core advantage. Through the synergistic effect of multiple microbial species, it precisely targets the survival and reproduction pathways of root-knot nematodes, not only effectively inhibiting nematode hatching and significantly reducing their survival rate, but also sustainably reducing the nematode parasitism density in the roots of cucumbers, tomatoes, and angelica, forming a long-lasting protective barrier. Compared with traditional chemical agents, this preparation uses natural microbial metabolites, leaving no residue and causing no pollution, effectively maintaining the soil microecological balance. At the same time, its unique growth-promoting mechanism can stimulate crop root vitality, significantly improving the fruit quality and yield of cucumbers and tomatoes while enhancing the control effect of root-knot nematodes, achieving the dual benefits of ecological protection and increased agricultural production. Attached Figure Description
[0011] To more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the drawings used in the embodiments will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present invention. For those skilled in the art, other drawings can be obtained based on these drawings without creative effort.
[0012] Figure 1 The effects of different fungicide treatments on nematode mortality.
[0013] Figure 2 The effects of different microbial agents on the population density of root-knot nematodes in soil. Detailed Implementation
[0014] Various exemplary embodiments of the present invention will now be described in detail. This detailed description should not be considered as a limitation of the present invention, but rather as a more detailed description of certain aspects, features, and embodiments of the present invention.
[0015] It should be understood that the terminology used in this invention is merely for describing particular embodiments and is not intended to limit the invention. Furthermore, with respect to numerical ranges in this invention, it should be understood that each intermediate value between the upper and lower limits of the range is also specifically disclosed. Every smaller range between any stated value or intermediate value within a stated range, and any other stated value or intermediate value within said range, is also included in this invention. The upper and lower limits of these smaller ranges may be independently included or excluded from the range.
[0016] Unless otherwise stated, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art. While only preferred methods and materials have been described herein, any methods and materials similar or equivalent to those described herein may be used in the implementation or testing of this invention. All references to this specification are incorporated by way of citation to disclose and describe methods and / or materials associated with those references. In the event of any conflict with any incorporated reference, the content of this specification shall prevail.
[0017] Various modifications and variations can be made to the specific embodiments described in this specification without departing from the scope or spirit of the invention, as will be apparent to those skilled in the art. Other embodiments derived from this specification will also be obvious to those skilled in the art. This application specification and embodiments are merely exemplary.
[0018] The terms “include,” “including,” “have,” “contain,” etc., used in this article are all open-ended terms, meaning that they include but are not limited to.
[0019] Unless otherwise specified, the technical solutions described in this invention are all conventional solutions in the field, and the reagents or raw materials used are all purchased from commercial channels or are publicly available unless otherwise specified.
[0020] This invention provides a composite microbial agent composed of Streptomyces rochei S36, Trichoderma sp. M2, and Bacillus velezensis Y10.
[0021] In some specific implementation schemes, the actinomycete S36 was deposited on December 5, 2024, at the China General Microbiological Culture Collection Center (CGMCC), located at No. 3, Courtyard 1, Beichen West Road, Chaoyang District, Beijing, with accession number CGMCC No. 32928; the Trichoderma M2 was deposited on December 5, 2024, at the China General Microbiological Culture Collection Center (CGMCC), located at No. 3, Courtyard 1, Beichen West Road, Chaoyang District, Beijing, with accession number CGMCC No. 41651; and the Bacillus belyesensis Y10 was deposited on December 5, 2024, at the China General Microbiological Culture Collection Center (CGMCC), located at No. 3, Courtyard 1, Beichen West Road, Chaoyang District, Beijing, with accession number CGMCC No. 32927.
[0022] The present invention also provides a method for preparing the composite microbial agent, comprising the following steps: inoculating Actinomycetes S36, Trichoderma M2 and Bacillus belye Y10 into a culture medium for culture to obtain three fermentation broths, and then mixing the three fermentation broths.
[0023] In some specific implementations, the culture medium is PDA medium; the culture conditions include: 180 r, cultured at 28°C for 3 days.
[0024] In some specific implementation schemes, the volume ratio of the three fermentation broths of Actinomycetes S36, Trichoderma M2 and Bacillus belyssus Y10 is (1-3):(1-2):(0.02-1).
[0025] This invention also provides the application of the compound microbial agent in the control of root-knot nematodes.
[0026] This invention also provides a method for controlling root-knot nematodes, which involves applying the compound microbial agent to the soil by fertigation or drenching the roots of plants.
[0027] In this embodiment of the invention, the root-knot nematodes were obtained from the Biocontrol Microbiology Research Laboratory of the Institute of Plant Protection, Beijing Academy of Agricultural and Forestry Sciences; Actinomycetes S36, Trichoderma M2 and Bacillus belye Y10 were isolated from nematode-contaminated soil and saline-alkali soil samples (sampling depth 5-15cm) in Qinghai Province.
[0028] Example 1
[0029] The S36 fermentation broth was prepared by inoculating actinomycete S36 into PDA medium and culturing at 180 rpm and 28°C for 3 days, until the viable cell count reached 5.0 × 10⁻⁶. 9 CFU / mL.
[0030] The M2 fermentation broth was prepared by inoculating Trichoderma M2 onto PDA medium and culturing at 180 rpm and 28°C for 3 days, until the viable cell count reached 5.0 × 10⁻⁶. 9 CFU / mL.
[0031] The Y10 fermentation broth was prepared by inoculating Bacillus belye Y10 onto PDA medium and culturing it at 180 rpm and 28°C for 3 days, until the viable cell count reached 5.0 × 10⁻⁶. 9 CFU / mL.
[0032] The preparation method of F1 fermentation broth is as follows: mix the volumes of three fermentation broths, S36, M2 and Y10, in a ratio of 3:1:1, and dilute them 50 times before use.
[0033] The control effects of different fungicide treatments on root-knot nematodes
[0034] Second-instar larvae of root-knot nematodes were isolated using the Bellman funnel method, and the nematode suspension concentration was adjusted to 100 larvae / mL with sterile water. The culture plate was divided into 5 groups, each containing 32 wells. Group 1 was the control group, with 50 μL of sterile water added to each well; Group 2 had 50 μL of S36 fermentation broth added to each well; Group 3 had 50 μL of M2 fermentation broth added to each well; Group 4 had 50 μL of LY10 fermentation broth added to each well; and Group 5 had 50 μL of LF1 fermentation broth added to each well. After addition, the culture plate was gently shaken to ensure even distribution of the inoculum or sterile water in the wells.
[0035] Nematode inoculation: Using a pipette, add 50 μL of nematode suspension at a concentration of 100 nematodes / mL to each well, ensuring an initial nematode count of approximately 5 nematodes per well. After adding the sample, gently shake the culture plate again to ensure thorough mixing of the nematodes with the bacterial agent (or sterile water). To prevent moisture evaporation, cover the culture plate with a breathable membrane.
[0036] Culture: Place the culture plate in a 25℃ constant temperature incubator and culture it. During the culture process, keep the environment inside the incubator stable and avoid vibration and temperature fluctuations.
[0037] During observation, the culture plate was removed from the incubator and placed on the microscope stage. First, a 4× objective lens was used to scan each well to roughly determine the distribution of the nematodes. Then, the objective lens was switched to 10× for detailed observation and counting of the nematodes within the wells. Since the nematodes may aggregate within the wells, the entire well must be carefully scanned during observation to ensure that no surviving nematodes are missed.
[0038] Data Recording: Prepare a recording table to record the number of surviving nematodes in each well at different time points for each treatment group. For nematodes whose survival is difficult to determine, gently blow the liquid in the well with a pipette and observe whether the nematodes move; if they move, they are considered alive. Calculate the average number of surviving nematodes for each treatment group.
[0039] Mortality rate (%) = (number of nematode deaths / total number of nematodes) × 100.
[0040] The results are as follows Figure 1 As shown, all of the several fungal agents can effectively increase the mortality rate of nematodes and achieve the effect of killing nematodes. However, the compound fungal agent F1 has the most significant nematode-killing effect and the largest increase in nematode mortality rate, which is significantly better than the three individual fungal agent treatments.
[0041] Example 2
[0042] The control effects of different inoculants on cucumber root-knot nematodes
[0043] This study investigated the efficacy of microbial agents against root-knot nematodes (Meloidogyne spp.) in farmland soils severely infested with cucumbers due to long-term continuous cropping, aiming to explore its impact on cucumber yield. A randomized controlled design was employed, with experimental and control groups (each group covering 5 m²). 2 (30 plants were planted), and each treatment was repeated 3 times.
[0044] The specific implementation process of the experiment is as follows: Five days before cucumber sowing, during the land preparation stage, the experimental group was treated with microbial agents by irrigation at a dosage of 10 L / mu. During the cucumber seedling stage, initial flowering stage, and fruiting stage, an additional 20 L / mu of microbial agents was applied, continuing until the cucumber plants were removed. The control group was treated with an equal amount of water instead of microbial agents at the same agricultural operation points. Throughout the experimental period, both the experimental and control groups implemented the same field management measures, including irrigation, fertilization, and pest and disease control. After the cucumbers were harvested and the vines were removed, root-knot nematodes were isolated from the soil, their population density was counted, and the control effect of the microbial agents was evaluated. Simultaneously, the actual yield of cucumbers in each group was recorded.
[0045] Disease severity grading standards:
[0046] Grade 0: No root knots on the root system; Grade 1: Mild infection, with only a few small root knots, and a root knot rate of less than 3%; Grade 2: Root knot rate of 3% to 25%; Grade 3: Numerous root knots, with a root knot rate of 25% to 50%; Grade 4: Secondary root knots on the root nodules, with 50% to 75% of the root system having root knots; Grade 5: Root knots interconnect to form root knot clumps, with more than 75% of the root system having root knots.
[0047] Calculation method:
[0048] Disease index (%) = Σ(number of diseased plants at each level × relative level value) / (total number of plants surveyed × 7) × 100;
[0049] Prevention and control effect (%) = (disease index of blank control area - disease index of treatment area) / (disease index of blank control area) × 100.
[0050] Table 1
[0051]
[0052] As shown in Table 1, compared with the control group, all treatment groups showed certain control effects, while the F1 treatment group was particularly outstanding. Its root-knot nematode density dropped to 2.98 nematodes / g, the lowest among all treatment groups; cucumber yield increased by 23.6%, higher than other groups; the disease index was only 19.28, and the control effect was as high as 73.42%, significantly better than the S36, M2, and Y10 treatment groups, which fully demonstrates the excellent effect of F1 in reducing nematode density, increasing cucumber yield, and controlling diseases.
[0053] Example 3
[0054] The control effects of different inoculants on tomato root-knot nematodes
[0055] Four-week-old tomato seedlings were transplanted into diseased soil and divided into experimental and control groups, with 30 seedlings in each group. The experimental group received root drenching with a microbial agent, followed by drenching again every 30 days, at a rate of 500 mL per seedling, for a total of three applications. Each treatment was planted in double rows of 15 seedlings, with three replicates per treatment. Routine management was maintained throughout the experiment, with normal watering and fertilization for all treatments. The experiment lasted 90 days. The population size of root-knot nematodes in the soil was measured 90 days after transplanting. (Second instar larvae of root-knot nematodes in the soil, J2)
[0056] After transplanting, the growth of tomatoes was continuously observed and recorded, along with any phytotoxicity caused by the applied inoculant. Thirty days after transplanting, tomato growth was assessed using a W-shaped sampling method at five points, with five plants measured per treatment. Plant height was measured with a tape measure, and stem diameter at 3cm from the base of the plant was measured with calipers. Three leaves were collected from each of the upper, middle, and lower parts of the plant, and the leaf area was calculated using a grid method. Harvesting and weighing were conducted weekly until fruit maturity. Ninety days after transplanting, the vines were pulled up, and the tomato yield for each treatment during the harvest period was recorded.
[0057] Root-knot nematode disease was investigated 90 days after tomato transplanting. Five samples were taken in a W-shape. Tomato roots were dug up, soil was washed away, and disease severity was graded, with five plants per treatment. Disease index and control efficacy were calculated.
[0058] Disease index = ∑(number of plants at each level × level) / (total number of plants surveyed × highest level) × 100; Control efficacy = (control disease index - treatment disease index) / control disease index × 100%.
[0059] Table 2
[0060] deal with Plant height / cm Stem diameter / cm <![CDATA[Leaf area / cm 2 > Severity index (90 days) Prevention and control efficacy (100%) CK 75.32 5.34 183.72 76 / S36 78.33 5.67 193.56 28 63.16 M2 76.53 5.32 190.87 32 57.89 Y10 79.81 5.89 208.37 24 68.42 F1 80.69 6.03 210.46 16 78.95
[0061] Depend on Figure 2 As shown in Table 2, the data and bar charts indicate that the soil J2 density in the CK (control group) was the highest, reaching approximately 400 nematodes / 100g. After different treatments, the soil J2 density in groups S36, M2, Y10, and F1 all decreased. Among them, the soil J2 density in the F1 treatment group was significantly lower than that in the other treatment groups, with the lowest value. This indicates that F1 can effectively inhibit root-knot nematodes J2, demonstrating outstanding effects in reducing nematode density and mitigating their damage to crops. It creates a healthier rhizosphere environment for crop growth, contributing to improved nematode control effectiveness and crop yield and quality.
[0062] Example 4
[0063] The control effects of different microbial agents on root-knot nematodes in Angelica sinensis
[0064] The bacterial fermentation liquid was used for root irrigation three times throughout the growing season (50 plants were planted in each experimental group). Each plant was irrigated with 300 mL of bacterial liquid to facilitate the investigation of the control effect of root-knot nematode disease during the harvest period and to determine the number of second-instar root-knot nematode larvae in the soil.
[0065] Grading standards for the investigation of Angelica sinensis root-knot nematode disease:
[0066] Level 0, no root knots on the root system;
[0067] Grade 1, mild infection, with only a few small root knots, and a root knot rate of less than 3%;
[0068] Grade 3, with numerous root knots, the root knot rate is 25% to 50%;
[0069] Grade 4, with secondary root knots on the root nodules, and 50%–75% of the root system having root knots;
[0070] Level 5: Root knots are interconnected to form root knot clusters, with more than 75% of the root system having root knots.
[0071] The following formula is used to calculate the prevention and control effect:
[0072] Disease index = [(Σ(number of disease-grade plants × number of representative grades)) / (total number of plants × highest representative grade value)] × 100;
[0073] Prevention and control effect (%) = [(disease index of control group - disease index of treatment group) / disease index of control group] × 100%.
[0074] Table 3
[0075] deal with Number of second-instar larvae / heads Nematode reduction rate / % Disease index Prevention and control efficacy / % CK 218 / 36 / S36 67 69.27 18 50 M2 93 57.34 20 44.44 Y10 56 74.31 13.6 62.22 F1 37 83.03 11.2 68.89
[0076] Table 3 shows that the F1 treatment outperformed the others in all indicators: the number of second-instar larvae was only 37, and the nematode reduction rate reached 83.03%, significantly higher than S36 (69.27%), M2 (57.34%), and Y10 (74.31%); the disease index was 11.2, and the control effect was 68.89%, the best among all treatment groups. This indicates that F1 can effectively suppress second-instar larvae, significantly reduce nematode density and disease severity, and has a significant effect on nematode control, effectively protecting crops and creating a healthier environment for crop growth, demonstrating a clear advantage in controlling root-knot nematodes.
[0077] Obviously, the above embodiments of the present invention are merely examples for clearly illustrating the present invention, and are not intended to limit the implementation of the present invention. For those skilled in the art, other variations or modifications can be made based on the above description. It is neither necessary nor possible to exhaustively describe all embodiments here. Any modifications, equivalent substitutions, and improvements made within the spirit and principles of the present invention should be included within the scope of protection of the claims of the present invention.
Claims
1. A compound microbial agent, characterized in that, It is composed of Streptomyces rochei S36, Trichoderma sp. M2 and Bacillus velezensis Y10.
2. The compound microbial agent according to claim 1, characterized in that, The actinomycete S36 was deposited on December 5, 2024, at the China General Microbiological Culture Collection Center (CGMCC), located at No. 3, Courtyard 1, Beichen West Road, Chaoyang District, Beijing, with accession number CGMCC No. 32928; the Trichoderma M2 was deposited on December 5, 2024, at the China General Microbiological Culture Collection Center (CGMCC), located at No. 3, Courtyard 1, Beichen West Road, Chaoyang District, Beijing, with accession number CGMCC No. 41651; and the Bacillus belyssus Y10 was deposited on December 5, 2024, at the China General Microbiological Culture Collection Center (CGMCC), located at No. 3, Courtyard 1, Beichen West Road, Chaoyang District, Beijing, with accession number CGMCC No. 32927.
3. The method for preparing the composite microbial agent as described in claim 1, characterized in that, Includes the following steps: Actinomycetes S36, Trichoderma M2, and Bacillus beleibrio Y10 were inoculated into a culture medium and cultured to obtain three fermentation broths, which were then mixed together.
4. The preparation method according to claim 3, characterized in that, The culture medium is PDA medium; the culture conditions include: 180 r, cultured at 28℃ for 3 days.
5. The preparation method according to claim 3, characterized in that, The volume ratio of the fermentation broths of Actinomycetes S36, Trichoderma M2 and Bacillus belysium Y10 was (1-3):(1-2):(0.02-1).
6. The application of the compound microbial agent as described in claim 1 or 2 in the control of root-knot nematodes.
7. A method for controlling root-knot nematodes, characterized in that, The compound microbial agent described in claim 1 or 2 can be used for soil drenching or for root irrigation of plants.