Wild biocontrol bacterium composition and application

Abstract

The invention discloses a wild biocontrol microbial agent, which is characterized in that the wild biocontrol microbial agent is bacillus and paecilomyces which are separated, purified and screened from Jiangpermanent xiangxiang ginger planting land in Yangzhou City of Hunan Province. The composition formed by the wild biocontrol inoculant, fosthiazate and abamectin has a synergistic effect on prevention and control of nematodes or nematode eggs, has the characteristics of small environmental pollution, safety and low cost, and is a green prevention and control method capable of playing a continuous prevention and control role.

Description

Technical Field

[0001] The invention belongs to the field of biotechnology, and in particular relates to a wild biocontrol fungus for preventing and treating root-knot nematode disease, a composition and an application thereof. Background Art

[0002] Root-knot nematode disease is a common soil-borne disease in crop cultivation, primarily caused by nematodes parasitizing crop roots. It affects a wide range of crops, including common fruits and vegetables such as ginger, taro, soybeans, cucumbers, tomatoes, potatoes, sweet potatoes, bananas, citrus fruits, and watermelons. The disease primarily harms crop roots, causing root knots in the diseased roots, which block and impair root conduction. This can affect plant growth, cause leaf yellowing, and reduce yields, even leading to crop failure in severe cases. It can also trigger other soil-borne fungal diseases caused by fungi such as Fusarium and Pythium.

[0003] Currently, the primary methods for controlling root-knot nematodes are agricultural and chemical control, supplemented by biological control. Agricultural control involves strengthening quarantine measures and planting robust, disease-resistant varieties; implementing a water-land rotation system and crop rotation with grasses; deep plowing of diseased soil after harvest to reduce the number of nematode eggs and larvae that overwinter; and increasing the use of high-phosphorus and high-potassium compound fertilizers to improve soil fertility and enhance crop resistance. Agricultural control can only reduce the incidence of nematodes, but its effectiveness is far from adequate. Chemical control typically involves fumigation, using agents such as chloropicrin or methyl bromide mixed in appropriate proportions and applied to the soil. Chloropicrin is volatile and, upon entering the nematode body, causes cell poisoning and death. Fumigation is effective against nematodes, but is less effective against nematode eggs and cannot eradicate root-knot nematodes. Other commonly used chemical pesticides on the market include chlorpyrifos, cypermethrin, oxamyl, Shennongdan, and carbofuran, which kill nematodes through contact, stomach poisoning, or systemic action. Long-term application of chemical pesticides can easily lead to drug resistance in nematodes and can be highly detrimental to beneficial bacteria in the soil, leading to pesticide residues, environmental pollution, and ecological degradation. Furthermore, the application of chemical pesticides is typically manual, which is not only costly but also harmful to the workers through volatilization.

[0004] Currently, new pesticides such as thiazolyl, abamectin, and emamectin benzoate demonstrate excellent control performance with high efficiency and low toxicity, but they also have some limitations. Thiazolyl is an insect neurotoxin that is ingested by pests through systemic uptake within plants, causing poisoning and death. It has broad-spectrum activity against pests such as nematodes, mites, and various pterosaurs, making it an excellent agent for controlling root-knot nematodes. However, due to its inherent ease of decomposition and ineffectiveness, thiazolyl has a short duration of effectiveness. Excessive use can easily cause phytotoxicity, which can range from inhibiting crop root growth to directly killing the crop. Abamectin also possesses broad-spectrum, high-efficiency insecticide properties with a long residual effect, resulting in even better control effectiveness. Therefore, thiazolyl is often used in combination with pesticides such as abamectin. The combination of the two achieves complementary effects while simultaneously killing nematodes, resulting in a significant synergistic effect. However, a significant drawback is that it does not kill eggs.

[0005] Biological control primarily utilizes naturally occurring biocontrol bacteria to antagonize harmful pathogens. It is characterized by minimal environmental pollution, safety, and low cost, making it a green, sustainable control method. The common antagonistic bacterium, Paecilomyces lilacinus, is well known for its effectiveness against nematodes and insect eggs. However, compared to chemical pesticides, it is expensive and has numerous practical limitations regarding soil porosity, temperature, humidity, pH, and nutrients.

[0006] Through searching, no patent publication documents related to the patent application of the present invention have been found. Summary of the Invention

[0007] The present invention aims to overcome the deficiencies in the prior art and provide a wild biocontrol bacterium, a composition and an application thereof for preventing and treating root-knot nematode disease.

[0008] The technical solution adopted by the present invention to solve its technical problem is:

[0009] A wild biocontrol bacterium, characterized in that the biocontrol bacterium is a mixed strain of Bacillus and Paecilomyces.

[0010] In the technical solution of the present invention, the wild biocontrol bacteria are characterized in that the wild biocontrol bacteria are Bacillus and Paecilomyces isolated, purified and screened from the Jiangyong fragrant ginger planting area in Yongzhou City, Hunan Province.

[0011] In the technical solution of the present invention, the ratio of the Bacillus and the Paecilomyces is 1:10 to 10:1, preferably 1:5 to 5:1, preferably 1:2 to 2:1, and more preferably 1:1.

[0012] In addition, the present invention also provides a wild biocontrol fungus agent, comprising the wild biocontrol fungus of the present invention and other components.

[0013] Furthermore, in the technical solution of the present invention, the other ingredients include any one of peanut bran powder, shrimp head powder, corn starch, and glycerin, or a combination thereof.

[0014] Furthermore, the present invention also provides a method for preparing a wild biocontrol agent, which is characterized by comprising the following steps:

[0015] The wild biocontrol bacteria of the present invention are prepared into a bacterial suspension, which is evenly mixed with sterilized peanut bran powder, shrimp head powder, corn starch, glycerin and water, and then dried to obtain the product.

[0016] In the technical solution of the present invention, the drying is blast drying, and the drying temperature is 20-100°C.

[0017] Furthermore, the present invention also provides a use of the wild biocontrol bacteria or wild biocontrol fungicide of the present invention in killing nematodes or nematode eggs.

[0018] In the technical solution of the present invention, the nematodes are Aphelenchoides, Meloidogyne, Heterodera, Globodera, Nacobbus, Pratylenchus, Ditylenchus, Xiphinema, Longidorus, and Trichodorus.

[0019] Furthermore, the present invention also provides a wild biocontrol fungus composition, comprising the wild biocontrol fungus agent of the present invention, thiazolyl and abamectin.

[0020] In the composition of the present invention, the volume percentage of the wild biocontrol fungus agent in the composition is 0.1% to 1.0%, the volume percentage of thiazolyl is 0.01% to 1.2%, the volume percentage of avermectin is 0.01% to 2.0%, and the balance is water.

[0021] The preferred formula includes 0.2% to 0.8% by volume of wild microbial agent, 0.05% to 0.8% of thiazolyl, and 0.01% to 1.0% of avermectin.

[0022] A more preferred formulation includes 0.4% by volume of wild microbial agent, 0.04% of thiazolyl, and 0.04% of abamectin.

[0023] The beneficial effects brought about by the technical solution provided by the present invention are:

[0024] 1. The present invention obtains a mixed biocontrol bacteria with good killing effect on root-knot nematodes and nematode eggs through in situ screening and isolation at the Jiangyong fragrant ginger planting site. The root-knot nematodes are controlled by contact killing, stomach poisoning or systemic killing, and the killing effect on nematode eggs is particularly good. The present invention can also be used to control root-knot nematode diseases in other crops including but not limited to taro, soybean, cucumber, tomato, potato, sweet potato, banana, citrus including grapefruit and watermelon.

[0025] 2. The raw materials of the wild microbial agent in the present invention are easily available, simple to prepare, low in cost, and in situ screened biocontrol bacteria to antagonize harmful pathogens, which conforms to the laws of nature itself and does not affect the sustainable development of the ecological environment.

[0026] 3. The thiazolylphos and abamectin in the present invention are effective in controlling nematodes quickly but do not kill eggs. Wild microbial agents not only have a good killing effect on nematodes but also have a good killing effect on nematode eggs. In addition, the action cycle is long-lasting and environmentally friendly. The combination of the three provides a reliable solution and idea for the control of root-knot nematode disease.

[0027] 4. The present invention provides a highly effective, low-toxic, inexpensive, and environmentally friendly root-knot nematode control composition for the effective and long-lasting control of root-knot nematodes and nematode eggs. The wild biocontrol bacteria, screened from the wild, are not only highly effective against nematode eggs, but also cost-effective and harmless to the ecological environment in the application area. Furthermore, they provide a long-lasting preventive effect after killing nematodes and nematode eggs.

[0028] 5. The present invention in situ screens out wild biocontrol bacteria with good control effects on root-knot nematodes and nematode eggs, and uses them in combination with the new, highly effective, and low-toxic pesticides thiazolyl and avermectin. The advantages of pesticide control being quick to take effect and wild microbial agents being effective and having a long-lasting action cycle are utilized, and the advantages of both complementing each other are achieved. This provides a new approach to the control of root-knot nematode disease.

[0029] 6. The present invention contains two wild biocontrol bacteria, identified by high-throughput sequencing as Bacillus and Paecilomyces. These two strains were screened in situ and shown to synergize with the low-toxic pesticides thiazolyl and avermectin, resulting in excellent nematode-killing efficacy. The addition of wild biocontrol bacteria significantly enhances the composition's effectiveness against nematode eggs. This composition has excellent control and prevention effects against root-knot nematode disease, can reduce pesticide use, save costs, reduce agricultural residues, and minimize pollution. It can be used to prevent and control root-knot nematode disease in a variety of vegetables and fruits, including ginger, taro, and citrus, and has broad application prospects. BRIEF DESCRIPTION OF THE DRAWINGS

[0030] Figures 1 to 5 This is a graph showing the growth inhibition of wild microbial strains by the dilution of thiazolyl in the present invention; wherein, Figure 1 This is a graph showing the growth inhibition of wild microbial strains by 10-fold dilution of thiazolyl. Figure 2This is a graph showing the growth inhibition of wild microbial strains by 100-fold dilution of thiazolyl; Figure 3 This is a graph showing the growth inhibition of wild microbial strains by 1000-fold dilution of thiazolyl; Figure 4 This is a graph showing the growth inhibition of wild microbial strains by 10,000-fold dilution of thiazolyl;

[0031] Figures 5 to 8 This is a graph showing the growth inhibition of wild microbial strains by the dilution of avermectin in the present invention; wherein, Figure 5 This is a graph showing the growth inhibition of wild microbial strains by 10-fold dilution of avermectin; Figure 6 This is a graph showing the growth inhibition of wild microbial strains by 100-fold dilution of avermectin; Figure 7 This is a graph showing the growth inhibition of wild microbial strains by 1000-fold dilution of avermectin; Figure 8 This is a graph showing the growth inhibition of wild microbial strains by 10,000-fold dilution of avermectin;

[0032] Figure 9 This is a diagram showing the growth of the wild microbial agent strains of the present invention on a plate containing an equal amount of water;

[0033] Figure 10 、 Figure 11 This is a comparison of nematode activity before and after treatment with No. 18 in the present invention; Figure 10 This is a graph of nematode vitality before treatment in test No. 18; Figure 11 This is a graph of nematode vitality after treatment in Experiment 18;

[0034] Figure 12 、 Figure 13 This is a comparison of nematode egg activity before and after treatment with No. 6 in the present invention; wherein, Figure 12 This is a graph of nematode egg vitality before treatment in test No. 6; Figure 13 This is a graph showing nematode egg vitality after treatment in Experiment 6;

[0035] Figure 14 This is a picture of nematode eggs being wrapped by mycelium of wild microbial inoculant in the present invention. DETAILED DESCRIPTION

[0036] The following is a detailed description of an embodiment of the present invention. It should be noted that this embodiment is descriptive rather than restrictive and should not be used to limit the scope of protection of the present invention.

[0037] Unless otherwise specified, the raw materials used in the present invention are conventional commercial products; the methods used in the present invention are conventional methods in the art unless otherwise specified.

[0038] Example 1: Strain screening

[0039] Wild biocontrol bacteria strains for controlling root knot nematodes were isolated, purified, and screened from a ginger planting area in Jiangyong, Yongzhou. The screening method is as follows:

[0040] (1) Isolation of strains: Soil samples were taken from the rhizosphere soil of the fragrant ginger plantation in Yongzhou. 10 g of soil was weighed and added to a triangular flask containing 90 mL of sterile water. The mixture was shaken at 37 °C and 180 rpm for 20 min and then allowed to stand for 15 min. The supernatant was diluted to 10 ﹣6 Concentration. Serial dilutions were plated onto NA medium and PDA medium, respectively, and incubated in a 37°C incubator for 48 hours. Different colonies were streaked onto NA medium and PDA medium to obtain purified Bacillus and Paecilomyces strains, respectively. The strains were stored in NB medium or PDB liquid medium in a refrigerator at 4°C.

[0041] Example 2. Isolation of Root-Knot Nematodes in Zingiber officinale: A diseased root of Zingiber officinale was collected from a Zingiber officinale plantation, rinsed with sterile water, and placed in a flask containing 250 mL of 0.5% sodium hypochlorite solution. The mixture was shaken for 3 minutes. The mixture was filtered through a 30 μm pore size sieve, and the filtrate was collected to obtain an egg suspension. The worm suspension was then incubated for 24 hours.

[0042] Example 3. Insecticidal Verification: 3 mL of the worm and egg suspensions were placed in a 6 cm diameter Petri dish. 1 mL of the strain culture medium or a 2-, 4-, or 8-fold dilution was added, along with an equal amount of normal saline as a control group. Three replicates were performed in each group. The mixtures were incubated at 28°C for 24 and 48 hours. The mortality of nematodes or eggs was examined microscopically, and the strain with the best insecticidal and egg-killing effects was selected.

[0043] Example 4: Determination of the toxicity of thiazolyl to strains in wild microbial inoculants

[0044] Prepare plates containing different pesticide concentrations: Dilute thiazolyl with sterile water to a series of concentrations, such as 10, 100, and 1000. Add 10 mL of the original solution and the diluted solution to a flask containing 90 mL of beef extract peptone medium. Pour the plates into the plates to obtain plates containing pesticide diluted 10, 100, 1000, and 10,000 times. Add an equal amount of sterile water as a blank control.

[0045] Preparation of bacterial suspensions of different repair agents: weigh 2g of wild microbial agent and add it to a centrifuge tube filled with 18mL of sterile water, shake it at 37℃ for 20min, and let it stand for 15min; then take 1mL of the supernatant and add it to a centrifuge tube filled with 9mL of sterile water, and dilute it to 10 －3 Dilution.

[0046] Plate coating: Take 100 μL 10 －3The diluted bacterial suspension was spread on the plate and cultured at 37℃ for 2 days. Figures 1 to 4 As shown, it can be seen that thiazolyl does not affect the growth of strains in the wild microbial inoculant after being diluted 100 times.

[0047] Example 5: Determination of the toxicity of avermectin against strains in wild microbial inoculants

[0048] Prepare plates containing different pesticide concentrations: Dilute avermectin with sterile water to a series of concentrations, such as 10, 100, and 1000. Add 10 mL of the original solution and the diluted solution to a flask containing 90 mL of beef extract peptone medium. Pour the plates into the plates to obtain plates containing the pesticide diluted 10, 100, 1000, and 10,000 times. Add an equal amount of sterile water as a blank control.

[0049] Preparation of bacterial suspensions of different repair agents: weigh 2g of wild microbial agent and add it to a centrifuge tube filled with 18mL of sterile water, shake it at 37℃ for 20min, and let it stand for 15min; then take 1mL of the supernatant and add it to a centrifuge tube filled with 9mL of sterile water, and dilute it to 10 －3 Dilution.

[0050] Plate coating: Take 100 μL 10 －3 The diluted bacterial suspension was spread on the plate and cultured at 37℃ for 2 days. Figures 2 to 9 As shown, it can be seen that the concentration of avermectin diluted 1000 times does not affect the growth of strains in wild microbial inoculants.

[0051] Example 6: Indoor control test of thiazolyl, avermectin and wild microbial agents on nematodes

[0052] Different ratios of thiazolyl, avermectin and wild microbial agents were tested for indoor control of nematodes. The test results are shown in Table 1.

[0053] Table 1: Indoor control effects of thiazolyl, avermectin and wild microbial agents on nematodes in different proportions (volume percentage)

[0054]

[0055]

[0056] The results showed that the ternary composition containing thiazolyl, avermectin, and microbial agent had a better control effect on root-knot nematodes than thiazolyl, avermectin alone, or a combination of the two. The best control effect on root-knot nematodes was achieved when the volume percentage of thiazolyl, avermectin, and wild microbial agent was 0.04%.

[0057] Example 7: Indoor control test of thiazolyl, avermectin and wild microbial agents on nematode eggs

[0058] Different ratios of thiazolyl, avermectin and wild microbial agents were tested for indoor control of nematode eggs. The test results are shown in Table 2.

[0059] Table 2: Indoor control effects of different proportions (volume percentage) of thiazolyl, avermectin and wild microbial agents on nematode eggs

[0060]

[0061]

[0062] The results showed that the ternary composition containing thiazolyl, avermectin, and microbial agent had a better control effect on root-knot nematode eggs than thiazolyl, avermectin, or a combination of the two.

[0063] Although the embodiments of the present invention are disclosed for illustrative purposes, those skilled in the art will understand that various substitutions, changes and modifications are possible without departing from the spirit and scope of the present invention and the appended claims. Therefore, the scope of the present invention is not limited to the contents disclosed in the embodiments.

Claims

1. A wild biocontrol bacterium, characterized in that: The wild biocontrol bacteria are mixed strains of Bacillus and Paecilomyces.

2. The wild biocontrol bacterium according to claim 1, wherein: The wild biocontrol bacteria are separated, purified and screened from a ginger planting area in Jiangyong, Yongzhou City, Hunan Province.

3. The wild biocontrol bacterium according to claim 1 or 2, wherein the ratio of the Bacillus to the Paecilomyces is 1:10 to 10:1, preferably 1:5 to 5:1, preferably 1:2 to 2:1, and more preferably 1:1, in terms of mass percentage.

4. A wild biocontrol fungicide comprising the wild biocontrol fungus according to any one of claims 1 to 3 and other ingredients.

5. The wild biocontrol agent according to claim 4, wherein the other ingredients comprise any one of peanut bran powder, shrimp head powder, corn starch and glycerin or a combination thereof.

6. The method for preparing the wild biocontrol agent according to claim 5, wherein: The following steps are involved: The wild biocontrol bacteria according to any one of claims 1 to 3 is prepared into a bacterial suspension, which is evenly mixed with sterilized peanut bran powder, shrimp head powder, corn starch, glycerin and water, and then dried to obtain the product.

7. Use of the wild biocontrol bacterium according to any one of claims 1 to 3 or the wild biocontrol agent according to any one of claims 4 to 5 in killing nematodes or nematode eggs.

8. The use according to claim 7, wherein the nematode is Aphelenchoides, Meloidogyne, Heterodera, Globodera, Nacobbus, Pratylenchus, Ditylenchus, Xiphinema, Longidorus, or Trichodorus.

9. A wild animal biocontrol fungus composition comprising the wild animal biocontrol fungus agent according to any one of claims 4 to 5, thiazolyl and abamectin.

10. The composition according to claim 9, wherein Calculated by volume percentage, the volume percentage of the wild biocontrol fungus agent in the composition is 0.1% to 1.0%, the volume percentage of thiazolyl is 0.01% to 1.2%, the volume percentage of avermectin is 0.01% to 2.0%, and the balance is water.

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