Clostridium beijerinckii for preventing and controlling plant parasitic nematode diseases and its application

Abstract

A Clostridium beijerinckii for preventing and treating plant parasitic nematode diseases and its application, belonging to the technical field of microbial pesticides. Clostridium beijerinckii ) Sneb518 or the fermentation liquid or the product of the fermentation liquid has strong toxic activity against the plant parasitic nematode J2. The present invention targets plant parasitic nematodes, screens microorganisms that have killing activity against plant parasitic nematodes, and prevents and controls plant parasitic nematode diseases, providing a new resource for the research and development of new biological nematicides.

Description

Technical Field

[0001] The present invention belongs to the technical field of microbial pesticides, and particularly relates to Clostridium beijerinckii for preventing and treating plant parasitic nematode diseases and an application thereof. Background Art

[0002] Plant parasitic nematodes occur and cause damage in many countries and regions around the world, and there are many different species. Among them, soybean cyst nematode disease and root knot nematode disease are particularly harmful to crops. Soybean cyst nematode disease (SCN) is caused by soybean cyst nematode ( Heterodera glycines ) infection, is one of the most serious diseases that harm soybean production worldwide. This disease is characterized by wide distribution, severe damage, a wide host range, multiple transmission routes, and long survival time of dormant bodies (cysts). It is a soil-borne disease that is extremely difficult to control. Meloidogyne spp.) is a common root disease in agricultural production. First discovered in cucumber roots in the UK (Sasser, 1989), it is a significant pathogenic nematode that causes economic losses in crops. In recent years, with improved living standards, the market demand for off-season vegetables has been steadily increasing, and the area of ​​greenhouses in northern China is increasing annually. Currently, chemical control is still the primary method for the prevention and control of plant-parasitic nematodes. However, the use of chemical nematicides has been associated with significant waste, environmental pollution, poor selectivity, and high lethality, disrupting soil ecological balance and causing environmental pollution. Highly toxic pesticide residues remain in agricultural products, seriously endangering human health. These drawbacks are attracting increasing attention. Therefore, research on the biological control of plant-parasitic nematodes has naturally become a hot topic.

[0003] Over the past 20 years, with the development of sustainable and organic agriculture, the focus of nematode control efforts has shifted to biological control. Biopesticides are a key initiative in the industrialization of high-tech agriculture worldwide in the 21st century and hold the key to addressing the sustainable development of modern agriculture. Their greatest advantage lies in their ability to mitigate the environmental pollution caused by chemical pesticides and reduce pesticide residues in agricultural and sideline products, thus enabling sustainable agricultural development. Consequently, research on the biological control of plant-parasitic nematodes has naturally become a hot topic. Biological control is minimally destructive to the environment, generally acting only on the target species and not causing harm to birds, insects, fish, or mammals. Microorganisms are diverse and abundant, offering enormous potential for development. Consequently, biological control is garnering increasing attention. Biological control of plant-parasitic nematodes primarily utilizes microorganisms such as fungi, bacteria, and actinomycetes, or their metabolites and volatile gases.

[0004] In order to enrich the diversity of nematode-killing microbial strains and better prevent and control plant parasitic nematode diseases, the present invention provides a Clostridium strain that is easy to reproduce, has strong environmental adaptability and is environmentally friendly, and can solve the problem of plant parasitic nematode diseases to a certain extent by using fermentation liquid seed coating or root irrigation treatment. Summary of the Invention

[0005] In order to better prevent and control plant parasitic nematode diseases and enrich the diversity of nematode-killing microbial species, the purpose of the present invention is to provide a Clostridium beijerinckii for preventing and controlling plant parasitic nematode diseases and its application, which does not pollute the soil environment, simplifies the prevention and control methods of plant parasitic nematode diseases, and has good development and application prospects.

[0006] The purpose of the present invention is to achieve the following technical solutions: a Clostridium beijerinckii for preventing and treating plant parasitic nematode diseases and its application

[0007] The Clostridium beijerinckii Sneb518 has the effect of preventing and treating plant parasitic nematodes and is classified as Clostridium beijerinckii ( Clostridium beijerinckii ), which was deposited in the General Microbiology Center of China Culture Collection Administration on June 6, 2022, with the deposit number CGMCC No.25009.

[0008] The Clostridium beijerinckii for preventing and treating plant parasitic nematode diseases and the application method thereof include the following steps.

[0009] (1) Liquid fermentation culture

[0010] The bacterial cells of Clostridium beijerinckii Sneb518 are inoculated onto a slant culture medium to obtain a test tube seed, which is then inoculated into a liquid culture medium to form a fermentation broth or a fermentation broth product.

[0011] In the above scheme, the slant culture medium is a beef extract peptone culture medium, and its ingredients include: 2.5-3.5g beef extract, 4.5-5.5g peptone, 9.5-10.5g glucose, 17-20g agar, and 1000mL distilled water.

[0012] In the above scheme, the method for obtaining the test tube strain is: Clostridium beijerinckii Sneb518 bacteria are inoculated on a slant culture medium and cultured at 27.5-28.5° C. for 2.5-3.5 days to obtain the test tube strain.

[0013] In the above scheme, by weight percentage, glucose is 1%-2%, peptone is 0.5%-0.6%, beef extract is 0.3%-0.4%, yeast extract is 0.1%-0.2%, and the rest is distilled water. The pH is 7.0-7.2, the fermentation temperature is 27.5-28.5°C, the shaking speed is 175-185 r·min-1, and the fermentation time is 2.5-3.5 days.

[0014] The above scheme comprises Clostridium beijerinckii Sneb518 or Clostridium beijerinckii fermentation broth or fermentation broth product.

[0015] (2) Coating treatment

[0016] A crop coating agent, the technical key point of which is that it contains the Clostridium beijerinckii Sneb518 described in claim 1 or the Clostridium beijerinckii fermentation liquid or fermentation liquid product described in claim 3. The Clostridium beijerinckii fermentation liquid or fermentation liquid product is used to coat soybean and tomato seeds at a ratio of 1:100. After drying, the treated soybean or tomato seeds are sown normally.

[0017] (3) Root irrigation treatment

[0018] A vegetable root irrigation agent, comprising Clostridium beijerinckii Sneb518 or a fermentation broth or fermentation broth product. During the planting period of tomatoes or cucumbers, the root irrigation agent is treated with a 10x dilution of the fermentation broth or fermentation broth product, with 20 mL applied to each plant.

[0019] The beneficial effects of the present invention are as follows: the Clostridium beijerinckii for preventing and treating plant parasitic nematode diseases and its application, Clostridium beijerinckii ( Clostridium beijerinckii ) Sneb518 or the fermentation liquid or the product of the fermentation liquid has strong toxic activity against the plant parasitic nematode J2. The present invention targets plant parasitic nematodes, screens microorganisms that have killing activity against plant parasitic nematodes, and prevents and controls plant parasitic nematode diseases, providing a new resource for the research and development of new biological nematicides. DETAILED DESCRIPTION

[0020] In order to make the above-mentioned objects, features and advantages of the present invention more obvious and easy to understand, the present invention is further described in detail below in conjunction with specific embodiments.

[0021] Example 1:

[0022] The present embodiment uses a Clostridium beijerinckii strain for preventing and controlling plant parasitic nematode diseases, with a deposit number of CGMCC No. 25009, deposited with the General Microbiology Center of the China Culture Collection Administration, and deposited at the Institute of Microbiology, Chinese Academy of Sciences, No. 3, Yard 1, Beichen West Road, Chaoyang District, Beijing, on June 6, 2022. It is classified as Clostridium beijerinckii ( Clostridium beijerinckii ), strain number Sneb518.

[0023] Clostridium beijerinckii Sneb518 was isolated using a dilution separation method from the root system of a large tree in the Imperial City Ruins Garden on Beiheyan Street in Dongcheng District, Beijing. This method uses bacterial fermentation broth to coat soybean and vegetable seeds before sowing them in fields infested with nematodes. During the onset of the disease, Clostridium beijerinckii Sneb518 or its metabolites demonstrated resistance to soybean cyst nematodes and vegetable root-knot nematodes, addressing the prevention and control of these diseases to a certain extent.

[0024] The preparation method of the Clostridium beijerinckii fermentation liquid for preventing and treating plant parasitic nematode diseases in this embodiment is as follows:

[0025] Clostridium beijerinckii ( Clostridium beijerinckii Sneb518 cells were inoculated onto a slant medium containing 3g beef extract and 5g peptone, 10g glucose, and 18g agar, along with 1000mL of distilled water. The culture was incubated at 28°C for 3 days to obtain a test tube culture. This culture was then inoculated into a 250mL Erlenmeyer flask (100mL per flask) containing the following liquid medium: by weight, 0.1% yeast extract, 0.5% peptone, 0.3% beef extract, and 1% glucose, with the remainder being distilled water. The pH was maintained at 7.0, the fermentation temperature was 28°C, and the shaker speed was 180 rpm. -1 , fermentation time 3d, the resulting fermentation liquid or fermentation liquid products can be used directly or indirectly to treat soybean and tomato seeds or irrigate roots.

[0026] In this example, Clostridium beijerinckii ( Clostridium beijerinckii ) Effect of Sneb518 fermentation liquid on plant parasitic nematodes: 2 mL of Sneb518 fermentation liquid was added to a sterilized crown dish, followed by 0.5 mL of soybean cyst nematode and vegetable root knot nematode J2 nematode liquid (containing approximately 99.11±15.06 nematodes), respectively. Sterile water treatment was used as a control. The dishes were placed in an incubator at 25°C, and the relative mortality of nematodes was recorded after 12 h, 24 h, and 48 h (see Table 1).

[0027] Table 1 Contact killing effect of Clostridium beijerinckii Sneb518 fermentation liquid on plant parasitic nematodes

[0028]

[0029] As shown in Table 1, with the increase of time, the contact killing rate of Sneb518 or its metabolites against soybean cyst nematodes increased from 78% to 90%, and the contact killing rate against vegetable root-knot nematodes increased from 83% to 92%, showing obvious effects.

[0030] Clostridium beijerinckii ( Clostridium beijerinckii The effects of Sneb518 fermentation liquid or fermentation liquid products as treatment agents on the control of soybean cyst nematode disease and vegetable root knot nematode disease in greenhouses and fields are as follows:

[0031] Sneb518 fermentation liquid or its products were used to treat coated soybean and tomato seeds at a ratio of 1:100. After drying, the treated soybean or tomato seeds were sown normally. When the soybean or tomato seeds germinated and seedled, they had a good control effect on soybean cyst nematode disease and vegetable root knot nematode disease. The inhibition rate of soybean cyst nematode disease reached 56.34%, and the control effect of vegetable root knot nematode disease reached 62.84% (see Table 2, Table 3).

[0032] Table 2 Inhibition rate of soybean cyst nematode disease by coating with Sneb518 fermentation broth

[0033]

[0034] Table 3 Control effect of Sneb518 fermentation liquid on vegetable root knot nematode disease

[0035]

[0036] Using Clostridium beijerinckii ( Clostridium beijerinckii The effects of Sneb518 fermentation liquid or its products as vegetable root irrigation agents on the prevention and control of root knot nematode disease in tomato plants are as follows:

[0037] During the tomato plant establishment period, 10 mL of Clostridium beijerinckii ( Clostridium beijerinckii Root irrigation with Sneb518 fermentation liquid or its products can achieve a good control effect of up to 73% on vegetable root-knot nematodes.

[0038] Example 2:

[0039] The difference between this embodiment and embodiment 1 is that the fermentation liquid of Clostridium beijerinckii for preventing and treating plant parasitic nematode diseases in this embodiment is prepared by:

[0040] Clostridium beijerinckii ( Clostridium beijerinckii Sneb518 cells were inoculated onto a slant medium containing 4g beef extract and 4g peptone, 9g glucose, and 17g agar, along with 1000mL of distilled water. The culture was incubated at 28°C for 3 days to obtain a test tube seed. This seed was then inoculated into a 250mL Erlenmeyer flask (100mL per flask) containing the following liquid medium: by weight, 0.15% yeast extract, 0.6% peptone, 0.35% beef extract, 1.5% glucose, and the remainder distilled water. The pH was maintained at 7.1, the fermentation temperature was 28°C, and the shaker speed was 185 rpm. -1, fermentation time is 3.5 days, the fermentation liquid or fermentation liquid products formed can be used to directly or indirectly treat soybean and tomato seeds or irrigate roots.

[0041] In this example, Clostridium beijerinckii ( Clostridium beijerinckii ) Effect of Sneb518 fermentation liquid on plant parasitic nematodes: 3 mL of Sneb518 fermentation liquid was added to a sterilized crown dish, followed by 0.5 mL of soybean cyst nematode and vegetable root knot nematode J2 nematode liquid (containing approximately 103.11 ± 9.53 nematodes), respectively. Sterile water treatment was used as a control. The dishes were placed in an incubator at 25°C, and the relative mortality of nematodes was recorded after 12 h, 24 h, and 48 h (see Table 4).

[0042] Table 4 Contact killing effect of Clostridium beijerinckii Sneb518 fermentation liquid on plant parasitic nematodes

[0043]

[0044] As shown in Table 1, with the increase of time, the contact killing rate of Sneb518 or its metabolites against soybean cyst nematodes increased from 80% to 92%, and the contact killing rate against vegetable root knot nematodes increased from 82% to 95%, showing obvious effects.

[0045] Clostridium beijerinckii ( Clostridium beijerinckii The effects of Sneb518 fermentation liquid or fermentation liquid products as treatment agents on the control of soybean cyst nematode disease and vegetable root knot nematode disease in greenhouses and fields are as follows:

[0046] Sneb518 fermentation liquid or its products were used to treat coated soybean and tomato seeds at a ratio of 1:70. After drying, the treated soybean or tomato seeds were sown normally. When the soybean or tomato seeds germinated and seedled, they had a good control effect on soybean cyst nematode disease and vegetable root knot nematode disease. The inhibition rate of soybean cyst nematode disease reached 53.14%, and the control effect of vegetable root knot nematode disease reached 60.64% (see Tables 5 and 6).

[0047] Table 5 Inhibition rate of soybean cyst nematode disease in seeds coated with Sneb518 fermentation liquid

[0048]

[0049] Table 6 Control effect of seeds coated with Sneb518 fermentation liquid on vegetable root knot nematode disease

[0050]

[0051] Using Clostridium beijerinckii ( Clostridium beijerinckii The effects of Sneb518 fermentation liquid or its products as vegetable root irrigation agents on the prevention and control of root knot nematode disease in tomato plants are as follows:

[0052] During the tomato plant establishment period, 20 mL of Clostridium beijerinckii ( Clostridium beijerinckii ) Using Sneb518 diluted 10× fermentation liquid or fermentation liquid products as root irrigation can achieve a good control effect of up to 70% on vegetable root-knot nematodes.

[0053] Example 3:

[0054] The difference between this embodiment and embodiment 1 is that the fermentation liquid of Clostridium beijerinckii for preventing and treating plant parasitic nematode diseases in this embodiment is prepared by:

[0055] Clostridium beijerinckii ( Clostridium beijerinckii Sneb518 cells were inoculated onto a slant medium containing 3.5 g beef extract and 5 g peptone, 9.5 g glucose, and 19 g agar, along with 1000 mL of distilled water. The culture was incubated at 28°C for 3 days to obtain a test tube culture. This culture was then inoculated into a 250 mL Erlenmeyer flask (100 mL per flask) containing the following liquid medium: by weight, 0.2% yeast extract, 0.55% peptone, 0.35% beef extract, and 2% glucose, with the remainder being distilled water. The pH was maintained at 7.0, the fermentation temperature was 28.5°C, and the shaker speed was 185 rpm. -1 , fermentation time is 3.5 days, the fermentation liquid or the fermentation liquid product can be used to directly or indirectly treat soybean and tomato seeds or irrigate roots.

[0056] In this example, Clostridium beijerinckii ( Clostridium beijerinckii ) Effect of Sneb518 fermentation liquid on plant parasitic nematodes: 4 mL of Sneb518 fermentation liquid was added to a sterilized crown dish, followed by 1 mL of soybean cyst nematode and vegetable root knot nematode J2 nematode liquid (containing approximately 207.76 ± 15.73 nematodes), respectively. Sterile water treatment was used as a control, and the dishes were placed in an incubator at 25°C. The relative mortality of nematodes was recorded after 12 h, 24 h, and 48 h (see Table 7).

[0057] Table 7 Contact killing effect of Clostridium beijerinckii Sneb518 fermentation liquid on plant parasitic nematodes

[0058]

[0059] As shown in Table 1, with the increase of time, the contact killing rate of Sneb518 or its metabolites against soybean cyst nematodes increased from 76% to 93%, and the contact killing rate against vegetable root-knot nematodes increased from 79% to 90%, showing obvious effects.

[0060] Clostridium beijerinckii ( Clostridium beijerinckii The effects of Sneb518 fermentation liquid or fermentation liquid products as coating agents on the prevention and control of soybean cyst nematode disease and vegetable root knot nematode disease in greenhouses and fields are as follows:

[0061] Sneb518 fermentation liquid or its products were used to treat coated soybean and tomato seeds at a ratio of 1:100. After drying, the treated soybean or tomato seeds were sown normally. When the soybean or tomato seeds germinated and seedled, they had a good control effect on soybean cyst nematode disease and vegetable root knot nematode disease. The inhibition rate of soybean cyst nematode disease reached 57.36%, and the control effect of vegetable root knot nematode disease reached 60.76% (see Tables 8 and 9).

[0062] Table 8 Inhibition rate of soybean cyst nematode disease by coating with Sneb518 fermentation broth

[0063]

[0064] Table 9 Control effect of Sneb518 fermentation liquid on vegetable root knot nematode disease

[0065]

[0066] Using Clostridium beijerinckii ( Clostridium beijerinckii The effects of Sneb518 fermentation liquid or its products as vegetable root irrigation agents on the prevention and control of root knot nematode disease in tomato plants are as follows:

[0067] During the tomato plant planting period, 100 mL of Clostridium beijerinckii ( Clostridium beijerinckii ) Root irrigation with 20× diluted fermentation liquid of Sneb518 or fermentation liquid products can achieve a good control effect of up to 67% on vegetable root-knot nematodes.

[0068] The above description is merely a specific embodiment of the present invention, but the scope of protection of the present invention is not limited thereto. Any modifications or substitutions that can be easily conceived by a person skilled in the art within the technical scope disclosed in the present invention should be included in the scope of protection of the present invention. Therefore, the scope of protection of the present invention should be based on the scope of protection of the claims.

Claims

1. A Clostridium beijerinckii Sneb518, which is classified as Clostridium beijerinckii ( Clostridium beijerinckii ), was deposited in the General Microbiology Center of China Culture Collection Administration on June 6, 2022, with the deposit number CGMCC No.25009.

2. Use of the Clostridium beijerinckii Sneb518 according to claim 1 in controlling soybean cyst nematode disease and vegetable root-knot nematode disease.

3. The use according to claim 2, characterized in that The Clostridium beijerinckii Sneb518 bacteria are used to form a Clostridium beijerinckii fermentation liquid or a fermentation liquid product for preventing and controlling plant parasitic nematode diseases. The preparation process of the Clostridium beijerinckii fermentation liquid or the fermentation liquid product is as follows: The bacterial cells of Clostridium beijerinckii Sneb518 are inoculated onto a slant culture medium to obtain a test tube seed, which is then inoculated into a liquid culture medium to form a fermentation broth or a fermentation broth product.

4. The use according to claim 3, characterized in that The slant culture medium is a beef extract peptone culture medium, and its ingredients include: 3-4g beef extract, 4.5-5.5g peptone, 9.5-10.5g glucose, 17-20g agar, and 1000mL distilled water.

5. The use according to claim 3, characterized in that: The method for obtaining the test tube strain is as follows: Clostridium beijerinckii Sneb518 bacteria are inoculated on a slant culture medium and cultured at 27.5-28.5° C. for 2.5-3.5 days to obtain the test tube strain.

6. The use according to claim 3, characterized in that: The liquid culture medium comprises, by weight percentage, 1%-2% glucose, 0.5%-0.6% peptone, 0.3%-0.4% beef extract, 0.1%-0.2% yeast extract, and the remainder distilled water; the pH value is 7.0-7.2; the fermentation temperature is 27.5-28.5°C; the shaking speed is 175-185 r·min-1; and the fermentation time is 2.5-3.5 days.

7. A soybean and vegetable seed treating agent, characterized in that: Comprising the Clostridium beijerinckii Sneb518 according to claim 1 or the Clostridium beijerinckii fermentation broth or fermentation broth product according to claim 3.

8. A vegetable planting root irrigation agent, characterized in that: Comprising the Clostridium beijerinckii Sneb518 according to claim 1 or the Clostridium beijerinckii fermentation broth or fermentation broth product according to claim 3.

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