A biocontrol bacterium OLJLGWLH-39 and its application

Abstract

The invention discloses a biocontrol bacterium OLJLGWLH-39 and its application. It relates to the field of biocontrol technology. The antagonistic strain OLJLGWLH-39 of the invention has different degrees of disease resistance to diseases of Solanaceae crops such as pepper, tomato and eggplant, as well as other crops such as cucumber, wild rice stem, cotton, citrus, strawberry and corn, such as the best antibacterial rate of pathogens is as high as 79%, and the disease prevention effect of pepper phytophthora is as high as 80%; and it has a significant growth-promoting effect on pepper. At the same time, the antagonistic bacteria OLJLGWLH-39 greatly enhances the resistance of pepper to phytophthora by enhancing the activity of proline (PRO), catalase (CAT) and superoxide dismutase (SOD) and weakening the activity of malondialdehyde (MDA) and peroxidase (POD), providing high-quality biocontrol strains for the biological control of pepper phytophthora, and laying a certain foundation for the green control of pepper diseases.

Description

Technical Field

[0001] The invention relates to the technical field of biocontrol, and more particularly to a biocontrol bacterium OLJLGWLH-39 and application thereof. Background Art

[0002] At present, the pepper cultivation area is constantly increasing, and pepper diseases are becoming more and more serious, seriously affecting the yield and quality of peppers; and the existing measures for preventing and controlling pepper diseases are also limited to chemical control, which has the advantages of fast effect, rapid control of disease spread, significant effect, simple use method, and wide application range.

[0003] However, long-term application of chemical pesticides will not only pollute the soil, water, air, etc., but may also kill non-target organisms such as natural enemies, leading to the destruction of ecological balance and more serious diseases. Peppers will also develop resistance to pesticides, reducing the effectiveness of prevention and control; and it poses a threat to the quality, flavor, yield and even human health of peppers themselves. Biocontrol bacteria is a type of measure that uses beneficial microorganisms to kill or reduce the number of pathogens to control the occurrence and development of plant diseases. Its essence is to inhibit the survival and activity of certain pathogens by using the antibiotic, competitive, hyperparasitic and bacteriolytic effects between or within microorganisms, or by inducing plant disease resistance through microbial metabolites; it not only does not cause destructive effects on the environment, but also can increase the activity of other microorganisms in the soil and promote the growth of peppers.

[0004] Therefore, providing a biocontrol bacterium OLJLGWLH-39 and its application to overcome the above technical deficiencies is an urgent problem to be solved by those skilled in the art. Summary of the invention

[0005] In view of this, the present invention provides a biocontrol bacterium OLJLGWLH-39 and an application thereof. The screened biocontrol bacteria capable of resisting pepper blight have the characteristics of high reproduction capacity, complex metabolic activities and multiple products, diverse modes of action on pathogens, short life cycle, easy artificial cultivation, etc. The screened antagonistic bacteria and their metabolites can play an important role in naturally occurring biological control and human application of biological control activities; the growth and disease resistance of pepper are effectively improved.

[0006] In order to achieve the above object, the present invention adopts the following technical solution:

[0007] The preservation information is:

[0008] A Bacillus sp. OLJLGWLH-39, deposited in China Center for Type Culture Collection, address: Wuhan University, Wuhan, China; deposit date: October 22, 2024; deposit number: CCTCC NO: M 20242292.

[0009] A Bacillus sp. OLJLGWLH-39, with a deposit number of CCTCC NO: M 20242292.

[0010] The present invention also provides a fermentation product of the above-mentioned Bacillus OLJLGWLH-39.

[0011] The present invention also provides a bacterial agent containing the above-mentioned Bacillus OLJLGWLH-39 or the above-mentioned fermentation product.

[0012] The present invention also provides a pesticide containing the above-mentioned Bacillus OLJLGWLH-39 or the above-mentioned fermentation product.

[0013] The present invention also provides the use of the above-mentioned bacterial agent in controlling or preventing plant diseases.

[0014] The present invention also provides the use of the above pesticide in controlling or preventing plant diseases.

[0015] Preferred: Plants are pepper, tomato, eggplant, cucumber, wild rice, cotton, citrus, strawberry and corn.

[0016] Preferred plant diseases are strawberry phytophthora, polygonatum anthracnose, citrus scaly bark disease, pepper wilt, wild rice stem wilt, cotton wilt, corn daiban disease, pepper white rot, pepper black spot, pepper phytophthora, pepper root rot, pepper anthracnose, tomato root rot, tomato gray mold, tomato early blight, eggplant wilt and cucumber wilt.

[0017] The present invention also provides the use of the Bacillus OLJLGWLH-39 or the fermentation product in promoting the growth of pepper and improving the disease resistance of pepper.

[0018] It can be seen from the above technical scheme that compared with the prior art, the present invention discloses a biocontrol bacterium OLJLGWLH-39 and its application, and the technical effect achieved is that the antagonistic strain OLJLGWLH-39 of the present invention has different degrees of disease resistance to diseases of Solanaceae crops such as pepper, tomato and eggplant, as well as other crops such as cucumber, wild rice stem, cotton, citrus, strawberry and corn, such as the best antibacterial rate of pathogens is as high as 79%, and the disease prevention effect of pepper phytophthora is as high as 80%; and it has a significant growth-promoting effect on pepper. At the same time, the antagonistic bacteria OLJLGWLH-39 greatly enhances the resistance of pepper to phytophthora by enhancing the activities of proline (PRO), catalase (CAT) and superoxide dismutase (SOD) and weakening the activities of malondialdehyde (MDA) and peroxidase (POD), providing a high-quality biocontrol strain for the biological control of pepper phytophthora, and laying a certain foundation for the green control of pepper diseases. DETAILED DESCRIPTION

[0019] The following will be described clearly and completely in conjunction with the technical solutions in the embodiments of the present invention. Obviously, the described embodiments are only part of the embodiments of the present invention, not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without creative work are within the scope of protection of the present invention.

[0020] The embodiment of the present invention discloses a biocontrol bacterium OLJLGWLH-39 and its application

[0021] In the examples, the experimental raw materials not mentioned are all commercially available, the pathogens not mentioned are conventional experimental fungi, and the experimental treatment processes not mentioned are all conventional operations, which will not be described in detail here.

[0022] Example 1

[0023] Isolation and screening of OLJLGWLH-39 strain

[0024] 1. Sample collection: rhizosphere soil of healthy pepper plants of high-resistant pepper varieties

[0025] Pepper variety: Jinchuan 298

[0026] 2. The pathogen used for screening antagonistic bacteria is Capsicum frutescens

[0027] 3. Culture medium configuration:

[0028] Phytophthora culture medium: Potato dextrose medium (PDA), add 200g potato, 20g glucose, 18-20g agar powder, add 1L distilled water, heat to dissolve, sterilize at 121℃ for 20min. Add 1ml of 0.1g / ml chloramphenicol solution (prepared with sterile water) to each 1L culture medium to inhibit bacterial growth.

[0029] Bacterial culture medium: NB medium, peptone 10g / L, sodium chloride 5g / L, beef extract 3g / L, agar 18-20g / L, sterilize at 121℃ for 20min, and cool to about 60℃.

[0030] Sterilization operation: After the agar powder is completely dissolved, the culture medium that has not yet solidified is dispensed into a conical flask, sterilized at 121°C for 20 minutes, dispensed into a culture dish under a sterile environment, and placed at 37°C for 24 hours after solidification. The culture medium that does not grow miscellaneous bacteria can be stored and used for the next test.

[0031] 4. Preparation of soil suspension:

[0032] After the three soil samples were mixed evenly, 10 g of soil was randomly weighed into a 250 ml conical flask, 90 ml of sterile water was added, and the flask was placed on a magnetic stirrer at 190 r / min and stirred for 1 h to prepare a soil suspension.

[0033] 5. Antagonistic bacteria isolation adopts dilution plate coating method:

[0034] Dilute the soil suspension to 10% of the original solution. -4 , 10 -5 , 10 -6 Multiple concentrations were added, 100 μl was spread on NB medium, and each concentration was repeated three times. The culture was placed at 30°C for 2 days, and single colonies were selected according to their morphology for streaking purification, numbered and stored at 14°C.

[0035] 6. Antagonistic bacteria plate confrontation test:

[0036] Taking pepper phytophthora as the target, the plate confrontation method was used to screen antagonistic bacteria. First, inoculate the phytophthora in the center of the PDA medium. After culturing a colony with a radius of 5 mm, draw two 5 cm long lines perpendicularly intersecting with the center of the plate as the intersection point, and inoculate the isolated bacteria at the four endpoints. The control group was only inoculated with pepper phytophthora without isolated bacteria, and each treatment was repeated 3 times. Cultivate at 30℃, and observe the antagonistic effect of the isolated bacteria when the colony of the control group fills the plate.

[0037] According to the above plate confrontation test, the strains with antagonistic effects were selected and the best antagonistic strain was OLJLGWLH-39, which was preserved by patent procedure.

[0038] 7. Research on the optimal environmental conditions for antagonistic bacteria:

[0039] (1) Optimum temperature test:

[0040] The strain was inoculated into NB medium and cultured at different temperatures (20°C, 30°C, 36°C, 37°C, 41°C, 45°C, 60°C) for 24 h, and then the growth of the bacteria was observed with the naked eye.

[0041] (2) Optimum pH test:

[0042] The NB medium was adjusted to different pH values ​​(5, 6, 7, 8, 9), inoculated with the strain, and cultured at 30°C for 24 h, and then the OD of the culture solution was measured. 600 .

[0043] (3) Optimum salt test:

[0044] Different inorganic salts (sodium chloride, magnesium sulfate, potassium sulfate) were added to the NB medium, and the strain was inoculated and cultured at 30°C for 24 hours. The OD of the culture medium was measured. 600 .

[0045] (4) Optimal carbon source test:

[0046] Different carbon sources (glucose, beef extract, sucrose) were added to the inorganic salt medium, and the strain was inoculated and cultured at 30°C for 24 hours. Then the OD of the culture medium was measured. 600 .

[0047] (5) Optimal nitrogen source test:

[0048] Different nitrogen sources (yeast powder, peptone, tryptone) were added to the NB medium, and after inoculation of the strain, the medium was cultured at 30°C for 24 h, and then the OD of the culture medium was measured. 600 .

[0049] The experimental results are shown in Table 1:

[0050] Table 1

[0051]

[0052] 8. Detect the physiological and biochemical characteristics of antagonistic bacteria.

[0053] Strain identification is one of the most important links in the research and production of biocontrol bacteria. Antagonistic bacteria must be reliably identified before production and application. The traditional method of strain identification is to start from the shape, size, surface characteristics, color and other aspects of the colony, and to determine the classification status of a certain strain through a series of physiological and biochemical reaction tests. Different microorganisms have different enzyme systems and different types of metabolism, so the metabolites produced after the utilization of various substances are also different. Therefore, the physiological and biochemical reactions of microorganisms can be used to determine the metabolites of microorganisms to identify microorganisms that are difficult to distinguish in morphology.

[0054] The results are shown in Table 2:

[0055] Table 2 Physiological and biochemical characteristics of antagonistic bacteria OLJLGWLH-39

[0056]

[0057] “+” indicates a positive reaction; “-” indicates a negative reaction.

[0058] The results of the physiological and biochemical tests showed that the antagonistic bacteria OLJLGWLH-39 has the ability to produce catalase, decompose harmful substances, participate in metabolism and enhance immunity; ferment glucose to produce acid, promote bacterial growth and reproduction, increase bacterial quantity and metabolic rate; produce amylase, decompose starch into maltose and glucose, and be used by microorganisms as a carbon source; produce IAA, which affects root structure, nutrient absorption and resistance to various abiotic stresses (such as drought, salinity and heavy metal toxicity), and enhances plant resistance; and has the ability to decompose and utilize lactose and casein.

[0059] Technical effect verification:

[0060] Antagonistic effect of antagonistic bacteria OLJLGWLH-39 on other crop diseases

[0061] The antagonistic strain OLJLGWLH-39 was used to conduct a confrontation test on the pathogens of crop diseases such as eggplant, cucumber, and tomato. First, the antagonistic bacterial strain to be tested was streaked and cultured on NB plate culture medium and cultured at 30℃ for 24 hours for use; the pepper and pathogenic bacteria single strains were cultured on PDA plate culture medium and cultured at 27℃ for about 5 days for use.

[0062] In the treatment group, a bacterial cake was punched in the center of the pathogen colony with a 5 mm diameter puncher and inoculated into the center of a new PDA plate culture medium. At the same time, a single colony of antagonistic bacteria was picked up with a bamboo stick and streaked on both sides of the PDA plate culture medium for culture (each treatment was repeated three times).

[0063] The control group consisted of PDA medium plates inoculated with only the crop pathogens.

[0064] The antagonistic effects of antagonistic bacteria OLJLGWLH-39 on other crops are shown in Table 3:

[0065] Table 3

[0066]

[0067] The results showed that the antagonistic bacteria OLJLGWLH-39 had different degrees of disease resistance to diseases of Solanaceae crops such as pepper, tomato and eggplant, as well as other crops such as cucumber, wild rice stem, cotton, citrus, strawberry and corn; among them, the best inhibition effect was on Polygonatum anthracnose and tomato early blight, with an inhibition rate of more than 70%, followed by pepper wilt, citrus sand bark disease, pepper anthracnose, tomato root rot and cucumber wilt, with an inhibition rate of 65%, and the inhibition effect on cotton wilt was poor, only 40%; this shows that the biocontrol bacteria OLJLGWLH-39 is a biocontrol bacterium with a broad antibacterial spectrum.

[0068] Application of antagonistic bacteria OLJLGWLH-39 fermentation liquid in pepper growth promotion and disease resistance:

[0069] Antagonistic bacteria OLJLGWLH-39 achieved growth-promoting and disease-resistant effects on pepper. The experiment was conducted in a matrix potted plant experiment. Four treatments were set up, each with 12 replicates.

[0070] They are:

[0071] Control group:

[0072] Pepper Phytophthora disease treatment (only inoculate pepper Phytophthora),

[0073] Pepper root rot disease treatment (only inoculated with pepper root rot bacteria);

[0074] Experimental Group:

[0075] Antagonistic treatment of pepper phytophthora (simultaneous inoculation of pepper phytophthora and antagonistic bacteria OLJLGWLH-39),

[0076] Antagonistic treatment of pepper root rot (simultaneous inoculation of pepper root rot pathogen and antagonistic bacteria OLJLGWLH-39),

[0077] The pepper variety used in the experiment was Xiangyan No. 15. The test potting medium was sterilized at high temperature. The pepper seedlings came from the Hunan Academy of Agricultural Sciences.

[0078] Preparation method of OLJLGWLH-39 fermentation broth: pick a single colony from the antagonistic bacteria OLJLGWLH-39NB solid culture plate that has been cultured for three days and inoculate it into a conical flask filled with NB liquid culture medium, shake and culture for 24 hours, adjust to zero with blank NB culture medium, and adjust the OD value to 1.0 at a wavelength of 600nm; draw 2 ml of bacterial liquid and inoculate it into each sterilized NB liquid culture medium conical flask, shake and culture at 37°C and 170rpm for 3 days, and dilute the cultured antagonistic bacteria fermentation broth 100 times with distilled water for use.

[0079] Experimental group: 10 days after pepper transplantation, the fermentation liquid of antagonistic bacteria OLJLGWLH-39 was inoculated into pepper substrate pots, and the inoculation volume of antagonistic bacteria was 10 mL (the concentration of antagonistic bacteria liquid was 1×10 7 ); Seven days later, pepper phytophthora and root rot pathogens were inoculated, with the pathogen inoculation volume of 5 mL (the concentration of pathogen spore liquid was 1×10 4 ).

[0080] Control group: 10 days after pepper transplantation, the control group was added with (antagonistic bacteria OLJLGWLH-39 fermentation liquid) and an equal amount of sterile water; seven days later, pepper phytophthora and root rot pathogens were inoculated, and the pathogen inoculation volume was 5mL (the concentration of pathogen spore liquid was 1×10 4 ).

[0081] After adding the pathogen solution, the addition of the pathogen solution was regarded as day 0, and the incidence of pepper phytophthora and root rot (prevention effect) was counted for 14 days; on the last day, the morphological indicators of pepper (plant height, stem thickness, leaf length, leaf width, SPAD, biomass) were measured. The disease resistance and growth promotion effects of the test are shown in Tables 4 to 10:

[0082] Table 4 Disease prevention effect:

[0083]

[0084] Table 5 Plant height

[0085]

[0086] Table 6 Stem diameter:

[0087]

[0088] Table 7 Leaf length

[0089]

[0090] Table 8 Leaf Width

[0091]

[0092] Table 9 SPAD

[0093]

[0094] Table 10 Biomass

[0095]

[0096] Tables 4 to 10 show that in the treatment of pepper phytophthora pathogen: compared with the treatment without inoculation of antagonistic bacteria OLJLGWLH-39, the disease prevention efficiency was as high as 80% after inoculation of the fermentation liquid of antagonistic bacteria OLJLGWLH-39. In the pepper morphological indicators, the height of the pepper plant increased by 12.15 cm, the stem diameter increased by 1.77 mm, the leaf length increased by 20.22 mm, the leaf width increased by 13.04 mm, the leaf chlorophyll increased by 6.82 SPAD, and the plant biomass increased by 1.04%. 5.38g; in the treatment of pepper root rot pathogens: compared with the non-inoculation of antagonistic bacteria OLJLGWLH-39, after inoculation of the fermentation liquid of the antagonistic bacteria OLJLGWLH-39, the disease prevention efficiency reached 40%, and in the pepper morphological indicators, the pepper plant height increased by 10.05cm, the stem diameter increased by 1.18mm, the leaf length increased by 2.81mm, the leaf width increased by 4.46mm, the leaf chlorophyll increased by 5.20SPAD, and the plant biomass increased by 2.71g. This shows that the antagonistic bacteria OLJLGWLH-39 can promote the growth and development of pepper plants, improve the fresh quality and weight of the plants, and significantly reduce the inhibitory effect of pathogens on the growth of peppers.

[0097] Determination of leaf enzyme activity after application of antagonistic bacteria OLJLGWLH-39 in pepper potted plants:

[0098] In order to explore the effect of antagonistic bacteria OLJLGWLH-39 on the enzyme activity of pepper leaves, the physiological and biochemical indicators of pepper leaves (proline (PRO), malondialdehyde (MDA), peroxidase (POD), catalase (CAT), superoxide dismutase (SOD)) were determined.

[0099] The experiment set up four treatments with 12 replications each:

[0100] Control group:

[0101] Pepper Phytophthora disease treatment (only inoculate pepper Phytophthora),

[0102] Pepper root rot disease treatment (only inoculated with pepper root rot bacteria);

[0103] Experimental Group:

[0104] Antagonistic treatment of pepper phytophthora (simultaneous inoculation of pepper phytophthora and antagonistic bacteria OLJLGWLH-39),

[0105] Antagonistic treatment of pepper root rot (simultaneous inoculation of pepper root rot pathogen and antagonistic bacteria OLJLGWLH-39),

[0106] The pepper variety used in the experiment was Xiangyan No. 15. The test potting medium was sterilized at high temperature. The pepper seedlings came from the Hunan Academy of Agricultural Sciences.

[0107] The results are shown in Tables 11 to 15:

[0108] (1) Proline (PRO)

[0109] The free proline content in plants reflects the plant's stress resistance. Drought-resistant varieties often accumulate more proline, which can be used as a physiological indicator of drought resistance.

[0110] Table 11

[0111]

[0112] (2) Malondialdehyde (MDA)

[0113] Malondialdehyde is produced due to biological aging or adverse conditions. For the same plant under different environmental conditions, the higher the MDA content means the greater the damage and the weaker the resistance.

[0114] Table 12

[0115]

[0116]

[0117] (3) Peroxidase (POD)

[0118] The level of peroxidase activity is closely related to resistance. The higher the POD content, the more the balance of active oxygen in the plant body is broken and the cell membrane is damaged.

[0119] Table 13

[0120]

[0121] (4) Catalase (CAT)

[0122] Catalase activity is related to the metabolic intensity and cold and disease resistance of the organism, providing an antioxidant mechanism for the body.

[0123] Table 14

[0124]

[0125] (5) Superoxide dismutase (SOD)

[0126] Superoxide dismutase has anti-aging effects, improves the body's resistance to various diseases, and can enhance the body's ability to adapt to the external environment. It is an important antioxidant enzyme in organisms that protects cells from oxidative damage.

[0127] Table 15

[0128]

[0129]

[0130] Tables 11 to 15 show that in the treatment of pepper phytophthora and root rot pathogens, compared with the uninoculated antagonistic bacteria OLJLGWLH-39, after inoculation with the fermentation liquid of the antagonistic bacteria OLJLGWLH-39, the disease resistance, stress resistance, drought resistance, cold resistance, oxidation resistance, aging resistance, metabolic intensity and the adaptability of the body to the external environment of the plant increased with the increase of the content of proline (PRO), catalase (CAT) and superoxide dismutase (SOD) in the plant, thereby protecting the pepper plants from external damage. And the reduction of malondialdehyde (MDA) and peroxidase (POD) in the pepper plants indicates that the less damage the pepper is, the stronger the resistance is. It shows that the antagonistic bacteria can improve the resistance of pepper plants to pepper phytophthora and root rot by enhancing the immune defense ability of pepper plants.

[0131] The various embodiments in this specification are described in a progressive manner, and each embodiment focuses on the differences from other embodiments. The same or similar parts between the various embodiments can be referenced to each other.

[0132] The above description of the disclosed embodiments enables one skilled in the art to implement or use the present invention. Various modifications to these embodiments will be apparent to one skilled in the art, and the general principles defined herein may be implemented in other embodiments without departing from the spirit or scope of the present invention. Therefore, the present invention will not be limited to the embodiments shown herein, but rather to the widest scope consistent with the principles and novel features disclosed herein.

Claims

1. A Bacillus ( Bacillus sp. ) OLJLGWLH-39, characterized in that, The deposit number is: CCTCC NO: M20242292.

2. A bacterial agent containing the Bacillus OLJLGWLH-39 according to claim 1.

3. A pesticide containing the Bacillus OLJLGWLH-39 according to claim 1.

4. Use of the microbial agent according to claim 2 in controlling or preventing plant diseases, characterized in that: The plant diseases are strawberry phytophthora, polygonatum anthracnose, citrus bark disease, pepper wilt, wild rice stem wilt, cotton wilt, corn daikon disease, pepper white rot, pepper black spot, pepper phytophthora, pepper root rot, pepper anthracnose, tomato root rot, tomato gray mold, tomato early blight, eggplant wilt and cucumber wilt.

5. Use of the pesticide according to claim 3 in controlling or preventing plant diseases, characterized in that: The plant diseases are strawberry phytophthora, polygonatum anthracnose, citrus bark disease, pepper wilt, wild rice stem wilt, cotton wilt, corn daikon disease, pepper white rot, pepper black spot, pepper phytophthora, pepper root rot, pepper anthracnose, tomato root rot, tomato gray mold, tomato early blight, eggplant wilt and cucumber wilt.

6. The use according to claim 4 or 5, characterized in that The plants are pepper, tomato, eggplant, cucumber, wild rice stem, cotton, citrus, strawberry and corn.

7. Use of the Bacillus OLJLGWLH-39 according to claim 1 in promoting growth and improving disease resistance of pepper, characterized in that: The pepper disease resistance is against pepper wilt, pepper white rot, pepper black spot, pepper phytophthora, pepper root rot and pepper anthracnose.

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