A biocontrol bacterium OLJLGWLH-41 and its application

Abstract

The present invention discloses a biocontrol bacterium OLJLGWLH-41 and its application, which relates to the field of biocontrol technology. The antagonistic strain OLJLGWLH-41 of the present invention has different degrees of disease resistance effects on Solanaceous crops such as peppers, tomatoes and eggplants, as well as other crops such as cucumbers, water bamboo, cotton, citrus, strawberries and corn. For example, the best antibacterial rate against pathogenic bacteria is as high as 73.97%, and the disease control effect against pepper root rot is as high as 63%. Moreover, it has a significant growth-promoting effect on peppers. At the same time, the antagonistic bacterium OLJLGWLH-41 greatly enhances the resistance of peppers to root rot 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 root rot and laying a certain foundation for the green prevention and control of pepper diseases.

Description

Technical Field

[0001] The present invention relates to the technical field of biological control, and more particularly to a biocontrol bacterium OLJLGWLH-41 and its application. Background Art

[0002] At present, the cultivated area of peppers is increasing continuously, and pepper diseases are becoming more and more serious, seriously affecting the yield and quality of peppers. At present, the existing measures for controlling pepper diseases are limited to chemical control, which has the advantages of quick effect, can quickly control the spread of diseases, has a significant effect, simple application method, and wide application range.

[0003] However, the long-term application of chemical pesticides will not only pollute the soil, water source, air, etc., but is more likely to kill non-target organisms such as natural enemies, resulting in the destruction of the ecological balance and more serious diseases. Peppers will also develop drug resistance and reduce the control effect as a result. Moreover, it poses a threat to a series of qualities, flavors, yields of peppers themselves and even human health. Biocontrol bacteria are a class of measures that use beneficial microorganisms to kill or reduce the number of pathogenic organisms to control the occurrence and development of plant diseases. Its essence is to use the antibiosis, competition, hyperparasitism, bacteriolysis between or within microbial species, or to induce plant disease resistance through microbial metabolites, etc., to inhibit the survival and activities of certain pathogens. It not only has no destructive impact on the environment but also can improve the activity of other soil microorganisms and promote the growth of peppers.

[0004] Therefore, providing a biocontrol bacterium OLJLGWLH-41 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-41 and its application. The screened biocontrol bacterium capable of resisting pepper root rot has the characteristics of high reproductive ability, complex metabolic activities and many products, diverse modes of action on pathogenic bacteria, short life cycle, easy to be artificially cultured, etc. In the activities of natural biological control and human application of biological control, the screened antagonistic bacteria and their metabolites can play an important role, and can effectively improve the growth and disease resistance ability of peppers.

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

[0007] The preservation information is as follows:

[0008] A Bacillus sp. OLJLGWLH-41, which is preserved in the China Center for Type Culture Collection, address: Wuhan University, Wuhan, China; preservation date: October 22, 2024; preservation number: CCTCC NO: M 20242293.

[0009] A Bacillus sp. OLJLGWLH-41, with the preservation number: CCTCC NO: M 20242293.

[0010] The present invention also provides a fermentation product of the above-mentioned Bacillus OLJLGWLH-41.

[0011] The present invention also provides a microbial agent containing the above-mentioned Bacillus OLJLGWLH-41 or the above-mentioned fermentation product.

[0012] The present invention also provides a pesticide containing the above-mentioned Bacillus OLJLGWLH-41 or the above-mentioned fermentation product.

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

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

[0015] Preferably, the plant is pepper, tomato, eggplant, cucumber, water bamboo, cotton, citrus, strawberry and corn.

[0016] Preferably, the plant diseases are Phytophthora cactorum of strawberry, Colletotrichum cirrhicola of polygonatum, Elsinoe fawcettii of citrus, Fusarium oxysporum f. sp. capsici of pepper, Fusarium oxysporum f. sp. zizaniae of water bamboo, Fusarium oxysporum f. sp. vasinfectum of cotton, Setosphaeria turcica of corn, Sclerotium rolfsii of pepper, Alternaria alternata of pepper, Phytophthora capsici of pepper, Rhizoctonia solani of pepper, Rhizoctonia solani of tomato, Botrytis cinerea of tomato, Alternaria solani of tomato, Fusarium oxysporum f. sp. melongenae of eggplant and Fusarium oxysporum f. sp. cucumerinum of cucumber.

[0017] The present invention also provides the application of the above-mentioned Bacillus OLJLGWLH-41 or the above-mentioned fermentation product in promoting the growth of pepper and improving the disease resistance of pepper.

[0018] Through the above technical solutions, compared with the prior art, the present invention discloses a biocontrol bacterium OLJLGWLH-41 and its application. The technical effect achieved is that the antagonistic strain OLJLGWLH-41 of the present invention has different degrees of disease resistance effects on diseases of solanaceous crops such as pepper, tomato and eggplant, and other crops such as cucumber, water bamboo, cotton, citrus, strawberry and corn. For example, the best antibacterial rate against pathogenic bacteria is up to 73.97%, and the disease control effect against pepper root rot is up to 63%. And it has a significant growth-promoting effect on pepper. At the same time, the antagonistic bacterium OLJLGWLH-41 greatly enhances the resistance of pepper to root rot 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 root rot and laying a certain foundation for the green prevention and control of pepper diseases. Detailed implementation manners

[0019] The technical solutions in the embodiments of the present invention will be clearly and completely described below. Apparently, the described embodiments are only a part of the embodiments of the present invention, rather than all of them. All other embodiments obtained by those of ordinary skill in the art based on the embodiments of the present invention without creative efforts shall fall within the protection scope of the present invention.

[0020] The embodiments of the present invention disclose a biocontrol bacterium OLJLGWLH-41 and its application

[0021] In the embodiments, the experimental raw materials not mentioned are all commercially available, the pathogenic bacteria not mentioned are conventional bacteria for experiments, and the experimental treatment processes not mentioned are all conventional operations, which will not be elaborated here.

[0022] Example 1

[0023] Isolation and screening of strain OLJLGWLH-41

[0024] 1. Sample collection: Rhizosphere soil of healthy pepper plants of highly resistant pepper varieties

[0025] The pepper variety is: Jinchuan 298

[0026] 2. The pathogenic bacteria for screening antagonistic bacteria are Phytophthora capsici

[0027] 3. Medium preparation:

[0028] Phytophthora medium: Potato dextrose agar (PDA). Add 200 g of potatoes, 20 g of dextrose, 18 - 20 g of agar powder to 1 L of distilled water, heat and dissolve, sterilize at 121 °C for 20 min. Add 1 ml of 0.1 g / ml chloramphenicol solution (prepared with sterile water) to each 1 L of the medium, and its function is to inhibit the growth of bacteria.

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

[0030] Sterilization operation: After the agar powder is completely dissolved, dispense the non-solidified medium into conical flasks, sterilize at 121 °C for 20 min, dispense into petri dishes in a sterile environment, and place in an environment at 37 °C for 24 h. The medium without the growth of contaminants is stored and used for the next experiment.

[0031] 4. Preparation of soil suspension:

[0032] After mixing 3 soil samples evenly, randomly weigh 10 g of soil into a 250 ml conical flask, add 90 ml of sterile water, and place on a magnetic stirrer at 190 r / min for 1 h to prepare a soil suspension.

[0033] 5. The antagonistic bacteria were isolated by the dilution plate coating method:

[0034] The soil suspension was diluted to 10 -4 , 10 -5 , 10 -6 times the concentration of the original solution. 100 μl was taken and coated on the NB medium, and each concentration was repeated three times. It was placed in an environment at 30 °C for 2 days, and single colonies were selected according to the colony morphology for streak purification, numbered respectively and stored in an environment at 14 °C.

[0035] 6. Antagonistic bacteria plate confrontation test:

[0036] Using Phytophthora capsici as the target, the antagonistic bacteria were screened by the plate confrontation method. First, Phytophthora capsici was inoculated in the center of the PDA medium. After a colony with a radius of 5 mm was obtained through cultivation, two perpendicular intersecting lines with a length of 5 cm were drawn with the center point of the plate as the intersection point. The isolated bacteria were inoculated at the 4 endpoints. In the control group, only Phytophthora capsici was inoculated without inoculating the isolated bacteria, and each treatment was repeated 3 times. It was cultured in an environment at 30 °C. When the colony in the control group covered the plate, the antagonistic effect of the isolated bacteria was observed.

[0037] According to the above plate confrontation test, the strain with the best antagonistic effect was selected from the strains with antagonistic effects, and the best antagonistic strain was OLJLGWLH-41, which was preserved through the patent procedure.

[0038] 7. Exploration of the optimal environmental conditions for antagonistic bacteria:

[0039] (1) Optimal temperature test:

[0040] The strain was inoculated in the NB medium and cultured at different temperatures (20 °C, 30 °C, 34 °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) Optimal pH test:

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

[0043] (3) Optimal salt test:

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

[0045] (4) Optimal carbon source test:

[0046] Add different carbon sources (glucose, beef extract, sucrose) to the inorganic salt medium respectively. After inoculating the strain, incubate at 30 °C for 24 h, and then measure the OD of the culture solution. 600 .

[0047] (5) Optimal nitrogen source test:

[0048] Add different nitrogen sources (yeast powder, peptone, tryptone) to the NB medium respectively. After inoculating the strain, incubate at 30 °C for 24 h, and then measure the OD of the culture solution. 600 .

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

[0050] Table 1

[0051]

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

[0053] Species identification is one of the most important links in the research and production of biocontrol bacteria. Before the production and application of antagonistic bacteria, reliable species identification must be carried out. The traditional method of species identification starts from the shape, size, surface characteristics, color, etc. of the colony, and the taxonomic status of a certain species can be determined through a series of physiological and biochemical reaction tests. The enzyme systems in different microorganisms are different, and the types of metabolism are different, 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 measure the metabolites of microorganisms to identify microorganisms that are difficult to distinguish morphologically.

[0054] The results are shown in Table 2:

[0055] Table 2 Physiological and biochemical characteristics of the antagonistic bacterium OLJLGWLH-41

[0056]

[0057]

[0058] "+" indicates a positive reaction; "-" indicates a negative reaction.

[0059] The results of this physiological and biochemical test show that the antagonistic bacterium OLJLGWLH-41 has the ability to produce catalase, decompose harmful substances, participate in metabolism and enhance immunity; the ability to ferment glucose to produce acid, promote the growth and reproduction of bacteria, increase the number of bacteria and the metabolic rate; the ability to produce amylase, decompose starch into maltose and glucose and be used as a carbon source by microorganisms; the ability to produce IAA, which affects root structure, nutrient absorption and resistance to various abiotic stresses (such as drought, salinity and heavy metal toxicity), and enhances the resistance of plants; the ability to decompose and utilize lactose and casein.

[0060] Verification of technical effects:

[0061] Antagonistic effect of antagonistic bacterium OLJLGWLH-41 against other crop diseases

[0062] A confrontation test was carried out on the pathogenic bacteria of crop diseases such as eggplant, cucumber, and tomato by the antagonistic bacterium strain OLJLGWLH-41. First, the antagonistic bacterium strain to be tested was streaked and cultured on an NB plate medium and incubated at 30 °C for 24 h for standby; the single-strain pathogenic bacteria of pepper were cultured on a PDA plate medium and incubated at 27 °C for about 5 d for standby.

[0063] For the treatment group, a bacterial cake with a diameter of 5 mm was punched from the center of the pathogenic bacteria colony and inoculated into the center of a new PDA plate medium. At the same time, a single colony of the antagonistic bacterium was picked with a bamboo stick and streaked on both sides of the PDA plate medium (each treatment was repeated three times).

[0064] The control group was a PDA medium plate inoculated only with the pathogenic bacteria of each crop.

[0065] The antagonistic effect of antagonistic bacterium OLJLGWLH-41 against other crops is shown in Table 3 below:

[0066] Table 3

[0067]

[0068]

[0069] The results showed that: the antagonistic bacterium OLJLGWLH-41 had different degrees of disease resistance effects on Solanaceae crops such as pepper, tomato, and eggplant, as well as other crops such as cucumber, water bamboo, cotton, citrus, strawberry, and corn; among them, the inhibitory effects on pepper fusarium wilt, citrus scab, and tomato early blight were the best, with an inhibition rate as high as about 70%, followed by the better inhibitory effect on cucumber fusarium wilt, with an inhibition rate of more than 65%; thus, it can be seen that the biocontrol bacterium OLJLGWLH-41 is a biocontrol bacterium with a relatively broad antibacterial spectrum.

[0070] Application of the fermentation broth of antagonistic bacterium OLJLGWLH-41 in promoting growth and disease resistance of pepper:

[0071] The effect of antagonistic bacterium OLJLGWLH-41 in promoting growth and disease resistance of pepper was a substrate pot experiment. Four treatments were set up, with 12 replicates for each treatment.

[0072] They were respectively:

[0073] Control group:

[0074] Treatment for Phytophthora capsici disease of pepper (only inoculated with Phytophthora capsici),

[0075] Treatment of pepper root rot disease (inoculated only with pepper root rot pathogen);

[0076] Experimental group:

[0077] Treatment of antagonistic bacteria against Phytophthora capsici (inoculated with Phytophthora capsici and antagonistic bacteria OLJLGWLH-41 at the same time),

[0078] Treatment of antagonistic bacteria against pepper root rot (inoculated with pepper root rot pathogen and antagonistic bacteria OLJLGWLH-41 at the same time),

[0079] The pepper variety used in this experiment is Xiangyan No. 15. The potting substrates for the experiment were all treated with high-temperature sterilization. The pepper seedlings were from the Hunan Academy of Agricultural Sciences;

[0080] Preparation method of OLJLGWLH-41 fermentation broth: Pick single colonies from the solid culture plate of antagonistic bacteria OLJLGWLH-41NB cultured for three days and inoculate them into a triangular flask containing NB liquid medium. Shake culture for 24 h, zero with blank NB medium, and adjust the OD value to 1.0 at a wavelength of 600 nm; Pipette 2 mL of the bacterial liquid and inoculate it into each sterilized triangular flask of NB liquid medium. Shake culture at 37 °C and 170 rpm for 3 days. Dilute the cultured antagonistic bacteria fermentation broth 100 times with distilled water for standby.

[0081] Experimental group: 10 days after pepper transplantation, inoculate the fermentation broth of antagonistic bacteria OLJLGWLH-41 into the pepper substrate potted plants. The inoculation amount of antagonistic bacteria is 10 mL (the concentration of antagonistic bacteria liquid is 1×10 7 ); Seven days later, inoculate the pathogens of Phytophthora capsici and root rot. The inoculation amount of pathogens is 5 mL (the concentration of pathogen spore liquid is 1×10 4 ).

[0082] Control group: 10 days after pepper transplantation, add an equal amount of sterile water to the control group (fermentation broth of antagonistic bacteria OLJLGWLH-41); Seven days later, inoculate the pathogens of Phytophthora capsici and root rot. The inoculation amount of pathogens is 5 mL (the concentration of pathogen spore liquid is 1×10 4 ).

[0083] After adding the pathogen bacterial liquid, consider the addition of the pathogen bacterial liquid as day 0, and count the incidence of Phytophthora capsici and root rot in peppers for 14 days (control effect); On the last day, measure the morphological indexes of peppers (plant height, stem diameter, leaf length, leaf width, SPAD, biomass). The disease resistance and growth promotion effects of the experiment are as shown in Tables 4-10 below:

[0084] Table 4 Disease control effect:

[0085]

[0086] Table 5 Plant height

[0087]

[0088] Table 6 Stem diameter:

[0089]

[0090] Table 7 Leaf length

[0091]

[0092] Table 8 Leaf width

[0093]

[0094] Table 9 SPAD

[0095]

[0096] Table 10 Biomass

[0097]

[0098] From Tables 4 to 10, it is shown that in the treatment with Phytophthora capsici pathogen: compared with the non-inoculated antagonistic bacterium OLJLGWLH-41, after inoculating with the fermentation broth of antagonistic bacterium OLJLGWLH-41, the disease control effect is as high as 70%. In the morphological indexes of pepper, the plant height of pepper increases by 13.43 cm, the stem diameter increases by 1.12 mm, the leaf length increases by 26.00 mm, the leaf width increases by 20.27 mm, the chlorophyll in the leaves increases by 4.15 SPAD, and the plant biomass increases by 10.73 g.

[0099] In the treatment with Fusarium solani f. sp. radicis-lycopersici pathogen: compared with the non-inoculated antagonistic bacterium OLJLGWLH-41, after inoculating with the fermentation broth of antagonistic bacterium OLJLGWLH-41, the disease control effect reaches 63%. In the morphological indexes of pepper, the plant height of pepper increases by 8.48 cm, the stem diameter increases by 1.44 mm, the leaf length increases by 7.30 mm, the leaf width increases by 9.24 mm, the chlorophyll in the leaves increases by 12.71 SPAD, and the plant biomass increases by 4.87 g. This shows that the antagonistic bacterium OLJLGWLH-41 can promote the growth and development of pepper plants, the fresh weight and weight of the plants are both increased, and the inhibitory effect of the pathogen on the growth of pepper is significantly weakened.

[0100] Determination of leaf enzyme activity in pepper potted plants after applying antagonistic bacterium OLJLGWLH-41:

[0101] To explore the effect of antagonistic bacterium OLJLGWLH-41 on the enzyme activity of pepper leaves, the physiological and biochemical indexes (proline (PRO), malondialdehyde (MDA), peroxidase (POD), catalase (CAT), superoxide dismutase (SOD)) of pepper leaves were measured.

[0102] Four treatments were set up in the experiment, with 12 replicates for each treatment, which were respectively:

[0103] Control group:

[0104] Treatment for Phytophthora capsici disease (only inoculated with Phytophthora capsici),

[0105] Treatment for Fusarium solani f. sp. radicis-lycopersici disease (only inoculated with Fusarium solani f. sp. radicis-lycopersici);

[0106] Experimental group:

[0107] Treatment for antagonistic effect against Phytophthora capsici disease (simultaneously inoculated with Phytophthora capsici and antagonistic bacterium OLJLGWLH-41),

[0108] Treatment for antagonistic effect against Fusarium solani f. sp. radicis-lycopersici disease (simultaneously inoculated with Fusarium solani f. sp. radicis-lycopersici and antagonistic bacterium OLJLGWLH-41),

[0109] The pepper variety used in this experiment was Xiangyan No. 15. The potting substrates for the experiment were all treated with high-temperature sterilization, and the pepper seedlings were from the Hunan Academy of Agricultural Sciences;

[0110] The results are shown in Tables 11 - 15:

[0111] (1) Proline (PRO)

[0112] The content of free proline in plants reflects the stress resistance of plants. Varieties with strong drought resistance often accumulate more proline, which can be used as a physiological index for drought resistance.

[0113] Table 11

[0114]

[0115] (2) Malondialdehyde (MDA)

[0116] Malondialdehyde is produced due to biological aging or under stress conditions. In the same plant under different environmental conditions, the higher the MDA content, the greater the damage and the weaker the resistance.

[0117] Table 12

[0118]

[0119] (3) Peroxidase (POD)

[0120] The level of peroxidase activity is closely related to resistance. The higher the POD content, the more the balance state of reactive oxygen species in the plant is disrupted, and the cell membrane is damaged.

[0121] Table 13

[0122]

[0123] (4) Catalase (CAT)

[0124] Catalase activity is related to the metabolic intensity and cold and disease resistance of organisms, providing an antioxidant mechanism for the body.

[0125] Table 14

[0126]

[0127]

[0128] (5) Superoxide Dismutase (SOD)

[0129] Superoxide dismutase has the functions of anti-aging, improving the resistance of the body to various diseases, enhancing the adaptability of the body to the external environment. It is an important antioxidant enzyme in the body, protecting cells from oxidative damage.

[0130] Table 15

[0131]

[0132] From Tables 11 to 15, it is shown that in the treatments with Phytophthora capsici pathogen and Fusarium solani pathogen: compared with the non-inoculation of the antagonistic bacterium OLJLGWLH-41, after inoculating the fermentation broth of the antagonistic bacterium OLJLGWLH-41: with the increase in the contents of proline (PRO), catalase (CAT) and superoxide dismutase (SOD) in the plant, the disease resistance, cold resistance, antioxidant and anti-aging abilities, metabolic intensity and the adaptability of the body to the external environment of the plant are also enhanced, thus protecting the pepper plants from external damage. And the decrease of malondialdehyde (MDA) and peroxidase (POD) in the pepper plants indicates that the pepper is less damaged and has stronger resistance. It shows that this antagonistic bacterium can improve the resistance of pepper plants to Phytophthora capsici root rot by enhancing the immune defense ability of pepper plants.

[0133] In this specification, each embodiment is described in a progressive manner. The key point of each embodiment is to illustrate the differences from other embodiments. For the same or similar parts among the embodiments, reference can be made to each other.

[0134] The above description of the disclosed embodiments enables those skilled in the art to implement or use the present invention. Various modifications to these embodiments will be obvious to those skilled in the art. The general principles defined herein can 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 these embodiments shown herein, but rather should be accorded the widest scope consistent with the principles and novel features disclosed herein.

Claims

1. A Bacillus Bacillus sp. sp. OLJLGWLH-41, characterized in that The preservation number is: CCTCC NO: M20242293.

2. A bacterial agent containing the Bacillus sp. OLJLGWLH-41 described in claim 1.

3. A pesticide containing the Bacillus sp. OLJLGWLH-41 described in claim 1.

4. Use of the microbial agent according to claim 2 in controlling or preventing plant diseases, characterized in that, The plants are pepper, tomato, eggplant, cucumber, water bamboo, cotton, citrus, strawberry and corn; the plant diseases are strawberry Phytophthora blight, polygonatum anthracnose, citrus scab, pepper fusarium wilt, water bamboo fusarium wilt, cotton fusarium wilt, corn northern leaf blight, pepper southern blight, pepper black spot, pepper Phytophthora blight, pepper root rot, pepper anthracnose, tomato root rot, tomato gray mold, tomato early blight, eggplant fusarium wilt and cucumber fusarium wilt.

5. Use of the pesticide according to claim 3 in controlling or preventing plant diseases, characterized in that, The plants are pepper, tomato, eggplant, cucumber, water bamboo, cotton, citrus, strawberry and corn; the plant diseases are strawberry Phytophthora blight, polygonatum anthracnose, citrus scab, pepper fusarium wilt, water bamboo fusarium wilt, cotton fusarium wilt, corn northern leaf blight, pepper southern blight, pepper black spot, pepper Phytophthora blight, pepper root rot, pepper anthracnose, tomato root rot, tomato gray mold, tomato early blight, eggplant fusarium wilt and cucumber fusarium wilt.

6. The application of the Bacillus sp. OLJLGWLH-41 described in claim 1 in promoting the growth of pepper and improving the disease resistance of pepper.

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