Bacillus cereus JBH-LY1 for preventing and treating corn ear rot and application of bacillus cereus JBH-LY1
The inhibition of Fusarium syllium fermentation broth by the fermentation broth of Bacillus cereus JBH-LY1 strain has solved the problem of unsatisfactory prevention and control of corn ear rot and environmental pollution, and achieved efficient and safe biological control effects.
Patent Information
- Application Number
- CN202411200230.2
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- Filing Date
- 2024-08-29
- Publication Date
- 2024-12-06
AI Technical Summary
The prior art has poor results in preventing and treating corn ear rot, and the use of chemical pesticides has brought about environmental pollution problems, and there is a lack of effective and safe biological control methods.
Provide a strain of Bacillus cereus JBH-LY1, which was obtained and identified as Bacillus cereus through screening, which can be easily cultivated on artificial culture medium. The fermentation broth has a significant inhibitory effect on Fusarium cystella, and the antibacterial rate is as high as 88.9%. , used to prepare microbial preparations to prevent and treat corn ear rot.
It effectively reduces the harm of corn ear rot to corn, avoids environmental pollution, and is easy to produce and promote and apply in factory.
Smart Images
Figure CN119081930A_ABST
Abstract
Description
Technical Field
[0001] The invention belongs to the technical field of biological control of plant diseases, and in particular relates to a Bacillus cereus JBH-LY1 for controlling corn ear rot and an application thereof. Background Art
[0002] Corn ear rot is an important disease caused by multiple pathogens including Fusarium and mildew that infect corn stems and ears. It occurs in all corn producing areas in China. The pathogens of Fusarium ear rot are mainly Fusarium pseudoverticillium, Fusarium laminarum and Fusarium graminearum, which are related to local climate conditions and geographical location, showing regional polymorphism.
[0003] Corn ear rot is the most serious in the late growth period of corn, mainly harming corn kernels and ears, resulting in serious loss of corn yield and great damage to quality. Corn stalk rot not only causes loss of corn yield, but also produces a variety of fungal toxins, seriously endangering food safety and human and animal health.
[0004] At present, the prevention and control of corn ear rot at home and abroad mainly adopts traditional means such as crop rotation, breeding of disease-resistant varieties, and chemical control, but the disease prevention effect is not ideal. These methods have certain limitations and shortcomings, poor operability and adaptability or unsatisfactory effects. Corn Fusarium ear rot is an airborne disease. Although it can be transmitted through seeds and soil, its pathogens are mainly transmitted through the air, infecting corn kernels through the silk channels or wounds caused by insects and birds. Although chemical pesticides can reduce the harm of corn ear rot to corn to a certain extent, the environmental problems caused by the use of pesticides are also receiving more and more widespread attention from the society. Biological control is the natural control of harmful organisms by beneficial organisms in nature in agricultural ecosystems. Biological control of plant diseases is increasingly attracting people's attention due to its safety, effectiveness, low toxicity and low residue. Biological control is considered to be the most promising prevention and control method. Summary of the invention
[0005] In order to solve the above technical problems, the present invention provides a Bacillus cereus JBH-LY1 strain that can prevent and control the pseudo-verticillium fusorium of corn ear rot, which has the advantages of easy cultivation, high control effect on pseudo-verticillium fusorium, and low environmental pollution, and its application. The Bacillus cereus JBH-LY1 strain was isolated and screened from the soil of the applicant's company and preserved in the China Center for Type Culture Collection with a preservation number of CCTCCNO: M 20241168. The JBH-LY1 strain can be cultured on an artificial culture medium and is easy to obtain and culture, so it is conducive to factory production and promotion and application. The JBH-LY1 strain can be used to inhibit pseudo-verticillium fusorium and prevent and control corn ear rot, and its fermentation liquid has a significant inhibitory effect on pseudo-verticillium fusorium, with an inhibition rate of up to 88.9%. The present invention uses biotechnology to effectively reduce the harm of corn ear rot fungi to corn, and at the same time, compared with chemical agents, it also effectively avoids problems such as environmental pollution.
[0006] In order to achieve the above object, the present invention adopts the following technical solutions:
[0007] One of the purposes of the present invention is to provide a strain of Bacillus cereus JBH-LY1 for preventing and controlling corn ear rot, which has been deposited in the China Center for Type Culture Collection (address: Wuhan University, Wuhan, China, i.e. Luojia Mountain, Wuchang, Wuhan City, Hubei Province) on June 6, 2024, with a deposit number of CCTCC NO: M 20241168.
[0008] The JBH-LY1 strain provided by the present invention was screened from a soil sample of Henan Jinbaihe Biotechnology Co., Ltd., and the isolation method thereof was as follows: 10 g of a soil sample of Henan Jinbaihe Biotechnology Co., Ltd. was added to a 250 mL triangular flask containing 90 mL of sterile water and shaken in a shaking table at 180 r / min for 30 min, and then 1 mL of the supernatant was taken with a pipette in an ultra-clean workbench and diluted by 10 times, and 10 3 ~10 6 The dilution was spread on nutrient agar medium and cultured in a 37°C incubator for 24 h. Different single colonies were then picked out with an inoculation loop and transferred to nutrient agar plates for purification for subsequent biological activity tests. The JBH-LY1 strain was thus screened.
[0009] After being stained with crystal violet and observed under an optical microscope at 100 times oil lens, the JBH-LY1 strain has rod-shaped cells with square ends in short or long chains. It produces spores, which are round or columnar, mesophotic or submesophotic, and the cysts have no obvious swelling. It is Gram-positive, capsule-free, and motile. It forms white, round or subcircular, raised, about 1-3mm in diameter, soft in texture, without pigment, and slightly shiny white colonies (candle-like color) on ordinary nutrient agar. And the 16S rRNA gene sequence was determined and the nucleic acid sequence homology was compared with other similar sequences included in the NCBI database. The results showed that the similarity between JBH-LY1 and Bacillus cereus strain P-6 accession number KY249126.1 was 100%. Therefore, the JBH-LY1 strain was identified as Bacillus cereus.
[0010] The culture method of the JBH-LY1 strain in the present invention is:
[0011] (1) In vitro culture
[0012] The formula of nutrient broth liquid culture medium is: 0.3% beef extract, 1% peptone, 0.5% sodium chloride, and the rest is water.
[0013] The JBH-LY1 bacteria were inoculated into the above culture medium, cultured in a shaking incubator at 37°C for 24 h at a rotation speed of 180 r / min to obtain test tube cultures.
[0014] (2) Liquid expansion culture
[0015] The formula of nutrient broth liquid culture medium is: 0.3% beef extract, 1% peptone, 0.5% sodium chloride, and the rest is water.
[0016] The test tube seeds obtained by culturing the above test tube seeds were inoculated into 250 mL triangular flask nutrient broth liquid culture medium, with 100 mL in each bottle, and cultured in a shaking incubator at 37° C. for 24 h at a rotation speed of 180 r / min.
[0017] (3) Plate inoculation culture
[0018] The formula of nutrient agar medium is: 0.3% beef extract, 1% peptone, 0.5% nutrient broth Cl, 2% agar, and the rest is water.
[0019] The JBH-LY1 strain obtained by the above liquid expansion culture was inoculated onto a nutrient agar medium and cultured at 37° C. for 24 h to obtain a plate strain.
[0020] The second object of the present invention is to provide a microbial preparation, which contains the Bacillus cereus JBH-LY1.
[0021] The third object of the present invention is to provide a method for preparing the microbial preparation, which comprises placing the Bacillus cereus JBH-LY1 on a nutrient agar medium, culturing at 35 to 39° C. for 22 to 26 hours, and obtaining its fermentation liquid.
[0022] Preferably, the culture conditions are: culture at 37°C for 24 hours.
[0023] A fourth object of the present invention is to provide an application of the Bacillus cereus JBH-LY1, the microbial preparation or the preparation method in the production of β-1,3 glucanase.
[0024] The fifth object of the present invention is to provide an application of the Bacillus cereus JBH-LY1, the microbial preparation or the preparation method in the production of protease.
[0025] The sixth object of the present invention is to provide an application of the Bacillus cereus JBH-LY1, the microbial preparation or the preparation method in inhibiting Fusarium verticillioides.
[0026] In the process of verifying the JBH-LY1 strain, its ability to produce β-1,3 glucanase and protease, and its inhibition of Fusarium spp., the present invention also provides a method for culturing a Fusarium spp. cake and a spore suspension of Fusarium spp.:
[0027] (1) Cultivation of Fusarium spp. cake
[0028] The formula of PDA medium is: 20% potato, 2% glucose, 2% agar, and the rest is water.
[0029] The strain of Pseudomonas verticillioides was transferred to the above-mentioned PDA liquid culture medium using a hole puncher with a diameter of 6 mm, and cultured at 26° C. for 72 h to obtain a Pseudomonas verticillioides cake.
[0030] (2) Preparation of Fusarium spore suspension
[0031] The formula of Czapek's liquid medium is: 3% sucrose, 0.3% sodium nitrate, 0.1% potassium dihydrogen phosphate, 0.05% magnesium sulfate, 0.05% potassium chloride, 0.001% ferrous sulfate, pH 7.0-7.2, and the rest is water.
[0032] The strain of Fusarium spp. was transferred to the above-mentioned Czapek liquid medium at a ratio of 1:100, and cultured in a shaking incubator at 26°C and 180 r / min for 72 h to obtain a spore suspension of Fusarium spp.
[0033] The seventh object of the present invention is to provide an application of the Bacillus cereus JBH-LY1, the microbial preparation or the preparation method in corn planting.
[0034] The eighth object of the present invention is to provide an application of the Bacillus cereus JBH-LY1, the microbial preparation or the preparation method in preventing and controlling corn ear rot.
[0035] Compared with the prior art, the present invention has the following technical effects:
[0036] (1) The JBH-LY1 strain obtained in the present invention can be cultured on an artificial culture medium and is easy to obtain and culture. The strain used for disease control must be cultured on an artificial culture medium, so the strain in the present invention is very conducive to industrialization and promotion and application.
[0037] (2) The fermentation liquid of the JBH-LY1 strain obtained in the present invention has a significant inhibitory effect on Fusarium spp., with an inhibition rate of up to 88.9%.
[0038] (3) The present invention utilizes biotechnology to effectively reduce the damage of Fusarium oxysporum to corn, and at the same time avoids problems such as environmental pollution compared with chemical agents.
[0039] Description of the deposit certificate:
[0040] Depository: China Center for Type Culture Collection;
[0041] Deposit number: CCTCC NO: M 20241168;
[0042] Deposit date: June 6, 2024;
[0043] Deposit address: Wuhan University, Wuhan, China;
[0044] Taxonomic name: Bacillus cereus. BRIEF DESCRIPTION OF THE DRAWINGS
[0045] Figure 1 The plate colony morphology of Bacillus cereus JBH-LY1 in Example 1 of the present invention;
[0046] Figure 2 The spore morphology of Bacillus cereus JBH-LY1 under oil microscope in Example 1 of the present invention;
[0047] Figure 3 This is a transparent circle picture of protease produced by Bacillus cereus JBH-LY1 in Example 5 of the present invention;
[0048] Figure 4This is a transparent circle picture of β-1,3-glucanase produced by Bacillus cereus JBH-LY1 in Example 5 of the present invention;
[0049] Figure 5 This is a plate confrontation diagram of the Bacillus cereus JBH-LY1 strain antagonizing Fusarium spp. in Example 6 of the present invention. DETAILED DESCRIPTION
[0050] The following examples are used to illustrate the present invention, but are not intended to limit the scope of the present invention. Without departing from the spirit and essence of the present invention, modifications or replacements made to the method, steps or conditions of the present invention all belong to the scope of the present invention. The reagents and instruments used in the following examples can all be obtained commercially, and the methods used in the examples are consistent with the conventional methods unless otherwise specified.
[0051] The culture medium formula used in the present invention is as follows:
[0052] The formula of nutrient broth liquid culture medium is: 0.3% beef extract, 1% peptone, 0.5% sodium chloride, and the rest is water.
[0053] The formula of nutrient agar medium is: 0.3% beef extract, 1% peptone, 0.5% sodium chloride, 2% agar, and the rest is water.
[0054] The formula of PDA medium is: 20% potato, 2% glucose, 2% agar, and the rest is water.
[0055] The formula of Czapek's liquid medium is: 3% sucrose, 0.3% sodium nitrate, 0.1% potassium dihydrogen phosphate, 0.05% magnesium sulfate, 0.05% potassium chloride, 0.001% ferrous sulfate, pH 7.0-7.2, and the rest is water.
[0056] β-1,3 glucanase detection culture medium formula: dextran 0.5%, NaNO3 0.2%, K2HPO3·3H2O 0.1%, KCl 0.05%, MgSO4·7H2O 0.05%, FeSO4 0.001%, Congo red 0.002%, agar 2%, pH 7.0-7.2, and the rest is water.
[0057] Protease detection culture medium formula: 0.3% skim milk powder, 1% peptone, 0.5% sodium chloride, 0.3% beef extract, 2% agar, pH 7.0-7.2, and the rest is water.
[0058] The test culture media β-1,3 glucanase detection medium and protease detection medium were sterilized at 115℃ for 15min, and other culture media were sterilized at 121℃ for 15min.
[0059] The technical solution of the present invention is further described in detail below in conjunction with embodiments.
[0060] Example 1 Isolation and identification of JBH-LY1 strain
[0061] 10g of soil sample from Henan Jinbaihe Biotechnology Co., Ltd. was added to a 250mL Erlenmeyer flask containing 90mL of sterile water and shaken at 180r / min for 30min. Then, 1mL of supernatant was taken with a pipette in a clean bench and diluted by 10 times. 3 ~10 6 The dilution was spread on nutrient agar medium and cultured in a 37°C incubator for 24 h. Different single colonies were then picked out with an inoculation loop and transferred to nutrient agar plates for purification for subsequent biological activity tests. The JBH-LY1 strain was thus screened.
[0062] After being stained with crystal violet and observed under an optical microscope at 100 times oil immersion, JBH-LY1 bacterial cells were rod-shaped with square ends in short or long chains. Spores were produced, which were round or cylindrical, mesogenic or submesogenic, and the cysts had no obvious swelling ( Figure 2 ). Gram-positive, no capsule, motile. Forms white, round or nearly round, raised, about 1-3 mm in diameter, soft, non-pigmented, slightly shiny white colonies (candle-like color, Figure 1 ). It can be seen that the biological characteristics of JBH-LY1 are consistent with those of Bacillus cereus.
[0063] Further 16S rRNA gene sequence determination (16S rRNA sequence is shown in SEQ ID NO.1) and nucleic acid sequence homology comparison with other similar sequences included in the NCBI database showed that the similarity between JBH-LY1 and Bacillus cereus strain P-6 accession number KY249126.1 was 100%.
[0064] Therefore, JBH-LY1 was identified as Bacillus cereus.
[0065] SEQ ID NO.1:
[0066]
[0067] Example 2 Cultivation of JBH-LY1 strain
[0068] The JBH-LY1 bacteria were inoculated into nutrient broth liquid culture tubes (5 mL per tube), and cultured at 37°C and 180 r / min shaking for 24 h to obtain test tube cultures.
[0069] Transfer the test tube culture into a fresh nutrient broth liquid culture medium test tube (5 mL per tube), and culture at 37°C and 180 r / min shaking for 24 hours to obtain a bacterial solution with strong activity.
[0070] Take 100 μL of the above bacterial solution and spread it on fresh nutrient agar medium, and culture it in a 37°C incubator for 24 hours to obtain plate bacteria.
[0071] Example 3 Obtaining JBH-LY1 fermentation broth
[0072] The test tube seed in Example 2 was inoculated into a 250 mL nutrient broth liquid culture medium in a triangular flask (100 mL per flask), and cultured at 37° C. and 180 r / min in a shaking incubator for 24 h to obtain a fermentation culture.
[0073] The fermentation culture was centrifuged at 12000 r / min for 15 min, the supernatant was taken, and filtered with a 0.22 um filter to obtain a sterile fermentation broth.
[0074] Example 4 Preparation of Fusarium pseudoverticillium spore suspension
[0075] The strain of Fusarium spp. (which was isolated and preserved by the R&D Center of Henan Jinbaihe Biotechnology Co., Ltd. from diseased corn plants in a corn field in Tangyin County, Anyang City, Henan Province) was transferred to the above-mentioned Czapek liquid medium at a ratio of 1:100 and cultured in a shaking incubator at 26°C and 180 r / min for 72 h to obtain 1×10 7 CFU / mL spore suspension of Fusarium spores is ready for use.
[0076] Example 5 Ability of JBH-LY1 strain to produce β-1,3 glucanase and protease
[0077] Prepare 100 mL of β-1,3 glucanase and protease detection medium, sterilize at 115℃ for 15 min, and pour into plates for later use. Use a sterilized 6 mm diameter hole puncher in the clean bench to punch holes in the center of the β-1,3 glucanase and protease detection plates, respectively. Use a micropipette to add 200 μl of the sterile fermentation liquid of Example 3 into the center holes of the β-1,3 glucanase and protease detection plates. Place in a 37℃ constant temperature incubator and culture for 48 hours, and observe the size of the enzymatic hydrolysis circle. Measure the size of the enzymatic hydrolysis circle and calculate the enzyme production capacity. The results are shown in Table 1 and Figure 3 , Figure 4shown.
[0078] Table 1 Protease and β-1,3 glucanase production ability of JBH-LY1 strain
[0079]
[0080] From Table 1 and Figure 3 , Figure 4 It can be seen that the JBH-LY1 strain can produce β-1,3 glucanase and protease with strong enzyme production ability. β-1,3 glucanase and protease can destroy the cell wall of Fusarium spp. and then kill Fusarium spp., thereby effectively preventing and controlling the occurrence of corn ear rot.
[0081] Example 6 Test on the inhibition of Fusarium spp. by JBH-LY1 strain
[0082] The fermentation liquid obtained in Example 3 was used to test the inhibitory effect of the JBH-LY1 strain on the hyphae growth of Fusarium spp. The Fusarium spp. in good growth state was removed from the original plate with a hole puncher with a diameter of 6 mm, and the cake was inoculated into the center of a new PDA solid culture medium in an ultra-clean workbench. A hole with a diameter of 6 mm was punched at 1.5 cm on both sides of the Fusarium spp. cake, and 200 μl of the sterile fermentation liquid of Example 3 was added into the holes on both sides with a micropipette, and the culture was placed in a 26°C constant temperature incubator for 5 days and the size of the inhibition zone was observed. The diameter of the inhibition zone was measured and the inhibition rate was calculated. Inhibition rate = (control group fungal colony growth radius - treatment group fungal colony growth radius) / control group fungal colony growth radius × 100%. The results are as follows Figure 5 shown.
[0083] like Figure 5 As shown, the fermentation liquid of JBH-LY1 strain had an inhibition rate of up to 88.9% against Fusarium spp., which showed a significant inhibitory effect, indicating that Bacillus cereus JBH-LY1 strain had great biocontrol potential for preventing and controlling corn ear rot.
[0084] Example 7 Experiment on the inhibition of spore germination of Fusarium pseudoverticillium by JBH-LY1 fermentation broth
[0085] The fermentation liquid of JBH-LY1 in Example 3 was inoculated into the suspension of Fusarium spp. in Example 4 at a ratio of 1:100, and a blank control was set. Sterile water was added to the blank control at a ratio of 1:100, and the germination of spores was observed under a microscope at 26°C and 180r / min for 24h. The germination number of spores was observed in 10 fields of view for each treatment and control, and the germination rate was calculated, germination rate = number of germinated spores / total number of spores. The results are shown in Table 2.
[0086] Table 2 Effect of JBH-LY1 strain on spore germination of Fusarium spores
[0087]
[0088]
[0089] From the test results in Table 2, it can be seen that after the spore suspension of F. pseudoverticillium was treated with JBH-LY1 fermentation broth for 24 hours, the germination rate of F. pseudoverticillium spores was only 25.9%, which was better than 49.0% of the blank control, showing a better inhibitory effect.
[0090] Example 8 Field Trial on Corn Ear Rot
[0091] The experiment adopted the silk channel injection method: 1-2 days after the corn silking and pollen shedding, the inoculum was injected into the space between the bracts and the cob from the top of the female ear along the silk channel using a 5mL syringe, and the inoculum was allowed to infiltrate inward along the silk. For each corn plant, only 2mL of inoculum was quantitatively injected into the first female ear at the top, and the inoculum concentration was 1×10 7 cfu / mL. In this test, 2 mL of JBH-LY1 fermentation liquid of Example 3 was first inoculated, and 1 day later, the spore suspension of Fusarium pseudoverticillium of Example 4 was inoculated. A blank control was set up for the test, and the blank control was connected to sterile water. Three repetitions were set up, and 10 corn ears were inoculated in each treatment. After the corn matured, a field disease survey was conducted, and the survey data was recorded to calculate the incidence rate, disease index and control effect The results are shown in Table 3.
[0092] Table 3 Field efficacy of JBH-LY1 strain against corn ear rot in the field
[0093] Incidence % Disease index Control effect% Blank control 100 62.36 - JBH-LY1 fermentation broth 42.5 33.28 46.63
[0094] From the test results in Table 3, it can be seen that the fermentation broth of JBH-LY1 has a good control effect on corn ear rot caused by Pseudomonas aeruginosa, and the control efficiency reaches 46.63% compared with the control, indicating that the JBH-LY1 strain has good resistance to corn ear rot caused by Pseudomonas aeruginosa.
[0095] The embodiments described above are only descriptions of the preferred modes of the present invention, and are not intended to limit the scope of the present invention. Without departing from the design spirit of the present invention, various modifications and improvements made to the technical solutions of the present invention by ordinary technicians in this field should all fall within the protection scope determined by the claims of the present invention.
Claims
1. A strain of Bacillus cereus JBH-LY1 for controlling corn ear rot, It is characterized in that Its deposit number is CCTCC NO: M 20241168, and it is deposited in China Center for Type Culture Collection.
2. A microbial preparation, It is characterized in that It contains the Bacillus cereus JBH-LY1 described in claim 1.
3. The method for preparing the microbial preparation according to claim 2, It is characterized in that The method comprises the steps of placing the Bacillus cereus JBH-LY1 on a nutrient agar medium, culturing at 35-39° C. for 22-26 hours, and obtaining a fermentation liquid thereof.
4. Use of the Bacillus cereus JBH-LY1 according to claim 1, the microbial preparation according to claim 2 or the preparation method according to claim 3 in the production of β-1,3 glucanase.
5. Use of the Bacillus cereus JBH-LY1 according to claim 1, the microbial preparation according to claim 2 or the preparation method according to claim 3 in producing protease.
6. Use of the Bacillus cereus JBH-LY1 according to claim 1, the microbial preparation according to claim 2 or the preparation method according to claim 3 in inhibiting Fusarium verticillioides.
7. Use of the Bacillus cereus JBH-LY1 according to claim 1, the microbial preparation according to claim 2 or the preparation method according to claim 3 in planting corn.
8. Use of the Bacillus cereus JBH-LY1 according to claim 1, the microbial preparation according to claim 2 or the preparation method according to claim 3 in preventing and controlling corn ear rot.