Bacillus velezensis Gc8, compound microbial agent and application of compound microbial agent

Abstract

The invention belongs to the technical field of biological control, and particularly relates to the technical field of crop stem rot control. The invention provides a bacillus velezensis Gc8, and the preservation number of the bacillus velezensis Gc8 is CGMCC (China General Microbiological Culture Collection Center) No. 28991. The bacillus velezensis Gc8 is preserved in the China General Microbiological Culture Collection Center (CGMCC) on November 14, 2023, the preservation number of the bacillus velezensis Gc8 is CGMCC No.28991, the center is called CGMCC for short, and the address of the center is No.3, Yard 1, Beichen West Road, Chaoyang District, Beijing. The invention also provides a compound microbial agent, wherein the compound microbial agent comprises bacillus velezensis Gc8, kitasapora sp. Ak67 and pseudomonas fluorescens pf27, and the compound microbial agent comprises bacillus velezensis Gc8, kitasapora sp. Ak67 and pseudomonas fluorescens pf27, and the compound microbial agent comprises bacillus velezensis Gc8, kitasapora sp. Ak67, kitasapora fluorescens pf27, kitasapora fluorescens pf27 and kitasapora fluorescens pf27 and kitasapora fluorescens pf27 and kitasapora fluorescens pf27. The compound microbial agent can inhibit spore germination of pathogenic microorganisms, and has the effect of promoting growth of corn while resisting diseases.

Description

Technical Field

[0001] The invention belongs to the technical field of biological control, and in particular relates to the technical field of controlling crop stem base rot. Background Art

[0002] Crop stem rot is a typical soil-borne complex pathogenic disease, with multiple pathogenic fungi as the main pathogens, showing a high degree of complexity. The infection cycle of this disease is long, and the pathogen residues in the soil continue to accumulate, which in turn exacerbates the epidemic trend of stem rot.

[0003] Corn stalk rot is caused by a variety of pathogens acting alone or in concert, and can be divided into two categories: bacterial stalk rot and fungal stalk rot. The main pathogen of bacterial stalk rot is Erwinia chrysanthemi, while the pathogens of fungal stalk rot mainly include Fusarium and Pythium. Generally, the incidence of corn stalk rot in the field is about 15%, which can cause a 25% loss in corn yield. When the disease is severe, the yield reduction rate can be as high as 80% or more, especially in years with more rainfall, when the disease is more serious.

[0004] At present, the prevention and control strategies for stem rot of crops mainly include chemical control, biological control and breeding of resistant varieties. However, the existing crop varieties on the market have significant differences in resistance to stem rot, and there is a lack of highly resistant wheat and corn varieties. Although the use of chemical agents can control the disease to a certain extent, it may have adverse effects on the ecological environment and food safety. Therefore, there is an urgent need to explore and apply safer and more efficient biological control measures. Summary of the invention

[0005] The technical problem to be solved by the present invention is to provide a composite microbial agent for biological control of crop stem base rot, thereby overcoming the drawbacks of single agent application in the field. The composite microbial agent includes Bacillus velezensis Gc8, Kitasatospora sp. Ak67 and Pseudomonas fluorescens pf27. The composite microbial agent can inhibit the germination of pathogenic microbial spores, and promote the growth of corn while resisting diseases.

[0006] Bacillus velezensis Gc8 was deposited in the General Microbiology Center of China Culture Collection Administration on November 14, 2023. The culture deposit number is CGMCC No. 28991. The center is abbreviated as CGMCC. The address of the center is No. 3, No. 1 Beichen West Road, Chaoyang District, Beijing.

[0007] Kitasatospora sp. Ak67 was deposited in the General Microbiology Center of China Culture Collection Administration on November 21, 2023. The culture collection number is CGMCC No.29076. The center is abbreviated as CGMCC. The address of the center is No. 3, No. 1 Beichen West Road, Chaoyang District, Beijing.

[0008] Pseudomonas fluorescens pf27 was deposited in the General Microbiological Center of China Microbiological Culture Collection Administration on May 8, 2017, with the culture deposit number CGMCCNO.14105. The center is referred to as CGMCC, and the address of the center is No. 3, No. 1 Beichen West Road, Chaoyang District, Beijing. Pseudomonas fluorescens pf27 has been disclosed in the patent text of Chinese Patent Publication No. CN107236689A.

[0009] The primary purpose of the present invention is to provide a composite microbial agent, which comprises Bacillus velezensis Gc8, Kitasatospora sp. Ak67 and Pseudomonas fluorescens pf27.

[0010] The composite microbial agent is characterized in that the fermentation broths of Bacillus velezensis Gc8, Kitasatospora sp. Ak67 and Pseudomonas fluorescens pf27 in the composite microbial agent are mixed in equal volume ratios.

[0011] The second object of the present invention is to provide a method for preparing the above-mentioned composite biological agent, wherein Bacillus velezensis Gc8, Kitasatospora sp. Ak67 and Pseudomonas fluorescens pf27 are fermented separately, and the fermentation broths of the two are mixed in a volume ratio of 1:1:1 to obtain the above-mentioned composite microbial agent.

[0012] Furthermore, the culture and fermentation method of Bacillus Velez Gc8 is as follows: Bacillus Velez Gc8 is activated on an LB solid plate, cultured at 28°C for 2 days, a single colony is picked and inoculated into an LB seed medium, cultured at 28°C and 200 rpm / min for 1 day, inoculated into Landy modified medium at a ratio of 5%, and cultured at 28°C and 200 rpm / min for 4 days to obtain a fermentation liquid of Bacillus Velez Gc8, and the number of viable bacteria reaches 1×10 9 ~1×10 10 CFU / mL.

[0013] Furthermore, the bacterial preparation method of Kitasatosporium Ak67 is as follows: the strain Ak67 is activated on an ISP 2 solid plate, cultured at 28°C for 2 days, a single colony is picked and inoculated into an NB seed medium, cultured at 28°C and 200rpm / min for 1 day, inoculated into Gao's No. 1 liquid medium at a ratio of 5%, and cultured at 28°C and 200rpm / min for 5 days to obtain a fermentation liquid of the strain Ak67, and the number of viable bacteria reaches 1×10 9 ~1×10 10 CFU / mL.

[0014] Furthermore, the culturing and fermentation method of Pseudomonas fluorescens pf27 is as follows: Pseudomonas fluorescens pf27 is activated on an LB solid plate, cultured at 28°C for 2 days, a single colony is picked and inoculated into an LB seed culture medium, cultured at 28°C and 200 rpm / min for 1 day, inoculated into an LB culture medium at a ratio of 5%, and cultured at 28°C and 200 rpm / min for 4 days to obtain a fermentation liquid of Pseudomonas fluorescens pf27, wherein the number of viable bacteria reaches 1×10 9 ~1×10 10 CFU / mL.

[0015] The third object of the present invention is to provide an application of the above-mentioned composite microbial agent in preventing and controlling crop stem rot and promoting crop growth.

[0016] Furthermore, the crop diseases include corn stalk rot.

[0017] Furthermore, the effect of promoting crop growth includes increasing the plant height of corn.

[0018] Furthermore, the application frequency and dosage of the composite microbial agent are as follows: before sowing, the composite microbial agent is diluted 50 times and then soaked in corn seeds for 1 hour; tillers are sprayed with a sprayer, and the dosage is: the agent is diluted 50 times, 50L / mu. BRIEF DESCRIPTION OF THE DRAWINGS

[0019] Figure 1 Cultivation of pathogenic microorganisms of corn stalk rot: Use isolation culture medium to screen possible pathogenic microorganisms from corn leaves and stalks infected with stalk rot.

[0020] Figure 2 To verify the pathogenicity function of Fusarium graminearum: Fusarium graminearum was isolated from corn samples and then injected into corn seedlings through the stems. The pathogenicity of the pathogen was observed after 2-3 weeks to verify its pathogenicity.

[0021] Figure 3 . Screening of antagonistic bacteria to Fusarium graminearum: Use the soil microbial database to screen and culture antagonistic bacteria to obtain biocontrol microorganisms that can effectively kill Fusarium graminearum on plates.

[0022] Figure 4 Growth promotion comparison chart.

[0023] Figure 5 This is a comparison chart of field diseases. DETAILED DESCRIPTION

[0024] The following examples are provided for better understanding of the present invention, but are not intended to limit the present invention. The experimental methods in the following examples are conventional methods unless otherwise specified. The experimental materials used in the following examples are purchased from conventional biochemical reagent stores unless otherwise specified.

[0025] The culture medium used in the following examples is as follows:

[0026] LB solid medium: 10 g / L sodium chloride, 10 g / L tryptone, 5 g / L yeast extract, pH 7.2, add 15 g / L agar to the solid medium;

[0027] NB liquid medium: beef extract 3g / L, peptone 10g / L, sodium chloride 5g / L;

[0028] Landy modified medium: 5 g / L sodium L-glutamate, 10 g / L glucose, 0.5 g / L magnesium sulfate heptahydrate, 0.78 g / L potassium chloride, 1.0 g / L potassium dihydrogen phosphate, 0.05 mg / L ferrous sulfate, 5 mg / L manganese sulfate, 0.16 mg / L copper sulfate, pH 7.2;

[0029] ISP 2 medium: yeast extract powder 4g / L, malt extract powder 10g / L, glucose 4g / L, agar powder 20g / L;

[0030] Gao's No. 1 liquid medium: KNO3 1g / L, soluble starch 20g / L, NaCl 0.5g / L, K2HPO4·3H2O0.5g / L, MgSO4·7H2O 0.5g / L, FeSO4·7H2O 0.01g / L, agar powder 15g / L;

[0031] PDA solid medium: peeled potato 200g / L, glucose 20g / L, agar powder 15g / L;

[0032] PDA liquid culture medium: peeled potatoes 200 g / L, glucose 20 g / L.

[0033] Example 1

[0034] Isolation, screening and identification of pathogens causing stem rot in crops

[0035] (1) Isolation and cultivation of pathogens of crop stem rot

[0036] Maize plants showing typical symptoms of stem base rot were collected from maize fields in Changtu County, Liaoning Province, and samples were collected from the stem base and infected leaves of the diseased plants using sterilized scissors. Cut diseased maize leaves or stalks of a certain size, rinse with tap water, and cut from the healthy tissue 1 to 2 mm around the lesion. Then, after the small beaker is disinfected, the separation material is placed in it and disinfected. After disinfection, rinse with sterile water 2 to 3 times. Cut the separation material into 2 to 3 mm cubes, and each piece of tissue should be alternating diseased and healthy tissue. Use sterilized tweezers to pick up the cut material, place it on a PDA culture medium plate, and press gently. Incubate at a constant temperature of 28°C, and observe after hyphae grow in the PDA culture medium (such as Figure 1 The final concentration of the solution was 25% glycerol and stored in a -80°C refrigerator.

[0037] (2) Identification of pathogenic microorganisms

[0038] Observe the morphology of the colonies in the culture dish, including color, texture, and growth rate. Extract the DNA of the growing strain, amplify the ITS region using PCR technology, and perform sequencing analysis to confirm the specific type of pathogen. The isolated pathogens are shown in Table 1:

[0039] Table 1. Main pathogens of corn stalk rot isolated

[0040]

[0041] (3) Verification of pathogenicity of corn stalk rot pathogen

[0042] Take out the above-mentioned Fusarium graminearum, the pathogenic bacteria of corn stalk rot, from the -80℃ refrigerator, first reactivate the purified and preserved strain for subculture, and culture it in the dark at 25℃ in an incubator for 7 days, then use a toothpick to pierce the stem base of healthy corn seedlings of 3 months old and 20-30 cm in height, with 4 puncture points for each plant, 2 on the top and 2 on the bottom, use the base of a 1ml pipette tip to take a 6mm diameter bacterial cake, move it to the wound site with a clean toothpick, wrap it with moist cotton, and finally wrap it with sticky plastic wrap to keep it moist. Plants inoculated with clean PDA round cakes were used as controls, and the moisturizing method was the same as that of the treatment group. Observe after 7-14 days, and record the disease conditions in the treatment and control groups. Figure 2 As shown, the leaves of the plants inoculated with corn stem rot wilt and droop, turn green, and the stem bases show spots, browning, and signs of rot compared to the healthy control group, indicating that the isolated corn pathogenic microorganisms are pathogenic.

[0043] Example 2

[0044] Isolation and identification of microorganisms for effective prevention and control of the main pathogens of stem rot

[0045] (1) Isolation and preservation of biocontrol strains

[0046] Strain isolation method: The collector collected soil samples in Changtu County, Liaoning Province on August 9, 2022, weighed 5 g of soil samples, put them into 100 mL of sterile water, shook them on a shaker for 30 min, and gradiently diluted them to 10 -4 , 10 -5 , 100 μL of the dilution was respectively applied to the LB plate, incubated at 28°C, colonies of different morphology and color were picked for streak purification, and finally stored in a glycerol solution with a final concentration of 25%, stored in a -80°C refrigerator, and named strain Gc8.

[0047] (2) Biocontrol strains Gc8 and Ak67 can effectively inhibit Fusarium graminearum

[0048] The biocontrol microbial strains Aa2, Ak67, Gc8, Gf81, Ga31, Fk12, Fk15, 4.7800, 4.7808 and NMB4 were taken out from the -80°C refrigerator, streaked on LB solid culture medium, single colonies were picked and inoculated into LB liquid culture medium, and cultured in a shaking incubator at 28°C for 2 days to obtain biocontrol bacteria solution.

[0049] Take out the corn stalk rot pathogen Fusarium graminearum stored in the -80℃ refrigerator and apply it to the PDA plate, and culture it at 28℃ for 2 days. Scrape an appropriate amount of colonies into a sterile centrifuge tube, fully suspend them with sterile water, and adjust the OD600 to 1.0. Take 10μL and inoculate it in the center of the PDA plate, and inoculate 5μL of the biocontrol bacteria solution at a position about 1cm around it. Use the plate without biocontrol bacteria as the control, and observe the results after culturing at 28℃ for 2 days.

[0050] like Figure 3 As shown, on the control plate, Fusarium graminearum was white mycelium, and the mycelial growth of Fusarium graminearum was significantly inhibited in the colonies treated with biocontrol bacteria strains 4.7800, 4.7808, Gc8 and Ak67, with sparse mycelial growth and lightened color, indicating that these biocontrol bacteria have the effect of inhibiting the growth of Fusarium graminearum.

[0051] Example 3

[0052] Preparation of single microbial agents and composite microbial agents

[0053] Take out the stored strains Gc8, Ak67 and pf27 from the -80℃ refrigerator, streak them on LB solid medium and invert them at 28℃ for 24h, pick a single colony and inoculate it in LB liquid medium, and shake and culture it at 28℃ for 1 day as seed liquid. At a ratio of 5%, inoculate Gc8 in Landy modified medium, inoculate Ak67 in Gao's medium No. 1, and inoculate pf27 in LB medium. Cultivate at 28℃, 200rpm / min for 4 days to obtain single bacterial agents of Gc8, Ak67 and pf27, and the number of viable bacteria is 1×10 9 ~1×10 10 CFU / mL.

[0054] The single bacterial agents of Gc8, Ak67 and pf27 are mixed in equal volumes to obtain the composite bacterial agent. The genome information sequence of strain pf27 is recorded in the patent document with the authorized patent number ZL201710480486.7. The 16SrDNA information of the genome of strain Ak67 is SEQ ID NO.1, and the 16SrDNA information of the genome of strain Gc8 is SEQ ID NO.2.

[0055] Example 4

[0056] Effect of compound microbial agent on the control of corn stalk rot

[0057] This example uses the composite microbial agent prepared in Example 3 to verify its effect on promoting corn growth and preventing corn stalk rot in laboratory and field environment tests. The specific steps are as follows:

[0058] The laboratory experiment was conducted at the Institute of Microbiology, Chinese Academy of Sciences in Beijing. Four treatments were set up: composite microbial agent, Ak67, Gc8 and water control. The composite microbial agent in Example 3 was diluted 50 times to obtain the composite microbial agent used in the laboratory experiment. Figure 4 As shown, the plant height of corn treated with the composite bacterial agent in the laboratory environment was significantly higher than that of other controls and the treatment group treated with a single bacterial agent alone, indicating that the composite bacterial agent of the present invention has a significant growth-promoting effect on corn growth.

[0059] The field test was conducted in a corn field in Changtu County, Tieling City, Liaoning Province. Two treatments were set up: a composite microbial agent and a clean water control. The composite microbial agent in Example 3 was diluted 50 times to obtain the composite microbial agent used for the field test. Before sowing, the corn seeds were soaked with the composite microbial agent, and watering and fertilization were consistent with normal field management measures. The incidence of corn basal rot was counted for corn during the growing period, and 3 groups were counted for each treatment group, with 100 plants in each group.

[0060] Incidence rate = susceptible plants / total number of plants × 100% ± standard error, biological control effect = (control incidence rate - treatment incidence rate) / control incidence rate × 100%. The statistics of corn stalk rot incidence in each treatment are shown in Table 2.

[0061] Table 2. Incidence of corn stalk rot in the field after treatment with composite microbial agent

[0062]

[0063] Note: Value = mean ± standard error, different lowercase letters indicate significant difference (P<0.05).

[0064] From the results in Table 2, it can be seen that the composite bacterial agent of the present invention can significantly and effectively prevent and treat corn stalk rot, reducing the incidence of corn stalk rot by 53.9%. Figure 5 As shown, the composite bacterial agent treatment group demonstrated that the composite bacterial agent had a good biocontrol effect when applied in the field.

[0065] Although the above embodiment describes the present invention in detail, it is only a part of the embodiments of the present invention, not all of the embodiments. People can also obtain other embodiments based on this embodiment without creativity, and these embodiments all fall within the protection scope of the present invention.

Claims

1. Bacillus velezensis Gc8, whose deposit number is CGMCC No. 28991.

2. The use of Bacillus velezensis Gc8z according to claim 1 in the preparation of a bacterial agent for inhibiting the growth of Fusarium graminearum.

3. A composite microbial agent comprising the Bacillus velezensis Gc8, Kitasatospora sp. Ak67 and Pseudomonas fluorescens pf27 according to claim 1.

4. The composite microbial agent according to claim 3, characterized in that: The Kitasatosporas p. Ak67 has a preservation number of CGMCC No.29076.

5. The composite microbial agent according to claim 3, characterized in that: The Pseudomonas fluorescens pf27 has a preservation number of CGMCC No.14105.

6. The composite microbial agent according to claim 3, characterized in that: The Bacillus velezensis Gc8, Kitasatospora sp. Ak67 and Pseudomonas fluorescens pf27 are mixed in equal volumes.

7. The composite microbial agent according to claim 3, characterized in that: The viable counts of Bacillus velezensis Gc8, Kitasatospora sp. Ak67 and Pseudomonas fluorescens pf27 were all 1×10 9 ~1×10 10 CFU / mL.

8. Use of the composite microbial agent according to any one of claims 3 to 7 in the preparation of medicines or fertilizers for preventing and treating corn stalk rot.

9. Use of the composite microbial agent according to any one of claims 3 to 7 in the preparation of medicines or fertilizers for promoting corn growth.

10. Use of the composite microbial agent according to any one of claims 3 to 7 in the preparation of a drug or fertilizer for preventing and treating corn stalk rot, characterized in that: The corn stalk rot is caused by Fusarium graminearum.

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