Bacillus cereus for antagonizing multiple pathogenic fungi of potatoes and application of bacillus cereus

Abstract

The invention discloses bacillus cereus for antagonizing multiple pathogenic fungi of potatoes and application of the bacillus cereus, and relates to the field of biological bacterium agents, the bacterium agent is bacillus cereus Bc19, the bacillus cereus Bc19 is preserved in China General Microbiological Culture Collection Center, the preservation address is No.3, No.1 yard, Beichen West Road, Chaoyang District, Beijing, the preservation date is November 11, 2024, and the preservation number is CGMCC No.32578. The method is used for preparing a fungicide for antagonizing various pathogenic fungi of potatoes. The bacillus cereus Bc19 strain which has an inhibiting effect on six pathogenic fungi of potatoes is obtained, a high-quality strain is provided for development and research of a fungicide for widely inhibiting the pathogenic fungi of the soil-borne diseases of the potatoes, and a foundation is laid.

Description

Technical Field

[0001] The invention belongs to the field of biological microbial agents, and in particular relates to a Bacillus cereus strain capable of antagonizing multiple pathogenic fungi of potatoes and an application thereof. Background Art

[0002] my country ranks first in the world in terms of potato planting area and total output, and it is an important crop for both food and vegetables. With the intensification of agricultural land and changes in planting structure, the potato planting area has increased. In order to obtain higher economic benefits, farmers usually choose to plant potatoes continuously, that is, continuous cropping. Long-term continuous cropping leads to an imbalance in the structure of soil microbial communities, especially fungal communities, which are enriched with a variety of pathogenic fungi, making it easy for soil-borne diseases to break out, resulting in yield losses or even crop failures.

[0003] Potato fungal diseases mainly include early blight (also known as brown spot), dry rot, wilt, leaf / stem spot, etc. The pathogens are Alternaria solani, Alternaria alternata, Fusarium solani, Fusarium oxysporum, Fusarium equiseti, etc. They occur in a wide range and are prone to cause large-scale yield reduction. In addition, Didymella glomerata was isolated from potato lesions for the first time in this experimental area. It has a wide host and can cause leaf spot disease in nearly 100 plants. The pathogens of soil-borne diseases usually coexist in the soil. When water and heat conditions are suitable for a certain pathogen to multiply in large quantities, the pathogenic bacteria will cause the disease. Most of the antagonistic bacteria currently used contain only a single species and have a good antagonistic effect on only a certain pathogen, resulting in unstable control effects of soil-borne diseases in the field. Therefore, obtaining a biocontrol strain that is antagonistic to multiple pathogenic fungi is of great significance for solving soil-borne potato diseases and ensuring stable yields.

[0004] Bacillus is widely used in agriculture. Bacillus cereus is mainly used to degrade complex organic compounds. Therefore, this strain is also used to degrade allelopathic autotoxic substances, such as phthalates, and to alleviate soil continuous cropping obstacles, but it is less used in suppressing soil-borne diseases. Although reports have shown that it can inhibit a variety of pathogens, including the pathogens of Acanthopanax senticosus diseases Rhizoctonia solani, Fusarium solani, Fusarium oxysporum, Alternaria tenuissima, Alternaria panax, Cylindrocarpon destructans, the main pathogenic fungi of strawberry soil-borne diseases Fusarium oxysporum, Alternaria alternate and Verticillium dahliae, the pathogens of rapeseed sclerotinia Sclerotiorum and Sclerotinia minor, the pathogen of ginseng rust Ilyonectria robusta, and the pathogen of corn ear rot Fusarium verticillioides, etc. However, there are no reports on the inhibition of pathogenic fungi Alternaria solani, Fusarium equiseti and Didymella glomerata, especially no reports on Bacillus cereus that can simultaneously inhibit the pathogenic fungi of potato early blight, wilt, dry rot and leaf / stem spot diseases Alternaria solani, Alternaria alternata, Fusarium equiseti, Fusarium oxysporum, Fusarium equiseti and Didymella glomerata. Summary of the invention

[0005] In order to solve the above technical problems, the present invention provides a strain of Bacillus cereus antagonistic to multiple pathogenic fungi of potato and application thereof.

[0006] The present invention discloses a Bacillus cereus strain antagonistic to multiple pathogenic fungi of potato, which is Bacillus cereus Bc19, which is deposited in the General Microbiology Center of China Culture Collection Administration, with the deposit address at No. 3, Yard No. 1, Beichen West Road, Chaoyang District, Beijing, the deposit date at November 11, 2024, and the deposit number at CGMCC No. 32578.

[0007] The invention discloses an application of a Bacillus cereus strain for antagonizing various pathogenic fungi of potato. The Bacillus cereus Bc19 is used for preparing a bacterial agent for antagonizing various pathogenic fungi of potato.

[0008] Furthermore, the potato multiple pathogenic fungi are pathogens of soil-borne diseases.

[0009] Furthermore, the pathogens of the soil-borne diseases are Alternaria solani, Alternaria alternata, Fusarium solani, Fusarium oxysporum, Fusarium equisetum and grape stem blight.

[0010] Furthermore, the antibacterial rate of the Bacillus cereus Bc19 against Alternaria solani, Alternaria alternata, Fusarium solani, Fusarium oxysporum, Fusarium equisetum and grape stem blight pathogen is 50% to 69%.

[0011] Furthermore, the Bacillus cereus Bc19 has a high inhibition rate of 68-69% against grape stem blight pathogen.

[0012] The present invention has the following beneficial effects:

[0013] The present invention provides a strain of Bacillus cereus Bc19, which has improved antibacterial ability because it is isolated from potato continuous cropping soil in an extreme environment. Bc19 can antagonize pathogenic fungi Alternaria solani, Alternaria alternata, Fusarium solani, Fusarium oxysporum, Fusarium equisetum and grape stem blight pathogens isolated from potato diseased plants. Bc19 has an inhibitory effect on potato early blight pathogens Alternaria solani and Alternaria alternata, dry rot pathogens Fusarium solani, Fusarium oxysporum and Fusarium equisetum, and leaf / stem spot pathogens Grape stem blight pathogens, with an inhibition rate of 50% to 69%. Among them, the inhibition rate of grape stem blight pathogens is relatively high at 68.18%. The present invention obtains a strain of Bacillus cereus Bc19 that has an inhibitory effect on 6 potato pathogens, provides a high-quality strain for comprehensive prevention and control of potato soil-borne diseases, and has a good application prospect. BRIEF DESCRIPTION OF THE DRAWINGS

[0014] Figure 1 These are photos of the isolation and culture of pathogenic fungi; the photos of the top row from left to right are the isolation and culture of diseased plants, Alternaria alternata, Alternaria solanacearum, and grape stem blight pathogen; the photos of the bottom row from left to right are the diseased plants, Fusarium solani, Fusarium oxysporum, and Fusarium equisetum;

[0015] Figure 2 It is a BLAST comparison map of pathogenic fungi; among them, from top to bottom, they are Alternaria alternata, Alternaria solani, Grape stem blight, Fusarium solani, Fusarium oxysporum, and Fusarium equisetum;

[0016] Figure 3 The photos of the antibacterial confrontation of Bw34 plates; among them, the upper row from left to right are Alternaria alternata, Alternaria solanacearum, and grape stem blight pathogen; the lower row from left to right are Fusarium solani, Fusarium oxysporum, and Fusarium equisetum;

[0017] Figure 4 This is a photo of purified Bc19;

[0018] Figure 5 This is the BLAST comparison map of Bc19 strain. DETAILED DESCRIPTION

[0019] In order to make the objectives, technical solutions and advantages of the embodiments of the present invention more clearly understood, the spirit of the contents disclosed by the present invention will be described in detail below. After understanding the embodiments of the contents of the present invention, any technician in the relevant technical field can change and modify them according to the techniques taught by the contents of the present invention without departing from the spirit and scope of the contents of the present invention.

[0020] The exemplary embodiments of the present invention and the description thereof are used to explain the present invention but are not intended to limit the present invention.

[0021] Example 1 Isolation and purification of pathogenic fungi

[0022] Cut the diseased potato plants into small pieces according to their parts, disinfect their surfaces, and then culture them on PDA medium. Observe the growth of the colonies and isolate and purify the pathogenic fungi (see Figure 1 ). The purified fungus was extracted and sequenced, and compared with the NCBI database (see Figure 2 ) and stored in a -80°C refrigerator. Common potato pathogenic fungi Alternaria solani, Alternaria alternata, Fusarium solani, Fusarium oxysporum, Fusarium equisetum and the first isolated pathogenic fungus Grape stem blight were selected for use.

[0023] Example 2 Isolation and purification of bacteria

[0024] This example is located in Wuchuan County, Inner Mongolia Autonomous Region. The soil is chestnut soil, which is relatively poor, low in organic matter, and has a long-term potato continuous cropping location field test with little rain and drought all year round. Collect the potato continuous cropping soil, weigh 5.0g and put it into a sterile triangular flask filled with 95mL LB culture medium, cover it with a sealing film, place it on a shaker at 220rpm for 15min, let it stand at room temperature for 10min, take the supernatant and dilute it 10 -3 , 10 -4 , 10 -5100ul of dilution was spread on NA plates, and 3 replicates were set for each dilution gradient. After culturing in a 37℃ constant temperature incubator for 2 days, the culture was purified. Single colonies were picked and streaked on new NA plates to purify the target bacteria, and stored in -20℃ and -80℃ refrigerators for later use.

[0025] Example 3 Screening and identification of antagonistic bacteria

[0026] The strains that have inhibitory effects on Alternaria alternata, Alternaria alternata, Fusarium solani, Fusarium oxysporum, Fusarium equisetum and grape stem blight were screened by the plate confrontation culture method. First, mark the back of the center of the laid PDA solid culture medium, mark one point on the left and right at 2 cm, use a pipette to absorb 10uL of Ba45 bacterial solution adjusted to a concentration of 108 and apply it to the left and right points, then take the above pathogenic fungus cake and stick it on the center point, and repeat 3 times for each bacterium. Place the culture dish in a 28℃ incubator and invert it for 48h-7d, and then observe the inhibition zone ( Figure 3 ) and calculated the inhibition rate (Table 1). Bacteria that have antagonistic effects on the above six pathogenic fungi were selected for species identification. DNA was extracted, PCR reaction system was established, amplification was performed using universal primers, and gel excision sequencing was performed. The homology with Bacillus cereus.BF15 and Bacillus cereus.MOB-3 in the database was higher than 99.8% ( Figure 5 ). This indicates that the strain is Bacillus cereus and is named Bacillus cereus.Bc19.

[0027] Example 4 Bc19 strain antibacterial properties test:

[0028] The enzyme activity of Bc19 was measured by β-1,3 glucanase test kit and chitinase test kit. 8 After culturing in M9 medium for 24 hours, the strain produced 428.2 U / g of β-1,3 glucanase and 3.84 U / g of chitinase, which showed that the strain could degrade the fungal cell wall and inhibit its growth and reproduction.

[0029] The morphological characteristics of the Bc19 colony in this example: After culturing Bc19 on LB solid medium at 37°C for 2 days, the colonies were observed to be round or nearly round, opaque, slightly shiny white bacteria, resembling frosted glass or melted wax ( Figure 4 ).

[0030] Table 1 Bacillus cereus Bc19 inhibition rate

[0031] Pathogenic fungi Control (cm) Colony diameter (cm) Inhibition rate (%) Alternaria solani 5.15 2.08 59.61 Alternaria alternata 7.80 3.10 60.26 Fusarium solani 4.40 2.48 43.56 Fusarium oxysporum 3.83 3.13 18.26 Fusarium equiseti 5.60 2.75 50.89 Didymella glomerata 7.70 2.45 68.18

Claims

1. A strain of Bacillus cereus that is antagonistic to multiple pathogenic fungi of potato, characterized in that It is Bacillus cereus Bc19, deposited in the General Microbiology Center of China Culture Collection Administration, the deposit address is No. 3, No. 1 Beichen West Road, Chaoyang District, Beijing, the deposit date is November 11, 2024, and the deposit number is CGMCC No. 32578.

2. The use of a strain of Bacillus cereus for antagonizing multiple pathogenic fungi of potato as claimed in claim 1, characterized in that The Bacillus cereus Bc19 is used for preparing a bacterial agent for antagonizing various pathogenic fungi of potatoes.

3. The use according to claim 2, characterized in that The potato multiple pathogenic fungi are pathogenic fungi of soil-borne diseases.

4. The use according to claim 3, characterized in that The pathogens of the soil-borne diseases are Alternaria solani, Alternaria alternata, Fusarium solani, Fusarium oxysporum, Fusarium equisetum and grape stem blight.

5. The use according to claim 4, characterized in that The bacteriostasis rate of the Bacillus cereus Bc19 on Alternaria solani, Alternaria alternata, Fusarium solani, Fusarium oxysporum, Fusarium equisetum and grape stem blight pathogen is 50% to 69%.

6. The use according to claim 4, characterized in that The Bacillus cereus Bc19 has a high inhibition rate of 68-69% on grape stem blight pathogen.

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