Bacillus cereus for antagonizing multiple pathogenic fungi of potato and application thereof

Abstract

The application relates to a bacillus cereus for antagonizing multiple pathogenic fungi of potatoes and an application thereof, and relates to the field of biological fungicides.The fungicide is bacillus cereus Bc19, which is preserved in the China General Microbiological Culture Collection Center, the address of which is No. 3, Beichen West Road, Chaoyang District, Beijing, and the preservation date is November 11, 2024, and the preservation number is CGMCC No. 32578.The application is used for preparing the fungicide for antagonizing multiple pathogenic fungi of potatoes.A bacillus cereus Bc19 strain with inhibiting effects on six pathogenic fungi of potatoes is obtained, high-quality strains for developing and preparing the fungicide for widely inhibiting pathogenic fungi of soil-borne diseases of potatoes are provided, and a foundation is laid.

Description

Technical Field

[0001] This invention belongs to the field of biological agents, specifically relating to a strain of Bacillus cereus that antagonizes multiple pathogenic fungi of potatoes and its application. Background Technology

[0002] my country ranks first in the world in both potato planting area and total output, making it an important crop suitable for both grain and vegetable production. With the intensification of agricultural land and changes in planting structure, the potato planting area has increased. To obtain higher economic benefits, farmers often choose to continuously cultivate potatoes as a single crop, a practice known as continuous cropping. Long-term continuous cropping leads to an imbalance in the soil microbial community structure, especially the fungal community, resulting in the enrichment of various pathogenic fungi, making it prone to outbreaks of soil-borne diseases, causing yield losses or even crop failure.

[0003] Potato fungal diseases mainly include early blight (also known as brown spot), dry rot, wilt, and leaf / stem spot. The pathogen is *Alternaria alternata* (also known as solanaceous algae spp.). Alternaria solani Alternaria ( Alternaria alternata Fusarium solani ( ) Fusarium solani Fusarium oxysporum ( ), Fusarium oxysporum ), Horsetail ( Fusarium horsetail This fungus, *Vitis vinifera*, is widely distributed and can easily cause large-scale yield reductions. Furthermore, this experimental area is the first to isolate *Vitis vinifera* (also known as grape stem blight pathogen) from potato lesions. Didymella glomerata Soil-borne diseases have a wide host range and can cause leaf spot disease in nearly a hundred plant species. Pathogens of soil-borne diseases typically coexist in the soil, and when conditions such as water and heat are suitable for the proliferation of a particular pathogen, it leads to disease outbreaks. Most currently used antagonistic agents contain only a single species and are only effective against a specific pathogen, resulting in inconsistent control of soil-borne diseases in the field. Therefore, obtaining a biocontrol strain that antagonizes multiple pathogenic fungi is of great significance for solving soil-borne diseases in potatoes and ensuring stable yields.

[0004] Bacillus species are widely used in agriculture. Bacillus cereus (… Bacillus cereus This strain is primarily used for degrading complex organic compounds. Therefore, it is also used to degrade allelopathic autotoxic substances, such as phthalates, and to mitigate soil continuous cropping obstacles, but its application in suppressing soil-borne diseases is relatively limited. Although there are reports showing that it can inhibit a variety of pathogens, including the pathogen of Acanthopanax senticosus, Rhizoctonia solani (… Rhizoctonia solani Fusarium solani ( ), Fusarium solani Fusarium oxysporum ( Fusarium oxysporum ), Alternaria spp. ( Alternaria tenuissima Alternaria ( Alternaria panax ), Destroyer of Columnar Species ( Cylindrocarpon destructans The main pathogens of soil-borne diseases in strawberries are Fusarium oxysporum and Alternaria alterniflora. Alternaria alternata ) and Verticillium dahliae ( Verticillium wilt dahlias ), the pathogen of rapeseed sclerotinia stem rot, *Sclerotinia sclerotiorum* ( Sclerotinia sclerotiorum ) and S. sclerotiorum ( Sclerotinia minor ), Ginseng rust pathogen *Sphaerocarpus spp.* Ilyonectria robusta ), and the pathogen of corn ear rot, Fusarium verticillatum ( Fusarium verticillium ), etc. However, no inhibitory effect was observed against the pathogenic fungus *Alternaria solanacearum* (…). Alternaria solani ), Horsetail ( Fusarium horsetail ) and grape stem blight fungus ( Didymel clusters No reports have been found on Bacillus cereus, especially its ability to simultaneously inhibit the pathogenic fungi of potato early blight, wilt, dry rot, and leaf / stem spot, including Alternaria alternata, Alternaria alternata, Fusarium solani, Fusarium oxysporum, Fusarium equisetifolium, and Grape stem blight. Summary of the Invention

[0005] In order to solve the above-mentioned technical problems, the present invention provides a strain of Bacillus cereus that antagonizes multiple pathogenic fungi of potatoes and its application.

[0006] This invention relates to a strain of Bacillus cereus that antagonizes multiple pathogenic fungi of potatoes. Bacillus cereus Bc19 is deposited at the China General Microbiological Culture Collection Center (CGMCC), located at No. 3, No. 1 Beichen West Road, Chaoyang District, Beijing, on November 11, 2024, with accession number CGMCC No. 32578.

[0007] The present invention relates to the application of a strain of Bacillus cereus antagonistic to multiple potato pathogenic fungi, wherein the Bacillus cereus... Bacillus cereus Bc19 is used to prepare inoculants that antagonize various pathogenic fungi of potatoes.

[0008] Furthermore, the aforementioned potato pathogens are soil-borne pathogens.

[0009] Furthermore, the pathogens of the soil-borne diseases mentioned above are Alternaria alternata, Alternaria alternata, Fusarium solani, Fusarium oxysporum, Fusarium equisetifolium, and Grape stalk blight pathogen.

[0010] Furthermore, the aforementioned Bacillus cereus Bacillus cereus Bc19 has an inhibition rate of 50% to 69% against Alternaria alternata, Alternaria alternata, Fusarium solani, Fusarium oxysporum, Fusarium equisetifolium, and Grape sclerotium.

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

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

[0013] This invention provides a strain of Bacillus cereus, Bc19, whose antifungal ability is enhanced due to its isolation from potato continuous cropping soil in extreme environments. Bc19 can antagonize pathogenic fungi isolated from diseased potato plants, including *Alternaria alternata*, *Alternaria alternata*, *Fusarium solani*, *Fusarium oxysporum*, *Fusarium equisetifolium*, and *Botrytis cinerea*. Bc19 exhibits inhibitory effects against *Alternaria alternata* and *Alternaria alternata* (pathogens of early blight), *Fusarium solani*, *Fusarium oxysporum*, and *Fusarium equisetifolium* (pathogens of dry rot), and *Botrytis cinerea* (pathogen of leaf / stem spot disease), with inhibition rates ranging from 50% to 69%. Among these, it shows a high inhibition rate of 68.18% against *Botrytis cinerea*. This invention provides a *Bacillus cereus* strain Bc19 that inhibits all six pathogenic fungi of potato, offering a high-quality strain for the integrated control of soil-borne potato diseases and showing promising application prospects. Attached Figure Description

[0014] Figure 1 These are photos of the isolation and culture of pathogenic fungi; the top row, from left to right, shows photos of diseased plant culture and isolation, Alternaria alternata, Alternaria solanacea, and Grape blight pathogen; the bottom row, from left to right, shows photos of diseased plants, Fusarium solani, Fusarium oxysporum, and Fusarium equisetifolium.

[0015] Figure 2 This is a BLAST comparison image of pathogenic fungi; from top to bottom, they are Alternaria, Alternaria solanacea, Grape stem blight pathogen, Fusarium solani, Fusarium oxysporum, and Fusarium equisetifolium.

[0016] Figure 3 Photographs of Bw34 plates showing antibacterial action; in the top row, from left to right, are Alternaria, Alternaria solanacea, and Grape blight pathogens; in the bottom row, from left to right, are Fusarium solani, Fusarium oxysporum, and Fusarium equisetifolium.

[0017] Figure 4 Photograph of purified Bc19;

[0018] Figure 5 This is a BLAST comparison diagram of strain Bc19. Detailed Implementation

[0019] To make the objectives, technical solutions, and advantages of the embodiments of the present invention clearer, the spirit of the contents disclosed in the present invention will be described in detail below. After understanding the embodiments of the present invention, any person skilled in the art can make changes and modifications based on the technology taught in the present invention without departing from the spirit and scope of the present invention.

[0020] The illustrative embodiments and descriptions of the present invention 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] Diseased potato plants were cut into small sections according to their location, surface-sterilized, and then cultured on PDA medium. Colony growth was observed, and the pathogenic fungus was isolated and purified (see...). Figure 1 DNA extracted from purified fungi was sequenced and compared with the NCBI database (see [link]). Figure 2 Store at -80℃. Common potato pathogenic fungi such as *Alternaria alternata*, *Alternaria alternata*, *Fusarium solani*, *Fusarium oxysporum*, and *Fusarium equisetifolium*, as well as the first isolated pathogenic fungus *Botrytis cinerea*, were selected for later use.

[0023] Example 2: Isolation and purification of bacteria

[0024] This example was conducted in Wuchuan County, Inner Mongolia Autonomous Region, using chestnut-calcareous soil, which is relatively infertile with low organic matter content and characterized by long-term continuous potato cropping in arid and arid conditions. Soil samples from the continuously cropped potato fields were collected, and 5.0 g was placed in a sterile Erlenmeyer flask containing 95 mL of LB broth. The flask was sealed with a sealing film and incubated on a shaker at 220 rpm for 15 min. After standing at room temperature for 10 min, the supernatant was diluted 10 times. -3 10 -4 10 -5 Dilute 100 μL and spread it onto a nanoplate. Perform three replicates for each dilution gradient. Incubate at 37°C for 2 days, then purify. Pile a single colony onto a fresh nanoplate to purify the target bacteria. Store at -20°C and -80°C for later use.

[0025] Example 3: Screening and Identification of Antagonistic Bacteria

[0026] Strains exhibiting inhibitory effects against *Alternaria alternata*, *Alternaria alternata*, *Fusarium solani*, *Fusarium oxysporum*, *Fusarium equisetifolium*, and *Botrytis cinerea* causal agent were screened using a plate confrontation culture method. First, a mark was made on the back of the center of a prepared PDA solid medium, with two marks 2 cm to the left and right. Using a pipette, 10 μL of Ba45 bacterial suspension (adjusted to a concentration of 10⁸) was pipetted onto the two marks on either side. Then, a fungal disc of the aforementioned pathogens was placed on the center mark. Each bacterium was tested in triplicate. The culture dishes were incubated upside down at 28°C for 48 h–7 days, and the inhibition zone was observed. Figure 3 The inhibition rate was calculated (Table 1). Bacteria exhibiting antagonistic activity against all six pathogenic fungi were selected for species identification. DNA was extracted, a PCR reaction system was established, amplification was performed using universal primers, and gel sequencing was conducted. The results were compared with those in the database. Bacillus cereus. BF15 and Bacillus cereus. The homology of MOB-3 was higher than 99.8% ( Figure 5 This indicates that the strain is Bacillus cereus, and it is named... Bacillus cereus. Bc19.

[0027] Example 4: Antibacterial properties test of strain Bc19:

[0028] The corresponding enzyme activities of Bc19 were determined using a β-1,3-glucanase assay kit and a chitinase kit, respectively. The results showed that the 10-1 of Bc19... 8 After culturing in M9 medium for 24 hours, the bacterial cells produced 428.2 U / g of β-1,3-glucanase and 3.84 U / g of chitinase. This indicates that the strain can degrade the fungal spore wall, thereby inhibiting its growth and reproduction.

[0029] The morphological characteristics of Bc19 colonies in this embodiment are as follows: After Bc19 was cultured on LB solid medium at 37°C for 2 days, its colonies were observed to be round or nearly round, opaque, slightly glossy white, resembling frosted glass or molten wax. Figure 4 ).

[0030] Table 1. Inhibition rate of Bacillus cereus Bc19

[0031]

Claims

1. A strain of Bacillus cereus antagonistic to multiple pathogenic fungi of potato, characterized in that... It is Bacillus cereus ( Bacillus cereus Bc19 is deposited at the China General Microbiological Culture Collection Center (CGMCC), located at No. 3, No. 1 Beichen West Road, Chaoyang District, Beijing, on November 11, 2024, with accession number CGMCC No. 32578.

2. The application of the Bacillus cereus strain antagonistic to multiple potato pathogens as described in claim 1, characterized in that... The aforementioned Bacillus cereus ( Bacillus cereus Bc19 is used to prepare inoculants that antagonize Alternaria alternata, Alternaria alternata, Fusarium solani, Fusarium oxysporum, Fusarium equisetifolium, and Grape stalk blight.

3. The application according to claim 2, characterized in that... The aforementioned Bacillus cereus ( Bacillus cereus Bc19 against Alternaria solanacearum ( Alternaria solani The antibacterial rate of ) was 59.61%, and that of Alternaria ( Alternaria alternate The antibacterial rate of ) was 60.26%, and that of Fusarium solani ( Fusarium solani The antibacterial rate of ) was 43.56%, and that of Fusarium oxysporum ( Fusarium oxysporum The antibacterial rate of ) was 18.26%, and that of Fusarium equisetifolium ( Fusarium horsetail The inhibition rate of ) was 50.89%, and that of Grape stem blight pathogen ( Didymella glomerata The antibacterial rate was 68.18%.

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