Bacterial strain for antagonizing multiple pathogenic fungi of potatoes and application of bacterial strain

Abstract

The invention discloses a bacterial strain for antagonizing multiple pathogenic fungi of potatoes and application of the bacterial strain, and relates to the field of biological prevention and control, the bacterial strain is bacillus atrophaeus Ba45 and 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.32577. The bacillus atrophaeus Ba45 is used for preparing a microbial inoculum for antagonizing the pathogenic fungi of the potatoes, and the bacillus atrophaeus Ba45 is used for antagonizing the pathogenic fungi of the potatoes. According to the Ba45 disclosed by the invention, the comprehensive inhibition rate on alternaria solani, alternaria alternate, fusarium solani, fusarium oxysporum, fusarium equisetum and grape stem blight pathogen is greatly improved. Therefore, the Ba45 provides a high-quality strain with higher resistance for comprehensively preventing and treating the potato soil-borne diseases, and the application prospect is good.

Description

Technical Field

[0001] The invention belongs to the field of biological control, and in particular relates to a bacterial strain antagonistic to multiple pathogenic fungi of potato and application thereof. Background Art

[0002] Potatoes are a key crop for global food security, with my country ranking first in the world in both planting area and total output. With the rapid development of intensive agriculture, the area of ​​long-term continuous potato cropping has continued to increase, and the problem of continuous cropping obstacles has become prominent, restricting the development of the potato industry. Potatoes are rich in nutrients and are often planted in relatively poor soils. Their continuous cropping obstacles are usually manifested as the outbreak of soil-borne diseases, which reduces production and increases the risk of storage losses.

[0003] In my country, soil-borne diseases of potatoes are mainly fungal diseases, among which early blight, dry rot and leaf / stem spot are widely distributed. The pathogen of potato early blight (also known as brown spot) is Alternaria, mainly including Alternaria solani and Alternaria alternata, which mainly infect stems and leaves. The pathogen of potato dry rot is Fusarium, mainly including Fusarium solani, Fusarium equiseti, Fusarium oxysporum, etc., which mainly infect tubers and are important storage diseases (Du Miru, 2008). Leaf spot is a common disease of potato, such as early blight, late blight and anthracnose, which all cause lesions on leaves and affect photosynthesis. It occurs in a wide range and causes yield reduction in severe cases. Didymella glomerata is a pathogenic fungus with a wide range of hosts. It causes leaf spot disease in nearly 100 plants such as corn, wheat, potato, and pea. It is a quarantine plant pathogen in my country. In recent years, diseases caused by this fungus have been found in corn, kiwi fruit, Schisandra chinensis, Cornus officinalis, and Sophora flavescens. Climate change has led to a gradual expansion of the infection range of pathogenic fungi and a more severe disease. Plant protection workers need to pay attention.

[0004] There are usually more than one pathogen of soil-borne diseases coexisting in the soil. When conditions are suitable and a certain pathogen accumulates to a certain number, the disease will occur. With the development of biotechnology, more and more culturable antagonistic bacteria have been obtained, but most of them are developed for a single pathogen, and there are few antagonistic strains that inhibit multiple pathogens at the same time. Therefore, obtaining a biocontrol strain that can antagonize multiple pathogenic fungi at the same time is of great significance to solving the problem of soil-borne potato diseases.

[0005] Bacillus is the most widely used antagonistic bacteria at present. For example, Bacillus subtilis and Bacillus atrophaeus have strong biocontrol advantages and have good inhibitory effects on different diseases of various crops. Among them, Bacillus atrophaeus is a variant of Bacillus subtilis, which is widely present in the natural environment and has significant biocontrol potential for a variety of soil-borne diseases. It has been widely used in plant disease prevention and control in recent years. The pathogenic fungi that can be inhibited include: Botrytis cinerea, Pythium sp., Verticillium dahliae, Sclerotinia sclerotiorum, Colletotrichum gloeosporioides, Fusarium oxysporum, Fusarium solani, Botrytis aquamosa, Phyllosticta zingiberi, Rhizoctonia sotani, Phytophthora capsici, Erysiphe cucurbitacearum, Alternaria sp., Colletotrichum sp., etc. There are few reports on the inhibition of potato early blight, dry rot and leaf spot, especially no reports on the simultaneous inhibition of their pathogenic fungi Alternaria solani, Alternaria alternata, Fusarium solani, Fusarium oxysporum, Fusarium equiseti and Didymella glomerata by Bacillus atrophaeus. Summary of the invention

[0006] In order to solve the above technical problems, the present invention provides a bacterial strain antagonistic to multiple pathogenic fungi of potato and its application.

[0007] The present invention discloses a bacterial strain antagonistic to multiple pathogenic fungi of potato, the strain is Bacillus atrophaeus Ba45, which is deposited in the General Microbiology Center of China National Committee for the Preservation of Microorganisms, with the deposit address at No. 3, Yard No. 1, Beichen West Road, Chaoyang District, Beijing, the deposit date is November 11, 2024, and the deposit number is CGMCC No. 32577.

[0008] The invention discloses an application of a bacterial strain for antagonizing multiple pathogenic fungi of potato. The Bacillus atrophaeus Ba45 is used for preparing a bacterial agent for antagonizing pathogenic fungi of potato.

[0009] Furthermore, the potato pathogenic fungi are pathogens of soil-borne diseases.

[0010] Furthermore, the potato pathogenic fungi are Alternaria solani, Alternaria alternata, Fusarium solani, Fusarium oxysporum, Fusarium equisetum and grape stem blight.

[0011] Furthermore, the bacteriostasis rate of the Bacillus atrophaeus Ba45 against Alternaria solani, Alternaria alternata, Fusarium solani, Fusarium oxysporum, Fusarium equisetum and grape stem blight pathogen is 44-80%.

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

[0013] The present invention provides a strain of Bacillus atrophaeus Ba45, which is isolated from potato continuous cropping soil in an extreme environment, and has improved antibacterial ability. Most of the reported inhibition rates of Bacillus atrophaeus against pathogenic fungi are less than 60%. At the same time, Ba45 is more targeted at pathogenic fungi of potato soil-borne diseases, and greatly improves the comprehensive inhibition rate of Alternaria solani, Alternaria alternata, Fusarium solani, Fusarium oxysporum, Fusarium equisetum and grape stem blight. Therefore, Ba45 provides a more resistant high-quality strain for comprehensive prevention and control of potato soil-borne diseases, and has good application prospects. BRIEF DESCRIPTION OF THE DRAWINGS

[0014] Figure 1 are photos of pathogenic fungi isolated and cultured; A1 is a photo of culture and isolation, A2-A4 are photos of Alternaria alternata, Alternaria solani, and grape stem blight pathogens; B1 is a photo of diseased plants of isolated pathogens, B2-B4 are photos of Fusarium solani, Fusarium oxysporum, and Fusarium equisetum;

[0015] Figure 2 The BLAST comparison results of pathogenic fungi are shown in Figure 1. 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 Ba45 plate against bacteria; 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 Ba45 after purification;

[0018] Figure 5 This is the BLAST comparison map of Ba45 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 fungus Grape stem blight were selected for use.

[0023] Example 2 Isolation and purification of bacteria

[0024] Collect potato continuous cropping soil, weigh 5.0 g, place in a sterile triangular flask containing 95 mL LB culture medium, cover with sealing film, place in a shaker at 220 rpm for 15 min, let stand at room temperature for 10 min, take the supernatant and dilute 10 -3 , 10 -4 , 10 -5 100uL of the dilution solution was spread on the NA plate, and each dilution gradient was repeated 3 times. After culturing in a 37℃ constant temperature incubator for 2 days, the culture was purified. A single colony was picked and streaked on a new NA plate to purify the target bacteria, and stored in a -20℃ and -80℃ refrigerator for later use.

[0025] Example 3 Screening and identification of antagonistic bacteria

[0026] The plate confrontation method was used to screen strains that had inhibitory effects on Alternaria solani, Alternaria alternata, Fusarium solani, Fusarium oxysporum, Fusarium equisetum and grape stem blight. First, mark the back of the center of the paved PDA solid medium and mark one point 2 cm to the left and right of it. Use a pipette to add a concentration of 10 810uL of Ba45 bacterial solution was applied to the left and right points, and then the pathogenic fungus cake was applied to the center point. Each bacterium was repeated 3 times. The culture dish was placed in an incubator at 28℃ and inverted for 48h-7d. After that, the inhibition zone ( Figure 3 ), and the inhibition rate was calculated (Table 1). The bacteria with the highest inhibition rate against the six pathogenic fungi were selected and cultured and purified ( Figure 4 ) for strain identification. DNA was extracted, PCR reaction system was established, amplification was performed using universal primers, and gel excision sequencing was performed. The obtained 16S rRNA gene sequence was compared with the NCBI database, indicating that the homology with Bacillus atrophaeus.SK3, Bacillus atrophaeus.XJUHX-35 and Bacillusatrophaeus.CNY01 in the database reached 99.93% ( Figure 5 ), the strain was identified as Bacillus atrophaeus 45 and named Ba45 (Bacillus atrophaeus 45).

[0027] The Ba45 (Bacillus atrophaeus 45) screened in this example was milky white in the early stage, milky yellow in the later stage, and dark brown in color. The surface of the colony was not smooth, opaque, slightly raised, and the edge was irregularly wavy.

[0028] Table 1 Bacillus atrophaeus Ba45 inhibition rate

[0029] Pathogenic fungi Control (cm) Colony diameter (cm) Inhibition rate (%) Alternaria solani 5.15 1.27 73.50 Alternaria alternata 7.00 1.60 77.14 Fusarium solani 4.33 2.33 46.15 Fusarium oxysporum 4.20 2.32 44.84 Fusarium equiseti 5.60 2.55 54.46 Didymella glomerata 7.70 1.60 79.22

[0030] As shown in Table 1, Ba45 (Bacillus atrophaeus 45) screened in this example has significant inhibitory effects on potato early blight pathogens Alternaria solani, Alternaria alternata, dry rot pathogens Fusarium solani, Fusarium oxysporum and Fusarium equisetum, as well as leaf spot pathogens Grape stem blight, especially Alternaria alternata and Grape stem blight. The average inhibition rate of the strain on the above 6 pathogens is 62.55%, among which the inhibition rates on Alternaria alternata and Grape stem blight are 77.14% and 79.22% respectively, and the inhibition rate on Fusarium oxysporum is the lowest at 44.84%.

Claims

1. A bacterial strain antagonistic to multiple pathogenic fungi of potato, characterized in that The strain is Bacillus atrophaeus Ba45, which is deposited in the General Microbiology Center of China Microorganism Culture Collection Administration, with the deposit address at No. 3, Yard No. 1, Beichen West Road, Chaoyang District, Beijing, the deposit date is November 11, 2024, and the deposit number is CGMCC No.32577.

2. The use of a bacterial strain antagonistic to multiple pathogenic fungi of potato as claimed in claim 1, characterized in that The Bacillus atrophaeus Ba45 is used for preparing a bacterial agent for antagonizing potato pathogenic fungi.

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

4. The use according to claim 2 or 3, characterized in that The potato pathogenic fungi 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 average antibacterial rate of the Bacillus atrophaeusBa45 against Alternaria solani, Alternaria alternata, Fusarium solani, Fusarium oxysporum, Fusarium equisetum and grape stem blight pathogen is 70-72%.

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