A bacterial strain antagonistic to multiple pathogenic fungi of potato and its application

Abstract

A bacterial strain antagonistic to multiple pathogenic fungi of potato and its application, which relate to the field of biological control, the strain is Bacillus atrophaeus Ba45, which is deposited in the General Microbiology Center of China Culture Collection Administration, with the deposit address: No. 3, Yard 1, Beichen West Road, Chaoyang District, Beijing, the deposit date: November 11, 2024, and the deposit number is CGMCC No. 32577. Bacillus atrophaeus Ba45 is used to prepare a bacterial agent antagonistic to potato pathogenic fungi. The Ba45 of the present invention 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 the comprehensive prevention and control of potato soil-borne diseases, and has good application prospects.

Description

Technical Field

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

[0002] Potatoes are a key crop for global food security, with my country leading the world in both cultivated area and total output. With the rapid development of intensive agriculture, the area of long-term continuous potato cropping has continued to increase, highlighting the problem of continuous cropping problems and hindering the development of the potato industry. Potatoes are rich in nutrients and are often grown in relatively poor soils. Continuous cropping problems often manifest as outbreaks of soilborne diseases, reducing yields and increasing the risk of storage losses.

[0003] The main soil-borne diseases of potatoes in my country are fungal diseases, among which early blight, dry rot and leaf / stem spot are widely distributed. The pathogens of potato early blight (also known as brown spot) are Alternaria fungi, mainly including Alternaria solani ( Alternaria solani ), Alternaria alternata ( Alternaria alternata ), mainly infecting stems and leaves. The pathogens of potato dry rot are Fusarium fungi, mainly including Fusarium solani ( Fusarium solani ), Fusarium equisetum ( Fusarium horsetail ), Fusarium oxysporum ( Fusarium oxysporum ) and other diseases that primarily infect tubers and are important storage diseases (Du Miru, 2008). Leaf spot diseases are common potato diseases, such as early blight, late blight, and anthracnose, all of which cause lesions on leaves, affecting photosynthesis. They occur widely and can cause yield reductions in severe cases. Didymella glomerata ) is a fungal pathogen with a wide range of hosts, causing leaf spot diseases in nearly 100 plant species, including corn, wheat, potatoes, and peas. It is a quarantine plant pathogen in my country. In recent years, diseases caused by this fungus have been found in corn, kiwifruit, Schisandra chinensis, Cornus officinalis, and Sophora flavescens. Climate change is causing the pathogen to infect a wider range of plants and increase the severity of the disease, necessitating the attention of plant protection professionals.

[0004] Soilborne disease pathogens often coexist in the soil with more than one species. Disease only occurs when conditions are favorable and a specific pathogen accumulates to a sufficient number. Advances in biotechnology have yielded a growing number of culturable antagonistic bacterial strains, but most are developed to target a single pathogen, while fewer strains can simultaneously inhibit multiple pathogens. Therefore, developing a biocontrol strain that simultaneously antagonizes multiple fungal pathogens is crucial for addressing soilborne potato disease.

[0005] Bacillus is the most widely used antagonistic bacteria, such as Bacillus subtilis, Bacillus atrophaeus ( Bacillus atrophied) has strong biocontrol advantages and has good inhibitory effects on various 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 various 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 ( Botrytis cinerea )、Pythium( Pythium sp.), Verticillium dahliae ( Verticillium dahliae ), Sclerotinia sclerotiorum ( Sclerotinia sclerotiorum ), Colletotrichum gloeosporioides ( Colletotrichum gloeosporioides ), Fusarium oxysporum ( Fusarium oxysporum ), Fusarium solani ( Fusarium solani ), Botrytis cinerea ( Botrytis water-loving )、Phyllostachys spp.( Phyllosticta zingiberi ), Rhizoctonia solani ( Rhizoctonia sotani )、Phytophthora capsici( Phytophthora capsicum )、Melon powdery mildew( Erysiphe cucurbitaceae ), Alternaria ( Alternaria sp.), Colletotrichum spp. Colletotrichum sp.) etc. There are few reports on the inhibition of potato early blight, dry rot and leaf spot, especially the simultaneous inhibition of their pathogenic fungi Alternaria solani ( Alternaria solani ), Alternaria alternata ( Alternaria alternata ), Fusarium solani ( Fusarium solani ), Fusarium oxysporum ( Fusarium oxysporum ), Fusarium equisetum ( Fusarium horsetail ) and grape stem blight ( Didymella glomerata ) has not been reported. 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 provides a bacterial strain for antagonizing multiple pathogenic fungi of potato, the bacterial strain being Bacillus atrophaeus ( Bacillus atrophaeus )Ba45, deposited in the General Microbiology Center of China Culture Collection Administration, deposit address: No. 3, Yard 1, Beichen West Road, Chaoyang District, Beijing, deposit date: November 11, 2024, deposit number: CGMCC No. 32577.

[0008] The present invention provides an application of a bacterial strain for antagonizing multiple pathogenic fungi of potato, wherein the Bacillus atrophaeus ( Bacillus atrophaeus ) Ba45 is used to prepare fungal agents to antagonize potato pathogenic fungi.

[0009] Furthermore, the potato pathogenic fungus is a pathogen 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 Bacillus atrophaeus ( Bacillus atrophaeus The inhibition rate of Ba45 against Alternaria solani, Alternaria alternata, Fusarium solani, Fusarium oxysporum, Fusarium equisetifolia and grape stem blight was 44-80%.

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

[0013] The present invention provides a strain of Bacillus atrophaeus Ba45, which has enhanced antibacterial properties due to its isolation from potato continuous cropping soil in an extreme environment. Currently reported inhibition rates of Bacillus atrophaeus against pathogenic fungi are mostly below 60%. Furthermore, Ba45 is more targeted against pathogenic fungi of soil-borne potato diseases, significantly improving its combined inhibition rate against Alternaria solani, Alternaria alternata, Fusarium solani, Fusarium oxysporum, Fusarium equisetum, and grape stem blight. Therefore, Ba45 provides a high-quality strain with enhanced resistance to soil-borne potato disease control and has promising application prospects. BRIEF DESCRIPTION OF THE DRAWINGS

[0014] Figure 1 These 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, respectively; B1 is a photo of diseased plants from which pathogens were isolated, B2-B4 are photos of Fusarium solani, Fusarium oxysporum, and Fusarium equisetum, respectively;

[0015] Figure 2 This is the BLAST comparison result of pathogenic fungi; 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 show the antibacterial confrontation. The top row from left to right shows Alternaria alternata, Alternaria solanacearum, and grape stem blight pathogen; the bottom row from left to right shows Fusarium solani, Fusarium oxysporum, and Fusarium equisetum.

[0017] Figure 4 This is a photo of purified Ba45;

[0018] Figure 5 This is the BLAST alignment 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 in 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 the contents of the present invention based on 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 sections according to their parts, disinfect the surface, and culture them on PDA medium. Observe the growth of the colonies and isolate and purify the pathogenic fungi (see Figure 1 The purified fungal DNA was sequenced and compared with the NCBI database (see Figure 2 ) and stored in a -80°C freezer. Common potato pathogenic fungi Alternaria solani, Alternaria alternata, Fusarium solani, Fusarium oxysporum, Fusarium equisetum, and the 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, and place in a sterile triangular flask containing 95 mL of LB culture medium. Cover with sealing film, place in a shaker at 220 rpm for 15 min, let it stand at room temperature for 10 min, and take the supernatant to dilute 10 -3 , 10 -4 , 10 -5 Pipette 100uL of the dilution solution onto a NA plate, with three replicates for each dilution gradient. Incubate at 37°C for 2 days before purification. Pick a single colony and streak it onto a new NA plate 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] The plate confrontation method was used to screen strains that had inhibitory effects on Alternaria solani, Alternaria alternata, Fusarium solani, Fusarium oxysporum, Fusarium equisetifolia and grape stem blight. First, mark the back of the center of the PDA solid medium and mark one point 2 cm to the left and right of it. Use a pipette to add a 10% 8 Place 10uL of Ba45 bacterial solution on the left and right points, then take the above pathogenic fungus cake and stick it on the center point. Repeat 3 times for each bacterium. Place the culture dish in a 28℃ incubator and invert it for 48h-7d. Observe 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, a PCR reaction system was established, and amplification was performed using universal primers. Gel excision and sequencing were performed. The obtained 16S rRNA gene sequence was compared with the NCBI database, showing a 99.93% homology with Bacillus atrophaeus SK3, Bacillus atrophaeus XJUHX-35, and Bacillusatrophaeus CNY01. Figure 5 ), the strain was identified as Bacillus atrophaeus and named Ba45 ( Bacillus atrophaeus 45).

[0027] The Ba45 ( Bacillus atrophaeus 45) On LB solid medium, the colony is milky white in the early stage and turns milky yellow and dark brown in the later stage. The surface of the colony is not smooth, opaque, slightly raised, and has irregular wavy edges.

[0028] Table 1 Inhibition rate of Bacillus atrophaeus Ba45

[0029]

[0030] As can be seen from Table 1, the Ba45 ( Bacillus atrophaeus 45) showed significant inhibitory effects against potato early blight pathogens Alternaria solani and Alternaria alternata; stem rot pathogens Fusarium solani, Fusarium oxysporum, and Fusarium equisetum; and leaf spot pathogen F. vitis vinifera. The strain exhibited particularly high inhibition rates against Alternaria alternata and F. vitis vinifera. The average inhibition rate against these six pathogens was 62.55%, with inhibition rates against Alternaria alternata and F. vitis vinifera reaching 77.14% and 79.22%, respectively. The lowest inhibition rate against F. oxysporum was 44.84%.

Claims

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

2. The use of a bacterial strain for antagonizing multiple pathogenic fungi of potato as claimed in claim 1, characterized in that The Bacillus atrophaeus ( Bacillus atrophaeus ) Ba45 is used to prepare a fungal agent for antagonizing potato pathogenic fungi; the potato pathogenic fungi is Alternaria solani ( Alternaria solani ), Alternaria alternata ( Alternaria alternate ), Fusarium solani ( Fusarium solani ), Fusarium oxysporum ( Fusarium oxysporum ), Fusarium equisetum ( Fusarium horsetail ) and grape stem blight ( Didymella glomerata ).

3. The use according to claim 2, characterized in that The Bacillus atrophaeus ( Bacillus atrophied ) Ba45 is effective against Alternaria solani ( Alternaria solani ), Alternaria alternata ( Alternaria alternate ), Fusarium solani ( Fusarium solani ), Fusarium oxysporum ( Fusarium oxysporum ), Fusarium equisetum ( Fusarium horsetail ) and grape stem blight ( Didymella glomerata ) had an average antibacterial rate of 62.55%.

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