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

Abstract

The invention discloses a bacterial strain for antagonizing multiple pathogenic fungi of potatoes and simultaneously promoting growth and application of the bacterial strain, and relates to the field of microbial agents, the bacterial strain is bacillus wiedmannii Bw34, the bacterial strain 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. The preservation number is CGMCC (China General Microbiological Culture Collection Center) The compound is used for preparing a fungicide for antagonizing potato pathogenic fungi and a fungicide for promoting potato growth. The Bw34 has the effect of comprehensively preventing and treating potato soil-borne diseases, meanwhile, potato growth and nutrient absorption are promoted, and the application prospect is good.

Description

Technical Field

[0001] The invention belongs to the field of bacterial agents, and particularly relates to a bacterial strain capable of antagonizing multiple pathogenic fungi of potatoes and promoting their growth at the same time, and an application thereof. Background Art

[0002] Potatoes are the world's fourth largest food crop, and are also a dual-purpose crop for food, feed, and industrial raw materials. In order to maximize the utilization rate of cultivated land and obtain the highest economic benefits, farmers often adopt a continuous cropping system. However, long-term continuous cropping can easily lead to soil degradation, and the problem of continuous cropping obstacles has become prominent. In particular, continuous planting of a single crop causes an imbalance in soil nutrients and microbial community structure, which is not conducive to crop growth. At the same time, it is more susceptible to diseases, leading to the risk of soil-borne diseases and threatening food security.

[0003] Soil-borne diseases usually involve multiple pathogens coexisting in the soil. When conditions are suitable and a certain pathogen accumulates to a certain number, the disease will occur. Soil-borne diseases in my country's main potato producing areas mainly include early blight, wilt, dry rot and leaf / stem spot. The pathogens mainly include Alternaria, which infects stems and leaves; Fusarium, which infects tubers. Pathogenic fungi usually have a certain dependence on climatic conditions. Therefore, climate change in recent years has further led to large-scale outbreaks of soil-borne diseases, and some areas have experienced diseases that were not common in the past. For example, grape stem blight (Didymella glomerata) is a pathogenic fungus with a wide range of hosts and was previously considered a weak pathogen. However, in recent years, my country has successively discovered diseases caused by this fungus in plants such as corn, kiwifruit, Schisandra chinensis, Cornus officinalis, and Sophora flavescens, and has caused a certain reduction in production. This experimental area also isolated this strain from potato lesions for the first time. It can be seen that obtaining biocontrol strains that antagonize multiple pathogenic fungi is crucial to the development of the potato industry under the background of climate change. In addition, promoting plant growth is conducive to improving its stress resistance, thereby reducing the chance of infection. Therefore, obtaining a biocontrol strain that antagonizes multiple pathogenic fungi and promotes growth at the same time is of great significance to solving potato soil-borne diseases and increasing production and efficiency.

[0004] Bacillus is the most widely used strain in agriculture, including strains used to antagonize pathogens, such as Bacillus subtilis, and strains used to promote crop growth, such as Bacillus megaterium. Among them, Bacillus wiedmannii has the function of degrading insoluble inorganic phosphorus in the soil and promoting phosphorus absorption by crops. It is also reported that it can dissolve potassium, produce iron carriers, and produce long-term IAA. Therefore, this strain is mostly used for crop growth promotion or soil improvement, and is less used in inhibiting soil-borne diseases. It has been reported that it can inhibit brown root rot (Phellinus lamaensis) and anthracnose (Colletotrichum gloeosporioides) of tea trees, wilt (Pantoea ananatis) of strawberries, and rot (Fusarium oxysporum) of gastrodia. However, there are no reports on the inhibition of potato early blight, dry rot and leaf / stem spot, especially there are no reports on the inhibition of pathogenic fungi such as Alternaria solani, Alternaria alternata, Fusarium solani, Fusarium oxysporum, Fusarium equiseti and Didymella glomerata while promoting growth. Summary of the invention

[0005] In order to solve the above technical problems, the present invention provides a strain capable of antagonizing multiple potato pathogenic fungi and promoting their growth at the same time and its application.

[0006] The present invention discloses a strain that antagonizes the simultaneous growth promotion of multiple potato pathogenic fungi, Bacillus wiedmannii Bw34, which is deposited in the General Microbiological Center of China National Committee for the Preservation of Microorganisms, with a deposit address of No. 3, Yard No. 1, Beichen West Road, Chaoyang District, Beijing, a deposit date of November 11, 2024, and a deposit number of CGMCC No. 32580.

[0007] The invention discloses an application of a strain for antagonizing multiple potato pathogenic fungi and promoting their growth simultaneously. The Bacillus wiedmannii Bw34 is used for preparing a bacterial agent for antagonizing potato pathogenic fungi.

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

[0009] Further, Alternaria solani, Alternaria alternata, Fusarium solani, Fusarium oxysporum, Fusarium equisetum and grape stem blight.

[0010] Furthermore, the bacteriostasis rate of the Bacillus wiedmannii Bw34 against Alternaria solani, Alternaria alternata, Fusarium solani, Fusarium oxysporum, Fusarium equisetum and grape stem blight pathogen is 3% to 63%.

[0011] Furthermore, the Bacillus wiedmannii Bw34 has a high inhibition rate of 62-63% against grape stem blight pathogen.

[0012] The invention discloses an application of a strain capable of antagonizing multiple pathogenic fungi of potato and promoting their growth simultaneously. The Bacillus wiedmannii Bw34 is used for preparing a bacterial agent for promoting potato growth.

[0013] The present invention has the following beneficial effects:

[0014] The present invention provides a strain of Widmann's bacillus Bw34, which is isolated from potato continuous cropping soil in an extreme environment, improves its growth-promoting ability and has a certain antibacterial ability, and the antagonistic strain screened from potato planting soil is more targeted to potato soil-borne diseases, has a stronger inhibitory ability, and a more stable field effect. Bw34 can antagonize pathogenic fungi Alternaria solani, Alternaria alternata, Fusarium solani, Fusarium oxysporum, Fusarium equisetum, and grape stem blight pathogens isolated from potato diseased plants. Bw34 has a certain inhibitory effect on potato early blight pathogens Alternaria solani and Alternaria alternata, dry rot pathogens Fusarium solani, Fusarium oxysporum, and Fusarium equisetum, as well as stem / leaf spot pathogens Grape stem blight pathogens, with an inhibition rate of 3% to 63%. Among them, it has a higher inhibition rate of 62.18% for grape stem blight pathogens. In addition, Bw34 has the functions of dissolving inorganic / organic phosphorus, producing protein, starch, cellulase, siderophore, indoleacetic acid (IAA), gibberellin (GA) and ACC deaminase (ACCD). Therefore, Bw34 provides a high-quality strain for the comprehensive prevention and control of potato soil-borne diseases, while promoting potato growth and nutrient absorption. The obtained Widmann's Bacillus not only promotes growth, but also has greater application potential in solving potato soil-borne diseases. The application prospects are good. BRIEF DESCRIPTION OF THE DRAWINGS

[0015] 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;

[0016] Figure 2It 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;

[0017] Figure 3 This is a picture of the antibacterial effect of the Bw34 plate. Among them, the upper row from left to right are Alternaria alternata, Alternaria solani, and grape stem blight pathogen; the lower row from left to right are Fusarium solani, Fusarium oxysporum, and Fusarium equisetum.

[0018] Figure 4 This is a photo of Bw34 after purification;

[0019] Figure 5 This is the BLAST alignment of the Bw34 strain;

[0020] Figure 6 These are photos of the verification of the growth-promoting function of Bw34; among them, the upper row, from left to right, are photos of inorganic phosphorus culture medium, Montana organic phosphorus culture medium, and potassium-lysing culture medium; the lower row, from left to right, are photos of protease detection culture medium, amylase detection culture medium, cellulase detection culture medium, and siderophore detection culture medium. DETAILED DESCRIPTION

[0021] 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.

[0022] Example 1 Isolation and purification of pathogenic fungi

[0023] 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.

[0024] Example 2 Isolation and purification of bacteria

[0025] 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 -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.

[0026] Example 3 Screening and identification of antagonistic bacteria

[0027] The plate confrontation culture 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 culture medium, mark one point on the left and right at 2 cm, and use a pipette to draw a 10 8 10uL of Bw34 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°C and incubated upside down for 48h-7d. The inhibition zone was observed and the inhibition rate was calculated (Table 1). Antagonistic bacteria that antagonize the six pathogenic fungi were selected and purified after culture ( Figure 4 ) for bacterial species identification. Select bacteria with higher antibacterial rate for bacterial species identification. Extract DNA, establish PCR reaction system, amplify using universal primers, and perform gel excision sequencing. Compare the obtained 16S rRNA gene sequence in the NCBI database ( Figure 5 ), the homology with Bacillus wiedmannii.KLS4 in the database reached 100%, and it was identified as Bacillus wiedmannii and named Bw34.

[0028] The physiological and biochemical characteristics of Bw34 in this example are as follows:

[0029] Colony morphology: After Bw34 was cultured on LB solid medium at 37°C for 2 days, its colonies were milky white, round, smooth, with neat edges, sticky, and convex in the middle ( Figure 4 ). Physiological and biochemical characteristics: The suitable culture temperature is 15-50℃. It is a Gram-positive bacterium with the growth-promoting functions of phosphate solubilization, protease production, amylase, cellulase and siderophore production ( Figure 6 ), and its concentration was 10 8After culturing Bw34 in M9 medium for 24 hours, the ability to produce IAA was 4.72 pmol / L, GA was 54.78 pmol / L, and ACCD was 439.5 U / L.

[0030] Table 1 Inhibition rate of Widmann's bacillus Bw34

[0031] Pathogenic fungi Control (cm) Colony diameter (cm) Inhibition rate (%) Alternaria solani 5.15 3.23 37.38 Alternaria alternata 7.70 3.90 49.35 Fusarium solani 4.40 3.27 25.76 Fusarium oxysporum 4.27 4.13 3.13 Fusarium equiseti 5.60 3.25 41.96 Didymella glomerata 7.80 2.95 62.18

[0032] Note: The antibacterial test results of Fusarium oxysporum and Fusarium solani were obtained after 3 days of cultivation; the antibacterial test results of Alternaria solani, Alternariaalternata, Fusarium equiseti and Didymella glomerata were obtained after 7 days of cultivation.

Claims

1. A bacterial strain that antagonizes multiple pathogenic fungi of potato and promotes their growth simultaneously, characterized in that It is Bacillus wiedmannii Bw34, 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.32580.

2. The use of a bacterial strain for antagonizing multiple pathogenic fungi of potato and promoting their growth at the same time as claimed in claim 1, characterized in that The Bacillus wiedmannii Bw34 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, characterized in that 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 wiedmannii Bw34 against Alternaria solani, Alternaria alternata, Fusarium solani, Fusarium oxysporum, Fusarium equisetum and grape stem blight pathogen is 3% to 63%.

6. The use according to claim 4, characterized in that The Bacillus wiedmannii Bw34 has a high inhibition rate of 62-63% on grape stem blight pathogen.

7. The use of a bacterial strain for antagonizing multiple pathogenic fungi of potato and promoting their growth simultaneously as claimed in claim 1, characterized in that The Bacillus wiedmannii Bw34 is used for preparing a bacterial agent for promoting potato growth.

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