Bacterial strain antagonizing multiple pathogenic fungi of potato and promoting growth of potato simultaneously and application thereof

Abstract

The application discloses a bacterial strain capable of antagonizing multiple potato pathogenic fungi and promoting growth of the potato simultaneously and application thereof, and relates to the field of bacterial agents.The bacterial strain is Bacillus wiedmannii Bw34, which is preserved in the China General Microbiological Culture Collection Center, located at No.3, Xibahe Road, Beijing Chaoyang District, on November 11, 2024, and has a preservation number of CGMCC No.32580.The bacterial strain is used for preparing a bacterial agent for antagonizing potato pathogenic fungi and a bacterial agent for promoting growth of the potato.The Bw34 has the effect of comprehensively preventing and treating soil-borne diseases of the potato, and can promote growth and nutrient absorption of the potato, and has a good application prospect.

Description

TECHNICAL FIELD

[0001] The application belongs to the field of microbial agents, and particularly relates to a bacterial strain antagonizing multiple pathogenic fungi of potatoes and promoting growth of the potatoes and application thereof. BACKGROUND

[0002] Potato is the fourth largest food crop in the world, and is a crop for food, feed and industrial raw material. In order to maximize the utilization of arable land and obtain the highest economic benefit, farmers often adopt the continuous cropping system. However, long-term continuous cropping is easy to cause soil degradation, and the problem of continuous cropping obstacles is highlighted. Especially, continuous planting of a single crop causes imbalance of soil nutrients and microbial community structure, which is not conducive to crop growth, and is more susceptible to diseases, threatening food security.

[0003] Soil-borne diseases usually have multiple pathogenic fungi coexisting in the soil. When the conditions are suitable, a certain pathogenic fungus accumulates to a certain amount, and then the disease occurs. The soil-borne diseases in the main potato producing areas in China mainly include early blight, fusarium wilt, dry rot and leaf / stem spot diseases. The pathogenic fungi mainly include Alternaria, which infects stems and leaves, and Fusarium, which infects tubers. Didymella glomerata is a host-specific pathogenic fungus, which was previously considered to be a weak pathogenic fungus. However, in recent years, the disease caused by this fungus has been found in corn, kiwi, schisandra, cornus, and sophora in China, and has caused a certain reduction in yield. This strain was isolated from potato disease spots for the first time in the test area. Therefore, it is of great importance to obtain a biocontrol strain antagonizing multiple pathogenic fungi for the development of potato industry under the background of climate change. In addition, promoting plant growth is conducive to improving its stress resistance, and thus reducing the disease incidence. Therefore, it is of great significance to obtain a biocontrol strain antagonizing multiple pathogenic fungi and promoting growth for solving potato soil-borne diseases, increasing yield and efficiency.

[0004] Bacillus is the most widely used bacteria in agriculture, including bacteria used to antagonize pathogenic bacteria, such as Bacillus subtilis, bacteria used to promote crop growth, such as Bacillus megaterium. Among them, Bacillus wiedmannii has the effect of degrading soil insoluble inorganic phosphorus and promoting the absorption of phosphorus by crops, and it has also been reported that it can dissolve potassium, produce siderophores, and produce auxin IAA. Therefore, this strain is often used for crop growth promotion or soil improvement, and its application in inhibiting soil-borne diseases is less. There are reports that it can inhibit the brown root rot (Phellinus lamaensis) and anthracnose (Colletotrichum gloeosporioides) of tea trees, the wilt (Pantoea ananatis) of strawberries, and the rot (Fusarium oxysporum) of Gastrodia elata. However, there is no report on the inhibition of early blight, dry rot, and leaf / stem spot of potatoes, especially Bacillus wiedmannii that promotes growth while inhibiting pathogenic fungi Alternaria solani, Alternaria alternata, Fusarium solani, Fusarium oxysporum, Fusarium equiseti, and Didymella glomerata. SUMMARY

[0005] The present application provides a strain of Bacillus wiedmannii Bw34 that can antagonize multiple pathogenic fungi of potatoes while promoting growth, and its application.

[0006] The strain of Bacillus wiedmannii Bw34 that can antagonize multiple pathogenic fungi of potatoes while promoting growth is deposited at the China General Microbiological Culture Collection Center, located at No. 1, Beichen West Road, Beijing, China, on November 11, 2024, and has the accession number CGMCC No. 32580.

[0007] The application of the strain of Bacillus wiedmannii Bw34 that can antagonize multiple pathogenic fungi of potatoes while promoting growth is used to prepare a bacterial agent that can antagonize pathogenic fungi of potatoes.

[0008] Further, the pathogenic fungi of potatoes are pathogenic bacteria of soil-borne diseases.

[0009] Further, Alternaria solani, Alternaria alternata, Fusarium solani, Fusarium oxysporum, Fusarium equiseti, and Didymella glomerata.

[0010] Further, the Bacillus wiedmannii Bw34 has an inhibition rate of 3-63% on Alternaria solani, Alternaria alternata, Fusarium solani, Fusarium oxysporum, Fusarium equiseti and Botryodiplodia theobromae.

[0011] Further, the Bacillus wiedmannii Bw34 has a higher inhibition rate of 62-63% on Botryodiplodia theobromae.

[0012] The strain of the application can be applied to promote the growth of potatoes.

[0013] The application has the following advantages:

[0014] The application provides a Bacillus wiedmannii Bw34, which is isolated from potato continuous cropping soil in an extreme environment, has improved growth-promoting ability and certain antibacterial ability, and is more targeted, stronger in inhibition and more stable in field effect on potato soil-borne diseases. The Bw34 can antagonize pathogenic fungi such as Alternaria solani, Alternaria alternata, Fusarium solani, Fusarium oxysporum, Fusarium equiseti and Botryodiplodia theobromae isolated from potato diseased strains. The Bw34 has a certain inhibitory effect on the pathogenic fungi of potato early blight, such as Alternaria solani and Alternaria alternata, the pathogenic fungi of dry rot, such as Fusarium solani, Fusarium oxysporum and Fusarium equiseti, and the pathogenic fungi of stem / leaf spot, such as Botryodiplodia theobromae, with an inhibition rate of 3-63%. The Bw34 has a higher inhibition rate of 62.18% on Botryodiplodia theobromae. In addition, the Bw34 has the functions of dissolving inorganic / organic phosphorus, producing proteinase, starch, cellulase, producing siderophore, indole acetic acid (IAA), gibberellin (GA) and ACC deaminase (ACCD). Therefore, the Bw34 provides a high-quality strain for comprehensive prevention and control of potato soil-borne diseases, promotion of potato growth and nutrient absorption, and has application potential in solving potato soil-borne diseases. BRIEF DESCRIPTION OF DRAWINGS

[0015] Figure 1 The photos are of pathogenic fungi isolated and cultured, wherein A1 is a photo of isolated culture, A2-A4 are photos of Alternaria alternata, Alternaria solani and Botryodiplodia theobromae respectively, B1 is a photo of a diseased strain of pathogenic fungi, and B2-B4 are photos of Fusarium solani, Fusarium oxysporum and Fusarium equiseti respectively.

[0016] Figure 2Figure 1 is a BLAST alignment chart of pathogenic fungi; wherein, from top to bottom, Alternaria alternata, Alternaria solani, Eutypa lata, Fusarium solani, Fusarium oxysporum, Fusarium equiseti;

[0017] Figure 3 Figure 2 is a Bw34 flat confrontation antibacterial effect chart; wherein, from left to right in the upper row, Alternaria alternata, Alternaria solani, Eutypa lata; from left to right in the lower row, Fusarium solani, Fusarium oxysporum, Fusarium equiseti;

[0018] Figure 4 Figure 3 is a photo of purified Bw34;

[0019] Figure 5 Figure 4 is a BLAST alignment chart of Bw34 strain;

[0020] Figure 6 Figure 5 is a Bw34 growth-promoting function verification photo; wherein, from left to right in the upper row, inorganic phosphorus culture medium, Mengjina organic phosphorus culture medium, potassium-removing culture medium photo; from left to right in the lower row, protease detection culture medium, amylase detection culture medium, cellulase detection culture medium, siderophore detection culture medium photo. DETAILED DESCRIPTION

[0021] In order to make the purpose, technical solutions and advantages of the embodiments of the present application more clear and apparent, the spirit of the present application will be described in detail below, and any person skilled in the art can make changes and modifications to the technology taught by the present application after understanding the embodiments of the present application, without departing from the spirit and scope of the present application.

[0022] Example 1: Isolation and purification of pathogenic fungi

[0023] Potato diseased stems were cut into small sections according to the parts, and after surface disinfection, were cultured on PDA medium. The growth state of the colonies was observed, and the pathogenic fungi were isolated and purified (see Figure 1 ). The purified fungi were extracted for DNA sequencing, and after comparison with the NCBI database (see Figure 2 ), were stored in a -80℃ refrigerator. The common pathogenic fungi of potatoes, Alternaria solani, Alternaria alternata, Fusarium solani, Fusarium oxysporum, Fusarium equiseti and the first isolated pathogenic fungus Eutypa lata were selected for standby 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, is poor, low organic matter content, perennial drought and potato long-term continuous cropping field test. Collection of potato continuous cropping soil, weighing 5.0g placed in a sterile flask with 95ml LB medium, cover with sealing film, placed in a shaking bed 220rpm vibration culture 15min, room temperature for 10min, take supernatant dilution 10 -3 、10 -4 、10 -5 Times, take 100uL diluent coated on NA plate, each dilution gradient set 3 repeats, placed in 37℃ incubator for 2 days, purification culture. Pick single colony in a new NA plate on line purification target bacteria, respectively, -20℃ and -80℃ refrigerator preservation for standby.

[0026] Example 3 antagonistic bacteria screening and identification

[0027] Through the plate confrontation culture method to screen the strain which has inhibitory effect on Alternaria solani, Alternaria alternata, Fusarium solani, Fusarium oxysporum, Fusarium equiseti and Botryosphaeria dothidea. First mark the back of the center of the PDA solid medium, mark a point on both sides of 2cm, take 10uL Bw34 bacteria liquid with concentration of 10 8 , point on both sides of the two points, and then take the above pathogenic fungus cake and paste on the center point, each bacteria do 3 times. Place the culture dish in 28℃ incubator for 48h-7d, then observe the inhibition zone and calculate the inhibition rate (Table 1). Select the antagonistic bacteria which can antagonize the six kinds of pathogenic fungi, and identify the strain after culture and purification. Figure 4 ) The bacteria with higher inhibition rate were selected for strain identification. Extract DNA, establish PCR reaction system, use universal primer for amplification, cut gel sequencing. The obtained 16S rRNA gene sequence was compared in NCBI database( Figure 5 ), and the homology with Bacillus wiedmannii.KLS4 in the database reached 100%, which was identified as Bacillus wiedmannii, 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℃ for 2d, the colony was observed to be milky white, round, smooth surface, neat edge, viscous, and convex in the middle. Figure 4 Physiological and biochemical characteristics: the optimum culture temperature is 15-50℃, it is gram-positive bacteria, has the functions of phosphorus solubilization, protease, amylase, cellulase production and siderophore production( Figure 6 ), and its concentration is 10 8The IAA production ability of Bw34 was 4.72 pmol / L, the GA production ability was 54.78 pmol / L, and the ACCD production ability was 439.5 U / L after the Bw34 was cultured in M9 medium for 24 h.

[0030] Table 1 Inhibition rate of Bacillus weihenstetterae 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 inhibition test of Fusarium oxysporum and Fusarium solani was determined after 3 days of culture; the inhibition test of Alternaria solani, Alternaria alternata, Fusarium equiseti and Didymella glomerata was determined after 7 days of culture.

Claims

1. A bacterial strain that antagonizes multiple potato pathogenic fungi while simultaneously promoting their growth, characterized in that... It is Bacillus videmannii ( Bacillus wiedmannii Bw34 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. 32580.

2. The application of the bacterial strain that antagonizes multiple potato pathogenic fungi and promotes their growth as described in claim 1, characterized in that... The aforementioned Bacillus videmannii ( Bacillus wiedmannii Bw34 is used to prepare an inoculant against the potato pathogenic fungus, wherein the potato pathogenic fungus is *Alternaria alternata* (…). Alternaria solani Alternaria ( Alternaria alternata Fusarium solani () Fusarium solani Fusarium oxysporum ( Fusarium oxysporum ), Horsetail ( Fusarium horsetail ) and grape stem blight fungus ( Didymella glomerata ).

3. The application of the bacterial strain that antagonizes multiple potato pathogenic fungi and promotes their growth as described in claim 1, characterized in that... The aforementioned Bacillus videmannii ( Bacillus wiedmannii Bw34 is used to prepare microbial agents that promote potato growth.

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