A strain of Bacillus subtilis 20Q2B-18-1 antagonistic to potato soft rot and its application

Abstract

The present invention discloses a potato soft rot antagonistic Bacillus subtilis 20Q2B‑18‑1 and its application, and relates to the field of biotechnology. The Bacillus subtilis 20Q2B‑18‑1 was deposited in the Guangdong Provincial Microbial Culture Collection Center on September 26, 2022, with a preservation address of 5th Floor, Building 59, No. 100, Xianlie Middle Road, Guangzhou, and a preservation number of GDMCC No: 62836. The Bacillus subtilis 20Q2B‑18‑1 provided by the present invention can be well colonized on Qingshu No. 9 and Qingshu No. 2, and it has an inhibitory effect on the soft rot pathogens of potato in both treatment and protection modes, and can induce SOD (superoxide dismutase), CAT (catalase), PPO (polyphenol oxidase) and POD (peroxidase) enzyme activities to increase.

Description

Technical Field

[0001] The invention relates to the field of biotechnology, and in particular to a potato soft rot antagonistic Bacillus subtilis 20Q2B-18-1 strain and an application thereof. Background Art

[0002] Potato is an annual herbaceous plant of the Solanaceae family, Solanum. It is rich in nutritional value and has wide adaptability. However, soft rot seriously affects the yield and quality of potatoes. Potato soft rot is the second largest bacterial disease in the world and is prevalent in some countries around the world, resulting in a significant reduction in potato production. At present, the commonly used prevention and control method for potato soft rot is chemical prevention, but long-term use of chemical prevention will cause serious environmental pollution. Therefore, it is particularly important to seek safer and more effective prevention and control methods.

[0003] Potato soft rot has the characteristics of rapid spread and great harm. The infection of potato tubers will not only cause them to rot during transportation and storage, but also may cause potato black leg disease in the middle and late stages of field growth, resulting in a large reduction in potato production. The storage period after harvesting potatoes is generally 3-6 months. Due to unsuitable storage environment and improper management after harvesting, the loss rate of potatoes due to rot reaches 20%-25%, of which the loss rate caused by soft rot is about 15%. The reported pathogens causing potato soft rot mainly include: Pectobacterium carotovorum, Pectobacterium atrosepticum, P. carotovorum subsp. carotovorum, Dickea chrysanthemi and Erwinia persicina, among which the annual yield loss rate of potato soft rot caused by Pectobacterium spp. and Dickea spp. is as high as 30%, which greatly reduces the commercial value of potatoes and has a great impact on the production of seed potatoes, becoming one of the bottlenecks restricting the healthy production of potatoes. In addition, Wang et al. discovered for the first time in China that Bacillus amyloliquefaciens can also cause potato soft rot, and Bathily et al. discovered for the first time that Bacillus pumilus can cause potato soft rot. Summary of the invention

[0004] The purpose of the present invention is to provide a potato soft rot antagonistic Bacillus subtilis 20Q2B-18-1 and its application to solve the problems existing in the above-mentioned prior art. The Bacillus subtilis 20Q2B-18-1 provided by the present invention can effectively inhibit the pathogenic bacteria of potato soft rot.

[0005] To achieve the above object, the present invention provides the following solutions:

[0006] The present invention provides a potato soft rot antagonistic Bacillus subtilis 20Q2B-18-1, which was deposited in the Guangdong Provincial Microbiological Culture Collection Center on September 26, 2022, with the deposit address being 5th Floor, Building 559, No. 100 Xianlie Middle Road, Guangzhou, and the deposit number being GDMCC No: 62836.

[0007] The present invention also provides a microbial agent, comprising the above-mentioned Bacillus subtilis 20Q2B-18-1.

[0008] The present invention also provides a medicament for antagonizing potato soft rot, comprising the fermentation liquid or the fermentation liquid extract of the above-mentioned Bacillus subtilis 20Q2B-18-1.

[0009] Furthermore, the extraction solvent of the fermentation broth extract is ethyl acetate or n-butanol.

[0010] Furthermore, the medicament also includes pharmaceutically acceptable excipients.

[0011] The present invention also provides use of the substance described in any one of the following (1)-(3) in preventing and / or treating potato soft rot:

[0012] (1) the above-mentioned Bacillus subtilis 20Q2B-18-1;

[0013] (2) the fermentation broth or fermentation broth extract of the above-mentioned Bacillus subtilis 20Q2B-18-1;

[0014] (3) The above-mentioned microbial agents.

[0015] Furthermore, the pathogen of potato soft rot is Erwinia persicina.

[0016] The present invention also provides use of the substance described in the following (1) or (2) in preparing a product for preventing and / or treating potato soft rot:

[0017] (1) the above-mentioned Bacillus subtilis 20Q2B-18-1;

[0018] (2) The fermentation broth or fermentation broth extract of the above-mentioned Bacillus subtilis 20Q2B-18-1.

[0019] Furthermore, the pathogen of potato soft rot is Erwinia persica.

[0020] The present invention also provides the use of the following substance (1) or (2) in the preparation of a product for inhibiting Erwinia persica:

[0021] (1) the above-mentioned Bacillus subtilis 20Q2B-18-1;

[0022] (2) The fermentation broth or fermentation broth extract of the above-mentioned Bacillus subtilis 20Q2B-18-1.

[0023] The present invention discloses the following technical effects:

[0024] The results of the colonization of Bacillus subtilis 20Q2B-18-1 on potatoes provided by the present invention show that the strain can be well colonized on Qingshu No. 9 and Qingshu No. 2. The in vivo protective effect and related defense enzyme activities of the active strain fermentation liquid were measured, and the results showed that it had an inhibitory effect on the soft rot pathogens of potato in both treatment and protection modes, and could induce the increase of SOD (superoxide dismutase), CAT (catalase), PPO (polyphenol oxidase) and POD (peroxidase) enzyme activities.

[0025] The antibacterial activity of petroleum ether, chloroform, ethyl acetate, n-butanol and aqueous fermentation broth extracts of Bacillus subtilis 20Q2B-18-1 was determined. The results showed that at a concentration of 100 mg / mL, the antibacterial activity of the ethyl acetate and n-butanol fermentation broth extracts of the strain was higher. The EC 50 The value is 67.60 mg / mL, and the EC of the n-butanol fermentation broth extract solution is 50 The value was 97.44 mg / mL. The stability of the extract of ethyl acetate fermentation broth of Bacillus subtilis 20Q2B-18-1 was evaluated. The results showed that the extract of ethyl acetate fermentation broth of this strain had good thermal stability, acid-base stability and ultraviolet stability when the concentration was 100 mg / mL. The research results are expected to provide a new theoretical basis for the development of biocontrol agents for potato soft rot and lay a foundation for its practical application in agriculture. BRIEF DESCRIPTION OF THE DRAWINGS

[0026] In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings required for use in the embodiments will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present invention. For ordinary technicians in this field, other drawings can be obtained based on these drawings without paying creative work.

[0027] Figure 1 The colony morphology and Gram staining characteristics of strain 20Q2B-18-1; A is the colony morphology of strain 20Q2B-18-1 after 48 hours of culture; B is the Gram staining result of strain 20Q2B-18-1;

[0028] Figure 2 Phylogenetic analysis of strain 20Q2B-8-1 based on 16S rDNA sequence;

[0029] Figure 3 The changes in the colonization amount of the active strain 20Q2B-18-1 on two potato varieties;

[0030] Figure 4 are the changes in the activities of defense enzymes related to Qingshu No. 9; among them, A is the change in the activity of SOD enzyme in the treatment mode of Qingshu No. 9; B is the change in the activity of SOD enzyme in the protection mode of Qingshu No. 9; C is the change in the activity of CAT enzyme in the treatment mode of Qingshu No. 9; D is the change in the activity of CAT enzyme in the protection mode of Qingshu No. 9; E is the change in the activity of PPO enzyme in the treatment mode of Qingshu No. 9; F is the change in the activity of PPO enzyme in the protection mode of Qingshu No. 9; G is the change in the activity of POD enzyme in the treatment mode of Qingshu No. 9; H is the change in the activity of POD enzyme in the protection mode of Qingshu No. 9;

[0031] Figure 5 The effects of temperature (A), pH (B) and ultraviolet light (C) on the antibacterial activity of the ethyl acetate fermentation broth extract of strain 20Q2B-18-1. DETAILED DESCRIPTION

[0032] Various exemplary embodiments of the present invention will now be described in detail. This detailed description should not be considered as limiting the present invention, but should be understood as a more detailed description of certain aspects, features, and embodiments of the present invention.

[0033] It should be understood that the terms described in the present invention are only for describing a particular embodiment and are not intended to limit the present invention. In addition, for the numerical range in the present invention, it should be understood that each intermediate value between the upper and lower limits of the scope is also specifically disclosed. The intermediate value in any stated value or stated range, and each smaller range between any other stated value or intermediate value in the described range is also included in the present invention. The upper and lower limits of these smaller ranges can be independently included or excluded in the scope.

[0034] Unless otherwise indicated, all technical and scientific terms used herein have the same meanings as those generally understood by those skilled in the art. Although the present invention describes only preferred methods and materials, any methods and materials similar or equivalent to those described herein may also be used in the implementation or testing of the present invention. All documents mentioned in this specification are incorporated by reference to disclose and describe the methods and / or materials associated with the documents. In the event of a conflict with any incorporated document, the content of this specification shall prevail.

[0035] It will be apparent to those skilled in the art that various modifications and variations may be made to the specific embodiments of the present invention description without departing from the scope or spirit of the present invention. Other embodiments derived from the present invention description will be apparent to those skilled in the art. The present invention description and examples are exemplary only.

[0036] The words “include,” “including,” “have,” “contain,” etc. used in this document are open-ended terms, meaning including but not limited to.

[0037] The pathogen used in the following examples is Erwinia persicina, which was purchased from Shanghai Luwei Technology Co., Ltd.

[0038] The culture medium formula used in the following examples is as follows:

[0039] Beef extract peptone medium (NA): beef extract 3g / L, peptone 10g / L and sodium chloride 5g / L, natural pH.

[0040] LB liquid medium: 10 g / L tryptone, 5 g / L yeast powder and 10 g / L sodium chloride, pH 7.0-7.4.

[0041] Potato dextrose agar (PDA): 200 g / L potato, 20 g / L glucose and 15 g / L agar, natural pH.

[0042] Example 1

[0043] 1. Separation

[0044] A strain of bacteria, numbered 20Q2B-18-1, was isolated from a potato tuber sample of Qingshu No. 2. The tuber sample was collected from a storage cellar at the Qinghai Potato Alpine Experiment Station (101°04′12″E, 36°73′31″N).

[0045] 2. Identification

[0046] 2.1 Morphological and physiological and biochemical identification

[0047] The activated strain 20Q2B-18-1 was streaked onto NA medium and cultured for 3 days before identification according to Bergey's Bacterial Identification Manual and Common Bacterial Systematic Identification Manual.

[0048] 2.1.1 Morphological identification

[0049] Depend on Figure 1 As shown in Figures A and B, the colonies of strain 20Q2B-18-1 on NA medium are round, milky white, opaque, and rough in surface. Gram staining is purple, positive, and rod-shaped.

[0050] 2.1.2 Physiological and biochemical identification

[0051] As shown in Table 1, the results of physiological and biochemical characteristics analysis of strain 20Q2B-18-1 showed that the gelatin hydrolysis test, catalase test and starch hydrolysis test were positive, and the indole test was negative.

[0052] Table 1 Physiological and biochemical characteristics of strain 20Q2B-18-1

[0053]

[0054] 2.2 Molecular Biological Identification

[0055] The strain 20Q2B-18-1 was activated and operated using the procedure of the column bacterial DNA extraction kit (Shanghai Bioengineering Co., Ltd.). The bacterial universal primers 27F (5′-AGAGTTTGATCCTGGCTCAGG-3′) and 1492R (5′-GGTTACCTTGTTACGACTT-3′) were used for PCR amplification. The PCR amplification system was: 10×PCR Buffer, 2.5 μL; dNTP, 2 μL; TaqDNA polymerase, 0.5 μL; DNA template, 1 μL; primers, 1 μL each; ddH20, 25 μL. The PCR reaction conditions were: 94°C, 5 min; denaturation 94°C, 30S; annealing 55°C, 45S; extension 72°C, 80S; 72°C, 10 min; a total of 35 cycles.

[0056] After the reaction, 5 μL of the reaction product was taken and subjected to agarose gel electrophoresis. The PCR reaction product was sent to Sangon Biotech (Shanghai) Engineering Co., Ltd. for sequencing. After the sequencing results were returned, the sequences with homology greater than 95% were selected and downloaded from https: / / www.ezbiocloud.net / . The Neighbor-Joining (Bootstrap = 1000) method was used to construct a phylogenetic tree on MEGA7.0. The results are shown in Figure 2 .

[0057] Depend on Figure 2It can be seen that strain 20Q2B-18-1 and Bacillus subtilis are clustered into one branch with a homology of 99.36%, and the closest relationship. Combining colony morphology, physiological and biochemical characteristics and molecular biological identification, strain 20Q2B-18-1 can be identified as Bacillus subtilis. The Genbank accession number is OK614043.

[0058] 3. Preservation

[0059] Strain 20Q2B-18-1 was deposited in the Guangdong Provincial Microbiological Culture Collection on September 26, 2022. The deposit address is 5th Floor, Building 59, No. 100 Xianlie Middle Road, Guangzhou, and the deposit number is GDMCC No: 62836.

[0060] Example 2 In vivo inhibitory effect of active strain fermentation broth on potatoes

[0061] 1 Experimental Materials

[0062] 1.1 Test strains

[0063] Pathogen: Erwinia persicina

[0064] Active strain: 20Q2B-18-1

[0065] 1.2 Test culture medium

[0066] Potato dextrose agar medium (PDA); beef extract peptone medium (NA); LB liquid medium.

[0067] 1.3 Experimental instruments

[0068] Electronic balance (JA2003N), clean bench (SW-CJ-1C), artificial climate chamber (MGC-100), biochemical incubator (BPC-250F), UV-visible spectrophotometer (T6 New Century), pH meter (pHSJ-5), high pressure sterilizer (HVA-50), high and low temperature constant temperature shaking incubator (HZQ-FI60A).

[0069] 2 Experimental methods

[0070] 2.1 Preparation of active strains and pathogenic bacteria fermentation broth

[0071] Active strains and pathogens were taken out from a 4°C refrigerator, streaked in the center of a NA plate, placed in a 37°C incubator, and incubated upside down for 3 days. Single colonies were inoculated into LB liquid culture medium, placed in a shaking incubator at 37°C and 180 r / min for 2 days to obtain fermentation broth of active strains and pathogens.

[0072] 2.2 Colonization of active strains on two potato varieties

[0073] Select 30 healthy green potatoes No. 9 and No. 2 of uniform size (about 90g), disinfect them, and air-dry them for later use.

[0074] The concentration of active strain fermentation broth was adjusted to 1.0×10 8 CFU / mL, the fermentation liquid was evenly sprayed on the surface of potatoes until there was no droplet (about 5mL of fermentation liquid), and then placed in a sterile ziplock bag and cultured in an artificial climate box at a temperature of 27°C and a humidity of 80%-90% for 1, 2, 3, 4, 5, 6, 7, 8, and 9 days. 1g of potato was cut from each treatment and placed in a sterile conical flask containing 10mL of 1wt% NaCl, and cultured in a shaker at 37°C and 180r / min for 30min. 100μL of bacterial solution was taken, diluted 10 times, and then evenly spread on NA culture medium with a sterile coating rod, placed in a 37°C incubator, and cultured upside down for 3d, and the number of colonies was recorded. Each treatment was repeated 3 times.

[0075] 2.3 Determination of the in vivo efficacy of active strain fermentation broth on potatoes

[0076] 250 healthy and uniform-sized green potatoes (about 90g) were selected and disinfected in the following two modes:

[0077] Treatment mode: Use a 0.5mm diameter puncher to punch holes on the surface of sterilized potatoes. For the first time, use a pipette to add 50μL of pathogen fermentation liquid to each hole. After the original potato tissue is stuffed back, place it in an artificial climate box with a temperature of 27℃ and a humidity of 80%-90% for incubation for 1d, then take it out and take out the potato tissue at the hole. For the second time, use a pipette to add 50μL of active strain fermentation liquid to each hole. Then stuff the original potato tissue back and place it in an artificial climate box with a temperature of 27℃ and a humidity of 80%-90% for incubation for 7d. Record its weight loss rate and rotten fruit rate, and calculate the control effect according to the following formula:

[0078] Control effect (%) = (1-treatment lesion diameter / control lesion diameter) × 100.

[0079] Protection mode: Use a 0.5mm diameter puncher to punch holes on the surface of the sterilized potatoes. For the first time, use a pipette to add 50μL of active strain fermentation liquid to each hole, stuff the original potato tissue back, place it in an artificial climate box with a temperature of 27℃ and a humidity of 80%-90% and culture it for 1d, then take it out and take out the potato tissue at the hole. For the second time, use a pipette to add 50μL of pathogen fermentation liquid to each hole, stuff the original potato tissue back, place it in an artificial climate box with a temperature of 27℃ and a humidity of 80%-90% and culture it for 7d. The recording method is the same as above.

[0080] Experimental treatment: There are 22 treatments in total, with 10 replicates for each treatment, as shown in Table 2:

[0081] Table 2 Experimental treatments for determination of in vivo efficacy of active strain fermentation liquid on Qingshu No. 9

[0082]

[0083] 2.4 Determination of related defense enzyme activities

[0084] Pretreatment: Take 1g of potatoes treated in 2.3 in a clean bench and place them in sterile EP tubes. Method for determining the activity of superoxide dismutase (SOD), catalase (CAT), polyphenol oxidase (PPO) and peroxidase (POD) The crude enzyme solution was extracted and determined according to the instructions of the SOD kit, CAT kit, PPO kit and POD kit (Suzhou Keming Biotechnology Co., Ltd.).

[0085] 2.5 Statistics and Analysis

[0086] Microsoft Excel 2016 and SPSS 24.0 were used for data analysis, and Duncan's new multiple range method was used to test the significance of differences (P < 0.05).

[0087] 3 Results and analysis

[0088] 3.1 Colonization of active strains on two potato varieties

[0089] In order to determine the colonization of the selected active strain 20Q2B-18-1 on two potato varieties, its colonization amount was measured. The results are as follows: Figure 3 As shown in the figure, the colonization of strain 20Q2B-18-1 on Qingshu No. 2 was the highest on the 5th day, which was 1.62×10 8 CFU / mL; in terms of the colonization of strain 20Q2B-18-1 on Qingshu No. 9, its colonization amount was the highest on the 5th day, which was 1.64×10 8CFU / mL, the colonization of the strain on Qingshu No. 2 and Qingshu No. 9 showed an overall trend of first increasing and then decreasing. The colonization amount gradually increased from 1 to 5 days after spraying, reached a peak on the 5th day, and gradually decreased from 5 to 9 days after spraying. In summary, the active strain 20Q2B-18-1 can colonize well on Qingshu No. 2 and Qingshu No. 9, and the colonization amount is the best on the 5th day.

[0090] 3.2 Effectiveness of active strain fermentation liquid on the in vivo control of potato Qingshu No. 9

[0091] The in vivo control effect of the fermentation liquid of the active strain 20Q2B-18-1 on Qingshu No. 9 was measured, and the results are shown in Table 3, where treatment a is a treatment mode and treatment b is a protection mode. As for the control, the addition of sterile water (CK) and the fermentation liquid of the active strain (CK1) did not cause the rotten fruit of Qingshu No. 9, and the infection of the pathogen (CK2) caused a rotten fruit rate of 30.00% on Qingshu No. 9, and the weight loss rate caused by the pathogen infection of Qingshu No. 9 was 18.36%. Since there was no lesion on the potatoes treated with CK and CK2, and the diameter of the lesion was 0.00mm, the control effect was 100.00%.

[0092] In terms of treatment mode, the weight loss rate of treatment a was higher than that of sterile water (CK) and only active strain fermentation liquid (CK2), and lower than that of pathogen infection (CK2). The weight loss rate of treatment a was higher, 12.10%, and the rotten fruit rate was 30.00%. The control effect of treatment a was better than that of the immersion group (CK2).

[0093] In terms of protection mode, the weight loss rate of treatment b was higher than that of adding sterile water (CK) and adding only active strain fermentation broth (CK2), and lower than that of pathogen infection (CK2). The weight loss rate of b was 11.92%, and the rotten fruit rate was lower than that of pathogen infection (CK2).

[0094] In summary, the fermentation broth of the active strain 20Q2B-18-1 had a good protective and therapeutic effect on the occurrence of potato soft rot on Qingshu No. 9.

[0095] Table 3 The in vivo control effect of the fermentation liquid of active strain 20Q2B-18-1 on Qingshu No. 9

[0096]

[0097]

[0098] 3.3 Determination of the defense activity of Qingshu No. 9

[0099] In order to study the changes in the activities of related defense enzymes in the fermentation liquid of the active strain 20Q2B-18-1 in the in vivo protection effect determination of Qingshu No. 9, the activities of related defense enzymes were determined using SOD kit, CAT kit, PPO kit and POD kit.

[0100] The changes in the activity of SOD enzyme in Qingshu No. 9 are as follows Figure 4 As shown in A and B. In treatment mode ( Figure 4 In A), the peak value of a is higher than that of each CK. The a treatment reaches the peak value on the 7th day. In the protection mode ( Figure 4 In B), the peak values ​​of b were higher than those of CK, and the peak values ​​appeared on the 14th day. In summary, the fermentation liquid of active strain 20Q2B-18-1 can induce the increase of SOD enzyme activity of Qingshu No. 9, thereby improving the resistance of Qingshu No. 9 to potato soft rot pathogens.

[0101] The changes in CAT enzyme activity of Qingshu No. 9 are as follows Figure 4 As shown in C and D, the CAT enzyme activities of all treatments were higher than those of the control in both treatment and protection modes. Figure 4 In C), the peak of treatment a occurs on the 7th day. In the protection mode ( Figure 4 In (D), the peak of treatment b occurred on the 7th day.

[0102] The changes in PPO enzyme activity of Qingshu No. 9 are as follows Figure 4 As shown in E and F, in treatment mode ( Figure 4 In E), the peak value of treatment a is higher than that of CK, and its peak value appears on the 14th day. Figure 4 In (F), the peak value of treatment b was higher than that of each CK, and its peak value appeared on the 7th day.

[0103] The changes in POD enzyme activity of Qingshu No. 9 are as follows Figure 4 As shown in G and H, in the treatment mode ( Figure 4 G) and protection mode ( Figure 4 In (H), the peak values ​​of treatment b were all higher than those of the controls.

[0104] In summary, the fermentation broth of the active strain 20Q2B-18-1 can induce the increase of SOD, CAT, PPO and POD enzyme activities in Qingshu No. 9, thereby improving the resistance of Qingshu No. 9 to potato soft rot pathogens.

[0105] Example 3 Determination of the activity of the fermentation broth extract in inhibiting soft rot pathogens and stability analysis

[0106] 1 Experimental Materials

[0107] 1.1 Test strains:

[0108] Pathogen: Erwinia persicina

[0109] Active strain: 20Q2B-18-1

[0110] 1.2 Test culture medium

[0111] Potato dextrose agar medium (PDA); beef extract peptone medium (NA); LB liquid medium.

[0112] 1.3 Experimental instruments

[0113] Clean bench (SW-CJ-1C), biochemical incubator (BPC-250F), electronic balance (JA2003N), rotary evaporator (EYELAN-1300), ice maker (DL-005653-181), high pressure sterilizer (HVA-50), high and low temperature constant temperature shaking incubator (HZQ-FI60A).

[0114] 2 Test methods

[0115] 2.1 Preparation of fermentation broth extract and determination of antibacterial activity

[0116] Use an inoculation needle to pick up 1 ring of strain 20Q2B-18-1, inoculate it in a triangular flask filled with LB liquid culture medium, place it in a shaker at a temperature of 37°C and a speed of 180r / min for 5 days, and obtain the strain fermentation liquid. After filtering through a Buchner funnel, the bacteria-free fermentation liquid can be obtained for standby use. Take 600mL of the bacteria-free fermentation liquid of strain 20Q2 B-18-1 and place it in a separating funnel. Use equal amounts of petroleum ether, chloroform, ethyl acetate and n-butanol to extract in turn. After the extraction is completed, the organic phase extract and the remaining aqueous phase extract can be obtained. The extract can be obtained by rotary evaporation. After pre-dissolving it in dimethyl sulfoxide (≤0.1%), it is prepared with sterile water to prepare extract solutions with mass concentrations of 6.25, 12.5, 25, 50, and 100 mg / mL, respectively. The extract solution is filtered with a microporous filter membrane with a diameter of 0.22μm and stored for standby use. A dimethyl sulfoxide solution (≤0.1%) with equal volume concentration was used as a control, and each treatment was repeated 3 times.

[0117] The activated pathogen was inoculated into LB liquid medium using the agar diffusion method and cultured at 28°C for 2 days. The concentration of the fermentation liquid was adjusted to 1.0×10 8CFU / mL, 150 μL of pathogen fermentation liquid was evenly spread on NA medium with a sterile coating stick, the bottom of NA medium was marked with a "cross method", a hole was punched at the center of the medium and 2.5 cm away from the center with a 0.5 mm diameter puncher, and agar in the hole was removed with a sterile toothpick, 200 μL of different extract solutions of the same concentration were added to the four holes except the center hole with a pipette, and a dimethyl sulfoxide solution of the same concentration was added to the center hole as a control, and each treatment was repeated 3 times. The calculation formula is as follows:

[0118] Relative inhibition zone / mm = inhibition zone diameter - punch diameter.

[0119] According to the method in "Zhang Zhixiang. Application of EXCEL in Virulence Regression Calculation", the inhibitory concentration (EC 50 ).

[0120] 2.2 Evaluation of the stability of the fermentation broth extract solution

[0121] The stability of 100 mg / mL ethyl acetate extract of the fermentation broth of strain 20Q2B-18-1 was evaluated according to the method in “Shi Hongmei. Stability of fermentation broth of Toxicodendron nivalis C41 and antibacterial activity of crude extract”.

[0122] (1) Thermal stability

[0123] The fermentation broth extract solution of strain 20Q2B-18-1 was treated in a water bath at 40, 60, 80 and 100°C, respectively, and sterilized under high pressure at 121°C (60 min). The untreated fermentation broth extract solution was used as a control. The stability of the fermentation broth extract solution was determined based on the size and change of the relative inhibition zone.

[0124] (2) Acid-base stability

[0125] The pH of the fermentation broth extract solution of strain 20Q2B-18-1 was adjusted with 1 mol / mL hydrochloric acid and 1 mol / mL sodium hydroxide solution to 2, 4, 6, 8 and 10, respectively. The untreated fermentation broth extract solution was used as a control, and the stability of the fermentation broth extract solution was determined based on the size and change of the relative inhibition zone.

[0126] (3) Photostability

[0127] The fermentation broth extract solution of strain 20Q2B-18-1 was irradiated with a 245nm ultraviolet lamp at 20cm for 20, 40, 60, 80, and 100min. The untreated fermentation broth extract solution was used as a control. The stability of the fermentation broth extract solution was determined based on the size and change of the relative inhibition zone.

[0128] 2.3 Statistical analysis

[0129] Microsoft Excel 2016 and SPSS 24.0 were used for data analysis, and Duncan's new multiple range method was used to test the significance of differences (P < 0.05).

[0130] 3 Results and analysis

[0131] 3.1 Inhibition of potato soft rot pathogens by the fermentation broth extract of the strain

[0132] As shown in Tables 4 and 5, the antibacterial activity of the extract solution of the active strain 20Q2B-18-1 increases with the increase of concentration. When the concentration of the extract solution of the fermentation broth of strain 20Q2B-18-1 is 100 mg / mL, the antibacterial activity of the ethyl acetate and n-butanol extract solutions is higher, and the relative inhibition zone sizes are 55.02 mm and 47.99 mm, respectively. The antibacterial activity of the oil ether, chloroform and aqueous extract solutions is lower, among which the aqueous extract solution has the lowest antibacterial activity, and the relative inhibition zone is as small as 9.97 mm. As for the extract solution, the ethyl acetate and n-butanol extract solutions of the active strain have higher inhibition effects, while the petroleum ether, chloroform and aqueous extract solutions have lower inhibition effects.

[0133] Table 4 Antibacterial activity of the fermentation broth extract of active strain 20Q2B-18-1 against potato soft rot pathogens

[0134]

[0135]

[0136] Table 5 The inhibitory concentration (EC) of the fermentation broth extract of strain 20Q2B-18-1 on potato soft rot pathogens 50 )

[0137]

[0138] As shown in Table 5, according to the results of significance analysis, when the concentration of the strain 20Q2B-18-1 extract solution was 100 mg / mL, it was significantly different from that of 6.25, 12.50, 25, and 50 mg / mL (P < 0.05). According to the toxicity regression equation, the EC values ​​of the n-butanol and ethyl acetate extract solutions of strain 20Q2B-18-1 against the pathogen of potato soft rot were 50 They are 97.44 mg / mL and 67.60 mg / mL respectively.

[0139] In summary, the ethyl acetate extract solution of strain 20Q2B-18-1 has high antibacterial activity against pathogenic bacteria.

[0140] 3.2 Evaluation of the stability of ethyl acetate extract of fermentation broth

[0141] Depend on Figure 5 It can be seen that the relative inhibition zone of the ethyl acetate fermentation broth extract of strain 20Q2B-18-1 is above 50 mm when the temperature is 121°C; when the pH is 8, the relative inhibition zone is larger; and it is relatively stable under ultraviolet irradiation.

[0142] Depend on Figure 5 As shown in Figure A, when the temperature rises to 121°C, the relative inhibition zone of the ethyl acetate extract solution of strain 20Q2B-18-1 is larger. This shows that strain 20Q2B-18-1 has better stability under high temperature. Figure 5 From B, we can see that in strong acid and strong base environment, the ethyl acetate extract solution of strain 20Q2B-18-1 has good stability; Figure 5 As shown in Figure C, the ethyl acetate extract of strain 20Q2B-18-1 showed no obvious downward or upward trend under ultraviolet irradiation, which was not much different from its initial antibacterial activity. In summary, the antibacterial activity of the ethyl acetate extract of strain 20Q2B-18-1 was not much different at a concentration of 100 mg / mL.

[0143] The embodiments described above are only descriptions of the preferred modes of the present invention, and are not intended to limit the scope of the present invention. Without departing from the design spirit of the present invention, various modifications and improvements made to the technical solutions of the present invention by ordinary technicians in this field should all fall within the protection scope determined by the claims of the present invention.

Claims

1. A potato soft rot antagonistic Bacillus subtilis 20Q2B-18-1, characterized in that: It was deposited in the Guangdong Provincial Microbiological Culture Collection on September 26, 2022. The deposit address is 5th Floor, Building 59, No. 100 Xianlie Middle Road, Guangzhou City, and the deposit number is GDMCC No: 62836.

2. A microbial agent, characterized in that: The invention comprises the Bacillus subtilis 20Q2B-18-1 according to claim 1.

3. A medicament for antagonizing potato soft rot, characterized in that: A fermentation broth or a fermentation broth extract comprising the Bacillus subtilis 20Q2B-18-1 according to claim 1.

4. The medicament according to claim 3, characterized in that The extraction solvent of the fermentation broth extract is ethyl acetate or n-butanol.

5. The medicament according to claim 3, characterized in that The medicament further includes pharmaceutically acceptable excipients.

6. Use of a substance described in any one of the following (1) to (3) for preventing and / or treating potato soft rot: (1) The Bacillus subtilis 20Q2B-18-1 according to claim 1; (2) The fermentation broth or fermentation broth extract of Bacillus subtilis 20Q2B-18-1 according to claim 1; (3) The microbial agent according to claim 2; The pathogen of potato soft rot is Erwinia persicina.

7. Use of the substance described in (1) or (2) below in the preparation of a product for preventing and / or treating potato soft rot: (1) The Bacillus subtilis 20Q2B-18-1 according to claim 1; (2) The fermentation broth or fermentation broth extract of Bacillus subtilis 20Q2B-18-1 according to claim 1; The pathogen of potato soft rot is Erwinia persica.

8. Use of the substance described in (1) or (2) below in the preparation of a product for inhibiting Erwinia persica: (1) The Bacillus subtilis 20Q2B-18-1 according to claim 1; (2) The fermentation broth or fermentation broth extract of Bacillus subtilis 20Q2B-18-1 according to claim 1.

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