A preparation method of Paenibacillus polymyxa fermentation liquid

Abstract

The present invention relates to a method for preparing a Paenibacillus polymyxa fermentation broth, comprising the following steps: fermenting and culturing Paenibacillus polymyxa DH3-8, wherein the preservation number of Paenibacillus polymyxa DH3-8 is CGMCC No. 28156. The Paenibacillus polymyxa fermentation broth prepared by the present invention can be used to inhibit the activity of pathogenic bacteria and prevent and treat diseases caused by pathogenic bacteria, and has the advantages of good prevention and treatment effect, safety and environmental protection, etc.

Description

[0001] This invention is a divisional application. The original Chinese patent application number is: 202311686083.X, the application date is: December 11, 2023, and the patent name at the time of application is: A strain of Bacillus polymyxa and its application. Technical Field

[0002] The invention belongs to the technical field of microorganisms, and in particular relates to a method for preparing a Paenibacillus polymyxa fermentation liquid. Background Art

[0003] Plant fungal diseases are the largest category of diseases that threaten plant growth. Common symptoms include spotting, rotting, wilting, discoloration, deformity, lodging, etc., which can easily lead to reduced yields or even crop death. They are usually controlled by selecting excellent disease-resistant varieties, strengthening cultivation management, and applying chemical pesticides.

[0004] Paenibacillus polymyxa (P.polymyxa), Firmicutes, Bacillaceae, Paenibacillus. Bacteria of the genus Paenibacillus are widely distributed in nature. They are a class of rod-shaped bacteria with diverse physiological characteristics. They are also one of the dominant populations in soil and plant microecology and can be used for biological control of plant diseases. Compared with chemical control methods, the use of Paenibacillus polymyxa for control has the advantages of no drug damage and residue, pollution-free, green, and environmentally friendly. However, different strains of Paenibacillus polymyxa have different types of diseases, types of plants, and control effects. At present, in the process of biological control, there are problems such as unsatisfactory control effect and few types of diseases. Summary of the invention

[0005] In view of the problems existing in the prior art, the present invention provides a strain of Paenibacillus polymyxa and its application. The Paenibacillus polymyxa provided by the present invention can be used to inhibit the activity of pathogenic bacteria and prevent and treat diseases caused by pathogenic bacteria, and has the advantages of good prevention and treatment effect, safety and environmental protection, etc.

[0006] The technical solution of the present invention to solve the above technical problems is as follows:

[0007] The present invention provides a strain of Paenibacillus polymyxa, the strain name is Paenibacillus polymyxa DH3-8, and the preservation number is CGMCC No.28156.

[0008] The strain was isolated from healthy larch branches collected from Mudangang Forest Farm in Dunhua. The morphological characteristics and 16SrDNA identification results showed that it belonged to Paenibacillus polymyxa. The strain can be used to inhibit the activity of pathogenic bacteria and prevent and control diseases caused by pathogenic bacteria.

[0009] The present invention provides a bacterial agent, comprising the above-mentioned Paenibacillus polymyxa and / or fermentation product. In addition to the above-mentioned components, the bacterial agent may also include other components added in the art for preparing the bacterial agent. The present invention has no special restrictions on the dosage form of the bacterial agent, which may be solid, liquid or other dosage forms.

[0010] The fermentation product may be a fermentation broth of Paenibacillus polymyxa DH3-8. The fermentation broth may be obtained by fermenting and culturing Paenibacillus polymyxa DH3-8. The fermentation broth may also be further dried (e.g., freeze-dried) to prepare a solid bacterial agent.

[0011] The present invention provides a method for preparing a bacterial agent, comprising the following steps: fermenting and culturing Paenibacillus polymyxa DH3-8. The culture medium for fermentation and culturing may include: 3.0 g beef extract, 5.0 g peptone, 1.0 g yeast extract, 10.0 g glucose, 1000 mL distilled water, pH 7.0-7.2, the inoculation amount may be 5%, and the fermentation and culturing conditions may be 28° C., 150 r / min, and shake flask fermentation for 3 days. The above method is conducive to the growth of Paenibacillus polymyxa DH3-8, so as to obtain a bacterial agent with better activity.

[0012] The invention provides a method for preparing a Paenibacillus polymyxa fermentation liquid, comprising the following steps: fermenting and culturing Paenibacillus polymyxa DH3-8, wherein the preservation number of Paenibacillus polymyxa DH3-8 is CGMCC No.28156.

[0013] The culture medium for fermentation culture may include: 3.0 g beef extract, 5.0 g peptone, 1.0 g yeast extract, 10.0 g glucose, and 1000 mL distilled water. The inoculation amount may be 5%. The temperature of fermentation culture may be 28° C. Shake flask fermentation may be adopted, the rotation speed is 150 r / min, and the shake flask fermentation time is 3 days.

[0014] Furthermore, the method may further include the step of obtaining the supernatant of the fermentation broth.

[0015] The Paenibacillus polymyxa fermentation liquid prepared by the method can be used to inhibit the activity of pathogenic bacteria and prevent and treat diseases caused by pathogenic bacteria, and has the advantages of good prevention and treatment effect, safety and environmental protection, etc.

[0016] The present invention provides the use of the above-mentioned Paenibacillus polymyxa and / or bacterial agent in resisting pathogenic bacteria.

[0017] The pathogenic bacteria can be selected from one or a combination of the genus Aspergillus, apple rot pathogen, poplar canker pathogen, corn leaf blight pathogen, eggplant brown streak pathogen, citrus rot pathogen, cucumber sclerotinia pathogen, tobacco brown spot pathogen, blueberry canker pathogen, larch dieback pathogen, spruce damping-off pathogen, and apple fruit rot pathogen.

[0018] The present invention provides the use of the above-mentioned Paenibacillus polymyxa and / or bacterial agent in preventing and controlling plant diseases.

[0019] The plant disease can be caused by pathogenic bacteria, and the pathogenic bacteria are selected from one or a combination of the genus Aspergillus, apple rot bacteria, poplar canker bacteria, corn leaf blight bacteria, eggplant brown streak pathogen, citrus rot pathogen, cucumber sclerotinia pathogen, tobacco brown spot pathogen, blueberry canker pathogen, larch dieback pathogen, spruce blight pathogen, and apple fruit rot pathogen.

[0020] The polymyxa bacillus and / or bacterial agent provided by the present invention has significant antibacterial activity against the above pathogenic bacteria, especially has the strongest inhibitory effect on the polyphytic fungus, and also has a strong inhibitory effect on corn leaf blight, tobacco brown spot pathogen, apple fruit rot, apple fruit rot, eggplant brown streak disease and spruce damping-off. It can be used for diseases caused by the above pathogenic bacteria. It has the advantages of good prevention and control effect, safety and environmental protection, etc. BRIEF DESCRIPTION OF THE DRAWINGS

[0021] Figure 1 This is a photo of live DH3-8 bacteria (grown on NA medium at 28°C for 3 days).

[0022] Figure 2 This is the morphology of strain DH3-8 under optical microscope.

[0023] Figure 3 This is the phylogenetic tree of strain DH3-8.

[0024] Figure 4 This is the result of the live bacteria DH3-8 against larch dieback disease. The left picture is the confrontation picture, and the right picture is the control.

[0025] Figure 5 This is the result of the DH3-8 fermentation liquid against larch dieback disease. The left picture is the confrontation picture, and the right picture is the control. DETAILED DESCRIPTION

[0026] The principles and features of the present invention are described below in conjunction with the accompanying drawings. The examples given are only used to explain the present invention and are not used to limit the scope of the present invention.

[0027] In the present invention, unless otherwise specified, the experimental methods used are conventional experimental methods in the art. The materials, reagents, and instruments used are conventional materials, reagents, and instruments in the art, which can be obtained through commercial channels or prepared by conventional methods.

[0028] Test plant pathogens: blueberry canker pathogen Botryosphaeria dothidea was donated by Associate Professor Xu Chengnan of the School of Life Sciences of Yan'an University; corn leaf spot pathogen Exserohilum turcicum, tobacco brown spot pathogen Alternaria alternata, citrus fruit rot pathogen Pythium aphanidermatum, eggplant brown streak pathogen Phomopsis vexans, and cucumber sclerotinia pathogen Sclerotinia sclerotiorum were donated by the Plant Virus Research Laboratory of Shenyang Agricultural University; poplar canker pathogen Botryosphaeria dothidea, spruce blight pathogen Rhizoctonia solani, larch blight pathogen Neofusicoccum laricinum, Dothiorella gregari, apple rot pathogen Valsamali, and apple fruit rot pathogen Monilinia polystroma were isolated and preserved by the laboratory of the inventor of the present invention. The above test plant pathogens are available to the public for non-commercial purposes only to repeat the embodiments described in the present invention.

[0029] The test samples were collected from healthy Changbai Larix olgensis branches from the Larix olgensis forest of Mudangang Forest Farm in Dunhua in June 2022.

[0030] Test medium:

[0031] NA medium nutrient agar (Nutrient Agar) medium, the formula includes: beef extract 3.0g, peptone 10.0g, sodium chloride 5.0g, agar 15.0g, distilled water 1000mL, pH7.0-7.2.

[0032] The formula of Potato Dextrose Agar (PDA) medium includes: 300g of potato (peeled and cut into pieces), 20g of glucose, 20g of agar, and 1000mL of distilled water; the preparation method includes the following steps: peel and cut the potato into pieces, add 1000mL of distilled water, and boil for 10-20min. Filter with gauze, and add distilled water to 1000mL. Add glucose and agar, heat to dissolve, divide into packages, and sterilize at 121℃ for 20min.

[0033] The fermentation medium includes: 3.0 g beef extract, 5.0 g peptone, 1.0 g yeast extract, 10.0 g glucose, 1000 mL distilled water, pH 7.0-7.2.

[0034] Reagents: Ezup column bacterial genomic DNA extraction kit was purchased from Shanghai Bioengineering Co., Ltd.; Taq PCR Master Mix and DNA Marker DL2000 were purchased from BBI Life Sciences; others were domestic analytical grade.

[0035] Instrument: BX53 Olympus optical microscope was purchased from Olympus Corporation.

[0036] In the embodiments, the data analysis involved was performed using SPSS 26.0 for data statistics.

[0037] The following is an introduction through specific embodiments.

[0038] Example 1 Strain Screening

[0039] Healthy larch branches were collected from the Mudangang Forest Farm in Dunhua. The separation method was based on the Plant Disease Research Methods edited by Fang Zhongda (Published on April 2007, Published by China Agricultural Press, CIP Approval No.: 2007038168, ISBN: 978-7-109-05255-0). The separation was carried out by step-by-step plate dilution and coating. The strains with different colony morphology were observed and selected every day and transferred to the slant for culture in time, and then numbered and preserved. In order to test the effect of surface disinfection of larch tissue, the tissue blotting method was used for testing. 100 μL of the last sterile water rinse was applied to the NA medium, repeated 3 times, and cultured for 3 days. If there was no colony growth in the culture dish, it means that the surface of the test material was thoroughly disinfected, otherwise, the separation result cannot be used. After repurification 3-5 times by the dilution separation method, the samples were numbered and stored in a 4°C refrigerator for later use.

[0040] After purification, 24 purified endophytic strains with consistent morphology, size and color were obtained and numbered respectively. One of the strains was numbered DH3-8, and its strain photo is shown in Figure 1 As shown, the colonies of DH3-8 are round, 1-2 mm in diameter, light yellow, with a smooth and raised surface, and feel sticky when touched with an inoculation loop.

[0041] Example 2 Determination of antibacterial activity of endophytes

[0042] All strains isolated in Example 1 (i.e., 24 strains obtained in Example 1) were cultured on NA medium at 28°C for 1-3 days, and the antibacterial activity of the endophytic bacteria isolated in Example 1 was determined by the plate confrontation culture method with the larch dieback pathogen as the target bacteria. Details are shown in Table 1. The pathogens to be tested were made into 7mm cakes and inoculated on a 90mm diameter PDA plate at 1.5cm from the center. The bacteria cultured for 24h were picked with an inoculation loop at 3cm from the cake, and parallel lines were drawn. After culturing at 28°C for 72h, the width of the inhibition zone between the bacteria and the pathogenic fungi was measured, and each treatment was repeated 3 times. According to the size of the inhibition zone, the strain DH3-8 with the most vigorous growth and antibacterial effect was selected as the selected strain for subsequent experiments.

[0043] Table 1 Antibacterial activity of live bacteria against larch dieback pathogen

[0044]

[0045] Note: The data in the table are mean ± standard deviation.

[0046] Example 3 Morphological observation of strain DH3-8

[0047] The strain DH3-8 was inoculated onto a plate of NA medium, and a sterilized cover slip was inserted into the medium at an angle of 45°. The culture was carried out at 28°C for 1 day, and the cover slip was removed. The strain DH3-8 grew vigorously on the NA medium. The morphology of the strain was observed under a BX53 Olympus optical microscope. The morphology of the strain DH3-8 under the optical microscope is shown in the figure below. Figure 2 shown.

[0048] Example 4 DH3-8 strain identification

[0049] Extract genomic DNA of strain DH3-8. DNA extraction was performed according to the instructions of Ezup column-type bacterial genomic DNA extraction kit. PCR amplification was performed using genomic DNA as a template.

[0050] The PCR reaction system was 50 μL: PCR Mixture 25 μL, Template 2 μL, Universal Primer 27F, Universal Primer 1429R 2 μL each, ddH2O 19 μL.

[0051] Universal primer 27F and universal primer 1429R were synthesized by Changchun Kumei Biotechnology Co., Ltd.

[0052] PCR reaction conditions: denaturation at 94°C for 4 min; denaturation at 94°C for 30 s, annealing at 58°C for 30 s, extension at 72°C for 1 min, 35 cycles; extension at 72°C for 10 min, and storage at 4°C.

[0053] The PCR product of sterile water was used as a negative control, and the PCR product of the strain whose 16SrDNA results had been determined in the laboratory was used as a positive control. The sample volume was 2 μL, and 2% agarose gel electrophoresis was performed. All operations were performed according to the operating procedures of the kit, and the recovered products were sent to Jilin Kumei Biotechnology Co., Ltd. for sequencing and analysis. According to the sequencing results, the phylogenetic tree was established using MEGA version 6.0, as shown in Figure 3 shown.

[0054] According to the characteristics of Example 3 and the sequencing comparison results, the strain DH3-8 was determined to be Paenibacillus polymyxa. The strain was deposited in the General Microbiological Center (CGMCC) of the China Microbiological Culture Collection Administration on August 11, 2023, and the deposit address is: Institute of Microbiology, Chinese Academy of Sciences, No. 3, Beichen West Road, Chaoyang District, Beijing, China. The deposit name is Paenibacillus polymyxa DH3-8, and the deposit number is CGMCC No. 28156.

[0055] Example 5

[0056] Referring to the method of Example 2, blueberry canker pathogen Botryosphaeria dothidea, corn leaf blight pathogen Exserohilum turcicum, tobacco brown spot pathogen Alternaria alternata, citrus fruit rot pathogen Pythiumaphanidermatum, eggplant brown streak pathogen Phomopsis vexans, cucumber sclerotinia pathogen Sclerotiniasclerotiorum, poplar canker pathogen Botryosphaeria dothidea, spruce blight pathogen Rhizoctoniasolani, larch dieback pathogen Neofusicoccumlaricinum, Dothiorella gregari, apple rot pathogen Valsa mali, and apple fruit rot pathogen Monilinia polystroma were used as test strains to detect the antibacterial spectrum of the antagonistic bacteria DH3-8 live body. Using the cup-and-plate method, all the test plant pathogens were activated and made into a spore suspension with 30-40 spores / field of view under a 10×15x microscope. A mixed bacterial plate was prepared by pipetting 300 μL and adding 70 mL of PDA culture medium. An Oxford cup was placed in the center of the culture dish, and 100 μL of fermentation liquid was added to the Oxford cup. After constant temperature culture at 28°C for 72 hours, the diameter of the inhibition zone was measured by the cross method.

[0057] According to the test results in Table 2, the antagonistic bacteria DH3-8 has a good inhibitory effect on all the above-mentioned test strains. Among them, DH3-8 has the strongest inhibitory effect on the polymorphic burrows, with an inhibition zone width of 14.83 mm; it also has a strong inhibitory effect on corn leaf blight, tobacco red star pathogen, apple fruit rot pathogen, and larch blight pathogen, with inhibition zone widths of 14.81 mm, 13.57 mm, 13.53 mm, and 12.05 mm, respectively ( Figure 4 ). The inhibitory effect on cucumber sclerotinia is slightly weaker than the above pathogens, but the width of the inhibition zone can reach 7.42mm.

[0058] Table 2 Antibacterial spectrum of antagonistic bacteria DH3-8

[0059]

[0060] Note: The data in the table are mean ± standard deviation.

[0061] Example 6 Preparation of fermentation broth and determination of its antibacterial spectrum

[0062] The antagonistic strain DH3-8 obtained by screening in Example 2 was subjected to liquid fermentation. The culture medium for fermentation culture may include: 3.0 g beef extract, 5.0 g peptone, 1.0 g yeast extract, 10.0 g glucose, 1000 mL distilled water, pH 7.0-7.2. In the liquid fermentation, the inoculation amount is 5%, the fermentation culture conditions are 28°C, 150 r / min, and the shake flask fermentation is 3 days to obtain a fermentation liquid.

[0063] The test bacteria were D. gregari, V. mali Miyabe, B. dothidea, E. turcicum, P. vexans, Pythium aphanidermatum, S. sclerotiorum, A. alternata, B. dothidea, N. laricinum, R. solani, M. polystroma as the apple rot pathogen, and the cup-disc method was used to determine the antibacterial spectrum of the supernatant of the fermentation broth of the antagonistic strain DH3-8.

[0064] The test results showed that the fermentation broth of the antagonistic bacteria DH3-8 had a good inhibitory effect on all the tested pathogens (Table 3); among them, the fermentation broth of the strain had the strongest antibacterial effect on the polymorphic burrows, reaching 25.85mm; it had a strong antagonistic effect on corn leaf blight and tobacco brown spot pathogen, with an inhibition diameter of more than 23.18mm; it also had a good inhibitory effect on apple fruit rot, eggplant brown streak disease and spruce damping-off disease, with inhibition diameters of 19.73mm, 18.98mm and 18.87mm respectively; it had a good inhibitory effect on apple rot pathogen, poplar canker pathogen, blueberry canker pathogen, larch dieback pathogen ( Figure 5 ) has an inhibition diameter between 15.26-16.67 mm; its inhibitory effect on cucumber sclerotinia is slightly weaker than that on the above pathogens, but the minimum inhibition diameter can still reach 13.00 mm.

[0065] Table 3 Antibacterial spectrum of fermentation broth of DH3-8

[0066]

[0067] Note: The data in the table are mean ± standard deviation.

[0068] In summary, the DH3-8 strain has fungicidal activity, and its live bacteria and fermentation liquid both show strong inhibitory effects on Aspergillus, apple rot pathogen, poplar canker pathogen, corn leaf blight pathogen, eggplant brown streak pathogen, citrus rot pathogen, cucumber sclerotinia pathogen, tobacco brown spot pathogen, blueberry canker pathogen, larch dieback pathogen, spruce damping-off pathogen, and apple fruit rot pathogen. The inventors speculate that this may be because the strain produces antifungal substances during its physiological metabolism.

[0069] The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. Any modifications, equivalent substitutions, improvements, etc. made within the spirit and principle of the present invention should be included in the protection scope of the present invention.

Claims

1. A method for preparing a polymyxa bacillus fermentation broth, characterized in that: The following steps are involved: Paenibacillus polymyxa DH3-8 was fermented and cultured. The deposit number of Paenibacillus polymyxa DH3-8 is CGMCC No.28156.

2. The preparation method according to claim 1, characterized in that: The culture medium for fermentation culture is: 3.0 g beef extract, 5.0 g peptone, 1.0 g yeast extract, and 10.0 g glucose per 1000 mL distilled water.

3. The preparation method according to claim 1 or 2, characterized in that: The inoculation amount was 5%.

4. The preparation method according to claim 1 or 2, characterized in that: The fermentation temperature was 28°C.

5. The preparation method according to claim 1 or 2, characterized in that: Shake flask fermentation was adopted with a rotation speed of 150r / min.

6. The preparation method according to claim 1 or 2, characterized in that: Shake flask fermentation was used and the fermentation time was 3 days.

7. The preparation method according to claim 1 or 2, characterized in that: The method also includes the step of obtaining the fermentation broth supernatant.

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