Bacillus velezensis sjd33 and application thereof

Abstract

The present application relates to the field of microorganisms, and particularly to a broad-spectrum antagonistic bacterium Bacillus velezensis SJD33 and application thereof. The present application provides Bacillus velezensis SJD33, which has a preservation number of CGMCC No. 29456. The strain has a significant inhibitory effect on Pythium aphanidermatum, Penicillium expansum, Alternaria alternata, Diplodia natalensis, Botrytis cinerea, Colletotrichum gloeosporioides and other various fruit pathogens (Valsa mali, Monilinia fructicola and Diplodia natalensis). SJD33 can significantly reduce the natural rot rate of pears; SJD33 can rapidly colonize in the wound of fruits; SJD33 does not affect the quality of fruits, and can delay the decrease of fruit hardness and L-ascorbic acid; the secondary metabolites of SJD33 are stable in activity, and have great application potential. The strain provides a new strain resource for the fruit disease biocontrol resource library.

Description

TECHNICAL FIELD

[0001] The present application relates to the field of microorganisms, in particular to a broad-spectrum antagonistic bacterium Bacillus velezensis SJD33 and its application. BACKGROUND

[0002] China is the largest pear production and export country in the world. In 2023, the total output of pear in China reached 1985.26 million tons, an increase of about 3% over 2022. In the past two decades, China's pear production has shown a steady growth trend. However, in the process of pear production in China, the management level of pear orchards is backward, and the lack of professional post-harvest processing links leads to frequent post-harvest diseases of pear, among which the decay of post-harvest fruits caused by pathogenic fungi is one of the main reasons for the loss of pear post-harvest, which seriously restricts the healthy development of the pear industry.

[0003] During the storage and transportation of pear, a variety of pathogenic fungi such as Botryosphaeria berengeriana (B. berengeriana), Alternaria alternata (A. alternata), Penicillium expansum (P. expansum), and Colletotrichum gloeosporioides (C. gloeosporioides) often occur. Among them, B. berengeriana and A. alternata can harm branches, leaves and fruits, while P. expansum mainly harms fruits during the near-mature period and storage period. Most of these pathogenic fungi are latent infections during the growth period of the fruit, and they cause disease during the post-harvest storage and shelf life of the fruit, affecting the storage quality of the fruit and greatly reducing the economic value of the pear, causing heavy economic losses to the pear industry.

[0004] Currently, the main methods for preventing post-harvest diseases of pear are the use of chemical fungicides and the cultivation of disease-resistant varieties. However, long-term use of chemical fungicides can pollute the environment, cause pathogen resistance, and leave pesticide residues, which can severely limit the safety and export of fruit. In addition, the selection of disease-resistant varieties is a long process. Therefore, there is an urgent need to find a green control method that is efficient, environmentally friendly, and safe. Microbial control has shown great application prospects in post-harvest disease control due to its efficiency, environmental friendliness, safety, and sustainability, and has become one of the main alternatives to chemical control. Biocontrol bacteria can colonize on the surface of fruit for a long time to form biofilms and have no effect on fruit quality, and can secrete antibacterial metabolites to inhibit the growth of pathogenic fungi or induce fruit resistance. However, existing biocontrol strains generally have unstable disease prevention effects and narrow inhibition spectrum. The wide screening and exploration of biocontrol strains with stable disease prevention effects and broad inhibition spectrum will provide a key resource base for post-harvest disease control. SUMMARY

[0005] The purpose of the present application is to provide a broad-spectrum antagonistic bacterium Bacillus velezensis SJD33 and its application, so as to solve the problems existing in the prior art.

[0006] To achieve the above-mentioned purpose, the present application provides the following solutions:

[0007] The present application provides a broad-spectrum antagonistic bacterium Bacillus velezensis SJD33, and the preservation number of the Bacillus velezensis SJD33 is CGMCC No. 29456.

[0008] The present application provides the application of the above-mentioned Bacillus velezensis SJD33 in preparing a microbial agent for resisting fungal diseases, and the fungal diseases include one or several of pear ring rot, pear black spot, pear gray mold, pear penicillium disease, pear anthracnose, pear fruit rot of atahualpa, apple core rot, plum brown rot and pomegranate dry rot.

[0009] The present application provides a microbial agent for resisting fungal diseases, and the microbial agent includes the above-mentioned Bacillus velezensis SJD33; and the fungal diseases include one or several of pear ring rot, pear black spot, pear gray mold, pear penicillium disease, pear anthracnose, pear fruit rot of atahualpa, apple core rot, plum brown rot and pomegranate dry rot.

[0010] The present application provides the application of the above-mentioned Bacillus velezensis SJD33 or the above-mentioned microbial agent in preventing and treating fungal diseases, and the fungal diseases include one or several of pear ring rot, pear black spot, pear gray mold, pear penicillium disease, pear anthracnose, pear fruit rot of atahualpa, apple core rot, plum brown rot and pomegranate dry rot.

[0011] The present application provides a method for preventing and treating fungal diseases, including the step of treating the fruit to be treated by using the above-mentioned microbial agent; and the fungal diseases include one or several of pear ring rot, pear black spot, pear gray mold, pear penicillium disease, pear anthracnose, pear fruit rot of atahualpa, apple core rot, plum brown rot and pomegranate dry rot.

[0012] The present application provides the application of the above-mentioned Bacillus velezensis SJD33 in preparing a microbial agent for reducing the fruit rot rate and / or improving the fruit quality.

[0013] Preferably, the improvement of the fruit quality includes delaying the reduction of the fruit hardness and the content of L-ascorbic acid (vitamin C).

[0014] The present application provides a microbial inoculant for reducing the rot rate of fruits and / or improving the quality of fruits, which comprises the above-mentioned Bacillus velezensis SJD33.

[0015] The present application provides the use of the above-mentioned Bacillus velezensis SJD33 or the above-mentioned microbial inoculant in reducing the rot rate of fruits and / or improving the quality of fruits.

[0016] The present application provides a method for reducing the rot rate of fruits and / or improving the quality of fruits, which comprises the step of treating the fruits to be treated with the above-mentioned microbial inoculant.

[0017] The present application discloses the following technical effects:

[0018] The present application separates and identifies a broad-spectrum antagonistic bacterium Bacillus velezensis SJD33, which has a significant inhibitory effect on common pathogenic fungi of various fruits such as pears, apples, plums and pomegranates. Therefore, the strain has a broad-spectrum inhibitory effect. The results of the specific embodiments of the present application show that the secondary metabolites of Bacillus velezensis SJD33 have stable antagonistic activity, are resistant to acid and alkali, high temperature, protease and ultraviolet light, etc.; the extract of the fermentation liquor of Bacillus velezensis SJD33 has a significant inhibitory effect on Physalospora piricola; Bacillus velezensis SJD33 can quickly colonize in the wound of the fruit; Bacillus velezensis SJD33 can significantly reduce the natural rot rate of pear fruits and delay the reduction of the hardness and L-ascorbic acid of the pear fruits during the shelf life, and has no negative effect on other quality indicators of the pear fruits. In summary, Bacillus velezensis SJD33 has a broad inhibitory spectrum, is efficient and stable in disease prevention, can delay the reduction of the hardness and L-ascorbic acid of the pear fruits during the shelf life, and has great application potential. Bacillus velezensis SJD33 provides a new strain resource for the fruit disease biocontrol resource library, and its popularization and application will reduce the use of chemical fungicides in the disease prevention process, realize efficient and safe, and sustainable and green fruit production, and provide more basis. The microbial inoculant provided by the present application is efficient and stable, safe to humans and animals, and has no pollution advantage. BRIEF DESCRIPTION OF DRAWINGS

[0019] In order to more clearly illustrate the technical solutions in the embodiments of the present application or the prior art, the drawings needed in the embodiments will be briefly introduced below. Obviously, the drawings in the following description are only some embodiments of the present application, and other drawings can be obtained by those skilled in the art without creative labor.

[0020] Figure 1Antagonistic effect of strain SJD33 on V. pirina; wherein, A is the antagonistic effect of strain SJD33 on V. pirina in the confrontation experiment; B is the mycelial growth inhibition rate of strain SJD33 on V. pirina; SJD33 is strain SJD33; CK is the control group;

[0021] Figure 2 Colony phenotype of strain SJD33 cultured on LBA culture plate for 24 h and 48 h;

[0022] Figure 3 Physiological and biochemical identification results of strain SJD33; wherein, A is the detection result of amylase; B is the detection result of protease; C is the detection result of caseinase; D is the detection result of citrate hydrolysis activity;

[0023] Figure 4 Phylogenetic tree constructed based on 16S rDNA (A) and gryA (B) sequences of strain SJD33;

[0024] Figure 5 Antagonistic effect of Bacillus velezensis SJD33 on 8 common pathogenic fungi of fruits in the confrontation experiment;

[0025] Figure 6 Antagonistic activity of Bacillus velezensis SJD33 against V. pirina (A), P. piricola (B), and A. kikuchiana (C); SJD33 is Bacillus velezensis SJD33;

[0026] Figure 7 Control effect of Bacillus velezensis SJD33 on V. pirina (A) and anthracnose (B) of three-season pears during shelf life; SJD33 is Bacillus velezensis SJD33;

[0027] Figure 8 Control effect of Bacillus velezensis SJD33 on natural rot of pear fruits; wherein, SJD33 is Bacillus velezensis SJD33; CK is the control group;

[0028] Figure 9 Colonization of Bacillus velezensis SJD33 in the wound of pear fruits during the shelf life of pear fruits;

[0029] Figure 10 Effect of Bacillus velezensis SJD33 on fruit firmness (A), soluble solids (B), titratable acid (C), and L-ascorbic acid (D) of pear fruits during the shelf life of pear fruits; SJD33 is Bacillus velezensis SJD33; CK is the control group;

[0030] Figure 11Figure 1 is the antagonistic effect of Bacillus velezensis SJD33 sterile fermentation broth on the mycelium of Physalospora piricola; wherein A is water; B is Bacillus velezensis SJD33 sterile fermentation broth; C is normal mycelium of Physalospora piricola; D is mycelium of Physalospora piricola treated by Bacillus velezensis SJD33 sterile fermentation broth;

[0031] Figure 12 Figure 4 is the stability of Bacillus velezensis SJD33 sterile fermentation broth under 5 different conditions; wherein A is the stability of ultraviolet treatment; B is the stability of heat treatment; C is the stability of protease treatment; D is the stability of acid-base treatment; E is the stability of metal ion treatment; CK is the control group;

[0032] Figure 13 Figure 5 is the plate inhibition effect of crude extract of antibacterial metabolites of Bacillus velezensis SJD33 on Physalospora piricola; wherein A is the control; B is crude extract of antibacterial metabolites of Bacillus velezensis SJD33; SJD33 crude extract is crude extract of antibacterial metabolites of Bacillus velezensis SJD33. DETAILED DESCRIPTION

[0033] A number of exemplary embodiments of the present application are described in detail below, which should not be considered limiting on the present application, but rather as a description of certain aspects, features and embodiments of the present application.

[0034] It should be understood that the terms used in the present application merely describe particular embodiments, and are not intended to limit the present application. In addition, for numerical ranges in the present application, it should be understood that every intermediate value between the upper limit and the lower limit of the range is specifically disclosed. Each intermediate value within any stated value or stated range, and any other stated value or intermediate value within the stated range, is also included within the present application. The upper and lower limits of these smaller ranges can be independently included or excluded from the ranges.

[0035] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this application belongs. Although methods and materials similar or equivalent to those described herein can be used in the practice or testing of the present application, preferred methods and materials are described. All publications mentioned in this specification are herein incorporated by reference to disclose and describe the methods and / or materials in connection with which the publications are cited. The citation of any reference in this specification is not intended as an admission that the reference is prior art, but rather that the reference is part of the state of the art.

[0036] Many modifications and variations of the specific embodiments of the application can be practiced in accordance with the teachings of the description of the application. Other implementations of the application will be apparent to those skilled in the art from consideration of the specification and practice of the application disclosed herein. The specification and examples given are exemplary only.

[0037] As used herein, the terms "comprises", "comprising", "includes", "including", "has", "having" or variants thereof are open-ended, and specifically do not exclude additional, unrecited elements or method steps.

[0038] The test strains, culture medium and instrument equipment involved in the present embodiment are as follows:

[0039] 1 Test pathogenic bacteria

[0040] Botryosphaeria berengeriana (B. berengeriana), Alternaria alternata (A. alternata), Penicillium expansum (P. expansum), Botrytis cinerea (B. cinerea), Colletotrichum gloeosporioides (C. gloeosporioides) and Athelia bombacina (A. bombacina) were all preserved in the laboratory in the early stage.

[0041] Trichothecium roseum (T. roseum), Monilinia fructigena (M. fructigena) and Zythia versoniana (Z. versoniana) were all preserved in the laboratory in the early stage.

[0042] The above strains are all conventional strains in the art, and have been reported in articles. Among them, Athelia bombacina (A. bombacina) has been disclosed in the literature "First report of Athelia bombacinacausing postharvest fruit rot on pear", and a promise to issue 20 years to the outside.

[0043] 2 Culture medium

[0044] (1) Luria-Bertani medium (LB medium): yeast extract 5 g, tryptone 10 g, sodium chloride 10 g, and distilled water 1000 mL, pH 7.2.

[0045] (2) Luria-Bertani agar medium (LBA medium): same as the LB medium, with the addition of agar powder 20 g.

[0046] (3) Potato dextrose agar medium (PDA medium): potato extract powder 6 g, glucose 20 g, agar powder 20 g, and distilled water 1000 mL, pH 5.6.

[0047] (4) Acid-producing medium: 1 wt.% maltose, 1 wt.% xylose, 1 wt.% sucrose, 1 wt.% mannitol, and 1 wt.% fructose were added to the LB medium, respectively, and the pH was adjusted to 7.0, and sterilized.

[0048] (5) Starch-hydrolyzing medium: 1.7 wt.% tryptone, 0.2 wt.% beef extract, 1.5 wt.% agar powder, and 0.2 wt.% soluble starch.

[0049] (6) Casein-hydrolyzing medium: skim milk powder was mixed with water at a ratio of 1:10 (w / v), and after dissolution, sterilized in a water bath at 100°C for 30 min; another agar solution containing 4 wt.% agar powder was sterilized at 121°C for 20 min; the two sterilized solutions were mixed in equal volumes and uniformly mixed at about 50°C, and then poured into plates.

[0050] (7) Cellulose-hydrolyzing medium: 2 wt.% carboxymethyl cellulose sodium, 0.05 wt.% MgSO4, 0.05 wt.% NaCl, 0.1 wt.% KH2PO4, 0.2 wt.% (NH4)2SO4, 2 wt.% agar powder, and 0.02 wt.% Congo red.

[0051] (8) Protein-hydrolyzing medium: 0.1 wt.% beef extract, 0.2 wt.% tryptone, 0.1 wt.% NaCl, 2 wt.% skim milk powder, and 2 wt.% agar powder.

[0052] (9) Tween 80-hydrolyzing medium: 1 wt.% tryptone, 0.5 wt.% NaCl, 0.9 wt.% agar powder, 0.01 wt.% CaCl2, and 1 wt.% Tween 80.

[0053] (10) Citrate agar medium: 0.1 wt.% NaCl, 0.05 wt.% NH4H2PO4, 0.02 wt.% MgSO4•7H2O, 0.2 wt.% sodium citrate, 2 wt.% phenol red solution, and 0.5 wt.% agar powder.

[0054] 3 Test instrument

[0055] Super-clean workbench (SW-CJ-2FD, Shanghai Hushi);Thermostatic incubator (MIR-254-PC, Panasonic, Japan);Thermostatic shaker (HZP-150, Shanghai Jinghong);Microscope (CX31, Olympus, Japan);Tabletop high-speed refrigerated centrifuge (TGL-18M, Shanghai Luxiang centrifuge instrument Co., Ltd.);Automatic potentiometric titrator (Metrohm808 titrando, Metrohm, Switzerland);Refractometer (PR-101a, ATAGO, Japan);Texture analyzer (GS-15, FTA2, South Africa);UV spectrophotometer (TU-1901, Shanghai Yuanzhi instrument Co., Ltd.).

[0056] Example 1 Isolation and screening of strains

[0057] 1.1 Isolation of strains

[0058] In August 2021, rhizosphere soil samples of pear trees were collected from a pear orchard in Suizhong County, Huludao, Liaoning Province. Plate dilution method was used for bacterial isolation. 20 g of ground soil sample was weighed and added to a conical flask containing 180 mL of sterile distilled water. The conical flask was placed in a 4℃ constant temperature shaker at 120 rpm for 1 h. After taking out, it was placed for 30 min. In the clean bench, the soil sample suspension was aspirated and gradient diluted with sterile water. Then, 100 μL of the diluted solution was uniformly coated on the LBA culture plate, which was incubated at 28℃ for 48 h. Different morphological and colored single colonies were picked and transferred to fresh LBA medium plates for purification. The purified 95 single colonies were transferred to fresh LB medium and stored with 20% glycerol for future use.

[0059] 1.2 Screening of active strains

[0060] The active strain is screened by flat plate confrontation method, and the Valsa mali is used as the test pathogenic fungus. From 95 strains of bacteria isolated and purified, a strain with significant antagonistic activity against Valsa mali is screened. The specific operation is as follows: 4 mm Valsa mali fungus cake is punched on a PDA plate with a diameter of 90 mm and cultured for 3 days, and a 4 mm Valsa mali fungus cake is punched on the edge of the colony and placed in the center of a new PDA plate. The bacterial strain grown for 24 h is inoculated in four directions at a distance of 2 cm from the Valsa mali fungus cake, and placed in a constant temperature environment at 28°C. The confrontation culture is carried out, and it is recorded as the treatment group. Each treatment is repeated three times. The plates without bacterial inoculation are used as the control group (CK). When the colonies of the control group grow on the plate, the colony diameters of Valsa mali in the control group and the treatment group are observed and recorded, and the inhibition rate is calculated. The inhibition rate formula is: inhibition rate (%) = (colony diameter of the control group - colony diameter of the treatment group) / colony diameter of the control group * 100%, and the results are shown in Figure 1 . The results show that the strain SJD33 has a significant inhibitory effect on Valsa mali (A in Figure 1 , and the mycelial growth inhibition rate reaches 84.7% (B in Figure 1 ).

[0061] 1.3 Identification of strain SJD33

[0062] 1.3.1 Identification of colony morphological characteristics

[0063] The strain SJD33 is streaked on an LBA plate and cultured at 28°C for 24 h, and the colony morphological characteristics are observed. The results are shown in Figure 2 . The results show that the single colony of the strain is round, translucent, smooth on the surface, and milky white. After continuous culture for 48 h, the colony surface is flat and forms obvious white wrinkles.

[0064] 1.3.2 Physiological and biochemical identification

[0065] The physiological and biochemical tests are carried out according to the “Common Bacteria System Identification Manual” and the “Berger System Bacteriology Manual”. The culture method of the strain SJD33 suspension in the following tests is as follows: the strain SJD33 is activated in LB medium, transferred to fresh LB medium, and cultured at 28°C and 180 rpm until OD 600 =0.6 for standby.

[0066] (1) Contact enzyme test

[0067] 300 μL of the strain SJD33 suspension to be tested is inoculated into 5 mL of fresh LB medium, and cultured at 28°C for 2 d. 300 μL of 10 (v / v)% hydrogen peroxide solution is added to one tube, and whether bubbles are generated is observed. If a large amount of bubbles is generated, it is a positive reaction.

[0068] (2) Starch hydrolysis test

[0069] From the center of the starch hydrolysis culture plate, 5 μL of the SJD33 strain suspension to be tested was inoculated at four equidistant points, and the same volume of sterile water was inoculated at the other two points as a control. Three repeats were performed, and the culture was incubated at 28°C for 3 days. Iodine solution was added dropwise. If a transparent circle was produced, it was a positive reaction.

[0070] (3) Casein hydrolysis test

[0071] The SJD33 strain suspension to be tested was inoculated on a plate containing a casein hydrolysis medium. Two of the four equidistant points were selected, and 5 μL of the SJD33 strain suspension was inoculated at each point. The same volume of sterile water was inoculated at the other two points as a control. Three repeats were performed, and the culture was incubated at 28°C for 3 days. If a transparent circle was produced around the SJD33 strain colony, it was a positive reaction.

[0072] (4) Cellulose hydrolysis test

[0073] The SJD33 strain suspension to be tested was inoculated on a plate containing a cellulose hydrolysis medium. The operation method was the same as that of the casein hydrolysis test in (3). The culture was incubated at 28°C for 3 days. If a transparent circle was produced around the SJD33 strain colony, it was positive, otherwise it was negative.

[0074] (5) Protein hydrolysis test

[0075] The SJD33 strain suspension to be tested was inoculated on a plate containing a protein hydrolysis medium. The operation method was the same as that of the casein hydrolysis test in (3). The culture was incubated at 28°C for 3 days. If a transparent circle was produced, it was positive.

[0076] (6) Tween 80 hydrolysis test

[0077] The SJD33 strain suspension to be tested was inoculated on a plate containing a Tween 80 hydrolysis culture. The operation method was the same as that of the casein hydrolysis test in (3). The culture was incubated at 28°C for 3 days. If a halo was produced, it was positive.

[0078] (7) Citrate hydrolysis test

[0079] The SJD33 strain suspension to be tested was inoculated on a plate containing a citrate agar medium. The operation method was the same as that of the casein hydrolysis test in (3). The culture was incubated at 28°C for 3 days. If the colony of the SJD33 strain turned pink around, it was positive, otherwise it was negative.

[0080] (8) Acid production test

[0081] 600 μL of the strain SJD33 bacterial suspension was inoculated into 10 mL of acid-producing medium, and the medium containing the same components and not inoculated with the strain SJD33 bacterial suspension was used as a control. The culture was incubated at 28°C with constant temperature and 5000 rpm shaking for 2 days, centrifuged at 5000 rpm for 10 min, and 5 mL of the supernatant was taken into a sterilized test tube. 2-3 drops of 0.04 wt.% bromocresol purple solution were added dropwise. If the color of the strain SJD33 bacterial suspension changed, it was positive, and if it was purple, it was negative.

[0082] The results of the physiological and biochemical characteristics of the strain SJD33 are shown in Table 1 and Figure 3 The results show that the strain SJD33 can decompose maltose, mannitol and fructose to produce acid, and can hydrolyze protein, casein, starch and citrate.

[0083] Table 1 Results of physiological and biochemical experiments of the strain SJD33

[0084]

[0085] 1.3.3 Molecular sequence identification

[0086] The 16S rDNA and gyrA genes of the strain were amplified by PCR, and NCBI comparison was performed. The synthesis of all primers and gene sequencing were completed by Shengong Bioengineering (Shanghai) Co., Ltd.

[0087] The total DNA of the strain SJD33 was extracted according to the method of Ezup column bacterial genomic DNA extraction kit (SK8255), and the universal primers 27F (5'-AGTTTGATCMTGGCTCAG-3', SEQ ID NO. 1, M is A or C) / 1492R (5'-GGTTACCTTGTTACGACTT-3', SEQ ID NO. 2) and GyrA-F (5'-CAGTCAGGAAATGCGTACGTCCTT-3', SEQ ID NO. 3) / GyrA-R (5'-CAAGGTAATGCTCCAGGCATTGCT-3', SEQ ID NO. 4) were used to amplify the 16S rDNA and gyrA gene of the strain SJD33, respectively. 25.0 μL of the PCR reaction system included: 10x PCR Buffer 2.0 μL, primers 0.5 μL each, template DNA 2.0 μL, dNTP 0.5 μL, Taq Plus DNA Polymerase 0.5 μL, and sterile water to 20 μL. The PCR reaction conditions were as follows: 95℃ for 5min, 35 amplification cycles (95℃ for 30s, 57℃ for 30s, 72℃ for 30s), and 72℃ for 8min. The sequencing results were spliced using Vector NTI Advance 11.5 software, and the spliced sequence was subjected to BLAST comparison in NCBI, and it was found that the homology of the strain SJD33 with Bacillus sp. was as high as 99.87%. The phylogenetic tree of the 16S rDNA sequence (A) and the gyrA (B) sequence was constructed by using the Neighbor Joint (NJ) method of MEGA5.0 software, and the correction value was set to 1000, and it was found that the strain SJD33 was most closely related to Bacillus velezensis. Figure 4 Figure 4

[0088] According to the results of morphology, physiology, biochemistry detection and molecular identification, the strain SJD33 is Bacillus velezensis, and the classification and naming is Bacillus velezensis. The strain SJD33 is named Bacillus velezensis SJD33, and the strain is preserved in the China General Microbiological Culture Collection Center (CGMCC) on December 29, 2023, and the address is No. 1, Beichen West Road, Haidian District, Beijing, China Institute of Microbiology, Chinese Academy of Sciences, and the preservation number is CGMCC No. 29456.

[0089] ​​Example 2. The prevention effect of Bacillus velezensis SJD33 on common pathogenic fungi of pear, apple, plum and pomegranate fruits

[0090] The antagonistic effect of Bacillus velezensis SJD33 on 8 common fruit pathogenic fungi, including Valsa mali, Botrytis cinerea, Penicillium expansum, Guignardia bidwellii, Glomerella cingulata, Monilinia fructicola, Monilinia laxa and Diplodia natalensis, was determined by the flat plate confrontation method. The specific steps were as follows: 8 pathogenic fungi with a diameter of 4 mm were inoculated on fresh PDA culture plates with a diameter of 90 mm, and incubated at 28°C. When the colony diameter of the test pathogenic fungi reached 2 / 3 of the PDA culture plate diameter, a 4 mm diameter disc was punched from the edge of the 8 pathogenic fungi using a puncher, and placed in the center of a fresh PDA plate. Bacillus velezensis SJD33 colonies cultured on LBA plates for 24 h were streaked on both sides of the pathogenic fungus disc at a distance of 3 cm from the disc, and incubated at 28°C. This was recorded as the treatment group. Each treatment was repeated 3 times. The PDA plate without Bacillus velezensis SJD33 was used as the control group (Control). When the colonies in the control group covered the plate, the colony diameters of the control and treatment groups were observed and recorded, and the mycelial growth inhibition rate was calculated. The results are shown in Table 2. Bacillus velezensis SJD33 had significant antagonistic effects on 8 common fruit pathogenic fungi, except Valsa mali, with an inhibition rate of more than 44%. The mycelial growth inhibition rate of Monilinia fructicola was nearly 80% (Table 2), indicating that Bacillus velezensis SJD33 had a broad spectrum of inhibition. Figure 5

[0091] Table 2. Inhibition rate of Bacillus velezensis SJD33 on 8 common fruit pathogenic fungi (postharvest pathogenic fungi)

[0092]

[0093] Note: SJD33 is Bacillus velezensis SJD33; the values in the table are mean ± standard deviation, and * represents P<0.01

[0094] Example 3. The in vivo prevention effect of Bacillus velezensis SJD33 on common postharvest diseases of pear fruits

[0095] Huangguan pears were used as test materials to detect the in vivo prevention effect of Bacillus velezensis on postharvest diseases of pear fruits, including Valsa mali, Botrytis cinerea and Guignardia bidwellii. The specific method was as follows: Bacillus velezensis SJD33 was activated on fresh LBA medium for 24 h, a single colony was picked and transferred to 150 mL fresh LB medium, and incubated at 150 rpm and 28°C for 48 h. The spore suspension was adjusted to 1.0×10 9 ​cfu / mL for later use. Soak the test pear fruit in 0.2wt.% sodium hypochlorite solution for 3 minutes, rinse with tap water, and dry naturally in a clean bench. Use a sterilized puncher to create a 4mm (diameter) × 3mm (depth) wound on each side of the equator of the pear fruit, add 30µL of Bacillus Velez SJD33 spore suspension to each wound, and add 30µL of sterile water to each wound of the pear fruit in the control group. Place the treated fruit in a plastic box covered with moist filter paper and seal it with plastic wrap. After moisturizing and culturing at 20℃ for 24 hours, inoculate the wound with pear ring rot, pear black spot and pear penicillium fungus cakes respectively. After continuing to culture for 2 days, remove the pathogenic fungal cakes, continue moisturizing and culturing, and measure the diameter of the lesions regularly. The results are as follows Figure 6 As shown, inoculation with a spore suspension of Bacillus Velez-SJD33 significantly inhibited the expansion of lesions on the fruit of the three tested fungal pathogens. The highest efficacy against the ring rot pathogen reached 82.09% on the seventh day after inoculation, and remained at 53.44% on the eleventh day. On the eleventh day after inoculation, the efficacy of Bacillus Velez-SJD33 against Penicillium spp. reached 46.48%, and on the fifteenth day after inoculation, the efficacy against Alternaria spp. reached 56.68%. These results indicate that Bacillus Velez-SJD33 exhibited significant control effects against all three tested fungal pathogens, but showed the greatest efficacy against the living organisms of the pear ring rot pathogen.

[0096] Example 4 Control Effect of Bacillus Velez SJD33 on Shelf-Stage Ring Rot and Anthracnose of Three-Season Pear

[0097] Three-season pears are prone to ring rot and anthracnose during shelf life, causing significant economic losses. In this example, three-season pears were used as test materials and a spore suspension of Bacillus Velezii SJD33 (1.0×10 9 cfu / mL) in the same manner as in Example 3, with sterile water treatment as the control, and the lesions were measured regularly. The results are as follows: Figure 7 Results showed that after inoculation with both pathogens, lesion expansion in the treated groups was significantly inhibited. Specifically, by the seventh day after inoculation with Pear Ring Rot, Bacillus Velezii SJD33 achieved a 91.31% control effect against Pear Ring Rot during the shelf life of three-season pears. Meanwhile, by the seventh day after inoculation with Pear Anthracnose, the control effect reached 52.82%.

[0098] Example 5 Bacillus Velez SJD33 can reduce the natural decay rate of pear fruit

[0099] The three-season pear fruits were used as test materials. They were harvested when they reached commercial maturity. The fruits with intact surface, free of mechanical damage and pests and diseases were selected. The fruits were treated with spore suspension of Bacillus Velezii SJD33 (1×10 8The treated group was soaked for 30S, and the control group (CK) was soaked in sterile water. After treatment, the fruits were placed in a plastic box lined with moist filter paper, sealed with plastic wrap, and incubated at 20°C in a humid environment. Each repeat had 10 fruits, and each treatment was repeated 3 times. On the 12th day, the rotting of the control and treated groups was recorded, and the rotting rate was calculated. The formula for calculating the rotting rate is: rotting rate (%) = actual rotting number / total number of pear fruits x 100, and the results are shown in Figure 8 The results show that on the 12th day, the rotting rate of the control group was 70%, and the rotting rate of the treated group was 26.67%. The treated group significantly reduced the natural rotting rate of pear fruits during the shelf life. Therefore, Bacillus velezensis SJD33 can significantly reduce the natural rotting rate of pear fruits.

[0100] Example 6: Colonization of Bacillus velezensis SJD33 in the Wounds of Pear Fruits

[0101] Pear fruits were sterilized and wounded according to the method of Example 3. 30µL of Bacillus velezensis SJD33 spore suspension (1×10 8 The treated pear fruits were placed in a plastic box containing moist filter paper and sealed with plastic wrap, and incubated at 20°C in a humid environment. On 0d (3h), 1d, 2d, 3d, 4d, 5d, 6d, 7d, 8d, and 9d after inoculation, approximately 0.5g of flesh from the wound and surrounding area was taken using an 8mm puncher, placed in a mortar, and ground with an equal amount of sterile water. Gradient dilution was performed, and 100µL of the diluted solution was evenly spread on LBA culture medium plates, which were incubated at 28°C for 2d. The number of colonies was counted, and the results are shown in Figure 9 The results show that the number of Bacillus velezensis SJD33 in the wound of pear fruits 3h after inoculation was 2.35×10 5 cfu / mL, which increased to 1.13×10 6 cfu / mL after 5d of culture, and the number remained at 1.11×10 6 cfu / mL on the 9th day, indicating that Bacillus velezensis SJD33 had successfully colonized the wound of pear fruits.

[0102] Example 7: Effect of Bacillus velezensis SJD33 on the Quality of Pear Fruits

[0103] Pear fruits were harvested when they reached commercial maturity, and fruits with intact surfaces, no mechanical damage, and no pests or diseases were selected. Bacillus velezensis SJD33 spore suspension (1×10 8The treated fruits were stored in a constant temperature box at 20°C, and the quality indicators (fruit firmness, soluble solids, titratable acid and L-ascorbic acid (vitamin C)) were measured every two days. 15 fruits were detected for each treatment each time, and the results are shown in Figure 10 The results show that Bacillus velezensis SJD33 treatment has no negative effect on the quality of Sanjili pear fruits, and can delay the decrease of fruit firmness and L-ascorbic acid. The fruit firmness and L-ascorbic acid of the pear fruits treated by Bacillus velezensis SJD33 are higher than those of the control group from the 2nd day to the 6th day. The soluble solids and titratable acid do not change significantly during the observation period.

[0104] Example 8 Identification of the antibacterial activity of the sterile fermentation broth of Bacillus velezensis SJD33

[0105] The antibacterial activity of the sterile fermentation broth of Bacillus velezensis SJD33 was determined by the Oxford cup method. Bacillus velezensis SJD33 was streaked on LBA plates, and single colonies were picked into a 50 mL LB medium in a triangular flask and cultured overnight at 180 rpm and 28°C. The culture was inoculated into a fresh LB medium at a ratio of 1% (v / v) and cultured at 180 rpm and 28°C for 24 h. The culture was centrifuged at 8000 rpm for 20 min, and the supernatant was filtered with a 0.2 μm sterile filter to obtain the sterile fermentation broth of Bacillus velezensis SJD33. 100 μL of the sterile fermentation broth of Bacillus velezensis SJD33 was added to the Oxford cup on the PDA plate, and a cultured pear ring rot fungus cake was placed on the same PDA plate with a puncher, which was recorded as the treatment group. Sterile water was added as the control group. Then, the morphological changes of the mycelium in the treatment group and the control group were observed under a light microscope, and the results are shown in Figure 11 The results show that an obvious antibacterial circle is produced around the Oxford cup with the sterile fermentation broth of Bacillus velezensis SJD33, and the mycelium at the edge of the antibacterial circle is distorted. Therefore, the sterile fermentation broth of Bacillus velezensis SJD33 also has antibacterial activity.

[0106] Example 9 Stability evaluation of the cell-free fermentation broth of Bacillus velezensis SJD33

[0107] Bacillus velezensis SJD33 was streaked on LBA plates, and single colonies were picked and cultured in 100 mL of LB liquid medium in a flask at 180 rpm and 28°C for 48 h. The obtained fermentation broth was centrifuged at 8000 rpm for 20 min, and the supernatant was filtered through a 0.22 μm filter to obtain a cell-free fermentation broth of Bacillus velezensis SJD33, which was used as the cell-free fermentation broth of Bacillus velezensis SJD33. The antagonistic activity of the cell-free fermentation broth of Bacillus velezensis SJD33 was determined by the plate method, and the stability of the cell-free fermentation broth of Bacillus velezensis SJD33 after ultraviolet treatment, heat treatment, acid-base treatment, metal ion treatment, and protease treatment was determined using Valsa mali as the test pathogenic fungus. The results are shown in Fig. 1. Figure 12

[0108] Ultraviolet treatment: Under sterile conditions, the cell-free fermentation broth of Bacillus velezensis SJD33 was irradiated with a 30 W ultraviolet lamp for 10, 20, 30, 40, 50, 60, 90, and 120 min, respectively. The cell-free fermentation broth of Bacillus velezensis SJD33 without irradiation was used as a control. The change in the antagonistic activity of the cell-free fermentation broth of Bacillus velezensis SJD33 was determined by the plate method. The results are shown in Fig. 2A. The results showed that the antagonistic activity of the cell-free fermentation broth of Bacillus velezensis SJD33 was not significantly affected after 2 h of ultraviolet irradiation. Figure 12

[0109] Heat treatment: The cell-free fermentation broth of Bacillus velezensis SJD33 was incubated at 40°C, 50°C, 60°C, 70°C, 80°C, 90°C, and 100°C for 20 min, respectively. The cell-free fermentation broth of Bacillus velezensis SJD33 without treatment was used as a control. The change in the antagonistic activity of the cell-free fermentation broth of Bacillus velezensis SJD33 was determined by the plate method. The results are shown in Fig. 2B. The results showed that the antagonistic activity of the cell-free fermentation broth of Bacillus velezensis SJD33 after heat treatment was not significantly affected compared with the control. Figure 12

[0110] Protease treatment: Pepsin, trypsin, and proteinase K were prepared into 1 mg / mL solutions, respectively. A certain amount of the above enzyme solution was added to the cell-free fermentation broth of Bacillus velezensis SJD33 to make the final enzyme concentration 0.1 mol / L. The reaction was carried out at 37°C for 1 h, and then the sample was immediately cooled on ice after 30 min of 80°C treatment. The change in the antagonistic activity was determined by the plate method. The cell-free fermentation broth of Bacillus velezensis SJD33 without treatment was used as a control. The results are shown in Fig. 2C. The results showed that the antagonistic activity of the cell-free fermentation broth of Bacillus velezensis SJD33 treated with proteinase K, trypsin, and pepsin was not significantly different from that of the control. Figure 12

[0111] ​​​​Acid-base treatment: the pH of the B. velezensis SJD33 cell-free fermentation broth was adjusted to 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, respectively, using 1 mol / L HCl or NaOH, and the B. velezensis SJD33 cell-free fermentation broth with pH 7 was used as a control. After 24 h of rest, the changes in the antagonistic activity of the treated B. velezensis SJD33 cell-free fermentation broth were detected by the plate method, and the results are shown as D in Figure 12 . The results show that the B. velezensis SJD33 fermentation filtrate has strong antagonistic activity at pH 2-10. At pH 2-9, the antagonistic activity of the fermentation broth has no significant difference from the control. At pH 10, the antagonistic activity of the fermentation broth against pathogenic fungi still reaches more than 57%.

[0112] Metal ion treatment: lmol / L solutions of CaCl2, ZnS04·7H2O, CuS04·5H2O, NaCl, MgSO4·7H2O, MnCl2·4H2O, FeS04·7H2O, and KCl were prepared. 1000 μL of the B. velezensis SJD33 cell-free fermentation broth was added with 100 μL of the above metal ion solution to make the final concentration of the metal ions 0.1 mol / L. After shaking and mixing, the mixture was treated in a water bath at 37°C for 1 h. The plate method was used to determine the effect of different metal ions on the antagonistic activity of the B. velezensis SJD33 cell-free fermentation broth. The untreated B. velezensis SJD33 cell-free fermentation broth was used as a control group, and the results are shown as E in Figure 12 . The results show that, compared with the control, the treatment of other metal ions does not affect the antagonistic activity of the B. velezensis SJD33 cell-free fermentation broth against pathogenic fungi, except for iron ions. The antagonistic activity of the B. velezensis SJD33 cell-free fermentation broth treated with iron ions against pathogenic fungi can still reach more than 50%.

[0113] Example 10 Extraction of B. velezensis SJD33 Antimicrobial Metabolite Crude Extract and Antibacterial Effect

[0114] The acid precipitation method is used to extract the antibacterial metabolites of Bacillus velezensis SJD33. The specific steps are as follows: the Bacillus velezensis SJD33 is activated by streaking on LBA plate, and a single colony is picked and inoculated in a triangular flask containing 100 mL of LB liquid medium, and then cultured at 180 r / min and 28℃ for 72 h. The supernatant is collected by centrifugation at 8000 rpm for 30 min, and the pH value of the supernatant is adjusted to 2.0 by HCl. The supernatant is placed in a refrigerator at 4℃ overnight, and then centrifuged at 8000 rpm for 30 min. The supernatant is discarded, and the precipitate is collected and dried at 50℃. The precipitate is dissolved in 2 mL of methanol, filtered through a 0.22 μm filter membrane, and the crude extract of the antibacterial metabolites of Bacillus velezensis SJD33, referred to as SJD33 crude extract, is obtained. The oxford cup method is used to detect the antagonistic activity of the crude extract of the antibacterial metabolites of Bacillus velezensis SJD33 on the pathogenic fungus (Valsa mali). Water is used as a control, and the results are shown in Table 1. Figure 13 The results show that the crude extract of the antibacterial metabolites of Bacillus velezensis SJD33 has a significant effect on the inhibition of pathogenic fungi.

[0115] The above-described embodiments are only used to describe the preferred modes of the present application, and do not limit the scope of the present application. Without departing from the design spirit of the present application, various modifications and improvements to the technical solutions of the present application made by those skilled in the art shall fall within the protection scope of the present application as defined by the claims.

Claims

1. Use of Bacillus velezensis SJD33 in the preparation of a microbial agent for resisting fungal diseases, characterized in that: The deposit number of the Bacillus Velez SJD33 is CGMCC No. 29456; the fungal diseases are one or more of pear ring rot, pear black spot, pear gray mold, pear blue mold, pear anthracnose, pear Athea fruit rot, apple core rot, plum brown rot and pomegranate dry rot; The pathogen of the pear ring rot is Botryosphaeria berengeriana; the pathogen of the pear black spot is Alternaria alternata; the pathogen of the pear gray mold is Botrytis cinerea; the pathogen of the pear blue mold is Penicillium expansum; the pathogen of the pear anthracnose is Colletotrichum gloeosporioides; the pathogen of the pear Athelia fruit rot is Athelia bombacina; the pathogen of the apple core rot is Trichothecium roseum; the pathogen of the plum brown rot is Monilinia fructigena; and the pathogen of the pomegranate dry rot is Zythia versoniana.

2. A microbial agent for resisting fungal diseases, characterized in that: The microbial agent comprises the Bacillus Velez SJD33 described in claim 1; the fungal disease is one or more of pear ring rot, pear black spot, pear gray mold, pear blue mold, pear anthracnose, pear Athea fruit rot, apple core rot, plum brown rot and pomegranate dry rot; The pathogen of the pear ring rot is Botryosphaeria berengeriana; the pathogen of the pear black spot is Alternaria alternata; the pathogen of the pear gray mold is Botrytis cinerea; the pathogen of the pear blue mold is Penicillium expansum; the pathogen of the pear anthracnose is Colletotrichum gloeosporioides; the pathogen of the pear Athelia fruit rot is Athelia bombacina; the pathogen of the apple core rot is Trichothecium roseum; the pathogen of the plum brown rot is Monilinia fructigena; and the pathogen of the pomegranate dry rot is Zythia versoniana.

3. The use of the microbial agent according to claim 2 in preventing and treating fungal diseases, characterized in that: The fungal diseases are one or more of pear ring rot, pear black spot, pear gray mold, pear blue mold, pear anthracnose, pear Athea fruit rot, apple core rot, plum brown rot and pomegranate dry rot; The pathogen of the pear ring rot is Botryosphaeria berengeriana; the pathogen of the pear black spot is Alternaria alternata; the pathogen of the pear gray mold is Botrytis cinerea; the pathogen of the pear blue mold is Penicillium expansum; the pathogen of the pear anthracnose is Colletotrichum gloeosporioides; the pathogen of the pear Athelia fruit rot is Athelia bombacina; the pathogen of the apple core rot is Trichothecium roseum; the pathogen of the plum brown rot is Monilinia fructigena; and the pathogen of the pomegranate dry rot is Zythia versoniana.

4. A method for preventing and controlling fungal diseases, characterized in that: The method comprises the steps of treating the fruit to be treated with the microbial agent according to claim 2; wherein the fungal diseases are one or more of pear ring rot, pear black spot, pear gray mold, pear blue mold, pear anthracnose, pear Athea fruit rot, apple core rot, plum brown rot and pomegranate dry rot; The pathogen of the pear ring rot is Botryosphaeria berengeriana; the pathogen of the pear black spot is Alternaria alternata; the pathogen of the pear gray mold is Botrytis cinerea; the pathogen of the pear blue mold is Penicillium expansum; the pathogen of the pear anthracnose is Colletotrichum gloeosporioides; the pathogen of the pear Athelia fruit rot is Athelia bombacina; the pathogen of the apple core rot is Trichothecium roseum; the pathogen of the plum brown rot is Monilinia fructigena; and the pathogen of the pomegranate dry rot is Zythia versoniana.

5. Use of the Bacillus Velezii SJD33 according to claim 1 in the preparation of a microbial agent for reducing fruit rot rate and / or improving fruit quality, characterized in that: The improvement of fruit quality is to delay the reduction of fruit firmness and L-ascorbic acid content.

6. A microbial agent for reducing fruit decay rate and / or improving fruit quality, characterized in that: The microbial agent includes the Bacillus Velez SJD33 described in claim 1; and the improvement of fruit quality is to delay the reduction of fruit hardness and L-ascorbic acid content.

7. Use of the Bacillus Velezii SJD33 of claim 1 or the microbial agent of claim 6 for reducing the decay rate of fruits and / or improving the quality of fruits, characterized in that: The improvement of fruit quality is to delay the reduction of fruit firmness and L-ascorbic acid content.

8. A method for reducing fruit decay rate and / or improving fruit quality, characterized in that: The method comprises the step of treating the fruit to be treated with the microbial agent according to claim 6; wherein the improvement of the fruit quality is to delay the reduction of the fruit hardness and L-ascorbic acid content.

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