Broad-spectrum antagonistic bacterium bacillus velezensis SJD33 and application thereof

Abstract

The invention relates to the field of microorganisms, in particular to a broad-spectrum antagonistic bacterium bacillus velezensis SJD33 and application thereof. The preservation number of the bacillus velezensis SJD33 provided by the invention is CGMCC (China General Microbiological Culture Collection Center) No.29456. The bacillus velezensis SJD33 provided by the invention has the advantages that the preservation number is CGMCC No.29456; the strain has a remarkable inhibition effect on pear fruit rot pathogens (physalospora piricola, penicillium, alternaria alternata, athelia fruit rot pathogens, botrytis cinerea and colletotrichum gloeosporioides) and other fruit pathogenic bacteria (apple core pathogens, brown rot pathogens and pomegranate dry rot pathogens). The SJD33 can obviously reduce the natural rotting rate of the pear fruits; the SJD33 can be rapidly colonized in fruit wounds; the SJD33 does not influence the fruit quality and can delay the reduction of the fruit hardness and the L-ascorbic acid; the SJD33 secondary metabolite is stable in activity and has great application potential. The strain provides a new strain resource for a fruit disease biocontrol resource library.

Description

Technical Field

[0001] The invention relates to the field of microorganisms, in particular to a broad-spectrum antagonistic bacterium Bacillus Velezii SJD33 and application thereof. Background Art

[0002] my country is the world's largest pear producer and exporter. In 2023, my country's total pear production reached 19.8526 million tons, an increase of about 3% over 2022. In the past two decades, my country's pear production has shown a steady growth trend, but in the process of pear production in my country, the management level of pear orchards is backward and professional post-harvest processing links are missing, resulting in frequent post-harvest diseases of pears. Among them, post-harvest fruit rot caused by pathogenic fungi infection is one of the main causes of post-harvest losses of pears, which seriously restricts the healthy development of the pear industry.

[0003] During the storage and transportation of pears, a variety of pathogenic fungal infectious diseases, mainly 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 near maturity and during storage. Most of these pathogens infect the fruit latently during the growth period, and become ill during the storage period and shelf life of the pear fruit after harvest, affecting the storage quality of the fruit, greatly reducing the economic value of the pear fruit, and causing heavy economic losses to the pear fruit industry.

[0004] At present, the main methods for the prevention and control of postharvest diseases of pear fruit are the application of chemical fungicides and the cultivation of disease-resistant varieties. However, the long-term application of chemical fungicides will pollute the environment, cause pathogen resistance and pesticide residues, among which the pesticide residue problem will seriously limit the safety and export of fruits; and the breeding of disease-resistant varieties has the problem of a long cycle. Therefore, it is urgent to find an efficient, environmentally friendly, long-lasting and safe green prevention and control method. Microbial control has shown great application prospects in the field of postharvest disease prevention and control with its high efficiency, environmental protection, safety and sustainability, and has become one of the main alternatives to chemical control. Biocontrol bacteria can colonize on the surface of fruits for a long time to form biofilms without affecting the quality of fruits, and can secrete antibacterial metabolites that inhibit the growth of pathogenic fungi or induce fruit resistance. However, the existing biocontrol strains generally have the problems of unstable disease prevention effect and narrow antibacterial spectrum. Extensive screening and discovery of biocontrol strains with stable disease prevention effect and broad antibacterial spectrum will provide a key resource basis for postharvest disease prevention and control. Summary of the invention

[0005] The purpose of the present invention is to provide a broad-spectrum antagonistic bacterium Bacillus Velez subtilis SJD33 and application thereof, so as to solve the problems existing in the above-mentioned prior art.

[0006] To achieve the above object, the present invention provides the following solutions:

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

[0008] The present invention provides the use of the above-mentioned Bacillus Velezii SJD33 in preparing a microbial agent for resisting fungal diseases, wherein the fungal diseases include one or more of pear ring rot, pear black spot, pear gray mold, pear penicillium, pear anthracnose, pear Athea fruit rot, apple rot, plum brown rot and pomegranate dry rot.

[0009] The invention provides a microbial agent for resisting fungal diseases, wherein the microbial agent comprises the above-mentioned Bacillus Velez SJD33; the fungal diseases comprise one or more of pear ring rot, pear black spot, pear gray mold, pear penicillium, pear anthracnose, pear Ateia fruit rot, apple core rot, plum brown rot and pomegranate dry rot.

[0010] The present invention provides the use of the above-mentioned Bacillus Velez SJD33 or the above-mentioned microbial agent in preventing and controlling fungal diseases, wherein the fungal diseases include one or more of pear ring rot, pear black spot, pear gray mold, pear penicillium, pear anthracnose, pear Ateia fruit rot, apple rot, plum brown rot and pomegranate dry rot.

[0011] The invention provides a method for preventing and controlling fungal diseases, comprising the step of treating to-be-treated fruits with the above-mentioned microbial agent; the fungal diseases include one or more of pear ring rot, pear black spot, pear gray mold, pear penicillium, pear anthracnose, pear Athea fruit rot, apple core rot, plum brown rot and pomegranate dry rot.

[0012] The present invention provides the use of the above-mentioned Bacillus Velezii SJD33 in the preparation of a microbial agent for reducing the fruit decay rate and / or improving the fruit quality.

[0013] Preferably, the improving fruit quality comprises delaying the reduction of fruit firmness and L-ascorbic acid (vitamin C) content.

[0014] The present invention provides a microbial agent for reducing the decay rate of fruits and / or improving the quality of fruits. The microbial agent comprises the above-mentioned Bacillus Velezii SJD33.

[0015] The present invention provides the use of the above-mentioned Bacillus Velez SJD33 or the above-mentioned microbial agent in reducing the fruit decay rate and / or improving the fruit quality.

[0016] The present invention provides a method for reducing the decay rate of fruits and / or improving the quality of fruits, comprising the step of treating the fruits to be treated by using the above-mentioned microbial agent.

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

[0018] The present invention isolates 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. It can be seen that the strain has a broad-spectrum antibacterial effect. The results of the specific embodiments of the present invention show that the secondary metabolites of Bacillus velezensis SJD33 have stable antagonistic activity, and are resistant to acid, alkali, high temperature, protease and ultraviolet light; the fermentation broth extract of Bacillus velezensis SJD33 has a significant inhibitory effect on pear ring rot bacteria; Bacillus velezensis SJD33 can quickly colonize in fruit wounds; Bacillus velezensis SJD33 can significantly reduce the natural decay rate of pear fruits, and can delay the reduction of hardness and L-ascorbic acid of pear fruits during the shelf life, and has no negative impact on other quality indicators of pear fruits. In summary, Bacillus Velez SJD33 has a wide antibacterial spectrum, an efficient and stable disease prevention effect, and can delay the reduction of hardness and L-ascorbic acid during the shelf life of pear fruit, and has great application potential. Bacillus Velez SJD33 provides a new strain resource for the fruit disease biological control resource library. Its promotion and application will reduce the use of chemical fungicides in the process of disease prevention and control, and provide more basis for achieving efficient, safe, and sustainable green fruit production. The microbial agent provided by the present invention has the advantages of high efficiency, stability, safety for humans and animals, and no pollution. BRIEF DESCRIPTION OF THE DRAWINGS

[0019] 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.

[0020] Figure 1This is the detection of the antagonistic effect between strain SJD33 and mycelium of pear ring rot pathogen; A is the antagonistic effect of strain SJD33 on pear ring rot pathogen in the plate confrontation experiment; B is the inhibition rate of strain SJD33 on mycelium growth of ring rot pathogen; SJD33 is strain SJD33; CK is the control group;

[0021] Figure 2 The colony phenotype of strain SJD33 cultured on LBA plates for 24 h and 48 h;

[0022] Figure 3 The physiological and biochemical identification results of strain SJD33; A is the test result of amylase; B is the test result of protease; C is the test result of casein; D is the test result of citrate hydrolysis activity;

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

[0024] Figure 5 The antagonistic effect of Bacillus Velez SJD33 on eight common fruit pathogenic fungi in the plate confrontation experiment;

[0025] Figure 6 Antagonistic activity of Bacillus velez SJD33 against pear ring spot pathogen (A), pear penicillium (B) and pear black spot pathogen (C); SJD33 is Bacillus velez SJD33;

[0026] Figure 7 The control effect of Bacillus Velez SJD33 on shelf-life ring rot (A) and anthracnose (B) of three-season pears; SJD33 is Bacillus Velez SJD33;

[0027] Figure 8 The control effect of Bacillus Velez SJD33 on natural rot of pear fruit; SJD33 is Bacillus Velez SJD33; CK is the control group;

[0028] Fig. 9 The results of colonization of Bacillus Velez SJD33 in pear fruit wounds during the shelf life of pear fruit.

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

[0030] Fig.11The antagonistic effect of the aseptic fermentation liquid of Bacillus Velez SJD33 on the mycelium of pear ring rot pathogen; wherein A is water; B is the aseptic fermentation liquid of Bacillus Velez SJD33; C is the normal mycelium of the pear ring rot pathogen; and D is the mycelium of the pear ring rot pathogen treated with the aseptic fermentation liquid of Bacillus Velez SJD33.

[0031] Fig.12 The stability of the aseptic fermentation broth of Bacillus Velez SJD33 under five different treatment conditions; 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 and alkali treatment; E is the stability of metal ion treatment; CK is the control group;

[0032] Fig.13 The figure shows the plate inhibitory effect of the crude extract of antibacterial metabolites of Bacillus Velez SJD33 on pear ring rot pathogen; A is the control; B is the crude extract of antibacterial metabolites of Bacillus Velez SJD33; SJD33 crude extract is the crude extract of antibacterial metabolites of Bacillus Velez SJD33. DETAILED DESCRIPTION

[0033] 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.

[0034] 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.

[0035] 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.

[0036] 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.

[0037] The words “include,” “including,” “have,” “contain,” etc. used in this document are open-ended terms, meaning including but not limited to.

[0038] The test strains, culture media and instruments and equipment involved in this embodiment are as follows:

[0039] 1 Test pathogens

[0040] Pear ring rot (Botryosphaeria berengeriana, B.berengeriana), pear black spot (Alternaria alternata, A.alternata), pear blue mold (Penicillium expansum, P.expansum), pear gray mold (Botrytis cinerea, B.cinerea), pear anthracnose (Colletotrichum gloeosporioides, C.gloeosporioides), and pear Athelia bombacina (A.bombacina) were all stored in the laboratory in the early stage.

[0041] Apple core rot pathogen (Trichothecium roseum, T.roseum), plum brown rot pathogen (Moniliniafructigena, M.fructigena) and pomegranate stem rot pathogen (Zythia versoniana, Z.versoniana) were previously stored in the laboratory.

[0042] The above strains are all conventional strains in this field and have been reported in articles. Among them, the pear Athelia bombacina (A. bombacina) has been disclosed in the document "First report of Athelia bombacina causing postharvest fruit rot on pear" and has been promised to be released to the public for 20 years.

[0043] 2 Culture medium

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

[0045] (2) Luria-Bertani agar medium (LBA medium): Same as LB medium, but add 20 g of agar powder.

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

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

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

[0049] (6) Casein hydrolysis medium: skim milk powder and water were mixed in a ratio of 1:10 (w / v), dissolved, and sterilized in a 100°C water bath for 30 min. Separately, an agar aqueous solution containing 4 wt.% agar powder was prepared and sterilized at 121°C for 20 min. The two sterilized cultures were cooled to about 50°C, mixed in equal volumes, and poured onto plates.

[0050] (7) Cellulose hydrolysis medium: 2wt.% sodium carboxymethyl cellulose, 0.05wt.% MgSO 4 ,0.05wt.%NaCl,0.1wt.%KH 2 PO 4 、0.2wt.%(NH 4 ) 2 SO 4 , 2wt.% agar powder and 0.02wt.% Congo red.

[0051] (8) Protein hydrolysis medium: 0.1wt.% beef extract, 0.2wt.% peptone, 0.1wt.% NaCl, 2wt.% skimmed milk powder and 2wt.% agar powder.

[0052] (9) Tween 80 hydrolysis medium: 1wt.% peptone, 0.5wt.% NaCl, 0.9wt.% agar powder, 0.01wt.% CaCl 2 and 1wt.% Tween 80.

[0053] (10) Citrate agar medium: 0.1wt.% NaCl, 0.05wt.% NH 4 H 2 PO 4 、0.02wt.%MgSO 4 7H 2 O, 0.2wt.% sodium citrate, 2wt.% phenol red solution and 0.5wt.% agar powder.

[0054] 3. Test instruments

[0055] Clean bench (SW-CJ-2FD, Shanghai Hujing); constant temperature incubator (MIR-254-PC, Panasonic, Japan); constant temperature shaker (HZP-150, Shanghai Jinghong); microscope (CX31, Olympus, Japan); desktop high-speed refrigerated centrifuge (TGL-18M, Shanghai Luxiangyi Centrifuge Instrument Co., Ltd.); automatic potentiometric titrator (Met rohm808 titrando, Metrohm, Switzerland); refractometer (PR-101a, ATAGO, Japan); texture analyzer (GS-15, FTA2, South Africa); UV spectrophotometer (TU-1901, Shanghai Yuanxi Instrument Co., Ltd.).

[0056] Example 1 Isolation, screening and identification of strains

[0057] 1.1 Isolation of strains

[0058] In August 2021, rhizosphere soil samples of pear trees were collected in a pear orchard in Suizhong County, Huludao, Liaoning Province, and bacterial isolation was performed using the plate dilution method. Weigh 20 g of ground soil sample, add it to a conical flask containing 180 mL of sterile distilled water, shake it at 120 rpm in a 4 ° C constant temperature shaker for 1 hour, and let it stand for 30 minutes after taking it out. In the clean bench, after absorbing the soil sample suspension, it was gradiently diluted with sterile water. After that, 100 μL of the dilution solution was evenly spread on the LBA culture plate, incubated at 28 ° C for 48 hours, and single colonies with different morphology and color were picked and transferred to a fresh LBA culture medium plate for purification. After purification, 95 single colonies were transferred to fresh LB culture medium and stored with 20% glycerol for standby.

[0059] 1.2 Screening of active strains

[0060] The plate confrontation method was used to screen active strains, and pear ring spot pathogen was used as the test pathogenic fungus. From the 95 isolated and purified bacterial strains, strains with significant antagonistic activity against pear ring spot pathogen were screened. The specific operation is as follows: a 4mm pear ring spot pathogen cake was punched and cultured on a 90mm diameter PDA plate for 3 days. A 4mm diameter pear ring spot pathogen cake was punched at the edge of the colony with a hole puncher and placed in the center of a new PDA plate. The bacterial strain grown for 24 hours was inoculated in four directions of the pear ring spot pathogen cake and 2cm away from the pear ring spot pathogen cake. The plate was placed in a constant temperature environment of 28°C for confrontation culture, which was recorded as the treatment group. Each treatment was repeated 3 times, and the plate without bacteria inoculation was used as the control group (CK). When the colony of the control group filled the plate, the colony diameter of the pear ring spot pathogen in the control group and the treatment group was observed and recorded, and the inhibition rate was calculated. The inhibition rate formula is: Inhibition rate (%) = (control group colony diameter - treatment group colony diameter) / control group colony diameter * 100%. The results are as follows Figure 1 The results showed that strain SJD33 had a significant inhibitory effect on pear ring rot pathogen ( Figure 1 A), the mycelium growth inhibition rate reached 84.7% ( Figure 1 B).

[0061] 1.3 Identification of strain SJD33

[0062] 1.3.1 Identification of colony morphology

[0063] The strain SJD33 was streaked on the LBA plate and cultured at 28°C for 24 h. The colony morphology was observed. Figure 2 The results showed that the single colony of the strain was round, translucent, smooth, and milky white. After continued cultivation for 48 hours, the surface of the colony was flat and formed obvious white wrinkles.

[0064] 1.3.2 Physiological and biochemical identification

[0065] Refer to the "Common Bacterial System Identification Manual" and "Berger's Manual of Systematic Bacteriology" for physiological and biochemical determinations. The culture method of the strain SJD33 bacterial suspension in the following experiment is: activate the strain SJD33 in LB medium, transfer to fresh LB medium, and culture at 28°C and 180rpm until the OD 600 =0.6 for standby use.

[0066] (1) Catalase test

[0067] 300 μL of the test strain SJD33 bacterial suspension was inoculated into 5 mL of fresh LB medium and cultured at 28°C for 2 days. 300 μL of 10 (v / v)% hydrogen peroxide solution was added to one tube to observe whether bubbles were generated. If a large number of bubbles were generated, it was a positive reaction.

[0068] (2) Starch hydrolysis test

[0069] From the center of the starch hydrolysis culture plate, divide it into 4 points at equal distances in 4 directions. Pipette 5 μL of the test strain SJD33 bacterial suspension and inoculate it into two of the points respectively. Inoculate the other two points with an equal volume of sterile water as a control. Repeat 3 times and culture at a constant temperature of 28℃ for 3 days. Add iodine solution. If a transparent circle is produced, it is a positive reaction.

[0070] (3) Casein hydrolysis test

[0071] The bacterial suspension of the test strain SJD33 was inoculated on a plate containing casein hydrolyzed medium. Two points were selected from the four points at equal distances, and 5 μL of the bacterial suspension of strain SJD33 was inoculated at each point. An equal volume of sterile water was used as a control and inoculated at the other two points. The test was repeated 3 times and cultured at a constant temperature of 28°C for 3 days. A positive reaction was considered if a transparent circle was produced around the colony of strain SJD33.

[0072] (4) Cellulose hydrolysis test

[0073] The strain SJD33 suspension to be tested was inoculated onto a cellulose hydrolysis medium plate, and the operation was the same as that of the casein hydrolysis test (3). The plate was cultured at 28°C for 3 days. If a transparent zone was formed around the colony of the strain SJD33, it was positive, otherwise it was negative.

[0074] (5) Protein hydrolysis test

[0075] Inoculate the test strain SJD33 suspension onto the protein hydrolysis medium plate. The operation is the same as (3) casein hydrolysis test. Incubate at 28°C for 3 days. If a transparent circle is produced, it is positive.

[0076] (6) Tween 80 hydrolysis test

[0077] Inoculate the test strain SJD33 suspension onto a Tween 80 hydrolysis culture plate. The operation is the same as (3) casein hydrolysis test. Culture at 28°C for 3 days. If a halo is produced, it is positive.

[0078] (7) Citrate hydrolysis test

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

[0080] (8) Acid production test

[0081] 600 μL of the test strain SJD33 bacterial suspension was inoculated into 10 mL of acid-producing culture medium, and a culture medium containing the same components but not inoculated with the strain SJD33 bacterial suspension was used as a control. The culture was cultured at 28°C and 5000 rpm for 2 days, and centrifuged at 5000 rpm for 10 min. 5 mL of the supernatant was taken into a sterilized test tube, and 2 to 3 drops of 0.04 wt.% bromocresol purple solution were added. 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 strain SJD33 are shown in Tables 1 and Figure 3 The results showed that strain SJD33 could decompose maltose, mannitol and fructose to produce acid, and could hydrolyze protein, casein, starch and citrate.

[0083] Table 1 Physiological and biochemical test results of strain SJD33

[0084] project result project result Catalase + Citrate Utilization + Amylase test + Maltose Utilization + Casein Utilization + Xylose Utilization - Cellulose Utilization - Sucrose Utilization - Protease detection + Mannitol Utilization + Tween 80 Utilization - Fructose Utilization +

[0085] 1.3.3 Molecular sequence identification

[0086] The 16S rDNA and gyrA genes of the strains were amplified by PCR and compared with NCBI. All primer synthesis and gene sequencing were completed by Sangon Biotech (Shanghai) Co., Ltd.

[0087] The total DNA of strain SJD33 was extracted according to the Ezup column bacterial genomic DNA extraction kit (SK8255), and the 16S rDNA and gyrA gene of strain SJD33 were amplified using 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), respectively. The 25.0μL PCR reaction system included: 10×PCRBuffer 2.0μL, primers 0.5μL each, Template DNA 2.0μL, dNTP 0.5μL, Taq Plus DNA Polymerase 0.5μL, and sterile water to 20μL. PCR reaction conditions: 95℃5min, 35 amplification cycles (95℃30S, 57℃30S, 72℃30S), 72℃8min. The sequencing results were spliced ​​using VectorNTIAdvance11.5 software, and the spliced ​​sequences were compared by BLAST at NCBI. It was found that the homology between strain SJD33 and Bacillus sp. was as high as 99.87%. The 16S rDNA sequence ( Figure 4 A) and gyrA( Figure 4 B) Phylogenetic tree of the sequences in , the calibration value was set to 1000, and the results showed that strain SJD33 was most closely related to Bacillus velez.

[0088] The results of comprehensive morphological, physiological and biochemical tests and molecular identification indicate that strain SJD33 is Bacillus velezensis of the genus Bacillus, and is classified and named Bacillus velezensis. The present invention names strain SJD33 as Bacillus velezensis SJD33, which was deposited in the General Microbiological Center (CGMCC) of the China Microbiological Culture Collection Committee on December 29, 2023, and the deposit address is the Institute of Microbiology, Chinese Academy of Sciences, No. 3, No. 1 Beichen West Road, Chaoyang District, Beijing, and the deposit number is CGMCC No. 29456.

[0089] Example 3 Control effect of Bacillus Velezii SJD33 on common pathogenic fungi of pear, apple, plum and pomegranate fruits

[0090] The plate confrontation method was used to determine the antagonistic effects of Bacillus Velez SJD33 on eight common fruit pathogenic fungi, including pear black spot, pear gray mold, pear penicillium, pear anthracnose, pear Atai fruit rot, apple core mildew, plum brown rot and pomegranate dry rot. The specific steps were as follows: 8 pathogenic fungi cakes with a diameter of 4 mm were inoculated onto fresh PDA culture plates with a diameter of 90 mm, and cultured at a constant temperature of 28°C. When the diameter of the test pathogenic fungi colonies grew to 2 / 3 of the diameter of the PDA culture plate, a punch was used to punch out 4 mm diameter cakes on the edges of the eight pathogenic fungi, and they were placed in the center of the fresh PDA plates respectively. Then, the colonies of Bacillus Velez SJD33 cultured on the LBA plate for 24 h were picked and streaked on both sides of the pathogenic fungi cakes at a distance of 3 cm from the cakes, and cultured at a constant temperature of 28°C, which were recorded as the treatment group. Each treatment was repeated 3 times, and the PDA plate without inoculation of Bacillus Velezii SJD33 was used as the control group (Control). When the colonies of the control group filled the plate, the colony diameters of the control group and the treatment group were observed and recorded, and the mycelial growth inhibition rate was calculated. Figure 5 As shown in the results, Bacillus Velez SJD33 had significant antagonistic effects on 8 common fruit disease fungi except pear ring rot pathogen, and the inhibition rate of pathogenic fungi reached more than 44%, among which the inhibition rate of mycelial growth of apple core pathogen was nearly 80% (Table 2), which indicated that Bacillus Velez SJD33 had a broad antibacterial spectrum.

[0091] Table 2 Inhibition rate of Bacillus Velezii SJD33 against 8 common fruit pathogens (fruit postharvest pathogens)

[0092]

[0093]

[0094] Note: SJD33 is Bacillus Velezii SJD33; the values ​​in the table are mean ± standard deviation, * represents P < 0.01

[0095] Example 4 In vivo control effect of Bacillus Velez SJD33 on common postharvest diseases of pear fruit

[0096] Huangguan pear was used as the test material to test the in vivo efficacy of Bacillus Velez on postharvest pear ring rot, penicillium and black spot. The specific method is as follows: first activate Bacillus Velez SJD33 on fresh LBA medium for 24 hours, pick a single colony and transfer it to 150mL fresh LB medium, culture at 150rpm and 28℃ for 48 hours, and adjust the spore suspension to 1.0×10 9cfu / 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 hole puncher to create a 4mm (diameter) × 3mm (depth) wound on both sides 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 fungus, pear black spot fungus 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 in the results, the spore suspension of Velez Bacillus SJD33 can significantly inhibit the expansion of the lesions of the three pathogenic fungi tested on the fruit. The control effect on the ring rot fungus was the highest at 7 days after inoculation, which could reach 82.09%. The control effect on the ring rot fungus at 11 days after inoculation could still reach 53.44%. At 11 days after inoculation, the control effect of Velez Bacillus SJD33 on Penicillium could reach 46.48%. At 15 days after inoculation, the control effect on the black spot fungus reached 56.68%. The results showed that Velez Bacillus SJD33 had significant control effects on the three pathogenic fungi tested, but had the best control effect on the living body of pear ring rot fungus.

[0097] Example 5 Control effect of Bacillus Velezii SJD33 on ring rot and anthracnose of three-season pears during shelf life

[0098] Three-season pears are prone to ring rot and anthracnose during the shelf life, causing great 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 4, with sterile water treatment as control, and the lesions were measured regularly. The results are as follows: Figure 7 The results showed that after inoculation with the two pathogens, the expansion of the lesions in the treatment group was significantly inhibited. Among them, on the 7th day after inoculation with pear ring rot, the control effect of Bacillus Velezii SJD33 on pear ring rot during the shelf life of three-season pears reached 91.31%; on the 7th day after inoculation with pear anthracnose, the control effect on pear anthracnose reached 52.82%.

[0099] Example 6 Bacillus Velezii SJD33 can reduce the natural decay rate of pear fruit

[0100] The three-season pear fruits were used as the test material. They were harvested when they reached commercial maturity. The fruits with intact surface, no mechanical damage, and no diseases or insect pests were selected. The fruits were treated with spore suspension of Bacillus Velezii SJD33 (1×10 8cfu / mL) were soaked for 30S as the treatment group, and the three-season pear fruits soaked in sterile water were used as the control group (CK). After treatment, they were placed in a plastic box covered with moist filter paper, sealed with plastic wrap, and placed in a moisturizing culture at 20℃. Each replication had 10 fruits, and each treatment was repeated 3 times. On the 12th day, the rottenness of the pears in the control group and the treatment group was recorded, and the rottenness rate of the pears was calculated. The calculation formula of the rottenness rate is: rottenness rate (%) = actual number of rotten fruits / total number of pears × 100. The survey results are shown in Figure 8 The results showed that on the 12th day, the rot rate of pear fruit in the control group was 70%, and the rot rate of pear fruit in the treatment group was 26.67%. The treatment group significantly reduced the natural rot rate of pear fruit during the shelf life. It can be seen that Bacillus Velezii SJD33 can significantly reduce the natural rot rate of pear fruit.

[0101] Example 7 Colonization of Bacillus Velezii SJD33 in Pear Fruit Wounds

[0102] The pear fruit was disinfected and wounded according to the method of Example 4, and 30 μL of Velezella SJD33 spore suspension (1×10 8 cfu / mL), the treated pear fruits were placed in a plastic box containing moist filter paper, sealed with plastic wrap, and cultured at 20°C. On 0d (3h), 1d, 2d, 3d, 4d, 5d, 6d, 7d, 8d, and 9d after inoculation, approximately 0.5g of pulp from the wound and the surrounding area was taken using an 8mm puncher, put into a mortar, added with an equal amount of sterile water, and ground, and gradient dilution was performed. 100μL of the dilution was evenly applied on the LBA medium plate, cultured at 28°C for 2d, and the number of clones was calculated. The results are shown in the figure. Fig. 9 The results showed that the number of Velez Bacillus SJD33 inoculated in the wound of pear fruit for 3 hours was 2.35×10 5 cfu / mL, and increased to 1.13×10 6 cfu / mL, and the number remained at 1.11×10 6 cfu / mL, indicating that Bacillus Velezii SJD33 had successfully colonized the pear fruit wounds.

[0103] Example 8 Effect of Bacillus Velez SJD33 on Pear Fruit Quality

[0104] The three-season pear fruits were harvested when they reached commercial maturity. The fruits with intact surface, no mechanical damage, and no pests and diseases were selected and spore suspension of Bacillus Velezii SJD33 (1×10 8cfu / mL) for 30 seconds. The three-season pear fruits soaked in sterile water were used as the control group (CK). After treatment, they were stored in a constant temperature box at 20℃. The quality indicators (fruit firmness, soluble solids, titratable acid and L-ascorbic acid (vitamin C)) were measured every two days. 15 fruits were tested for each treatment each time. The results are shown in the figure. Fig.10 The results showed that the treatment with Bacillus Velez SJD33 had no negative impact on the fruit quality of Pyrus tricolor, and could delay the decrease of pear fruit firmness and L-ascorbic acid. The fruit firmness and L-ascorbic acid of pear fruit treated with Bacillus Velez SJD33 were higher than those of the control group from the 2nd to the 6th day, and the soluble solids and titratable acid did not change significantly during the observation period.

[0105] Example 9 Identification of antibacterial activity of sterile fermentation broth of Bacillus velez SJD33

[0106] The Oxford cup method was used to determine the antibacterial activity of the sterile fermentation broth of Bacillus Velez subtilis SJD33. Bacillus Velez subtilis SJD33 was streaked and activated on an LBA plate, and a single colony was picked in a triangular flask containing 50 mL of LB medium. It was cultured overnight at 180 rpm and 28°C, and inoculated into a triangular flask containing fresh LB medium at a ratio of 1% (v / v). It was cultured at 180 rpm and 28°C for 24 hours, centrifuged at 8000 rpm for 20 minutes, and the supernatant was taken and filtered with a 0.2 μm sterile filter membrane to obtain the sterile fermentation broth of Bacillus Velez subtilis SJD33. 100 μL of the sterile fermentation broth of Bacillus Velez subtilis SJD33 was added to the Oxford cup on the PDA plate, and the cultured pear ring rot fungus cake was punched out with a hole punch and placed on the same PDA plate, which was recorded as the treatment group; the control group was added with sterile water. Afterwards, the changes in the mycelial morphology of the treatment group and the control group were observed under an optical microscope. The results are as follows Fig.11 The results showed that an obvious antibacterial zone was formed around the Oxford cup with the sterile fermentation liquid of Bacillus Velez SJD33, and the hyphae at the edge of the inhibition zone were distorted. It can be seen that the sterile fermentation liquid of Bacillus Velez SJD33 also has antibacterial activity.

[0107] Example 10 Evaluation of stability of cell-free fermentation broth of Bacillus velez SJD33

[0108] Bacillus Velez SJD33 was streaked and activated on an LBA plate, and a single colony was picked and placed in a triangular flask containing 100 mL of LB liquid culture medium, and cultured at 180 rpm and 28°C for 48 hours. The obtained fermentation liquid was then centrifuged at 8000 rpm for 20 minutes. The supernatant was filtered with a 0.22 μm filter membrane to obtain a cell-free fermentation liquid, which was the cell-free fermentation liquid of Bacillus Velez SJD33. Using Pear Ring Spot Pathogen as the test pathogen, the plate method was used to determine the stability of the antagonistic activity of the fermentation liquid of Bacillus Velez SJD33 after ultraviolet treatment, heat treatment, acid-base treatment, metal ion and protease treatment. The results are as follows: Fig.12 shown.

[0109] Ultraviolet treatment: In a sterile environment, the cell-free fermentation broth of Bacillus Velez subtilis SJD33 was irradiated with a 30W ultraviolet lamp for 10, 20, 30, 40, 50, 60, 90, and 120 minutes, respectively. The antagonistic activity of the cell-free fermentation broth of Bacillus Velez subtilis SJD33 was determined by the plate method with the non-irradiated cell-free fermentation broth of Bacillus Velez subtilis SJD33 as the control. The results are shown in Fig.12 As shown in A. The results showed that the antagonistic activity of the fermentation broth of Bacillus velez SJD33 was not significantly affected by ultraviolet irradiation for 2 hours.

[0110] Heat treatment: The cell-free fermentation broth of Bacillus Velez SJD33 was incubated at 40°C, 50°C, 60°C, 70°C, 80°C, 90°C, and 100°C for 20 min, respectively. The untreated cell-free fermentation broth of Bacillus Velez SJD33 was used as the control group. The changes in the antibacterial activity of the cell-free fermentation broth of Bacillus Velez SJD33 were detected by the plate method. The results are as follows: Fig.12 The results showed that the antagonistic activity of the cell-free fermentation broth of Bacillus velez SJD33 after heat treatment was not significantly affected compared with the control.

[0111] Protease treatment: pepsin, trypsin and proteinase K were prepared into 1 mg / mL solutions respectively. A certain amount of the above enzyme solutions were added to the cell-free fermentation broth of Bacillus Velez subtilis SJD33 to make the final enzyme concentration of 0.1 mol / L. The reaction was carried out at 37°C for 1 hour. After treatment at 80°C for 30 minutes, it was immediately cooled on ice and the changes in its antagonistic activity were determined by the plate method. The untreated cell-free fermentation broth of Bacillus Velez subtilis SJD33 was used as the control group. The results are as follows: Fig.12 As shown in C. The results showed that the antagonistic activity of the cell-free fermentation broth of Bacillus velez SJD33 treated with proteinase K, trypsin and pepsin was not significantly different from that of the control group.

[0112] Acid-base treatment: The pH of the cell-free fermentation broth of Bacillus Velez SJD33 was adjusted to 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13 with 1 mol / L HCl or NaOH, and after 24 hours of rest, the cell-free fermentation broth of Bacillus Velez SJD33 with pH 7 was used as the control, and the changes in the antagonistic activity of the cell-free fermentation broth of Bacillus Velez SJD33 after treatment were detected by plate method. The results are shown in the figure. Fig.12 The results showed that the fermentation filtrate of Bacillus Velezii SJD33 had strong antagonistic activity at pH 2-10. When the pH was 2-9, the antagonistic activity of the fermentation broth had no significant difference from that of the control. When the pH was 10, its antagonistic activity against pathogenic fungi still reached more than 57%.

[0113] Metal ion treatment: CaCl 2 、ZnS0 4 7H 2 O、CuS0 4 ·5H 2 O, NaCl, MgSO 4 7H 2 O、MnCl 2 ·4H 2 O、FeS0 4 7H 2 O and KCl were prepared into 1 mol / L solution, 100 μL of the above metal ion solution was added to 1000 μL of Velez SJD33 cell-free fermentation broth to make the final concentration of metal ions 0.1 mol / L, the mixture was shaken and mixed, and the mixture was treated in a 37°C water bath for 1 h. The effects of different metal ions on the antagonistic activity of Velez SJD33 cell-free fermentation broth were determined by plate method. The untreated Velez SJD33 cell-free fermentation broth was used as the control group. The results are shown in Fig.12 As shown in E. The results showed that compared with the control, except for iron ions, other metal ion treatments did not affect the antagonistic activity of the cell-free fermentation broth of Bacillus Velez SJD33 against pathogenic fungi; the antagonistic activity of the cell-free fermentation broth of Bacillus Velez SJD33 treated with iron ions against pathogenic fungi could still reach more than 50%.

[0114] Example 11 Extraction and antibacterial effect of crude extract of antibacterial metabolites of Bacillus velez SJD33

[0115] The acid precipitation method was used to extract the antibacterial metabolites of Bacillus Velez subsp. SJD33. The specific steps were as follows: Bacillus Velez subsp. SJD33 was streaked and activated on an LBA plate, a single colony was picked and placed in a conical flask containing 100 mL of LB liquid culture medium, cultured at 180 r / min and 28°C for 72 h, centrifuged at 8000 rpm for 30 min, the supernatant was collected, and the pH value of the supernatant was adjusted to 2.0 with HCl, placed in a 4°C refrigerator overnight, centrifuged again at 8000 rpm for 30 min, the supernatant was discarded, the precipitate was collected, the precipitate was dried at 50°C, and dissolved with 2 mL of methanol, filtered with a 0.22 μm filter membrane, and the crude extract of the antibacterial metabolites of Bacillus Velez subsp. SJD33 was obtained, referred to as SJD33 crude extract. The antagonistic activity of the crude extract of the antibacterial metabolites of Bacillus Velez subsp. SJD33 against pathogenic fungi (pear ring spot pathogen) was detected by the Oxford cup method, with water as the control. The results are as follows Fig.13 The results showed that the crude extract of antimicrobial metabolites of Bacillus Velezii SJD33 had a significant inhibitory effect on pathogenic fungi.

[0116] 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 broad-spectrum antagonistic bacterium Bacillus velezensis SJD33, characterized in that: The deposit number of the Bacillus Velez SJD33 is CGMCC No.29456.

2. The use of the Bacillus Velezii SJD33 according to claim 1 in the preparation of a microbial agent for resisting fungal diseases, characterized in that: The fungal diseases include one or more of pear ring rot, pear black spot, pear gray mold, pear blue mold, pear anthracnose, pear Ateia fruit rot, apple rot, plum brown rot and pomegranate dry rot.

3. A microbial agent for resisting fungal diseases, characterized in that: The microbial agent includes the Bacillus Velez SJD33 described in claim 1; the fungal diseases include one or more of pear ring rot, pear black spot, pear gray mold, pear penicillium, pear anthracnose, pear Athea fruit rot, apple core rot, plum brown rot and pomegranate dry rot.

4. Use of the Bacillus Velez SJD33 according to claim 1 or the microbial agent according to claim 3 in preventing and controlling fungal diseases, characterized in that: The fungal diseases include one or more of pear ring rot, pear black spot, pear gray mold, pear blue mold, pear anthracnose, pear Ateia fruit rot, apple rot, plum brown rot and pomegranate dry rot.

5. A method for preventing and controlling fungal diseases, characterized in that: The method comprises the step of treating the fruit to be treated with the microbial agent described in claim 3; the fungal diseases comprise one or more of pear ring rot, pear black spot, pear gray mold, pear penicillium, pear anthracnose, pear Athea fruit rot, apple core rot, plum brown rot and pomegranate dry rot.

6. Use of the Bacillus Velez SJD33 described in claim 1 in the preparation of a microbial agent for reducing fruit rot rate and / or improving fruit quality.

7. The use according to claim 6, characterized in that: The improved fruit quality includes delaying the reduction of fruit firmness and L-ascorbic acid content.

8. A microbial agent for reducing fruit decay rate and / or improving fruit quality, characterized in that: The microbial agent includes the Bacillus Velezii SJD33 described in claim 1.

9. Use of the Bacillus Velez SJD33 described in claim 1 or the microbial agent described in claim 8 in reducing the fruit rot rate and / or improving the fruit quality.

10. 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 8.

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