Bacillus velezensis and application thereof

Abstract

The invention discloses bacillus velezensis and application thereof, the bacillus velezensis is bacillus velezensis BVZ0942, the preservation number of the bacillus velezensis BVZ0942 is CCTCC NO: M 20231560, the preservation time is August 31, 2023, and the bacillus velezensis BVZ0942 is a new strain and is preserved. Experiments prove that the strain BVZ0942 has an inhibiting effect on various pathogenic bacteria, so that the strain BVZ0942 can be used as a biocontrol bacterium to be applied to a biocontrol bacterium agent; the strain can also be prepared into a bacteriostatic agent for inhibiting phytopathogen. The strain can also be prepared into a disease-resistant agent for resisting phytopathogen; a sterile metabolite produced by the strain can prevent and treat plant diseases; a sterilization metabolite produced by the strain can be used for preventing and treating plant diseases; volatile substances generated by the strain can prevent and treat plant diseases; volatile substances generated by the strain are prepared into biopesticide which can prevent and control plant diseases.

Description

Technical Field

[0001] The present application relates to the technical fields of microbiology, biotechnology and biological control, and in particular to a strain of Bacillus Velezii and its application. Background Art

[0002] Bacillus is a type of aerobic or facultative anaerobic Gram-positive bacteria that can produce spores. The bacteria are straight rod-shaped and arranged in pairs or chains. Bacillus is widely found in soil, water, air, etc. The genus Bacillus can be divided into the following subgroups: Bacillus polymyxa, Bacillus subtilis (including Bacillus cereus and Bacillus licheniformis), Bacillus brevis and Bacillus anthracis.

[0003] In the study of plant disease control, Bacillus has high safety evaluation, good control effect, and is easy to make into preparations. Studies have shown that the main types of Bacillus suitable for pathogen control are: Bacillus subtilis, Bacillus amyloliquefaciens, Bacillus methylotrophicus, Bacillus licheniformis, Bacillus pumilus, Bacillus megeterium, and Bacillus velezensis. At present, it is difficult to discover new species in the genus Bacillus, and it is even more difficult to discover new species with citation value. Summary of the invention

[0004] In view of this, the present application provides Bacillus Velez and its application, aiming to improve the problem that it is difficult to discover new species in the existing Bacillus genus, and it is even more difficult to discover new species with citation value.

[0005] The embodiment of the present application is implemented as follows: a strain of Bacillus velezensis, the Bacillus velezensis (Bacillus velezensis) BVZ0942, is deposited in the China Center for Type Culture Collection, with a deposit number of CCTCC NO: M20231560, and a deposit date of August 31, 2023.

[0006] Optionally, in some embodiments of the present application, the 16SrDNA sequence of the Bacillus velezensis BVZ0942 is as shown in the sequence listing SEQ ID No: 1.

[0007] Optionally, in some embodiments of the present application, the Bacillus Velezii BVZ0942 is from fish pond mud.

[0008] Correspondingly, the embodiments of the present application also provide the use of the Bacillus Velez in the preparation of a biocontrol agent, wherein the biocontrol agent is a biocontrol agent for strawberry gray mold (Botrytis cinerea), a biocontrol agent for pepper root rot (Fusarium solani), a biocontrol agent for kiwifruit anthracnose (Colletotrichum sp.) or a biocontrol agent for rapeseed sclerotinia (Sclerotinia sclerotiorum).

[0009] Correspondingly, the embodiments of the present application also provide the use of the Bacillus Velez in the preparation of an antibacterial agent, which is a bacterial agent for inhibiting strawberry gray mold (Botrytis cinerea), pepper root rot (Fusarium solani), kiwi anthracnose (Colletotrichum sp.) or rapeseed sclerotinia (Sclerotinia sclerotiorum).

[0010] Correspondingly, the embodiments of the present application also provide the use of the Bacillus Velez in the preparation of a plant disease-resistant agent, wherein the disease-resistant agent is a disease-resistant agent for plant diseases caused by strawberry gray mold (Botrytis cinerea), pepper root rot (Fusarium solani), kiwifruit anthracnose (Colletotrichum sp.) or rapeseed sclerotinia (Sclerotinia sclerotiorum).

[0011] Accordingly, the embodiments of the present application also provide the use of the sterile metabolites produced by the Bacillus Velez in preventing and controlling plant diseases, wherein the plant diseases are plant diseases caused by strawberry gray mold (Botrytis cinerea), pepper root rot (Fusarium solani), kiwifruit anthracnose (Colletotrichum sp.) or rapeseed sclerotinia (Sclerotinia sclerotiorum).

[0012] Accordingly, the embodiments of the present application also provide the use of the sterilized metabolites produced by the Bacillus Velez in preventing and controlling plant diseases, wherein the plant diseases are plant diseases caused by strawberry gray mold (Botrytis cinerea), pepper root rot (Fusarium solani), kiwifruit anthracnose (Colletotrichum sp.) or rapeseed sclerotinia (Sclerotinia sclerotiorum).

[0013] Correspondingly, the embodiments of the present application also provide the use of the volatile substances produced by the Bacillus Velez in preventing and controlling plant diseases, wherein the plant diseases are plant diseases caused by strawberry gray mold (Botrytis cinerea), pepper root rot (Fusarium solani), kiwifruit anthracnose (Colletotrichum sp.) or rapeseed sclerotinia (Sclerotinia sclerotiorum).

[0014] Accordingly, the embodiments of the present application also provide the use of the volatile substances produced by the Bacillus Velez in the preparation of biological pesticides for preventing and controlling plant diseases, wherein the plant diseases are plant diseases caused by strawberry gray mold (Botrytis cinerea), pepper root rot (Fusarium solani), kiwifruit anthracnose (Colletotrichum sp.) or rapeseed sclerotinia (Sclerotinia sclerotiorum).

[0015] In this application: Bacillus velezensis BVZ0942, deposit number CCTCC NO:M 20231560, deposit time: August 31, 2023, deposited in China Center for Type Culture Collection, deposit location: Wuhan University, Wuhan, China.

[0016] LB liquid medium: 5 g yeast extract, 10 g tryptone, 10 g sodium chloride, 1 L deionized water, pH 7.0, sterilized by high-pressure steam at 121°C for 30 min.

[0017] LB solid medium: 10 g tryptone, 10 g sodium chloride, 5 g yeast extract, 20 g agar, 1 L deionized water, pH 7.0, 121°C high pressure steam sterilization for 30 min.

[0018] PDA medium: 200 g potato (peeled), 20 g agar, 20 g glucose, 1 L deionized water, pH 7.0, high pressure steam sterilization at 121°C for 30 min.

[0019] NA medium: 5 g beef extract, 10 g tryptone, 5 g yeast extract, 10 g sodium chloride, 20 g agar, 1 L deionized water, pH 7.0, high-pressure steam sterilization at 121°C for 30 min.

[0020] In the present application, strawberry gray mold (Botrytis cinerea), pepper root rot (Fusarium solani), kiwi anthracnose (Colletotrichum sp.) and rapeseed sclerotinia (Sclerotiniasclerotiorum) are all existing strains, which are preserved in the Biological Control and Rapeseed Disease Laboratory of the Institute of Plant Protection, Sichuan Academy of Agricultural Sciences.

[0021] In this application,

[0022] The present application separates, purifies and screens strain BVZ0942 from fish pond sediment, identifies it as Bacillus velezensis through morphological identification, molecular biological identification and physiological and biochemical characteristics research, and preserves it as a new strain. The experiment verified that the strain BVZ0942 has an inhibitory effect on strawberry gray mold (Botrytis cinerea), pepper root rot (Fusarium solani), kiwi anthracnose (Colletotrichum sp.) and rapeseed sclerotinia (Sclerotinia sclerotiorum), so it can be used as a biocontrol bacterium in biocontrol agents; the strain BVZ0942 can also inhibit plant pathogens, and can be prepared into an antibacterial agent for inhibiting plant pathogens; the strain BVZ0942 can also resist plant pathogens, and can be prepared into an antidisease agent for resisting plant pathogens; the sterile metabolites produced by the strain BVZ0942 can prevent and control plant diseases; the sterilized metabolites produced by the strain BVZ0942 can prevent and control plant diseases; the volatile substances produced by the strain BVZ0942 can prevent and control plant diseases; the volatile substances produced by the strain BVZ0942 are prepared into biological pesticides to prevent and control plant diseases. BRIEF DESCRIPTION OF THE DRAWINGS

[0023] In order to more clearly illustrate the technical solutions in the embodiments of the present application, the drawings required for use in the description of the embodiments will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present application. For those skilled in the art, other drawings can be obtained based on these drawings without paying any creative work.

[0024] Figure 1 It is a growth state diagram of the strain BVZ0942 of the present application on three plates;

[0025] Figure 2 It is the electrophoresis band diagram of the strain BVZ0942 of the present application;

[0026] Figure 3 is the phylogenetic tree of strain BVZ0942 of the present application;

[0027] Figure 4 This is the antibacterial spectrum effect diagram of the strain BVZ0942 of the present application;

[0028] Figure 5 It is a schematic diagram of the double-layer flat plate confrontation of this application;

[0029] Figure 6 This is a diagram showing the effect of the volatile substances of the strain BVZ0942 of the present application on inhibiting the mycelial growth of Sclerotinia sclerotiorum;

[0030] Figure 7 This is a diagram showing the effect of the sterile filtrate of the strain BVZ0942 of the present application on inhibiting the mycelial growth of Sclerotinia sclerotiorum;

[0031] Figure 8 This is a diagram showing the effect of the sterilized metabolites of the strain BVZ0942 of the present application on inhibiting the growth of mycelium of Sclerotinia sclerotiorum. DETAILED DESCRIPTION

[0032] The technical solutions in the embodiments of the present application will be clearly and completely described below in conjunction with the drawings in the embodiments of the present application. Obviously, the described embodiments are only part of the embodiments of the present application, rather than all of the embodiments. Based on the embodiments in the present application, all other embodiments obtained by those skilled in the art without making creative work belong to the scope of protection of the present application. In addition, it should be understood that the specific implementation methods described herein are only used to illustrate and explain the present application, and are not used to limit the present application.

[0033] In this application, "and / or" describes the association relationship of associated objects, indicating that there may be three relationships. For example, A and / or B can represent: A exists alone, A and B exist at the same time, and B exists alone. A and B can be singular or plural.

[0034] In the present application, "at least one" means one or more, and "plurality" means two or more. "One or several", "at least one of the following" or similar expressions refer to any combination of these items, including any combination of single items or plural items. For example, "at least one of a, b, or c", or "at least one of a, b, and c" can all mean: a, b, c, ab (i.e., a and b), ac, bc, or abc, where a, b, c can be single or multiple, respectively.

[0035] Various embodiments of the present application may be presented in the form of a range; it should be understood that the description in the form of a range is only for convenience and brevity, and should not be understood as a rigid limitation on the scope of the present application; therefore, the range description should be considered to have specifically disclosed all possible sub-ranges and single numerical values ​​within the range. For example, the range description from 1 to 6 should be considered to have specifically disclosed sub-ranges, such as from 1 to 3, from 1 to 4, from 1 to 5, from 2 to 4, from 2 to 6, from 3 to 6, etc., as well as single numbers within the range, such as 1, 2, 3, 4, 5 and 6, which apply regardless of the range. In addition, whenever a numerical range is indicated herein, it is meant to include any cited number (fractional or integer) within the indicated range.

[0036] The technical solution of this application is as follows:

[0037] See also Figure 1 , the present application example provides a strain of Bacillus velezensis, the Bacillus velezensis (Bacillus velezensis) BVZ0942, deposited in the China Center for Type Culture Collection, with a deposit number of CCTCC NO: M20231560, and a deposit date of August 31, 2023.

[0038] In the present application, strain BVZ0942 was isolated, purified and screened from fish pond sediment, and identified as Bacillus velezensis through morphological identification, molecular biological identification and physiological and biochemical characteristics research, which was a new strain and was preserved. The experiment verified that the strain BVZ0942 has an inhibitory effect on strawberry gray mold (Botrytis cinerea), pepper root rot (Fusarium solani), kiwi anthracnose (Colletotrichum sp.) and rapeseed sclerotinia (Sclerotinia sclerotiorum), so it can be used as a biocontrol bacterium in biocontrol agents; the strain BVZ0942 can also inhibit plant pathogens, and can be prepared into an antibacterial agent for inhibiting plant pathogens; the strain BVZ0942 can also resist plant pathogens, and can be prepared into an antidisease agent for resisting plant pathogens; the sterile metabolites produced by the strain BVZ0942 can prevent and control plant diseases; the sterilized metabolites produced by the strain BVZ0942 can prevent and control plant diseases; the volatile substances produced by the strain BVZ0942 can prevent and control plant diseases; the volatile substances produced by the strain BVZ0942 are prepared into biological pesticides to prevent and control plant diseases.

[0039] It can be understood that BVZ0942 is the strain number of the Bacillus sp.

[0040] In some embodiments, the 16S rDNA sequence of the Bacillus velezinii BVZ0942 is shown in the sequence listing SEQ ID No: 1.

[0041] In some embodiments, the Bacillus Velez subtilis BVZ0942 comes from fish pond mud, the sampling site is Qushui Fish Pond, Gonghe Road, Xindu District, Chengdu City, Sichuan Province, and the sampler is Yu Yaoying.

[0042] In some embodiments, the Bacillus Velezii is used in the preparation of a biocontrol agent.

[0043] Furthermore, the biocontrol agent is a biocontrol agent for strawberry gray mold (Botrytis cinerea), a biocontrol agent for pepper root rot (Fusarium solani), a biocontrol agent for kiwifruit anthracnose (Colletotrichum sp.) or a biocontrol agent for rapeseed sclerotinia (Sclerotinia sclerotiorum).

[0044] In some embodiments, the Bacillus Velezii is used in the preparation of an antibacterial agent.

[0045] Furthermore, the antibacterial agent is a fungicide that inhibits strawberry gray mold (Botrytis cinerea), pepper root rot (Fusarium solani), kiwi anthracnose (Colletotrichum sp.) or rapeseed sclerotinia (Sclerotiniasclerotiorum).

[0046] In some embodiments, the Bacillus Velez is used in the preparation of a plant disease resistance agent.

[0047] Furthermore, the disease-resistant agent is an agent for resisting plant diseases caused by strawberry gray mold (Botrytis cinerea), pepper root rot (Fusarium solani), kiwifruit anthracnose (Colletotrichum sp.) or rapeseed sclerotinia (Sclerotinia sclerotiorum).

[0048] In some embodiments, the sterile metabolites produced by Bacillus Velez are used in preventing and controlling plant diseases.

[0049] Furthermore, the plant disease is a plant disease caused by Sclerotinia sclerotiorum.

[0050] In some embodiments, the sterilizing metabolites produced by Bacillus Velez are used in preventing and controlling plant diseases.

[0051] Furthermore, the plant disease is a plant disease caused by Sclerotinia sclerotiorum.

[0052] In some embodiments, the volatile substances produced by Bacillus Velez are used in preventing and controlling plant diseases.

[0053] Furthermore, the plant disease is a plant disease caused by Sclerotinia sclerotiorum.

[0054] In some embodiments, the volatile substances produced by the Bacillus Velez subtilis are used in the preparation of biological pesticides for controlling plant diseases.

[0055] Furthermore, the plant disease is a plant disease caused by Sclerotinia sclerotiorum.

[0056] Test example

[0057] Isolation, purification and screening of BVZ0942 strain

[0058] Take 10g of soil sample (from fish pond mud) and mix with 90mL of sterile water to dilute to 10 -1 , and then serially diluted to obtain 10 -3 , 10 -4 , 10 -5 , 10 -6 , 10 -7 , 10 -8 Use a pipette to take 400 μL of 10 -3 , 10 -4 , 10 -5 , 10 -6 , 10 -7 , 10 -8 The soil dilution was placed on the LB plate and evenly spread with a spreading rod. Three replicates were performed for each concentration, and the plates were cultured at 25°C for 72 hours. The morphology of the single colonies was observed, and the single colonies with different morphological characteristics were selected for isolation and purification, and numbered and stored.

[0059] Activation of pathogenic bacteria: Use a cork puncher to obtain the outer bacterial disc of the Sclerotinia sclerotiorum pathogen and inoculate it into the center of the PDA plate. Invert and culture at 20°C for later use.

[0060] Plate confrontation for initial and rescreening of antagonistic bacteria:

[0061] The activated fresh Sclerotinia sclerotiorum outer edge bacterial disc was obtained by punching with a cork puncher and inoculated in the center of the PDA plate. The different bacteria isolated above were streaked at 4 cm away from the Sclerotinia sclerotiorum bacterial disc, and the PDA plate inoculated with only the Sclerotinia sclerotiorum bacterial disc was used as a control. Three replicates were made for each group, and the culture was carried out at 25°C for 48 hours. The effective antagonistic bacteria BVZ0942 strain obtained by screening was purified and preserved. The antagonistic bacteria BVZ0942 strain was purified by the four-zone streaking method.

[0062] Strain preservation: Use a pipette to take 100 μL of the seed solution of the BVZ0942 strain after shaking culture and add it to a 2 mL centrifuge tube containing 1.5 mL of 30% glycerol, and store it at -80°C for later use.

[0063] Morphological identification

[0064] The BVZ0942 strain was streaked onto a PDA plate, an LB plate, and a NA plate at the same time and incubated upside down at 30°C for 48 h. The morphological characteristics of the BVZ0942 strain on the three different plates were observed and photographed for preservation.

[0065] See also Figure 1 , the strain BVZ0942 was streaked on PDA plates, LB plates and NA plates, respectively, and cultured at 30°C for 2 days to observe its morphological characteristics, such as Figure 1 As shown, the BVZ0942 strain grew well on the three plates. Figure 1 In the figure, a is the strain BVZ0942 streaked and grown on a PDA plate, b is the strain BVZ0942 streaked and grown on an LB plate, and c is the strain BVZ0942 streaked and grown on a NA plate. Figure 1 It can be seen that the colonies of strain BVZ0942 grown on the PDA plate have white serrated protrusions around them; the colonies of strain BVZ0942 grown on the LB plate also have a concave top in the center of the circle, and the bacteria are relatively dry; the colonies of strain BVZ0942 grown on the NA plate have more wrinkles, and picking up a small number of colonies with an inoculation needle will result in a wire-drawing phenomenon.

[0066] Molecular Biology Identification

[0067] Extract strain DNA: Extract the DNA of strain BVZ0942 according to the steps of the bacterial DNA extraction kit provided by Chengdu Fuji Biotechnology Co., Ltd.

[0068] Strain 16S rDNA sequence amplification: Universal primers (provided by Qingke Biotechnology Co., Ltd.): upstream primer 27F (5'-AGAGTTTGATCCTGGCTCAG-3'), downstream primer 1492R (5'-GGTTACCTTGTTACTT-3'). The DNA extraction product of strain BVZ0942 was PCR amplified and then subjected to agarose gel electrophoresis. The length of the electrophoresis product band was observed with DL 5000 Marker as a control, and the gel imager was used to take pictures and retain them. The PCR product was sent to Qingke Biotechnology Co., Ltd. for 16S rDNA sequencing. The PCR reaction system (20 μL) is shown in Table 1, and the PCR reaction procedure is shown in Table 2.

[0069] Table 1 PCR reaction system (20 μL)

[0070]

[0071] Table 2 PCR reaction program

[0072]

[0073] The 16S rDNA sequence of the Bacillus velezinii BVZ0942 is shown in the sequence table as SEQ ID No: 1.

[0074] SEQ ID No: 1 is

[0075]

[0076] Strain sequence comparison and analysis and phylogenetic tree construction: The 16SrDNA sequencing results of strain BVZ0942 were compared and analyzed on the official website of NCBI, and sequence homology was compared and the phylogenetic tree of strain BVZ0942 was constructed using MEGA 11.

[0077] 16S rDNA sequence analysis and phylogenetic tree construction

[0078] See also Figure 2 and Figure 3 ,like Figure 2 As shown in Figure 2, the electrophoresis band size of strain BVZ0942 was between 1000bp and 2000bp. The 16S rDNA sequencing results showed that the DNA fragment size of the strain was 1480bp. Figure 3 It can be seen that the homology similarity with Bacillus velezensis strain SH2 recorded in the NCBI database is the highest, reaching 99%.

[0079] Physiological and biochemical characteristics

[0080] With reference to the research methods of Bergey's Manual of Bacterial Identification and Manual of Identification of Common Bacterial Systems, the physiological and biochemical characteristics of strain BVZ0942 were studied through motility assay, catalase reaction test, sugar alcohol fermentation test, gelatin liquefaction test, VP reaction test, MP reaction test, starch hydrolysis test, citrate test, and nitrate reduction test.

[0081] The results of physiological and biochemical tests of strain BVZ0942 are shown in Table 3. From Table 3, it can be seen that strain BVZ0942 can decompose and utilize glucose, mannitol, etc., can hydrolyze starch and gelatin, motility and MP tests are negative, and VP test is positive.

[0082] Table 3 Physiological and biochemical test results

[0083]

[0084] In summary, based on the phylogenetic tree of strain BVZ0942 and combined with the above-mentioned colony morphological characteristics and physiological and biochemical properties, strain BVZ0942 was determined to be Bacillus velezensis.

[0085] Determination of antibacterial spectrum of strain BVZ0942

[0086] The strain BVZ0942 was streaked and inoculated 2 cm from the right edge of the PDA plate, and cultured at 30°C for 1 day. A hole puncher was used to obtain the outer edge of the pathogens (Botrytis cinerea, Fusarium solani, Colletotrichum sp., and Sclerotinia sclerotiorum) that were activated in advance and involved in the antibacterial spectrum experiment and inoculated at 2 cm from the left edge of the PDA plate. The PDA plate of the control group was only inoculated with pathogens, and the experiment was repeated 3 times for each group. Incubate at 25°C and stop culturing when the mycelium of the control group covers the entire PDA plate. Measure and record the colony diameter of the pathogen, and calculate the growth inhibition rate of the antagonistic strain BVZ0942 on different pathogens (see Table 4). Take photos and keep them. Figure 4 , Figure 4 Figure 1 shows the inhibition of strawberry gray mold by strain BVZ0942; Figure 2 shows the inhibition of pepper root rot by strain BVZ0942; Figure 3 shows the inhibition of kiwifruit anthracnose by strain BVZ0942; Figure CK1 shows the inoculation of strawberry gray mold only; Figure CK2 shows the inoculation of pepper root rot only; Figure CK3 shows the inoculation of kiwifruit anthracnose only; Figure 4 shows the inhibition of rapeseed sclerotinia by strain BVZ0942; Figure CK4 shows the inoculation of rapeseed sclerotinia only. Inhibition rate (%) = (average colony diameter of control group - average colony diameter of treatment group) / (average colony diameter of control group - bacterial plate diameter) × 100.

[0087] Table 4 Inhibition rate of the antibacterial spectrum of strain BVZ0942

[0088]

[0089] As shown in Table 4, strain BVZ0942 had an inhibitory effect on all the tested pathogens, and the inhibition rates against strawberry gray mold, pepper root rot, kiwi anthracnose and rapeseed sclerotinia were 38.42%, 23.08%, 12.57% and 46.70%, respectively.

[0090] Study on the antibacterial activity of volatile substances against bacteria

[0091] The strain can produce a special smell of gas when it is cultured. The double-layer plate confrontation culture method was used to explore the inhibitory effect of the strain on the mycelium of Sclerotinia sclerotiorum. The antagonistic strain BVZ0942 was placed in a constant temperature incubator at 30°C for 1 day in advance for activation and culture. Single colonies of the activated strain BVZ0942 were picked and inoculated into LB liquid culture medium after high temperature sterilization. The culture was carried out at 30°C and 180r·min. -1The fermentation broth of strain BVZ0942 was obtained by shaking culture for 1 day. -1 , 10 -2 , 10 -3 , 10 -4 The dilution was evenly spread on plate 1 containing 20 mL of LB solid medium and cultured at 35°C for 1 day. Fresh sclerotinia sclerotiorum with a diameter of 0.5 cm was inoculated in the center of plate 2 of the same specifications containing 20 mL of PDA medium, and blank LB medium was used as a control. Figure 5 After removing the lids of plate 1 and plate 2, plate 2 was placed upside down on top of plate 1, and the two plates were sealed with sealing film. Three groups of repeated experiments were set for each concentration, and the culture was carried out at 25℃ for 2 days. The diameter of the colony of Sclerotinia sclerotiorum was measured by the cross method. Table 5 shows that the fermentation seed solution of strain BVZ0942 and the 10 -1 , 10 -2 , 10 -3 , 10 -4 The inhibition rates of volatile substances produced by the dilutions on Sclerotinia sclerotiorum were 64.89%, 66.67%, 61.56%, 59.78% and 87.11% respectively, indicating that the volatile substances of strain BVZ0942 can effectively inhibit the growth of Sclerotinia sclerotiorum hyphae, but its inhibition rate has no significant correlation with the dilution multiple of the seed solution. Figure 6 ,from Figure 6 It can be seen that the volatile substances of strain BVZ0942 can effectively inhibit the growth of mycelium of Sclerotinia sclerotiorum, among which, Figure 6 The stock solution in the sample is the fermentation seed solution of strain BVZ0942.

[0092] Table 5 Effects of volatile substances of strain BVZ0942 on the mycelial growth of Sclerotinia sclerotiorum

[0093]

[0094] Study on the antibacterial activity of sterile filtrate of biocontrol bacteria

[0095] The antagonistic strain BVZ0942 was placed in a constant temperature incubator at 30°C for 1 day for activation culture. Single colonies of the activated strain BVZ0942 were picked and inoculated into LB liquid medium after high temperature sterilization at 30°C and 180 r·min. -1 The fermentation broth of strain BVZ0942 was obtained by shaking culture for 1 day. -1Centrifuge for 15 minutes, and filter the supernatant obtained by centrifugation with a 0.22μm bacterial filter to obtain the sterile filtrate of the antagonistic strain BVZ0942. Dilute the sterile filtrate of strain BVZ0942 with PDA medium 5 times, 10 times and 20 times respectively, then take 20mL of PDA medium of each dilution multiple, mix thoroughly and pour into the plate. Inoculate fresh Sclerotinia sclerotiorum with a diameter of 0.5cm in the center of the plate. Set up a control group with the same concentration gradient, and set up 3 repeated experiments for each concentration. Culture at 25℃ for 2 days, observe the growth status of the colonies and then measure the colony diameter of Sclerotinia sclerotiorum with the cross method, take pictures and keep them. Figure 7 , Figure 7 CK5 times, CK10 times and CK20 times are the control groups with the same concentration gradient.

[0096] The results are shown in Table 6. Figure 7 It can be seen that the inhibitory effect of the sterile filtrate of strain BVZ0942 on the growth of Sclerotinia sclerotiorum hyphae showed regular changes. With the increase of the dilution concentration of the sterile filtrate of strain BVZ0942, the inhibition rate gradually decreased. After the seed liquid of the sterile filtrate of strain BVZ0942 was diluted 5 times, 10 times, and 20 times, the inhibition rates of Sclerotinia sclerotiorum hyphae were 100.00%, 77.06%, and 54.79%, respectively.

[0097] Table 6 Effect of sterile filtrate of strain BVZ0942 on mycelial growth of Sclerotinia sclerotiorum

[0098]

[0099] Study on the antibacterial activity of the metabolites of biocontrol bacteria

[0100] The antagonistic strain BVZ0942 was placed in a constant temperature incubator at 30°C for 1 day for activation culture. Single colonies of the activated strain BVZ0942 were picked and inoculated into high temperature sterilized LB liquid culture medium at 30°C and 180 r·min. -1 After 5 days of oscillation culture, the fermentation broth of strain BVZ0942 was obtained. The fermentation broth of strain BVZ0942 was sterilized at 120℃ for 30 minutes to obtain the sterilized metabolites of the antagonistic strain BVZ0942. The sterilized metabolites of strain BVZ0942 were diluted 4 times, 5 times, 10 times and 20 times with PDA medium, respectively. Then 20 mL of PDA medium of each dilution multiple was taken, mixed thoroughly and poured into a plate. Fresh Sclerotinia sclerotiorum with a diameter of 0.5 cm was inoculated in the center of the plate. A control group was set up with the same concentration gradient, and 3 repeated experiments were set up for each concentration. The experiment was cultured at 25℃ for 2 days, and the growth status of the colonies was observed. The colony diameter of Sclerotinia sclerotiorum was measured by the cross method, and photos were taken for preservation. Figure 8 , Figure 8 CK4 times, CK5 times, CK10 times and CK20 times are used as controls with the same concentration gradient.

[0101] The results are shown in Table 7. Figure 8 It can be seen that the inhibitory effect of the sterilized metabolites of strain BVZ0942 on the mycelial growth of Sclerotinia sclerotiorum shows regular changes. With the increase of the dilution concentration of the sterilized metabolites of strain BVZ0942, the inhibition rate gradually decreases. After the seed liquid of the sterilized metabolites of strain BVZ0942 was diluted 4 times, 5 times, 10 times, and 20 times, the inhibition rates of Sclerotinia sclerotiorum were 100.00%, 97.71%, 37.45%, and 26.02%, respectively.

[0102] Table 7 Effects of sterilized metabolites of strain BVZ0942 on hyphal growth of Sclerotinia sclerotiorum

[0103]

[0104] The above is a detailed introduction to a strain of Bacillus and its application provided in the examples. Specific examples are used herein to illustrate the principles and implementation methods of the present application. The description of the above examples is only used to help understand the method and core idea of ​​the present application. At the same time, for those skilled in the art, according to the idea of ​​the present application, there will be changes in the specific implementation method and application scope. In summary, the content of this specification should not be understood as limiting the present application.

Claims

1. A strain of Bacillus Velez, characterized in that: The Bacillus velezensis BVZ0942 is deposited in the China Center for Type Culture Collection, with the deposit number CCTCC NO:M 20231560, and the deposit time is August 31, 2023.

2. The Bacillus Velezii according to claim 1, characterized in that: The 16S rDNA sequence of the Bacillus velezinii BVZ0942 is shown in the sequence table as SEQ ID No:

1.

3. The Bacillus Velezii according to claim 1, characterized in that: The Bacillus Velez BVZ0942 comes from fish pond sediment.

4. The application of Bacillus Velez according to claim 1 in the preparation of a biocontrol agent, wherein the biocontrol agent is a strawberry gray mold (Botrytis cinerea) biocontrol agent, a pepper root rot (Fusarium solani) biocontrol agent, a kiwifruit anthracnose (Colletotrichum sp.) biocontrol agent or a rapeseed sclerotinia (Sclerotiniasclerotiorum) biocontrol agent.

5. Use of the Bacillus Velezii according to claim 1 in the preparation of an antibacterial agent, wherein the antibacterial agent is a bacterial agent that inhibits strawberry gray mold, pepper root rot, kiwi anthrax or rapeseed sclerotinia.

6. Use of the Bacillus Velez subtilis according to claim 1 in the preparation of a plant disease-resistant agent, wherein the disease-resistant agent is an agent for resisting plant diseases caused by strawberry gray mold, Zanthoxylum root rot, kiwifruit anthracnose or rapeseed sclerotinia.

7. Use of the sterile metabolites produced by Bacillus Velezii according to claim 1 in preventing and controlling plant diseases, wherein the plant diseases are plant diseases caused by gray mold of strawberry, root rot of Zanthoxylum bungeanum, kiwifruit anthracnose or Sclerotinia sclerotiorum of rape.

8. Use of the sterilized metabolites produced by Bacillus Velezii according to claim 1 in preventing and controlling plant diseases, wherein the plant diseases are plant diseases caused by gray mold of strawberry, root rot of Zanthoxylum bungeanum, kiwifruit anthracnose or Sclerotinia sclerotiorum of rape.

9. Use of the volatile substances produced by Bacillus Velezii according to claim 1 in preventing and controlling plant diseases, wherein the plant diseases are plant diseases caused by gray mold of strawberry, root rot of Zanthoxylum bungeanum, kiwifruit anthracnose or Sclerotinia sclerotiorum of rape.

10. Use of the volatile substances produced by Bacillus Velezii according to claim 1 in the preparation of biological pesticides for preventing and controlling plant diseases, wherein the plant diseases are plant diseases caused by strawberry gray mold, Zanthoxylum bungeanum root rot, kiwifruit anthracnose or rapeseed sclerotinia.

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