Bacillus subtilis and its use

Abstract

The present disclosure relates to a new Peribacillus sp. and the use for controlling plant diseases or promoting plant growth.

Description

TECHNICAL FIELD

[0001] The present application relates to the technical field of plant disease control, and in particular to a new Peribacillus sp. X-13, a microbial agent containing the Peribacillus sp., inactivates, cultures or metabolites thereof, and applications thereof in controlling plant diseases or promoting growth. BACKGROUND

[0002] Crop diseases are an important factor causing food reduction and quality decline, and mycotoxins produced by plant fungal diseases, such as fusarium produced by fusarium toxin and aspergillus produced by aspergillus toxin, mixed into food and feed, seriously threaten the food safety and the life and health of human and animals in China.

[0003] In addition to planting disease-resistant varieties, chemical control is still the most economical and effective control measure in production, but long-term use of chemical pesticides has also brought problems such as increased pathogen resistance, excessive pesticide residues, and environmental pollution, and it is urgent to explore microbial pesticide products that can replace chemical pesticides and reduce dependence on traditional chemical pesticides.

[0004] Bacillus is a dominant biological population in soil and plant microecological flora, is non-toxic to humans and animals, does not pollute the environment, can produce various antibacterial substances and molds, has broad-spectrum antibacterial activity and strong stress resistance, and thus has become a research hotspot at home and abroad. Bacillus achieves the purpose of biological control by nutrient competition, induction of resistance, inhibition of pathogen growth, and promotion of crop growth. SUMMARY

[0005] The biocontrol agent X-13 is isolated from rotten wheat straw, and is identified by morphological characteristics, physiological and biochemical reaction characteristics, 16S gene sequence analysis, and second-generation and third-generation whole genome sequencing analysis by the China Center for Type Culture Collection. The bacteria are significantly different from the reported Bacillus, and are only identified as Peribacillus sp., and the species cannot be identified. The biocontrol agent X-13 has great research potential and can be used to control various plant diseases.

[0006] According to an aspect of the present application, there is provided a Peribacillus sp., which is any one or more of the following:

[0007] (1) Peribacillus sp. X-13, which was deposited with the China Center for Type Culture Collection on August 12, 2024, and has the accession number CCTCC NO: M20241776;

[0008] (2) 16sRNA comprises a nucleotide sequence as set forth in SEQ ID NO: 3, or a nucleotide sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 98.65%, 99%, 99.5%, 99.6%, 99.7%, 99.8%, 99.9%, 99.99%, or 100% identity thereto;

[0009] (3) a strain having at least 95%, 96%, 97%, 98%, 98.65%, 99%, 99.5%, 99.6%, 99.7%, 99.8%, 99.9%, 99.99%, or 100% average nucleotide identity (ANI) to the genome of (1) or (2);

[0010] (4) a strain having at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 98.65%, 99%, 99.5%, 99.6%, 99.7%, 99.8%, 99.9%, 99.99%, or 100% DNA-DNA hybridization (DDH) value to (1) to (3).

[0011] In some embodiments, the Peribacillus sp. comprises any one or more of the following properties or abilities: reduction of nitrate; decomposition of hevea; ability to hydrolyze gelatin; assimilation of glucose; assimilation of arabinose; assimilation of mannitol; assimilation of N-acetyl-glucosamine; assimilation of maltose; assimilation of gluconate; assimilation of malate; assimilation of citrate; and, assimilation of phenylacetic acid.

[0012] According to another aspect of the present disclosure, there is provided a microbial agent, wherein the active ingredient of the microbial agent comprises the above-mentioned Peribacillus sp. of the present disclosure, an inactivated form thereof, a culture thereof, or a metabolite thereof.

[0013] In some embodiments, the microbial agent can further comprise an additional active agent and / or an excipient.

[0014] In some embodiments, the additional active agent can comprise other active substances for controlling plant diseases and / or plant insect pests.

[0015] In some embodiments, the excipient can comprise any excipient acceptable in agriculture or horticulture.

[0016] In some embodiments, the culture can be a fermentation broth of the Peribacillus sp.

[0017] According to another aspect of the present disclosure, there is provided use of the above-mentioned Peribacillus sp., inactivates thereof, cultures thereof, or metabolites thereof, or the above-mentioned microbial inoculants in controlling plant diseases or promoting plant growth.

[0018] In some embodiments, the plant diseases can include diseases caused by pathogens, such as fungi, bacteria, viruses, nematodes, and the like.

[0019] In some embodiments, the plant diseases can include, but are not limited to, corn southern leaf blight, wheat foot rot, brassica sclerotinia, wheat sharp eyespot, apple canker, tomato gray mold, wheat scab, wheat powdery mildew, brassica sclerotinia, and strawberry gray mold.

[0020] In some embodiments, the pathogenic fungi causing the plant diseases can include, but are not limited to, corn southern leaf blight fungus, wheat foot rot fungus, brassica sclerotinia fungus, wheat sharp eyespot fungus, apple canker fungus, tomato gray mold fungus, wheat scab fungus, wheat powdery mildew fungus, brassica sclerotinia fungus, and strawberry gray mold fungus.

[0021] In some embodiments, the plants can include plants of the family Poaceae, plants of the family Solanaceae, plants of the family Apiaceae, plants of the family Brassicaceae, plants of the family Rosaceae, and the like.

[0022] In some embodiments, the plants of the family Poaceae can include, but are not limited to, corn, wheat, rice, sorghum, barley, oat, rye, millet, foxtail millet, broomcorn millet, buckwheat, and the like.

[0023] In some embodiments, the plants of the family Solanaceae can include, but are not limited to, tomato, pepper, potato, eggplant, black nightshade, belladonna, wolfberry, and the like.

[0024] In some embodiments, the plants of the family Apiaceae can include, but are not limited to, carrot, angelica, radix angelicae dahuricae, radix saposhnikoviae, radix bupleuri, coriander, celery, water celery, radix ligustici wallichii, and the like.

[0025] In some embodiments, the plants of the family Brassicaceae can include, but are not limited to, cabbage, cauliflower, broccoli, kale, Chinese cabbage, leaf mustard, rape, preserved vegetable, pricklyash, turnip, radish, Arabidopsis thaliana, and the like.

[0026] In some embodiments, the plants of the family Rosaceae can include, but are not limited to, apple, rose, plum, Chinese rose, flowering crabapple, wild apple, and the like.

[0027] According to another aspect of the present disclosure, there is provided a method for controlling plant diseases or promoting plant growth, the method comprising applying the above-mentioned Peribacillus sp., inactivates thereof, cultures thereof or metabolites thereof, or the above-mentioned microbial agent to a plant.

[0028] It should be understood by those skilled in the art that the application can be applied to any part of the plant as long as the above-mentioned Peribacillus sp., inactivates thereof, cultures thereof or metabolites thereof, or the above-mentioned microbial agent can control plant diseases or promote plant growth.

[0029] In a specific embodiment, the application can include applying to the leaves, roots, flowers, fruits and the like of the plant.

[0030] In some embodiments, the promotion of plant growth includes one or more of the following: promoting the absorption of nutrients by the plant; promoting the growth or elongation of stems, leaves or roots; increasing the dry weight or fresh weight of the plant.

[0031] Deposit Information:

[0032] Strain X-13 was deposited with the China Center for Type Culture Collection (CCTCC) and has the deposit name Peribacillus sp. X-13, the deposit number CCTCC NO: M20241776, the deposit date August 12, 2024, and the deposit address Wuhan, China, Wuhan University, postal code 430072. The deposited strain was identified by the deposit center and was alive. BRIEF DESCRIPTION OF DRAWINGS

[0033] Figure 1 The morphological observation results of the biocontrol strain X-13 strain are shown. Figure 1 A is a colony morphology plate photo of X-13; Figure 1 B is a microscope (1000X) observation photo of X-13 strain.

[0034] Figure 2 The Neighbor-Joining phylogenetic tree of the biocontrol strain X-13 strain is shown.

[0035] Figure 3 The inhibition activity diagram of strain X-13 on corn small spot pathogen is shown. Figure 3 A is CK (control group); Figure 3 B is the 10-fold diluted treatment group of X-13 fermentation liquid.

[0036] Figure 4 The inhibition activity diagram of strain X-13 on wheat stem base rot pathogen is shown. Figure 4 A is CK (control group);Figure 4 B is the treatment group of 10-fold dilution of fermentation broth of strain X-13.

[0037] Figure 5 A chart showing the inhibitory activity of strain X-13 on Sclerotinia sclerotiorum is shown. Figure 5 A is CK (control group); Figure 5 B is the treatment group of 10-fold dilution of fermentation broth of strain X-13.

[0038] Figure 6 A chart showing the inhibitory activity of strain X-13 on Gaeumannomyces graminis is shown. Figure 6 A is CK (control group); Figure 6 B is the treatment group of 10-fold dilution of fermentation broth of strain X-13.

[0039] Figure 7 A chart showing the inhibitory activity of strain X-13 on Botryosphaeria dothidea is shown. Figure 7 A is CK (control group); Figure 7 B is the treatment group of 10-fold dilution of fermentation broth of strain X-13.

[0040] Figure 8 A chart showing the inhibitory activity of strain X-13 on Botrytis cinerea is shown. Figure 8 A is CK (control group); Figure 8 B is the treatment group of 10-fold dilution of fermentation broth of strain X-13.

[0041] Figure 9 A chart showing the inhibitory activity of strain X-13 on Gibberella zeae is shown. Figure 9 A is CK (control group); Figure 9 B is the treatment group of 10-fold dilution of fermentation broth of strain X-13.

[0042] Figure 10 A chart showing the inhibitory activity of strain X-13 on Blumeria graminis is shown. Figure 10 A is CK (control group); Figure 10 B is the treatment group of 10-fold dilution of fermentation broth of strain X-13.

[0043] Figure 11 A chart showing the inhibitory activity of strain X-13 on Sclerotinia sclerotiorum is shown. Figure 11 A is CK (control group); Figure 11 B is the treatment group of 10-fold dilution of fermentation broth of strain X-13.

[0044] Figure 12 A chart showing the inhibitory activity of strain X-13 on Botrytis cinerea is shown. Figure 12 A is CK (control group); Figure 12 B is the treatment group of 10-fold dilution of fermentation broth of strain X-13. DETAILED DESCRIPTION

[0045] In order to make the objectives, technical solutions, and advantages of the present application clearer, the present application will be described in further detail below with reference to embodiments. The specific embodiments described herein are intended for illustration only and are not intended to be limiting on the present application. In addition, in the following description, descriptions of well-known structures and techniques are omitted to avoid obscuring the concept of the present disclosure. Such structures and techniques are described in many publications.

[0046] Definitions

[0047] 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. The following definitions are applied to the descriptions of the present application and throughout this document, and apply equally to both the singular and plural form of the terms.

[0048] As used herein, the terms "a" and "an" include plural references unless the context clearly indicates otherwise. For example, reference to "a cell" includes a plurality of such cells, equivalents, and the like, as will be apparent to one of ordinary skill in the art.

[0049] As used herein, the term "about" means a range of ±20% of the value that follows. In some embodiments, the term "about" means a range of ±10% of the value that follows. In some embodiments, the term "about" means a range of ±5% of the value that follows.

[0050] The term "average nucleotide identity (ANI)" as used herein refers to the similarity of homologous genes between two bacterial genomes. ANI values can be calculated by methods such as BLAST. In the field of bacterial taxonomy, it is generally accepted that a threshold of 95% for ANI homology. In other words, an ANI value below 95% will be identified as belonging to different bacterial species (Yoon, S. H., Ha, S. M., Lim, J. M., Kwon, S. J. & Chun, J. (2017). A large-scale evaluation of algorithms to calculate average nucleotide identity. Antonie van Leeuwenhoek. 110: 1281-1286; Lee, I., Kim, Y. O., Park, S. C., & Chun, J. (2015). OrthoANI: An improved algorithm and software for calculating average nucleotide identity. Int J Syst Evol Microbiol. 66: 1100-1103).

[0051] "Digital DNA-DNA hybridization (dDDH)" or "DNA-DNA hybridization (DDH)" can be used interchangeably, which is used to measure the locus similarity of bacterial species. In the field of bacterial taxonomy, it is generally accepted that a threshold of 70% for dDDH homology. In other words, a dDDH value below 70% will be identified as belonging to different bacterial species (Meier-Kolthoff, J. P., Carbasse, J., Peinado-Olarte, R. L., M. (2022). TYGS and LPSN: a database tandem for fast and reliable genome-based classification and nomenclature of prokaryotes. Nucleic Acid Res 50: D801-D807; Meier-Kolthoff, J. P., Auch, A. F., Klenk, H.-P., M. (2013). Genome sequence-based species delimitation with confidence intervals and improved distance functions. BMC Bioinformatics 14: 60).

[0052] The "percent sequence identity" or "percent identity" between two polynucleotide or polypeptide sequences refers to the number of identical matches between the sequences in the comparison window, taking into account any additions or deletions (i.e., gaps) that must be introduced for optimal alignment of the two sequences. A match is any position in the target sequence and the reference sequence where the same nucleotide or amino acid is present. Gaps are not counted as nucleotides or amino acids. Likewise, gaps present in the reference sequence are not counted, as are nucleotides or amino acids from the reference sequence, since they are not counted.

[0053] The percent sequence identity can be calculated by determining the number of positions at which the same amino acid residue or nucleic acid base occurs in both sequences to yield the number of matched positions, dividing the number of matched positions by the total number of positions in the comparison window, and multiplying the result by 100 to yield the percent sequence identity. Comparison of sequences and determination of percent sequence identity between two sequences can be accomplished using software readily available for online use and download. Suitable software programs are available from various sources for alignment of protein and nucleotide sequences. One suitable program for determining percent sequence identity is bl2seq, which is part of the BLAST suite of programs available from the National Center for Biotechnology Information's BLAST website (blast.ncbi.nlm.nih.gov). Bl2seq uses either the BLASTN or BLASTP algorithm for comparison between two sequences. BLASTN is used for comparing nucleic acid sequences, while BLASTP is used for comparing amino acid sequences. Other suitable programs are, for example, Needle, Stretcher, Water, or Matcher, which are part of the EMBOSS suite of bioinformatics programs, and are also available from the European Bioinformatics Institute (EBI) at www.ebi.ac.uk / Tools / psa.

[0054] The following embodiments and accompanying drawings are provided to aid in understanding the present invention. However, it should be understood that these embodiments and drawings are for illustrative purposes only and do not constitute any limitation. The actual scope of protection of the present invention is set forth in the claims. It should be understood that any modifications and changes can be made without departing from the spirit of the invention. The reagents and / or kits used in the following embodiments are commercially available or can be synthesized by known methods.

[0055] It should be noted that, unless specific conditions are specified in the examples, experimental conditions should be performed according to standard conditions, manufacturer recommendations, or publicly reported experimental conditions. Reagents or instruments whose manufacturers are not specified are all commercially available, standard products. For reagents whose manufacturers are specified, similar products from other manufacturers are substitutes.

[0056] Example

[0057] Example 1: Isolation of biocontrol bacteria X-13

[0058] Twenty samples each of decaying corn, wheat, and rice straw were collected from four different ecological regions in Hubei Province: the western mountainous area, the Jianghan Plain, the eastern hilly area, and the central hilly area. The straw was first surface-washed with sterile water, then ground in a sterile mortar with an appropriate amount of sterile water. The supernatant was removed, and the mixture was diluted to 10⁻⁶. -1 ~10 -6 The bacteria were evenly spread on a solid culture medium and incubated at 30°C. Bacteria with different colony morphologies were then isolated and purified. The mycelial growth rate method was used to determine the bacterial activity against wheat stem rot pathogens, and the biocontrol strain X-13 was screened.

[0059] Example 2: Identification of biocontrol bacteria strain X-13

[0060] 1. Strain morphology

[0061] Strawberry strain X-13 was streaked onto Luria-Bertani solid medium plates (10 g peptone, 5 g yeast extract, 10 g NaCl, 1 L distilled water, 14 g agar powder, pH adjusted to 7.0) and incubated at 30°C. The morphology was as follows: Figure 1 As shown in Figure A. The photograph observed under a microscope (1000×) is as follows. Figure 1 As shown in B.

[0062] 2. Identification of 16S rRNA in the strain: Genomic DNA was extracted using the SteadyPure Bacterial Genomic DNA Extraction Kit (AccurateBiotechnology (Hunan) Co., Ltd.). The procedure is as follows:

[0063] (1) Bacterial lysis

[0064] 12,000rpm centrifugation for 2 minutes to collect 0.5-2.0 x 10 9 bacteria, and discard the supernatant (culture medium).

[0065] Add 500 μl of Buffer BP to resuspend the bacteria, add 50 μl of Lysozyme (20 mg / ml), mix well, and incubate at 37°C for 60 minutes.

[0066] Centrifuge at 12,000 rpm for 5 minutes at room temperature, and discard the supernatant.

[0067] Add 180 μl of Buffer LS-2, then add 20 μl of Proteinase K (20 mg / ml) and 10 μl of RNase A (10 mg / ml), mix well by shaking or vortexing, and incubate at 56°C for 10 minutes until the solution becomes transparent.

[0068] Add 200 μl of Buffer BS-2 and 200 μl of 100% ethanol to the solution, and mix well.

[0069] Transfer the above solution to a Bacterial DNA Mini Column, stand for 1 minute at room temperature, centrifuge at 12,000 rpm for 1 minute at room temperature, and discard the filtrate.

[0070] (2) Add 500 μl of Buffer WA to the Mini Column, centrifuge at 12,000 rpm for 1 minute at room temperature, and discard the filtrate.

[0071] (3) Add 750 μl of Buffer WB to the Mini Column, centrifuge at 12,000 rpm for 1 minute at room temperature, and discard the filtrate.

[0072] (4) Repeat step 3) once.

[0073] (5) Place the Mini Column on a new 2 ml collection tube, and centrifuge at 12,000 rpm for 2 minutes at room temperature.

[0074] (6) Place the Mini Column on a new 1.5 ml centrifuge tube, add 50 μl of Elution Buffer or sterilized water at the center of the Mini Column membrane, and stand for 1 minute at room temperature.

[0075] (7) Centrifuge at 12,000 rpm for 2 minutes at room temperature to elute the DNA.

[0076] (8) The 16S-PCR reaction system is shown in Table 1 below.

[0077] Table 1: PCR reaction system.

[0078]

[0079]

[0080] Bio-Rad T100 Thermal Cycler reaction conditions: 94℃ 10min; 94℃ 1min, 55℃ 1min, 72℃ 1min, 34 cycles; 72℃ 10min, 10℃∞.

[0081] The primer sequences used in Table 1 are shown in Table 2 below.

[0082] Table 2: Primer sequences

[0083] Primer Sequence SEQ ID NO: 27F 5'-GAGTTTGATCCTGGCTCAG-3' 1 1540R 5'-AAGGAGGTGATCCAGCC-3' 2

[0084] The 16S sequence PCR product was sent for sequencing (Wuhan Benya Technology Co., Ltd.), and the sequencing results are as follows:

[0085]

[0086] The BLAST results of the 16S rRNA gene of the X-13 strain are shown in Table 3 below.

[0087] Table 3: BLAST results of the 16S rRNA gene of the X-13 strain.

[0088]

[0089] Further alignment of the 16S rRNA sequence of the X-13 strain with multiple sequences found that the closest species was Peribacillus frigoritolerans, with a similarity of about 99.72%. The Neighbor-Joining phylogenetic tree was constructed with Joncia denitrificans NCTC10816 (X78420) as the outgroup, and the results are shown in Figure 1. Figure 2

[0090] 3. Physiological and biochemical characteristics of the strain

[0091] (1) Culturing the microorganism

[0092] The obtained pure culture strain was inoculated on a BUG+B culture medium and cultured at 33°C for 4-24 hours.

[0093] (2) Inoculating the micro-well plate

[0094] The bacterial suspension was poured into a V-shaped sample loading tank.

[0095] An 8-channel pipette was used to suck the bacterial suspension into the pipette tip.

[0096] The bacterial suspension was sequentially added to all the wells of the micro-well plate in an amount of 100ul per well, and the cover of the micro-well plate was closed.

[0097] (3) Incubating the micro-well plate

[0098] The micro-well plate was directly placed into the incubation and reading instrument of the OmniLog, and the culture temperature was 33°C.

[0099] (5) Reading the plate

[0100] The BIOLOG microbial identification system was used to read the detection results, and the results are shown in Tables 4-6.

[0101] Table 4: Physiological and biochemical characteristics of the strain X-13 - enzyme activity, carbon source assimilation

[0102]

[0103] +: positive reaction; -: negative reaction.

[0104] ​Table 5: Physiological and biochemical characteristics of strain X-13 - carbon source utilization

[0105]

[0106]

[0107]

[0108] + positive reaction; - negative reaction; W: weak positive reaction.

[0109] Table 6: Physiological and biochemical characteristics of strain X-13 - acid production from carbon sources

[0110]

[0111]

[0112] + positive reaction; - negative reaction.

[0113] 4. Whole genome sequencing of strain X-13

[0114] 4.1 DNA library preparation by next generation sequencing:

[0115] (1) DNA quality check

[0116] 1% agarose gel electrophoresis to check DNA sample for degradation and impurities;

[0117] Spectrophotometer to check sample purity (IMPLEN, CA, USA);

[0118] 3.0 Flurmeter (Life Technologies, CA, USA) to check DNA sample concentration.

[0119] (2) DNA library preparation

[0120] Using lug gDNA template, library preparation was performed according to the TruSeq DNA Sample Preparation Guide (Illumina 15026486 Rev. C) method and procedure.

[0121] (3) Library check

[0122] After the library is constructed, use Qubit 3.0 for preliminary quantification, dilute the library to 1 ng / ul, then use Agilent 2100 to detect the fragment size of the library. After the fragment size meets the expected value, use Bio-RAD CFX 96 fluorescence quantitative PCR instrument, Bio-RAD KITiQ SYBR GRN for Q-PCR, and accurately quantify the effective concentration of the library (library effective concentration > 10 nM) to ensure the quality of the library.

[0123] (4) Sequencing

[0124] The qualified library is run on the NovaSeq X plus sequencing platform for double-end sequencing program (PE150) to obtain 150bp sequence reads.

[0125] 4.2 Third-generation sequencing method

[0126] (1) Take 2-2.5ug of DNA samples that have passed quality inspection, add 1* magnetic bead purification, and take 1ul sample for quantification;

[0127] The damage and end repair system is shown in the following table.

[0128] Table 7

[0129]

[0130]

[0131] Incubation conditions: 20℃ for 10min, 65℃ for 10min, 4℃ for storage;

[0132] (3) 1* magnetic bead purification of DNA, 25ul EB elution of DNA, taking 1ul sample Qubit quantification;

[0133] (4) Barcode label connection (taking repaired DNA)

[0134] Table 8

[0135] DNA after repair 22.5ul Native Barcode (EXP-NBD196) 2.5ul Blunt / TA Master Mix (NEB) 25ul Total 50ul

[0136] Incubation conditions: 25℃ for 10min;

[0137] (5) * magnetic bead purification, 25ul EB elution of DNA, taking 1ul Qubit quantification;

[0138] (6) Pooling library

[0139] Mix different samples with the same proportion after adding labels into a total volume of 65ul;

[0140] (7) Sequencing adapter connection

[0141] Table 9

[0142] Pooling DNA 65ul Quick ligation buffer 5x (NEB) 20ul Adapter Mix II (EXP-AMII001) 5ul Quick T4 DNA ligase (NEB) 10ul Total 100ul

[0143] Incubation condition: 25℃ for 10 min;

[0144] (8) 0.5* magnetic bead purification DNA, 25ul Elution buffer (SQK-LSK110) to elute DNA, take 1ul sample for Qubit quantification;

[0145] (9) Configure the library for sequencing

[0146] Table 10

[0147]

[0148]

[0149] (10) Sequencing

[0150] Load the library into R9.4.1 sequencing chip, and sequence on PromethION sequencer (Oxford Nanopore Technologies, Oxford, UK) for 48-72h.

[0151] By comparing the whole genome data of strain X-13 with Peribacillus simplex (DSM1321) model strain, the average nucleotide similarity (ANI) value is about 94.33%; the dDDH value is 68.6%, 55.9%, and 67.9%.

[0152] According to the above test results, strain X-13 is identified as Peribacillus sp. (Peribacillus), named Peribacillus sp. X-13, and preserved in China Center for Type Culture Collection on August 12, 2024, with the preservation number CCTCC NO: M20241776.

[0153] Example 3: Obtaining of fermentation broth of strain X-13

[0154] Under sterile conditions, the strain X-13 was streaked onto Luria-Bertani solid medium (10 g of proteose peptone, 5 g of yeast extract, 10 g of NaCl, 1 L of distilled water, 14 g of agar powder, and adjusted to pH 7.0) plates, and incubated at 30°C. When single colonies were formed, a single colony was picked and transferred to Luria-Bertani liquid medium, and incubated at 30°C, 120 rpm for 3 days. The supernatant was obtained by centrifugation of the fermentation broth at 10,000 rpm for 5 min, and filtered through a 0.22-μm filter for use.

[0155] Example 4: Activity determination of the strain X-13 against Bipolaris maydis

[0156] In this experiment, Bipolaris maydis was used, and the treated 1 mL of the fermentation broth of the strain X-13 was added to 9 mL of PDA medium (250 g of potato, 20 g of glucose, 9 g of agar powder, and 1 L of distilled water) cooled to 50°C. Each treatment was repeated three times. After the medium solidified, fresh Bipolaris maydis cakes were inoculated in the middle of the plates, and incubated in an incubator at 25°C. When the mycelium of the control grew rapidly and filled the plates, the diameter of the mycelium was measured twice using a vernier caliper at right angles. Figure 3 A and Figure 3 B shows the inhibition activity of the strain X-13 against Bipolaris maydis.

[0157] Inhibition rate (%) = (control colony diameter - treated colony diameter) / control colony diameter x 100%

[0158] Table 11: Activity results of the fermentation broth of the strain X-13 against Bipolaris maydis in the laboratory

[0159] Treatment Average diameter (mm) Inhibition rate (%) X-13 fermentation broth 10 times 18.40 61.83 CK (control group) 48.20 -

[0160] As shown in Table 11, the fermentation broth of the strain X-13 diluted 10 times had good antibacterial activity against Bipolaris maydis, with an inhibition rate of about 61.83%.

[0161] Example 5: Activity determination of the strain X-13 against Fusarium pseudograminearum

[0162] In this experiment, Fusarium pseudograminearum was used, and the treated 1 mL of the fermentation broth was added to 9 mL of PDA medium (250 g of potato, 20 g of glucose, 9 g of agar powder, and 1 L of distilled water) cooled to 50°C. Each treatment was repeated three times. After the medium solidified, fresh Fusarium pseudograminearum cakes were inoculated in the middle of the plates, and incubated in an incubator at 25°C. When the mycelium of the control grew rapidly and filled the plates, the diameter of the mycelium was measured twice using a vernier caliper at right angles.Figure 4 A and Figure 4 B shows the inhibition activity of strain X-13 against C. herbarum.

[0163] Inhibition rate (%) = (control colony diameter - treated colony diameter) / control colony diameter x 100%

[0164] Table 12: Results of the activity of strain X-13 fermentation broth against C. herbarum in the laboratory

[0165] Treatment Average diameter (mm) Inhibition rate (%) X-13 fermentation broth 10 times 16.32 65.19 CK 46.88 -

[0166] As shown in Table 12, the 10-fold diluted fermentation broth of strain X-13 had good antibacterial activity against C. herbarum, with an inhibition rate of about 65.19%.

[0167] Example 6: Activity determination of strain X-13 against Sclerotinia sclerotiorum

[0168] In this experiment, Sclerotinia sclerotiorum (Lib.) de Bary was used as the pathogen of Sclerotinia sclerotiorum, and the growth rate method was used. 1 mL of treated fermentation broth was added to 9 mL of PDA medium (potato 250 g, glucose 20 g, agar powder 9 g, distilled water 1 L) cooled to 50°C, and each treatment was repeated 3 times. After the medium solidified, fresh Sclerotinia sclerotiorum cake was inoculated in the middle of the plate, and placed in a constant temperature incubator at 25°C. When the mycelium of the blank control grew rapidly and filled the culture dish, it was measured twice vertically with a vernier caliper. Figure 5 A and Figure 5 B shows the inhibition activity of strain X-13 against Sclerotinia sclerotiorum.

[0169] Inhibition rate (%) = (control colony diameter - treated colony diameter) / control colony diameter x 100%

[0170] Table 13: Results of the activity of strain X-13 fermentation broth against Sclerotinia sclerotiorum in the laboratory

[0171] Treatment Average diameter (mm) Inhibition rate (%) X-13 fermentation broth 10 times 8.61 82.92 CK 50.4 -

[0172] As shown in Table 13, the 10-fold diluted fermentation broth of strain X-13 had good antibacterial activity against Sclerotinia sclerotiorum, with an inhibition rate of about 82.92%.

[0173] Example 7: Activity determination of strain X-13 against Gaeumannomyces graminis

[0174] In this experiment, the wheat sheath blight pathogen Rhizotonia cerealis was used, and the growth rate method was used. 1 mL of treated fermentation liquor was added to 9 mL of PDA medium (potato 250 g, glucose 20 g, agar powder 9 g, distilled water 1 L) cooled to 50°C, each treatment was repeated 3 times, after the medium was solidified, fresh wheat sheath blight fungus cake was inoculated in the middle of the plate, and placed in a constant temperature incubator at 25°C. When the mycelium of the blank control grew fast and filled the culture dish, it was measured twice vertically with a vernier caliper. Figure 6 A and Figure 6 B exemplarily shows the inhibition activity diagram of strain X-13 on wheat sheath blight fungus.

[0175] Inhibition rate (%) = (control colony diameter - treated colony diameter) / control colony diameter x 100%

[0176] Table 14: Inhibition activity results of strain X-13 fermentation liquor on wheat sheath blight fungus in vitro

[0177] Treatment Average diameter (mm) Inhibition rate (%) X-13 fermentation broth 10 times 12.30 75.15 CK 49.5 -

[0178] According to Table 14, the fermentation liquor of strain X-13 diluted 10 times has good antibacterial activity on wheat sheath blight fungus, and the inhibition rate is about 75.15%.

[0179] Example 8: Activity determination of strain X-13 on Valsa mali Miyabe & G. Yamada

[0180] In this experiment, the apple canker pathogen Valsa mali Miyabe & G. Yamada was used, and the growth rate method was used. 1 mL of treated fermentation liquor was added to 9 mL of PDA medium (potato 250 g, glucose 20 g, agar powder 9 g, distilled water 1 L) cooled to 50°C, each treatment was repeated 3 times, after the medium was solidified, fresh apple canker fungus cake was inoculated in the middle of the plate, and placed in a constant temperature incubator at 25°C. When the mycelium of the blank control grew fast and filled the culture dish, it was measured twice vertically with a vernier caliper. Figure 7 A and Figure 7 B exemplarily shows the inhibition activity diagram of strain X-13 on Valsa mali Miyabe & G. Yamada.

[0181] Inhibition rate (%) = (control colony diameter - treated colony diameter) / control colony diameter x 100%

[0182] Table 15: Inhibition activity results of strain X-13 fermentation liquor on Valsa mali Miyabe & G. Yamada in vitro

[0183] Treatment Average diameter (mm) Inhibition rate (%) X-13 fermentation broth 10 times 9.54 79.91 CK 47.48 -

[0184] According to Table 15, the fermentation broth of X-13 has good inhibitory activity against V. mali at a dilution of 10 times, with an inhibition rate of about 79.91%.

[0185] Example 9: Activity determination of strain X-13 against Botrytis cinerea

[0186] In this experiment, the pathogen of tomato gray mold Botrytis cinerea was used, and the growth rate method was used. 1 mL of treated fermentation broth was added to 9 mL of PDA medium (potato 250 g, glucose 20 g, agar powder 9 g, distilled water 1 L) cooled to 50°C, and each treatment was repeated 3 times. After the medium solidified, fresh tomato gray mold cake was inoculated in the middle of the plate, and placed in a constant temperature incubator at 25°C. When the mycelium of the control grew fast and filled the culture dish, it was measured twice vertically with a vernier caliper. Figure 8 A and Figure 8 B exemplarily shows the inhibition activity diagram of strain X-13 against Botrytis cinerea.

[0187] Inhibition rate (%) = (control colony diameter - treated colony diameter) / control colony diameter x 100%

[0188] Table 16: Activity results of strain X-13 fermentation broth against Botrytis cinerea in vitro

[0189] Treatment Average diameter (mm) Inhibition rate (%) X-13 fermentation broth 10 times 10.57 74.38 CK 41.25 -

[0190] According to Table 16, the fermentation broth of X-13 has good inhibitory activity against Botrytis cinerea at a dilution of 10 times, with an inhibition rate of 74.38%.

[0191] Example 10: Activity determination of strain X-13 against Fusarium graminearum

[0192] In this experiment, the pathogen of tomato gray mold Botrytis cinerea was used, and the growth rate method was used. 1 mL of treated fermentation broth was added to 9 mL of PDA medium (potato 250 g, glucose 20 g, agar powder 9 g, distilled water 1 L) cooled to 50°C, and each treatment was repeated 3 times. After the medium solidified, fresh tomato gray mold cake was inoculated in the middle of the plate, and placed in a constant temperature incubator at 25°C. When the mycelium of the control grew fast and filled the culture dish, it was measured twice vertically with a vernier caliper. Figure 9 A and Figure 9 B exemplarily shows the inhibition activity diagram of strain X-13 against Botrytis cinerea.

[0193] Inhibition rate (%) = (control colony diameter - treated colony diameter) / control colony diameter x 100%

[0194] Table 17: Results of the in vitro activity of the fermentation broth of strain X-13 against Fusarium graminearum

[0195] Treatment Average diameter (mm) Inhibition rate (%) X-13 fermentation broth 10 times 15.42 66.95 CK 46.66 -

[0196] According to Table 17, the fermentation broth of X-13 diluted 10 times has good inhibitory activity against Fusarium graminearum, with an inhibition rate of 66.95%.

[0197] Example 11: Determination of the activity of strain X-13 against Blumeria graminis

[0198] In this experiment, the Blumeria graminis f. sp. tritici (Blumeria graminis) was used as the strong pathogenic strain E21, and the wheat was the susceptible wheat variety "chancellor". The in vitro leaf segment method was used. The wheat was cultured to 1 leaf 1 heart, the first leaf was cut into 30 mm long leaf segments, and placed on 0.5% water agar plates containing 50 μg / mL benzimidazole, 6 leaf segments per dish (diameter 60 mm). The fermentation broth of X-13 was diluted to 5 x 10 -4 The Tween 20 and the fermentation broth were mixed thoroughly and applied in a spray tower, with a spray amount of 3 mL / cm 2 At the same time, 5 x 10 -4 The LB medium containing Tween 20 was used as the negative control, and was naturally air-dried. After 4 h, the wheat Blumeria graminis E21 conidia (inoculation amount of 10-20 conidia per 10x field) were inoculated. Then, it was placed in a 17 ± 1 °C air-conditioned room for culture, first in the dark for 12 h, and then under the illumination of a 24 h 40 W daylight lamp (culture room). After the blank control was fully diseased (7 days), the disease severity of each leaf of each treatment was investigated. Figure 10 A and Figure 10 B exemplarily shows the inhibition activity of strain X-13 against Blumeria graminis.

[0199] Average inhibition rate (%) = (disease severity of the control Blumeria graminis - disease severity of the treated Blumeria graminis) / disease severity of the control Blumeria graminis x 100%.

[0200] Table 18: Results of the in vitro activity of the fermentation broth of strain X-13 against Blumeria graminis

[0201] Treatment Severity (%) Inhibition rate (%) X-13 fermentation broth 0 100 CK 95 -

[0202] According to Table 18, the fermentation broth of X-13 has good inhibitory activity against Blumeria graminis, with an inhibition rate of 100%.

[0203] Example 12: In vitro pot activity determination of strain X-13 against Sclerotinia sclerotiorum

[0204] In this experiment, Sclerotinia sclerotiorum (Lib.) de Bary was used as the pathogen of Sclerotinia sclerotiorum, and Brassica chinensis was used as the susceptible variety "Improved Jinqin 28". At the stage of the Brassica chinensis plant, 5x10 -4 Tween 20 and the fermentation liquor were mixed thoroughly and applied in a spray tower, with a spray amount of 3 mL / cm 2 At the same time, 5x10 -4 The LB medium containing Tween 20 was used as a negative control, and the fresh Sclerotinia sclerotiorum cake was inoculated on the leaf surface, and the moisture was maintained. After 7 days, the severity of the inoculated leaves was investigated. Figure 11 A and Figure 11 B exemplarily shows the inhibition activity of strain X-13 on Sclerotinia sclerotiorum.

[0205] Average inhibition rate (%) = (severity of control treatment - severity of treatment) / severity of control treatment x 100%

[0206] Table 19: X-13 fermentation liquor indoor activity results on Sclerotinia sclerotiorum indoor potting

[0207] Treatment Treatment severity (%) Inhibition rate (%) X-13 fermentation broth 0 100 CK 16.2 -

[0208] According to Table 19, the X-13 fermentation liquor has good antibacterial activity on Sclerotinia sclerotiorum, with an inhibition rate of 100%.

[0209] Example 13: In vitro flower activity determination of strain X-13 on strawberry gray mold

[0210] In this experiment, Botrytis cinerea was used as the pathogen of strawberry gray mold, and the in vitro flower method was used. The flowers of the strawberry at the full bloom stage were picked and placed in a culture dish with a wet paper towel to maintain moisture. According to 5x10 -4 Tween 20 and the fermentation liquor were mixed thoroughly and applied in a spray tower, with a spray amount of 3 mL / cm 2 At the same time, 5x10 -4 The LB medium containing Tween 20 was used as a negative control, and the fresh Sclerotinia sclerotiorum cake was inoculated on the leaf surface, and the moisture was maintained. After 7 days, the severity of the inoculated leaves was investigated. -4 The Botrytis cinerea spore solution containing Tween 20 was uniformly sprayed on the surface of the flowers, and the moisture was maintained. After 5 days, the severity of the petal disease was investigated. Figure 12 A and Figure 12 B exemplarily shows the inhibition activity of strain X-13 on strawberry gray mold.

[0211] Average inhibition rate (%) = (severity of control treatment - severity of treatment) / severity of control treatment x 100%

[0212] Table 20: X-13 fermentation liquor indoor activity results on strawberry gray mold

[0213] Treatment Treatment severity (%) Inhibition rate (%) X-13 fermentation broth 6.1 93.90 CK 100 -

[0214] According to Table 20, the X-13 fermentation liquor has good antibacterial activity on strawberry gray mold, and the inhibition rate is 93.90%.

[0215] The technical scheme of the present application is not limited to the above specific embodiments, and any technical modification made according to the technical scheme of the present application falls within the protection scope of the present application.

Claims

1. Peribacillus sp. characterized in that, The Peribacillus sp. is Peribacillus sp. X-13, which was preserved in China Center for Type Culture Collection on August 12, 2024, and the preservation number is CCTCC NO: M20241776.

2. A microbial agent, wherein the active ingredient of the microbial agent comprises the Peribacillus sp. according to claim 1, or a culture thereof.

3. The bacterial agent of claim 2, wherein The microbial agent further comprises additional active agents and / or adjuvants.

4. The bacterial agent of claim 3, characterized in that, The additional active agents are other active substances for preventing and treating plant diseases and / or plant insect pests.

5. The bacterial agent of claim 3, wherein The adjuvants comprise any agriculturally or horticulturally acceptable adjuvant.

6. The bacterial agent of claim 2, wherein The culture is a fermentation broth of the Peribacillus sp.

7. Use of the Peribacillus sp. according to claim 1, or the microbial agent according to any one of claims 2-6, in preventing plant diseases or promoting plant growth; The plant diseases are corn small spot, wheat foot rot, Sclerotinia sclerotiorum, wheat sharp eyespot, apple canker, tomato gray mold, wheat scab, wheat powdery mildew, Sclerotinia sclerotiorum, and strawberry gray mold; The pathogenic fungi causing the plant diseases are Bipolaris zeicola, Guignardia bidwellii, Sclerotinia sclerotiorum, Septoria tritici, Valsa mali, Botrytis cinerea, Fusarium graminearum, Erysiphe graminis, Sclerotinia sclerotiorum, and Botrytis cinerea; The plants are corn, wheat, rape, apple, tomato, and strawberry.

8. Use according to claim 7, characterized in that, The Peribacillus sp. or the microbial agent can inhibit the growth or reproduction of the pathogenic fungi.

9. Use according to claim 7, characterized in that, The Peribacillus sp. or the microbial agent is applied to the leaf, root, flower, or fruit part of the plant.

10. Use according to claim 7, characterized in that, The promotion of plant growth is one or more of the following: Promoting the absorption of nutrients by the plant; Promoting the growth or elongation of stems, leaves, or roots; Increasing the dry weight or fresh weight of the plant.

11. A method for controlling plant diseases or promoting plant growth, characterized by, The method comprises applying the Peribacillus sp. according to claim 1, or the microbial agent according to any one of claims 2-6, to the plant; The plant diseases are corn small spot, wheat foot rot, Sclerotinia sclerotiorum, wheat sharp eyespot, apple canker, tomato gray mold, wheat scab, wheat powdery mildew, Sclerotinia sclerotiorum, and strawberry gray mold; The pathogenic fungi causing the plant diseases are Bipolaris zeicola, Guignardia bidwellii, Sclerotinia sclerotiorum, Septoria tritici, Valsa mali, Botrytis cinerea, Fusarium graminearum, Erysiphe graminis, Sclerotinia sclerotiorum, and Botrytis cinerea; The plants are corn, wheat, rape, apple, tomato, and strawberry.

12. The method of claim 11, wherein, The Peribacillus sp. or the microbial agent can inhibit the growth or reproduction of the pathogenic fungi.

13. The method of claim 11, wherein, The Peribacillus sp. or the microbial agent is applied to the leaf, root, flower, or fruit part of the plant.

14. The method of claim 11, wherein, The promotion of plant growth is one or more of the following: Promoting the absorption of nutrients by the plant; promoting the growth or elongation of stems, leaves or roots; increasing the dry weight or fresh weight of the plant.

Images