A Bacillus species and its applications

Abstract

This invention discloses a Bacillus species and its applications, belonging to the field of microbial technology. The Bacillus species is a new species of the genus Bacillus (Bacillus sp. RP12), with the strain name RP12 and preservation number CCTCC No: M20221018. The Bacillus RP12 provided by this invention not only broadly antagonizes kiwifruit canker pathogens in multiple kiwifruit producing areas in China, and can be widely used to control kiwifruit canker in major kiwifruit producing areas of China; but also broadly antagonizes various plant pathogenic fungi, and can be used to control various plant diseases such as kiwifruit postharvest rot and wheat stem rot.

Description

Technical Field

[0001] This invention belongs to the field of microbial technology, specifically relating to a Bacillus species and its applications. Background Technology

[0002] Bacterial canker of kiwifruit is a devastating disease caused by *Pseudomonas syringaepv. actinidiae*, a pathogenic species of *Actinidia*. This disease is characterized by its high pathogenicity, rapid spread, wide distribution, and difficulty in control. It causes the death of a large number of plants within a short period after infection, resulting in significant losses in kiwifruit quality and yield. Currently, the control of bacterial canker in kiwifruit mainly relies on chemical control. However, the pathogen's resistance to chemical pesticides is gradually increasing, reducing the effectiveness of chemical control. Furthermore, increasing the amount of chemical pesticides used increases the risk of environmental pollution.

[0003] Biological control of plant diseases utilizes organisms and their metabolites, offering advantages such as high safety and environmental friendliness. Using Psa antagonistic microorganisms and their metabolites for biological control is an important method for the green control of kiwifruit canker. Among Psa antagonistic microorganisms, Bacillus and Paenibacillus, which produce various antibacterial substances and generate endogenous spores for stress resistance, have attracted the most attention. For example, Shao Baolin et al. isolated and screened a strain of Bacillus B2 from the rhizosphere soil of kiwifruit, which can antagonize Psa, and its fermentation broth can reduce the damage of kiwifruit canker (Shao Baolin et al., Identification and application of biocontrol Bacillus B2 for kiwifruit canker [J]. Chinese Agricultural Science Bulletin, 2015, 31(26):103-108); For example, Chinese patent document with publication number CN114591876A disclosed a strain of Paenibacillus polymyxa YLC1, which was isolated from the rhizosphere soil of kiwifruit, and its cells and fermentation broth can inhibit the growth of kiwifruit canker pathogen. The timely alternation of wettable powders made from Bacillus and Bacillus subtilis with chemical pesticides can effectively control kiwifruit canker in the main kiwifruit producing areas of Shaanxi (Qin Huqiang et al., Demonstration and effect evaluation of key technologies for the control of bacterial canker in kiwifruit [J]. Shaanxi Agricultural Sciences, 2020, 66(05):20-25).

[0004] However, the problem with existing biocontrol technologies is that when screening for Psa antagonistic microorganisms, only one Psa strain is often targeted. Psa has genetic diversity, and Psa with different genetic backgrounds show different sensitivities to the same antagonistic Psa microorganism. Most antagonistic Psa microorganisms actually only antagonize individual Psa strains. As a result, the microbial agents prepared in this way have regional limitations and low versatility in controlling kiwifruit canker, which limits the effectiveness of biocontrol agents. Summary of the Invention

[0005] To address the technical problems of regional limitations and low versatility of existing biological control technologies for the prevention and control of kiwifruit canker, this invention provides a broad-spectrum antagonistic bacillus RP12 against the kiwifruit canker pathogen Psa, and the bacillus RP12 can also antagonize a variety of plant pathogenic fungi.

[0006] The Bacillus RP12 is deposited at the China Center for Type Culture Collection (CCTCC) with accession number M20221018 and deposit date of July 4, 2022.

[0007] The Bacillus RP12 was isolated from root nodules of Robinia pseudoacacia planted in Anshan Town, Jiangxia District, Wuhan City, Hubei Province. It belongs to a new species of the genus Bacillus in taxonomy and is named Bacillus.sp.RP12. Through genome similarity comparison, it is most closely related to Bacillus pumilus.

[0008] The 16S rRNA gene sequence of the Bacillus RP12 is shown in SEQ ID NO.1, and it has the nucleotide sequence encoding the specific protein shown in SEQ ID NO.2.

[0009] The genome of Bacillus RP12 contains multiple gene clusters that synthesize antimicrobial compounds, including antibacterial lichenysin and subtilin, antifungal fengycin and bacillin, and antibacterial and antifungal zwittermicin A and bacillibactin.

[0010] This invention provides the application of Bacillus RP12 in inhibiting plant pathogens. Bacillus RP12 exhibits antagonistic activity against multiple strains of *Actinidia cuspidatum* Psa, the causal agent of kiwifruit canker, originating from various kiwifruit-producing regions in China, demonstrating broad-spectrum antibacterial activity. It also shows antagonistic activity against a variety of plant pathogenic fungi. These plant pathogenic fungi include: *Botrytiscinerea*, *Sclerotinia sclerotiorum*, *Fusarium oxysporum*, *Fusarium graminearum*, or *Magnaporthe oryzae*.

[0011] The present invention also provides a biological agent, wherein the active ingredient of the biological agent comprises Bacillus RP12.

[0012] The present invention also provides the application of the Bacillus RP12 in the prevention and control of plant diseases caused by the plant pathogen.

[0013] Preferably, Bacillus RP12 and its biological agents can be used to prevent and control kiwifruit canker, kiwifruit postharvest rot, or wheat stem base rot.

[0014] Preferably, when Bacillus RP12 is used to inhibit plant pathogens or prevent plant diseases caused by plant pathogens, the culture, fermentation broth, or supernatant of the Bacillus RP12 strain obtained by culturing the strain in a culture medium is used to inhibit plant pathogens or prevent plant diseases caused by plant pathogens.

[0015] The beneficial effects of this invention are as follows:

[0016] The Bacillus RP12 provided by this invention not only has a broad spectrum of antagonism against Psa, the pathogen of kiwifruit canker, and is highly versatile, making it applicable to the prevention and control of kiwifruit canker in multiple kiwifruit producing areas in China; but also has a broad spectrum of antagonism against plant pathogenic fungi, especially Botrytis cinerea, Sclerotinia sclerotiorum, Fusarium oxysporum, Fusarium graminearum, and Magnaphorthe oryzae, making it applicable to the prevention and control of various plant diseases such as postharvest rot in kiwifruit and stem rot in wheat. Attached Figure Description

[0017] Figure 1 The colony morphology of Bacillus RP12 grown on LB solid medium.

[0018] Figure 2 These are Bacillus RP12 cells stained with uranium acetate as observed by transmission electron microscopy.

[0019] Figure 3 This image shows the plate antagonistic effect of Bacillus RP12 against Psa, the causal agent of kiwifruit canker, isolated from multiple kiwifruit producing areas in China.

[0020] Figure 4 This is a plate antagonistic effect diagram of Bacillus RP12 against various plant pathogenic fungi.

[0021] Figure 5The graph shows the effect of Bacillus RP12 and its fermentation broth supernatant on inhibiting the infection of Actinidia kiwifruit canker pathogen Psa on kiwifruit leaves. In the graph, A is the sterile water blank control group, B is the experimental group inoculated with Actinidia kiwifruit canker pathogen Psa alone, C is the experimental group inoculated with both Actinidia kiwifruit canker pathogen Psa and Bacillus RP12, and D is the experimental group inoculated with both Actinidia kiwifruit canker pathogen Psa and Bacillus RP12 fermentation broth supernatant.

[0022] Figure 6 The diagram shows the effect of Bacillus RP12 and its fermentation broth supernatant on inhibiting gray mold infection on kiwifruit. In the diagram, A is the sterile water blank control group, B is the experimental group inoculated with gray mold alone, C is the experimental group inoculated with both gray mold and Bacillus RP12, and D is the experimental group inoculated with both gray mold and Bacillus RP12 fermentation broth supernatant.

[0023] Figure 7 The image shows the effect of Bacillus RP12 in inhibiting Fusarium graminearum infection on the stem base of wheat seedlings. In the image, A is the sterile water blank control group, B is the experimental group inoculated with Fusarium graminearum alone, and C is the experimental group inoculated with both Fusarium graminearum and Bacillus RP12. Detailed Implementation

[0024] The present invention will be further illustrated below with reference to the accompanying drawings and embodiments. It should be understood that these embodiments are for illustrative purposes only and are not intended to limit the scope of the invention.

[0025] Example 1: Isolation and Identification of Bacterial Strains

[0026] (1) Isolation of bacteria from black locust root nodules

[0027] Root nodules were collected from *Robinia pseudoacacia* trees growing in Anshan Town, Jiangxia District, Wuhan City, Hubei Province. The nodules were soaked in 70% ethanol for 1 minute, then immersed in 2% (w / v) sodium hypochlorite solution for 10 minutes, and washed 6 times with sterile water. The surface-sterilized nodules were crushed in a sterile mortar, and the juice was collected using an inoculation loop and streaked onto YMA medium (containing 10.0 g mannitol, 0.8 g yeast extract, 0.25 g K₂HPO₄, 0.25 g KH₂PO₄, 0.2 g MgSO₄·7H₂O, 0.1 g NaCl, and 15.0 g agar per liter; pH 7.0). After incubation at 30°C for 3 days, single colonies were streaked again on YMA medium for purification. Single colonies were also streaked again on LB solid medium for purification. Single colonies were then incubated overnight in LB liquid medium to the late logarithmic phase, and 50% glycerol was added to a final concentration of 15%, and stored at -80°C.

[0028] On LB solid medium, Bacillus RP12 colonies are pale yellow, opaque, with a smooth surface and regular edges. Figure 1The cells of strain RP12 are slender rods, 2.0–3.0 μm long and 0.5–0.8 μm wide, with polar flagella, Gram-positive, and containing endospores. Figure 2 ).

[0029] (2) Bacterial 16S rRNA gene amplification, sequencing and analysis

[0030] Genomic DNA was extracted from bacteria using the TIANGEN Bacterial Genome Kit. The 16S rRNA gene sequence was amplified by PCR using universal primers 27F and 1492R. The amplified product was sequenced using the Sanger method, yielding a 1385 bp DNA sequence (SEQ ID NO. 1). The 16S rRNA gene sequence was entered into the EzBioCloud website (www.ezbiocloud.net) for comparison with the 16S rRNA gene sequences of typical strains of known species. The compared 1385-base DNA sequence was compared with that of *Bacillus pumilus* ATCC7061. T and Bacillus zhangzhouensis DW5-4 T It differs from Bacillus australimaris NH7I_1 by only one base, with a similarity of 99.93%; T It differs from Bacillus safensis FO-36bT by only two bases, showing 99.86% similarity; therefore, RP12 belongs to the genus Bacillus and is closely related to species such as Bacillus pumilus and Bacillus zhangzhouensis.

[0031] (3) Bacterial whole genome sequencing, assembly and analysis

[0032] Using NEBNext Ultra TMThe DNA library construction kit was used to construct a 350-bp insert library from 1 μg of genomic DNA. The DNA library sequence was determined using an Illumina NovaSeq PE150 platform, and Readfq 10 analysis was used to remove low-quality sequences from the raw sequence data. High-quality 965 Mb sequencing data from both ends were analyzed and assembled using SOAPdenovo 2.04, SPAdes 3.11.1, and ABySS 2.0.2. The assembly results were integrated using CISA 4.0, assembling the Bacillus RP12 genome sequence into 11 scaffold fragments with a scaffold N50 of 1796852 bp. The assembled sequence covered 102 times the genome. The total length of the Bacillus RP12 genome draft is 3590437 bp, with a G+C content of 41.74%. The whole genome sequence is stored in the DDBJ / EMBL / GenBank database, accession number JANBZT000000000. Pangenomic analysis revealed that Bacillus RP12 has a unique protein encoding a nucleotide sequence SEQ ID NO.2, and the amino acid sequence of this unique protein is SEQ ID NO.3.

[0033] The complete genome sequence of Bacillus RP12 was submitted online to the Type-Strain Genome Server (https: / / tygs.dsmz.de / ). Bacillus RP12 was identified as a novel species, similar to the typical strain NCTC 10337 of the known species Bacillus pumilus. T The genome similarity was the highest, with a DDH value of 64.1% (>70% belong to the same species).

[0034] The whole genome sequence of Bacillus RP12 was analyzed using the antiSMASH 6.0 online platform. The genome of Bacillus RP12 contains multiple gene clusters that synthesize various antimicrobial compounds. These antimicrobial compounds include antibacterial lichenysin and subtilin, antifungal fengycin and bacillin, and antibacterial and antifungal zwittermicin A and bacillibactin.

[0035] Compounds in Bacillus RP12 culture and its fermentation broth supernatant were detected using matrix-assisted laser desorption / ionization time-of-flight mass spectrometry (MALDI-TOF MS). Based on the mass-to-charge ratio (m / z) of the antibacterial compound ions, it was determined that Bacillus RP12 produces antibacterial lichenin in LB medium and antifungal fenestrations on PDA medium.

[0036] Example 2: The broad-spectrum antagonistic effect of Bacillus RP12 against kiwifruit canker pathogens.

[0037] (1) Highly virulent strains of Actinidia kiwifruit canker pathogen Psa were collected from major kiwifruit producing areas in China, including strains T6-1 and ML2-12 from Zhejiang Province, strain GZ5 from Guizhou Province, strains SC530, SC540 and SCP2 from Sichuan Province, strain XA1 from Shaanxi Province, strain YN1 from Yunnan Province, and strain SYT1 from Anhui Province.

[0038] Table 1. Names and sources of *Psa* strains, the pathogen causing kiwifruit canker.

[0039] Psa strain name source T6-1 Zhejiang Province ML2-12 Zhejiang Province GZ5 Guizhou Province SC530 Sichuan Province SC540 Sichuan Province SCP2 Sichuan Province XA1 Shaanxi Province YN1 Yunnan Province SYT1 Anhui Province

[0040] (2) Prepare LB medium according to standard procedure: each liter of medium contains 10g tryptone, 5g yeast extract, 10g NaCl, pH 7.0, 1.5% (w / v) agar for solid medium, and 0.8% (w / v) agar for semi-solid medium. Sterilize at 121°C for 20 min by high pressure steam.

[0041] (3) Activation of the cryopreserved bacterial strain by streaking on LB solid medium: Take a single colony of the above-mentioned kiwifruit canker disease strain, inoculate it into LB liquid medium, and incubate at 25℃ and 180 rpm in the dark for 24 h. After centrifugation, adjust the bacterial pellet to 1×10⁻⁶ with sterile water. 8 CFU·mL -1 A single colony of Bacillus RP12 was taken and inoculated into LB liquid medium. The culture was carried out at 30°C and 180 rpm in the dark for 24 hours. The bacterial pellet after centrifugation was adjusted to 1×10⁻⁶ with sterile water. 8 CFU·mL -1 .

[0042] (4) Pour 10 mL of LB solid medium into a 90 mm petri dish, then add the kiwifruit canker pathogen suspension obtained in step (3) (1 × 10⁻⁶). 8 CFU·mL -1 Mix the 1:10 solution with LB semi-solid medium at 45–50°C and pour it onto LB solid medium to solidify. Add 5 μL of the Bacillus RP12 suspension obtained in step (3) (1×10⁻⁶) to the double-layer medium. 8 CFU·mL -1 Incubate at 25°C for 3 days.

[0043] like Figure 3As shown, *Psa*, the pathogen causing kiwifruit canker, grew in the upper layer of a double-layer LB medium; *Bacillus RP12* grew on the surface of the LB medium, producing inhibition zones, indicating that *Bacillus RP12* can antagonize all the tested kiwifruit canker pathogens.

[0044] Example 3: The antagonistic effect of Bacillus RP12 against plant pathogenic fungi

[0045] Prepare potato dextrose agar (PDA) solid medium as follows: each liter of PDA contains 200g peeled and cooked potatoes, 20g glucose, 15g agar, pH 6.5, and is sterilized by autoclaving at 121°C for 20 minutes.

[0046] Table 2. Names and sources of plant pathogenic fungi

[0047] plant pathogenic fungi name source Botrytis cinerea (gray mold) grapevines Sclerotinia sclerotiorum rape Fusarium oxysporum watermelon Fusarium graminearum wheat Rice blast fungus (Magnaporthe oryzae) rice

[0048] The aforementioned plant pathogenic fungi were subjected to confrontation culture with Bacillus RP12 using the conventional confrontation culture method. Fresh plant pathogenic fungal mycelium (5 mm in diameter) was inoculated into the center of a 90-mm PDA medium. 5 μL of Bacillus RP12 suspension (1×10⁻⁶) was then added dropwise from a distance of 3 cm from the fungal mycelium. 8 CFU·mL -1 Incubate at 30°C in the dark for 7 days.

[0049] like Figure 4 As shown, the growth of the aforementioned plant pathogenic fungal colony front edge near Bacillus RP12 is inhibited, and an inhibition zone is generated around the Bacillus RP12 colony, indicating that the growth of the aforementioned plant pathogenic fungi is inhibited.

[0050] Example 4: The effect of Bacillus RP12 in preventing and controlling kiwifruit canker.

[0051] The biocontrol effect of Bacillus RP12 and its fermentation broth supernatant on kiwifruit canker was detected by inoculating detached kiwifruit leaves with kiwifruit canker pathogen Psa strain ML2-12 or GZ5 using the needle inoculation method.

[0052] Disinfect fresh, healthy kiwifruit leaves by soaking them in 75% ethanol for 1 minute, then in 3% sodium hypochlorite for 6 minutes, washing them 6 times with sterile water, and air-drying them in a laminar flow hood. Place two layers of 125mm diameter sterile filter paper on the bottom of a 150mm petri dish, add 20mL of sterile water, and wrap the air-dried kiwifruit petioles with moist cotton to retain moisture, placing them on the filter paper in the petri dish. Select three evenly distributed sites on the back of the kiwifruit leaf, and use a sterile syringe needle to puncture two 3mm-spaced holes in the leaf epidermis. Add 20μL of sterile water and 20μL of Bacillus RP12 suspension (1×10⁻⁶) to each hole.8 CFU·mL -1 ), 20 μL of Bacillus RP12 fermentation broth supernatant diluted 10 times, and then add 20 μL of kiwifruit canker pathogen Psa suspension (1×10⁻⁶) dropwise onto sterile water, Bacillus RP12 suspension, and fermentation broth supernatant. 8 CFU·mL -1 20 μL of sterile water was used as a sterile control. The experiment was conducted in triplicate, with three leaves used for each treatment. After placing the petri dishes in an artificial climate chamber at 26℃, 16 h light-8 h dark, and 75% relative humidity for 10 days, the area of ​​leaf canker was measured, and the inhibition rate of Bacillus RP12 and its fermentation broth supernatant against kiwifruit canker was calculated.

[0053] Experimental results are as follows Figure 5 As shown, A is the sterile water blank control group, B is the experimental group inoculated with *Actinidia kiwifruit canker pathogen* Psa alone, C is the experimental group inoculated with both *Actinidia kiwifruit canker pathogen* Psa and *Bacillus RP12*, and D is the experimental group inoculated with both *Actinidia kiwifruit canker pathogen* Psa and the supernatant of fermentation broth diluted 10 times with *Bacillus RP12*. The experimental results demonstrate that the *Bacillus RP12* and its 10-fold diluted fermentation broth supernatant provided by this invention can significantly inhibit the infection of kiwifruit leaves by *Actinidia kiwifruit canker pathogen* Psa, with inhibition rates of 92% and 72%, respectively, significantly reducing the severity of kiwifruit canker.

[0054] Example 5: The effect of Bacillus RP12 in controlling postharvest rot in kiwifruit.

[0055] Botrytis cinerea was inoculated onto kiwifruit using the needle-punch inoculation method to detect the biocontrol effect of Bacillus RP12 and its fermentation broth supernatant on postharvest rot of kiwifruit.

[0056] Disinfect the surface of fresh, healthy kiwifruit by soaking in 75% ethanol for 1 minute, then in 3% sodium hypochlorite for 6 minutes, washing 6 times with sterile water, and air-drying in a laminar flow hood. Place the kiwifruit flat in a sterile, transparent polypropylene plastic box, and use a sterile syringe needle to make a 3mm × 3mm cross-shaped incision on the fruit's skin with the fruit facing upwards. Add 20μL of sterile water and 20μL of Bacillus RP12 suspension (1×10⁻⁶) at the intersection of the cross-shaped incisions. 8 CFU·mL -120 μL of the supernatant from the Bacillus RP12 fermentation broth, diluted 10-fold, was air-dried in a clean bench. A 5 mm diameter piece of fresh gray mold was placed on each of the sterile water, Bacillus suspension, and fermentation broth supernatant sites. 20 μL of sterile water served as a sterile control. The experiment was conducted in six replicates, with six kiwifruit fruits used for each treatment. The plastic boxes were inverted and placed in a 26°C, dark incubator for seven days. The area of ​​fruit rot was measured, and the inhibition rate of Bacillus RP12 and its fermentation broth supernatant against postharvest rot in kiwifruit was calculated.

[0057] Experimental results are as follows Figure 6 As shown, A is the sterile water blank control group, B is the experimental group inoculated with *Botrytis cinerea* alone, C is the experimental group inoculated with both *Botrytis cinerea* and *Bacillus RP12*, and D is the experimental group inoculated with the supernatant of fermentation broth from both *Botrytis cinerea* and *Bacillus RP12*. The experimental results demonstrate that the *Bacillus RP12* and its 10% diluted fermentation broth supernatant provided by this invention can significantly inhibit *Botrytis cinerea* infection of kiwifruit, with inhibition rates of 100% and 90%, respectively, significantly reducing the incidence of postharvest rot in kiwifruit.

[0058] Example 6: The effect of Bacillus RP12 in controlling wheat stem rot

[0059] Wheat seeds were surface-sterilized by soaking in 75% ethanol for 1 minute, soaking in 3% sodium hypochlorite for 6 minutes, and washing 6 times with sterile water. The surface-sterilized wheat seeds were then immersed in a Bacillus RP12 bacterial suspension (1×10⁻⁶). 8 CFU·mL -1 Soak the inoculated wheat seeds in sterile water for 4 hours, using sterile water as a blank control. Air-dry the inoculated wheat seeds in a clean bench. Place two layers of 125-mm diameter sterile filter paper on the bottom of a 150-mm petri dish, add 10 mL of sterile water, and spread the air-dried wheat seeds on the filter paper in the petri dish, 12 seeds per dish, 3 dishes per treatment. Germinate and grow in the dark at 25℃ for 3 days. Cut off the 2-3 mm coleoptile of the wheat seedling with sterile scissors, and place a 5 mm diameter fresh Fusarium graminearum mycelium cake on the coleoptile wound, using a sterile PDA block as a negative control. Place the wheat seedlings in the petri dishes in an artificial climate chamber at 26℃, 16 h light-8 h dark, and 75% relative humidity. Eight days after fungal inoculation, measure the length of the coleoptile and calculate the inhibition rate of Bacillus RP12 against wheat stem rot.

[0060] Experimental results are as follows Figure 7 As shown, A is the sterile water blank control group, B is the experimental group inoculated with Fusarium graminearum alone, and C is the experimental group inoculated with both Fusarium graminearum and Bacillus RP12. The experimental results demonstrate that the Bacillus RP12 provided by this invention can significantly inhibit Fusarium graminearum infection, with an inhibition rate of 60%, and reduce the severity of wheat stem rot.

[0061] The embodiments described above provide a detailed explanation of the technical solutions of the present invention. It should be understood that the above descriptions are merely specific embodiments of the present invention and are not intended to limit the present invention. Any modifications, additions, or similar substitutions made within the scope of the principles of the present invention should be included within the protection scope of the present invention.

Claims

1. A type of Bacillus ( Bacillus sp.)RP12, characterized in that, A new species belonging to the genus Bacillus in systematic classification. Bacillus sp. nov., strain name RP12, is deposited at the China Center for Type Culture Collection, accession number CCTCC No: M20221018.

2. The Bacillus RP12 as described in claim 1, characterized in that, The 16S rRNA gene sequence of the Bacillus RP12 is shown in SEQ ID NO.

1.

3. The Bacillus RP12 as described in claim 1, characterized in that, It has the encoding nucleotide sequence of the specific protein shown in SEQ ID NO.

2.

4. A biological agent, characterized in that, The active ingredient comprises Bacillus RP12 as described in claim 1.

5. The application of Bacillus RP12 as described in claim 1 in inhibiting plant pathogens, characterized in that, The plant pathogen is: *Pseudomonas syringae*, a pathogenic strain of Actinopterygium in kiwifruit (…). Pseudomonas syringae pv . actinidiae ), gray mold ( Botrytis cinerea ), Sclerotinia sclerotiorum ( Sclerotinia sclerotiorum Fusarium oxysporum ( Fusarium oxysporum Fusarium graminearum ( ), Fusarium graminearum ) or rice blast fungus ( Magnaporthe oryzae ).

6. The application of Bacillus RP12 as described in claim 1 in the prevention and control of plant diseases caused by plant pathogens, characterized in that, Plant diseases caused by plant pathogens include kiwifruit canker, kiwifruit postharvest rot, or wheat stem base rot.

7. The application as described in claim 5 or 6, characterized in that, In application, the fermentation broth or supernatant obtained by culturing the Bacillus RP12 strain in a culture medium is used to inhibit plant pathogens or prevent plant diseases caused by plant pathogens. The plant pathogen inhibited by the supernatant of the fermentation broth is *Pseudomonas syringae*, a pathogen causing disease in kiwifruit. Pseudomonas syringae pv . actinidiae ) or gray mold ( Botrytis cinerea The supernatant of the fermentation liquid is used to prevent and control plant diseases such as kiwifruit canker or kiwifruit postharvest rot.

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