Bacillus velezensis MY-8 and application thereof in preventing and treating plant diseases

Abstract

The bacillus velezensis MY-8 disclosed by the invention has obvious broad-spectrum antibacterial activity, and has an obvious inhibition effect on various plant pathogenic bacteria including cabbage alternaria alternata, rhizopus arrhizus, cladosporium very fine, fusarium tripteroides, alternaria alternata, Columbia cladosporium, botryosphaeria dothidea, fusarium oxysporum, geotrichum candidum, colletotrichum gloeosporioides and A. arundinis, and the bacillus velezensis MY-8 has an obvious inhibition effect on various plant pathogenic bacteria such as bacillus velezensis MY-8. The compound has a remarkable control effect on downy mildew, is expected to be prepared into a microbial preparation for biological control, and can be used as a biopesticide, a bio-fertilizer, a plant growth promoter and the like. The bacillus velezensis MY-8 disclosed by the invention can also express a structural domain containing protein of DUF4393, so that the bacillus velezensis MY-8 has potential bacteriophage infection resistance.

Description

Technical Field

[0001] The invention belongs to the technical field of microorganisms, and in particular relates to Bacillus Velezii MY-8 and application thereof in preventing and controlling plant diseases. Background Art

[0002] The frequent occurrence of plant diseases has brought huge losses to global agricultural production. It is estimated that more than 20% of crops in the world have reduced or no harvest due to diseases every year, which seriously threatens food security and economic development. Fungal diseases caused by plant pathogens such as Botrytis cinerea, Fusarium graminearum, and Pythiumaphanidermatum are widely present in agricultural ecosystems and can infect a variety of crops, causing them to rot, wither, reduce yields, and even die. It is estimated that fungal diseases reduce the output of crops such as rice, wheat, corn, soybeans, and potatoes by 125 million tons per year worldwide. Among them, the damage to rice, wheat, and corn causes an annual economic loss of US$60 billion to global agriculture. This situation has a particularly serious impact on some developing regions. Experts' calculations show that if, in extreme cases, the above five major food crops are hit by fungal disease pandemics at the same time in a year, the global food production reduction in that year may be as high as 900 million tons, which will cause 4.2 billion people to starve and cause global famine. Therefore, plant disease prevention and control is one of the most critical links in agricultural production.

[0003] At present, chemical pesticides are still the main means of controlling plant diseases, but the long-term use of chemical pesticides has caused increasingly serious environmental pollution, food safety problems and pathogen resistance. Some commonly used fungicides, such as chlorothalonil and mancozeb, have been proven to cause environmental residues and destroy the ecological balance of the soil. In addition, more and more plant pathogens have developed resistance to chemical pesticides, limiting the effectiveness of traditional fungicides.

[0004] Natural biological control products can not only reduce chemical residues, but also protect the quality and yield of crops. Bacillus spp. is a type of Gram-positive bacteria that can form spores that tolerate environmental stress. It has strong stress resistance and broad-spectrum antibacterial activity and has become one of the main microorganisms for the development of biological control agents. Studies have shown that Bacillus can inhibit the growth of plant pathogens by producing a variety of antimicrobial substances (such as lipopeptides, enzymes and volatile organic compounds), thereby effectively preventing and controlling plant diseases. In addition, Bacillus can colonize in the rhizosphere of plants, form biofilms, and improve the resistance of plants to pathogenic bacteria. Due to its advantages of being efficient, environmentally friendly and sustainable, biological control agents based on Bacillus have received more and more attention in the agricultural field and have become an alternative or supplement to chemical pesticides. With people's attention to food safety and environmental protection, the use of microbial agents such as Bacillus to replace chemical pesticides has become a green, safe and sustainable means of plant disease control.

[0005] Bacillus Velez is a species of Bacillus. Although it also has the stress resistance and broad-spectrum antibacterial activity of Bacillus, the antibacterial spectrum of different species of Bacillus Velez and the inhibitory effect on the same plant pathogen may still be significantly different. Therefore, it is still necessary to develop Bacillus Velez with a broader antibacterial spectrum and stronger inhibitory effect on specific plant pathogens. Summary of the invention

[0006] In order to meet the current demand for Bacillus velezensis with better antibacterial performance, the present invention provides a Bacillus velezensis MY-8 with broad-spectrum antibacterial activity.

[0007] In one aspect, the present invention provides a Bacillus velezensis MY-8, and the deposit number of the Bacillus velezensis MY-8 is CCTCC NO: M20241654.

[0008] In one or more embodiments, the Bacillus Velez MY-8 has genes expressing one or more proteins selected from the following:

[0009] dynein heavy chain, V-phase sporulation protein AE, SnoaL-like domain-containing protein, transcription activator GCR1-like domain-containing protein, RING-type domain-containing protein, synaptotagmin-1X13 isoform, RDD domain-containing protein, bacteriophage protein, RNA polymerase subunit β, 2'-hydroxyisoflavone reductase, RCAR3-interacting f-box protein 1, PHD finger protein 21B, protein CBG18524, type 1 ABC transmembrane domain-containing protein, haloacid dehalogenase domain-containing protein, NADH dehydrogenase subunit 5, urea-active transporter , replication initiation protein, SANTA domain-containing protein, α-1,2-fucosyltransferase, DUF3784 domain-containing protein, PQQ enzyme repeat domain protein, tRNA(isoleucine)-lysine synthetase, DUF1878 family protein, Flp family type IVb fimbriae protein, type II secretion system protein GspF domain-containing protein, LPXTG cell wall anchor domain-containing protein, pre-fimbriae type IV endopeptidase peptidase domain-containing protein, BRO domain-containing protein, DUF1140 family protein, and DUF4393 domain-containing protein.

[0010] In one or more embodiments, the domain-containing protein of DUF4393 comprises the amino acid sequence shown in SEQ ID NO:1.

[0011] In another aspect, the present invention provides a bacterial agent, comprising the Bacillus Velezii MY-8 or a culture solution thereof as described in any embodiment herein.

[0012] In one or more embodiments, the bacterial agent is bacterial powder obtained after drying.

[0013] Preferably, the bacterial powder is freeze-dried bacterial powder or spray-dried bacterial powder.

[0014] In another aspect, the present invention provides the use of Bacillus Velez MY-8 or a bacterial agent as described in any embodiment of the present invention in controlling plant diseases, wherein the plant diseases are caused by plant pathogens, and the plant pathogens are selected from one or more of Alternaria brassicicola, Rhizopus arrhizus, Cladosporium tenuissimum, Fusarium tricinctum, Alternaria alternat, Cladosporium colombiae, Botryosphaeria dothidea, Fusarium oxysporum, Geotrichum candidum, Colletotrichum gloeosporioides and Apiospora arundinis.

[0015] In another aspect, the present invention provides the use of Bacillus Velez MY-8 or the bacterial agent as described in any embodiment herein in controlling plant downy mildew.

[0016] Preferably, the plant is Shanghai Green.

[0017] In another aspect, the present invention provides a method for preparing the bacterial agent as described in any embodiment of the present invention, the method comprising inoculating Bacillus Velez MY-8 into a liquid culture medium for culturing, and diluting the culture solution by an appropriate multiple to obtain the bacterial agent.

[0018] In one or more embodiments, the method comprises inoculating Bacillus Velezii MY-8 into a liquid culture medium, culturing at a temperature of 30 to 45° C. for 4 to 24 hours until the late logarithmic growth phase; and diluting the culture fluid obtained by culturing by 0 to 1000 times to obtain a bacterial agent.

[0019] In one or more embodiments, the culture temperature is 35-40° C., and the culture time is 7-12 hours.

[0020] Preferably, the culture temperature is 37° C., and the culture time is 10 h.

[0021] Preferably, the culture solution is diluted 0 times, 10 times, 20 times, 50 times, 100 times, 200 times, 300 times, 400 times, 500 times or 1000 times to obtain the bacterial agent.

[0022] In one or more embodiments, the culture solution is diluted 0 to 500 times to obtain a bacterial agent.

[0023] In one or more embodiments, the method further comprises freeze-drying or spray-drying the bacterial agent to prepare freeze-dried bacterial powder or spray-dried bacterial powder.

[0024] In one or more embodiments, the inlet temperature of the spray drying is 90-150°C, the feed rate is 400-1000 mL / h, and the air inlet rate is 10-60 M 3 / h, the spray pressure is 0.1~1.0MPa.

[0025] Preferably, the inlet temperature of the spray drying is 100°C, 110°C or 120°C; preferably, the feed rate is 500mL / h, 600mL / h or 700mL / h; preferably, the air inlet rate is 20M 3 / h,25M 3 / h or 30M 3 / h; preferably, the spray pressure is 0.2MPa, 0.25MPa or 0.3MPa.

[0026] The Bacillus velezensis MY-8 of the present invention has obvious broad-spectrum antibacterial activity, and has significant inhibitory effects on various plant pathogens including Alternaria brassicae, Rhizopus arrhizus, Cladosporium exigua, Fusarium trilineatum, Alternaria alternata, Cladosporium columbiae, Botrytis cinerea, Fusarium oxysporum, Geotrichum candidum, Colletotrichum gloeosporioides, and A. arundinis, and also has significant control effects on downy mildew, and is expected to be prepared into a microbial preparation for biological control, and play the role of biological pesticides, biological fertilizers, plant growth promoters, etc. BRIEF DESCRIPTION OF THE DRAWINGS

[0027] Figure 1 This is the colony morphology of strain MY-8.

[0028] Figure 2 This is the cell morphology of strain MY-8 under a microscope.

[0029] Figure 3 This is the cell morphology of strain MY-8 under scanning electron microscope.

[0030] Figure 4 This is the phylogenetic tree of strain MY-8 based on the 16S rDNA sequence.

[0031] Figure 5 This is the phylogenetic tree of strain MY-8 based on the gyrB sequence.

[0032] Figure 6The full genome map and plasmid map of strain MY-8. Figure 6 A is the whole genome map of strain MY-8; Figure 6 B and Figure 6 C is a plasmid circle map of strain MY-8.

[0033] Figure 7 It is the structure of the domain-containing protein of DUF4393 encoded by the Chromosome_ORF2405 gene of strain MY-8. Figure 7 A is the structure of the domain-containing protein of DUF4393 predicted by Alphafold 3; Figure 7 B is a structural comparison diagram of the DUF4393 domain-containing protein and the DUF4393 protein of Ureibacillus terrenus.

[0034] Figure 8 This is a graph showing the inhibitory effect of the fermentation solution of strain MY-8 on 11 plant pathogenic fungi.

[0035] Fig. 9 These are the results of the gradient inhibition experiment of the fermentation broth of strain MY-8 against Cladosporium tenuissima.

[0036] Fig.10 These are the results of the gradient inhibition experiment of the fermentation broth of strain MY-8 against Rhizopus arrhizus.

[0037] Fig.11 These are the results of the gradient inhibition experiment of the fermentation broth of strain MY-8 against Apiospora arundinis.

[0038] Fig.12 This is a picture of the effect of using clean water, chemical pesticides and fermentation liquid of strain MY-8 to control downy mildew in the field. Fig.12 A is a graph showing the effect of downy mildew control using clean water in the field; Fig.12 B is a graph showing the effect of downy mildew control using chemical pesticides in the field; Fig.12 C is a graph showing the effect of downy mildew control using the fermentation liquid of strain MY-8 in the field.

[0039] Preservation matters

[0040] The Bacillus velezensis MY-8 obtained by the present invention was submitted to the China Type Culture Collection (CCTCC) located at Wuhan University, Wuhan, China for preservation on July 22, 2024, with the preservation number being CCTCCNO: M20241654. DETAILED DESCRIPTION

[0041] The present invention is further described in detail below in conjunction with specific examples. It should be understood that the following examples are only used to illustrate the present invention and are not used to limit the scope of the present invention.

[0042] The inventor of the present application isolated a strain from farmland soil in Qingpu District, Shanghai. Combined with colony morphology observation and 16S rDNA detection, it was identified as Bacillus velezensis and named MY-8. Further research found that Bacillus velezensis MY-8 has broad-spectrum antibacterial activity against pathogenic bacteria and has broad application prospects. The Bacillus velezensis has been submitted to CCTCC for deposit.

[0043] Example 1: MY-8 colony morphology

[0044] Take MY-8 out of the -80℃ freezer, inoculate it on the LB medium plate in three-zone stripe pattern, and place it in a 37℃ incubator for 24 hours. After it grows into a distinct single colony, observe the colony morphology and take photos, including the colony shape, color, surface, edge, transparency, and ridge shape.

[0045] Colony morphology Figure 1 As shown, the colonies are 2.5-5 mm in diameter, and are raised, milky white, opaque, round, with irregular edges and wrinkled surfaces.

[0046] Example 2: MY-8 microscopic morphology

[0047] The bacterial strain cultured on the LB medium plate for 12 h in Example 1 was selected for Gram staining microscopy, and the method was referred to the Manual of Identification of Common Bacterial Systems. Figure 2 As shown, the bacteria are Gram-positive and short rod-shaped.

[0048] MY-8 was inoculated into LB liquid culture medium at an inoculum rate of 2% (v / v), cultured in a shaking incubator at 37°C for 10 hours, activated for two generations, and the activated MY-8 was cultured to the logarithmic growth phase. Take the activated MY-8 bacterial solution, centrifuge at 4000rpm for 10 minutes, discard the supernatant, and rinse with PBS three times; then add 2.5% glutaraldehyde to fix the cells for 16 hours, and then rinse with PBS three times. The fixed and rinsed cells were dehydrated in 30%, 50%, 60%, 70%, 80%, 90% and 100% ethanol in a gradient manner for 10 minutes; among them, 100% ethanol dehydration was repeated 3 times. After dehydration, the bacteria were resuspended in an appropriate amount of 100% ethanol. The dehydrated bacterial solution was aspirated and dropped onto a coverslip, pre-frozen in a -80°C refrigerator for 15 minutes, and then freeze-dried for 2 hours. After freeze-drying, the surface of the sample was sprayed with gold, and observed and photographed using an S-3400N scanning electron microscope. The results are as follows Figure 3shown.

[0049] Depend on Figure 3 It can be seen that MY-8 has a rod-shaped morphology observed under a scanning electron microscope, which is consistent with the typical characteristics of Bacillus.

[0050] Example 3: Identification of MY-8 strain

[0051] Strain identification method: The DNA of strain MY-8 was extracted using a bacterial DNA extraction kit, and the 16S rDNA and gyrB conservative sequences were selected for PCR amplification of the DNA of strain MY-8. The resulting PCR products were sequenced by Shanghai Qingke Biotechnology Co., Ltd. The sequences of 16S rDNA or gyrB were compared with the NCBI database, and the sequencing results were analyzed using MEGA11 software. The maximum likelihood method was used to construct a multi-gene phylogenetic tree to clarify the classification status of strain MY-8.

[0052] 3.1 Construction of 16S rDNA evolutionary tree

[0053] 16S rDNA sequence analysis is a commonly used method for bacterial identification. The genome of the strain was amplified using 16S primers 27F (5'-AGAGTTTGATCCTGGCTCAG-3') and 1492R (5'-GGTTACCTTGTTACGACTT-3').

[0054] Reaction system: 0.5 μL each of primer 27F and primer 1492R with a concentration of 10 μM, 12.5 μL of mixTaq enzyme (2×, purchased from Yisheng Biotechnology, product number 10102ES03), 1 μL of DNA, and add water to a total volume of 25 μL.

[0055] Reaction conditions: pre-denaturation at 95°C for 5 min; denaturation at 98°C for 10 s, annealing at 65°C for 1 min, extension at 72°C for 2 min, 30 cycles; final extension at 72°C for 8 min.

[0056] The amplified products were sent to Shanghai Qingke Biotechnology Co., Ltd. for sequencing and splicing. The determined 16S rDNA sequence was analyzed with the known nucleic acid sequences in GenBank by BLSAT, and sequences homologous to the strain were obtained. The phylogenetic tree was constructed using MEGA11 software, as shown in Figure 4 shown.

[0057] 3.2 Construction of the gyrB gene evolutionary tree

[0058] GyrB sequence analysis is a commonly used method for bacterial identification. The genome of the strain was amplified using gyrB primers UP1 (5'-GAAGTCATCATGACCGTTCTGCAYGCNGGNGGNAARTTYGA-3') and UP2R (5'-AAACTGGTCTGACCCGTGTC-3').

[0059] Reaction system: 0.5 μL of 10 μM primer UP1 and primer UP2R, 12.5 μL of mixTaq enzyme, 1 μL of DNA, and add water to 25 μL.

[0060] Reaction conditions: pre-denaturation at 95°C for 5 min; denaturation at 98°C for 10 s, annealing at 65°C for 1 min, extension at 72°C for 2 min, 30 cycles; final extension at 72°C for 8 min.

[0061] The amplified products were sent to Shanghai Qingke Biotechnology Co., Ltd. for sequencing and splicing. The determined gyrB sequence was analyzed with the known nucleic acid sequence in GenBank by BLSAT, and the sequence homologous to the strain was obtained. The phylogenetic tree was constructed using MEGA11 software, as shown in Figure 5 shown.

[0062] 3.3 Identification results of MY-8 strain

[0063] According to the multi-gene phylogenetic tree of 16S rDNA and gyrB of strain MY-8, the strain was located on the same branch as Bacillus velezensis and had the highest similarity.

[0064] According to the above analysis results, combined with the strain morphological characteristics and phylogenetic tree construction and identification, it can be known that strain MY-8 belongs to Bacillus velezensis.

[0065] Example 4: MY-8 genome profile

[0066] Bacillus velezensis MY-8 was cultured to its exponential growth phase, and the bacterial pellet was obtained by centrifugation. The bacterial pellet was cryopreserved and sent to Meiji Biotechnology Co., Ltd. (Shanghai) for DNA extraction, whole genome sequencing and assembly.

[0067] The results showed that the whole genome of Bacillus velezensis MY-8 was a circular chromosome of 4,104,034 bp in length, with 2 natural plasmids, a GC content of 46.15%, and 3916 coding genes. Figure 6A; the plasmid A genome circle diagram is as shown Figure 6 B shows the genome circle of plasmid B. Figure 6 As shown in C.

[0068] Example 5: Analysis of MY-8 anti-phage infection functional genes

[0069] After whole genome analysis of MY-8, gene Chromosome_ORF2405 was found, and its corresponding amino acid sequence is:

[0070] MDNEKKKDILETSTGTIVDAIVDAASSTGADVVKELTSNLVGEFLVEVGASAIPGVSGAVSSYKRVRFEKNIKKLVEGLANRIEELKSSLEDKSETQQHKINTLFTYVLDYVIDEKQELKIDIMVNGFINISHHEDISE DFVLIYYDVLRDLLRLIDISVLRFYSKLKNYDQEGQITIIDILEEYEVSYEQYESVRHNLLRFGLLTTKKDYKVIDDIEELSQSISNIHKYFKAIKEGKKRLPTLKDPKIKSTDKFELSKFGKDFVWFFLNDTTRL(SEQ ID NO: 1).

[0071] Comparison with Uniprot and NCBI databases revealed that the sequence was not found in the existing Bacillus Velez genome. The function of the amino acid sequence predicted by Uniprot database was: DUF4393 domain-containing protein. A search in Interpro database revealed that it was a type of protein related to the bacterial defense system (anti-phage infection) and was commonly found in lactic acid bacteria.

[0072] The above amino acid sequence was used to predict its protein structure online using Alphafold3. The predicted structure is as follows Figure 7 A. The structural similarity was compared with the DUF4393 protein structure from Ureibacillus terrenus provided in Uniprot. The comparison results are shown in Figure 7 As shown in Figure B, its RMSD value is 1.256, indicating that the two proteins are highly similar, and the protein structures are very close in spatial arrangement, with only very small local differences. Therefore, it can be judged that Bacillus velezensis MY-8 has the potential to resist phage infection, which promotes its survival ability in agricultural planting environments.

[0073] Example 6: Plasmid gene analysis of strain MY-8

[0074] The genes on the MY-8 plasmid were analyzed using the Uniprot and NCBI databases, and a total of 55 genes were found that were not found in the existing Bacillus velez genome in the database. Among them, there are 30 genes with confirmed functions as shown in Table 1, including proteins related to spore formation (pA_gene0014), transcription activator domain (pA_gene0019), RNA polymerase subunit (pA_gene0047), 2-hydroxyisoflavone reductase (pA_gene0049), dehalogenase domain (pA_gene0061), NADH dehydrogenase subunit (pA_gene0064), replication initiation protein (pA_gene0074), α-1,2-fucosyltransferase (pA_gene0077), tRNA (isoleucine)-lysine synthetase (pA_gene0083), Flp family IVb pilus protein (pA_gene0088), etc., which play an important role in the physiological metabolism of the strain, as well as enzymes with application value; there are 25 genes with undetermined functions, among which, according to the prediction of the Uniprot database, the genes that can correspond to proteins are shown in Table 1 below. It can be seen that Bacillus velezensis MY-8 has relatively significant differences at the genetic level from the currently reported Bacillus velezensis.

[0075] Table 1: Characteristic genes in the plasmid of Bacillus velez MY-8

[0076]

[0077]

[0078]

[0079] Example 7: Broad-spectrum antibacterial experiment of MY-8 against plant pathogens

[0080] Prepare PDA solid culture medium, sterilize it at 121°C, wait for it to cool to about 55°C, take 15 mL and pour it into a sterile culture dish, wait for it to solidify to make a PDA culture medium plate; prepare a certain number of plates according to this method. A small amount of mycelium of 11 pathogenic fungi screened from the soil of Qingpu area, including Alternaria brassicicola, Rhizopus arrhizus, Cladosporium tenuissimum, Fusarium tricinctum, Alternaria alternat, Cladosporium colombiae, Botryosphaeria dothidea, Fusarium oxysporum, Geotrichum candidum, Colletotrichum gloeosporioides, and Apiospora arundinis, was picked up with a sterile inoculation needle and inoculated on PDA plates, and cultured in a constant temperature incubator at 28±1℃ until the mycelium covered the entire plate for later use.

[0081] MY-8 was inoculated in LB liquid culture medium at an inoculum volume of 2% (v / v), and cultured in a shaking incubator at 37°C for 10 hours to activate two generations. The MY-8 bacterial solution was diluted with unsolidified PDA culture medium cooled to 55±1°C at a volume ratio of 1:9 (bacterial solution: PDA culture medium), and after sufficient mixing, 10 mL was transferred to a sterile culture dish with a sterile pipette, and the culture dish was gently shaken to make it evenly flat, and after solidification, a test plate was made; a certain number of test plates were prepared according to this method. A plate prepared by mixing LB liquid culture medium and PDA culture medium at a volume ratio of 1:9 was used as a negative control plate; a certain number of negative control plates were prepared according to this method.

[0082] Use a sterile hole puncher (6mm inner diameter) to punch holes at the same radius edge in each test plate and the corresponding negative control plate. Use sterile tweezers to take the bacterial cakes from the PDA plates where the pathogenic fungi grow, and place the bacterial cakes in the middle of the prepared test plate and the corresponding negative control plate. Repeat each treatment 3 times. Place the plates in a constant temperature incubator and incubate at 28±1℃ for 24h~48h. Figure 8 The results showed that the fermentation liquid containing MY-8 in the plate had a significant inhibitory effect on the growth of the above 11 plant pathogenic strains.

[0083] Example 8: Gradient antibacterial experiment of MY-8 against plant pathogen Cladosporium tenuissima

[0084] PDA culture medium plates, test plates and negative control plates were prepared according to the method of Example 7.

[0085] Use a sterile hole puncher (6mm inner diameter) to punch holes at the same radius edge in each test plate and the corresponding negative control plate. Use sterile tweezers to take the bacterial cake from the PDA plate with Cladosporium spp. and place the bacterial cake in the middle of the prepared test plate and the corresponding negative control plate. Repeat each treatment 3 times. Place the plate in a constant temperature incubator and incubate at 28±1℃ for 24-48h before taking it out. The growth of Cladosporium spp. on each plate is shown in Figure 1. Fig. 9 The colony diameter on the plate was measured by the cross method, and the average value of the two measurements was the colony diameter. The inhibition rate of MY-8 against Cladosporium tenuissima was calculated based on the measured colony diameter.

[0086] Inhibition rate (%) = [(control colony diameter - 6) - (treated colony diameter - 6)] / (control colony diameter - 6) × 100%.

[0087] The antibacterial rates of the MY-8 bacterial solution in the plate were 100% (500 times dilution), 100% (400 times dilution), 100% (300 times dilution), 100% (200 times dilution), 100% (100 times dilution) and 100% (original solution) in the order of dilution multiples. It can be seen that the MY-8 bacterial solution of different dilutions has a very significant inhibitory effect on the growth of Cladosporium tenuissima.

[0088] Example 9: Gradient antibacterial experiment of MY-8 on plant pathogen Rhizopus arrhizus

[0089] Prepare PDA culture medium plates, test plates and negative control plates according to the method of Example 7. Use a sterile hole puncher (inner diameter 6mm) to punch holes at the same radius edge in each test plate and the corresponding negative control plate. Use sterile tweezers to take the bacterial cake on the PDA plate where Rhizopus arrhizus grows, and place the bacterial cakes in the middle of the prepared test plate and the corresponding negative control plate. Repeat each treatment 3 times. Place the plates upright in a constant temperature incubator and culture at 28±1℃ for 24 to 48 hours, then take them out. The growth of Rhizopus arrhizus on each plate is shown in the figure below. Fig.10 The calculation method of the antibacterial rate is the same as that of Example 8.

[0090] The inhibition rates of the MY-8 bacterial solution in the plate were 71% (400 times dilution), 80% (300 times dilution), 88% (200 times dilution), 93% (100 times dilution) and 99% (stock solution) in the order of dilution multiples. It can be seen that the MY-8 bacterial solution of different dilutions has a significant inhibitory effect on the growth of Rhizopus arrhizus.

[0091] Example 10: Gradient antibacterial experiment of MY-8 against plant pathogen Apiospora arundinis

[0092] Apiospora arundinis is a plant pathogen reported for the first time in 2024, which mainly causes wet root rot of Psoralea corylifolia.

[0093] Prepare PDA culture medium plates, test plates and negative control plates according to the method of Example 7. Use a sterile hole puncher (inner diameter 6mm) to punch holes at the same radius edge in each test plate and the corresponding negative control plate. Use sterile tweezers to take the bacterial cake from the PDA plate where Apiospora arundinis grows, and place the bacterial cakes in the middle of the prepared test plate and the corresponding negative control plate. Repeat each treatment 3 times. Place the plates upright in a constant temperature incubator and culture at 28℃±1℃ for 24h~48h, then take them out. The growth of Apiospora arundinis on each plate is shown in the figure. Fig.11 The calculation method of the antibacterial rate is the same as that of Example 8.

[0094] The inhibition rates of the MY-8 bacterial solution in the plate were 68% (400 times dilution), 71% (300 times dilution), 74% (200 times dilution), 80% (100 times dilution) and 87% (original solution) in the order of dilution multiples. It can be seen that the MY-8 bacterial solution of different dilutions has a significant inhibitory effect on the growth of Apiospora arundinis.

[0095] Example 11, MY-8 control effect on downy mildew

[0096] This example explores the control effect of Bacillus Velez MY-8 on downy mildew through field experiments.

[0097] Experimental location: Vegetable greenhouse in Qingpu District, Shanghai.

[0098] Experimental variety: Shanghai green (also known as "chicken feather vegetable").

[0099] In the laboratory, the MY-8 strain was inoculated into LB liquid medium at a 2% (v / v) inoculation amount, cultured at 37°C in a shaking incubator for 10 hours, and activated for two generations. The activated MY-8 bacterial solution cultured to the late logarithmic growth stage was used as the fermentation stock solution.

[0100] The stock solution was diluted 10 times with water as the test group solution, clean water as the negative control group solution, and chemical pesticides as the positive control group solution. Use a manual sprayer to evenly spray a layer of solution on the front and back sides of the Shanghai Green leaves, and spray once every 5 days. The experimental group sprayed about 0.1 kg of the test group solution per square meter of land each time, the negative control group sprayed about 0.1 kg of the negative control group solution (clean water) per square meter of land each time, and the positive group sprayed about 0.1 kg of the positive control group solution (chemical pesticide) per square meter of land each time, and sprayed continuously for 2 months. The control effect is as follows. Fig.12 shown.

[0101] Depend on Fig.12 It can be seen that compared with the negative control group ( Fig.12 A), chemical pesticides ( Fig.12 B) and MY-8 fermentation broth dilution ( Fig.12 C) has a significant control effect on downy mildew.

[0102] Example 12: Preparation of bacterial powder by spray drying of MY-8 fermentation broth

[0103] MY-8 was fermented in a 5L fermenter (liquid volume 3L) for 18h to obtain a fermentation solution, which could be directly placed in the feed port of a spray dryer for drying.

[0104] Since spray drying is a high-temperature stress process, the number of viable bacteria in the product after spray drying is affected by many factors. After preliminary screening, four factors, namely, inlet temperature (A), feed rate (B), air inlet rate (C) and spray pressure (D), were selected for orthogonal experiments at three levels to determine the optimal spray drying conditions. The parameters are shown in Table 2.

[0105] Table 2: Orthogonal experimental parameters for spray drying conditions

[0106]

[0107] 1L of MY-8 fermented bacterial liquid was placed in the feed port of the spray dryer for spray drying. The number of viable bacteria in the obtained bacterial powder was used as the evaluation standard. The results are shown in Table 3. The spray temperature corresponding to No. 3 is 110°C, the feed rate is 600mL / h, and the air inlet rate is 25M 3 / h and a spray pressure of 0.25MPa were the optimal spray conditions. After testing, the number of live bacteria in the spray-dried bacterial powder reached 8.23×10 9 CFU / g, the number of live bacteria in the other serial numbered powders can also be maintained at the same order of magnitude. It can be seen that after MY-8 is made into spray-dried powder, it can still maintain most of the activity of the strain, which is conducive to further improving its stability in storage and transportation, and has significant application potential.

[0108] Table 3: Results of orthogonal test for spray drying parameter optimization

[0109]

[0110] Note: n=3, SPSS19.0 software was used for Duncan multiple range test and one-way analysis of variance. Different letters indicate significant differences among treatments (p<0.05)

[0111] According to the above embodiments, the Bacillus velezensis MY-8 of the present invention has obvious broad-spectrum antibacterial activity, and has significant inhibitory effects on various plant pathogens including Alternaria brassicae, Rhizopus arrhizus, Cladosporium exigua, Fusarium trilineatum, Alternaria alternata, Cladosporium columbiae, Botrytis cinerea, Fusarium oxysporum, Geotrichum candidum, Colletotrichum gloeosporioides, and A.arundinis, and also has significant control effects on downy mildew, and is expected to be prepared into a microbial preparation for biological control, and play the role of biological pesticides, biological fertilizers, plant growth promoters, etc.

[0112] The above embodiments are only for illustrating the technical concept and features of the present invention, and their purpose is to enable those skilled in the art to understand the contents of the present invention and implement them accordingly, but they cannot be used to limit the protection scope of the present invention. All equivalent transformations or modifications based on the spirit of the present invention, such as using the Bacillus velezensis MY-8 of the present invention to control other plant pathogens, or making the Bacillus velezensis MY-8 of the present invention into freeze-dried bacterial powder or other bacterial agents, etc., should be covered within the protection scope of the present invention.

Claims

1. A Bacillus velezensis MY-8, characterized in that The deposit number of the Bacillus Velez MY-8 is CCTCC NO: M20241654.

2. The Bacillus Velez MY-8 according to claim 1, characterized in that The Bacillus Velez MY-8 has genes expressing one or more proteins selected from the following: dynein heavy chain, V-phase sporulation protein AE, SnoaL-like domain-containing protein, transcription activator GCR1-like domain-containing protein, RING-type domain-containing protein, synaptotagmin-1X13 isoform, RDD domain-containing protein, bacteriophage protein, RNA polymerase subunit β, 2'-hydroxyisoflavone reductase, RCAR3-interacting f-box protein 1, PHD finger protein 21B, protein CBG18524, type 1 ABC transmembrane domain-containing protein, haloacid dehalogenase domain-containing protein, NADH dehydrogenase subunit 5, urea-active transporter , replication initiation protein, SANTA domain-containing protein, α-1,2-fucosyltransferase, DUF3784 domain-containing protein, PQQ enzyme repeat domain protein, tRNA(isoleucine)-lysine synthetase, DUF1878 family protein, Flp family type IVb fimbriae protein, type II secretion system protein GspF domain-containing protein, LPXTG cell wall anchor domain-containing protein, pre-fimbriae type IV endopeptidase peptidase domain-containing protein, BRO domain-containing protein, DUF1140 family protein, and DUF4393 domain-containing protein.

3. The Bacillus Velezii MY-8 according to claim 2, characterized in that The domain-containing protein of DUF4393 comprises the amino acid sequence shown in SEQ ID NO:

1.

4. A bacterial agent, characterized in that The bacterial agent comprises the Bacillus Velez MY-8 or a culture solution thereof according to any one of claims 1 to 3.

5. The bacterial agent according to claim 4, characterized in that The bacterial agent is bacterial powder obtained after drying.

6. Use of the Bacillus Velez MY-8 according to any one of claims 1 to 3 or the bacterial agent according to claim 4 in preventing and controlling plant diseases, characterized in that: The plant disease is caused by a plant pathogen, and the plant pathogen is selected from one or more of Alternaria brassicicola, Rhizopus arrhizus, Cladosporium tenuissimum, Fusarium tricinctum, Alternaria alternat, Cladosporium colombiae, Botryosphaeria dothidea, Fusarium oxysporum, Geotrichum candidum, Colletotrichum gloeosporioides and Apiospora arundinis.

7. Use of the Bacillus Velez MY-8 according to any one of claims 1 to 3 or the bacterial agent according to claim 4 in preventing and controlling plant downy mildew.

8. A method for preparing the bacterial agent according to claim 4, characterized in that: The method comprises the steps of inoculating Bacillus Velez MY-8 into a liquid culture medium for culturing, and diluting the culture medium to a suitable multiple to obtain the bacterial agent.

9. The method according to claim 8, characterized in that The method comprises inoculating Velez bacillus MY-8 into a liquid culture medium, culturing at 30-45°C for 4-24 hours to the late logarithmic growth stage, and taking the culture fluid obtained by culturing and diluting it 0-1000 times to obtain a bacterial agent.

10. The method according to claim 8, characterized in that The culture temperature is 35-40° C., and the shaking culture time is 7-12 hours.

11. The method according to claim 8, characterized in that The culture solution is diluted 0 to 500 times to obtain a bacterial agent.

12. The method according to any one of claims 8 to 11, characterized in that The method further comprises freeze-drying or spray-drying the bacterial agent to prepare freeze-dried bacterial powder or spray-dried bacterial powder.

13. The method according to claim 12, characterized in that The inlet temperature of the spray drying is 90-150°C, the feed rate is 400-1000 mL / h, and the air inlet rate is 10-60 M 3 / h, the spray pressure is 0.1~1.0MPa.

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