Bacillus velezensis BV1 and application thereof in enhancing basal stem rot resistance of wheat

Abstract

The invention belongs to but not limited to the technical field of biological control of plant diseases, and discloses bacillus velezensis BV1 and application thereof in enhancing the resistance of wheat to basal stem rot. The bacillus velezensis BV1 provided by the invention can inhibit the growth of fusarium pseudograminearum, and the inhibition rate is 66.67%. The BV1 can enable hyphae of the fusarium pseudograminearum to expand, deform and generate apoptosis. According to the invention, the mechanism of inducing the apoptosis of the fusarium pseudograminearum by the BV1 is analyzed. Meanwhile, the main pathogenic factor RPL22 in the fusarium pseudograminearum is found. A foundation is laid for developing RPL22 gene silencing biological agents to reduce toxins generated by the fusarium pseudograminearum, so that the wheat yield and the food safety are guaranteed.

Description

Technical Field

[0001] The present invention belongs to but is not limited to the technical field of biological control of plant diseases, and in particular relates to Bacillus velezensis BV1 and its application in enhancing wheat resistance to stem base rot. Background Art

[0002] Base stem rot (FCR) is a global, soil-borne disease that significantly impacts wheat yield and quality. In some severely affected fields in China, yield losses can reach 38%-61%. The main pathogens of wheat FCR include Fusarium graminearum, Fusarium graminearum, and Fusarium erythrorhizium. FCR is harmful to wheat throughout its entire growth cycle, infecting all parts of the wheat plant, from roots to spikes. During infection, it produces various toxins, such as deoxynivalenol (DON), which pose a health risk to humans and livestock.

[0003] FCR, caused by Fusarium graminearum, was first reported in Qinyang City, Henan Province, China in 2012. Due to the widespread infection of wheat varieties in production and the large-scale implementation of straw return in recent years, the disease has been spreading and worsening in China. In June 2022, the China Association for Science and Technology listed the question, "Why have FCR outbreaks occurred in my country's major wheat-producing regions in recent years, and how can it be scientifically and effectively controlled?" as one of 10 key industrial and technological issues that will guide China's industrial development. Because the pathogen can survive in the soil for extended periods, it can spread rapidly and cause disasters when the climate is favorable, making its control difficult. Currently, the main methods for controlling wheat FCR include agricultural control, chemical control, breeding of disease-resistant varieties, and biological control. However, agricultural control methods are ineffective in effectively controlling the occurrence and development of wheat FCR; chemical control agents are generally ineffective and are associated with environmental pollution; and there is a lack of FCR-resistant wheat varieties in production. Biological control methods, however, are gaining increasing attention due to their safety, effectiveness, and environmental friendliness. Few studies have investigated the biological control of wheat FCR caused by F. graminearum. Therefore, there is an urgent need to identify biocontrol resources with high utilization value against F. graminearum. Rhizobacteria and endophytic bacteria can be used for biological control. Rhizobacteria form biofilms on plant roots and occupy ecological niches to protect plants from pathogens. Endophytic bacteria inhibit pathogen invasion through antagonism, competition, and induction of plant disease resistance. Plant rhizobacteria and endophytic bacteria have high colonization rates and minimal harm to host plants, making them suitable as biocontrol agents. Currently, Bacillus, Pseudomonas, Rhizobium, and Streptomyces have excellent biocontrol effects. Among them, Bacillus has strong stress resistance and is widely used in the biological control of plant diseases.

[0004] Bacillus is a rod-shaped, aerobic or facultatively anaerobic, Gram-positive bacterium that produces endospores. It does not produce toxins and is harmless to humans and animals. Currently, the main Bacillus species used for biological control of plant diseases include Bacillus Velez, Bacillus amyloliquefaciens, and Bacillus subtilis. Bacillus Velez is a novel biocontrol bacillus with excellent control effects against plant diseases. Studies have shown that B. Velez has inhibitory effects on Verticillium dahliae, Aspergillus niger, Alternaria brassicae, Botrytis cinerea, Fusarium oxysporum, Aspergillus graminearum, and Fusarium graminearum. However, little research has been conducted on how B. Velez inhibits the growth of F. pseudograminearum, necessitating an urgent need to analyze the mechanism by which this bacterium inhibits F. pseudograminearum. Summary of the Invention

[0005] In view of the problems existing in the prior art, the present invention provides a Bacillus Velezii BV1 and its application in enhancing the resistance of wheat to stem base rot.

[0006] The present invention is achieved by providing a Bacillus velezensis BV1, wherein the deposit number of the Bacillus velezensis BV1 is CCTCC NO: M 20251216, and the deposit time is May 28, 2025.

[0007] Another object of the present invention is to provide a biocontrol agent for preventing and treating wheat stem base rot, wherein the biocontrol agent comprises the Bacillus Velezii BV1.

[0008] Another object of the present invention is to provide a ribosomal protein L22 (RPL22) gene silencing biological preparation, which contains the Bacillus Velezii BV1.

[0009] Another object of the present invention is to provide a use of Bacillus Velezii BV1 in enhancing the resistance of wheat to stem base rot.

[0010] Furthermore, the stem base rot is caused by Pseudomonas graminearum.

[0011] Furthermore, the Bacillus Velezii BV1 enhances wheat resistance to stem base rot by inducing apoptosis of Fusarium graminearum cells.

[0012] Furthermore, ribosomal protein RPL22 and ribosomal protein L32 (RPL32) responded to BV1 stress through indirect interactions.

[0013] The ribosomal protein RPL22 is a potential pathogenic factor of Fusarium graminearum.

[0014] In combination with the above technical solutions and the technical problems solved, the advantages and positive effects of the technical solutions to be protected by the present invention are as follows:

[0015] The Bacillus velezensis BV1 provided by the present invention can inhibit the growth of Fusarium graminearum with an inhibition rate of 66.67%. BV1 can cause the hyphae of Fusarium graminearum to swell, deform and undergo apoptosis. The present invention studies the mechanism of action of BV1 in inhibiting the growth of Fusarium graminearum. Through transcriptome analysis, it was found that after BV1 was co-cultured with Fusarium graminearum for 4 hours, the structure, composition and translation process of ribosomes in Fusarium graminearum were significantly affected. After BV1 was co-cultured with Fusarium graminearum for 16 hours, transcriptome analysis showed that the metabolism of a large number of amino acids and the function of the cell membrane in Fusarium graminearum were significantly affected. The main pathogenic ribosomal protein RPL22 gene in Fusarium graminearum was further explored. After knocking out the RPL22 gene, the pathogenicity of the strain was significantly reduced, while the pathogenicity of the complemented strain was restored. Yeast two-hybrid and bimolecular fluorescence complementation experiments showed that RPL22 and RPL32 responded to BV1 stress through indirect interactions. Therefore, RPL22 is proposed to be a potential pathogenic factor of F. graminearum.

[0016] The technical solution of this invention fills a technological gap in the industry, both domestically and internationally. Wheat stem rot is a new disease that has caused serious damage to wheat production in recent years. Research on wheat stem rot is weak both domestically and internationally, lacking efficient and safe technical means suitable for large-scale production. The Bacillus velezinii used in this study is environmentally safe, highly effective against wheat stem rot, and has the potential to be used as a biocontrol agent. This research will fill a gap in the biological control of wheat stem rot. BRIEF DESCRIPTION OF THE DRAWINGS

[0017] Figure 1 Schematic diagram of the evolutionary tree analysis of Bacillus velezensis BV1 provided in an embodiment of the present invention;

[0018] Figure 2 Schematic diagram of the inhibitory effect of BV1 on Fusarium graminearum provided by an embodiment of the present invention;

[0019] Figure 3 Schematic diagram of the expression of related genes after co-cultivation of BV1 and Pseudomonas graminearum provided in the embodiment of the present invention

[0020] Figure 4 Schematic diagram of transcriptome results provided by an embodiment of the present invention;

[0021] Figure 5 Schematic diagram of GO enriched functional items and KEGG enriched metabolic pathways in the transcriptome provided by the embodiment of the present invention;

[0022] Figure 6The heat map of the main significantly differentially expressed genes after 4 hours and 16 hours of co-culture and the metabolic pathway diagram of the candidate gene RPL22 provided in the embodiments of the present invention are provided;

[0023] Figure 7 Schematic diagram of pathogenicity detection after knockout and complementation of the ribosomal protein synthesis gene RPL22 in F. graminearum provided in an embodiment of the present invention;

[0024] Figure 8 Schematic diagram of the interaction between RPL22 and RPL32 proteins in Fusarium graminearum provided in an embodiment of the present invention in response to BV1 stress;

[0025] Figure 9 Schematic diagram of the RPL22 and RPL32 proteins in Fusarium graminearum responding to BV1 stress through indirect interaction provided by an embodiment of the present invention;

[0026] Figure 10 1 is a diagram showing the mechanism by which Bacillus velezensis inhibits the growth of Fusarium graminearum provided by an embodiment of the present invention;

[0027] Figure 11 This is a comparative verification diagram of RT-qPCR and RNA-seq of gene expression under different treatment conditions provided in the embodiments of the present invention. DETAILED DESCRIPTION

[0028] In order to make the purpose, technical solutions and advantages of the present invention more clearly understood, the present invention is further described in detail below in conjunction with the embodiments. It should be understood that the specific embodiments described herein are only used to explain the present invention and are not intended to limit the present invention.

[0029] Example 1:

[0030] Seeds of wheat (common wheat) varieties Jimai 22 and Aikang 58 were sown in petri dishes. The seedlings were cultured in a greenhouse with a photoperiod of 8 hours dark / 16 hours light, a temperature of 25°C during darkness and 25°C during light, and a relative humidity of 60%. After one week, the seedlings were treated with a concentration of 1×10 5 Wheat plants were inoculated with a spore suspension containing 10 spores / mL. Ten days later, the disease index of the wheat was measured.

[0031] After 10 days, the disease index of F. graminearum was investigated. The disease severity of the seedlings was scored 10 days after inoculation according to the following criteria: 0 = healthy, with no symptoms on the outer sheath; 1 = the area of ​​the lesion on the first sheath is less than 1 / 4 of the sheath length; 2 = the area of ​​the lesion on the first sheath is 1 / 4-1 / 2 of the sheath length; 3 = the area of ​​the lesion on the first sheath is 1 / 2-3 / 4 of the sheath length, and the disease affects the inner sheaths; 4 = the first sheath is completely chlorotic and rotten, or the second sheath has obvious browning; 5 = the third sheath has obvious brown spots or the entire plant is dead. The overall disease index and control effect are calculated as follows:

[0032] Disease index = ((0n0+1n1+2n2+3n3+4n4+5n5) / 5n)×100

[0033] where n0-n5 are the number of plants with the corresponding disease score and n is the total number of plants evaluated.

[0034] For transcriptome screening data, genes meeting the following criteria were considered differentially expressed genes: log2|(foldchange (FC))|≥1 and the P value ≤0.05. Gene Ontology (GO) terms and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathways were considered significantly enriched if their P values ​​were less than 0.05. Based on RNA sequencing (RNA-seq) results, 14 genes were selected from the National Center for Biotechnology Information (NCBI) database. Gene-specific primers were designed using this information and validated by real-time fluorescence quantitative reverse transcription polymerase chain reaction (qRT-PCR).

[0035] For yeast two-hybrid experiments, the coding sequences of ribosomal protein RPL32 and ribosomal protein RPL22 were recombined into pGADT7 or pGBKT7 vectors. The obtained plasmids were transformed into yeast strain AH109. Yeast cells were cultured at 30°C for 2 days in a selective medium containing 0.67% (w / v) yeast nitrogen base, 2% (w / v) glucose, and auxotrophic amino acids required for growth. For bimolecular fluorescence complementation (BiFC) experiments, the coding sequences of RPL22 and RPL32 without stop codons were inserted into the PacI and SpeI restriction sites of YC and Yn vectors, respectively. The constructed vectors were transformed into Agrobacterium strain GV3101 and transiently expressed in tobacco leaves. Fluorescence signals were detected using confocal laser scanning microscopy (CLSM).

[0036] All experiments and data presented in this article were repeated at least three times. Data were analyzed using SPSS 19.0 software (SPSS Inc.). Student's t-test and one-way ANOVA (α = 0.05) were used, followed by Duncan's test for multiple comparisons. Significant differences are reported in the text and presented in the figures.

[0037] Example 2: Phylogenetic tree of Bacillus velezinis BV1

[0038] Several biocontrol strains were isolated from wheat rhizosphere soil, and a biocontrol bacterium with inhibitory effect on FCR was screened by plate confrontation test. After 16S rRNA sequencing and phylogenetic tree construction, it was found that the strain was Bacillus velezensis and was named BV1 ( Figure 1 ).

[0039] Example 3: Inhibitory effect of Bacillus velezensis BV1 on FCR

[0040] The strain has a strong inhibitory effect on F. graminearum, with an inhibition rate of 66.67%, and the hyphae around BV1 swell and deform at both ends ( Figure 2 Propidium iodide staining results showed that BV1 induced apoptosis of F. graminearum hyphae. Evans blue staining results indicated that BV1 could directly form pores in the cell membrane, leading to necrosis of F. graminearum. These results indicate that BV1 has a strong bactericidal effect against F. graminearum.

[0041] Example 4: Response of genes closely related to cell wall and cell membrane synthesis, antioxidant and stress resistance to BV1 stress

[0042] To verify whether the genes GEL, ERG, FAS, ELOA, CHI, SOD1, CAT, and FUS3 responded to BV1 stress after co-culture with F. graminearum. RT-PCR results showed that after BV1 was co-cultured with F. graminearum for 4 and 16 hours, all genes except FAS and FUS3 were significantly increased compared with the control group ( Figure 3 These results indicate that genes closely related to cell wall and membrane synthesis, antioxidant and stress resistance responded to BV1 stress.

[0043] Example 5: Transcriptome analysis of co-culture of BV1 and F. graminearum

[0044] 14 genes were randomly selected from the RNA-seq results and the transcriptome results were verified by qRT-PCR. The results showed that the expression trends of the 14 genes were consistent with the trends identified in the transcriptome data, with an accuracy of 100%. This finding indicates that the transcriptome results are reliable. The results of the principal component analysis (PCA) of the transcriptome showed that after 4 hours and 16 hours of co-culture of BV1 with F. graminearum, there was good reproducibility between the treatments, indicating that the results are reliable ( Figure 4 -A and B). After 4 hours of co-culture of BV1 with F. graminearum, there were 1073 up-regulated differentially expressed genes and 1396 down-regulated differentially expressed genes. After 16 hours of co-culture of BV1 with F. graminearum, there were 453 up-regulated differentially expressed genes and 697 down-regulated differentially expressed genes ( Figure 4 -C). There were 533 differentially expressed genes after 4 and 16 hours of co-culture. There were 1936 specific differentially expressed genes after 4 hours of co-culture and 617 specific differentially expressed genes after 16 hours of co-culture ( Figure 4 -D). At 4 hours of differential gene expression, FPSE_05873 was most significantly down-regulated, and FPSE_03817 was most significantly up-regulated (Table 1, Figure 4 -E and G). At 16 hours of differential gene expression, FPSE_04073 was most significantly down-regulated, and FPSE_02102 was most significantly up-regulated (Table 1, Figure 4 -F and H). These genes may play an important role in the response of F. graminearum to BV1 stress.

[0045] Table 1 The top 20 genes with the largest up-regulated and down-regulated folds after 4 and 16 hours of co-culture

[0046]

[0047]

[0048] Example 6: GO function and KEGG metabolic pathway analysis after co-culture of BV1 and F. graminearum

[0049] Transcriptome analysis showed that after 4 h of co-culture of BV1 with F. graminearum, the most significantly enriched GO terms were translation, cytoplasmic large ribosomal subunits, and ribosomal structural composition ( Figure 5 -A). KEGG enrichment analysis showed that the most significant pathway was the ribosome pathway ( Figure 5 -C). Transcriptome analysis showed that after 16 h of co-culture of BV1 with F. graminearum, the most significant GO enrichment terms were amino acid transmembrane transport, membrane integral components, and inorganic phosphate transmembrane transporter activity ( Figure 5 -B). KEGG enrichment analysis showed that the most significant pathways were valine, leucine, and isoleucine biosynthesis pathways ( Figure 5-D). In addition, regardless of whether co-cultured for 4 hours or 16 hours, KEGG enrichment analysis significantly enriched a large number of amino acid metabolic pathways. These results suggest that BV1 may first destroy the ribosomal metabolic pathway of F. graminearum, resulting in abnormalities in a large number of amino acid metabolic pathways, and then affect the function of the cell membrane, leading to apoptosis of F. graminearum. After 4 hours of co-culture of BV1 and F. graminearum, the expression level of the RPL22 gene was the highest ( Figure 6 ), which may play an important role in responding to BV1 stress.

[0050] Example 7: Functional verification of the RPL22 gene in Fusarium graminearum

[0051] In order to verify whether the ribosomal protein synthesis gene RPL22 is a key pathogenic factor of F. graminearum. After knocking out the ribosomal protein synthesis gene RPL22 in F. graminearum using CRIPR-Cas9 technology, the present invention found that the pathogenicity of the knockout strain (KO) was significantly reduced compared with the control group, and the disease index of Aikang 58 and Jimai 22 was reduced by 42.56% and 10.95% respectively. Figure 7 The virulence of the complemented strain (EC) was restored to the level of the control. These results indicate that the ribosomal protein synthesis gene RPL22 is a key pathogenic factor of F. graminearum.

[0052] Example 8: RPL22 and RPL32 proteins in F. graminearum respond to BV1 stress through interaction

[0053] In order to verify the interaction between RPL22 and RPL32 proteins, the present invention used AlphaFold-Multimer docking technology to find the interaction between the two proteins ( Figure 8 ). A hydrogen bond is formed between the hydroxyl group of threonine and the ornithine group of arginine. Hydrogen bonds can be formed between the carboxyl group of aspartic acid and the amino group of lysine because the amino group of lysine is positively charged and the carboxyl group of aspartic acid is negatively charged. Valine is a hydrophobic, non-polar amino acid with a simple side chain structure consisting of two methyl groups branching from a central carbon atom. It is usually involved in the formation of a hydrophobic core in proteins and helps to stabilize the three-dimensional structure of proteins. Asparagine is a polar but uncharged amino acid with an amide functional group in its side chain that can form hydrogen bonds with valine residues through its oxygen or nitrogen atoms. To further verify the interaction between RPL22 and RPL32 proteins, yeast two-hybrid dot-to-dot verification experiments revealed that RPL22 and RPL32 could not interact directly ( Figure 9 -A). BiFC experiments revealed that RPL22 and RPL32 can interact with each other ( Figure 9 -B), suggesting that RPL22 and RPL32 may respond to BV1 stress through indirect interactions ( Figure 10 ).

[0054] The conclusion obtained by the present invention is that BV1 inhibits the growth of Fusarium graminearum. BV1 first destroys the structure, composition and translation process of the ribosome of Fusarium graminearum. RPL22 is the main pathogenic gene of Fusarium graminearum. RPL22 and RPL32 may respond to BV1 stress through indirect interactions. This causes the metabolism of a large number of amino acids and the function of cell membranes to be significantly affected, ultimately leading to apoptosis. The present invention analyzes the mechanism by which BV1 induces apoptosis of Fusarium graminearum. At the same time, the main pathogenic factor RPL22 in Fusarium graminearum was discovered. This lays the foundation for the development of RPL22 gene silencing biological agents to reduce the toxins produced by Fusarium graminearum, thereby ensuring wheat yield and food safety.

[0055] The specific strain preservation information of the present invention is:

[0056] China Center for Type Culture Collection

[0057] Notification (receipt) of acceptance of culture deposit for patent procedure

[0058] Address: Wuhan University, Wuhan, China, Postal Code: 430072

[0059] Tel: (027) 68754052 Fax: (027) 68754833 Email: cctcc@whu.edu.cn

[0060] Requesting depositor: Zhou Heng, Crop Research Institute, Anhui Academy of Agricultural Sciences

[0061] Name of the culture for which deposit is requested and its identifying characteristics:

[0062] Fusarium pseudograminearum Fpg1

[0063] The accession number of this collection center

[0064] CCTCC NO:M 20251216

[0065] The preservation time is: May 28, 2025.

[0066] like Figure 11As shown, the present invention discloses a Bacillus velezensis BV1 strain, which, through morphological, physiological, biochemical, and 16S rRNA gene sequence identification, is consistent with Bacillus velezensis and has been successfully deposited with the China Center for Typical Microorganisms under the deposit number CCTCC NO: M 20251216. This strain has a rod-shaped cell shape, is Gram-positive, can produce spores, and exhibits typical homogenous growth on common selective media.

[0067] To reveal the secondary metabolic potential of the BV1 strain, the present invention combined RT-PCR and RNA-seq techniques to analyze the expression of representative antimicrobial factor synthesis genes under control (CK) and induced treatment (BV1) conditions (see attached figure). The results showed that genes such as aznB, cefD2, pksF, rpl22, prnC, aat2, and pho5 were significantly upregulated in response to BV1 treatment (RNA-seq FPKM and RT-PCR fold changes were consistent), while secondary metabolic genes such as ustaY, mpaA, het6, tdiE, RSB1, and aflT showed varying degrees of downregulation or expression inhibition, fully demonstrating that the BV1 strain can preferentially synthesize a series of active substances with inhibitory effects on plant pathogens under specific conditions.

[0068] The present invention further formulates the BV1 strain into a biocontrol agent, using a pollution-free bentonite and sodium alginate composite as the carrier, with a bacterial content of no less than 1×10^9 CFU / g, for spraying or seed dressing. Field and greenhouse tests on wheat stem rot showed that, compared with untreated controls, treatment with the BV1 agent reduced the incidence of diseased plants by an average of over 65%, significantly increased plant height and root vitality, and produced no residual pesticide residue.

[0069] The Bacillus Velez BV1 bacterial agent of the present invention has the advantages of clear preservation resources, clear disease resistance mechanism, convenient and safe use, and wide adaptability. It can provide an efficient and sustainable biological solution for the green prevention and control of wheat stem base rot.

[0070] The above description is only a specific embodiment of the present invention, but the scope of protection of the present invention is not limited thereto. Any modifications, equivalent substitutions and improvements made by any technician familiar with this technical field within the technical scope disclosed by the present invention and within the spirit and principles of the present invention should be covered by the scope of protection of the present invention.

Claims

1. A Bacillus velezensis BV1, characterized in that The deposit number of the Bacillus velezensis BV1 is CCTCC NO: M 20251216.

2. A biocontrol agent for preventing and treating wheat stem base rot, characterized in that: The biocontrol agent comprises the Bacillus Velezii BV1 according to claim 1.

3. A ribosomal protein RPL22 gene silencing biological agent, characterized in that: The biological preparation comprises the Bacillus Velezii BV1 according to claim 1.

4. Use of the Bacillus Velez BV1 according to claim 1 in enhancing the resistance of wheat to stem base rot.

5. The use according to claim 4, characterized in that The stem base rot is caused by the fungus Fusarium graminearum.

6. The use according to claim 4, characterized in that The Bacillus Velez BV1 enhances wheat resistance to stem base rot by inducing apoptosis of Fusarium graminearum cells.

7. The use according to claim 4, characterized in that Ribosomal proteins RPL22 and RPL32 respond to BV1 stress through indirect interactions.

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