Bacillus velezensis BVDT-1 and application thereof

Abstract

The invention provides bacillus velezensis BVDT-1 and application thereof, and belongs to the technical field of biological agents. The bacillus velezensis BVDT-1 strain is obtained through separation and identification, has an inhibition effect on various plant pathogenic fungi such as waxberry wilting germs, persimmon colletotrichum gloeosporioides and persimmon leaf spot germs, provides a new resource for biological prevention and control of related fruit tree diseases, and compared with part of similar strains, the bacillus velezensis BVDT-1 strain has the advantages that the antibacterial effect of the plant pathogenic fungi is not mentioned, and the bacillus velezensis BVDT-1 strain has a good application prospect. The strain is excellent in genome characteristic, high in degradation capacity, low in drug-resistant gene risk and large in secondary metabolite synthesis potential, can secrete unknown antibiotics possibly, provides powerful support for application in the fields of biological control and microbial fertilizer, and has wide application prospects in the aspects of biological control, agricultural production and the like.

Description

Technical Field

[0001] The invention relates to the technical field of biological bacterial agents, and in particular to Bacillus Velezii BVDT-1 and applications thereof. Background Art

[0002] In modern agriculture, plant diseases seriously affect the yield and quality of crops. Although traditional chemical pesticides can control pests and diseases, long-term use will cause environmental pollution and agricultural product residues, prompting people to seek environmentally friendly, efficient and low-residue biological control methods.

[0003] Biocontrol bacteria refer to a type of beneficial microorganisms that can inhibit or prevent the growth, reproduction and infection of plant pathogens, thereby promoting plant growth and improving plant resistance. They mainly play a biocontrol role through a variety of mechanisms, such as competition, competing with pathogens for nutrients and living space, antagonism, producing antibacterial substances to inhibit the growth of pathogens, inducing plant resistance and enhancing the plant's own defense system, etc. Common biocontrol bacteria include Bacillus and Trichoderma.

[0004] However, the existing technology has certain defects. On the one hand, the antibacterial spectrum of some biocontrol bacteria is narrow, and they can only work on specific pathogens, making it difficult to cope with the complex situation of simultaneous infection by multiple pathogens. For example, some biocontrol bacteria only have an inhibitory effect on a single type of fungi or bacteria. In actual agricultural production, crops are often threatened by multiple pathogens, which limits the widespread application of such biocontrol bacteria. On the other hand, the activity of some biocontrol bacteria is unstable, and their antibacterial effect is easily affected by environmental factors (such as temperature, humidity, soil pH, etc.). Under different environmental conditions, the growth, reproduction and production of antibacterial substances of biocontrol bacteria may be inhibited, so that they cannot effectively play a biological control role. In addition, some biocontrol bacteria have safety risks. For example, some strains may carry more antibiotic resistance genes, which may spread resistance genes to other microorganisms during application, leading to the aggravation of drug resistance problems and posing a potential threat to the ecological environment and human health. Therefore, it is urgent to screen out biocontrol bacteria with a wider antibacterial spectrum, stable activity and high safety. Summary of the invention

[0005] The purpose of the present invention is to provide a Bacillus Velezii BVDT-1 with better performance.

[0006] In order to achieve the above-mentioned object of the invention, the present invention provides the following technical solutions: The present invention provides a Bacillus Velez, wherein the Bacillus Velez is Bacillus velezensis BVDT-1, the deposit number is CGMCC No.30693.

[0007] The 16S rDNA gene sequence of the Bacillus Velezii is shown in SEQ ID NO.1.

[0008] The present invention also provides a method for culturing the above-mentioned Bacillus Velez subtilis, comprising the following steps: inoculating Bacillus Velez subtilis BVDT-1 into the above-mentioned culture medium, culturing, and obtaining Bacillus Velez subtilis culture fluid.

[0009] Preferably, the culture temperature is 15-80°C; Preferably, the culture temperature is 35-38°C.

[0010] The present invention also provides the Bacillus Velez subtilis culture solution obtained by the above-mentioned culture method.

[0011] The present invention also provides the use of the above-mentioned Bacillus Velez subtilis, the culture method or the above-mentioned Bacillus Velez subtilis culture solution in the preparation of crop pathogenic fungi inhibitors.

[0012] Preferably, the crop pathogenic fungus is Pseudomonas microsporus, Pseudomonas carinii or Fusarium equisetum.

[0013] The present invention also provides the use of the above-mentioned Bacillus Velez subtilis or the culture solution of Bacillus Velez subtilis in producing antibacterial active substances.

[0014] The present invention also provides the above-mentioned Bacillus Velez, the culture method or the use of the Bacillus Velez culture solution in preparing a plant disease biocontrol agent.

[0015] The present invention also provides a plant disease biocontrol fungicide, which contains the above-mentioned Bacillus Velezii; The plant diseases are bayberry wilt, sweet persimmon anthracnose and sweet persimmon leaf spot.

[0016] Beneficial effects of the present invention: The present invention isolated and identified Bacillus velezinifolia (Bacillus velezinifolia) from the culture residue of Cicadae convolvulus in Dongtou, Wenzhou. Bacillus velezensis) BVDT-1 strain. In terms of antibacterial activity, it showed inhibitory effects on various plant pathogenic fungi such as sweet persimmon anthracnose, sweet persimmon leaf spot and bayberry wilt, and its plate resistance to the above pathogens reached a high level, showing broad-spectrum antibacterial properties, providing new resources for biological control of related fruit tree diseases, and its antibacterial effect was better than that of some similar strains. Its genome characteristics are excellent, including chromosomes and plasmids, with clear genome length, GC content, number of coding genes and RNA types, and it also contains multiple genomic islands, prophages and CRISPR Cas systems; it has rich functional genes and many metabolic pathway-related genes, such as amino acid and carbon metabolic pathway genes, and there are many antibacterial-related pathway genes. It has strong degradation ability, and there are many genes in the genome that belong to the CAZy family that encode protein domains, covering a variety of hydrolases, transferases, etc., which can degrade a variety of carbohydrates and inhibit mycelial growth. It has development potential in the treatment of crop residues. The risk of drug-resistant genes is low, and it contains only a small number of antibiotic resistance genes, and the risk of spreading drug-resistant genes is low. It has great potential for the synthesis of secondary metabolites, contains multiple secondary metabolite synthesis gene clusters, can synthesize a variety of known antibacterial substances, and some gene clusters have low similarity with those that have been identified, and may secrete unknown antibiotics, providing strong support for the application in the field of biological control and microbial fertilizer. This strain has broad application prospects in biological control, agricultural production and many other aspects. BRIEF DESCRIPTION OF THE DRAWINGS

[0017] Figure 1 This is the colony morphology of strain BVDT-1; Figure 2 This is the phylogenetic tree of strain BVDT-1 constructed based on the 16S rDNA gene sequence; Figure 3 The results of the inhibitory effect of strain BVDT-1 on fruit tree pathogens are shown in Figure 1, among which A is C. fructicola; B is F. equiseti; C is P. microspora; Figure 4 The circle diagram of the genome of strain BVDT-1, where the outermost circle indicates the genome size; the second and third circles are the genes on the positive and negative strands of the genome, respectively; the fourth circle is the repetitive sequence; the fifth circle is tRNA and rRNA; the sixth circle is the GC content; and the innermost circle is the GC-skew; Figure 5 This is the eggNOG annotation map of the genome of strain BVDT-1; Figure 6 This is the GO annotation map of the genome of strain BVDT-1; Figure 7 This is the statistical analysis diagram of the KEGG database of the genome of strain BVDT-1; Figure 8This is the distribution ratio of carbohydrate enzymes of strain BVDT-1; Fig. 9 This is a petal diagram of gene families among species. Biological Deposit Description

[0018] The Bacillus Velez subsp. provided by the present invention is named Bacillus Velez subsp. BVDT-1 ( Bacillus velezensis BVDT-1 ） , deposited on May 20, 2024 in the General Microbiology Center of China Microorganism Culture Collection, referred to as CGMCC, address: No. 3, Yard No. 1, Beichen West Road, Chaoyang District, Beijing. Institute of Microbiology, Chinese Academy of Sciences, Postal Code: 100101, China. The deposit number is CGMCC No.30693. DETAILED DESCRIPTION

[0019] The 16S rDNA gene sequence of Bacillus Velezii BVDT-1 provided by the present invention is as follows:

[0020] The technical solutions provided by the present invention are described in detail below in conjunction with the embodiments, but they should not be construed as limiting the protection scope of the present invention.

[0021] Example 1 Materials and methods 1.1 Sample collection The residues of Cordyceps sinensis culture were obtained from Zhejiang Chengyi Pharmaceutical Co., Ltd. in Dongtou District, Wenzhou City. Samples were taken from three points and stored at 4°C after sampling.

[0022] 1.2 Sources of test materials and strains in the examples Plant pathogenic fungi: sweet persimmon anthracnose, fruit-dwelling spores ( Colletotrichum fructicola )、Persimmon leaf spot, Fusarium equisetum ( Fusarium equiseti ) and the causative fungus of bayberry wilt, Pseudomonas microsporus ( Pestalotiopsis microspora ) was provided by Zhejiang Subtropical Crops Research Institute. Main culture medium (LB solid and liquid medium).

[0023] 1.3 Test methods 1.3.1 Isolation and identification of Bacillus velez Weigh 2.0 g of cicada fungus culture residue into a 100 mL conical flask, add 20 mL of distilled water, shake well, oscillate for 20 minutes, and centrifuge at 1000 rpm. Take 200 μL of supernatant and apply it to LB solid medium, invert it in a 37 ℃ constant temperature incubator for 12-24 hours. Select single colonies with different morphology from the LB plate, and after three streaking purifications, select pure colonies and inoculate them in LB liquid medium for 24 hours. Add glycerol to the bacterial solution and store it in a refrigerator at -20 ℃. After the bacterial solution was expanded and cultured, the bacterial solution was collected and sent to Shenggong Bioengineering Co., Ltd. for 16SrDNA sequence analysis and detection. The 16S rDNA gene sequence obtained by sequencing was submitted to the NCBI database. After selecting the BLAST comparison results, the orthomcl software was used to cluster the gene family and screen out the common single-copy genes. The muscle tool was then used to align and align these sequences, and finally the phylogeny was constructed by phyML software.

[0024] 1.3.2 Inhibitory effect of test strains on plant pathogenic fungi 1.3.2.1 Inhibitory effect of test strains on plant pathogenic fungi In the plate confrontation test, the sweet persimmon anthracnose pathogen, sweet persimmon leaf spot pathogen and bayberry pathogen were placed in the center of the culture dish, and the strains were inoculated in a cross-shaped symmetrical manner at a distance of about 2 cm from the pathogen block. Each group of experiments was repeated three times, and a control group without BVDT-1 inoculation was set up. The culture was carried out at a constant temperature of 28°C for 5 days. The growth inhibition percentage (inhibition rate) of BVDT-1 on sweet persimmon anthracnose pathogen, sweet persimmon leaf spot pathogen and bayberry wilt pathogen was calculated according to the following formula.

[0025] Inhibition rate (%) = (control colony diameter - treated colony diameter) / (control colony diameter - bacterial cake diameter) × 100% 1.3.2.2 Effect of test strains on the control of anthracnose infection of sweet persimmon twigs of “Taiqiu” A. Artificial inoculation and control The spore liquid used for infection was artificially cultured anthrax bacteria in a 25°C fungus incubator for 7 days after identification in the laboratory to produce conidia, and then a spore suspension was prepared with 2% sucrose solution. The spore liquid concentration was 1×10 6 Each treatment selected 50 new young branches with the same growth conditions and used the single-branch area spore suspension drip method for artificial inoculation. Each branch was divided into 5 areas with an interval of 1.5 cm. The center of each area was inoculated with 5 μL of spore suspension dripped with a pipette, bagged, and removed the next day. Three days later, BVDT-1 (concentration of 1×10 8 ), observe and record for 15 consecutive days, recording the onset time and the growth of lesions within 15 days.

[0026] B. Evaluation of control effects Use a vernier caliper to record the size of the lesions, investigate each plant and record the disease level. The standard for disease level classification is: the ratio of the lesion diameter to the total length of the branch is 0-20% and is recorded as level 1; the ratio of the lesion diameter to the total length of the branch is 20%-40% and is recorded as level 2; the ratio of the lesion diameter to the total length of the branch is 40%-60% and is recorded as level 3; the ratio of the lesion diameter to the total length of the branch is 60%-80% and is recorded as level 4; the ratio of the lesion diameter to the total length of the branch is 80%-100% and is recorded as level 5.

[0027] 1.3.3 DNA extraction and whole genome sequencing The strain was inoculated into LB liquid medium and cultured for 12 to 14 hours until it reached the logarithmic growth phase, and then the bacteria were collected by centrifugation. The whole genome sequencing task was then handed over to BioMed Biotechnology Co., Ltd.

[0028] 1.3.4 Genome assembly and prediction 1.3.4.1 Genome assembly The sequencing data of the strain was assembled using the assembly software Hifiasm for the integration of the three-generation sequences. Subsequently, the Circlator v1.5.5 tool was used to circularize the assembly results and adjust the start site. During the assembly process, Pilon v1.22 software was also introduced to perform sequence correction, thereby obtaining a complete chromosome sequence.

[0029] 1.3.4.2 Coding gene prediction Prodigal v2.6.3 software was used to predict coding genes in newly sequenced genomes. The software can identify genes with high accuracy by virtue of its dynamic programming algorithm. Non-coding genes such as tRNA and rRNA were predicted using tRNAscan-SEv2.0 and Infernal v1.1.3 software, respectively. Mobile elements such as gene islands, prophages, and CRISPR were predicted using IslandPath-DIMOB v0.2, PhiSpy v2.3, and CRT v1.2 software, respectively.

[0030] 1.3.5 Genome functional annotation The predicted gene sequences were compared with NR, Swiss-Prot, Pfam, eggNOG, GO and KEGG databases by BLAST. The gene functions were annotated based on the comparison results, and the eggNOG, KEGG metabolic pathways and GO functional categories were further enriched. The CAZyme database and Hmmer software were used to annotate and analyze the gene functions of carbohydrate active enzymes. At the same time, based on the Virulence Factor Database (VFDB) and the Comprehensive Antibiotic Resistance Gene Database (CARD), rgi software was used to compare virulence factors and resistance genes to obtain corresponding annotation information.

[0031] 1.3.6 Comparative genome analysis of strains We used OrthoMCL software to perform family cluster analysis on the protein sequences predicted by the strains and the protein sequences of reference genomes (such as EN01, UB2017, etc.), and conducted in-depth discussions on gene families. Based on these analyses, we constructed a petal diagram to intuitively display the number of genes shared by the strains and the number of genes unique to each strain.

[0032] In the analysis of the secondary metabolite synthesis gene cluster of the strain, we used antiSMASH v5.0.0 software to predict it. Combining the gene annotation results with the BLAST comparison results of the NCBI database, we made a comprehensive inference on the secondary metabolite encoding gene cluster of the strain and its potential antibacterial substances.

[0033] 2. Results and Analysis 2.1 Isolation and identification of strains Purification and separation on LB solid plate resulted in 14 strains of Bacillus, namely DTYM-1~14, with rough colony surface, irregular edges, wrinkles, off-white color, and opaque ( Figure 1 ), the antibacterial activity of the isolated strains against Colletotrichum spp. was determined by plate confrontation method, among which DTYM-1 strain had the strongest antibacterial activity against Colletotrichum spp. The 16SrDNA sequence was analyzed and submitted to the NCBI database, and the BLAST program was selected for comparison to construct a phylogenetic tree ( Figure 2 ). The results showed that the strain B. velezensis UB2017, MG-2 and LQ-3 clustered together with Bacillus amyloliquefaciens ( B. amyloliquefaciens )、Bacillus( B. sp ) were clustered into different branches, and the strain was 100% consistent with the Bacillus Velez subtilis LQ-3 strain, indicating that the strain was most closely related to Bacillus Velez subtilis. Therefore, it was identified as Bacillus Velez subtilis and named BVDT-1 in subsequent studies. It was sent to the General Microbiology Center of China Microbiological Culture Collection and obtained the preservation number: CGMCC No.30693.

[0034] 2.2 Antagonistic effect of strain BVDT-1 on pathogens 2.2.1 Test results of the antagonistic effect of strain BVDT-1 on persimmon anthracnose, persimmon leaf spot and bayberry wilt Antagonism test results ( Figure 3 ) showed that the strain BVDT-1 had a good inhibitory effect on the growth of persimmon anthracnose, persimmon leaf spot and bayberry wilt, with the inhibition rates being 71.01%, 45.71% and 80.70%, respectively.

[0035] 2.2.2 Effect of strain BVDT-1 on the control of artificial anthracnose infection of sweet persimmon branches According to the results in Table 1, the strain BVDT-1 has a good control effect on the anthracnose of the tender branches of "Taiqiu" sweet persimmon, and can be used for early prevention in the field in the future.

[0036] Table 1 The control effect of BVDT-1 on the anthracnose of sweet persimmon twigs of “Taiqiu”

[0037] 2.3 Overview of the genome of strain BVDT-1 The genome sequencing results showed that strain BVDT-1 has 1 chromosomal gene sequence, the total genome length is 3.916789Mb, the chromosome size is 3.916789Mb, the GC content is 46.53%, the total length of the predicted coding gene sequence is 3.468417Mb, and there are 3742 genes in total. BVDT-1 has 86 tRNAs and 27 rRNAs, of which 5S rRNA, 16SrRNA and 23S rRNA genes are 9 each. We found 4 genomic islands, 3 prophages and 4 CRISPR Cas systems in strain BVDT-1. Based on the basic characteristics of the genome, we mapped out B. velezensis The genome circle map ( Figure 4 ).

[0038] 2.4 Genome functional annotation results We compared the protein sequences of the sequenced genes with the NR, eggNOG, GO, KEGG, Swiss-Prot and Pfam functional databases using Diamond (E-value ≤ e -5 ) and Hmmer 3.0 software were used for analysis, and the alignment results with the highest score (default Identity ≥ 40%, Coverage ≥ 40%) were selected for annotation. The number of genes with functional annotation in NR, eggNOG, GO, KEGG, SwissProt and Pfam databases were 3729, 2983, 2895, 2159, 2823 and 3345, accounting for 87.63%, 79.72%, 77.37%, 57.70%, 75.44% and 89.39, respectively.

[0039] 2.4.1 eggNOG annotation results Based on the eggNOG database (which is an extension of the homologous protein cluster COG), we performed a detailed alignment and taxonomic annotation of the genome of strain BVDT-1. Figure 5As shown in the figure, there are 2983 protein-coding genes in the genome of strain BVDT-1, which are classified into 21 eggNOG subclasses, accounting for 79.72%. The three most abundant categories are: Amino acid transport and metabolism, which contains 280 genes, accounting for 7.48%; General function prediction only, which contains 272 genes, accounting for 7.27%; and Carbohydrate transport and metabolism, which contains 224 genes, accounting for 5.99%. In addition, there are a large number of genes with unknown functions in the genome, totaling 476, accounting for 12.72% of the total sequence.

[0040] 2.4.2 GO annotation results like Figure 6 As shown in the figure, the gene functions of strain BVDT-1 can be divided into three categories: biological process, cellular component and molecular function, but a single gene often participates in multiple functions. In the biological process category, a total of 448 genes were annotated, of which the most important were genes involved in metabolic processes, with a total of 1447 functions. For the cellular component category, 708 genes were annotated, of which the largest number of genes were related to membrane and its components, with 932 functions and 881 functions, respectively. In the molecular function category, 1739 genes were annotated, of which the largest number were genes related to catalytic activity and binding, with 1711 functions and 1181 functions, respectively.

[0041] 2.4.3 KEGG annotation results According to the KEGG annotation results (see Figure 7), there are 2159 protein-coding genes in the genome of strain BVDT-1, which are classified into four categories: cellular processes, metabolism, genetic information processing, and environmental information processing. Among them, the number of genes in the metabolic pathway is the largest, mainly concentrated in the two pathways of amino acid biosynthesis and carbon metabolism, with 121 and 99 genes annotated respectively. The environmental information processing pathway is closely followed, mainly concentrated in the ABC transporters and two-component system pathways, with 127 and 115 genes annotated respectively. The five common pathways related to antibacterial activity and their gene numbers are: carbohydrate metabolism (99), terpenoid backbone biosynthesis (14), streptomycin biosynthesis (9), ketone body synthesis and degradation (8), and penicillin and cephalosporin biosynthesis (2). It can be seen that strain BVDT-1 has rich metabolic pathways, very active metabolic activities, and more antibacterial genes.

[0042] 2.4.4 CAZy (Carbohydrate Active Enzyme) Functional Analysis As shown in Table 2, Figure 8As shown in the figure, the genome of strain BVDT-1 contains 143 protein domains encoded by genes belonging to the CAZy family, which are distributed as follows: 46 glycoside hydrolases (GHs), 38 glycosyltransferases (GTs), 30 carbohydrate esterases (CEs), 20 carbohydrate binding modules (CBMs), 6 auxiliary oxidoreductases (AAs) and 3 polysaccharide lyases (PLs). Among them, GHs and GTs account for a large proportion, accounting for 32.16% and 26.57% respectively. Specific genes in these families, such as GH18, GH23 and CBM50, are related to the encoding of chitinase; GH1, GH3 and GH51 are associated with cellulose and hemicellulose degrading enzymes; GH126 is related to the encoding of amylase; at the same time, GH1, GH18, GH26, GH3 and GH51 are also involved in the encoding of xylan degrading enzymes; CBM50, CE4, GH18, GH23, GH73 and GT51 are related to the encoding of peptidoglycan degrading enzymes; and GH1, GH3, GH16, GH26 and GH51 are also related to the encoding of glucan degrading enzymes, and GH23 is related to the encoding of lysozyme.

[0043] Table 2 CAZyme statistics in the genome of strain BVDT-1

[0044] 2.4.5 Antibiotic resistance gene prediction To evaluate the safety of strain BVDT-1 from the risk of transmission of antibiotic resistance genes, we used the CARD database to perform homology comparison analysis on all predicted genes of BVDT-1. As shown in Table 3, a total of 3 resistance genes were identified in strain BVDT-1 in the CARD database.

[0045] Table 3 CARD annotation results of strain BVDT-1

[0046] 2.5 Analysis results of gene clusters involved in the synthesis of secondary metabolites Using the antiSMASH v5.0.0 software prediction analysis, we found that the genome of strain BVDT-1 contains 12 secondary metabolite synthesis gene clusters. Table 4 shows that the secondary metabolites encoded by these gene clusters include Surfactin, Fengycin, Bacillibactin and Bacilysin synthesized by the non-ribosomal pathway (NRPS), and Butirosin A / B, Macrolactin H, Bacillaene and Difficidin synthesized by the polyketide synthase pathway (PKS). In the comparison with known gene clusters, we found that 6 gene clusters had high similarity (100%) with the identified gene clusters, among which the similarity of the Surfactin synthesis gene cluster reached 82%. Another gene cluster (Cluster 2) can be compared with the identified gene clusters, but the similarity is low, only 7%. In addition, there are 4 gene clusters that failed to find matching identified gene clusters. These results suggest that strain BVDT-1 may have the potential to synthesize new antibacterial substances, thus showing good application prospects in the field of agricultural plant disease control.

[0047] Table 4 Comparison of secondary metabolite synthesis gene clusters in strain BVDT-1

[0048] 2.6 Analysis of novelty of the genome of strain BVDT-1 2.6.1 Comparative genome analysis of BVDT-1 and 12 strains of Bacillus velez OrthoMCL software was used to perform family clustering on the protein sequences predicted by the BVDT-1 strain and the protein sequences of 12 strains including the reference genomes MG-2, LQ-3, EN01, and UB2017, and then the gene families were analyzed. The results are as follows Fig. 9 The results showed that the 13 strains had 3116 gene families in common, accounting for 84.6% of the total gene families of strain BVDT-1. There was one gene family unique to strain BVDT-1. There was one gene family unique to strains EN01, GHI-13, JNUCC-22, LPL-KI03, R-71003, SRCM102741, Sam8H1, and AP202 respectively. There was zero gene family unique to strains UB2017, MG-2, LQ-3, and UFLA258, indicating that there was a high similarity between these four strains, which was consistent with the result that UB2017, MG-2, LQ-3, and BVDT-1 were clustered into one branch in the 2.1 evolutionary tree.

[0049] 2.6.2 SNP analysis of BVDT-1 and KOF112 strains The NCBI genome database retrieved strain KOF112, which has the same genome size as the BVDT-1 strain, but the two strains have different characteristics. The KOF112 strain contains a 13003bp plasmid, while this genome does not contain a plasmid. The two strains were isolated from different locations and environmental crops, as shown in Table 5. Whole-genome SNP (Single Nucleotide Polymorphism) analysis showed that the two strains had 20 single base differences, as shown in Table 6.

[0050] Table 5 Comparison of characteristics between BVDT-1 and KOF112 strains

[0051] Table 6 Distribution of single base difference sites between the genomes of BVDT-1 and KOF112 strains

[0052] 3. Discussion 3.1 Antibacterial effect of BVDT-1 By Colletotrichum fructicola ) caused by sweet persimmon anthracnose, Fusarium equisetum ( Fusarium equiseti ) caused by sweet persimmon leaf spot and Pseudomonas microsporus ( Pestalotiopsis microspora ) is a serious threat to production, affecting the quality and yield of fruits. Bayberry wilt disease induces the death of the entire plant and is a difficult fruit tree disease to prevent and control. In order to effectively prevent and control these diseases, in addition to the need to adopt traditional agricultural and chemical integrated prevention and control measures, the use of beneficial microorganisms for biological control is a new, sustainable green agricultural technology. Bacillus is widely used in the field of biological control of plant diseases due to its rapid reproduction rate, low nutritional requirements and strong colonization ability, especially the newly discovered Bacillus Velez, which shows greater application potential.

[0053] 3.2 BVDT-1 has new antibacterial activity Our research results show that the Velez bacteria BVDT-1 isolated from the cicada culture medium in Dongtou District has a positive effect on C. fructicola , F. equiseti and P.microspora All showed inhibitory effects, showing broad-spectrum antibacterial activity. This discovery has not been reported before, and it provides new microbial resources for the biological control of sweet persimmon and bayberry fungal diseases, and also expands the application possibilities of Bacillus Velezii BVDT-1 as a new type of biological control agent.

[0054] 3.3 BVDT-1 has good carbohydrate degradation effect The prediction of CAZyme database revealed that the genome of strain BVDT-1 contained 143 genes belonging to CAZyme. The protein domains encoded by the genes of the family are specifically distributed as 46 glycoside hydrolases (GHs), 38 glycosyltransferases (GTs), 30 carbohydrate esterases (CEs), 20 carbohydrate binding modules (CBMs), 6 auxiliary oxidoreductases (AAs) and 3 polysaccharide lyases (PLs). Therefore, it can be inferred that strain BVDT-1 has the ability to degrade chitin, cellulose, hemicellulose, starch, xylan, peptidoglycan and glucan, showing its potential to inhibit mycelial growth, and also has the function of developing and degrading crop residues such as straw and producing sugars.

[0055] 3.4 Antibiotic resistance genes Using the CARD database to perform homology comparison analysis on all predicted genes of strain BVDT-1, it was found that strain BVDT-1 has 3 antibiotic resistance genes, while the existing strain Bacillus Velez MC-1 has 208 resistance genes, including multiple multidrug resistance genes. This indicates that strain BVDT-1 contains fewer highly homologous antibiotic resistance genes and has a low safety risk of spreading resistance genes.

[0056] 3.5 Secondary metabolite gene cluster 3.5.1 Antibiotic gene clusters Through the analysis of AntiSMASH v5.0.0, we found 12 secondary metabolite gene clusters in the genome of BVDT-1. Among them, three lipopeptide antibiotic gene clusters (surfactin, fengycin, bacillibactin) and three polyketide antibiotic gene clusters (macrolactin, bacillaene, difficidin) have high similarity with the identified gene clusters, and there is one gene cluster with only 7% similarity to the antibiotic Butirosin A / B. These gene clusters are highly consistent with the prediction results of secondary metabolite synthesis gene clusters of Bacillus velezine with antibacterial effect, indicating that these gene clusters are relatively conserved in Bacillus velezine.

[0057] 3.5.2 Function of antibiotic gene cluster It is worth noting that surfactin is associated with biofilm formation; fengycin can effectively inhibit the growth of fungal hyphae such as Aspergillus flavus ABA-31; bacilysin has a wide range of antibacterial activity and has been reported as a key component of Bacillus velezensis against Gram-negative foodborne pathogens; bacillaene, as a polyene antibiotic, resists bacteria and fungi by synthesizing inhibitory proteins, which can inhibit the biofilm formation of Campylobacter jejuni; macrolactin and difficidin belong to macrolide antibiotics and are found in many types of Bacillus. Among them, difficidin can enhance the antibacterial spectrum of Bacillus amyloliquefaciens; the direct antibacterial toxin (bacillibactin) has greatly broadened the B. amyloliquefaciens Antimicrobial range of MBI600.

[0058] 3.5.3 Novelty of Antibiotic Gene Clusters It is worth noting that the similarity between the inferred product butirosin A / B of cluster 2 and the known sequences in the database is only 7%. The BVDT-1 gene cluster also metabolizes 4 substances that have no similarity with the known gene clusters, which indicates that in addition to the known antibiotics, the strain may also secrete other unknown antibiotics. Therefore, in-depth research on the secondary metabolite synthesis gene cluster of BVDT-1 will help discover more unknown antibiotics and functional genes, and provide stronger support for its application in the fields of biological control and microbial fertilizers.

[0059] As can be seen from the above examples, the present invention has confirmed that Bacillus Velezii BVDT-1 has a certain inhibitory effect on pathogenic fungi such as sweet persimmon anthracnose, Fusarium equisetum and bayberry wilt through antibacterial tests and whole genome determination. The disease-resistant genes and antibacterial products mined by whole genome sequencing technology, as well as the relationship between BVDT-1 and the model strains of the same genus, provide evidence for its high degree of horizontal gene transfer in long-term evolution. The present invention not only provides a theoretical basis for the biological control research of sweet persimmon and bayberry diseases, but also enriches the resources of biocontrol bacteria, and provides a strong basis for the development and application of new biological pesticides.

[0060] The above is only a preferred embodiment of the present invention. It should be pointed out that for ordinary technicians in this technical field, several improvements and modifications can be made without departing from the principle of the present invention. These improvements and modifications should also be regarded as the scope of protection of the present invention.

Claims

1. A Bacillus Velez, characterized in that The Bacillus velezensis is Bacillus velezensis BVDT-1, with a preservation number of CGMCC No. 30693, and was deposited in the General Microbiology Center of the China National Microbiological Culture Collection Administration on May 20, 2024.

2. The Bacillus Velezii according to claim 1, characterized in that The 16SrDNA gene sequence of the Bacillus Velezii is shown in SEQ ID NO.

1.

3. A method for culturing Bacillus Velezii according to claim 1, characterized in that: The following steps are involved: Bacillus Velez subtilis BVDT-1 is inoculated into a culture medium and cultured to obtain a Bacillus Velez subtilis culture solution.

4. The method for culturing Bacillus Velezii according to claim 3, characterized in that: The culture time is 12 to 14 hours; The culture temperature is 15-80°C; Preferably, the culture temperature is 35-38°C.

5. A culture solution of Bacillus velezensis obtained by the culture method according to claim 3 or 4.

6. Use of the Bacillus Velez subtilis according to claim 1, the culture method according to claim 3 or 4, or the culture solution of Bacillus Velez subtilis according to claim 5 in the preparation of a crop pathogenic fungus inhibitor.

7. The use according to claim 6, characterized in that: The crop pathogenic fungi are Pseudomonas microsporus, Pseudomonas carinii or Fusarium equisetum.

8. Use of the Bacillus Velez subtilis according to claim 1 or the culture solution of the Bacillus Velez subtilis according to claim 5 in producing antimicrobial active substances.

9. Use of the Bacillus Velez subtilis according to claim 1, the culture method according to claim 3 or 4, or the culture solution of Bacillus Velez subtilis according to claim 5 in preparing a biocontrol agent for plant diseases.

10. A plant disease biocontrol agent, characterized in that: The plant disease biocontrol agent contains the Bacillus Velezii described in claim 1; The plant diseases are bayberry wilt, sweet persimmon anthracnose and sweet persimmon leaf spot.

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