A Bacillus velezensis strain isolated from oats with disease-preventing and growth-promoting properties

Endophytes have been shown to promote plant growth and health. In the present study, a Bacillus velezensis CH1 (CH1) strain was isolated and identified from high-quality oats, which was capable of producing indole-3-acetic acid (IAA) and strong biofilms, and capabilities in the nitrogen-fixing and iron carriers. CH1 has a 3920 kb chromosome with 47.3% GC content and 3776 code genes. Compared genome analysis showed that the largest proportion of the COG database was metabolism-related (44.79%), and 1135 out of 1508 genes were associated with the function “biosynthesis, transport, and catabolism of secondary metabolites.” Furthermore, thirteen gene clusters had been identified in CH1, which were responsible for the synthesis of fifteen secondary metabolites that exhibit antifungal and antibacterial properties. Additionally, the strain harbors genes involved in plant growth promotion, such as seven putative genes for IAA production, spermidine and polyamine synthase genes, along with multiple membrane-associated genes. The enrichment of these functions was strong evidence of the antimicrobial properties of strain CH1, which has the potential to be a biofertilizer for promoting oat growth and disease resistance.

As a type of grain, oats are not only one of the main food sources but also an important feed for livestock globally.The main planting areas of oats are mountainous, plateaus, and high cold areas in China.The Ulanqab region of Inner Mongolia is one of the main oat-producing areas in China.Its planting area and output both rank first among prefectural-level cities in China, and it is an important local food crop.B. velezensis has been found in many plants with specific functions, but it is not well-studied in oats.Therefore, in the present study, we screened, isolated, purified, and identified B. velezensis from high-quality oats and characterized its phenotypic features by applying a series of biochemical procedures.We then conducted a whole genome comparison analysis of B. velezensis to explore the molecular mechanism by which B. velezensis promoted the growth and pest control of oats from a molecular perspective.

Results
Identification and biochemical features of the CH1 strain CH1 has a protruding, irregularly round shape with concentric circles in the center and white mucus inside (Fig. 1a).It is a Gram-positive bacillus (Fig. 1a) and exhibits a series of beneficial microorganism features such as aerobic or facultative anaerobic respiration, motion, catalase and VP positive, gelatin liquefaction positive, and indole negative, and is able to ferment glucose, arabinose, xylose, mannose, and starch.The 16s RNA sequence analysis showed that there was a 100% homology similarity of the CH1 16s DNA sequence to Bacillus velezensis strain GD-1 (MH373536.1)and Bacillus velezensis strain SN337 (MW857238.1)(Fig. 1c).CH1 grew weakly in an agar medium with ACC as the only nitrogen source (Supplementary Fig. S1) and produced a blue color in the Nfb solid medium compared with the blank medium (Supplementary Fig. S2).The present study confirmed that CH1 did not demonstrate phosphate solubility in NBRIP solid medium (Supplementary Fig. S3), but did produce iron carriers (Supplementary Fig. S4).In addition, CH1 had a significant capacity for generating IAA (Table 1) and biofilms (Table 2) and had a strong inhibitory effect on Bipolaris sorokiniana, Powdery mildew, and Tilletia controversa (Table 3, Fig. 1b).

Whole-genome sequencing profiling and COG functional annotation of strain CH1
A total of 494,097 reads were sequenced with an average length of 882 bp.The CH1 strain was assembled into a round chromosome with a size of 392,0437 bp, and the guanine-cytosine (GC) content of the total genome was 47.3% (Table 4).There are 3776 code genes in the genome of the CH1 strain, 27 in rRNA, 86 in tRNA, and 93 in ncRNA.The genome circle map was constructed based on the COG database annotation results (Fig. 2a).Metabolism accounted for the largest proportion of annotations (44.79%), and this category was considered to be related to predicted secondary metabolic activity proteins.Among them, 1508 genes were annotated into 1135 COGs, which were divided into eight types of functions (Fig. 2b).Proteins related to "secondary metabolite biosynthesis, transport, and catabolism" accounted for 7.36%.The enrichment of these functions was strong evidence for the antibacterial properties of strain CH1.
A genome-based phylogenetic tree was constructed between strain CH1 and 201 B. velezensis strains in the NCBI genome database.The dendrogram showed that strain CH1 was closely related to strains KS04AU Table 1.The results of determination of IAA (n = 3, X ± SD).The culture medium TSB of uninoculated bacteria was used as blank control to remove the influence of the medium on the results, and Bacillus subtilis was the positive control group.The ability of CH1 to produce IAA was significantly higher than that of the blank control group and the positive control group (P < 0.05).This showed that CH1 had a significant capacity for generating IAA.Each value represents the mean value standard deviation (SD) from three trials under the same experimental conditions.*The significant difference compared with blank control (P < 0.05), **Indicates the extremely significant difference compared with blank control (P < 0.01).2. Results of CH1 biofilm formation (n = 3, X ± SD).The culture medium TSB of uninoculated bacteria was used as blank control to remove the influence of the medium on the results, Bacillus subtilis was the positive control group.The ability of CH1 to produce biofilm was extremely significant higher than that of the blank control group and positive control group (P < 0.01).Each value represents the mean value standard deviation (SD) from three trials under the same experimental conditions.*Indicates the significant difference compared with blank control (P < 0.05), **Indicates the extremely significant difference compared with blank control (P < 0.01).www.nature.com/scientificreports/(CP092750.1)and Bacillus velezensis YL2021 (YL2021) (CP120641.1)and clustered with YYC (CP075055.1)and Bacillus velezensis LZN01 (LZN01) (CP081488.1)(Fig. 1d).It has been reported that the Ortho ANI value of 95% (corresponding to 70% DDH) can be used as a cut-off point for bacterial species classificatio 9,10 .Using the genome sequences of strain CH1 and six Bacillus genera, OrthoANI values were calculated.Strain CH1 showed the highest similarity with KS04AU, YYC and Bacillus velezensis 26.3 (26.3) (99.34%), followed by LZN01 (99.33%),Bacillus velezensis VCN56 (VCN56) (99.31%),YL2021 (99.31%), and FZB42 (98.92%) (Fig. 1e).Additionally, the dDDH values between strain CH1 and YYC, KS04AU, LZN01, 26.3, VCN56, YL2021, and FZB42 ranged from 95.70% to 97.90% (Supplementary Table S1).Based on the above results, six strains of B. velezensis with the closest affinity were selected to construct a phylogenetic tree based on the full-length 16S rRNA gene with the target strains, and the results showed that strain CH1 was the closest affinity to YYC. (Supplementary Fig. S5).
Taking it all together, we selected KS04AU and YYC as well as the Bacillus model strains for comparative analysis.

Comparative genomics analysis of CH1
Bases from the core and pan-gene cluster analysis showed that strain CH1 formed a close genetic relationship with strain KS04AU and had obvious branching with four other strains:YYC, FZB42, DSM7 and 168 (Fig. 1f).Core-and pan-genome analysis showed that the number of non-redundant pan-genes between CH1 and the five reference strains was 4382, among which the number of shared core genes was 2736 and the number of non-shared dispensable genes was 1646.The number of specific genes in CH1 accounted for 3.9% of the total number of genes, and accounting for 3.2% and 0.9% of the total number of genes in strain KS04AU and strain YYC, respectively (Fig. 3a,b).These data suggested that the number of specific genes held by CH1 was relatively large and was scattered in the genome, while the function of most specific genes was unknown.These data  demonstrated that strain CH1 was potentially a newly discovered antifungal strain.The genome collinearity analysis showed that the CH1 has a high genome similar to strain YYC (96.49%),KS04AU (95.65%) and FZB42 (95.63%) , followed by the Bacillus amyloliquefaciens DSM7 (DSM7) (87.95%), and 168 (67.31%) (Fig. 4).Among all the sequenced genomes, CH1 showed the highest homology with YYC, KS04AU and FZB42 indicating that they had a close relationship at a genomic scale.

Prediction of CH1 genome-wide secondary metabolites
The CH1 strain encoded a total of 13 secondary metabolite synthesis gene clusters, among which eight gene clusters were identified with complete similarity or high similarity, and another three gene clusters could not be identified (Table 5).The secondary metabolites encoded by the CH1 strain included five non-ribosomal peptide synthases (NRPSs), four transAT-PKSs, two terpenes, two T3PKSs, one Lanthipeptide class III, and one PKS-like.Most of these were related to the production of the NRPS and PKS systems.Cluster 13 is a unique metabolite pathway, which is associated with metabolite and synthesis of the bacilysin.CH1 was found to produce bacinapeptin, surfactin, fengycin, bacillibactin, bacillaene, macolactin, difficidin, bacilysin, locillomycin, and other peptide glycans and polyketosaccharide resistance compounds.The synthesis of these secondary metabolites is one of the main mechanisms of disease prevention.By comparing the gene clusters of CH1 with those of five other strains of Bacillus (KS04AU, YYC, FZB42, DSM7, and 168), it was found that six gene clusters (2, 4, 6-8, and 13) were conserved among the six strains.Two clusters (3 and 9) were conserved among CH1, KS04AU, YYC, and FZB42; two clusters (5 and 10) were conserved among CH1, KS04AU, YYC, and FZB42; and one cluster (1) was conserved between CH1 and YYC (Table 5), which encodes andalusicin.Based on comparative genomics research, we identified three clusters (4, 8, and 9) encoding terpenes and T3PKs, which were not described as previously reported due to the limited similarity of the compounds deposited in the Minimal Information on Biosynthesis Genes in Clusters (MIBiG) database (Fig. 5).Although the core biosynthetic genes of the four strains CH1, KS04AU, YYC, and FZB42 are very similar, they have different genetic structures.In the surfactin cluster, xy02 genes were only present in CH1, ycxC and ycxD genes were lacking in DSM7, and the yx01 gene was lost in 168.The Fengycin cluster in DSM7 lacked fenB, fenC, and fenD, and the bmyC, bmyB, bmyA, ituA, ituB, ituD, bmyD, YxjF, scoB, scoA, and YxjC genes were missing in 168 (Supplementary Table S2).

Antimicrobial activity of CH1
The CH1 genome was found to encode a variety of antimicrobial active compounds by genetic comparison of secondary metabolite gene clusters with the MIBiG database (Table 6).We selected the ones with more than 80% homology for analysis.Bacillaene (BGC0001089, BGC0001090), Difficidin (BGC0000176), and Amylocyclicin (BGC0000616) were antimicrobial compounds encoded by gene clusters 6, 10, and 12, respectively.In addition, CH1 had gene clusters (2, 5, 6, 7) involved in the production of some antifungal compounds such as Surfactin (BGC0000433), Macrolactin H (BGC0001383, BGC0000181), Bacillomycin D (BGC0001090), iturin (BGC0001098), and fengycin (BGC0001095).Some active compounds detected in clusters 7and13 had dual antibacterial-antifungal activity, such as Pipastatin (BGC0000407) and Bacilysin (BGC0001184).It was also notable that Bacillibactin (BGC0001185) was a gene cluster encoding an iron carrier product.CH1 hindered the  www.nature.com/scientificreports/growth of plant-treating bacteria and fungi by competing for essential iron ions and thus played an important role in accelerating the acquisition of iron ions (Fe 3+ ) from minerals and inter-root organic compounds 11 .

Plant growth-promoting activity of CH1
In this study, The CH1 genome harbors numerous genes encoding proteins linked to plant growth-promoting activities, prominently including seven genes involved in the synthesis of IAA (Table 7).These genes are clustered within the tryptophan biosynthesis pathway, suggesting their participation in tryptophan-dependent IAA biosynthetic pathways.Moreover, some genes encode putative spermidine acetyltransferases and cytochrome P450 synthetases, which are predicted to produce spermidine and polyamines.Spermidine, a type of polyamine, has been shown to enhance plant growth and improve salt tolerance when accumulated within plants.Notably, the CH1 genome also comprises 12 genes that play a role in the development and regulation of biological membranes.

CH1 is involved in symbiotic nitrogen fixation and tumorigenesis
The CH1 genome contains genes predicted to produce arabinogalactanase, pectate lyase, and xylanase, as well as the guaB gene encoding an IMP dehydrogenase, which is predicted to be involved in the nodulation process in conjunction with rhizobia.In addition, three genes (cysK, cysE, and ilvE) were detected in the CH1 genome and are predicted to be involved in cysteine biosynthesis.An enhanced plant nitrogen fixation ability can promote plant growth and development.

Discussion
NCBI's genomic phylogenetic analysis and 16S rDNA homology analysis reveal that CH1 exhibits a high degree of homology with KS04AU, YL2021, YYC, and LZN01, originating from diverse sources.The homology analysis based on 16S rDNA sequencing is a commonly employed taxonomic method for bacteria 25 .Among the six Bacillus velezensis strains analyzed, CH1 shows the closest relationship to YYC based on 16S rDNA homology, which is further supported by the Average Nucleotide Identity (ANI) analysis.Bacillus velezensis YYC was isolated from tomato rhizosphere soil.Given the aforementioned findings, there appears to be no significant correlation between the strains and their host sources.In this study, biochemical characterizations and a comparative genome analysis were performed to survey the potential biology characteristics of CH1.B. velezensis was found to be nitrogen-fixing and capable of forming biofilms 6 .It has been shown that the antagonistic activity and biocontrol effects of Bacillus are related to its ability to form biofilms 38,39 .B. velezensis was also shown to produce IAA to promote plant growth 4,5 .It was also confirmed in this study that CH1 produced IAA.Before this study, there was little research on whether B. velezensis encoded an ACC deaminase, produced an iron carrier, or solubilized phosphate.ACC deaminase directly promotes plant growth and indirectly inhibits plant diseases 40 .Recently, ACC deaminase has been discovered in various Gram-negative bacteria, fungi, endophytes, and rhizobia 39 .However, the expression level of ACC deaminase varies among different organisms.Currently, the entire genetic composition and functions of ACC deaminase genes have only been described in a limited number of bacterial species, while most of the relevant bacterial genes are unknown 41 .Early studies also found that some genes encoding ACC deaminase activity also encode D-cysteine desulfhydrase, suggesting that changes in specific gene loci could potentially be attributed to D-cysteine desulfhydrase 42 .Reports have documented that B. velezensis possesses ACC deaminase activity 43 .In this study, we also observed that CH1 exhibits a certain level of ACC deaminase activity, yet a complete gene (acdS) related to this activity was not identified in the CH1 genome.Instead, we found a homocysteine desulfhydrase gene, which may play a significant role in the expression of ACC deaminase activity in B. velezensis.IAA biosynthesis involves both tryptophan-dependent and tryptophan-independent pathways, with the former being the most dominant and common pathway 44,45 .In addition, iron carriers make iron useful as an enzyme cofactor for the shuffling of electrons, therefore enabling transition metals to be involved in many basic biological processes, such as respiration, photosynthesis, and nitrogen fixation 46 .Most of the inorganic phosphorus salts applied as fertilizers in soil consolidate quickly after application and become unavailable.Inoculation of seeds or soil with bacteria that increase phosphorous could improve the solubility of soil phosphorus and exert an equivalent effect of phosphorus fertilization 47 .However, although CH1 bacteria grew in the NBRIP solid medium, there was no transparent trend compared with positive control, which confirmed that CH1 did not solubilize phosphate.
CH1 demonstrated significantly higher inhibition than the negative control group (PBS) on Bipolaris sorokiniana, Powdery mildew, and Tilletia controversa in this study.Bipolaris sorokiniana is a worldwide plant pathogenic fungus, which can infect the underground and above-ground parts of wheat and cause wheat root rotis.Tilletia controversa is a fungal disease characterized by a black whiplash (black spike) growing from the apex of the stem.Tilletia controversa causes smut in wheat.Powdery mildew occurs in leaves, stems, stalks, buds, and petals.The affected area turns gray, the leaves shrink and become smaller when the damage is severe, the tender ends are twisted and deformed, and the flower buds do not open.These results confirmed that B. velezensisto inhibited the growth of the main pathogenic bacteria of whea 20,29,48,49 .The inhibition by CH1 of these pathogens might be through the production of metabolites.The secondary metabolites might be important for plant growth and defense 29 ; volatile organic compounds of B. velezensis were found to be involved in biocontrol 49 .Moreover, antimicrobial active substances were found in B. velezensis 20,49 .Antibacterial compounds not only inhibit the growth of plant pathogens and fungi but also stimulate the induction of systemic resistance in plant 50 .The largest www.nature.com/scientificreports/proportion of annotations in the COG database was metabolism (44.79%), including biosynthesis, transport, and catabolism of secondary metabolites.The enrichment of these functions was strong evidence of the antimicrobial properties of strain CH1.The secondary metabolites are potentially associated with participation in the cluster production of antimicrobials, including bacillin, surfactin, metallatin, arosetin, bacillin, dysgentin, and terpenes.However, clusters that predict the production of antipeptides are not typical in other bacillus.Antipeptides are ribosomally synthesized peptides that undergo extensive post-translational modifications and are antimicrobial compounds produced by gram-positive bacteria 51 .Biological nitrogen fixation is an important factor in promoting plant growth 52 .B. velezensis is able to secrete a rich variety of secondary metabolites, which is an important prerequisite for its beneficial effects on plants such as growth promotion and disease prevention 53 .Wu et al. found that the genome of B. velezensis NAU-B3 contains eight genes clusters coding for secondary metabolites with known functions, such as bacillomycin D, bacilysin, and bacillaene, and one genes cluster with unknown functions, which are synthesized in the NPRS, PKS, and PRS pathways, and have the effects of inhibition of bacteria and fungi, formation of biofilm, and accumulation of ferric iron ions, etc 54 .In the present study, we found that the genome of CH1 is rich in gene clusters, which are effective for promoting the growth of the plant and defense.Thirteen gene clusters were identified in the CH1 genome, which were responsible for the synthesis of 15 secondary metabolites, including those encoding andalusicin, macrolactin, bacillaene, fengycin, difficidin, and bacillothiazol, bacillbactin, bacilysin, etc.The gene clusters encoding the synthesis of eight inhibitory products were identical to the identified gene clusters, and the similarity between the surfactin synthesis gene cluster and the identified gene clusters was as high as 91%.In the surfactin cluster, the xy02 gene is unique to CH1 in the surface protein cluster and is a derivative of CA4P.Furthermore, xy02 exhibited a certain growth inhibition effect on human non-small cell lung cancer NCI-H460 cells 55 .However, it was also shown that although CH1 had a large number of specific genes distributed in the genome, the function of most of the specific genes was unknown.Further investigation will help to understand the molecular mechanisms of the prebiotic properties of strain CH1.These secondary metabolites have been reported to antagonize phytopathogenic bacteria, fungi, and viruses, induce plant disease resistance, accumulate iron ions, and form biofilms, which suggests that the bioprophylactic bacterium CH1 has a good application value.In addition, three unknown clusters of secondary metabolites synthesizing genes were predicted from the genome of CH1, which will provide a basis for exploring the potential of CH1 for bioprevention and discovering novel bacterial inhibitors in the future.
A number of genes that may be related to plant growth-promoting activity have been identified in the CH1 genome.The CH1 genome has trpA, trpB, trpC, trpD, trpE, trpF, and trpG genes involved in the synthesis of IAA from indole-3-acetonitrile 24 .Therefore, we suggest that CH1 contains genes functionally related to growth hormone synthesis and plays an important role in promoting plant growth and development 56 .Moreover, CYP102A2/ CYP102A3 are genes encoding cytochrome P450, while speE, msmX, and speG are genes encoding enzymes involved in spermidine and polyamine synthesis, which are believed to play a role in plant development and growth promotion through participation in the synthesis of active metabolites 57 .The development of biofilms in the strain promotes plant growth and protects the plant from infecting microorganisms through the secretion of antimicrobial compounds 28 .CH1 has several genes involved in biofilm development.It has been shown that many genes are involved in the synthesis of Fengycin, Bacillibactin, while Bacilysin is upregulated during biofilm formation 34 .We expected that CH1 might promote rhizoma formation because some genes associated with rhizoma formation were detected in the CH1 genome, and these genes were predicted to produce arabinogalactanase, pectate lyase, and xylanase, which may weaken the root hair cell walls, facilitate bacterial invasion, and contribute to form rhizomes 58 .Another study shows that the guaB gene encoding guanine is efficiently expressed during the early stages of rhizoma formation 59 .

Conclusion
In the study, CH1 was isolated, purified, and identified from high-quality oat varieties.The physiological indexes of CH1 and the whole genome of CH1 were determined to verify the effect of CH1 on promoting the growth of oat and inhibiting the main diseases of oat.It was confirmed that CH1 might be a potential biofertilizer for promoting oat growth and disease resistance.

Isolation and purification of the strain
The oat plants used in this experiment were Baiyan No.2, supplied by Ulanqab Agricultural and Animal Husbandry Academy, and Inner Mongolia Academy of Agriculture and Animal Husbandry Sciences in China, which possess a strong ability to inhibit disease.The oat plants were rinsed well with tap water and then rinsed five times with sterile PBS.They were cut into smaller pieces with sterile scissors, homogenized in a small homogenizer, and drained into a paste.The plant tissue paste was eluted with PBS into a sterilized glass bead flask and fully oscillated.Cells from plant tissues were cultured in trypticase soy broth (TSB) solid medium using multiple dilutions and the pour culture method.Then, the plates were cultured in an anaerobic incubator and thermostatic incubator at 37°C for 24-36 h.After the bacteria grew, a single colony was selected that had a white color, wrinkled surface, dry texture, and filamentous mucus inside the colonies, and the bacteria were positive upon Gram staining.The bacteria were marked until the colonies were single and typical.

Identification of the strain
The physiological and biochemical characteristics of the purified strain were identified using an HBI Bacillus Biochemical Identification strip (GB) (Qingdao Hope Bio-Technology Co., LTD., Qingdao, China), and 16s RNA sequence analysis based on universal primers (27F and 1492R) was used to confirm the strain.The physiological and biochemical experiment process is detailed as follows: The bacteria were cultured in an anaerobic incubator, a constant temperature incubator, and an anaerobic bag in a constant temperature incubator to test whether the bacteria grew in an anaerobic state, an aerobic state, or a facultative anaerobic state.The experimental bacteria were inoculated with a straight needle in a semi-solid medium and cultured in an anaerobic incubator, and their motility could be observed through the optical eye.If the culture grew on the puncture line of the inoculation and the edge was very clear, it indicated that the test bacteria had no motility; if the growth of the culture spread from the puncture line to the periphery in a cloud shape, and the edge was blurred into a cloud shape, it indicated that the bacteria had motility.Take a ring of culture coated on a clean slide, and then add a drop of 3-15% hydrogen peroxide onto it, if there is a bubble, it is a positive reaction, if there is no bubble is a negative reaction.The culture was inoculated in a gelatin medium for culture, and the results were observed by low-temperature treatment.The inoculated and uninoculated pairs were placed in the refrigerator for solidification and then removed, and their liquefaction status was compared.For example, when the inoculated tubes were solidified, the results were positive for liquefaction and negative for coagulation or liquefaction at the same time.The culture was mixed with 40% NaOH and 1 mg creatine at room temperature for 30 to 60 minutes, and the red reaction was positive and unchanged into a negative reaction.The bacteria were inoculated in a sugar fermentation medium, and if the result turned yellow, it indicated that the carbohydrate acid production was positive, and if it did not change color, it was negative.Each experiment was repeated three times.Then, the amplified products were sequenced, and phylogenetic analysis was performed with the software MEGA version 6.0.

CH1 biochemical features
The biochemical features of strain CH1 were detected by a series of biochemical tests, including assays for ACC deaminase, nitrogen, solubility of phosphate, iron carrier formation, IAA production, biofilm formation, and antibacterial activity.All biochemical tests were according to standard experimental procedures and followed the manufacturer's instructions.In addition, the Oxford cup method was used for detecting antibacterial activity 60 .The diameter of the bacteriostatic zone was measured accurately with vernier calipers to determine the bacteriostatic ability.

Whole-genome sequencing and functional annotation
After activation of strain CH1, bacteria were collected by centrifugation.Following the manufacturer's instructions, the genomic DNA of the strain was extracted using the bacterial DNA extraction kit (Omega Bio-Tek, USA).Subsequently, the purified DNA samples underwent quality control.The genomic DNA was quantified using the TBS-380 fluorometer (Turner BioSystems Inc., Sunnyvale, CA).Illumina libraries were constructed using the TruSeq™ Nano DNA Sample Prep Kit (Illumina, USA) and sequenced on the Illumina novaseq6000 platform.For PacBio sequencing, 20k insert whole-genome shotgun libraries were generated and sequenced using standard methods on the PacBio RS instrument.Illumina sequencing data were employed for correcting PacBio sequencing data and simultaneously assessing the complexity of the genome.Following genome assembly and result evaluation, genome composition analysis was conducted.Gene prediction for the strain CH1 genome was performed using GeneMarkS (Version 4.17, http:// topaz.gatech.edu/ GeneM ark/) software.The prediction of rRNA, tRNA, and other ncRNA contained in the genome was accomplished using RNAmmer (version 1.2, http:// www.cbs.dtu.dk/ servi ces/ RNAmm er/), tRNAscan-SE (version 2.0.4,http:// lowel ab.ucsc.edu/ tRNAs can-SE), and Infernal (version 1.1.14)softwar [61][62][63] .The eggNOG (http:// www.ncbi.nlm.nih.gov/ COG/) database was applied to the CH1 genome-wide data for orthologous (COG) functional annotation.The SwissProt (http:// unipr ot.org) database was used to annotate growth-promoting genes in CH1.

Comparative genomics analysis
Based on the whole genome sequence of strain CH1, the whole genome sequences and annotations of 201 B. velezensis strains isolated from different hosts and environments were obtained from the GenBank genome database (https:// www.ncbi.nlm.nih.gov/ genome/).Genome phylogenomic analysis of 206 B. velezensis strains was performed using REALPHY (http:// realp hy.unibas.ch/ realp hy).Orthologous Average Nucleotide Identity Tool (v0.93.1) was used to calculate OrthoANI and type the CH1 genom 64 .Digital DNA-DNA hybridization (dDDH) analysis was performed by Genome-to-Genome Distance Calculator (GGDC v 3.0, https:// ggdc.dsmz.de/ ggdc.php) 65 .Utilizing the MEGA version 6.0 software, a phylogenetic tree was constructed based on the fulllength 16S rRNA gene sequences of six closely related Bacillus velezensis strains.A comparative whole-genome analysis was further conducted, including strains closely affiliated to CH1 as well as Bacillus reference strains (KS04AU 66 , YYC 67 , FZB42 17 , DSM7 68 , and 168 69 ).The complete genome sequences of strains KS04AU, YYC, FZB42, DSM 7, and 168 were downloaded from NCBI Genome and then analyzed together using comparative genomics with the complete genome sequences of CH1.CD-HIT 4.6.6 (http:// cd-hit.org) software was used for cluster analysis, core genes and specific genes of the genome were determined 70 , and the number of common and specific gene petals was constructed.A phylogenetic tree was constructed using PhyML (Maximum Likelihood, version 3.0, http:// www.atgc-montp ellier.fr/ phyml/) software.AntiSMASH version 7.1.0software (https:// antis mash.secon darym etabo lites.org) was used to predict the scope of secondary metabolite synthesis 71 .Meanwhile, the predicted gene clusters of secondary metabolite synthesis were compared and analyzed.Using antiSMASH 7.1.0on the default settings, the structure of secondary metabolism was detected against MIBiG.

Figure 2 .
Figure 2. Whole Genome Structure and Functional Analysis of Strain CH1.(a) Circle diagram of CH1 genome.(b) The statistical results of the Clusters of Orthologous Groups of proteins (COG) functional classification of genomic protein.

Figure 3 .
Figure 3. Comparative genomic analysis of strain CH1.(a) CorePan genome analysis of bacteria strains.(b) Venn diagram showing the distribution of orthologous gene families in the genomes of CH1, KS04AU, YYC, FZB42, DSM7 and 168.This analysis exploits all CDS of the genomes and is not restricted to the core genome.The number above each part represents the number of gene families, while the number below (in parentheses) represents the number of genes.

Figure 4 .
Figure 4. Global alignment of CH1 and Bacillus spp.chromosomes.Indels are depicted by gray rectangles.Dark blue indicates a sequence with synteny, red indicates forward collinearity and light blue indicates negative collinearity.

Figure 5 .
Figure 5. Comparative analysis of secondary metabolic gene clusters.Comparative analysis of the location and products of secondary metabolite gene clusters in CH1, KS04AU, YYC, FZB42, DSM7 and 168.

Table 3 .
Antimicrobial activity analysis of CH1 (n = 3, X ± SD).Phosphate buffer (PBS) was used as the blank control group, and XinSheng, a product on the market for inhibiting Bipolaris sorokiniana, Powdery mildew, and Tilletia controversa, was used as the positive control group in this experiment.The inhibition of CH1 and positive control on pathogens were extremely significant different from that of the blank control group (P < 0.01), and CH1 decreased significantly to Powdery mildew compared with positive control group (P < 0.05), CH1 increased significantly in Bipolaris sorokiniana compared with the positive control group (P < 0.05), CH1 had no significant difference in Tilletia controversa compared with positive # The significant difference compared with the positive control group (P < 0.05), ## The extreme significant difference compared with the positive control group (P < 0.01).

Table 4 .
Statistical table of CH1 genome components.

Table 6 .
Predicting genes and gene clusters encoding active metabolites in the genome of CH1.*% of gene cluster similarity against MIBiG database.

Table 7 .
Bioactivity-related genes in the CH1 genome.
37 Vol:.(1234567890)Scientific Reports | (2024) 14:12950 | https://doi.org/10.1038/s41598-024-63756-8 This study was approved by the Plant Protection Association of Inner Mongolia Autonomous Region and the Professional Committee of Jining Normal University of Inner Mongolia Autonomous Region (Ulanqab, China; Approval No. 20221012), and complied with the IUCN Policy Statement on Research Involving Species at Risk of Extinction and the Convention on the Trade in Endangered Species of Wild Fauna and Flora.