Complete Genome Analysis of Bacillus Velezensis ES2-4: A Novel Bacillus Strain with Potential for Biofitilizer and Biocontrol Agent

Abstract

Bacillus velezensis is a new species of Bacillus sp recently discovered. It is considered a new plant growth promoting rhizobacteria because of its ability to inhibit a variety of pathogens and promote plant growth. In this study, we isolated a novel Bacillus velezensis strain ES2-4 in orchard soil. Based on comparative analysis, including average nucleotide polymorphism (ANI), digital DNA-DNA hybridization (dDDH), and phylogenetic analysis, ES2-4 was designated as Bacillus velezensis. Genomic analysis revealed that Bacillus velezensis contains a series of genes closely related to promoting plant growth directly and indirectly. Genomic secondary metabolite analyses indicated there are 9 identified gene clusters involved in the biosynthesis of various secondary metabolites (Surfactin, Iturin, Fengycin, Bacillibactin, Bacilysin, Macrolactin, Bacillaene, Difficidin, Amylocyclicin), and five clusters encoding polyketides, lanthipeptide, terpenes, but the specific substances are unclear. In addition, Bacillus velezensis ES2-4 showed good antagonistic activity against various plant pathogens, especially Rhizoctonia solani. These findings suggest that Bacillus velezensis ES2-4 could be used as a PGPR strain to develop new biofertilizers and biocontrol agents.

Introduction

It is anticipated that the human population will reach 9 billion by 2050, which will pose a major challenge for agricultural systems to produce enough food to feed the global population(Chapman et al. 2021). However, in agricultural production, biotic and abiotic stresses are key constraints to food quality, crop yield and global food security. Actions must be taken to increase food production against global threats posed by abiotic and biotic stresses(Prosekov and Ivanova 2018). Abiotic stresses include decreased precipitation, increased drought, salinization, inappropriate temperature, heavy metals, and nutrient deficiencies(Singh et al. 2011). Biological stress is mainly caused by the invasion of plant pathogenic microorganisms (such as bacteria, viruses, and fungi), which can reduce crop productivity(Khoshru et al. 2020). Plants are severely affected by these stresses and function impaired Tor to increase agricultural production, the abuse of chemical fertilizers and other agrochemicals is inevitable(Glick 2014). However, the excessive use of chemical fertilizers will cause a series of negative impacts on the environment, such as soil pollution, water pollution, and air pollution(Adesemoye et al. 2009). The study found that an important class of beneficial root-colonizing bacteria exists in the plant rhizosphere and soil, which exhibit synergistic and antagonistic roles with soil microbiota and participate in a range of ecologically significant activities. They directly or indirectly increase biotic and abiotic stress tolerance and thereby promote plant growth, which we collectively refer to as plant growth promoting rhizobacteria(PGPR)(Basu et al. 2021). Reported PGPRs include members of the Aeromonas, Agrobacterium, Azotobacter, Bacillus, Burkholderia, Enterobacter, Flavobacterium, Pseudomonas, Rhizobium, and others(Khoshru et al. 2020). PGPR have attracted worldwide attention in recent years in the control of various plant pathogens and promoting plant growth due to their low cost, easy production, environmental friendliness, and no residue effects.(Vignesh et al. 2022)

Bacillus species is one of the main bacteria found in soil(Saxena et al. 2020). Compared to other types of PGPR, Bacillus species has the outstanding property of being able to tolerate heat, radiation and some chemicals due to its ability to form spores(Setlow 2010), and is convenient to make various bacterial fertilizers or biocontrol agent for easy transportation and storage, which makes Bacillus species one of the most commercially useful bacteria in the agricultural biotechnology industry(Rabbee et al. 2019; Saxena et al. 2020). It is currently recognized that Bacillus species can directly or indirectly promote plant growth(Fan et al. 2018). On the one hand, Bacillus species use nitrogen fixation, phosphorus and potassium solubilization, siderophore, plant hormone synthesis (auxin, cytokinin, gibberellin, ethylene), ACC deaminase, etc(Ahmed et al. 2022; Alijani et al. 2022; Khan et al. 2020; Sibponkrung et al. 2020), directly promote plant growth, on the other hand indirectly promote plant growth through nutrient competition with pathogens, niche exclusion, induction of systemic resistance and production of antimicrobial metabolites and lytic enzyme(Chowdhury et al. 2015; Fan et al. 2018; Peng et al. 2019; Yin et al. 2021)

In 2005, Cristina Ruiz-García isolated two strains of Bacillus CR-502T and CR-14b capable of synthesizing surfactants in the Vélez River, Spain. It was confirmed by DNA-DNA hybridization that its hybridization rate with Bacillus subtilis, Bacillus vallismortis and Bacillus amyloliquefaciens was less than 20%, so these strains were classified into a new species of Bacillus and named Bacillus velezensis sp.nov. However, the determination of its taxonomic status and species name has been the focus of debate among bacterial taxonomists until 2016, when scientists made it possible to determine the effective species name of Bacillus velezensis based on systematic genomics methods(Dunlap et al. 2016; Ruiz-Garcia et al. 2005). As a new species of Bacillus. spp, with the completion of the sequencing of the model strain FZB42 of the first Gram-positive plant growth promoting bacteria and biocontrol rhizosphere bacteria(Tayeb et al. 2008), more and more strains of Bacillus velezensis have been isolated from different environments, such as plant rhizosphere soil and animal dung (Ye et al. 2018), and now there are more than 400 genomes of different strains uploaded to GeneBank. Compared with other Bacillus. spp, Bacillus velezensis can synthesize more abundant secondary metabolites, including cyclolipopeptides (Surfactin, Iturin, Fengycin, Bacillibactin), polyketides (Macrolactin, Bacillaene, and Difficidin), a kind of dipeptide Bacilysin, and two kinds of bacteriocin, Plantazolicin, Amylocyclicin found in FZB42 strain(Basu et al. 2021; Rabbee et al. 2019; Scholz et al. 2011; Scholz et al. 2014). Since many strains of this species strongly inhibit the growth of microbial pathogens, including bacteria, fungi, and nematodes, Bacillus velezensis has attracted attention as a valuable biological control agent and an effective alternative to synthetic agrochemicals(Ye et al. 2018).

In this study, we isolated a strain of Bacillus velezensis ES2-4 from the garden soil of Sichuan Normal University and obtained the whole genome sequence of the strain by whole genome sequencing. Through genome-wide analysis, it was found that the strain contains a large number of 14 huge gene clusters related to the synthesis of secondary metabolites, and can synthesize multiple substances that inhibit various familiar plant pathogens such as R. Solani, B. dothidea, Gibberella zeae, F.oxysporum. In addition, the strain contains a large number of coding genes related to the promotion of plant growth. The results indicated that Bacillus velezensis ES2-4 has the potential for further application as a biological control agent in agricultural production.

Materials And Methods

Microbial Strains and Related Medium

In the early stage of the experiment, we collected part of the soil from the orchard and vegetable garden soil of Sichuan Normal University and referred to the Manli Zhu’s separation method(Zhu et al. 2019), and screened out a bacterium that can strongly inhibit Botrytis cinerea and named it ES2-4. The strain was preliminarily identified as Bacillus spp by some methods of physiology and biochemistry. Strain ES2-4 was incubated in liquid LB medium (10g tryptone, 5g yeast extract, and 10g NaCl per liter) at 37℃ for 24h in a rotatory shaker (200rpm). The ability of ES2-4 to form biofilms can be tested under no shaking. For secondary metabolite production, strain ES2-4 was incubated in Landy medium (MgSO₄ 0.5g, KH₂PO₄ 1g, KCl 0.5g, MnSO₄·5mg, FeSO₄ 0.15mg, CuSO₄ 0.16 mg, glucose 20g, sodium hydrogen glutamate 5g per liter) at 30℃ and 200 rpm for 72h. The plant pathogens (R. Solani, B. dothidea, Gibberella zeae, F.oxysporum) preserved in the laboratory were incubated at 28℃ in PDA medium (200g potato,20g glucose, and 20g agar per liter).

Genomic DNA Extraction and Sequencing

Genomic DNA was extracted using Wizard® Genomic DNA Purification Kit (Promega) according to the manufacturer’s protocol. Purified genomic DNA was quantified by TBS-380 Fluorometer (Turner BioSystems Inc., Sunnyvale, CA). High-quality DNA (OD260/280 = 1.8 ~ 2.0, > 1µg) was used to do further research. For Illumina sequencing, at least 1µg genomic DNA was used for each strain in sequencing library construction. DNA samples were sheared into 400–500 bp fragments using a Covaris M220 Focused Acoustic Shearer following the manufacturer’s protocol. Illumina sequencing libraries were prepared from the sheared fragments using the NEXTflex™ Rapid DNA-Seq Kit. Briefly speaking, 5’ prime ends were first end-repaired and phosphorylated. Next, the 3’ ends were A-tailed and ligated to sequencing adapters. The third step is to enrich the adapters-ligated products using PCR. The prepared libraries then were used for paired-end Illumina sequencing (2×150bp) on an Illumina HiSeq X Ten machine at Shanghai Majorbio Biopharm Technology Co., L td.

Data Quality Control and Splicing and Assembly

For quality control of raw data, the specific steps are as follows: remove the adapter sequence in the reads; cut and remove the bases containing non-A, G, C, and T at the 5' end; trim the ends of the reads with lower sequencing quality (sequencing quality value is less than Q20); remove reads with a ratio of N containing up to 10%; discard adapters and small fragments with a length of less than 25bp after quality trimming. After the above series of quality control, high-quality clean data was obtained. Use the assembly software SOAP denovo 2. to splice clean data to get the best assembly results(Luo et al. 2012).

Functional annotation and Data Analysis

The coding sequence (CDS) in the genome was predicted by Glimmer(http://ccb.jhu.edu/software/glimmer/index.shtml)(Delcher et al. 2007), and the plasmid gene was predicted by GeneMarkS software(http://topaz.gatech.edu/GeneMark), tRNA was predicted by tRNAscan-SE v2.0(http://trna.ucsc.edu/software/), and rRNA was predi-cted by Barrnap(https://github.com/tseemann/barrnap). Using sequence alignment tools such as BLAST, Dia-mond, HMMER, etc., the predicted CDS was annotated with protein functions through NR, Swiss-Prot, Pfa-m, GO, COG, and KEGG databases(Amos and Rolf 1999; Bateman et al. 2004). Secondary metabolite gene clusters were predicted by antiSM-ASH v4.0.2(Kai et al. 2019). Carbohydrate-related enzymes and their functions are annotated by the carbohydrate-active enzyme database(CAZy)(Cantarel et al. 2009).

Construction of Molecular Phylogenetic Tree

We molecularly identified ES2-4 using the housekeeping gene gyrA gene, which is more conserved relative to 16S rRNA and has a stronger resolution in Bacillus species identification using this method. Genomic DNA was first extracted according to the kit's instructions and amplified using gyrA universal primers (gyrA-F: 5`-GCAATGAGCGTTATCGTATCCCGG-3, gyrA-R: 5`-TCAATCTTTTCGCGCTCCAGATCC). Thermo cycler settings were:2 min at 94℃; 30 cycles of 94℃ for 30 s, 52℃ for 30 s, and 72℃ for 2min; 72℃ for 10 min. PCR products were sequenced by BGI Genomics Co., Ltd., Beijing, China, and the resultant sequences were blasted against the NCBI nucleotide collection database. Highly homologous sequences were selected for multiple sequence alignment with the MEGA X software. A phylogenetic tree was constructed via a Neighbor-Joining approach, with 1,000 replicate Bootstrap analyses being used to calculate node support.

Comparative Analysis of ES2-4 with Other Bacillus spp.

To assess the relatedness of ES2-4 to other Bacillus spp., we firstly downloaded the whole genome sequence, from the GenBank genome database (https://www.ncbi.nlm.nih.gov/genome/, accessed on 20 May 2022). ANI(Average Nucleotide Identity)values were found using Jspecies WS (http://jspecies.ribohost.com/jspeciesws/,accessed on 20 May 2022)(Choi et al. 2021a). dDDH(digital DNA-DNA Hybridization)analysis was carried out by the Genome-to-Genome Distance Calculator (GGDC v3.0) (https://ggdc.dsmz.de/, accessed on 20 May 2022) (Choi et al. 2021b). Comparative genomic analysis was performed by BLAST Ring Image Generator software (BRIG) (https://sourceforge.net/projects/brig/, accessed on 20 May 2022)(Alikhan et al. 2011).

Plant Pathogens’ Growth Inhibition Assays

In order to study the potential of ES2-4 as a biocontrol bacteria, we selected four plant pathogens, R. Solani, D. gregaria, Gibberella zeae, and F. oxysporum, and observed the inhibitory ability of ES2-4 on fungi using a plate confrontation. At the same time, we selected some hyphae whose growth was inhibited and fixed and observed the morphological characteristics of the hyphae of pathogenic bacteria in the inhibited state by electron microscope(Hitachi SU8100, Japan) by Wuhan servicebio technology CO., LTD, China The specific operations are as follows: using PDA and LB medium to activate the pathogenic fungi and ES2-4, respectively, and after the colonies cover the entire plate, use a punch to make a bacterial cake, and transfer it to a new PDA plate. The pathogenic fungus cake was placed in the middle of the plate, four ES2-4 bacteria cakes were placed symmetrically around the plate, and the blank control only placed the pathogenic fungi cake in the center of the plate. Culture in a constant temperature incubator at 28°C. When the fungal mycelium of the blank control covers the entire petri dish, the width of the inhibition zone (the average distance between colonies), is measured, and calculating the inhibition rate. the formula is as follows:

inhibition rate(%)=(control colony diameter-treatment colony diameter)/control colony diameter×100%

Detached-leaf and in Fungi Inhibition Assays

Detached leaf bioassays were conducted with slight modifications as described by Murthy Vignesh(Vignesh et al. 2022). Carefully remove some leaves of the same size from the potted tomato plants (20day), disinfect the leaves with 1% sodium hypochlorite, rinse with sterile water three times, and place them on a Vertical superclean bench for a period to dry the surface. The bacterial liquid cultured for 3 days was centrifuged at 8000 rpm for 15 min, and the supernatant was taken and filtered with a 0.22 µm filter to obtain a sterile fermentation broth and diluted 0-fold, 5-fold, 10-fold, 20-fold, and 40-fold, respectively, and sprayed on the surfaces of both sides of the leaves, and treated with sterile water as a negative control, repeated three times. After a little drying, use a hole puncher to take a 5mm-diameter bacterial cake from the medium and inoculate the center of all leaves with the colony face down. All leaves were placed on wet filter paper in Petri dishes and then cultured aseptically for seven days in a 28°C incubator. The condition of the leaves was checked after seven days, and the damaged diameter of the leaves was measured. The formula for calculating the inhibition rate is as follows:

Inhibition rate=(lesion diameter of the control group - lesion diameter of experimental group)/lesion diameter of control group × 100%

Result

Genome Feature of B.velezensis ES2-4

The genomic structure information showed that the ES2-4 genome is composed of a single circular chromosome with a length of 3,929,792 bp and the GC content is 46.5%(Fig. 1). The whole genome of ES2-4 was predicted to contain 4016 coding sequences (CDSs). The average length is 872bp, and the coding sequence accounts for 89.1% of the entire genome sequence.The chromosome contained 27 rRNA ,86 tRNA ,81 sRNA ,70 tandem repeats and 46 interspersed repeat(Table.1).The COG annotation results showed that 3047 gene annotations were finally obtained (Fig. 2), accounting for 75.9% of the total genes, and the genes with unknown functions accounted for 24.1% of the total gene predictions. There were 20 types of gene annotation results(Table.2). Among them, 289 are amino acid metabolism and transport, accounting for 9.5% of the annotated genes, 232 are transcribed, accounting for 7.6% of the annotated genes, 222 are carbohydrate transport and metabolism, accounting for 7.3% of the annotated genes, and there are 761 more Genes with unknown functions need mining, accounting for 24.9% of the annotated genes. Its function needs further confirmation. The GO annotation results showed that: GO functions were divided into three categories: 1991 genes in biological processes, 1363 genes in cellular components, and 2115 genes in molecular functions. The KEGG annotation results showed that there were a total of 2519 genes, accounting for about 55.3% of the total bacterial genes, with a total of 41 metabolic pathways. The results of carbohydrate activity enzyme analysis showed that: the B.velezensis ES2-4 strain encodes a total of 126 CAZy enzyme gene families, which are divided into six categories of proteins: Glycoside Hydrolases, GlycosylTransferases, Polysaccharide Lyases, Carbohydrate Esterases, Carbohydrate-Binding Modules, and Auxiliary Activities family. Among them, Glycoside hydrolases contain 40 genes related to the hydrolysis of glycosidic bonds for the hydrolysis of sugars and their derivatives mainly including chitinase. Numerous studies have demonstrated that bacterial chitinases display an important role in inhibiting hyphal growth of phytopathogenic fungi. At the same time, some studies have shown that chitinase has been proved to have the activity of inhibiting the random hatching of nematodes(Choub et al. 2021; Tran et al. 2022; Won et al. 2021).

Comparative Genome Analysis of Strain ES2-4 with Bacillus Strains

In the early stage of the experiment, we observed through morphological observation that ES2-4 is a kind of gram-positive bacteria that can produce resistant endospores, which are rod-shaped and milky-white colonies. In the early stage of the growth of plate culture, the edge of the colony was round and viscous, and in the later stage of growth, the edge was irregular, the middle was slightly raised and folded, and it was easy to dry. When growing statically in a liquid medium, it forms wrinkles and is an aerobic bacteria. So we preliminarily identified ES2-4 as Bacillus. A BLAST comparison with B. velezensis and B. subtilis strains is illustrated in Fig. 5 with annotation of the gene involved in promoting plant growth and triggering plant immunity and secondary metabolite synthesis. In general, it is inaccurate to distinguish B. subtilis, B. amyloliquefaciens, and B. velezensis according to 16S rRNA gene sequences(Dong et al. 2022). As a housekeeping gene, the gyrA gene is highly conserved and has only a single copy of all bacteria, which can avoid the heterogeneity of the 16S rRNA gene due to multiple copies, and its nucleotide sequence length and evolution speed are both high. Much higher than the 16S rRNA gene, showing higher genetic differences(Tayeb et al. 2008). According to gyrA sequencing analysis, ES2-4 had 99% sequence homology with B.velezensis. The phylogenetic analysis of the gyrA sequence showed that strain ES2-4 also clustered with B.velezensis LS69 and B.velezensis SQR9 as Fig. 3. In addition, as Fig. 3 and Fig. 4 show, the ANI and dDDH values show that ES2-4 shares 99.72% and 99.9% with LS69 respectively. According to the generally accepted ANI value of 95% and dDDH value of 70% as a threshold for species classification, this strain can be classified as Bacillus. velezensis, and has a recent evolutionary relationship with LS69. In conclusion, based on cytomorphological features, gyrA, and ANI values, ES2-4 was identified as a new member of B.velezensis.At the same time, it also guides us that when facing Bacillus velezensis and its relatively closely related species, we can use a variety of housekeeping genes such as gyrA, rpoB, cheA, and bioinformatics methods such as ANI and dDDH for comprehensive identification(Kim et al. 2014; Rossello-Mora et al. 2011; Wang et al. 2007).

Table.3 Comparative genomic analysis of Bacillus velezensis ES2-4 with Bacillus genomes

Analysis of gene clusters related to secondary metabolite synthesis

Genomic analysis showed that Bacillus velezensis ES2-4 would use no less than 8.3% of the entire genome for the synthesis of secondary metabolites, a capacity that exceeds that of the closely related model Gram-positive bacterium Bacillus subtilis 168 by more than 2-fold (Chen et al. 2009). Through antiSMASH software analysis, we found that ES2-4 contains 14 gene clusters for directing non-ribosomal peptide synthase (NRPS) and polyketide synthase (PKS) to synthesize bioactive peptides and polyketides with antibiotic properties. Secondary metabolites could be identified (Fig. 5, Table 4). Surfactin, iturin, and fengycin are all lipopeptides, and the cyclic structure formed by fat and polypeptide chains accounts for about 2.6% of the entire genome. Due to their amphiphilic structure, lipopeptides have a variety of biological activities, including antibacterial, antifungal, antiviral, and antitumor activities, and are used in chemical, agricultural, pharmaceutical, and food industries(Crouzet et al. 2020; Jiang et al. 2014; Ongena and Jacques 2008). Not only that, but lipopeptides can also act as inducing factors of plant systemic resistance to enhance the defense ability of plant pathogenic bacteria(J. et al. 2018). Surfactin is synthesized under the guidance of four gene clusters of srfABCD, composed of 7 amino acids and aliphatic segments, and has strong surface activity. In addition to having antibacterial and antiviral activities, it also participates in the formation of biofilms and plays an important role in the chemotaxis of PGPR and the process of colonizing the rhizosphere of plants(Vanittanakom et al. 1986). Iturin is composed of four gene clusters ituABCD. Like surfactin, the peptide chain part is also composed of 7 amino acids, but it has a strong inhibitory ability against a variety of fungi recently, with no obvious antibacterial effect and no antiviral activity(Ongena and Jacques 2008). Fengycin is synthesized by four gene clusters of fenABCDE. Unlike the peptide chain of surfactin and iturin, the peptide chain part of fengycin contains 10 amino acids. Fengycin can significantly inhibit the growth of plant fungi, especially filamentous fungi(Vanittanakom et al. 1986). The siderophore Bacillibactin is synthesized by the dhb gene cluster and has a similar structure to the siderophore Enterobactin produced by Gram-negative bacteria. It incorporates a trilactone ring and three catecholate moieties(Chen et al. 2007; Dertz et al. 2006). Bacillibactin can help host bacteria compete for Fe element to inhibit the growth of pathogenic bacteria, and its chelated Fe element can also be absorbed by plants, thereby promoting plant growth(Qiao et al. 2011). Bacilysin is one of the simplest antifungal dipeptides, which contains an L-Ala residue at the N-terminus and a non-proteinogenic amino acid, L-anticapsin, at the C-terminus. Bacilysin formation is catalyzed by an amino acid Ligase, whose synthesis is directed by the bacABCDE gene cluster (Inaoka et al. 2003; Steinborn et al. 2005). Bacilysin was first discovered in Bacillus subtilis, and it has attracted much attention due to its strong inhibitory properties against various bacteria such as Albicans(Walker and Abraham 1970). About 19kb DNA region of ES2-4 encodes polyketide synthase bae mln and dfn, which are used to synthesize bacillaene, macrolactin, and difficidin, respectively. Polyketides are a large class of secondary metabolites, including numerous bioactive compounds with antibacterial, immunosuppressive, antitumor, or other physiologically relevant biological activities. Amylocyclicin is a novel cyclic bacteriocin first discovered in FZB42 and synthesized by the ribosome under the guidance of the acnBACDE gene cluster. This compound has high antibacterial activity against closely related gram-positive bacteria(Scholz et al. 2014).

In addition, there are 4 secondary metabolite synthesis-related gene clusters in ES2-4, with a total length of not less than 15.6kb, including a polyketide compound whose specific substance is unknown. This compound is synthesized by chalcone synthase and is a potential antibiotic. Two gene clusters are responsible for the synthesis of terpenoids. By prediction, there is another gene cluster that may be responsible for the synthesis of Butirosin, but the similarity of this gene cluster is only 7%. Through gene annotation, it was found that the gene cluster contains a large number of genes related to protein transport and amino acid transport. It can be speculated that this gene cluster is responsible for the synthesis of a similar or novel secondary metabolite to Butirosin. ES2-4 also contains a gene cluster associated with the synthesis of lanthiopeptide compounds. Lanthipep-tides are a large class of natural peptides synthesized by ribosomes and post-translationally modified. These compounds are widely produced from different kinds of bacteria, have a rich structural and biological activity diversity, and provide an important source for active drug research and development. Among them, lanM is responsible for encoding the second-class lantipeptide synthase, which is involved in the translation and modification of lantipeptide compounds, such as dehydrogenation and cyclization. In addition, through gene annotation, we found that ES2-4 also contains lcnC, lcnDR2, nisFEGRK, nukEFG, and other gene clusters responsible for the transport, modification, and regulation of lantipeptide compounds(Repka et al. 2017; Zhang et al. 2012). The above results show that ES2-4 can synthesize various kinds of antibiotic substances, and has great potential for biological control.

In addition to producing a variety of secondary metabolites with antibacterial and antifungal activities, ES2-4 also contains a series of interactions with colonizing plants, synthesizing phytohormones, inducing systemic responses, etc. to enhance plant nutrition and trigger plant defense responses(Table.5). ES2-4 contains genes required for swarming movement, including genes involved in chemotaxis and biofilm formation. ES2-4 contains the protein encoded by the swrA gene, which is necessary for bacterial colony movement, and its hydrophilic and surfactant properties facilitate colonization of plant cell surfaces and uptake of nutrients(Compant et al. 2005), in addition, including fliD, hag, and other genes. srfABCD, sinR, resE, espA-O, etc are involved in the formation of biofilm, which is beneficial to reducing the surface tension of ES2-4 during chemotaxis(Kearns et al. 2005). Recently, it was found that L-sucrase synthesized by sacB can convert sucrose to levan, which stimulates strong production of surfactin and hyperflagellation, and promote solid surface motility (SSM) and Bacillus subtilis Root colonization(Idris et al. 2007). The direct role of PGPR in the mechanism of promoting plant growth is the secretion of plant hormones such as IAA. Through genome analysis, it was found that ES2-4 also contains genes yhcX, dhaS, and ysnE related to the synthesis of indole triacetic acid. These three genes have been confirmed to be involved in the biosynthesis of IAA through experiments(Idris et al. 2007). Interestingly, we found that ES2-4 contains the gene encoding the alkaline serine protease aprE, which is highly similar (98%) to bace16 of Bacillus sp. B16 encodes an alkaline serine protease which can degrade nematode cuticle and kill it (Qiuhong et al. 2006). We speculate that ES2-4 also has the effect of killing nematodes, but it needs experimental verification. In addition, ES2-4 is capable of synthesizing acetoin and 2,3-butanediol, which have been shown to promote plant growth and induce systemic resistance(He et al. 2012). In summary, ES2-4 has a relatively abundant genetic material basis in promoting plant growth and enhancing sensitivity and immunity and shows great potential in promoting plant growth.

To explore the control potential of strain ES2-4 against plant pathogens, the antagonistic activity was determined in PDA media. As is shown in Fig. 6 and Fig. 7, the strain ES2-4 was cultured with R. Solani, B. dothidea, Gibberella zeae, and F. oxysporum and the results showed that the strain ES2-4 could significantly inhibit the growth of these fungal pathogens. Among them, the inhibitory efficiency against R. solani reached 74.4%, showing a strong inhibitory ability. This result indicated that strain ES2-4 exhibited a broad spectrum against plant pathogens. To further study the inhibitory mechanism of ES2-4 against these four pathogens, we collected some samples from the area of the fungus closest to ES2-4 colonies and observed the morphological characteristics of the hyphae by scanning electron microscopy. As shown in Fig. 8, A-B represents the morphological characteristics of the four fungal hyphae under normal culture conditions, and the hyphae surface is smooth and regular. Compared with the control group, C-D represented the morphological characteristics of the four fungal hyphae in the inhibited state. The hyphae of the four fungi were atrophied, deformed, or twisted to varying degrees. Fig. H The apical atrophy of the hyphae affects the growth of the hyphae. Therefore, we can infer that ES2-4 can change the morphological structure of hyphae to affect the growth activity of fungi.

Detached Leaf Bioassays of Crude Lipopeptides Against A. solani in Tomato

In order to determine the inhibitory ability of strain ES2-4 to plant pathogenic infecting plant leaves, we first prepared a sterile fermentation broth of strain ES2-4 with different dilution ratios and treated in vitro tomato leaves, and then selected the strongest antibacterial ability in the plate confrontation experiment. Rhizoctonia solani was inoculated on tomato leaves, and the diameter of the lesions on the leaves was observed. As shown in Fig. 9 and Fig. 10, after 7 days of culture, the leaf necrosis area of the control group without fermentation broth basically occupied the entire leaf. However, the leaves treated with different dilution times of fermentation broth showed lesions with different diameters, which were significantly different from those of the control group. Among them, when the dilution ratio is 0, the diameter of the lesions is the smallest, which may be the most antibacterial substances and the strongest inhibitory ability against Rhizoctonia solani. Therefore, we can infer that ES2-4 can synthesize and secrete secondary metabolites that can inhibit the further infection of tomato leaves by Rhizoctonia solani, and has a strong inhibitory effect.

Conclusions

In this study, we isolated a strain ES2-4 with broad-spectrum antibacterial activity from soil and classified it as Bacillus velezensis by the housekeeping gene gyrA and bioinformatics methods. Through whole genome sequencing and analysis, it was found that ES2-4 contains 14 gene clusters related to the synthesis of giant secondary metabolites, and also contains a large number of genes related to the promotion of plant growth. The study revealed the PGPR mechanism of Bacillus velezensis ES2-4 through genome analysis and antagonism experiments. Our findings provide a potential resource for biological control agent or biofertilizer.

Declarations

Confllicts of interest

No conflict of interest exists in the submission of this manuscript.

Funding

This work was supported by General Project of National Natural Science Foundation of China(No. 31271332).

Author contribution

All authors contributed to the study conception and design. Material preparation, data collection and analysis were performed by Qun He, Maohua Wu , Feng Liu, Wei Li. The first draft of the manuscript was written by Feng Liu and all authors commented on previous versions of the manuscript. All authors read and approved the final manuscript.”

Repositories

Nucleotide sequence accession: The complete genome sequence of Bacillus velezensis ES2-4 was deposited in the GeneBank database under accession numbers CP097328(BioProject: PRJNA837401, BioSample: SAMN28200750).

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