The Genome of Bacillus velezensis SC60 Provides Evidence for Its Plant Probiotic Effects

Root colonization and plant probiotic function are important traits of plant growth-promoting rhizobacteria (PGPR). Bacillus velezensis SC60, a plant endophytic strain screened from Sesbania cannabina, has a strong colonization ability on various plant roots, which indicates that SC60 has a preferable adaptability to plants. However, the probiotic function of the strain SC60 is not well-understood. Promoting plant growth and suppressing soil-borne pathogens are key to the plant probiotic functions. In this study, the genetic mechanism of plant growth-promoting and antibacterial activity of the strain SC60 was analyzed by biological and bioinformatics methods. The complete genome size of strain SC60 was 3,962,671 bp, with 4079 predicted genes and an average GC content of 46.46%. SC60 was designated as Bacillus velezensis according to the comparative analysis, including average nucleotide polymorphism (ANI), digital DNA–DNA hybridization (dDDH), and phylogenetic analysis. Genomic secondary metabolite analyses indicated two clusters encoding potential new antimicrobials. The antagonism experiments revealed that strain SC60 had the ability to inhibit the growth of a variety of plant pathogens and its closely related strains of Bacillus spp., which was crucial to the rhizospheric competitiveness and growth-promoting effect of the strain. The present results further suggest that B. velezensis SC60 could be used as a PGPR strain to develop new biocontrol agents or microbial fertilizers.


Introduction
Due to the abuse of chemical fertilizers and pesticides in agricultural production, the quality of soil and agricultural products has been reduced, which also negatively affects the ecology and sustainability of agricultural development. Plant growth-promoting rhizobacteria (PGPR) are a kind of beneficial microbes that stably survive and colonize in the rhizosphere of plants. PGPR are the major source of biofertilizer strains, which show beneficial effects on crops, such as growth promotion, inhibition of soil-borne pathogens, and enhancement of plant tolerance [1]. The beneficial functions of PGPR on plants largely rely on root colonization ability, for which chemotactic motility and biofilm formation on the rhizoplane are the most important colonization processes [2,3]. Rhizospheric soil is rich in microbial communities, including microbes that exert plant growth-promoting properties [4]. Among the rhizosphere microbial communities, the Bacillus genus is among the most ubiquitous soil microbes and possesses several plant growth-promoting traits [5,6]. Members of the Bacillus genus are ubiquitous in soil due to their endospore-forming properties that allow them to survive in different ecological niches [7]. Members of the Bacillus genus use several mechanisms to improve plant growth and enhance plant pathogen resistance under various environmental conditions.
Another important application of PGPR is to protect the plant from pathogens by producing antimicrobial compounds or competing nutrients. Bacillus velezensis, a representative strain of the genus Bacillus, uses more than 8% of the genome to synthesize secondary metabolites in response to competing microbes in the plant rhizosphere [17]. A dozen non-ribosomal polypeptides (bacillaene, bacillibactin, macrolactin, bacillomycin D, fengycin, bacilysin, difficidin, and surfactin) with antifungal and antibacterial activities are synthesized by Bacillus spp. [18]. Biosynthetic gene clusters (BGCs) are responsible for the synthesis of various secondary metabolites [19]. Due to genetic variety, different strains of Bacillus exhibit unique differences in metabolites and antimicrobial capacity, and even among the closely related Bacillus spp. strains, there are many variations in the BGCs [20]. Therefore, based on the genome sequencing, it is possible to discover new BGCs encoding potential secondary metabolites by comparative analysis. This will provide a basis for screening new PGPR strains. Furthermore, these bacteria also can induce systemic resistance responses of plants to pathogens, improve abiotic stress tolerance, and suppress the growth of fungal and bacterial pathogens [21][22][23].
Sesbania cannabina is a multipurpose leguminous crop native to tropical and subtropical regions such as Asia, Africa, and Australia. Due to its wide adaptability and outstanding stress resistance to drought, waterlogging, and salinity, Sesbania cannabina is widely planted as a green manure to improve soil fertility [24,25]. In China, Sesbania cannabina as a pioneer plant has been successfully introduced to the saline-alkaline land restoration of the Yellow River Delta (YRD) [26]. Furthermore, Sesbania cannabina has strong disease and insect resistance, so it is an excellent material for isolating PGPR.
Strain SC60, which was isolated from the seeds of Sesbania cannabina, showed a strong colonization ability on different plant roots. However, the plant probiotic function of strain SC60 is unclear. This study intends to reveal the probiotic potential of the strain SC60 through the genome sequencing information mining, and to elucidate whether the strain SC60 can be used as a PGPR strain in agriculture through plant growth-promoting and pathogen-inhibiting experiments. Here, a series of gene clusters associated with promoting plant growth and inhibiting soil-borne pathogens were identified based on genome sequencing, and we confirmed that SC60 can promote plant root development and inhibit the growth of some pathogens. In addition, we further revealed that the strain SC60 can enhance its rhizosphere competitiveness by inhibiting the growth of closely related species. Thus, B. velezensis SC60 could be used as a PGPR strain to develop new microbial fertilizers.

Genomic DNA Preparation, Sequencing and Analysis
Genomic DNA was extracted from strain SC60 using the Bacteria Genomic DNA Extraction Kit (AxyGen, China, Hangzhou), and the genomic DNA of SC60 was sequenced at Shanghai Majorbio Biopharm Technology Co., Ltd. using an Illumina HiSeq×10 system and PacBio high-throughput sequencing technology. Quality control of the raw reads was performed, including base quality, base error rate, and base distribution. Raw Q20 is an important quality filtering parameter, and a total of 6,815,634 filtered paired-end reads with a length of 150 bp were obtained. The filtered reads were assembled using SOAPdenovo2 (http://soap.genomics.org.cn/, accessed on 20 September 2020) with a K-mer size of 17 bp to generate contigs [27]. The complete genome was assembled by Unicycler (v0.4.8) [28]. The coding sequence (CDS) of strain SC60 was predicted using Glimmer (http://ccb.jhu.edu/software/glimmer/index.shtml, accessed on 22 September 2020) [29]. Genome annotation of the CDS was accomplished using NR, Swiss-Prot, Pfam, COG, GO, and KEGG databases [30][31][32][33]. Gene islands (GIs) were predicted in the strain SC60 genome using IslandViewer (v1.2) [34].

Root Colonization of Strain SC60
The colonization abilities of strain SC60 on sesbania, maize, tomato, and pepper root were conducted according to previously reported methods [37]. Briefly, we germinated the sterilized seeds in a sterile environment. The normally germinated seeds were then transferred into sterile vermiculite until two cotyledons emerged. The plants were washed with sterile water to remove the vermiculite on the surface of plant roots and transfer to 1/5 sterile Hoagland nutrient solution for culture. Overnight cultures of strain SC60 were transferred into 100 mL of LB medium at 1% inoculum and incubated at 37 • C and 170 rpm until an OD 600 value of 1.0 was achieved. Cells were collected at 8000 rpm at 4 • C. The bacterial cells were washed in 1/5 Hoagland solution 3 times and then resuspended in sterile 1/5 Hoagland nutrient solution. Subsequently, the strains were transferred to sterile seedlings with 3 true leaves to a final concentration of 0.5 × 10 6 cells mL −1 . After three days of cultivation, the roots of plants were washed with sterilized water, and the cells colonized on roots were collected by shaking and measured by plate counting.

Plant Growth-Promoting Assay of Strain SC60
Sesbania cannabina seeds were surface-sterilized and germinated in sterile conditions. The normally germinated seeds were then transferred to a square plate covered with sterile filter paper. Then, 15 mL of 1/5 Hoagland solution containing a final concentration of 0.5 × 10 6 cells mL −1 was added to the treatment group, while 15 mL of sterile 1/5 Hoagland solution was added to the control group. The plates were incubated in a climatic chamber at 12 h light (26 • C)/12 h dark (22 • C) and 70% relative humidity. After seven days of cultivation, root development was recorded.

Plant Pathogens' Growth Inhibition Assays
The antagonistic activities of strain SC60 against fungal and bacterial pathogens were measured in vitro on PDA and NA agar, respectively. Fungi preserved at −80 • C were activated on PDA plates; after spore germination, the fungal pieces were transferred to new culture medium with an inoculation shovel to completely restore their activity. The antagonistic fungal activity was carried out on 9 cm plates. First, the strain SC60 was activated in LB medium until the OD 600 reached 1.0, and then a 5 mm-diameter fungal piece was inoculated onto the center of a PDA plate. Then, 2.5 µL of strain SC60 suspension was pipetted onto 2 cm from each of the fungal pieces, and the plates were incubated for 5 days at 28 • C. The antagonistic effect of strain SC60 against fungal pathogens was evaluated by whether there was an inhibition zone [38]. The activity of antagonistic bacteria was detected by a similar method. All antagonistic experiments were performed in triplicate.

Closely Related Species' Growth Inhibition Assays
The strain SC60 was fermented in Landy medium and the culture was collected by centrifugation at 8000 rpm for 10 min. The supernatant was dehydrated in a freeze dryer, and then extracted five times with methanol (100 mL of methanol to extract 1 L of supernatant). The extraction was filtered by a 0.22 µm sterile filter. SC60 were detected on LB medium, and a sterile Oxford cup with an average of 150 µL extraction was placed on a plate with the tested Bacillus spp. strains, and 150 µL methanol as CK. The plates were stored at room temperature for 36 h [17].

Comparative Genome Analysis of Strain SC60 with Bacillus Strains
The genomic structure information showed that the chromosome length of strain SC60 was 3,962,671 bp, and a BLAST comparison with B. velezensis and B. subtilis strains is shown in Figure 1. The SC60 genome is composed of a single circular chromosome with a length of 3,962,671 bp, and the average G + C content is 46.46%. The whole genome of SC60 was predicted to contain 4079 coding sequences (CDSs), 27 rRNA, 89 tRNA, and 83 sRNA genes ( Table 1). The classification of SC60 genes into clusters of orthologous groups (COGs) assigned 2986 CDSs to at least one COG group (73.2%). A total of 1093 CDSs were not classified into COGs. PHAST analysis of the strain SC60 genome identified 3 prophage regions (Table 1) depicted on the green ring (Figure 1), which represents 3.6% of the entire genome. Sixteen gene islands (GIs) are depicted on the blue ring in Figure 1, which represent 3.3% of the entire genome.
In general, it is inaccurate to distinguish B. subtilis, B. amyloliquefaciens, and B. velezensis according to 16 S rRNA gene sequences. At present, distinguishing and detecting novel species in Bacillus taxa is mostly based on phylogenetic analysis of the core genome. To identify the species of strain SC60 in Bacillus, the phylogenomic NJ tree was constructed based on 2968 homologous genomes. The comparative phylogenomic analysis indicated that strain SC60 showed the highest homology with Bacillus velezensis (Figure 2). Indeed, ANI and dDDH analyses between strain SC60 and Bacillus velezensis were ≥97.45% and ≥91.20%, respectively, which indicated that the strain SC60 was finally identified as Bacillus velezensis (Table 2).  In general, it is inaccurate to distinguish B. subtilis, B. amyloliquefaciens, and B. velezensis according to 16 S rRNA gene sequences. At present, distinguishing and detecting novel species in Bacillus taxa is mostly based on phylogenetic analysis of the core genome. To identify the species of strain SC60 in Bacillus, the phylogenomic NJ tree was constructed based on 2968 homologous genomes. The comparative phylogenomic analysis indicated that strain SC60 showed the highest homology with Bacillus velezensis (Figure 2). Indeed, ANI and dDDH analyses between strain SC60 and Bacillus velezensis were ≥97.45% and ≥91.20%, respectively, which indicated that the strain SC60 was finally identified as Bacil-

Plant Growth-Promoting Traits of SC60
Bacillus spp. is a Gram-positive soil bacterium that is commonly found in the rhizosphere environment, the area of soil surrounding plant roots. Root colonization of microbes determines the efficiency of their beneficial effects on plants. In this study, the colonization ability of the strain on the root of Sesbania cannabina, maize, pepper, and tomato was tested, and the results indicated that strain SC60 can colonize the root surface of many plants ( Figure 3A). Moreover, we found that strain SC60 can promote the root development of Sesbania cannabina ( Figure 3B,C). Further genomic analysis showed that strain SC60 contained multiple genes related to root colonization and plant growth promotion, and it has even been postulated that strain SC60 has potential as a biocontrol bacterium in the rhizosphere (Table 3).

Secondary Metabolite Cluster Analysis
Thirteen BGCs of secondary metabolites were identified in the SC60 genome by the antiSMASH tool (Table 4), including two encoding NRPS, two transAT-PKS, three transATPKS-NRPS, one ladderane, two terpene, one lantipeptide, one T3PKS, and one PKS-like. This analysis showed that about 19% of the SC60 genome is responsible for the regulation, biosynthesis, and transport of antimicrobials. Seven clusters were clearly identified to participate in the synthesis of surfactin, plantazolicin, macrolactin, bacillaene, difficidin, bacillibactin, and bacilysin. Furthermore, Cluster 7 is responsible for the synthesis of both bacillomycin D and fengycin. All clusters have antimicrobial/antibacterial activities or are related to antibiosis. For the 8 clusters, compared with the reference strain B. velezensis FZB42, it was found that the gene cluster arrangement of SC60 was very similar to that of FZB42 ( Figure 4, Table 4). The secondary metabolite cluster in strain SC60 was compared with the cluster of four Bacillus strains (FZB42, SQR9, DSM7, and 168) ( Table 4), and we found cluster 3 and cluster 12 encoding potential novel metabolites. Cluster 3, encoding PKS-like, showed 7% gene similarity with butirosin A, but the biosynthetic genes of strain SC60 are inconsistent with the butirosin A biosynthetic genes in the MIBiG database. Cluster 12, encoding lantipeptide class II, was not present in any of the four Bacillus strains.

Antagonistic Activity against Plant Pathogens
To explore the control potential of strain SC60 against soil-borne plant pathogens, the antagonistic activity was determined in PDA and NA media. The strain SC60 was cultured with Fusarium verticillioides, Phytophthora capsica, Fusarium oxysporum sp. cucumebrium Owen, Fusarium graminearum schw, Botrytis cinerea Pers. ex Fr, Aspergillus niger Van Tiegh, and Fusarium solani, and the results showed that the strain SC60 could significantly inhibit the growth of these fungal pathogens. Moreover, strain SC60 also strongly inhibited the growth of the plant pathogenic bacterium Ralstonia solanacearum. This result indicated that strain SC60 exhibited a broad spectrum against soil-borne plant pathogens ( Figure 5).

Antagonistic Activity against Bacillus spp.
Plant roots can secrete a variety of root exudates to provide nutrition for the growth of microbes and allow beneficial microbes to grow and colonize. However, the nutrition provided by plant root exudates is limited. Beneficial bacteria should not only compete with pathogenic bacteria but also compete with closely related strains because of the similar nutritional utilization among the closely related strains. To enhance the rhizosphere competitiveness of PGPRs, some strains have evolved to produce a variety of antibiotics that inhibit or kill other rhizosphere microbes. In this study, the antagonistic ability of strain SC60 against closely related species was verified by the Oxford cup method. The results suggested that the fermentation broth of strain SC60 significantly inhibited the growth of a variety of Bacillus spp., including Bacillus megaterium, Bacillus amyloliquefaciens, Bacillus velezensis, Bacillus cereus, Bacillus halotolerans, Bacillus brevis, Bacillus fordii, and Bacillus subtilis ( Figure 6). This characteristic can significantly enhance the rhizosphere competitiveness of strain SC60 and play a major role in rhizosphere biocontrol.

Discussion
PGPR are the major source of biofertilizer strains, which show beneficial effects on crops, such as growth promotion and inhibition of soil-borne pathogens. However, their beneficial functions largely depend on efficient colonization on roots, for which chemotactic motility and biofilm formation on the rhizoplane are the most important colonization processes. There is a fla-che operon responsible for chemotactic motility and seven methylaccepting chemotaxis proteins in the SC60 genome. Biofilm matrices are controlled by several synthesis genes, such as EPS, which is synthesized by the epsA-O operon [39]. The amyloid fibers, encoded by the tapA-sipW-tasA operon, are primarily composed of the TasA protein [40]. BslA is a biofilm-surface layer protein, which is responsible for the hydrophobic layer on the biofilms' surface encoded by bslA [41]. In addition, γ-polyglutamate plays an important role in enhancing the structure of biofilms [42]. The genome of strain SC60 contains the genes encoding the extracellular matrix of the biofilm. The beneficial effect of rhizospheric Bacillus results from the synergy of multiple factors, including the production of phytohormones and the secretion of phytase and the volatile compound acetoin [43][44][45]. The genome of SC60 contains the coding gene cluster trpABFCDE of tryptophan, the precursor of IAA synthesis, the coding gene phy of phytase, and the coding gene cluster alsDSR of acetoin. BGCs are responsible for the synthesis of various secondary metabolites, which contribute to the competitive role between different microbes and resistance to self-encoded antibiotics [46]. In the soil environment, there are various pathogenic fungi and bacteria as well as a large number of closely related species of biocontrol strains. However, due to the lack of nutrition around the plant rhizosphere, PGPR strains compete with these strains. Therefore, PGPR strains should have strong rhizosphere competitiveness. Bacillus spp. devote a large percentage of their genomes to encoding bioactive secondary metabolites that are crucial to competition in the rhizosphere. A large number of secondary metabolites with antibacterial activity were reported in Bacillus velezensis strain FZB42 to inhibit plant pathogens. For example, surfactin, fengycin, and bacillomycin D have strong growthinhibitory activities against cucumber Fusarium wilt [47]. The known secondary metabolite gene cluster in SC60 was observed to be similar to the corresponding gene cluster in FZB42, with high homology at the nucleotide level ( Figure 4). This shows that strain SC60 also has the potential to prevent and control a variety of plant diseases.
Competition with closely related species also plays a crucial role in the effective biocontrol function of PGPR strains. In recent years, it has been found that the plantazolicin and amylocyclicin produced by B. velezensis FZB42 inhibited the growth of Bacillus megaterium and Bacillus subtilis. Bacillus velezensis SQR9 was also found to encode novel antimicrobial fatty acids to inhibit the growth of closely related Bacillus [17]. Recent studies have shown that the BGCs distance of the Bacillus genome is significantly positively correlated with their phylogenetic distance; moreover, the interspecies antagonism of Bacillus spp. was also significantly positively correlated with the distance of BGCs, suggesting that BGCs profiling may play a role in regulating interspecies antagonism [48]. Therefore, we can speculate that the antagonism between the same Bacillus species should be weak. In the present study, the strain SC60 encoded 13 BGCs, indicating that it is rich in secondary metabolites. Additionally, we found that strain SC60 can not only inhibit the growth of distantly related strains such as Bacillus megaterium and Bacillus cereus, but also inhibit the growth of closely related strains such as Bacillus amyloliquefaciens and Bacillus velezensis (FZB42 and SQR9) (Supplementary Figure S1). The results imply that strain SC60 may produce a new antibacterial compound. In the future, we will further excavate the coding gene cluster in strain SC60 that inhibits the growth of closely related strains, and determine the properties and structure of the secondary metabolite through chemical methods. Although the secondary metabolites of strain SC60 can inhibit the growth of many kinds of Bacillus, according to the existing research, we speculated that some Bacillus species that are distantly related to strain SC60 may also have inhibition on the growth of strain SC60. Our findings will provide an important theoretical basis for the agricultural application of strain SC60.

Conclusions
In this study, the colonization ability of the Bacillus velezensis SC60 on the root surface of various plants was tested and the effects of SC60 on the root development of Sesbania cannabina were analyzed. We found that the Bacillus velezensis SC60 has strong colonization and growth-promoting functions. This research further revealed the probiotic mechanism of the Bacillus velezensis SC60 through genomic analysis and antagonism experiments. In addition, the results of the study also showed that the Bacillus velezensis SC60 can enhance its rhizosphere competitiveness by inhibiting the growth of closely related species. Our findings provide a potential resource for biocontrol agents or biofertilizers.

Institutional Review Board Statement: Not applicable.
Informed Consent Statement: Not applicable.

Data Availability Statement:
The assembled genome sequences for strain SC60 were uploaded to the NCBI GenBank, project number PRJNA716758.

Conflicts of Interest:
The authors declare that they have no conflict of interest.