The genus Saccharibacillus belongs to the family Paenibacillaceae and is mainly isolated as plant endophytes and from the phyllosphere from plants such as cotton, wheat, vegetables and fruits, sugar cane, and barley seed. Furthermore, this genus can also be found in desert soil and mine tailings (Rivas et al., 2008, Yang et al., 2009, Han et al., 2016, Kampfer et al., 2016, Sun et al., 2016, Besauryand Remond 2020, Jiang et al., 2020, Darji et al., 2021). Plant-associated bacteria, especially plant endophytes, play an important role in PGP and productivity (Hardoim et al., 2008, Santoyo et al., 2016, Chen et al., 2019, Singh et al., 2021). To the best of our knowledge, there have been no studies on interactions between bacteria of the genus Saccharibacillus and plants. The genus Saccharibacillus has been proven to have strong cellulase activity (Rivas et al., 2008, Darji et al., 2021). Cellulase, a cell wall-degrading enzyme, can affect the structural integrity of the host plants, thereby indirectly promoting host plant growth, suggesting a role for bacteria of the genus Saccharibacillus in plant growth and/or biocontrol. This finding is significant in light of the increasing applications being identified for biofertilizers in modern agriculture (e.g., the GRAS Bacillus strain) (Bhardwaj et al., 2014, Majeed et al., 2018, Chen et al., 2019). In the present study, we found that the strain ATSA2T, which is an endophyte of kimchi cabbage, contains no antibiotic resistance genes or virulence genes. Therefore, this bacterial strain is likely safe for both humans and the environment.
Endophyte bacteria promote plant growth via the regulation of various plant hormones (e.g., IAA) or increasing nutrient uptake. IAA is a natural auxin that aids bacterial biosynthesis via the L-tryptophan metabolism pathway or via the L-tryptophan independent pathway. IAA is the most common plant auxin and regulates various aspects of plant growth and development. IAA also enhances both cell elongation and cell division and is essential for plant tissue differentiation. More importantly, IAA also induces auxin-dependent lateral root formation, root hair development, and primary root growth, which contribute to PGP. Many bacterial species (e.g., Pseudomonas (Singh et al., 2021) and Enterobacter (Guo et al., 2020)) promote plant growth via IAA synthesis. Siderophore and phosphate solubilization are also very important to plant growth-promoting traits. Bacteria produce and secrete siderophores for iron absorption, iron transfer into cells, and iron scavenging from the host to inhibit plant pathogens. These mechanisms act indirectly to promote plant growth. Phosphorous is an essential macronutrient for plant growth, but the majority of phosphate is not bioavailable. As such, phosphate-solubilizing bacteria enhance plant production by solubilizing insoluble phosphorus to increase its bioavailability and improve phosphorus nutrition. Some PGP microbes improve plant growth by solubilizing insoluble phosphates in the soil, particularly in phosphorus-deficient environments, thereby increasing phosphorus cycling and improving soil quality. Many bacterial strains, including Pseudomonas (Asaf et al., 2018, Singh et al., 2021), Enterobacter (Guo et al., 2020), and Serratia (Adam et al., 2016), promote plant growth by improving nutrient availability. In the present study, we showed that an isolated strain ATSA2T from surface-sterilized seeds produces a relatively high IAA content in the presence of L-tryptophan, with detectable siderophore and phosphate solubilization activity (Table 1). We showed that these mediators promote growth in kimchi cabbage, bok choy, and pepper under greenhouse conditions (Fig. 1, Supplementary Fig. 1).
We used whole-genome sequencing and annotation to provide useful insights into the mechanisms governing plant growth. In a previous study, many plant growth-promoting bacteria were analyzed at the whole-genome level to gain an in-depth understanding of PGP mechanisms in bacteria such as Bacillus pumilus strain SF-4 (Iqbal et al., 2021b), B. subtilis BS87 and B. megaterium BM89 (Chandra et al., 2021), Pseudomonas aeruginosa B18 (Singh et al., 2021), Klebsiella variicola UC4115 (Guerrieri et al., 2021), and Streptomyces (Subramaniam et al., 2020). Many genes have been identified by whole-genome sequencing and have been implicated in direct mechanisms of plant growth (e.g., chitinase, phosphate solubilization, auxin production, iron acquisition, and nitrogen fixation) (Subramaniam et al., 2020, Chandra et al., 2021, Guerrieri et al., 2021, Iqbal et al., 2021b, Singh et al., 2021). In the present study, the strain ATSA2T showed high IAA synthesis. In agreement with this observation, the trpABCDEFG gene cluster—which is involved in IAA production—was annotated in the ATSA2T genome. The trp gene cluster is involved in tryptophan biosynthesis and associated with IAA biosynthesis (Gupta et al., 2014, Asaf et al., 2018, Guo et al., 2020, Babalola et al., 2021, Singh et al., 2021). Furthermore, the occurrence of the following gene clusters, which are responsible for IAA production, also supports our findings: trpABCDEG, trpBCDES, trpABCDR, trpABD, and trpBE from whole-genome analysis of strains Pseudomonas aeruginosa B18 (Singh et al., 2021), Rhizobacteria (Gupta et al., 2014), Bacillus cereus T4S (Babalola et al., 2021), Sphingomonas sp. LK11 (Asaf et al., 2018), and Enterobacter roggenkampii ED5 (Guo et al., 2020), respectively. A key gene, ipdC, is involved in the IAA biosynthetic pathway and has been identified in Klebsiella sp. D5A (Liu et al., 2016). Moreover, the patB gene, which constitutes a potential biosynthetic IAA pathway in B. amyloliquefaciens Ba13, was also found in the ATSA2T genome (Ji et al., 2021). Importantly, these bacterial strains have been proven to have plant growth-promoting activity. The strain ATSA2T used in this study also showed consistent plant growth-promoting activity in kimchi cabbage, bok choy, and pepper.
Regarding siderophore acquisition, iron is a known essential nutrient that promotes bacterial virulence but must be scavenged by the microbe. Bacteria have several iron transporters (e.g., Ybt, Feo, Efe, Yfe, Fet, and Fhu in Yersinia pestis) (Forman et al., 2007). Among those iron transporters, the Fhu system was first identified in Escherichia coli (Kammler et al., 1993) and participates in siderophore (hydroxamate)-dependent iron (III) transport, with FhuD being a siderophore receptor. In the present study, FhuABD, which is part of the Fhu family, was detected in the ATSA2T genome. Furthermore, similar to our previous study, FhuBCD was also identified in Streptomyces as a siderophore transport system (Subramaniam et al., 2020). In addition, other genes implicated in the iron-siderophore transport system, including but not limited to SirABC, FecBCD, CbrABC, FeoB, FtsABC, SiuABD, EfeO, and FagABC (Table 2), were found in the ATSA2T genome. Similar observations were made in Staphylococcus (SirABC) (Dale et al., 2004), Shigella flexneri (FecIRABCDE) (Luck et al., 2001), E. coli (FeoAB) (Kammler et al., 1993)d pestis (Efe, Yfe, and Fet) (Forman et al., 2007). Genome analysis revealed that the strain ATSA2T also contains siderophore acquisition activity, which agrees with our phenotypic data and previous findings. We also identified some genes involved in phosphate uptake and solubilization, some of which have been extensively studied, including phoA (alkaline phosphatase), pst (Pi-specific transporter), phn (alkaline phosphatase affinity transport system), and ugp (glycerol-3-P uptake) (Gebhard et al., 2006, Martinand Liras 2021). We identified the phosphate solubilization-related gene clusters phnCDEP, phoABHLU, pstABCSH, and ugpABE in the ATSA2T genome (Table 2). Furthermore, pstSCAB, phoACX, and phnCDE have all been found in Streptomyces and Mycobacterium tuberculosis genomes (Gebhard et al., 2006, Martinand Liras 2021).
Although some extracellular enzyme phenotypes of the strain ATSA2T (e.g., cellulase, amylase, and chitinase activities) were negative, this strain contains genes encoding cellulase (bglBX, ramA), amylase (glgA), and chitinase (nagZ) activities, which have been reported in Streptomyces and B. cereus and B. subtilis (Guo et al., 2015, Subramaniam et al., 2020, Adeleke et al., 2021). Similarly, we identified genes involved in nitrogen metabolism in the strain ATSA2T, including genes implicated in nitrate transport (narGIJKQWVHYZI, narGZ, and nasA) and nitrite reduction (nirBCD) (Supplementary Table S1). Interestingly, cellulase activity is strain-dependent, with S. sacchari and S. alkalitolerans showing significant cellulase isozyme activity (Rivas et al., 2008, Darji et al., 2021), while S. kuerlensis showed no cellulase activity (Yang et al., 2009). Our data agree with these previously published findings. In addition to plant-related traits in the present analysis, using whole-genome analysis, we also found that the strain ATSA2T produces volatile compounds via whole-genome analysis. Increasing evidence suggests that volatile compounds produced by bacteria and fungi can stimulate plant growth via processes that are dependent on changes in the metabolome and/or proteome. Furthermore, volatile compounds, including 2,3-butanediol and methanethiol isoprene, have been shown to promote plant growth (Jardine et al., 2016, Yi et al., 2016) in the most efficient plant growth-promoting bacteria, Bacillus sp. (Guo et al., 2015, Yi et al., 2016) and Pseudomonas sp. In the present study, the 2,3-butanediol-related gene cluster ilvABCDEGHLN was annotated from the whole genome, similar to the occurrence of the gene cluster ilvABCDEH, ilvHC in the whole genome of Bacillus sp. and P. aeruginosa B18 (Guo et al., 2015, Singh et al., 2021). The methanethiol isoprene-related gene cluster metABCEGINKQXY in the genome of ATSA2T was similar to that in Pseudomonas aeruginosa B18 (Singh et al., 2021). The production of 3-butanediol and methanethiol isoprene from strain ATSA2T must be further studied.
Plant growth-promoting bacterial strains are often mediated by producing important secondary metabolites that act as a reservoir of bioactive metabolites, such as inhibiting pathogen growth (Kiesewalter et al., 2021). These biocontrol compounds, including fengycin, are produced by B. subtilis and B. velezensis strains that inhibit Rhizoctonia disease and Aspergillus flavus, respectively (Deleu et al., 2008, Chen et al., 2019). Iturin A is produced by B. amyloliquefaciens and suppresses the biopathogen Rhizoctonia solani and PGP (D'Aes et al., 2011, Murata et al., 2013, Kushwaha et al., 2020). Identification and characterization of secondary metabolites is the traditional approach for elucidating the complete chemical structure (Deleu et al., 2008, D'Aes et al., 2011, Blin et al., 2013). This process can be accelerated by using antiSMASH bioinformatic analysis to predict secondary metabolite clusters (Blin et al., 2013) and better characterize the genetic determinants related to plant growth and/or biocontrol activity (Nelson et al., 2014, Nasrin et al., 2015). There are seven published species of the genus Saccharibacillus; however, limited secondary metabolites have been studied, and the potential source of natural products in this genus remains untapped. Bacteriocin, thiopeptide, terpene, and nonribosomal peptide (NRP) synthase clusters have been identified in the S. alkalitolerans VR-M41T genome (Darji et al., 2021). Among these compounds, some bacteriocin is produced by plant growth-promoting bacteria and can promote plant growth (Gray et al., 2006, Lee et al., 2009). Moreover, thiopeptides and terpenes also have potent antibiotic (Awolope et al., 2021) and plant growth-promoting activities (Abdel-Hamid et al., 2021, Brookbank et al., 2021), which emphasizes the potential plant growth-promoting activity of Saccharibacillus.
In the present study, the strain ATSA2T was investigated for biosynthetic secondary metabolites. Eight notable BGC regions were detected. These encode terpenes, siderophores, proteusins, NRPs and NRP-like compounds such as bacillaene, staphylobactin, carotenoids, cerecidin and isocomplestatin. Some of these BGCs were previously unknown. Four BCGs, terpene, carotenoid, siderophore (staphylobactin), and bacillaene, are known to be related to direct or indirect plant growth mechanisms. Terpenes include ABA, GAs, phytoalexins, and membrane-related sterols (Bottini et al., 2004, Piccoli 2013, Huangand Osbourn 2019). ABA helps plants maintain their cell turgor to preserve water and indirectly stimulates plant growth, while GAs promote root and shoot growth (Piccoli 2013, Fiodor et al., 2021). In addition, phytoalexins can protect plants against pathogens, thereby directly promoting plant growth (Masunaka et al., 2011, Salas-Marina et al., 2011). Carotenoids are a group of isoprenoid metabolites that are vital for diverse plant functions, such as pigmentation and signaling. Increasing evidence shows that carotenoids play an important role in plant growth and improve both plant yield and nutritional value (Yuan et al., 2015, Swapnil et al., 2021). Siderophores are low-molecular-mass compounds that have been shown to promote plant growth via suppression of pathogen growth and by increasing iron from the environment (Sulochana et al., 2014, Lurthy et al., 2020). Bacillaene is a polyene synthesized by trans-acyltransferase polyketide synthases via inhibition of prokaryotic protein biosynthesis. Furthermore, this compound has antibacterial activity, which indirectly promotes plant growth (Chen et al., 2019, Iqbal et al., 2021a, Ji et al., 2021). Additional research on these secondary metabolites is required to fully elucidate the functions of each metabolite in plant growth.
An endophytic bacterium, strain ATSA2T, was isolated from seeds of kimchi cabbage (Jiang et al., 2020). We demonstrated tryptophan-dependent IAA production in this strain along with phosphate solubilization and siderophore activity, all of which have contributed to mechanisms of plant growth in kimchi cabbage, bok choy, and pepper in a greenhouse test. Whole-genome sequencing was performed to mine functional genes and IAA-, phosphate solubilization-, and siderophore-related gene clusters. These genes were all identified and highly correlated with our phenotypic data. In addition, secondary metabolites, including carotenoids, siderophores (staphylobactin), and bacillaene, underlining PGP were also identified in the ATSA2T genome by antiSMASH. These data show that genomic analysis offers comprehensive insights into the plant growth-promoting mechanisms of the strain ATSA2T, thereby suggesting a role for this bacterial strain in biotechnological applications in agriculture for promoting growth in kimchi cabbage, bok choy, and pepper.