The growing of plants for food production is affected by environmental factors (biotic and abiotic) (Dimkpa et al. 2009). Biotic factor such as microorganisms establishing mutualism or antagonism relationship with the host plants can significantly express their importance in mitigating ecological stressors (Yu et al. 2019). Our interest is targeted at cooperation between plants and microbes. Many beneficial microbes occupy different ecological niches, such as the rhizosphere, phyllosphere, spermatosphere, and endosphere (Adeleke and Babalola 2020). Endosphere represents the entire internal tissue of plants colonized by endophytic microbes with utmost beneficial effects on plant growth (Asaf et al. 2017). The beneficial endosphere inhabitants with PGP traits, as demonstrated by in vivo assay in this study, confirm their agricultural importance. The coevolution of endophytic microbes with the hosts has prompted complementary dynamics in their functions, such that, the host plant houses, protect, and supply nutrients to microbes. In return, these microorganisms enhance plant growth and survival (Omomowo and Babalola 2019).
The design of the study was to isolate and characterize endophytic bacteria strains from the sunflower root with PGP potentiality. Generally, Stenotrophomonas species are linked to human pathogens with opportunistic tendencies (Rojas-Solís et al. 2018). In recent times, their potential in plant growth promotion has significantly widened the research scope of this species, thus providing knowledge based on their environmental and agricultural importance (Alexander et al. 2019). So far, whole-genome analysis for detecting diverse genes involved in cellular metabolism and phytohormone production from S. indicatrix has not been studied. Hence, we present the first report on the genomic information on S. indicatrix isolated from sunflower roots.
S. indicatrix was isolated on Luria Bertani medium, screened on growth-promoting media, and then sequenced. From the annotation results, it was revealed that the type of genes detected in strain BOVIS40 corroborate the in vitro screening. Nevertheless, other genes found aside from those screen in vitro were also detected. The results from plant growth-promoting tests showed that strain BOVIS40 could modulate plant growth in diverse ways. For instance, in phosphate solubilization, exopolysaccharide production, IAA, and siderophore biosynthesis. The results obtained corroborate with earlier documentation on plant growth-promoting attributes of Stenotrophomonas strains and other root-associated endophytic bacteria (Ngoma et al. 2013; Mukherjee and Roy 2016; Perez-Perez et al. 2020).
The genome-based phylogeny classification showed that strain BOVIS40 forms 100% identity to S. indicatrix WS40 and S. lactitubi M15 in a closely related cluster clade. The average nucleotide identity (ANI) of S. indicatrix WS40 (97.05%) further confirms that strain BOVIS40 belongs to indicatrix species based on high ANI value (Goris et al. 2007). As reported in this study, researchers have also demonstrated the phylogenetic relationship of novel S. cyclobalanopsidis with 100% similarity to S. indicatrix WS40 and S. lactitubi M15 in a close cluster clade (Bian et al. 2020).
The ability of microbes to fix atmospheric nitrogen for plant use depends on certain genes and enzymes produced by them (Valentine et al. 2018). The protein nature of enzyme coding genes such as nifH and nifDK involved in nitrogenase production in a bacterial cell has contributed to their functions in the biotransformation of bound nitrogen in the soil into absorbable form (Guerrieri et al. 2021). The enzyme cysteine desulfurase coding for gene nifS involving in the nitrogen fixation pathway was identified in strain BOVIS40. Other genes involved in nitrogen regulatory protein NR (I) (ntrC) and flavodoxin production (nifF), together with the ammonia transport gene amt identified, could contribute to an increase in nitrogen level in the soil. The presence of nitrogen fixation (NF) genes such as nifABDEFHJKLMNSTUVWXYZ in the genome of Klebsiella species function in the formation of complex membrane structure and transport of molecules in the presence of nitrogenase have been employed as a model in the NF ability of Rhodobacter capsulatus (Jeong and Jouanneau 2000). The detection of nifF in the genome of rhizobacterium Azospirillum brasilense Sp7 and K. variicola UC4115, contributing to their nitrogen fixation ability for yield improvement in tomato, has been identified with success in recent findings (Guerrieri et al. 2021). A similar gene identified in this study could contribute to bacterial functions in improving sunflower yield. Also, the expression of fix genes such as fixABCX in the genome of Azorhizobium caulinodans and Bradyrhizobium japonicum with alternate nifJF and their functions in the electron transport chain system have contributed to the transfer of proteins in rhizobia (Tsoy et al. 2016). Also, identification of flavodoxin and ammonia transport gene (amtB) in the genome of strain MSR2 has been reported (Nascimento et al. 2020a), with similar results documented in this study.
The number of nif genes produced by the bacterial cell may be due to their physiological nature and source of isolation. However, unidentified genes as reported in other literature might be responsible for the replacement of these genes with other nif products (Shen et al. 2013). Interestingly, information relating to the nitrogen fixation potential of S. indicatrix isolated from the root of oilseed sunflower based on their genome studies has not been documented.
Phosphate solubilizing potential of strain BOVIS40 under in vitro assay on tricalcium medium was validated by identification of copious genes involved in phosphate solubilization and transport. Gluconic acid (GA) is known as a precursor that facilitates phosphate solubilization in diverse bacterial phyla. The synthesis of GA due to the catalytic actions of enzyme glucose-1-dehydrogenase and its non-protein chemical compound pyrroloquinolone quinine (pqq) have contributed to microbial functions and applications (Ramachandran et al. 2006). Despite non-detection of pqq genes in strain BOVIS40, reports have shown the expression of heterologous pqq genes (pqqABCDEF) that confer phosphate solubilization potential in diazotroph endophytic bacterium Herbaspirillum seropedicae Z67 associated with economic plants (Baldotto et al. 2011; Wagh et al. 2014). Similarly, the absence of pqq genes has been reported in the genome of Klebsiella sp. D5A (Guerrieri et al. 2021).
Phosphate uptake by strain BOVIS40 may be enhanced due to their high affinity and presence of phosphate transport genes, pstABCS, which corroborate the findings earlier documented by Guerrieri et al. (2021) and Shariati et al. (2017) on the expression of phosphate transport genes, pstABCS on the whole-genome analysis of K. variicola UC4115 and Pantoea agglomerans P5. The presence of pst may enhance phosphate bioavailability in the soil and uptake by plants. The detection of phoA gene coding for enzyme alkaline phosphatase involved in various phosphorus metabolism has been identified in Burkholderia multivorans WS-FJ9 (Liu et al. 2020). Furthermore, the detection of pgl, ppx, and ppa in the genome of BOVIS40 that code for 6-phosphogluconolactonase, exoployphosphatase, and inorganic pyrophosphatase for the synthesis of D-gluconate and degradation of inorganic pyrophosphatase could contribute to the ability of the bacterial strain to solubilize phosphate in the soil. Interestingly, our results corroborate the findings of Nascimento et al. (2020b), who reported phoAD genes, coding for alkaline phosphatase in the genome of Bacillus megaterium STB1, that enhance phosphate solubilization ability in the bacterial strain.
In line with the previous studies on the PGP properties of endophytic bacteria colonizing plant root (Stoltzfus et al. 1997; Tariq et al. 2014; Eida et al. 2021), expression of genes involved in secretion system, siderophore, IAA synthesis, acetoin, and 2,3-butanediol was detected in endophytic bacterial strain BOVIS40. The synthesis of IAA in plants and microorganisms has been postulated through multidimensional pathways, which include, indole-3-acetonitrile (IAN), tryptamine, indole-3-pyruvate (IPA), and indole-3-acetamide (Spaepen and Vanderleyden 2011). IAA, a major occurring auxin in plants can signally mediate gene expression and biosynthesis in microorganisms. Thus, plant stimuli against natural pathogenic microbes may be linked to the synthesis of auxin signal molecules by the microorganisms (Spaepen and Vanderleyden 2011). To this premise, two major pathways, namely IPA and IAN were identified in the genome of BOVIS40 as previously reported in the genome of K. variicola UC4115 (Guerrieri et al. 2021). The biotransformation of IPyA - tryptophan to indole pyruvic acid in the presence of aminotransferases that convert IPyA to IAAld - indole acetaldehyde and gene ipdC encoding indole pyruvate decarboxylase in Pantoea spp has been documented (Nascimento et al. 2020a). Also, the presence of ipdC gene in Enterobacter cloacae with similar functions has been identified (Koga, et al. 1991). Furthermore, the presence of aldehyde dehydrogenase in the genome of strain BOVIS40 has been identified to facilitate the conversion of intermediate IAAid to IAA (Koga et al. 1991).
Interestingly, strain BOVIS40 harbors tryptophan synthase that reversibly catalyzes indole-3-glycerol phosphate to form glyceraldehyde-3-phosphate and irreversibly involved in pyridoxal phosphate pathway to form tryptophan through the condensation of serine and indole (Ireland et al. 2008). The presence of amidase enzymes could contribute to bacterial functions in the production of IAA along the IAN pathway. Additionally, the involvement of strain BOVIS40 in two different IAA metabolic pathways significantly revealed their IAA production ability to play a vital role in plant root formation and development.
Subsequently, miaAB genes involving cytokinin biosynthesis and transformation were detected in the genome of BOVIS40. Enzyme coding for tRNA dimethylallyltransferase genes in the bacterial genome could enhance iPR - N6-(dimethylallyl)adenosine production. The expression of other cytokinin genes such as miaBE that convert iPR to 2-methylthio-N6-(dimethylallyl) adenosine and further to 2-methylthio-cis-ribozeatin has been identified in the genome of P. phytobeneficialis MSR2 (Nascimento et al. 2020a). The direct effects of these genes in plant growth enhancement suggest that cytokinin synthesis could play a vital role in plant growth promotion and plant health (Wani et al. 2016). Nevertheless, further comparative research into agriculturally important S. indicatrix strains will help provide information on their novel genes, as reports on cytokinin genes from this endophytic bacterium strain BOVIS40 have not been documented.
The detection of siderophore genes in Stenotrophomonas strain BOVIS40 can enhance plant accessibility to soil mineral nutrients and contribute to their growth. Genes coding for iron transport found in strain BOVIS40 could play a vital role in the mineralization of insoluble iron and bioavailability for plant use. Furthermore, the synthesis of 2,3-butanediol by endophytic bacteria has been linked to their biocontrol ability, induction of systemic resistance, and improve plant tolerance to drought (Samaras et al. 2020). Reports on the biocontrol activity of endophytic bacteria such as Pseudomonas spp against Phytophthora capsici that cause rot and blight disease in pepper (Aravind et al. 2009), Bacillus spp against wilt disease in chili (Dowarah et al. 2021), Burkholderia cenocepacia against Fusarium wilt in banana (Ho et al. 2015), and Stenotrophomonas spp against Sclerotium rolfsii that cause collar rot in tomato (Sahu et al. 2019) have been studied.
Endophytic microbes have employed indirect mechanisms in the production of metabolic compounds in the control of phytopathogens, and their pathogen suppressive actions rely on their ability to produce biocontrol agents (Santoyo et al. 2016). The biocontrol ability of strain BOVIS40 may be due to siderophore synthesis that enhances their antibiosis activity against plant pathogens (Maheshwari et al. 2019). Notably, strain BOVIS40 harbors the siderophore gene fiu. Siderophore catecholate found in the bacterial genome has been recognized to play an important role in bacterial adherence to the receptor surfaces, iron chelation and transport (Pedraza et al. 2010). Conversely, other related genes such as entABCDEFGHS and fepABCDG are involved in siderophore production and transport that function in the conversion process of chorismate into enterobactin that was not detected in this study have been documented (Tortora et al. 2011; Hubrich et al. 2021).
Additionally, based on the beneficial effects on plant growth promotion, strain BOVIS40 harboring biocontrol genes could contribute to disease suppression promote plant growth. Also, detection of genes modulating the biological activity of strain BOVIS40 can stimulate the secretion of antimicrobial compounds such as g-aminobutyric acid (GABA) and 4-hydroxybenzoate, thus revealing their genome response in plant health and exploration as bioinoculants for the control of microbial infections.
The survival of endophytic microbes in the root endosphere is affected by the number of exudates released from the plant roots. Majorly, root exudate is composed of amino acids, sugars, phenolics, organic acids, etc. (Baudoin et al. 2003). Various organic compounds such as sugars serve as an energy source for microbial metabolism. The release of carbon-containing compounds from the rhizo-compartment as exudate could supply endo-rhizobiome the required energy for various cellular activities (Zhalnina et al. 2018). Sugar assimilation potential of strain BOVIS40, as reported in Table 1 was validated based on the detection of various genes involved in carbohydrate metabolism and transport. Carbohydrate utilization response of strain BOVIS40 can enhance their biofilm production and colonization in the host plants. Several genes involved in the degradative pathway of organic compounds such as carbohydrates may contribute to the biotransformation of various organic substrates (Nascimento et al. 2020a). Aside from carbohydrates, the expression of genes participating in organic acid metabolism may contribute to bacterial affinity with the host plant. Also, various genes involved in the degradation and transport of amino acids and their derivatives contributing to amino acid metabolism and bacterial colonization with the host plants have been documented (Mavrodi et al. 2021).
Prephenate hydratase gene (pheA) involved in catechol metabolism was found in strain BOVIS40. Genes involved in gallate catabolism were also detected in strain BOVIS40. The presence of these genes could suggest their ability to modulate phenolic degradation, which plays a major role in endophytic lifestyle. Phenolics are classified as vital compounds that regulate plant growth and stimulate resistance stimuli in plants (Nascimento et al. 2020a). Furthermore, phenolic metabolism can enhance the colonization efficiency of strain BOVIS40 in plants. The ability of bacteria to degrade lignin due to their inherent lignin-degrading enzymes is known to functionally contribute to bacterial infiltration and colonization in the host endosphere compartment (Shi et al. 2015).
Bacterial colonization within the plant endosphere to form biofilm can be engineered based on certain bacterial traits and the synthesis of biofilm production genes. Genes involved in bacterial attachment to plant surfaces such as bcsABCFGZ and yhjQ as expressed in the genome of strain BOVIS40 have been identified in the genome of Ghats1 (Shastry et al. 2020). The expression of colonization genes in the genome of endophytic bacteria may enhance their colonization potential, host selection and establishment of plant-bacterial interactions in a symbiotic manner (Eida et al. 2021). Furthermore, the presence of cellulose biosynthetic genes in strain BOVIS40 may help configure endophytic lifestyle for host-bacterium mutual dependence.
The presence of exopolysaccharide genes could enhance bacterial colonization to form a biofilm, mucilaginous active substances, and boost stress tolerance in plants (Santaella et al. 2008; Meneses et al. 2011). This suggests that bacterial strain BOVIS40 could form synergistic cooperation by colonizing the plant endosphere, thus benefiting the host plants. Endophytes can infiltrate the host plant either by tissue damage, natural opening or through enzyme action (Omomowo and Babalola 2019; Yadav et al. 2020). Hence, the ability of bacterial strain BOVIS40 to produces enzymes, and genes coding for exopolysaccharides and cellulose biosynthesis may mediate bacterial colonization and attachment for biofilm production in the root endosphere (Meneses et al. 2011).
Cellular organelles such as fimbriae, flagella, and pili play a vital role in bacterial attachment, movement, and response to chemicals (Zheng et al. 2015). The detection of chemotaxis and motility genes can mediate bacterial ability to infiltrate plant roots and establish a bacterial community for plant benefits. The results obtained from this study corroborate the findings of Shastry et al. (2020), who reported chemotaxis functional genes (cheAVY) and other genes involved in flagella biosynthesis in E. cloacae Ghats1. Similarly, identification of motAB and other genes responsible for chemotaxis actions such as cheABYWR in P. phytobeneficialis MSR2 have been reported (Nascimento et al. 2020a).
Plant-microbe interactions stimulating a lot of chemical reactions in the production of antioxidative and antimicrobial compounds such as phytoalexins, nitric oxide, and reactive oxygen species (ROS) have boost plant defense against osmotic stress (Gaber et al. 2006; Samaras et al. 2020). The expression of oxidative and osmotic stress genes in the bacterial genome could reveal their strong resistance tendencies to ROS, a chemical compound produced by bacteria in a stress environment (Scott et al. 2007; Nascimento et al. 2020b). Also, the expression of osmotic genes is known to boost host immune responses. The colonization and survival of endophytic bacteria in plants growing in an environment exposed to oxidative stress can be modulated by the synthesis of antioxidant lytic enzymes such as glutathione reductase, monodehydroascorbate reductase, superoxide dismutase, ascorbate peroxidase, and catalase. The function of these enzymes is paramount in scavenging free radicals in the plant endosphere and other regulators such as reduced glutathione and ascorbic acid (Das et al. 2015). Similar functional genes coding for catalase, superoxide dismutase, peroxiredoxin, glutathione peroxidase, alkyl hydroperoxide reductase, glutaredoxin 3, glutathione monothiol glutaredoxin, and glutathione-disulfide reductase were detected in strain BOVIS40. The detection of a catalase-peroxidase gene (katE) and catalase gene (katG) responsible for the degradation of toxic peroxide corroborates the findings of Nascimento et al. (2020a), who reported similar genes from Pantoea phytobeneficialis MSR2. Hence, their ability to stimulate defense against oxidative and osmotic stress in plants could be promising in the formulation of bioinoculants for plant benefit.
The presence of osmotic stress regulatory genes could enhance plant response to heat or cold shock. Similarly, the protein-coding genes that modulate cold and heat stressors can function through multiple gene families and differential regulation (Wani et al. 2016). Plant response to cold or heat stress has been attributed based on the expression of notable genes such csp, hsp, and a gene coding for the chaperone (Phadtare 2004). As presented in this study, these genes could contribute to the resistance and survival of sunflower in temperate environments. The presence of csp in the bacterial genome contributing to RNA and DNA stabilization for improved translational and transcriptional efficiency of plants under cold or heat conditions has been documented (Nascimento et al. 2020b). However, the expression of these genes in strain BOVIS40 corroborates the findings of Nascimento et al. (2020b), who had earlier reported chaperones and other heat stress genes such as groEL, groES, dnaK, dnaJ and hsp in the genome of Bacillus megaterium STBI.
Transcriptional regulator and amino acid transporter genes were found in train BOVIS40. These genes can mediate mechanisms involved in quorum-sensing and dynamics in the lifestyle and ecology of endophytic microbes. Various antimicrobial compounds secreted by bacterial strains can exert beneficial effects in sustaining plant health. For example, the role of bacteriocin produced by endophytic bacteria as a source of new antibacterial compounds have synergistically been employed to upsurge antibiotic resistance (Lopes et al. 2017; Oliveira et al. 2018). Organic compounds synthesized by bacteria naturally play a vital role in the adaptation and survival of the host plant. Although, Stenotrophomonas species are regarded as opportunistic pathogens that affect humans and plants, and in recent times, their biocontrol efficacy due to the ability to secrete vital secondary metabolite is promising. However, documentation on secondary metabolite gene clusters from S. indicatrix is rare. Interestingly, two prominent gene clusters such as arylpolyene (APE Vf) and bacillibactin NRP: siderophore with type arylpolyene and non-ribosomal peptides (NRPS) detected in the genome of strain BOVIS40 could contribute to plant protection against oxidative stress and other biological functions. For instance, the siderophore gene cluster detected in the genome of copious endophytic Streptomyces kebangsaanensis contributes to their biocontrol activity against pathogenic fungi Fusarium oxysporum and enhances plant survival (Remali et al. 2017; Siupka et al. 2020). The attribute of siderophore synthesis by bacterial strains can provide information on their ecological functions as biocontrol agents against plant pathogens. Hence, screening of secondary metabolites in the genome of strain BOVIS40 can enable researchers to understand their application in the control of phytopathogens.
Under greenhouse experimental conditions, an increase in yield parameters of inoculated sunflower compared to un-inoculated was observed. This can be linked to the pheno-genotypic attribute of endophytic bacteria to solubilize phosphate, synthesize IAA and siderophore, and detection of possible biocontrol genes in the bacterial genome, and this may contribute to bacterial activity in the improvement of below-and-aboveground crop yield. The presence of IAA genes seemed evident in the root formation in sunflower. Hence, results obtained as demonstrated significantly reveal the agricultural importance of S. indicatrix BOVIS40 upon inoculation. Nevertheless, the use of S. indicatrix under greenhouse experimental conditions in improving crop yield is rare in literature.