Skip to main content

Characterization of a novel root-associated diazotrophic rare PGPR taxa, Aquabacter pokkalii sp. nov., isolated from pokkali rice: new insights into the plant-associated lifestyle and brackish adaptation


Salinity impacts crop growth and productivity and lowers the activities of rhizosphere microbiota. The identification and utilization of habitat-specific salinity-adapted plant growth-promoting rhizobacteria (PGPR) are considered alternative strategies to improve the growth and yields of crops in salinity-affected coastal agricultural fields. In this study, we characterize strain L1I39T, the first Aquabacter species with PGPR traits isolated from a salt-tolerant pokkali rice cultivated in brackish environments. L1I39T is positive for 1-aminocyclopropane-1-carboxylate deaminase activity and nitrogen fixation and can promote pokkali rice growth by supplying fixed nitrogen under a nitrogen-deficient seawater condition. Importantly, enhanced plant growth and efficient root colonization were evident in L1I39T-inoculated plants grown under 20% seawater but not in zero-seawater conditions, identifying brackish conditions as a key local environmental factor critical for L1I39T-pokkali rice symbiosis. Detailed physiological studies revealed that L1I39T is well-adapted to brackish environments. In-depth genome analysis of L1I39T identified multiple gene systems contributing to its plant-associated lifestyle and brackish adaptations. The 16S rRNA-based metagenomic study identified L1I39T as an important rare PGPR taxon. Based on the polyphasic taxonomy analysis, we established strain L1I39T as a novel Aquabacter species and proposed Aquabacter pokkalii sp nov. Overall, this study provides a better understanding of a marine-adapted PGPR strain L1I39T that may perform a substantial role in host growth and health in nitrogen-poor brackish environments.

Peer Review reports


In nature, the plant rhizosphere hosts a large and phylogenetically diverse group of complex bacterial communities whose interactions and activities are increasingly recognized as important determinants of plant health, fitness, and productivity [1]. The rhizosphere-associated bacteria that confer health benefits to plants are collectively called plant growth-promoting rhizobacteria (PGPR). The well-characterized beneficial functions of PGPR include 1) increasing the availability of essential nutrients inaccessible to the plants, such as nitrogen via fixation of atmospheric nitrogen [2] and phosphate via solubilization of insoluble soil phosphates through organic acid production [3], 2) promote plant growth and development via the production of phytohormones [4], 3) protection against plant pathogens through secreting different classes of antimicrobial substances or by eliciting plant defense hormones [5], and 4) provide enhanced protection against abiotic stress such as salinity and drought through scavenging plant stress ethylene with the help of 1-aminocyclopropane 1-dicarboxylate (ACC) deaminase [6]. Despite having huge potential in sustainable agriculture, the current knowledge about the PGPR and its potential beneficial interactions across a wide range of plants, including crops of brackish-associated environments, an interface between marine and terrestrial ecosystems that are agriculturally important, is very limited.

Pokkali is a unique traditional rice variety cultivated organically in water-logged brackish tidal environments of coastal regions of central Kerala, southern India. Notably, most of the pokkali rice fields are nearer to the sea. As a result, seawater enters these fields during each tidal cycle, leading to rapid fluctuations in salinity levels and creating an unfavorable environment for crop growth. Despite these constantly changing saline conditions, the pokkali rice grows well and yields good productivity. Thanks to its naturally evolved genetic traits enabling them to thrive at higher coastal salinity than any inland terrestrial rice varieties [7]. The other possible reason for their adaptability and better growth performance under such coastal salinity could be their close symbiotic relationships with the beneficial native microbes inhabiting these brackish pokkali rice fields. Till now, relatively little information is available on pokkali rice and its associated beneficial native microbes. Moreover, understanding the PGPR of such ecosystems may offer valuable insights into their physiology, genomic traits, habitat-specific adaptations, and functional role in plant health and productivity. Further, it can provide sustainable solutions for improving rice productivity under coastal saline conditions. For instance, very few studies have shown that microbes from the pokkali rice can promote growth and confer tolerance to salt stress in salt-sensitive rice varieties, Fusarium sp., in IR64 rice variety [8], and Pseudomonas taiwanensis strain PK7 in Uma rice variety [9].

In an ongoing research study on the isolation and functional characterization of the rhizosphere-associated PGPR of this unique brackish-associated pokkali rice, we specifically targeted habitat-specific indigenous PGPR strains that possess ACC deaminase activity and can promote rice growth under brackish water conditions. To achieve this, we cultured the rhizosphere samples of different pokkali rice varieties collected from various sites using different minimal media compositions that contained ACC as the sole nitrogen source and different sugars or organic acids as the carbon source, and 50% natural or 50% artificial seawater (Himedia, India) to mimic brackish environmental conditions. This investigation resulted in the isolation of several potential ACC deaminase-producing indigenous rhizobacterial strains, including the genera Burkholderia, Herbaspirillum, and Rhizobium, and a few unclassified strains that are characterized as novel bacterial taxa; Vogesella oryzae L3B39T [10], Arthrobacter pokkalii P3B162T [11], Novosphingobium pokkalii L3E4T [12], Pokkaliibacter plantistimulans L1E11T [13], Sphingomonas pokkalii L3B27T [14], and Flavobacterium pokkalii L1I52T [15], and functionally some are shown to possess plant growth-promoting abilities [13, 15]. Additionally, Rhodoplanes pokkaliisoli JA415T, a novel phototrophic alphaproteobacterium, was reported from a pokkali paddy field by Lakshmi et al. [16].

This study characterizes another novel ACC deaminase-producing diazotrophic strain designated L1I39T. Initial 16S rRNA sequence analysis using the EzBiocloud server [17] indicated that L1I39T belongs to the genus Aquabacter. This genus belongs to the family Xanthobacteraceae, phylum Pseudomonadota, and was first described by Irgens et al., 1991 [18] with Aquabacter spiritensis SPL-1 T as the type species. Aquabacter species are Gram-negative rods, non-motile, and aerobic heterotrophic bacteria that form yellow to white chalk-pigmented colonies. In addition to their taxonomic description, the possible ecological significance and detailed genome characterization in relationships with their growth habitats are lacking. At present, the genus Aquabacter contains two validly described species (, A. spiritensis SPL-1 T isolated from Spirit Lake [18] and A. cavernae Sn-9-2 T from cave soil [19]. The taxon described in this study would be the third and first marine member of the Aquabacter species isolated from a native salt-tolerant pokkali rice traditionally grown in brackish tidal environments of the coastal region of central Kerala, India. Based on the detailed genetic (including multilocus sequence and whole genome analysis), phenotypic, and chemotaxonomic characteristics, we establish that L1I39T represents a novel Aquabacter species for which we named Aquabacter pokkalii sp nov. In addition, we functionally characterize its potential plant growth-promoting (PGP) properties, including ACC deaminase production, nitrogen fixation, and ability to colonize and promote rice growth under nitrogen-limiting seawater conditions. Through detailed physiological experiments, we demonstrate its eco-physiological adaptations relating to a brackish environment, and by analyzing the 16S rRNA amplicon metagenomic datasets of brackish-associated different native rice varieties of Kerala, we determined the occurrence and abundance of L1I39T in those habitats. Finally, the genome of L1I39T was sequenced and conducted an in-depth analysis to understand the molecular determinants that may contribute to its plant-associated lifestyle, plant-growth functions, and habitat-specific adaptations, including the brackish tidal environment. Further, we interrelate these genome data with results obtained from our phenotypic experiments.

Materials and methods

L1I39T isolation, preservation, and seawater collection

The strain L1I39T was isolated in 2014 from the rhizosphere of a salt-tolerant pokkali rice cultivated in water-logged brackish tidal environments of coastal Kerala, Kumbalangi (9°52′06.4’’N, 76°16′58.3’’E) India. For isolation, a traditional serial dilution method was followed [13]. The bacteriological medium A used for isolating L1I39T has the following composition (g/100 ml): 0.1 g of malate, 3 mM of ACC (Sigma), 50 ml of artificial seawater (HiMedia), 50 ml of distilled water, 1.8 g of agar. The Reasoners 2A (R2A) agar (HiMedia catalog no SMEB962D) with 0.5% NaCl (R2A-N) or 20% natural seawater was used for routine subculture and to maintain L1I39T at 4 °C. Cells were suspended in 20% glycerol and kept as glycerol stocks at -80 °C for long-term preservation. The natural seawater used in this study was collected from the Arabian Sea (~ 2 km from the Vizhinjam beach, Thiruvananthapuram, Kerala, India). The collected seawater was filter-sterilized in 8 µm cellulose nitrate filters (Sartorius Stedim Biotech, Germany) to remove particle traces and stored at 4ºC until used. The reference type strains Aquabacter cavernae KCTC 62308 T, Aquabacter spiritensis LMG 8611 T, Azorhizobium caulinodans LMG 6465 T, and Xanthobacter autotrophicus LMG 7043 T were used for comparative taxonomic characterization.

Polyphasic taxonomy

Molecular phylogeny

The extraction of genomic DNA, amplification of 16S rRNA gene, bi-directional Sanger sequencing, and sequence and phylogenetic analysis were performed following the described methods [13].

The high-quality DNA of L1I39T for whole genome sequencing was extracted using the QIAamp DNA mini kit (Qiagen, Germany). The genome sequencing of L1I39T was performed using the Illumina HiSeq 4000 sequencer at Bionivid Technology Pvt Ltd., Bengaluru, India. The raw sequencing reads were subjected to quality filtering (Phred score greater than 30) using the IlluQC tools available in the NGSQC toolkit [20]. The de novo assembly of the filtered high-quality reads was performed using the SPAdes v3.0.0 paired-end library pipeline [21]. A reference-based genome finishing was performed using the MeDuSa web server with Xanthobacter autotrophicus DSM 432 T as the reference genome. The L1I39T genome completeness, contamination, and heterogeneity were determined using CheckM v1.1.2 [22].

For multilocus sequence analysis (MLSA), six high-resolution phylogenetic taxonomic markers such as ATP synthase β subunit (atpD), recombinase A (recA), DNA gyrase subunit B (gyrB), molecular chaperone (dnaK), citrate (Si)-synthase (gltA) and RNA polymerase β subunit (rpoB) were selected. These protein-coding gene sequences were retrieved from the genomes of LI139T and its related phylogenetic neighbors of the family Xanthobacteraceae (Table S1). The individual protein-coding gene sequence and phylogenetic tree analysis were performed as described before [11]. For concatenated sequence analysis, the protein-coding sequences of six housekeeping genes were aligned individually using the MAFFT multiple alignment program [23] and concatenated using the Gblocks tool available within PhyloSuite v1.2.2 [24]. The resultant concatenated protein-coding sequences were used for the construction of phylogenetic trees using different tree-making algorithms available in MEGA v7.0.26 [25]. Likewise, a phylogenomic tree was constructed using 539 single-copy orthologous protein-coding gene sequences obtained using OrthoFinder v2.5.4 [26]. Escherichia coli DSM 30083 T served as the outgroup.

The genome-relatedness similarity analysis, including the average nucleotide identity (ANI), average amino acid identity (AAI), and digital DNA-DNA hybridization (dDDH) values, was performed between the genomes of L1I39T and related phylogenetic neighbors of the family Xanthobacteraceae as described before [14, 27,28,29].

Chemotaxonomy and phenotype characters

Cells were cultivated in trypticase soy agar (TSA) to analyze cellular fatty acids. After incubation of 48 h at 28 °C, whole-cell fatty acid methyl esters (FAME) were extracted, and analysis was performed as described before [30]. The bacterial identification service available at DSMZ, Germany, was availed for determining the polar lipids and respiratory quinones of L1I39T. This analysis was performed using the protocol described by Tindall, 1990; Krishnan et al., 2018 [13, 31].

The phenotypic characteristics such as cell morphology, motility, anaerobic growth, reduction of nitrate, production of indole, Methyl red and Voges Proskauer (MR-VP), hydrolysis of starch, xylan, carboxymethyl cellulose, esculin, casein, urea, and DNA, and the ability to grow in different standard heterotrophic media, including Luria Bertani (LB), nutrient agar, and trypticase soya agar were performed using methods as described by Rameshkumar et al., 2016 [10]. The growth at varying temperatures and pH ranges was assayed using the procedure described by Rameshkumar et al., 2016 [11], with modifications specified by Menon et al., 2020 [15]. The assimilation of different substrates was determined using an API 20NE strip as per the instructions provided by the manufacturer, and the results were noted after 48 h of incubation at 30 °C.

Genome functional analysis

The genome sequence was annotated using NCBI Prokaryotic Genome Annotation Pipeline (PGAP) and Rapid Annotation using Subsystem Technology (RAST) v.4 [32]. The different genes and gene clusters encoded in the L1I39T genome under different functional gene categories were identified through the subsystem feature analysis in RAST. The rRNAs were predicted using RNAmmer-1.2 [33], and tRNAs were identified using ARAGORN-v1.2.36 [34]. The gene organization of the important operons was constructed according to the size and orientation of the genes using the genoPlotR package in R v2.6.0 [35]. The circular genome was constructed using the blast ring image generator, BRIG v0.95 [36], utilizing various functional gene categories identified using the EGGNOG-mapper v5.0 [37].

Plant growth-promoting traits

ACC deaminase activity

The utilization of ACC as the sole nitrogen source and ACC deaminase quantification was determined by following the methods described by Krishnan et al., 2018 [13].

Growth in nitrogen-free medium and nitrogenase (nifH) gene amplification

The ability of L1I39T to grow in a nitrogen-free medium was determined using Jensen’s medium (HiMedia, catalog no M710) with 0.5% NaCl. The presence of the nifH gene in L1I39T was determined by polymerase chain reaction (PCR) based amplification of the nifH gene using the PolF and PolR universal nifH gene primers [38] and PCR cycling conditions: an initial denaturation step at 95 °C for 5 min (min), followed by 30 cycles consisting of denaturation at 95 °C for 45 s, annealing at 54 °C for 45 s, extension at 72 °C for 1 min, and a final extension step lasting 10 min. A single amplicon with a size of 360 bp indicated the presence of the nifH gene, and the genomic DNA of Azoarcus communis LMG 22127 T was used as the positive control.

In-planta detection of L1I39T-nifH

Overnight grown L1I39T cells from an R2A-N broth were centrifuged and washed, and the entire cell pellet was suspended in a sterile carbon and nitrogen-free minimal salt (MS) solution (consisting of 0.8 g/L of K2HPO4, 0.2 g/L of KH2PO4, 0.2 g/L of MgSO4.7H2O, 0.1 g/L Na2.MoO4, 5 g/L NaCl, and a trace amount of yeast), making a final OD600 of 0.4 (~ 10–6 cells). Five days old sterile pokkali rice seedling roots were incubated in this bacterial MS solution for 90 min. The seedlings incubated in MS solution without L1I39T cells were the control. These incubated seedlings (L1I39T-treated and control) were transferred aseptically to the respective phytajars containing semi-solid MS media (prepared with 0.2% agar to create a microaerophilic condition). The entire setup was incubated in plant growth racks maintained with light/dark (12 h/12 h) cycles under 28 °C for five days. The same procedure was followed for nitrogen-excess conditions, except the seedlings were incubated in a semi-solid MS media prepared with 20 mM NH4Cl. Total RNA was extracted from the incubated root samples using the RNeasy Plant Mini-Kit (Qiagen, Germany). The RevertAid First Strand cDNA Synthesis kit (Thermo Scientific, USA) was used for complementary DNA synthesis. The RNA-converted cDNA samples were subjected to PCR amplification using the universal nifH gene primers PolF/PolR [38] and amplification conditions (as mentioned above). A single amplicon at 360 bp was considered positive.

NifHDK phylogeny and nif gene cluster comparison

Annotated protein sequences of NifH, NifD, and NifK were obtained from the genomes of LI139T and its related phylogenetic neighbors, including the members of the genera Aquabacter, Azorhizobium, and Xanthobacter. These protein sequences were aligned individually using the MAFFT multiple alignment program [23] and concatenated using the Gblocks tool available within PhyloSuite v1.2.2 [24]. The phylogenetic trees were constructed using the concatenated protein sequences of NifHDK using MEGA v7.0.26 [25]. For the nif gene cluster comparison, the software program genoPlotR package was used [35]. The following reference strain genomes were included: Aquabacter cavernae Sn-9-2 T, Aquabacter spiritensis DSM 9035 T, Azorhizobium caulinodans ORS 571 T, Xanthobacter autotrophicus Py2 as well as four well-characterized diazotrophs associated with terrestrial rice plants namely, Azoarcus sp BH72, Azospirillum brasilense Sp7T, Azotobacter vinelandii CA, and Pseudomonas stutzeri DSM 4166.

Siderophore production

The production of siderophore was determined by following the protocol described by Krishnan et al., 2017 [12], except Luria Bertani was used as the basal growth medium. The development of a yellow-colored clearance zone surrounding the grown colonies after a week of incubation is considered positive.

In planta growth experiment

The ability of L1I39T to promote host growth under nitrogen-poor seawater conditions was investigated using pokkali rice. Briefly, three days old sterile germinated pokkali seeds were incubated with L1I39T (≥ 108 cells) suspensions for three hours, followed by a brief drying, and then aseptically transferred to pots containing sterile soil rite mix: sand (2:1) as growth substrates. Germinated seeds treated without L1I39T cells served as control. In order to maintain a brackish environmental condition, the growing seedlings were irrigated regularly with 20% natural seawater, and the nitrogen-free half-strength Hoagland solution containing 20% natural seawater was supplemented as a plant nutrient solution every 3-days once till the end of the experiment. After 18 days of growth, both L1I39T-treated and untreated control plants were uprooted and quantified for various plant growth-related parameters, including the shoot fresh weight, fresh and dry weight of the roots, shoot and root length. The nitrogen content of the shoots was estimated using the KjelTRON™ KDIGB 8 M system according to the manufacturer’s instructions (Tulin Equipments, India). To validate whether 20% natural seawater condition is critical for L1I39T plant growth promotion. A similar plant growth experiment was performed as detailed above, but this time, the germinated seeds, including the L1I39T-treated and control, were grown under nitrogen-free zero seawater conditions. The nitrogen-free half-strength Hoagland solution with zero seawater was supplemented as a plant nutrient solution. The plant parameters were recorded after 28 days post-inoculation. The comparative bar graphs were plotted for the quantified plant parameters to represent the differences between the L1I39T-treated and untreated control plants. The statistical significance was determined through the student’s t-test, and the significance was indicated in the graphs as follows: *, p-value < 0.05; **, p-value < 0.01; ***, p-value < 0.005.

Plant-associated traits

Hydrogen peroxide tolerance

Active L1I39T cells were inoculated to an LB broth containing 20% seawater and incubated at 30 °C, 180 rpm. When the cells reached an OD600 of 0.2 (~ 104 cells), different concentrations of hydrogen peroxide (5 mM and 10 mM) were added to the culture broths, respectively, and allowed to grow further for 24 h. The culture broth with no added hydrogen peroxide served as a control. The hydrogen peroxide tolerance was determined by monitoring the visible growth of the test against the control after 24 h.

Biofilm formation

The biofilm formation ability of L1I39T cells was tested in 96-well microtiter plates using the method reported by Krishnan et al., 2016 [11], except R2A-N broth was used as the growth medium. The formation of a crystal violet stained ring on the walls of the microtiter plate was regarded as positive for biofilm formation.

Metabolic capacity

The ability to utilize different plant-derived substrates, including various sugars, amino acids, and organic acids, was determined using methods described by Krishnan et al., 2017 [12]. The assimilation of other carbon sources was determined using the Biolog Gen III system, and the results were noted after 48 h of incubation at 30 °C.

Cell–cell contact-dependent killing assay

The ability of L1I39T cells to inhibit the neighboring cells via cell–cell contact was performed as described previously [39]. L1I39T was used as an attacker strain, and E. coli harboring pHC60-GFP plasmid with tetracycline resistance was used as a target strain. Pokkaliibacter plantistimulans L1E11T, known for its contact-dependent killing ability, was used as the positive control. Active cells of both attacker and target strains were suspended in 0.85% saline, normalized to an OD600 of ~ 0.9, and mixed in a volume ratio of 10:1 (attacker vs target). 20 µl this mixture was spotted on a solid LB agar medium and incubated for 36 h at 30 °C. Surviving E. coli target cells were enumerated by standard serial dilution and counting viable colonies on tetracycline-supplemented LB agar medium. The final results were quantified from the average of three independent experiments.

Ecophysiological conditions

Growth at different concentrations of NaCl (0.5, 1, 1.5, 2, 3, and 4%, w/v) was tested by inoculating the L1I39T cells in R2A broth (HiMedia catalog no LQ251X) containing respective NaCl concentrations and incubated for a week at 30 °C. The culture flasks showing good turbidity were considered positive.

The ability to grow in different natural seawater concentrations (0, 20, 40, 60, 80, 100%) was determined using R2A broth as the base growth medium. Active L1I39T cells were inoculated in R2A broth with respective seawater concentrations and incubated for a week at 30 °C. The optimal seawater concentration for growth was determined by measuring the OD 600 of R2A broth containing respective seawater concentration at 12 h intervals for a total period of 60 h.

The ability to grow in ZoBell marine medium was checked by streaking a single colony of L1I39T on ZoBell marine agar medium (HiMedia catalog no M385) and evaluating its growth after seven days of incubation at 30 °C.

L1I39T GFP tagging, root attachment, and colonization

The L1I39T was tagged with a pLH6000 plasmid harboring green fluorescent protein (GFP) by following the method described [40]. Briefly, the electrocompetent cells were prepared by suspending the late log phase of L1I39T cells in 10% ice-cold glycerol. The plasmid pLH6000 was transformed into electrocompetent L1I39T cells using the electroporation conditions: 2.5 kV, 200Ω, and 25µF. Immediately after electroporation, the L1I39T cells were grown in a fresh SOC broth [41] for 4–6 h. After this, the cells were centrifuged, and the resulting cell pellet was spread-plated on R2A-N agar plates containing 50 µg spectinomycin. After three days of incubation at 30 °C, the spectinomycin-resistant L1I39T colonies were picked, checked for stable GFP transformation, and later used for root colonization and imaging studies.

The ability to attach host roots under a brackish environmental condition was determined by incubating the sterile five days old pokkali rice seedling roots in GFP-tagged L1I39T suspension prepared in 20% seawater. After 3 h of incubation, the roots were excised and washed two to three times in sterile 20% seawater to remove loosely adhered cells. Following this, the roots were placed on a clean glass slide with 40% glycerol, and then a clean glass coverslip was placed over the roots and was used for visualization under the epifluorescent microscope. Imaging was performed using a Zeiss AXIO Imager M2 fluorescent imaging microscope fitted with a Zeiss Axiocam 503 mono camera (Zeiss, Germany), and the images were processed in ZEISS ZEN (version 2.6) software. The green fluorescence signals emitted from the GFP-tagged L1I39T cells attached along the pokkali seedling roots were considered positive for root attachment.

For root colonization, overnight-grown GFP-tagged L1I39T cells were suspended in a nitrogen-free half-strength Hoagland plant nutrient solution. 1 ml of this GFP-tagged L1I39T (~ 106 cells) suspension was inoculated into gnotobiotic phytajars containing five days old pokkali rice seedlings growing in a nitrogen-free half-strength Hoagland nutrient solution prepared with 20% seawater. This entire phytajar setup was incubated in the plant growth racks (12 h of light and 12 h of dark at 28 °C). Roots were collected on the 7th and 14th day post-inoculation and used to estimate the root colonization potential by following the described methods [12]. The roots were processed for imaging purposes, as mentioned above in the root attachment section. To determine the colonization ability of L1I39T under zero seawater conditions, the same experimental setup was carried out as described above, except the pokkali rice seedlings were grown in a nitrogen-free half-strength Hoagland nutrient solution with no seawater added.

Mapping of operational taxonomic units (OTUs) of L1I39T from brackish-associated rice metagenomic datasets

To identify OTUs of L1I39T, we mapped the full-length 16S rRNA gene sequence of L1I39T against the 16S rRNA amplicon metagenomic datasets of brackish-associated different rice varieties of coastal Kerala, India, generated from our earlier study (NCBI Accessions: pokkali rice—PRJNA970257 and kaipad—PRJNA970551, unpublished results). Only those 16S rRNA sequences showing > 99% similarity and > 400 bp length were considered as representative OTUs of L1I39T. For this analysis, we used the latest version of the NCBI-BLAST program (v2.7.1 +).

Statistical analysis

All experiments were performed with at least three technical replicates, and the data were analyzed using Student’s t-test and the software GraphPad Prism version 8. Error bars in the graph represent standard deviation within the replicates, and the statistical significance is represented with an asterisk (*) sign as follows: *, p-value < 0.05; **, p-value < 0.01; ***, p-value < 0.001.

Availability of data and materials

The 16S rRNA gene sequence of L1I39T was deposited in NCBI under accession number KX533957. The draft genome sequence of L1I39T was deposited in NCBI with accession number CP072392. The genome accession numbers of reference-type strains of the family Xanthobacteraceae used in this study are listed in Table S1.

Results and discussion

Proposal for Aquabacter pokkalii sp. nov.,

Phylogenetic analysis

The 16S rRNA gene analysis identified strain L1I39T as a member of the family Xanthobacteraceae, sharing the highest pairwise 16S rRNA gene sequence similarities with Aquabacter cavernae Sn-9-2 T (99.00%), followed by A. spiritensis DSM 9035 T (97.23%), Xanthobacter oligotrophicus 29kT (97.01%) and Xanthobacter autotrophicus 7cT (96.94%). Pairwise 16S rRNA sequence similarities to all other recognized members of the family Xanthobacteraceae were below 96.94%. Additionally, a 16S rRNA phylogenetic tree reconstructed using the maximum likelihood method showed that strain L1I39T clustered stably with A. cavernae Sn-9-2 T as the closest known Aquabacter species with a bootstrap support value of 99% (Fig. 1a). Notably, this cluster is well-positioned as an independent branch that was well-separated from a phylogenetic group comprising A. spiritensis DSM 9035 T and remaining all other known species of the genus Xanthobacter and Azorhizobium with a bootstrap support value of 100% (Fig. 1a). Furthermore, these results indicate that strain L1I39T is phylogenetically more closely related to the cluster comprising the members of the genera Aquabacter, Xanthobacter, and Azorhizobium rather than to the phylogenetic cluster containing other described members of the family Xanthobacteraceae; Ancyclobacter and Labrys (Fig. 1a). Similar phylogenetic positioning of L1I39T was obtained in other 16S rRNA tree construction methods, neighbor-joining and maximum parsimony (data not shown). Notably, the 16S rRNA similarity value between the strain L1I39T and its phylogenetic nearest type strain A. cavernae Sn-9-2 T was well above the standard boundary 16S rRNA value of 98.65% to differentiate two different bacterial species [42]. Therefore, high-resolution phylogenetic taxonomic markers such as atpD, recA, gyrB, dnaK, gltA, and rpoB were retrieved from the L1I39T genome and used further to clarify its taxonomic positioning in the family Xanthobacteraceae. The analysis of individual and concatenated sequences of the six housekeeping genes (atpD, recA, gyrB, dnaK, gltA, and rpoB) revealed that strain L1I39T shared relatively lower gene sequence similarities (< 93%, < 90%, < 86%, < 91%, < 91%, < 92% and < 90%, respectively) with its closest phylogenetic relatives (Table S2). Importantly, these gene sequence similarity values are relatively lower than the commonly proposed threshold cut-off similarity value of 94% for novel bacterial species description [43]. Furthermore, the phylogenetic trees inferred using both individual (data not shown) and concatenated amino acid sequences of the above six housekeeping genes showed that strain L1I39T was placed in a separate branch along with the type strains of A. cavernae Sn-9-2 T and A. spiritensis DSM 9035 T as its closest phylogenetic neighbors with a bootstrap support value of 91% (Fig. S1). Similar phylogenetic positioning of L1I39T was retrieved when we constructed a protein-based core phylogenomic tree using 539 genes (Fig. 1b). Altogether, distinct phylogenetic position and low levels of housekeeping gene sequence similarities with the described members of the genus Aquabacter indicate that strain L1I39T may represent a novel Aquabacter species.

Fig. 1
figure 1

a Maximum likelihood phylogenetic tree based on the 16S rRNA gene sequences of L1I39T and related members of the family Xanthobacteraceae. The significance of each branch is indicated by the bootstrap value (as a percentage) calculated for 1000 subsets. The bar indicates sequence divergence. Different shapes and color codes indicate the isolation habitats of the respective type strains, (b) maximum likelihood phylogenetic tree based on the protein-coding sequences of 539 core genes (overall 170,910 amino-acid positions) of L1I39T and related members of the family Xanthobacteraceae. Closed dark circles at each node represent a similar grouping obtained from the neighbor-joining and maximum parsimony algorithms. The significance of each branch is indicated by the bootstrap value (as a percentage) calculated from 1000 subsets. E. coli ATCC 11775 T was used as an outgroup. The scale bar indicates nucleotide substitution per site

Genome-based relatedness

Estimating the genome-relatedness similarity values, including ANI, AAI, and dDDH for species-level bacterial classifications, is considered the gold standard [44, 45]. Accordingly, we estimated the genome-relatedness similarity values between the genomes of L1I39T and its phylogenetically closest Aquabacter type strains and with other recognized members of the family Xanthobacteraceae. This result showed that the calculated values between the genomes of L1I39T and A. cavernae Sn-9-2 T and A. spiritensis DSM 9035 T and other recognized members of the family Xanthobacteraceae (Table S3) are relatively lower than the standard proposed ANI/AAI/dDDH values (< 95%, < 95%, and < 70%) for novel bacterial species delineation [44, 45], suggesting that strain L1I39T represent as a distinct Aquabacter species within the family Xanthobacteraceae.

Chemotaxonomic and phenotypic characterization

The fatty acids of the strain L1I39T were dominated by C18:1ω7c (88.79%), which is in accordance with the members of the family Xanthobacteraceae [46]. However, the fatty acids profiles that may be useful in differentiating the strain L1I39T from its nearest phylogenetic neighbors are mentioned in (Table S4). The strain L1I39T contained ubiquinone Q-10 as the predominant quinone and major polar lipids as phosphatidylglycerine, phosphatidylethanolamine, phosphatidylglycerol, and phospholipid (Fig. S2). All these chemotaxonomic data are in agreement with earlier published reports on the different species of the family Xanthobacteraceae [47].

The phenotypic characters that differentiate strain L1I39T from its closest phylogenetic neighbors, Aquabacter, Xanthobacter, and Azorhizobium, are colony pigmentation, growth in ZoBell marine medium, tolerance to 3% NaCl, and growth in the presence of full-strength seawater (Table 1).

Table 1 Major phenotypic characters differentiating strain L1I39T from its nearest phylogenetic neighbors

Plant growth-promoting properties

L1I39T can utilize ACC as the sole nitrogen source when grown in a defined mineral salts media (Fig. 2a) and exhibited ACC deaminase activity of 1.72 ± 0.89 µmol of α-ketobutyrate/h/mg of protein. Furthermore, the L1I39T genome contains a putative gene encoding for ACC deaminase (acdS), suggesting that the acdS gene is functional. Additional analysis showed that L1I39T tested positive for growth in a nitrogen-free Jensens medium, followed by a PCR amplification confirming the presence of the 360 bp region of the nifH gene (data not shown), a universal marker widely used for detecting diazotrophic bacteria [38]. Besides, the L1I39T genome carries a complete set of essential genes required for biological nitrogen fixation (detailed in the genome section), suggesting that nif regulon is functional. In support of this view, an in-planta nifH gene expression assay showed that the nifH gene transcripts of L1I39T were detected in the pokkali rice roots when grown under a nitrogen-deficient condition (Fig. 2b). However, this nifH gene expression was repressed by free excess ammonium present in the growth medium (Fig. 2b), indicating that L1I39T regulates its nifH gene expression according to the status of nitrogen availability in the growth environment [48]. Furthermore, these findings suggest the significant ability of L1I39T to fix atmospheric nitrogen in the host roots under a nitrogen-deficient growth condition.

Fig. 2
figure 2

The plant-growth-promoting traits of L1I39T: (a) positive growth of L1I39T on a defined minimal salts medium containing ACC as the sole nitrogen source, images taken after 4 days of incubation at 30 °C, (b) detection of In-planta L1I39T nifH gene expression, lanes, L, 100 bp plus DNA ladder; 1 & 2, nifH gene amplicon from total root RNA converted cDNA from nitrogen-free conditions; 3 & 4, nifH gene amplicon from total root RNA converted cDNA from nitrogen-excess conditions; 5, the total root RNA converted cDNA from pokkali seedlings roots incubated without L1I39T cells (negative control); 6, no reverse transcriptase (RT) control. Amplicon size of 360 bp is indicated in the gel image

Following these observations, we investigated the plant growth-promoting ability of L1I39T under a nitrogen-deficient seawater condition that mimics or is nearer to the physiological state of brackish pokkali rice fields. For this purpose, surface-sterilized pokkali rice seeds were treated with L1I39T and transferred to pots containing a sterile soil rite sand mixture (2:1 ratio). Untreated seeds were used as a control. The entire setup was irrigated with a 20% natural seawater concentration, and a nitrogen-free plant nutrient solution containing 20% natural seawater was provided to the rice plants once every three days until the experiment was complete. On the 7th dpi (days post inoculation), no visible growth difference was observed between the L1I39T-treated and untreated control plants (data not shown). However, on the 18th dpi, we observed better growth performance of the pokkali rice plants treated with L1I39T than the untreated control plants (Fig. 3a & b). This observation includes a significant increase in the plant parameters, such as the fresh and dry weight of the roots (Fig. 3c). Likewise, we also observed an increase in the root and shoot length and the shoot fresh weight in rice plants treated with L1I39T (Fig. 3c). Nevertheless, this data is not statistically found significant. Notably, a substantial increase in the nitrogen content of the shoot portions was observed in rice plants treated with L1I39T compared to untreated control plants (Fig. 3c), indicating its ability to provide fixed nitrogen inputs to the host pokkali rice when grown under a nitrogen-limiting condition corroborating above in planta nifH gene expression assay. Altogether, these results suggest the potential ability of L1I39T to enhance the pokkali rice growth by supplying fixed nitrogen, an essential plant macronutrient, in a nitrogen-limiting brackish environmental condition, thereby identifying strain L1I39T as an important PGPR taxon.

Fig. 3
figure 3

Plant growth-promoting effects of L1I39T under nitrogen-limiting seawater conditions, (a) pot images showing the growth of pokkali rice (L1I39T-treated and control plants) after 18 days post-inoculation, (b) uprooted images showing the growth differences between the L1I39T-treated and control plants after 18 days post-inoculation, (c) bar graphs represent the significant growth differences observed in plant parameters such as (i) shoot length, (ii) root length, (iii) shoot fresh weight, (iv) root fresh weight, (v) root dry weight, and (vi) nitrogen content (%) in shoots between (A) control and (B) L1I39.T-treated pokkali plants estimated after 18 days post-inoculation. Statistical significance represented by *, p-value < 0.05; **, p-value < 0.01; ***, p-value < 0.001

Most members of the family Xanthobacteriaceae described are associated with terrestrial plants [49]. However, only very few members have shown to possess PGP abilities, including the well-characterized stem nodulating nitrogen-fixing strain, Azorhizobium caulinodans ORS 571 T [50], and a few different strains of Xanthobacter autotrophicus [51, 52]. Moreover, none of these studies tested their plant growth promotion ability under a nitrogen-limiting seawater growth condition. Notably, no reports show direct experimental evidence that strains of Aquabacter possess PGPR properties. Hence, this study describes strain L1I39T as the first Aquabacter species from a brackish-associated pokkali rice shown to possess multiple PGPR traits, including biological nitrogen fixation and ACC deaminase production and can potentially promote pokkali rice growth in nitrogen-limiting seawater conditions.

Eco-physiological adaptations

Pokkali rice fields are highly prone to seawater intrusions, and to sustain in these habitats, native microbes need to be well-adapted to higher and constantly changing salinity conditions. Studies have shown that most strains belonging to the genera Aquabacter, Xanthobacter, and Azorhizobium of the family Xanthobacteraceae are from terrestrial environments, are more sensitive to NaCl, and can tolerate only up to 1% NaCl [19]. In this regard, a NaCl tolerance test was performed and found that L1I39T could grow in NaCl concentrations ranging from 0 to 3% NaCl. Furthermore, we show that L1I39T can grow in ZoBell marine medium, a synthetic marine medium specifically used for cultivating heterotrophic marine bacteria (Fig. S3). Notably, the nearest phylogenetic neighbors of L1I39T tested negative for growth at 3% NaCl and in ZoBell marine medium (Table 1). Besides, we also tested the ability of L1I39T to grow in R2A broth prepared with varying natural seawater concentrations ranging from 20 to 100%. This result showed that L1I39T could grow very well in 20 and 40% natural seawater but displayed weak and delayed growth at higher seawater concentrations (> 60%), suggesting that higher seawater concentrations had some negative growth effects on L1I39T (Fig. S4). Importantly, L1I39T showed an optimal growth at 20% natural seawater compared to zero-seawater conditions (Fig. S4), signifying that lower seawater concentrations can influence the L1I39T growth, a habitat-specific trait typically observed in bacteria originating from brackish-associated environments.

Next, we investigated the colonization ability of the L1I39T in the presence and absence of a seawater condition that was used earlier in our plant growth experiments. For this purpose, five days old sterile pokkali rice seedlings were grown in separate gnotobiotic phytajars containing respective nitrogen-free plant nutrient solutions containing 20% seawater and zero seawater. To this setup, 105 cells (CFUs/ml) of GFP-tagged L1I39T were inoculated and incubated in a plant growth chamber for 14 days. On 7th dpi, we recovered L1I39T-GFP cells from the roots of zero seawater (5.47 (± 1.10) X 106 CFUs/gm of roots) and 20% seawater (1.20 (± 0.08) X 107 CFUs/gm of roots) conditions. Further, at 14th dpi, L1I39T-GFP cells reached 7.10 (± 2.68) X 106 and 3.37 (± 1.54) X 107 CFUs/gm of roots in zero seawater and 20% seawater conditions, respectively (Fig. 4i). Notably, we observed that 20% seawater conditions enhanced the colonization ability of L1I39T-GFP cells by one log fold compared to zero seawater conditions, indicating its ability to interact with pokkali rice roots more efficiently in a brackish environmental condition. This view is in agreement with the above in planta growth experiments, where L1I39T-treated plants showed enhanced growth when grown under a 20% seawater condition (Fig. 3a, b & c). In contrast, no significant difference in the plant growth parameters was observed between the L1I39T-treated and untreated control plants when grown under zero seawater conditions (Fig. S5). These results potentially indicate that a brackish environment plays a vital role in L1I39T-pokkali rice symbiosis. Also, it suggests that L1I39T is well-adapted physiologically to its brackish growth habitat, which is critical for pokkali rice interactions.

Fig. 4
figure 4

i The comparative pokkali rice root colonization potential of GFP-tagged L1I39T cells between 20% seawater and zero-seawater conditions quantified after the 7th and 14th days post-inoculation is shown as a bar graph. Statistical significance is represented by *, p-value < 0.05; **, p-value < 0.01. ii Epi-fluorescent images showing the pokkali rice root colonization of GFP-tagged L1I39T cells under 20% seawater conditions, (a & d) the primary roots (7th dpi), (b & c) the root hairs of primary root (7th dpi), (e) the lateral root (7th dpi), (f & h) the root hairs of primary root (14th dpi), and (g) the primary root-lateral root junction (14th dpi). Scale bars (20 µm) are indicated in all the microscopic images

Through epifluorescence microscopy, we show that L1I39T-tagged GFP cells colonize the pokkali rice seedlings primarily on the root surfaces, including the primary roots, lateral root junctions, and root hairs (Fig. 4ii). However, the colonization pattern of L1I39T cells on the root surfaces is not uniform but occurs as patches and clusters, and in certain portions, we found cells anchoring and foraging the root hairs. Importantly, we did not observe cells colonizing inside parts of the roots under our tested seawater growth conditions. A similar finding was reported in a terrestrial halotolerant PGPR Azospirillum halopraeferens, where the colonization was observed more on the root surfaces of black mangrove seedlings under seawater growth conditions [53].

Metagenomic survey of L1I39T-specific OTUs in brackish-associated rice varieties

Our attempts to re-isolate L1I39T-like strains from the pokkali rice using different cultivation methods were unsuccessful. Therefore, we determined the L1I39T prevalence by mapping its full-length 16S rRNA gene sequence against the 16S rRNA amplicon metagenomic datasets of two native salt-tolerant rice cultivars collected from brackish environments of central and northern regions of Kerala. This analysis revealed limited operational taxonomic units (OTUs) having a read length of 400 bp sharing > 99% 16S rRNA sequence similarities with L1I39T from both rice cultivars (Table 2), suggesting its occurrence is very low abundance in those ecosystems. Importantly, we noticed that L1I39T-specific OTUs represented more in the roots when compared to the rhizosphere samples where we found, except in one rhizosphere sample, all other rhizosphere samples were negative (Table 2). Moreover, these findings provided us with essential information on L1I39T that 1) it has a preferential colonization of the roots over the rhizosphere region, thereby signifying its close relationship with the plant host, 2) it is an integral part of the root-associated indigenous microbiota of brackish-associated rice varieties and 3) it occurs with a low abundance. Studies have shown that bacterial taxa of low-abundance populations in a habitat can form the most physiologically significant symbiotic interactions [54] and, in some instances, fulfill essential environmental functions, for example, nitrogen fixation by diazotrophic bacteria in the ocean [55]. Based on the findings of in-vitro PGPR traits and in-planta nifH expression and growth experiments, we hypothesize that L1I39T might play a similar significant functional role in pokkali rice growth and health in nitrogen-poor brackish environments despite their low abundance, thus, conferring L1I39T as a rare important PGPR taxon.

Table 2 List of L1I39T OTUs recovered from 16S rRNA amplicon metagenomics datasets of brackish-associated rice varieties of central and northern coastal regions of Kerala

General genome features

The L1I39T genome comprises a single circular chromosome of 5,388,224 bp with no extra plasmids and a GC content of 66.9% (Fig. 5). Genome quality estimation determined using checkM showed that the assembled genome is of high quality with 99.65% completeness with no contamination and strain heterogeneity. The general genome features of the L1I39T and its closest phylogenetic neighbors, A. cavernae Sn-9-2 T and A. spiritensis DSM 9035 T, were summarized in Table S5.

Fig. 5
figure 5

A single circular genome representation of L1I39T. The inner circle represents the genome size, the second circle represents the GC content, and the following circles represent coding sequences of different functional gene categories, as indicated by different colors

Next, we investigated the genomic composition of L1I39T to relate its specific adaptations to plant-associated lifestyles and brackish tidal environments. For this purpose, we analyzed the SEED subsystem features available in the RAST server [32]. This analysis revealed that a high proportion of protein-coding genes were assigned to the functional category amino acids and derivatives (631), followed by carbohydrates (506). In contrast, genes listed under the protein metabolism (255) were less abundant, which points to a specialized adaptation of L1I39T to various plant-derived substrate metabolism. Notably, several protein-coding genes were categorized under the membrane transport (336), indicating the ability of L1I39T to sense and respond to various environmental stimuli, including host-derived nutrients and tolerance to different abiotic stress, including salinity. An overview of the SEED subsystem categories identified in the genomes of L1I39T, A. cavernae Sn-9-2 T, and A. spiritensis DSM 9035 T were listed in Table S6.

Functions related to plant-associated lifestyle

Motility, chemotaxis, and biofilm

The plant colonization traits such as motility, chemotaxis, and biofilm formation are essential prerequisites for potential PGPR to interact with their hosts efficiently. The L1I39T genome encodes a complete set of genes related to flagellar motility and chemotaxis organized into a single large operon of 42.15 kb size comprising 44 genes (Table S7). Importantly, L1I39T is negative for motility under our tested phenotypic conditions. This disagreement between the presence of genes encoding for flagellar motility and the non-motile phenotype indicates tight regulation of motility in L1I39T. Similar discrepancies in non-motile phenotype and genome information were observed in other plant-associated endophytic bacteria [56,57,58].

Further genome analysis identified 11 methyl-accepting chemotaxis proteins (MCP) with one MCP (WP_209103394.1) positioned within the flagellar-chemotaxis gene cluster. This MCP gene shared 68.04% amino acid identity with the transmembrane chemoreceptor tlpA1 gene (AZC_0660) of Azorhizobium caulinodans ORS 571 T. This chemoreceptor gene was identified as critical for A. caulinodans ORS 571 T host colonization, where the tlpA1 mutant strain was severely impaired with chemotaxis and competitive root colonization [59]. Likewise, the tlpA1 gene homolog in the L1I39T genome might indicate a similar role in host colonization. However, the number of MCP genes identified in the L1I39T genome is less compared to the A. caulinodans ORS 571 T genome, which contains 43 MCP genes [60]. The non-motile phenotype and a low number of MCP genes in L1I39T indicate a lifestyle confined to a specific environmental niche.

The strain LI139T is positive for biofilm formation (Fig. S6a) and a strong attachment of pokkali rice root seedlings under 20% seawater conditions was observed (Fig. S6b). Hence, we searched for putative genes and gene clusters critical for host tissue attachment and establishment. The L1I39T genome carries four genes organized as a single cluster participating in lipopolysaccharide-o-antigen biosynthesis. These genes include dTDP-4-dehydrorhamnose 3,5-epimerase (rfbC, WP_209104488.1), dTDP-glucose 4,6-dehydratase (rfbB, WP_209104489.1), dTDP-dehydrorhamonase reductase (rfbD, WP_209105319.1) and glucose-1-phosphate thymidylyltransferase (rfbA, WP_209104490.1). Also, genes coding for lipopolysaccharide (LPS) biosynthesis, including LPS heposyltransferase II (waaF, WP_209100376.1), LPS heposyltransferase I (waaC, WP_209100378.1), ADP-glyceromanno-heptose 6-epimerase (rfaD, WP_209100380.1) and D-glycero-beta-D-manno-heptose-7-phosphate kinase (rfaE1, WP_247657679.1) were present. Additionally, we identified two genes coding for prepilin peptidase (tadV, WP_209101036.1) and type IVb pilin (flp, WP_209101034.1) as part of the tight adherence (tad) locus. Also, genes for exopolysaccharide biosynthesis (exoD, WP_247657663.1), LPS assembly protein (lapA, WP_247657749.1), and putative surface layer protein (sapC, WP_247658490.1) were present. Further analysis revealed a symbiosis polysaccharide locus (syp) consisting of 18 genes spanning around 16.55 kb in size (Fig. S6c). Studies have shown that this syp locus is essential for bacteria host attachment and colonization [61]. Therefore, L1I39T might use these genes, including the syp locus, to efficiently attach and colonize the host.

Rhizosphere oxidative stress (ROS) detoxification

To establish an efficient colonization event, L1I39T must overcome the rhizosphere oxidative stress (reactive oxygen species, ROS) induced by the host plant [62]. Experimentally, we show that L1I39T can tolerate hydrogen peroxide up to 5 mM (Fig. S7). Accordingly, the L1I39T genome encodes multiple genes, including superoxide dismutase, catalase/peroxidase HPI (katG), catalase, alkyl hydroxy peroxide reductase, thioredoxin reductase, thiol peroxidase, non-heme chloroperoxidase, glutathione S-transferase, glutathione reductase (gorA), glutaredoxin, OsmC family protein, organic hydroperoxide resistance protein, and peroxiredoxin (Table S8), responsible for detoxifying the exogenous ROS resulting from plant interactions [63]. Also, genes that regulate ROS detoxification, including two copies of hydrogen peroxide-inducible activator, were identified. These results indicate the significant ability of L1I39T to detoxify exogenous ROS, which may affect several aspects of plant–microbe interactions, including plant growth and health.

Energy metabolism

The strain L1I39T is capable of aerobic respiration and encodes a complete set of genes required for oxidative phosphorylation (Table S9). These include NADH-quinone oxidoreductase (nuo genes, complex I), succinate dehydrogenase (sdh genes, complex II), and three-terminal respiratory oxidases belonging to a) cbb3-type cytochrome c oxidases (ccoNOQPGS operon), b) aa3-type cytochrome c oxidase (ctaD, coxB, coxC, and coxIV), and c) bd-type quinol oxidases; cytochrome o ubiquinol oxidase (cyoA, cyoBCD), and cytochrome d ubiquinol oxidase (bd I complex; cydABX operon and two copies of bd II complex; appC, cydB). Notably, these aerobic terminal oxidases have different oxygen affinities and are differentially regulated depending on the availability of varying oxygen concentrations in the growth habitat. For instance, cbb3-type cytochrome c oxidases and bd-type quinol oxidases have a high oxygen affinity critical for microaerobic respiration and nitrogen fixation [64, 65]. In contrast, aa3-type cytochrome c oxidases have a low oxygen affinity, predominantly expressed under aerobic growth conditions [66].

In addition to multiple terminal oxidases, the L1I39T genome encodes a putative dissimilatory nitrate reductase operon (narGHJI), class I fumarate hydratase (fumA), class II fumarate hydratase (fumC, two copies), formate dehydrogenase with four subunits, L-lactate utilization protein (lutB), LUD domain protein, L-lactate dehydrogenase (lldD) and its regulator (lldR) (Table S10). These genes may confer a metabolic growth advantage under low-oxygen conditions, with formate, lactate, and fumarate as electron donors and nitrate as an electron acceptor. Together, these findings suggest the significant genetic ability of L1I39T to adapt and thrive in water-logged brackish pokkali rice fields.

Central carbon metabolism

The L1I39T genome encodes a complete set of genes required for a functional pentose phosphate pathway and the Entner-Doudorff pathway (Table S11). However, it lacks a key regulatory enzyme of the glycolysis pathway (6-phosphofructokinase) and two rate-limiting enzymes of the gluconeogenesis pathway (phosphoglucoisomerase and glucose-6-phosphatase), indicating that glycolysis and gluconeogenesis pathways are incomplete. Although enzymes catalyzing the remaining steps of the glycolysis and gluconeogenesis pathways were present (Table S11), suggesting that carbohydrate metabolism is carried out through the Entner-Doudorff and pentose phosphate pathways, indicating that L1I39T may depend on the host for carbohydrate precursors. For oxidation of pyruvate to acetyl-coenzyme, the L1I39T genome encodes a three-component pyruvate dehydrogenase complex (two E1 component subunits, alpha and beta, and a dihydrolipoamide S-acetyltransferase). Further, we identified genes coding for enzymes catalyzing all eight essential steps of the complete citric acid cycle (Table S11). Also, the presence of citrate lyase (three copies) and a malate synthase indicate an active glyoxylate shunt pathway. Together, these genomic data suggest that L1I39T is likely to prefer dicarboxylates (metabolized via a critic acid cycle) as the source of energy for its growth and other metabolic functions rather than carbohydrates. In support, we show experimentally through phenotypic growth assays that L1I39T could grow well on dicarboxylates instead of carbohydrates (Fig. S8 & Table S12). Similar findings were reported in several well-characterized diazotrophic PGPR strains, notably of the genera Azospirillum, Azoarcus, and Herbaspirillum, which are often unable to utilize carbohydrates [67].

In addition, the L1I39T genome encodes > 600 transporter-linked genes, comprising about 15.59% of all the genes. The highest number of genes annotated to ATP binding cassette (ABC) type transporter (365 genes), which corresponds to 46% of the total transporter genes, 93 genes (12%) belong to Tripartite ATP-independent periplasmic (TRAP) transporters, including dicarboxylate and tricarboxylate, 63 genes (8%) belong to branched-chain amino acids transporter genes, and 46 genes (6%) belong to MFS transporters genes (Table S13). Moreover, the presence of a higher number of transporters than its related phylogenetic neighbors (Table S13), indicates its potential genetic ability to uptake and utilize diverse substrates. We also found genes involved in the degradation of various aromatic compounds through 3-oxoadipate and phenylacetate degradation pathways (Table S14).

Vitamins biosynthesis

Vitamins are essential growth cofactors required for several enzymatic processes in central metabolism. In addition, vitamins can also play a crucial role in maintaining the symbiotic status of two different organisms. For instance, bacterial symbionts capable of synthesizing vitamins can become key symbiotic partners with eukaryotic hosts, including legume-rhizobia symbiosis [68]. The L1I39T genome encodes all essential genes involved in the biosynthesis of multiple vitamins (Fig. S9 & Table S15), suggesting its genetic capability to synthesize necessary growth factors for growth. In support, we experimentally confirm that L1I39T can grow in a defined vitamin-free mineral salts medium without dependence on the external supply of vitamins under our tested conditions even after repeated subcultures (data not shown).

Several studies have shown that vitamin auxotrophs of diverse PGPR failed to establish an effective symbiosis with their respective plant hosts. For example, thiamine auxotrophic mutants a) PCL1079 of Pseudomonas fluorescens WCS 365 lost their ability to colonize the tomato roots [69] and b) Ao5-E01 and Ao55-D02 of A. caulinodans ORS 571 T formed defective nodules with reduced nitrogen fixation [70]. Likewise, riboflavin auxotrophic mutant of Rhizobium leguminosarum bv. trifolii strain T1 formed ineffective root nodules in clover [71], and the biotin auxotrophic mutant of Sinorhizobium meliloti strain 1021 showed poor alfalfa root colonization [72]. Based on these findings, we hypothesize that L1I39T would utilize these vitamin biosynthetic gene clusters for effective host interactions rather than supply vitamins to its host, which is unlikely.

Sulfur, phosphate, and nitrogen-assimilation

Plant-associated bacteria can assimilate sulfonates and sulfur esters as organic sulfur and sulfates as inorganic sulfur from the environments [73]. In the L1I39T genome, genes primarily involved in the metabolism of sulfonates were absent. However, a single-copy gene encoding aliphatic sulfonate ABC transporter substrate-binding protein (ssuA) was present. We also identified genes coding for reducing sulfur esters, including a sulfatase-like hydrolase and two copies of arylsulfatases. Additional analysis identified genes for the assimilation and transport of sulfates (Table S16). However, the homolog of cysB, the primary regulator of sulfate metabolism, was absent. Further, genes that participate in the assimilation and export of sulfite, including the three copies of sulfite reductase and seven copies of sulfite exporter TauE/SafE family proteins (Table S16), were identified. The putative genes that catalyze the oxidation of sulfide to sulfate were absent, but a gene coding for sulfite oxidase was present.

Phosphonate is considered one of the rich sources of phosphates in the soil. Microbes use a phosphonate-related gene system (phn) to break down phosphonates in the environments to release phosphates efficiently. Accordingly, the L1I39T genome encodes a complete gene cluster (phnNLKAFBJIHGFM genes) responsible for phosphonate degradation into phosphate (Table S17). In addition to this, we also identified two potential high-affinity phosphate transport systems crucial for phosphate mobilization, including pstBACS (transport) and phoUB (regulator) gene clusters for inorganic phosphates and phnE1E2DC for phosphonates (Table S17). These results suggest L1I39T might depend on these systems to mobilize inaccessible phosphorous during phosphate limitations, as observed in other plant-associated bacteria [74]. Further analysis identified potential enzymes, such as polyphosphate kinase and exopolyphosphatase, indicating that L1I39T likely stores energy and phosphorus in the form of polyphosphate.

In addition to examining the genome for sulfur and phosphate assimilation, we also identified several potential genes involved in the assimilation of nitrogen, including genes coding for ammonium transporters, reductases for nitrate and nitrite, regulators that sense nitrogen status, glutamate dehydrogenase, and a functional glutamine synthetase/glutamate synthase (GS/GOGAT) system as listed in (Table S18). In addition, the L1I39T genome possesses genes coding for the uptake and hydrolysis of urea, including a urease operon (ureABCEFGD) and the urea transport systems (urtEDC and urtBA) and a functional urea carboxylase/allophanate hydrolyase pathway, suggesting its ability to degrade urea into ammonia (Table S19).

Iron acquisition

The L1I39T genome lacks genes that participate in the biosynthesis and secretion of siderophores. In support, we experimentally prove that L1I39T is negative for siderophore production (data not shown). However, the L1I39T contains dedicated transport systems to capture and uptake external iron, including TonB-dependent siderophore receptor proteins, siderophore-interacting proteins, iron ABC transport substrate-binding proteins, iron ABC transport permease, iron ABC transport ATP-binding proteins, ferric hydroxamate ABC transport permease (fhuB), and siderophore-iron reductase (fhuF). Importantly, these genes are organized into different gene clusters (Table S20).

Further investigations revealed genes encoding for ferrochelatase (hemH), TonB-dependent hemoglobin/transferrin/lactoferrin family proteins, biliverdin-producing heme oxygenase, heme ABC transporter-ATP binding protein, and hemin uptake protein (hemP), responsible for acquiring host iron. Also, genes coding for iron storage proteins that include ferritin, bacterioferritin (bfr), and two copies of ferritin-like domain-containing proteins and fur-type iron response transcriptional regulator (three copies) were present. Based on these genome findings, we hypothesize that L1I39T cells may depend on siderophores produced by other root-inhabiting rhizobacteria and host heme to satisfy their need for iron, a similar feature documented in well-characterized diazotrophic PGPR strains such as Pseudomonas stutzeri A1051 [75] and Azoarcus sp. BH72 [76].

Plant growth functions

Nitrogen fixation and hydrogen metabolism

The L1I39T genome encodes all necessary genes required for nitrogen fixation (nif), organized in a 29.2 kb large nif region consisting of 32 genes distributed non-contiguously across two different loci (Fig. 6). The GC content of this nif region is similar to the average GC content of the whole genome (66.3% vs. 66.9%), suggesting that this nif region is not part of a genomic island that has been acquired recently through horizontal gene transfer, as it was observed in Pseudomonas stutzeri [75]. The nitrogenase system of L1I39T constitutes the molybdenum-iron (MoFe) type nitrogenase encoded by nifDK and an iron (Fe) protein encoded by nifH. Genes necessary for FeMo-co biosynthesis (nifB, nifQ, nifENX, and nifUSV), nitrogenase maturation (nifM, nifZ), electron transfer flavoproteins (fixABCX), and transcriptional regulation of nif operon (nifA) were present. Other essential genes likely to be involved in the regulatory cascade of nitrogen fixation are shown in Table S18. No genes coding for alternative vanadium (VFe) or iron only (FeFe)-dependent nitrogenase systems were present.

Fig. 6
figure 6

The nitrogen fixation (nif) gene cluster comparison between L1I39T and four well-characterized rice-associated diazotrophs: A. brasilense Sp7T, A. vinelandii CA, and Azoarcus sp. BH72 and P. stutzeri DSM 4166. Red open sharp triangles indicate the location of additional genes. Highlighted regions with continuous border lines indicate gene synteny oriented in the same direction, whereas highlighted regions with dotted lines indicate the gene synteny oriented in the opposite direction

A NifHDK phylogenetic analysis showed that the nitrogenase system of L1I39T is closely related to A. cavernae Sn-9-2 T (Fig. S10). However, it is distantly related to the nitrogenase system of A. spiritensis DSM 9035 T and other nitrogen-fixing members of the genera Xanthobacter and Azorhizobium (Fig. S10). Moreover, the nif gene cluster of L1I39T and A. cavernae Sn-9-2 T shared high gene synteny compared to A. spiritensis DSM 9035 T, A. caulinodans ORS 571 T, and Xanthobacter autotrophicus Py2 (Fig. S11). These results suggest that the nitrogenase system of L1I39T and A. cavernae Sn-9-2 T might have been acquired from a common ancestor but are evolutionary different from that of other members of the family Xanthobacteraceae.

We then compared the nif gene organization of L1I39T against the four other well-studied diazotrophs associated with rice plants: Azospirillum brasilense Sp7T, Azotobacter vinelandii CA, Azoarcus sp. BH72, and P. stutzeri 4166. This analysis revealed that the nif region of L1I39T shared a relatively close similarity to A. brasilense Sp7T than with other diazotrophs (Fig. 6). For instance, In A. brasilense Sp7T, the nif region that contained a set of genes (nifQ frdxN nifXNEKDH) were contiguous and arranged in the same order as observed in L1I39T, except a few additional protein-coding sequences were found in between the genes of nifQ frdxN nifXNE and nifKDH (Fig. 6). Similarly, the region encoding genes for nifUSVW and fixABCX were organized very similar to L1I39T, except this region is flipped (Fig. 6). In addition to this, an electron transport system (rnfABCDGEF operon) and NifL, a negative regulator of the nitrogenase system, were absent in both L1I39T and A. brasilense Sp7T but were present in A. vinelandii CA, Azoarcus sp. BH72, and P. stutzeri 4166. Importantly, genes encoding for DraT/DraG-mediated posttranslational regulation of nitrogenase by ADP-ribosylation were absent in L1I39T, suggesting a nitrogenase regulation mechanism different from that reported in A. brasilense Sp7T and Azoarcus sp. BH72.

During the nitrogen fixation process, hydrogen (H2) gas is released as a by-product of the nitrogenase reaction, making the nitrogen fixation process less efficient. However, the oxidation of H2 by the hydrogenase enzyme improves the efficiency of the nitrogen fixation process. Hence, to fix nitrogen more efficiently, the L1I39T genome encodes a complete set of genes required for H2 metabolism distributed along a 19.7 kb cluster comprising 19 potential genes (Fig. S12 & Table S21), arranged in the order of hupTUV, hypF, hupSLCDFGHJK, hypAB, hupR, and hypCDE. This cluster closely resembles the H2 uptake genetic system of A. caulinodans ORS 571 T, except it does not contain the hupE gene coding for nickel transporter. Notably, the H2 uptake genetic system is absent in the genomes of A. cavernae Sn-9-2 T and A. spiritensis DSM 9035 T.

ACC deaminase production

The L1I39T genome encodes a copy of the ACC deaminase (acdS) gene (WP_209104892.1) and a leucine-responsive regulatory protein (Lrp, WP_209104891.1) known to regulate the acdS gene expression were identified. The AcdS and Lrp proteins shared relatively low similarity (71.73 and 49.66%, respectively) with well-characterized ACC deaminase-producing PGPR, Paraburkholderia phytofirmans PsJNT. No transposable or mobile genetic elements nearer or to the flanking regions of the acdS gene and its regulatory protein were present, indicating that this gene is stably present in the L1I39T genome. Further, this data potentially suggests that this gene could have been acquired positively through a long-term evolution while interacting with its plant hosts in a brackish environment, where the expression of the acdS gene plays a valuable and positive plant function. In support of this view, the acdS gene was absent in the genomes of A. cavernae Sn-9-2 T and A. spiritensis DSM 9035 T (Table S13). Studies have shown that PGPR expressing ACC deaminase promotes plant growth under various stressful conditions, including salinity [77]. Similar to these findings, we show that L1I39T promotes the pokkali rice growth under a tested nitrogen-deficient seawater condition, indicating that the acds gene could be functional and can play a significant contributory role in pokkali rice growth and fitness in those stressful nitrogen-poor brackish growth environments.

Other PGP traits

The L1I39T genome also encodes genes for acetoin and 2,3-butanediol production (Table S22), well-characterized volatile compounds known to promote plant growth [78].

Plant interactions

Secretion systems

Protein secretion systems play a significant role in plant–microbe and environmental interactions. In the L1I39T genome, genes coding for different membrane proteins of the general secretion (Sec)-mediated pathway, including SecA (ATPase), SecY, SecE, SecG, SecDF-YajC (forming a putative protein complex), YidC, and SecB (export chaperone) were identified. Likewise, we also found two putative signal recognition particle (SRP)-binding proteins (FtsY and FfH). These findings suggest that LI139T may use the SRP and the Sec-mediated pathways for optimal translocation of proteins [79]. Additional analysis revealed the presence of a twin-arginine protein export (TatABC) complete system that delivers folded proteins across the cytoplasmic cell membrane, unlike the Sec-mediated pathway that translocates unfolded proteins. In addition to Sec and Tat-mediated protein translocating systems, the L1I39T genome also contains dedicated secretion systems, including the Types I (T1SS), IV (T4SS), and VI (T6SS) as well as a specific subset of effector molecules, suggesting that L1I39T can modulate host cellular functions as well as mediate host-specific interactions.

T1SS is a simple protein secretory apparatus that translocates a variety of proteins, including toxins, lipases, proteases, exopolysaccharides, and siderophores across the bacterial cell envelope into the external environment in a single step and plays a vital role in host interactions [80]. The L1I39T genome encodes all necessary genes as part of a functional T1SS (Table S23). Also, several membrane fusion proteins were found as part of the multidrug efflux resistance transport systems, suggesting a possible functional role in stress tolerance. Notably, we identified a single gene cluster (from WP_209105009.1 to WP_209105014.1) consisting of four genes, namely a type I secretion membrane fusion protein, a T1SS permease/ATPase, a putative glycosyltransferase and a putative surface layer protein (SapC), is probably involved in the secretion of exopolysaccharide necessary for the host attachment and biofilm formation.

T4SS is a versatile multiprotein translocation system involved in genetic exchange and the delivery of host-specific effector proteins or DNA–protein complexes directly into target prokaryotic or eukaryotic cells that may aid in establishing host interactions, including symbiosis or pathogenesis [81]. Most T4SSs described comprised a minimum of core 12 proteins (VirB1 to VirB11 and VirD4). The L1I39T genome encodes a complete set of VirB1 to VirB10 proteins along with VirD4 and VirD2 (Fig. S13 & Table S24), indicating that this T4SS is part of a conjugal transfer system. However, OriT and VirE were absent, suggesting that this system might not transfer DNA into the eukaryotic hosts [82]. Also, the homologs of T4SS effector proteins, VirJ, VirF, VirE1, VirE2, and VirD5, were absent. These findings suggest that T4SS might participate in host interactions rather than a conjugal transfer of DNA or any effector proteins.

Further genome investigations identified two different gene clusters comprising a minimum of 13 core subunits required to form a functional type 6 secretory apparatus (Fig. S14a & Table S25). Studies have shown that T6SS plays a central role in interbacterial interactions, where Gram-negative bacterial cells use this delivery system to kill other competing bacteria to gain fitness in their growth habitat, and this inhibition of cells requires cell–cell contact [83]. We checked whether this was true for L1I39T, and an in vitro contact-dependent growth inhibition assay was performed. This experiment showed a negative result for L1I39T (Fig. S14b), indicating that this system may not be involved in interbacterial competition but can mediate host-specific interactions. A similar finding was reported in A. caulinodans ORS 571 T, where T6SS is not engaged in interbacterial competition but is identified as important for its symbiotic interactions with the host [84].

In addition to the secretion systems, the L1I39T genome carries two copies of N-acyl homoserine lactonase (WP_209105105.1 and WP_209102178.1), a metalloenzyme that degrades bacterial quorum sensing (QS) signals. The genes involved in the N-acyl homoserine lactone (AHL)-mediated QS signaling, LuxI/LuxS-type autoinducer synthetase, and LuxR-type autoinducer-response regulator were absent. Therefore, the presence of AHL lactonase indicates that L1I39T can inactivate the AHL-based QS signaling molecules produced by other competing rhizobacteria, thereby disrupting their coordinated cellular functions, including biofilm formation, and thus can have a competitive advantage during the host root colonization. A similar finding has been reported in AHL lactonase-producing Azospirillum brasilense Az39, where degradation of AHL signaling compounds improves the root colonization competitiveness of Az39 [85]. Notably, this enzyme was absent in the genome of A. cavernae Sn-9-2 T.

Further, we identify several potential eukaryotic-like interactive proteins encoded in the L1I39T genome (Table S26), which may participate in the host interactions, including the plant defense system evasion [86].

Brackish adaptation

Ecophysiological data suggests optimal cell growth, root colonization, and plant growth promotion of L1I39T are greatly enhanced by the 20% seawater condition, implying that L1I39T might rely on sodium ions for energy generation to perform cellular functions, as observed in many marine bacteria [87]. However, our genome analysis indicated that the L1I39T genome has an incomplete sodium-dependent energy cycle. Since the genes encoding for Na+ translocating NADH: ubiquinone oxidoreductase (Na+NQR) and Na+ transporting ATPase were absent. Nevertheless, the L1I39T genome encodes several sodium transporters (Table S27), including a Na+ dependent transporter, multiple Na+/H+ antiporter subunits, Na+/Pi cotransporter family, Na+/bile acid symporter, Na+/alanine symporter, Na+/glutamate symporter, Na+/Pi symporter, Na+/dicarboxylate symporter and Na+ translocating pyrophosphatase. It is well known that Na+ pumps, especially Na+/H+ antiporters, play an essential role in sodium homeostasis [88]. Therefore, L1I39T might depend on these Na+ pumps to export excess toxic Na+ and import H+ when the external sodium levels are too high, thereby maintaining cellular osmotic homeostasis and tolerating hyperosmotic stress. Notably, fewer genes coding for sodium transporters were identified in the genomes of A. cavernae Sn-9-2 T and A. spiritensis DSM 9035 T compared to the L1I39T genome (Table S13).

Bacterial cells also accumulate more intracellular cations of K+ as an immediate transient response to high salinity stress [89]. The presence of genes encoding for three putative potassium uptake systems, including TrK, Kdp, and Kup (Table S27), could indicate the potential genetic capability of L1I39T to use these systems for hyperosmotic adaptation. This view agrees with the earlier observations that Sinorhizobium meliloti carrying single or double mutations in TrK and Kup systems lost their ability to grow at high salinity [90]. Additional analysis identified genes coding for magnesium transporters, two copies of mgtE, and a CorA responsible for maintaining ionic homeostasis under salt stress [91].

In addition to the Na+ extrusion and inorganic solute uptake mechanisms, bacteria can synthesize and accumulate diverse organic compatible solutes, which improves their ability to cope with high salinity stress [92, 93]. The L1I39T genome encodes genes for trehalose biosynthesis, including maltose alpha-D-glucosyltransferase (treS), trehalose-6-phosphate synthase (otsA), trehalose-phosphatase (otsB), malto-oligosyltrehalose synthase (treY), and malto-oligosyltrehalose trehalohydrolase (treZ). In addition, genes coding for glutamate synthesis, including glutamate dehydrogenase (gdh), glutamine synthetase (glnA), and glutamate synthase alpha and beta subunits (gtlBD), were present. Further analysis identified genes coding for enzymes that convert glutamate to proline, an important osmoprotectant. These enzymes include glutamate-5-semialdehyde dehydrogenase (ProA), glutamate-5-kinase (ProB), and pyrroline-5-carboxylate reductase (ProC). Also, one trifunctional transcriptional regulator/proline dehydrogenase/ pyrroline-5-carboxylate dehydrogenase (putA) was present. Putative genes coding for synthesizing betaine, choline, ecotine, hydroxyectoine, and dipeptide N-acetylglutaminylglutamine amide (NAGGN) were absent. However, a betaine/carnitine/choline (BCCT) transporter and N-acetylglutaminylglutamine amidotransferase (asnO) was present. These genome findings suggest that L1I39T may depend on these genes (Table S27) for survival and growth in brackish environments.


We isolated and functionally characterized a novel Aquabacter strain L1I39T from the rhizosphere region of a native salt-tolerant pokkali rice cultivated in water-logged brackish tidal environments of central Kerala, India. The strain L1I39T possesses multiple PGP properties, including biological nitrogen fixation and ACC deaminase production, and has the ability to promote pokkali rice growth in nitrogen-limiting seawater conditions. More importantly, optimal cell growth, rice root colonization, and plant growth promotion of L1I39T are greatly influenced by the presence of a 20% seawater condition compared to a zero-seawater condition, suggesting that a brackish environmental condition is essential for L1I39T-pokkali rice symbiosis. In addition, the ability to tolerate 3% NaCl, positive growth in ZoBell marine agar, and optimal growth at 20% seawater concentration indicates that L1I39T is physiologically well-adapted to its brackish growth habitat. Detailed genome characterization of L1I39T revealed an array of genetic traits as specific adaptations to plant-associated lifestyles and brackish environments (Fig. 7). The 16S rRNA amplicon sequence analysis identified L1I39T as part of the root-associated indigenous microbiota of brackish-associated native rice varieties but with a low abundance. Overall, the findings of this study show that L1I39T is an important rare PGPR taxon that would play a significant functional role in pokkali rice growth and health in nitrogen-limiting brackish environments, which needs further investigation. Finally, we establish L1I39T as a novel Aquabacter species by following a polyphasic taxonomy approach and named Aquabacter pokkalii sp.nov., (LMG 28413 T = MCC 4526 T).

Fig. 7
figure 7

Schematic Figure representing the genetic determinants of L1I39T critical for plant-associated lifestyle, plant growth functions, and brackish environmental adaptations

Description of Aquabacter pokkalii sp. nov

Aquabacter pokkalii (pok.ka'li.i. N.L. gen. n. pokkalii of pokkali (a variety of rice))

Cells are Gram-negative, non-motile, aerobic, and rod-shaped. Colonies are cream in color, smooth, and circular after 4 days on R2A-N agar medium at 30 °C. The strain is able to tolerate up to 3% NaCl and 37 °C. Able to grow in ZoBell marine agar and 100% seawater. Negative for hydrolysis of starch, pectin, CMC, and casein. Positive utilization for alanine, proline, glutamine, and methionine. Weak for tryptophan and putrescine. Negative for lysine, histidine, and arginine. Based on API 20NE strips, positive results were obtained for urease, hydrolysis of esculin, β-galactosidase, and assimilation for glucose, arabinose mannose, mannitol, maltose, potassium gluconate, malate, and trisodium citrate utilization; negative results were obtained for reduction of nitrates and nitrites, indole production, fermentation of glucose, arginine dihydrolase, hydrolysis of gelatin, N-acetyl-glucosamine, capric acid, adipic acid, phenylacetic acid and oxidase. According to Biolog GEN III MicroPlate, positive results were obtained for assimilation for L-galactonic acid lactone, bromo-succinic acid, β-hydroxy-D,L-butyric acid, L-glutamic acid, L-pyroglutamic acid, D-galacturonic acid, methylpyruvate, L-lactic acid, citric acid, D-malic acid, propionic acid, L-malic acid, sodium lactate and acetic acid, tetrazolium violet, sodium butyrate, and tetrazolium blue and growth at 1% NaCl, and pH 6, whereas negative results were obtained for assimilation for D-fucose, L-fucose, D-raffinose, α-D-glucose, α-D-lactose, D-mannose, D-maltose, D-melibiose, D-fructose, D-trehalose, D-galactose, D-cellobiose, gentiobiose, sucrose, D-turanose, L-rhamnose, stachyose, D-sorbitol, D-mannitol, D-arabitol, L-alanine, L-aspartic acid, L-arginine, D-aspartic acid, L-histidine, D-serine, L-serine, gelatin, glycyl-L-proline, glycerol, glucuronamide, α-ketoglutaric acid, pectin, tween 40, dextrin, D-lactic acid methylester, β-methyl-D-glucoside, myo-inositol, D-salicin, 3-methylglucose, N-acetyl-D glucosamine, D-glucose-6-PO4, N-acetyl-b-D-mannosamine, D-fructose-6-PO4, N-acetyl-D-galactosamine, inosine, D-saccharic acid, γ-aminobutyric acid, α-ketobutyric acid, α-hydroxybutyric acid, p-hydroxyphenylacetic acid, formic acid, D-gluconic acid, D-glucuronic acid, acetoacetic acid, mucic acid, quinic acid, N-acetyl neuraminic acid, guanidine HCl, lithium chloride, potassium tellurite, D-serine, and sodium bromate and growth at pH 5, 4% and 8% NaCl. Susceptible to antibiotics such as lincomycin, vancomycin, aztreonam, fusidic acid, and rifamycin SV. Resistant to troleandomycin, nalidixic acid, and minocycline. The major fatty acid is summed feature 8 (C18:1ω7c/C18:1ω6c), and the complete cellular fatty acid profile is given in Table S4. Other chemotaxonomy properties, including polar lipids and respiratory quinone, are given in the main text.

L1I39T is positive for ACC deaminase production, in planta nifH gene expression and can promote pokkali rice growth by supplying fixed nitrogen under a nitrogen-deficient seawater condition. Its genome possesses multiple gene systems contributing to its plant growth promotion ability, host association, and brackish adaptations. The 16S rRNA metagenomic analysis identified L1I39T as an integral part of the root-associated native microbiota of brackish-associated rice varieties but occurs with a lower abundance, conferring L1I39T as a rare PGPR taxa.

The type strain L1I39T (= LMG 28413 T = MCC 4526 T) was isolated from the rhizosphere region of a salt-tolerant pokkali rice grown in Ernakulam, Kerala, India. The DNA G + C content is 66.9 mol% (derived from genome).

Availability of data and materials

The 16S rRNA gene sequence of L1I39T was deposited in NCBI under accession number KX533957. The draft genome sequence of L1I39T was deposited in NCBI with accession number CP072392.


  1. Chepsergon J, Moleleki LN. Rhizosphere bacterial interactions and impact on plant health. Curr Opin Microbiol. 2023;73:102297.

    Article  CAS  PubMed  Google Scholar 

  2. Kuan KB, Othman R, Abdul Rahim K, Shamsuddin ZH. Plant Growth-Promoting Rhizobacteria Inoculation to Enhance Vegetative Growth, Nitrogen Fixation and Nitrogen Remobilisation of Maize under Greenhouse Conditions. PLoS ONE. 2016;11:e0152478.

    Article  PubMed  PubMed Central  Google Scholar 

  3. Lelapalli S, Baskar S, Jacob SM, Paranthaman S. Characterization of phosphate solubilizing plant growth promoting rhizobacterium Lysinibacillus pakistanensis strain PCPSMR15 isolated from Oryza sativa. Curr Res Microb Sci. 2021;2:100080.

    CAS  PubMed  PubMed Central  Google Scholar 

  4. Egamberdieva D, Wirth SJ, Alqarawi AA, Abd Allah EF, Hashem A. Phytohormones and Beneficial Microbes: Essential Components for Plants to Balance Stress and Fitness. Front Microbiol. 2017;8:2104.

    Article  PubMed  PubMed Central  Google Scholar 

  5. Singh D, Kumar YD. Potential of Bacillus amyloliquefaciens for Biocontrol of Bacterial Wilt of Tomato Incited by Ralstonia solanacearum. J Plant Pathol Microbiol. 2016;07(327):1–6.

    CAS  Google Scholar 

  6. Gupta A, Rai S, Bano A, Sharma S, Kumar M, Binsuwaidan R, et al. ACC Deaminase Produced by PGPR Mitigates the Adverse Effect of Osmotic and Salinity Stresses in Pisum sativum through Modulating the Antioxidants Activities. Plants. 2022;11:3419.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  7. Jose AE, Charitha N, Karde R, Bayskar A, Reddy YA. Pokkali Rice Cultivation: A Review on the Indigenous Rice Cultivation Method in Kerala. Int J Environ Climate Change. 2023;13:1090–5.

    Article  Google Scholar 

  8. Sampangi-Ramaiah MH, Jagadheesh, Dey P, Jambagi S, Vasantha Kumari MM, Oelmüller R, et al. An endophyte from salt-adapted Pokkali rice confers salt-tolerance to a salt-sensitive rice variety and targets a unique pattern of genes in its new host. Sci Rep. 2020;10:3237.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  9. Reshma TS, Dileep C. Interactive effects on ACC deaminase activity in salt-tolerant plant growth-promoting rhizobacteria and impacts on rice crop improvement. J Agron Crop Sci. 2024;210(2):e12688.

    Article  CAS  Google Scholar 

  10. Rameshkumar N, Lang E, Tanaka N. Description of Vogesella oryzae sp. nov., isolated from the rhizosphere of saline tolerant pokkali rice. Syst Appl Microbiol. 2016;39:20–4.

    Article  CAS  PubMed  Google Scholar 

  11. Krishnan R, Menon RR, Tanaka N, Busse H-J, Krishnamurthi S, Rameshkumar N. Arthrobacter pokkalii sp nov, a Novel Plant Associated Actinobacterium with Plant Beneficial Properties, Isolated from Saline Tolerant Pokkali Rice, Kerala. India PLoS One. 2016;11:e0150322.

    Article  PubMed  Google Scholar 

  12. Krishnan R, Menon RR, Likhitha, Busse H-J, Tanaka N, Krishnamurthi S, et al. Novosphingobium pokkalii sp nov, a novel rhizosphere-associated bacterium with plant beneficial properties isolated from saline-tolerant pokkali rice. Res Microbiol. 2017;168:113–21.

    Article  CAS  PubMed  Google Scholar 

  13. Krishnan R, Lang E, Midha S, Patil PB, Rameshkumar N. Isolation and characterization of a novel 1-aminocyclopropane-1-carboxylate (ACC) deaminase producing plant growth promoting marine Gammaproteobacteria from crops grown in brackish environments. Proposal for Pokkaliibacter plantistimulans gen. nov., sp. nov., Balneatrichaceae fam. nov. in the order Oceanospirillales and an emended description of the genus Balneatrix. Syst Appl Microbiol. 2018;41:570–80.

    Article  CAS  PubMed  Google Scholar 

  14. Menon RR, Kumari S, Kumar P, Verma A, Krishnamurthi S, Rameshkumar N. Sphingomonas pokkalii sp. nov., a novel plant associated rhizobacterium isolated from a saline tolerant pokkali rice and its draft genome analysis. Syst Appl Microbiol. 2019;42:334–42.

    Article  CAS  PubMed  Google Scholar 

  15. Menon RR, Kumari S, Viver T, Rameshkumar N. Flavobacterium pokkalii sp. nov., a novel plant growth promoting native rhizobacteria isolated from pokkali rice grown in coastal saline affected agricultural regions of southern India, Kerala. Microbiol Res. 2020;240:126533.

    Article  CAS  PubMed  Google Scholar 

  16. Lakshmi KVNS, Sasikala Ch, Ramana ChV. Rhodoplanes pokkaliisoli sp. nov., a phototrophic alphaproteobacterium isolated from a waterlogged brackish paddy soil. Int J Syst Evol Microbiol. 2009;59:2153–7.

    Article  CAS  PubMed  Google Scholar 

  17. Yoon S-H, Ha S-M, Kwon S, Lim J, Kim Y, Seo H, et al. Introducing EzBioCloud: a taxonomically united database of 16S rRNA gene sequences and whole-genome assemblies. Int J Syst Evol Microbiol. 2017;67:1613–7.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  18. Irgens RL, Kersters K, Segers P, Gillis M, Staley JT. Aquabacter spiritensis, gen. nov., sp. nov. an aerobic, gas-vacuolate aquatic bacterium. Arch Microbiol. 1991;155:137–42.

    Article  Google Scholar 

  19. Duo J-L, Cha Q-Y, Zhou X-K, Zhang T-K, Qin S-C, Yang P-X, et al. Aquabacter cavernae sp. nov., a bacterium isolated from cave soil. Int J Syst Evol Microbiol. 2019;69:3716–22.

    Article  CAS  PubMed  Google Scholar 

  20. Patel RK, Jain M. NGS QC Toolkit: A Toolkit for Quality Control of Next Generation Sequencing Data. PLoS ONE. 2012;7:e30619.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  21. Bankevich A, Nurk S, Antipov D, Gurevich AA, Dvorkin M, Kulikov AS, et al. SPAdes: A New Genome Assembly Algorithm and Its Applications to Single-Cell Sequencing. J Comput Biol. 2012;19:455–77.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  22. Parks DH, Imelfort M, Skennerton CT, Hugenholtz P, Tyson GW. CheckM: assessing the quality of microbial genomes recovered from isolates, single cells, and metagenomes. Genome Res. 2015;25:1043–55.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  23. Katoh K. MAFFT: a novel method for rapid multiple sequence alignment based on fast Fourier transform. Nucleic Acids Res. 2002;30:3059–66.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  24. Zhang D, Gao F, Jakovlić I, Zou H, Zhang J, Li WX, et al. PhyloSuite: An integrated and scalable desktop platform for streamlined molecular sequence data management and evolutionary phylogenetics studies. Mol Ecol Resour. 2020;20:348–55.

    Article  PubMed  Google Scholar 

  25. Tamura K, Stecher G, Peterson D, Filipski A, Kumar S. MEGA6: Molecular Evolutionary Genetics Analysis Version 6.0. Mol Biol Evol. 2013;30:2725–9.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  26. Emms DM, Kelly S. OrthoFinder: phylogenetic orthology inference for comparative genomics. Genome Biol. 2019;20:238.

    Article  PubMed  PubMed Central  Google Scholar 

  27. Richter M, Rosselló-Móra R, Oliver Glöckner F, Peplies J. JSpeciesWS: a web server for prokaryotic species circumscription based on pairwise genome comparison. Bioinformatics. 2016;32:929–31.

    Article  CAS  PubMed  Google Scholar 

  28. Rodriguez-R LM, Konstantinidis KT. The enveomics collection: a toolbox for specialized analyses of microbial genomes and metagenomes. 2016.

    Article  Google Scholar 

  29. Auch AF, Klenk H-P, Göker M. Standard operating procedure for calculating genome-to-genome distances based on high-scoring segment pairs. Stand Genomic Sci. 2010;2:142–8.

    Article  PubMed  PubMed Central  Google Scholar 

  30. Eder K. Gas chromatographic analysis of fatty acid methyl esters. J Chromatogr B Biomed Sci Appl. 1995;671:113–31.

    Article  CAS  Google Scholar 

  31. Tindall BJ. Lipid composition of Halobacterium lacusprofundi. FEMS Microbiol Lett. 1990;66:199–202.

    Article  CAS  Google Scholar 

  32. Aziz RK, Bartels D, Best AA, DeJongh M, Disz T, Edwards RA, et al. The RAST Server: Rapid Annotations using Subsystems Technology. BMC Genomics. 2008;9:75.

    Article  PubMed  PubMed Central  Google Scholar 

  33. Lagesen K, Hallin P, Rødland EA, Stærfeldt H-H, Rognes T, Ussery DW. RNAmmer: consistent and rapid annotation of ribosomal RNA genes. Nucleic Acids Res. 2007;35:3100–8.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  34. Laslett D. ARAGORN, a program to detect tRNA genes and tmRNA genes in nucleotide sequences. Nucleic Acids Res. 2004;32:11–6.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  35. Guy L, Roat Kultima J, Andersson SGE. genoPlotR: comparative gene and genome visualization in R. Bioinformatics. 2010;26:2334–5.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  36. Alikhan N-F, Petty NK, Ben Zakour NL, Beatson SA. BLAST Ring Image Generator (BRIG): simple prokaryote genome comparisons. BMC Genomics. 2011;12:402.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  37. Cantalapiedra CP, Hernández-Plaza A, Letunic I, Bork P, Huerta-Cepas J. eggNOG-mapper v2: Functional Annotation, Orthology Assignments, and Domain Prediction at the Metagenomic Scale. Mol Biol Evol. 2021;38:5825–9.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  38. Poly F, Ranjard L, Nazaret S, Gourbière F, Monrozier LJ. Comparison of nifH Gene Pools in Soils and Soil Microenvironments with Contrasting Properties. Appl Environ Microbiol. 2001;67:2255–62.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  39. Hachani A, Lossi NS, Filloux A. A Visual Assay to Monitor T6SS-mediated Bacterial Competition. J Vis Exp. 2013.

    Article  PubMed  PubMed Central  Google Scholar 

  40. Glaeser SP, Imani J, Alabid I, Guo H, Kumar N, Kämpfer P, et al. Non-pathogenic Rhizobium radiobacter F4 deploys plant beneficial activity independent of its host Piriformospora indica. ISME J. 2016;10:871–84.

    Article  PubMed  Google Scholar 

  41. Kámán-Tóth E, Pogány M, Dankó T, Szatmári Á, Bozsó Z. A simplified and efficient Agrobacterium tumefaciens electroporation method. 3 Biotech. 2018;8:148.

    Article  PubMed  PubMed Central  Google Scholar 

  42. Barco RA, Garrity GM, Scott JJ, Amend JP, Nealson KH, Emerson D. A genus definition for Bacteria and Archaea based on a Standard Genome Relatedness Index. mBio. 2020;11.

  43. Glaeser SP, Kämpfer P. Multilocus sequence analysis (MLSA) in prokaryotic taxonomy. Syst Appl Microbiol. 2015;38:237–45.

    Article  CAS  PubMed  Google Scholar 

  44. Qin Q-L, Xie B-B, Zhang X-Y, Chen X-L, Zhou B-C, Zhou J, et al. A Proposed Genus Boundary for the Prokaryotes Based on Genomic Insights. J Bacteriol. 2014;196:2210–5.

    Article  PubMed  PubMed Central  Google Scholar 

  45. Park M-J, Kim YJ, Park M, Yu J, Namirimu T, Roh Y-R, et al. Establishment of genome based criteria for classification of the family Desulfovibrionaceae and proposal of two Novel Genera, Alkalidesulfovibrio gen. nov. and Salidesulfovibrio gen. nov. Front Microbiol. 2022;13:738205.

  46. Tikhonova EN, Grouzdev DS, Kravchenko IK. Xanthobacter oligotrophicus sp.nov., isolated from paper mill sewage. Int J Syst Evol Microbiol. 2021;71(8).

  47. Oren A. The Family Xanthobacteraceae. In: The Prokaryotes. Berlin, Heidelberg: Springer Berlin Heidelberg; 2014. p. 709–26.

    Chapter  Google Scholar 

  48. Rabouille S, Staal M, Stal LJ, Soetaert K. Modeling the Dynamic Regulation of Nitrogen Fixation in the Cyanobacterium Trichodesmium sp. Appl Environ Microbiol. 2006;72:3217–27.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  49. Belova AA, Kaparullina EN, Agafonova N.V, Grouzdev DS, Kopitsyn DS, Machulin A.V, et al. Ancylobacter crimeensis sp. nov., a New Species of Aerobic Methylotrophic Bacteria Isolated from Oak Phyllosphere. Microbiology (N Y). 2023;92:598–608.

    CAS  Google Scholar 

  50. Lee K-B, De Backer P, Aono T, Liu C-T, Suzuki S, Suzuki T, et al. The genome of the versatile nitrogen fixer Azorhizobium caulinodans ORS571. BMC Genomics. 2008;9:271.

    Article  PubMed  PubMed Central  Google Scholar 

  51. Liu C, Sakimoto KK, Colón BC, Silver PA, Nocera DG. Ambient nitrogen reduction cycle using a hybrid inorganic–biological system. Proc Natl Acad Sci. 2017;114:6450–5.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  52. Manuel Sánchez-Yañez J. Xanthobacter autotrophicus an endophytic beneficial bacterium for wheat and other plants: a short review. In: Current Trends in Wheat Research. IntechOpen; 2022.

  53. Puente ME, Holguin G, Glick BR, Bashan Y. Root-surface colonization of black mangrove seedlings by Azospirillum halopraeferens and Azospirillum brasilense in seawater. FEMS Microbiol Ecol. 1999;29:283–92.

    Article  CAS  Google Scholar 

  54. D Ainsworth T, Krause L, Bridge T, Torda G, Raina J-B, Zakrzewski M, et al. The coral core microbiome identifies rare bacterial taxa as ubiquitous endosymbionts. ISME J. 2015;9:2261–74.

    Article  PubMed  PubMed Central  Google Scholar 

  55. Shiozaki T, Fujiwara A, Ijichi M, Harada N, Nishino S, Nishi S, et al. Diazotroph community structure and the role of nitrogen fixation in the nitrogen cycle in the Chukchi Sea (western Arctic Ocean). Limnol Oceanogr. 2018;63:2191–205.

    Article  CAS  Google Scholar 

  56. Xie C-H, Yokota A. Pleomorphomonas oryzae gen. nov., sp. nov., a nitrogen-fixing bacterium isolated from paddy soil of Oryza sativa. Int J Syst Evol Microbiol. 2005;55:1233–7.

    Article  CAS  PubMed  Google Scholar 

  57. Im W-T, Kim S-H, Kim MK, Ten LN, Lee S-T. Pleomorphomonas koreensis sp. nov., a nitrogen-fixing species in the order Rhizobiales. Int J Syst Evol Microbiol. 2006;56:1663–6.

    Article  CAS  PubMed  Google Scholar 

  58. Carlier A, Cnockaert M, Fehr L, Vandamme P, Eberl L. Draft genome and description of Orrella dioscoreae gen. nov. sp. nov., a new species of Alcaligenaceae isolated from leaf acumens of Dioscorea sansibarensis. Syst Appl Microbiol. 2017;40:11–21.

    Article  CAS  PubMed  Google Scholar 

  59. Liu W, Yang J, Sun Y, Liu X, Li Y, Zhang Z, et al. Azorhizobium caulinodans Transmembrane Chemoreceptor TlpA1 Involved in Host Colonization and Nodulation on Roots and Stems. Front Microbiol. 2017;8:1327.

    Article  PubMed  PubMed Central  Google Scholar 

  60. Nan Jiang WLYLZX. Comparative genomic and protein sequence analyses of the chemotaxis system of Azorhizobium caulinodans. Acta Microbiol Sin. 2016;56:1256–65.

    Google Scholar 

  61. Shibata S, Yip ES, Quirke KP, Ondrey JM, Visick KL. Roles of the Structural Symbiosis Polysaccharide ( syp ) Genes in Host Colonization, Biofilm Formation, and Polysaccharide Biosynthesis in Vibrio fischeri. J Bacteriol. 2012;194:6736–47.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  62. Ali M, Cheng Z, Ahmad H, Hayat S. Reactive oxygen species (ROS) as defenses against a broad range of plant fungal infections and case study on ROS employed by crops against Verticillium dahliae wilts. J Plant Interact. 2018;13:353–63.

    Article  CAS  Google Scholar 

  63. Reniere ML. Reduce, Induce, Thrive: Bacterial Redox Sensing during Pathogenesis. J Bacteriol. 2018;200(17):e00128–18.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  64. Preisig O, Zufferey R, Thöny-Meyer L, Appleby CA, Hennecke H. A high-affinity cbb3-type cytochrome oxidase terminates the symbiosis-specific respiratory chain of Bradyrhizobium japonicum. J Bacteriol. 1996;178:1532–8.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  65. Gier J-WL, Schepper M, Reijnders WNM, Dyck SJ, Slotboom DJ, Warne A, et al. Structural and functional analysis of aa 3 -type and cbb 3 -type cytochrome c oxidases of Paracoccus denitrificans reveals significant differences in proton-pump design. Mol Microbiol. 1996;20:1247–60.

    Article  PubMed  Google Scholar 

  66. Mahinthichaichan P, Gennis RB, Tajkhorshid E. Cytochrome aa 3 Oxygen Reductase Utilizes the Tunnel Observed in the Crystal Structures To Deliver O 2 for Catalysis. Biochemistry. 2018;57:2150–61.

    Article  CAS  PubMed  Google Scholar 

  67. Reinhold-Hurek B, Hurek T. The Genera Azoarcus, Azovibrio, Azospira and Azonexus. In: The Prokaryotes. New York, NY: Springer New York; 2006. p. 873–91.

    Chapter  Google Scholar 

  68. Randhawa GS, Hassani R. Role of rhizobial biosynthetic pathways of amino acids, nucleotide bases and vitamins in symbiosis. Indian J Exp Biol. 2002;40(7):755–64.

    CAS  PubMed  Google Scholar 

  69. Simons M. Gnotobiotic System for Studying Rhizosphere Colonization by Plant Growth-Promoting Pseudomonas Bacteria Gnotobiotic System for Studying Rhizosphere. Mol Plant Microbe Interact. 1996;9:600.

    Article  CAS  PubMed  Google Scholar 

  70. Tsukada S, Aono T, Akiba N, Lee K-B, Liu C-T, Toyazaki H, et al. Comparative Genome-Wide Transcriptional Profiling of Azorhizobium caulinodans ORS571 Grown under Free-Living and Symbiotic Conditions. Appl Environ Microbiol. 2009;75:5037–46.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  71. Pankhurst CE, Schwinghamer EA, Thorne SW, Bergersen FJ. The Flavin Content of Clovers Relative to Symbiosis with a Riboflavin-requiring Mutant of Rhizobium trifoli. Plant Physiol. 1974;53:198–205.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  72. Entcheva P, Phillips DA, Streit WR. Functional Analysis of Sinorhizobium meliloti Genes Involved in Biotin Synthesis and Transport. Appl Environ Microbiol. 2002;68:2843–8.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  73. Kertesz MA. Riding the sulfur cycle – metabolism of sulfonates and sulfate esters in Gram-negative bacteria. FEMS Microbiol Rev. 2000;24:135–75.

    CAS  PubMed  Google Scholar 

  74. Kafle A, Cope K, Raths R, Krishna Yakha J, Subramanian S, Bücking H, et al. Harnessing Soil Microbes to Improve Plant Phosphate Efficiency in Cropping Systems. Agronomy. 2019;9:127.

    Article  CAS  Google Scholar 

  75. Yan Y, Yang J, Dou Y, Chen M, Ping S, Peng J, et al. Nitrogen fixation island and rhizosphere competence traits in the genome of root-associated Pseudomonas stutzeri A1501. Proc Natl Acad Sci. 2008;105:7564–9.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  76. Krause A, Ramakumar A, Bartels D, Battistoni F, Bekel T, Boch J, et al. Complete genome of the mutualistic, N2-fixing grass endophyte Azoarcus sp. strain BH72. Nat Biotechnol. 2006;24:1384–90.

    Article  Google Scholar 

  77. Gupta S, Pandey S. ACC Deaminase Producing Bacteria With Multifarious Plant Growth Promoting Traits Alleviates Salinity Stress in French Bean (Phaseolus vulgaris) Plants. Front Microbiol. 2019;10:1506.

    Article  PubMed  PubMed Central  Google Scholar 

  78. Ryu C-M, Farag MA, Hu C-H, Reddy MS, Wei H-X, Paré PW, et al. Bacterial volatiles promote growth in Arabidopsis. Proc Natl Acad Sci. 2003;100:4927–32.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  79. Crane JM, Randall LL. The Sec System: Protein Export in Escherichia coli. EcoSal Plus. 2017;7(2):10.

    Article  PubMed Central  Google Scholar 

  80. Bui D-C, Luo T, McBride JW. Type 1 secretion system and effectors in Rickettsiales. Front Cell Infect Microbiol. 2023;13:1175688.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  81. Wangthaisong P, Piromyou P, Songwattana P, Wongdee J, Teamtaisong K, Tittabutr P, et al. The Type IV Secretion System (T4SS) Mediates Symbiosis between Bradyrhizobium sp. SUTN9–2 and Legumes. Appl Environ Microbiol. 2023;89(6):e0004023.

    Article  PubMed  Google Scholar 

  82. Zatyka M, Thomas CM. Control of genes for conjugative transfer of plasmids and other mobile elements. FEMS Microbiol Rev. 1998;21:291–319.

    Article  CAS  PubMed  Google Scholar 

  83. Russell AB, Wexler AG, Harding BN, Whitney JC, Bohn AJ, Goo YA, et al. A Type VI Secretion-Related Pathway in Bacteroidetes Mediates Interbacterial Antagonism. Cell Host Microbe. 2014;16:227–36.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  84. Lin H-H, Huang H-M, Yu M, Lai E-M, Chien H-L, Liu C-T. Functional Exploration of the Bacterial Type VI Secretion System in Mutualism: Azorhizobium caulinodans ORS571– Sesbania rostrata as a Research Model. Mol Plant Microbe Interact. 2018;31:856–67.

    Article  PubMed  Google Scholar 

  85. Gualpa J, Lopez G, Nievas S, Coniglio A, Halliday N, Cámara M, et al. Azospirillum brasilense Az39, a model rhizobacterium with AHL quorum-quenching capacity. J Appl Microbiol. 2019;126:1850–60.

    Article  CAS  PubMed  Google Scholar 

  86. Nishad R, Ahmed T, Rahman VJ, Kareem A. Modulation of Plant Defense System in Response to Microbial Interactions. Front Microbiol. 2020;11:1298.

    Article  PubMed  PubMed Central  Google Scholar 

  87. Kogure K. Bioenergetics of marine bacteria. Curr Opin Biotechnol. 1998;9:278–82.

    Article  CAS  PubMed  Google Scholar 

  88. Patiño-Ruiz M, Ganea C, Călinescu O. Prokaryotic Na+/H+ Exchangers—Transport Mechanism and Essential Residues. Int J Mol Sci. 2022;23:9156.

    Article  PubMed  PubMed Central  Google Scholar 

  89. Assaha DVM, Ueda A, Saneoka H, Al-Yahyai R, Yaish MW. The Role of Na+ and K+ Transporters in Salt Stress Adaptation in Glycophytes. Front Physiol. 2017;8:509.

    Article  PubMed  PubMed Central  Google Scholar 

  90. Domínguez-Ferreras A, Muñoz S, Olivares J, Soto MJ, Sanjuán J. Role of Potassium Uptake Systems in Sinorhizobium meliloti Osmoadaptation and Symbiotic Performance. J Bacteriol. 2009;191:2133–43.

    Article  PubMed  PubMed Central  Google Scholar 

  91. Payandeh J, Pfoh R, Pai EF. The structure and regulation of magnesium selective ion channels. Biochim Biophys Acta Biomembr. 2013;1828:2778–92.

    Article  CAS  Google Scholar 

  92. Gregory GJ, Boyd EF. Stressed out: Bacterial response to high salinity using compatible solute biosynthesis and uptake systems, lessons from Vibrionaceae. Comput Struct Biotechnol J. 2021;19:1014–27.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  93. Roberts MF. Organic compatible solutes of halotolerant and halophilic microorganisms. Saline Syst. 2005;1:5.

    Article  PubMed  PubMed Central  Google Scholar 

Download references


The authors, SK, RRM, GGS, and RK, acknowledge the Council of Scientific and Industrial Research (CSIR) for providing a research fellowship. We acknowledge Dr. Jafargholi Imani, Justus-Liebig University Giessen, Germany, for kindly providing the pLH6000 plasmid. We thank our Director, CSIR-NIIST, for providing infrastructure, resources, and laboratory facilities during the experiment. CSIR-NIIST manuscript reference number (NIIST/2023/Oct/587).


This research received no specific grant from funding agencies in the public, commercial, or not-for-profit sectors.

Author information

Authors and Affiliations



Conceived and designed the experiments: NR. Performed the experiments: SK, RRM, GGS, RK and NR. Analyzed the data: SK, RRM, GGS, RK and NR. Fund acquisition/contributed reagents/materials/analysis tools: NR. Wrote the paper NR with inputs from SK, RRM, GGS, and RK. All authors reviewed the manuscript.

Corresponding author

Correspondence to N. Rameshkumar.

Ethics declarations

Ethics approval and consent to participate

Not applicable.

Consent for publication

Not applicable.

Competing interests

The authors declare no competing interests.

Additional information

Publisher’s Note

Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.

Supplementary Information

Additional file 1:

Fig. S1. Maximum Likelihood phylogenetic tree based on the concatenated alignments of six highly conserved housekeeping genes recA, gyrB, rpoB, dnaK, atpD, and gltA (overall 3885 amino acid positions) of L1I39T and related members of the family Xanthobacteraceae. Closed dark circles at each node represent a similar grouping obtained from the neighbor-joining and maximum parsimony algorithms. The significance of each branch is indicated by the bootstrap value (as a percentage) calculated for 1000 subsets. Escherichia coli ATCC 11775T was used as an outgroup. The bar indicates sequence divergence. Fig. S2. Total polar lipid profile of strain L1I39T obtained after two-dimensional thin layer chromatography method. The total lipid spots were detected by spraying with 5% ethanolic molybdatophosphoric acid. Abbreviations: L, Lipid; AL, Aminolipid; GL, Glycolipid; PL, Phospholipid; PC, Phosphatidylglycerine; PE, Phosphatidylethanolamine; PG, Phosphatidylglycerol. Fig. S3. The positive growth of L1I39T on ZoBell Marine agar plate after 10 days of incubation at 30°C. Fig. S4. (a) Imaging showing the growth curve of L1I39T in R2A broth prepared with different seawater concentrations. (b) Phenotypic growth of L1I39T in R2A broth containing different seawater concentrations (A) zero, (B) 20%, and (C) 60% after 24 hours of incubation at 30°C, as represented by (i) visual culture broth turbidity, (ii) recovery of L1I39T cells on respective R2A agar and (iii) a comparative bar graph. Statistical significance represented by *, p-value < 0.05; **, p-value < 0.01; ***, p-value < 0.005. Fig. S5. Plant growth-promoting effects of L1I39T under nitrogen-limiting zero seawater conditions, (a) pot images showing the growth of pokkali rice (L1I39T-treated and control plants) after 28 days post-inoculation, (b) uprooted images showing the no growth differences between the L1I39T-treated and control plants after 28 days post-inoculation, (c) bar graphs represent no significant growth differences observed in plant parameters such as (i) shoot length, (ii) root length, (iii) root fresh weight, and (vi) root dry weight, between (A) control and (B) L1I39T-treated pokkali plants after 28 days post-inoculation. Fig. S6. (a) biofilm formation of L1I39T on a microtiter plate; (i) and (ii) represent replicates, (b) epi-fluorescent image showing a firm pokkali rice root attachment of GFP-tagged L1I39T cells after 3 hrs of incubation under 20% seawater conditions; (i) and (ii) represent different regions of the primary root, scale bar 20µm, (c) The symbiosis polysaccharide (syp) gene cluster of L1I39T. Fig. S7. Image showing the visual growth turbidity of L1I39T under different concentrations of hydrogen peroxide after 24 hours at 30°C. ++, good growth; +, weak growth. Fig. S8. (a) Image showing the visual growth of L1I39T in minimal media containing glucose or DL-malic acid as the sole carbon sources, (b) Bar graph representing the comparative growth differences of L1I39T in minimal media containing respective carbon sources. Images and data taken after 60 h time point. Fig. S9. Identified vitamin biosynthesis gene clusters in L1I39T (a) cobalamine, (b) biotin, and (c) thiamine biosynthesis. Fig. S10. Maximum likelihood phylogenetic tree based on the concatenated NifHDK protein-coding sequences of L1I39T and related members of the family Xanthobacteraceae. The significance of each branch is indicated by the bootstrap value (as a percentage) calculated for 1000 subsets. The scale bar indicates nucleotide substitution per site. Fig. S11. The nitrogen fixation (nif) gene cluster comparison between L1I39T and related members of the family Xanthobacteraceae; A. cavernae Sn-9-2T, A. spiritensis DSM 9035T, A. caulinodans ORS 571T, and X. autotrophicus Py2. Fig. S12. Putative hydrogenase gene cluster identified in (a) L1I39T and (b) A. caulinodans ORS 571T. Fig. S13. Putative T4SS gene cluster identified in L1I39T genome. Fig. S14. (a) Genetic organization of T6SS-1 and T6SS-2 gene clusters of L1I39T (b) Cell-cell contact-dependent assay. (i) Image showing the recovery of E. coli cells (target strain) after co-culturing with (A) E. coli (self-negative control), (B) L1I39T (attacker), and (C) P. plantistimulans L1E11T (attacker-positive control) on a solid LB agar plate with tetracycline. (ii) Box plot representing the recovery of E. coli cells (target strain) after co-culturing with (A) E. coli (self-negative control), (B) L1I39T (attacker), and (C) P. plantistimulans L1E11T (attacker-positive control).

Additional file 2


Rights and permissions

Open Access This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons licence, and indicate if changes were made. The images or other third party material in this article are included in the article's Creative Commons licence, unless indicated otherwise in a credit line to the material. If material is not included in the article's Creative Commons licence and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this licence, visit The Creative Commons Public Domain Dedication waiver ( applies to the data made available in this article, unless otherwise stated in a credit line to the data.

Reprints and permissions

About this article

Check for updates. Verify currency and authenticity via CrossMark

Cite this article

Sunithakumari, V.S., Menon, R.R., Suresh, G.G. et al. Characterization of a novel root-associated diazotrophic rare PGPR taxa, Aquabacter pokkalii sp. nov., isolated from pokkali rice: new insights into the plant-associated lifestyle and brackish adaptation. BMC Genomics 25, 424 (2024).

Download citation

  • Received:

  • Accepted:

  • Published:

  • DOI: