Pantoea tagorei sp. nov., a Rhizospheric Bacteria with Plant Growth-Promoting Activities

Abstract

A Gram-negative, short-rod, non-motile, facultatively anaerobic, potassium-solubilizing bacterium MR1 (Mine Rhizosphere) was isolated from rhizospheric soil of an open-cast coal mine of Jharia, Jharkhand, India. Isolate MR1 can grow in a broad range of temperature, pH, and NaCl concentrations. The 16S rRNA gene sequence of the strain showed 99.24% similarity with Pantoea septica LMG 5345^T. However, maximum-likelihood tree constructed using 16S rRNA gene sequence, multilocus sequence analysis using concatenated sequences of ten housekeeping genes, whole-genome based phylogenetic reconstruction, digital DNA–DNA hybridization, and average nucleotide identity (ANIm and ANIb) values indicated segregation of MR1 from its closest relatives. Fatty acid profile of MR1 also suggested the same, with clear variation in major and minor fatty acid contents, having C_13:0 anteiso (10-Methyldodecanoic acid) as the unique one. Thus, considering all polyphasic data, strain MR1^T (= MTCC 13265^T, where ‘T’ stands for Type strain) is presented as a novel species of the genus Pantoea, for which the name Pantoea tagorei sp. nov. is proposed.

Supplementary Information

The online version contains supplementary material available at 10.1007/s12088-023-01147-9.

Introduction

The family Enterobacteriaceae comprises a number of genera, including Escherichia, Enterobacter, Klebsiella, Salmonella, Shigella, Erwinia, Citrobacter, Flavobacterium, and Pantoea. Among them, the genus Pantoea that was initially described by Gavini et al. (1989) and currently has 26 species with valid names (http://www.bacterio.net/pantoea.html) [1]. Pantoea is a group of Gram-negative, facultative anaerobe, non-spore-forming, coccoid to rod-shaped bacteria. They are represented in a wide range of habitats, including soil, water, and are often associated with insects, plants, and animals. In fact, some species have been reported from the human gut and blood as well and were identified as disease-causing agents in various hosts. In immune-compromised patients, P. agglomerans, the most commonly reported species in humans, causes intravenous fluid contamination, nosocomial infections, and joint and soft tissue infection [2]. They have also been isolated from the human gut as opportunistic pathogens [3]. In case of plants, P. ananatis is reported to induce white spot disease in maize [4]. Another Pantoea species, P. stewartii, has been linked to wilting as well as leaf blight of corn [5]. In contrast to their above-mentioned harmful effects, many Pantoea species can actually be beneficial to plants. Several such isolates were reported to have multiple plant growth-promoting (PGP) traits such as phosphate solubilization, nitrogen fixation, indole acetic acid, siderophore, and HCN production; increased accumulation of soluble sugars; and colonization in rhizosphere [6]. Recently, members of this genera have been found to reside as an endophyte in flowers [7] and stems [8]. These endophytes also promote plant growth in several ways, thereby strengthening their potential use in agriculture as bio-inoculants. Furthermore, they can grow in a wide range of temperatures, pH, and salt concentrations and can also be used as a biocontrol agent against pathogenic fungi [9, 10]. Studies have also confirmed the adaptation of Pantoea species with epiphytic lifestyle. For instance, the genome sequencing of P. agglomerans 299R revealed the genes for high-affinity uptake and utilization of the photosynthates like sucrose, fructose, and glucose, as well as for repair of UV-damaged DNA, and production of the osmoprotectants, betaine, and trehalose [11].

Here, we report the taxonomic characterization of a newly isolated potassium-solubilizing, plant growth-promoting Pantoea strain MR1 using a polyphasic approach. Data from phenotypic, chemotaxonomic, 16S rRNA gene-based phylogenetic, and whole genome-based analysis revealed that the strain represents a novel species of genus Pantoea, for which the strain name Pantoea tagorei sp. nov. is proposed.

Materials and Methods

Isolation and Culture Condition

In the framework of a study that examines multi-trait PGP bacteria with a focus on their capacity to solubilize potassium, an open-cast coal mine in the Jharia district of Jharkhand (23.3857°N, 86.2829°E) was chosen as the sampling site. Sampling was done from the top 10 cm rhizospheric layer in April 2013. Available nitrogen (N), phosphate (P), potassium (K), and total organic carbon (C) contents in the soil sample were determined using standardized protocols [12]. For microbiological works, the samples were kept at 4 °C until further use.

To isolate potential K-solubilizing bacteria, Aleksandrov medium (Himedia, India) was modified by adding K-feldspar powder (0.5%) as a sole source of potassium. Bromophenol blue (100 mg l⁻¹) was added for better visibility of clear zone around K-solubilizing colonies. Soil samples were diluted, and appropriate dilutions were spread on modified Aleksandrov agar plates and incubated at 37 °C for 48 h for selecting suitable K solubilizing isolates. The most potent strain MR1, with elevated K solubilizing, P solubilizing, and N-fixing capabilities, was selected, and the culture is deposited in a bacterial culture collection center (MTCC, India) with accession number 13265. The pathogenicity of the isolate was evaluated using hemolysis testing on Blood agar (Himedia, India) containing defibrinated sheep blood (Sigma-Aldrich, USA), and DNase activity was assessed using DNase agar (Himedia, India) supplemented with Calf thymus DNA (Sigma-Aldrich, USA) [13].

16S rRNA Gene-Based Phylogeny

Genomic DNA from strain MR1 was extracted using GSure Bacterial DNA Isolation Kit (GCC Biotech, India) following manufacturer’s protocol. Using a universal primer set (Primer: 8_F and 1492_R), the 16S rRNA gene was amplified, and the purified PCR product was sequenced on Sanger sequencing platform [14]. The 16S rRNA gene sequence has been deposited at GenBank under the accession number OM698818. The similarity search of the 16S rRNA gene sequence was conducted using the latest EzBioCloud server with validated type strains [15]. All the available nearly complete 16S rRNA gene sequences of closely related type genera were retrieved from NCBI database and aligned using the MUSCLE algorithm in MEGA X program [16]. Gaps and missing data were treated with a complete-deletion option, and nucleotide substitution was performed based on Kimura 2-parameter with Gamma distribution model (K2 + G) [17]. The maximum-likelihood trees were reconstructed in MEGA X program, bootstrapping with 1000 replications [18].

Genome-Based Phylogeny

Genomic DNA was sequenced on an Ion PGM³¹⁸ chip with 400 bp chemistry at Thermo Fisher Scientific, Gurugram, India. The Whole Genome sequence has been deposited at DDBJ/ENA/GenBank under the Bioproject accession PRJNA892926.

Primarily, raw sequences were visualized in an updated version of FastQC v0.11.9 (https://www.bioinformatics.babraham.ac.uk/projects/fastqc) [19], followed by trimming and size selection (> 200 bp) in Trim_Galore v0.6.5 (https://www.bioinformatics.babraham.ac.uk/projects/trim_galore) [20]. High-quality reads (Phred score > 20) obtained after trimming were then used for downstream analysis. Unicycler v0.4.4 (https://www.github.com/rrwick/Unicycler/releases), a hybrid bacterial genome assembly pipeline [21], was used for de novo assembly. The assembly pipeline was optimized, which involved an error correction of sequenced reads with Bayes Hammer and assembly with SPAdes v3.15.3, with a k-mer value (up to 99) [22, 23]. Finally, the assembled sequences were annotated using Prokka v1.14 (https://www.github.com/tseemann/prokka) [24]. Assembled genome was visualized as a circular map using CGView server (https://www.cgview.ca) [25], and the assembly quality was visualized in Quast v5.2.0 (https://www.quast.sourceforge.net) [25, 26]. Additional graphic elements were arranged with Inkscape v0.92.4 (https://www.inkscape.org).

For MLSA analysis, complete sequences of ten housekeeping genes of MR1 were concatenated head-to-tail and compared with the closely related species [27, 28]. The genes used are ATP synthase beta subunit (atpD), DNA topoisomerase (ATP-hydrolyzing) subunit (gyrB), translation initiation factor IF-2 (infB), DNA-directed RNA polymerase subunit beta (rpoB), tryptophan synthase subunit beta (trpB), recombinase (recA), glyceraldehyde-3-phosphate dehydrogenase (gapA), phosphate acetyltransferase (pta), elongation factor G (fusA), and leucine–tRNA ligase (leuS). Representatives of the phylogenetically diverse members of Pantoea, Erwinia, Tatumella, and Brenneria (outgroup), for which all 10 full-length housekeeping genes are available in their respective draft/complete genome sequences, were included in the analysis. All these genes were concatenated head-to-tail (atpD-gyrB-infB-rpoB-trpB-recA-gapA-pta-fusA-leuS) and exported in FASTA format, providing a total of 40 strains (including the outgroup taxa) in the final dataset (Supplementary Table S1). The best-fit evolutionary model was the General Time Reversible with Gamma distribution evolutionarily invariable model (GTR + G + I), which calculated the pairwise distance. Finally, phylogenetic trees were reconstructed using MEGA X program [17].

Whole-genome-based taxonomic analysis, namely genome-to-genome distances (GGDs) and digital DNA-DNA hybridization (dDDH), were performed using the Type Strain Genome Server (TYGS) (https://www.tygs.dsmz.De) [29]. The whole-genome-based phylogenetic tree was constructed using FastME [30] from the genome blast distance phylogeny (GBDP). The trees were rooted at the midpoint [31]. The average nucleotide identity (ANI; using MUMmer and BLAST) values were calculated in JSpeciesWS (https://jspecies.ribohost.com/jspeciesws/) [32].

Phenotypic and Biochemical Characterization

Gram property, colony characteristics, and motility of the isolate MR1 were analyzed using standard protocols [33]. All growth parameters were checked in heterotrophic-rich media, Luria–Bertani medium (LB). Anaerobic growth was tested on LA at 37 °C for 7 days in an anaerobic jar using anaero gas pack and indicator tablet (Himedia, India). A 12 h old culture of MR1 was used for cell morphology analysis using field-emission scanning electron microscopy (FESEM; Gemini SEM 450-8216010130, Zeiss, Germany). Chemical fixation and dehydration were done, and critical drying point of the sample was achieved following standard methods [34]. The length and diameter of the cells were measured using FESEM inbuilt software. Growth of the isolate was investigated under various physiological conditions, including temperature (10–60 °C), pH (4–10), and salt (NaCl; 0.17–1.19 M, in excess of normal LB composition). Extracellular enzyme production, hydrolysis of esculin, nitrate reduction, gelatin liquefaction, and production of H₂S, catalase, oxidase, and indole acetic acid were examined using standard protocols [35, 36]. The strain was also tested for its ability to utilize different carbon sources using the Hicarbo kit, Himedia, India [37, 38]. Antibiotic sensitivity of the isolate was also tested using the disc diffusion method [39].

Fatty Acid Profiling

For whole-cell fatty acid analysis, cells were harvested from 48 h old culture of strain MR1 grown on tryptic soy agar (TSA) at 37 °C. Cellular fatty acids were saponified, methylated, and extracted according to the standard protocol of the MIDI Sherlock Microbial Identification System (MIDI ID No: 3247) by Royal Life Sciences Pvt. Ltd. (An ISO 9001: 2015 certified company affiliated with MIDI Sherlock, USA) [40]. Fatty acid methyl esters were analyzed by gas chromatography (Agilent GC-7820A, USA) and identified using the RTSBA6 database of the Microbial Identification System [41].

Results and Discussion

16S rRNA Gene-Based Phylogeny

The open-cast coal mine soil (sandy loam) sampled to isolate the plant growth-promoting (PGP) bacteria has a near-neutral pH of 6.5 and was poor in nutrients with available nitrogen (N), phosphate (P), potassium (K), and total organic carbon (C) contents of 231, 22, 10 (mg kg⁻¹), and 2.5% respectively.

After screening more than 200 colonies on Aleksandrov medium, 18 most potent K-solubilizing isolates with a solubilization zone of ≥ 3 mm were selected and checked for other PGP activities (data not presented here, manuscript under preparation). Eventually, the strain MR1, with elevated K-solubilizing, P-solubilizing, and N-fixing capabilities, was selected for further characterization. The strain MR1^T (where ‘MR1’ is the name of the strain isolated from Mine Rhizosphere, and ‘T’ designates the strain to be a Type strain for the said novel species) is deposited in a bacterial culture collection center (MTCC, India) with accession number 13265. The isolate is non-pathogenic for animals and humans, which was confirmed by negative results for hemolysis and DNase test (Supplementary Fig. 1).

The strain MR1 shared more than 98% similarity of 16S rRNA gene sequence with several members of the family Enterobacteriaceae that includes seven species of Pantoea, four of Enterobacter, two of Klebsiella, two of Tatumella, and one species each of Pseudescherichia, Erwinia, Citrobacter, and Leclercia (Supplementary Table S2). In addition, MR1 has 98.2% sequence similarity with the 16S rRNA gene of Flavobacterium acidificum, a member of the family Flavobacteriaceae. Among them, P. septica LMG 5345^T is the closest phylogenetic relative of strain MR1, and shares 99.24% sequence similarity. Although the highest 16S rRNA gene sequence homology is > 99%, the strain interestingly has more than 98% homology with seven distinct genera of Enterobacteriaceae and one genus of the family Flavobacteriaceae. Therefore, for its proper taxonomic positioning, a detailed polyphasic analysis is required to be performed with different molecular chronometers. The maximum-likelihood tree of the 16S rRNA gene sequence of MR1 with all its close relatives, clustered it on a separate branch with P. septica LMG 5345^T and P. latae AS1^T (Fig. 1). However, the low bootstrap support within this group makes it difficult to determine its proper taxonomic position but suggests that the isolate to be a member of Pantoea genus. It must be noted that similar phenomena were also reported in case of other novel Pantoea spp. such as P. vagans, P. eucalypti, P. deleyi, P. anthophila [42], and P. theicola [43]. This might be the result of a high degree of homoplasy in the 16S rRNA gene sequences of the Enterobacteriaceae family members [44]. Additionally, the polyphyletic nature of Pantoea spp. interferes heavily with its phylogenetic classification [42].

Fig. 1.

Construction of maximum likelihood phylogenetic tree elicited from the 16S rRNA gene sequences of Pantoea sp. MR1 and the type strains of Pantoea sp. Bootstrap values (> 50% are expressed as percentages of 1000 replications) are shown at branching points

Genome-Based Phylogeny

In order to accurately define the phylogeny of a new species and determine its genomic uniqueness, multilocus sequence analysis (MLSA) followed by whole genome comparison and digital DNA-DNA hybridization (dDDH) is recommended [45]. For this, the genome of strain MR1 was sequenced, and it could be assembled in 40 contigs, with a total size of 4,719,613 bp and G + C content of 58.68% (Supplementary Table S3) (Fig. 2). The G + C content of closely related reference strains lies within the range 53.0 to 59.6%, with the two closest representatives having almost similar values (Pantoea latae AS1, 59.60%; Pantoea septica LMG 5345, 59.10%). To observe clear differentiation with its neighbor strains, MLSA analysis was performed. Complete sequences of ten housekeeping genes of MR1 were concatenated head-to-tail (atpD-gyrB-infB-rpoB-trpB-recA-gapA-pta-fusA-leuS) and compared with the closely related species in MLSA. Maximum-likelihood phylogenetic analysis of the selected concatenated housekeeping genes generated a tree in which most nodes have > 90% bootstrap support. In MLSA phylogenetic analysis, similar to the 16S rRNA gene-based tree (Fig. 1), strain MR1 is placed in a separate clade (Fig. 3) with the closest member being P. septica LMG 5345^T followed by P. latae AS1^T. Strain MR1 exhibits MLSA sequence similarity of 71.75% and 71.67% to P. septica LMG 5345^T and P. latae AS1^T, respectively (Supplementary Table S4). These values are way lower than 97%, the recommended threshold of MLSA sequence similarity for the division of bacterial species [46].

Fig. 2.

Graphical presentation of strain MR1 genome (~ 4.7 mb) performed with CG view server. The 6 concentric circles represent the following (from outermost to innermost): circles 1 contigs; circles 2 and 3, protein-coding genes on forward and reverse strands; circle 4 (black): G + C content; circle 5: G + C skew; circle 6: DNA base position (Mbp)

Fig. 3.

Reconstructions of maximum-likelihood phylogenetic tree using MEGA X based on pairwise distance of head-to-tail concatenated atpD-gyrB-infB-rpoB-trpB-recA-gapA-pta-fusA-leuS gene sequences (full length) extracted from respective whole genome sequences. Multilocus sequence analysis (MLSA) distances were calculated in MEGA X program using General Time Reversible with Gamma distribution evolutionarily invariable model (GTR + G + I). Accession no. of housekeeping genes included in MLSA phylogenetic tree are provided in Supplementary Table S1.

Digital whole genome comparisons are considered as the gold standard for checking the systematic position of a particular species [47]. The whole-genome-based phylogenetic reconstruction data positions strain MR1 with P. septica LMG 5345^T as a separate cluster (Fig. 4). Stackebrandt and Ebers recommended mandatory DNA-DNA Hybridization to be performed to determine genomic uniqueness of a novel isolate with 16S rRNA gene sequence similarities up to 99% [48]. The average nucleotide identity, ANIm and ANIb (Supplementary Table S5) and dDDH (Supplementary Table S6) values of strain MR1 are 92.82%, 91.78%, and 68.0%, respectively, with its closest relative, P. septica LMG 5345^T. These values, which demarcate a bacterial isolate as a separate species, are way lower than the proposed ANI and dDDH threshold values (95–96% and 70%, respectively) [49]. Even the genome size and protein count of strain MR1 are bigger/higher by 9.79% and 30.26%, respectively than its closest relative, i.e., P. septica LMG 5345^T (Fig. 4). Thus, the genome-based phylogenetic parameters indicate that the strain MR1 cannot be grouped with any reported species of Pantoea and must be recognized as a new species.

Fig. 4.

TYGS tree designing based on whole genome sequence inferred with FastME 2.1.6.1 [59] from Genome BLAST Distance Phylogeny approach (GBDP); distances calculated from genome sequences. The branch lengths are scaled in terms of GBDP distance formula d₅. The numbers above branches are GBDP pseudo-bootstrap support values > 60% from 100 replications, with an average branch support of 94.6%. The tree was rooted at the midpoint [31]. Leaf labels are annotated by affiliation to species and subspecies clusters, genomic G + C content, δ values, and overall genome sequence length and number of proteins. Note: The synonym for Pantoea piersonii is Kalamiella piersonii

Phenotypic and Biochemical Characterization

Strain MR1 is a Gram-negative, short rod, a non-capsulated, non-spore-forming, facultatively anaerobic, and non-motile bacterium (Table 1; Fig. 5). Colonies are yellow, smooth, round, and convex with entire margins in Luria–Bertani Agar plate. The isolate can grow in the temperature range of 20–60 °C, pH of 4 to 7, and elevated salt concentrations (up to 1.19 M NaCl) in excess of normal heterotrophic media composition, with optimum values for these three parameters being 37 °C, pH 7, and 0.17 M NaCl, respectively. The strain can produce extracellular enzymes like protease, chitinase, catalase, and oxidase but shows negative results for amylase, gelatinase, and cellulase activities. Besides this, the isolate can produce β-galactosidase, indole acetic acid, and H₂S; reduce nitrate; and utilize citrate and malonate. Among the tested carbon sources, MR1 can effectively utilize lactose, xylose, maltose, fructose, dextrose, galactose, trehalose, melibiose, sucrose, l-arabinose, mannose, rhamnose, cellobiose, xylitol, and D-arabinose. However, the strain is unable to utilize raffinose, inulin, gluconate, glycerol, salicin, dulcitol, inositol, sorbitol, mannitol, adonitol, arabitol, erythritol, alpha-methyl-d-glucoside, alpha-methyl-d-mannoside, malonate and sorbose (Supplementary Table S7). The antibiotic sensitivity test confirms strain MR1 to be highly susceptible to ciprofloxacin and norfloxacin but resistant against bacitracin, cephalexin, cephaloridine, lincomycin, methicillin, novobiocin, oleandomycin, penicillin G, piperacillin, teicoplanin, and vancomycin (Supplementary Table S8).

Table 1.

Morphological, physiological, and biochemical characterization of MR1 and its closest phylogenetic neighbors

Characteristics		MR1	1	2	3	4	5	6	7	8	9	10	11	12	13	14	15	16	17	18	19	20
Cell morphology
Colony color		Y	Be	Y/NY	Y	Y	Y	Y	NP	NP	NP	NP	NP	Y	NP	NY	CC	LBe	Be	NP	Y	PW
Cell size (diameter × length; in μm)		0.7–0.9 × 2.0	0.9 × 1.5–3.0	0.5–1.0 × 1.0- 3.0	NP	NP	NP	0.9 × 1.2–2.5	0.9 × 1.5–3.0	0.9 × 1.5–3.0	0.5–1.0 × 1.0–3.0	0.9 × 1.5–3.0	NP	0.7–0.9 × 1.4- 1.7	0.9 × 1.5- 3.0	1.0 × 2.0–3.0	0.6 × 1.2	1 × 1.5–3	1.0 × 2.0–3.0	0.8–1.2 × 1.5–3.0	0.9 × 1.5–3.0	1.0 × 1.0–2.5
Cell shape		SR	NP	SR	Rod	NP	NP	SR	NP	NP	NP	SR	NP	Rod	NP	Rod	Rod	SR	SR	SR	SR	SR
Motility		–	+	−	+	+	+	+	NP	+	+	+	NP	NP	+	+	+	WM	−	+	+	+
Growth condition
pH range (optimum)		4.0–10.0 (7.0)	NP	NP	4.0–10.0 (7.0)	NP	NP	NP	NP	NP	NP	NP	NP	4.5–10	NP	NP	4–9 (7)	NP	NP	5–9 (7)	NP	NP
Temperature (°C) (optimum)		10–40 (37)	NP	30	7–48 (37)	30–40 (30)	4–41 (37)	NP	NP	NP	NP	NP	–	5–35 (28)	NP	10–44 (37)	15–45 (35)	NP	NP	10–40 (30)	NP	NP
NaCl (M) (optimum)		0.17–1.19 (0.17)	NP	NP	0–1.19% (0–0.34%)	NP	NP	NP	NP	NP	NP	NP	NP	0–1.02%	NP	NP	0–1.7 (0.085)	NP	NP	−	NP	NP
Hydrolysis of carbon utilization (1%)
Casein		+	NP	NP	+	+	NP	NP	NP	NP	NP	NP	NP	NP	NP	NP	−	NP	NP	NP	NP	NP
Starch		−	NP	NP	−	−	−	NP	NP	NP	NP	NP	+	+	NP	+	NP	NP	NP	−	NP	NP
Esculin		+	NP	+	+	+	NP	+	NP	NP	NP	+	NP	+	NP	+	+	NP	NP	+	NP	NP
Carbon utilization (1%)
d-glucose		+	NP	+	+	+	NP	+	NP	NP	NP	NP	+	+	NP	+	NP	+	+	+	+	+
d-xylose		+	+	+	+	+	−	+	+	+	+	+	+	+	+	+	NP	+	+	+	+	+
d-fructose		+	NP	+	+	+	NP	+	NP	+	+	+	+	+	NP	+	NP	+	+	+	+	+
Sucrose		+	NP	+	+	+	+	+	+	+	+	+	+	+	–	+	NP	+	−	+	+	NP
Other activities
Indole production		+	−	−	+	+	+	+	−	−	−	−	NP	+	−	+	NP	−	−	−	–	−
H2S production		+	–	−	−	−	NP	+	NP	NP	NP	−	NP	+	NP	+	NP	−	−	−	−	−
Nitrate reduction		+	NP	NP	+	NP	+	NP	NP	NP	NP	−	NP	NP	NP	NP	+	NP	NP	+	−	NP
Oxidase		+	NP	−	−	−	NP	NP	NP	NP	NP	−	NP	−	NP	−	−	−	−	−	−	−
Catalase		+	NP	NP	+	+	NP	NP	NP	NP	NP	NP	NP	+	NP	+	+	+	+	+	NP	+

Y: Yellow, CC: Cream color, Be: Beige, LBe: light beige, PW: Pale yellow, NY: Non-yellowish, WM: Weakly motile, SR: Short rod, NP: Not provided, (+): Positive, (−): Negative

Used strain: Pantoea sp. MR1, 1. P. septica LMG 5345 [51], 2. P. agglomerans LMG 1286 [1], 3. P. alhagi LTYR-11Z [52], 4. P. allii BD 390 [53], 5. P. ananatis LMG 2665 [54], 6. P. anthophila LMG 2558 [42], 7. P. brenneri LMG 5343 [51], 8. P. conspicua LMG 24534 [51], 9. P. cypripedii LMG 2657 [51], 10. P. deleyi LMG 24200 [42], 11. P. dispersa LMG 20603 [1], 12. P. endophytica 596 [55], 13. P. eucrina LMG 5346 [51], 14. P. gaviniae DSM 22758 [56], 15. P. intestinalis 29Y89B [3], 16. P. rodasii LMG 26273 [57], 17. P. rwandensis LMG 26275 [57], 18. P. theicola QC88-366 [43], 19. P. vagans LMG 24199 [42], 20. P. wallisii LMG 26277 [57]

Fig. 5.

Morphological view of strain MR1 under field-emission scanning electron microscopy (FESEM). Bacterial cells were grown for 12 h in LB medium at 37 °C before image acquisition. The length and diameter of the cells were measured using FESEM inbuilt software

Fatty Acid Profiling

The major cellular fatty acids in strain MR1 were identified as C_16:0 (25.89%), summed feature 8 (C_18:1ω7c and/or C_18:1ω6c; 17.8%), summed feature 3 (C_16:1ω7c and/or C_16:1ω6c; 13.7%), summed feature 2 (comprising any combination of C_12:0 aldehyde, an unknown fatty acid of equivalent chain length; 12.82%), C_14:0 3-OH/16:1 iso I (11.1%), C_12:0 (9%), C_14:0 (6.8%), cyclo-C_17:0 (4%), C_18:1ω9c (2.55%), and C_18:0 (1.44%) (Table 2). Notably, fatty acids like C_12:0, C_18:1ω9c, C_14:03-OH/16:1 isoI, summed feature 2 (combination of C_12:0 aldehyde and unknown fatty acid) and summed feature 3 (C_16:1ω7c and/or C_16:1ω6c) shows considerable variation in their proportions as compared to its phylogenetic neighbors (Table 2). Minor fatty acids (< 1%) like, C_10:0 (0.6%), C_19:00 (0.8%), C_13:0 anteiso (0.4%), and C_12:0 3-OH (0.7%) found in MR1 also differed from the nearest strains. Notably, C_13:0 anteiso (10-Methyldodecanoic acid), a methyl-branched saturated fatty acid is the unique one, and only present in the strain (Table 2). Apart from genetic makeup, the fatty acid composition of bacterial cells is strongly influenced by culture conditions and might be the reason for the observed variation among closely related species [50].

Table 2.

Cellular fatty acid compositions of strain MR1 and the available reference strains of genus Pantoea sp., expressed as percentages of total fatty acid peak areas

Name of fatty acid	Type of fatty acid	Percentage of total fatty acid (%)
		MR1	1	2	3	4	5	6	7	8	9	10	11	12
C10:0 (Decanoic acid)	Medium chain saturated	0.6	–	–	–	0.1	–	5.4	–	–	–	–	–	–
C12:00 (Dodecanoic acid)	Medium chain saturated	9.6	–	3.8	8.8	7.9	4.1	–	3.9	4.5	4.2	3.4–4.8	5.8	4.2
C13:00 (Tridecanoic acid)	Long chain saturated	0.3	–	–	0.1	0.4	–	–	0.2	–	–	–	–	–
C14:00 (Tetradecanoic acid)	Long chain saturated	6.8	3.8	6.0	6.3	9.7	5.7	6.6	5.3	6.7	6.9	0–5.1	6.4	6.9
C15:00 (Pentadecanoic acid)	Long chain saturated	–	1.3	1.1	–	–	–	–	–	–	–	–	–	–
C16:00 (Hexadecanoic acid)	Long chain saturated	25.9	34.7	27.1	25.3	24.4	29.8	38.9	29.4	27.4	26.1	24.3–29.6	38.9	26.1
C17:00 (Heptadecanoic acid)	Long chain saturated	0.5	–	< 1	–	0.5	–	–	0.9	–	–	< 1 (1)	–	–
C18:00 (Octadecanoic acid)	Long chain saturated	1.4	2.3	–	0.8	0.2	0.4	–	0.6	–	–	1.5–2.4	–	–
C19:00 (Nonadecanoic)	Long chain saturated	0.8	–	–	–	–	0.3	–	0.3	–	–	–	–	–
C13:0 anteiso (10-Methyldodecanoic acid)	Methyl branched saturated	0.4	–	–	–	–	–	–	–	–	–	–	–	–
C17:0 iso 3-OH	Long chain saturated	–	–	–	3.4	0.1	–	–	–	–	–	–	–	–
C17:0 cyclo	Long chain saturated	4	8.8	13.2	11.9	8.7	13.8	3.8	18.2	8.8	7.1	–	3.8	7.1
C19:0 cyclo ω8c	Long chain saturated	0.8	–	–	–	0.5	1.4	–	4.2	–	–	–	–	–
C16:01 (Palmitoleic acid)	–	–	–	17.2–26.7	–	–	–	15.1	–	–	–	–	15.1	–
C18:01 (Vaccenic acid)	–	–	–	–	–	–	–	16.6	–	–	–	–	16.6	–
C18:02 (Linoleic acid)	–	–	–	–	–	–	–	5.8	–	–	–	–	5.8	–
C16:1 ω5c ((11Z)-11-Hexadecenoic acid)	Long chain saturated	–	–	–	–	–	–	–	0.2	–	–	–	–	–
C17:1 ω7c ((10Z)-10-Heptadecenoic acid)	–	0.4	–	–	–	–	–	–	0.3	–	–	–	–	–
C17:1ω8c ((9Z)-9-Heptadecenoic acid)	Long chain saturated	–	–	–	–	0.1	–	–	–	–	–	–	–	–
C18:1 ω5c	–	–	–	1	–	–	–	–	0.1	–	–	–	–	–
C18:1 ω9c ((9Z)-9-Octadecenoic acid)	Monounsaturated	2.5	–	–	0.6	–	–	–	–	–	–	–	–	–
C12:0 3-OH	–	0.7	–	–	–	0.1	–	–	–	–	–	–	–	–
C15:0 3-OH	Long chain saturated	–	–	13.2	–	0.1	–	–	–	–	–	–	–	–
C16:0 3-OH	Long chain saturated	–	–	–	0.5	–	–	–	–	–	–	–	–	–
C14:0 3-OH/16:1 iso I	–	11.1	–	17.2–26.7	–	–	–	–	–	13.9	14.3	–	–	14.3
Summed feature 1* (C15:1 iso H and/or C13:0 3OH)	–	–	–	–	–	0.3	–	–	–	–	–	–	–	–
Summed feature 2* (C12:0 aldehyde and unknown fatty acid)	–	12.8	–	–	11.0	19.1	9.8	–	9.0	–	–	–	–	–
Summed feature 3* (C16:1ω7c/C16:1ω6c)	–	13.7	–	–	21.8	15.8	16.6	–	8.0	–	–	–	–	–
Summed feature 8* (C18:1ω7c/C18:1ω6c)	–	17.8	–	–	9.5	11.8	17.3	–	18.0	11.0	11.8	35.7–41.4	–	11.8

The fatty acid profile for P. septica LMG 5345^T (the closest phylogenetic relative of MR1) is not yet available

Used strain: Pantoea sp. MR1, 1. P. septica OOWS-10 [58] 2. P. agglomerans LMG 1286 [57], 3. P. alhagi LTYR-11Z [52], 4. P. endophytica 596 [55], 5. P. calida DSM 22759T5 [3], 6. P. gaviniae DSM 22758 [3], 7. P. intestinalis 29Y89B [3], 8. P. rodasii LMG 26273 [57], 9. P. rwandensis LMG 26275 [57], 10. P. stewartii subsp. stewarti [3], 11. P. theicola QC88-366 [3], 12. P. wallisii LMG 26277 [57]

*Summed Features are fatty acids that cannot be resolved reliably from another fatty acid using the chromatographic conditions chosen. The MIDI system groups these fatty acids together as one feature with a single percentage of the total

Description of Pantoea tagorei sp. nov.

Above results suggest that strain MR1^T represents a novel species of the genus Pantoea. Thus, based on phenotypic characteristics, an in-depth phylogenetic study, whole genome analysis, and cellular fatty acid composition analysis, strain MR1^T is contemplated as a novel species, so accordingly, the name is proposed to be Pantoea tagorei sp. nov.

Pantoea (ta.go.rei. N.L. masc. gen. n. tagorei of Tagore), named after Rabindranath Tagore, the first Asian Nobel laureate and founder of Visva-Bharati, Santiniketan, India; and his son Rathindranath Tagore, an agriculture scientist and the first Vice-Chancellor of Visva-Bharati. The naming of this beneficial plant growth-promoting rhizospheric bacteria after Tagore is pertinent as the founder of Visva-Bharati and his son highly promoted indigenous agriculture for rural benefits. Moreover, the timing of this publication also coincides with UNESCO declaring Santiniketan as a World Heritage site.

P. tagorei is a Gram-negative, facultatively anaerobic, short rod (0.7–0.9 µm of diameter × 2.0 µm of length), non-motile, non-spore-forming bacteria, occurring singly or in pairs (Fig. 1). After 24 h of aerobic incubation at 37 °C on Luria agar medium, colonies are observed as yellow-pigmented, circular, and convex, with a diameter of approximately 1–2 mm. The isolate grows optimally at 37 °C, pH 7.0, in an LB medium supplemented with 0.17 M NaCl. The strain is positive for protease, catalase, gelatinase, oxidase, and chitinase; but negative for amylase, cellulase, and DNase. It is unable to hemolyzed blood. MR1^T can utilize several carbon monomers and polymers, which include lactose, xylose, maltose, fructose, dextrose, galactose, raffinose, trehalose, melibiose, sucrose, l-arabinose, mannose, rhamnose, cellobiose, and sorbose. Isolate is highly susceptible to ciprofloxacin and norfloxacin. The primary cellular fatty acids of the strain are C_16:0 (25.89%), summed feature 8 (C_18:1ω7c and/or C_18:1ω6c; 17.8%), summed feature 3 (C_16:1ω7c and/or C_16:1ω6c; 13.7%), summed feature 2 (comprising any combination of C_12:0 aldehyde, an unknown fatty acid of equivalent chain length; 12.82%), C_14:0 3-OH/16:1 iso I (11.1%), C_12:0 (9%), C_14:0 (6.83%), cyclo-C_17:0 (4%), C_18:1ω9c (2.55%), and C_18:0 (1.44%). Notably, C_13:0 anteiso (10-Methyldodecanoic acid), a methyl-branched saturated fatty acid is the unique one, and only present in the strain. The DNA has a G + C content of 58.68 mol% and a draft genome size of ~ 4.72 Mb.

Conclusion

The strain MR1^T (= MTCC 13265^T) was isolated from open-cast coalmine rhizospheric soil at Jharia, Jharkhand, India, while exploring potential potassium solubilizing bacteria in these K-deficient mine soils. Preliminary data indicates that the isolate MR1^T can efficiently solubilize K from the insoluble K-bearing complex compound and also possess other potential PGP activities. While performing a detailed characterization of the isolate, it was noticed that the strain has some unique characteristics in terms of its phenotypic, phylogenetic, and fatty acid profile, with variations in its genome features from closely related organisms. Based on detailed molecular phylogenetic characterization, the strain is claimed to be a novel species and named as Pantoea tagorei sp. nov. Further in-depth study of the biofertilizer potential of the strain in different environmental conditions with variable K-source can be helpful for field application to promote crop growth in K-deficient agricultural soil.

Supplementary Information

Below is the link to the electronic supplementary material.

Abbreviations

WGS

Whole-genome sequencing

MLSA

Multilocus sequence analysis

TYGS

Type Strain Genome Server

dDDH

Digital DNA–DNA hybridization

FAME

Fatty acid methyl ester

Author contributions

BD, RB, and AM designed the study. RB, and AM analyzed the data. RB, AM, SG, AC, PM, and BD wrote the manuscript. All authors read and approved the final manuscript.

Data availability

The Whole Genome sequence has been deposited at DDBJ/ENA/GenBank under the Bioproject accession PRJNA892926.

Declarations

Conflict of interest

The authors declare no conflict regarding financial or any academic interests.