An exopolysaccharide-producing novel Agrobacterium pusense strain JAS1 isolated from snake plant enhances plant growth and soil water retention

A peculiar bacterial growth was very often noticed in leaf-initiated tissue cultures of Sansevieria trifasciata, a succulent belonging to the Asparagaceae family. The isolate left trails of some highly viscous material on the walls of the suspension vessels or developed a thick overlay on semisolid media without adversities in plant growth. FTIR identified this substance to be an extracellular polysaccharide. Various morphological, biochemical tests, and molecular analyses using 16S rRNA, atpD, and recA genes characterized this isolate JAS1 as a novel strain of Agrobacterium pusense. Its mucoidal growth over Murashige and Skoog media yielded enormous exopolysaccharide (7252 mg l−1), while in nutrient agar it only developed fast-growing swarms. As a qualifying plant growth-promoting bacteria, it produces significant indole-3-acetic acid (86.95 mg l−1), gibberellic acid (172.98 mg l−1), ammonia (42.66 µmol ml−1). Besides, it produces siderophores, 1-aminocyclopropane-1-carboxylic acid deaminase, fixes nitrogen, forms biofilms, and productively solubilizes soil inorganic phosphates, and zinc. Under various treatments with JAS1, wheat and chickpea resulted in significantly enhanced shoot and root growth parameters. PGP effects of JAS1 positively enhanced plants’ physiological growth parameters reflecting significant increments in overall chlorophyll, carotenoids, proline, phenols, flavonoids, and sugar contents. In addition, the isolated strain maintained both plant and soil health under an intermittent soil drying regime, probably by both its PGP and EPS production attributes, respectively.

Enzyme activity assays. All enzyme activity screening assays employed standard methods. For screening cellulolytic (= cellulose degradation) activity, carboxymethylcellulose (CMC) agar plates were spot inoculated with the bacterial isolate and incubated for 24 h (at 28 °C) following which media was overlaid with iodine solution. A clear halo zone infers positive cellulolytic activity 91 . For proteolytic activity screening, the bacterial isolate was spot inoculated on plates with skim milk agar media (Himedia, Mumbai, India) which if develops a clear halo zone around its colony(s) (upon incubation at 28 °C for 24 h) would indicate positive proteolytic activity 92 . Lipase activity was assayed by spot inoculating the isolate on TBA (Tributyrin agar) base supplemented with 1% Tributyrin (Himedia, Mumbai, India). A positive lipase activity would be inferred from a clear zone around the bacterial colony(s) following an incubation regime of 24-48 h (at 28 °C). Pectinase assay employed inoculating the bacterial isolate over Pectinase Screening Agar Medium (PSAM). Plates were incubated at 28 °C for 2 to 3 days and later flushed with 3-4 ml of iodine solution. A halo zone around the bacterial growth indicates positive pectinase activity 93 . Amylase activity was screened on starch agar medium (SAM) spot inoculated with the bacterial isolate (and incubated at 28 °C for 1 to 2 days). Plates were flooded with 1% iodine solution for 20 min. A clear yellow zone around the growth indicates positive amylase activity 94 . Other details on media composition are provided elsewhere (see media and reagents in supplementary file).
Bacterial motility tests. The motility test was performed using two standard methods: the semi-solid agar and the wet mount method 95 . The former used a fine loop to stab bacteria vertically deep into an agar butt (SIM Medium Butt; Himedia, Mumbai, India) which was incubated overnight (at 28 °C) following the manufacturer's recommendations. The other method used a wet mount of bacteria over a glass slide and viewed for growth patterns under a light microscope (Metzer, Vision plus-5000 DPCT). Both methods used fresh inoculums raised from a single colony on NB media. Antibiotic sensitivity tests. Antibiotic sensitivity assays for the bacterial isolate used a standard disc diffusion test 96 following the CLSI guidelines 97 . Bacterial starter culture raised overnight from a single colony (in NB, 28 °C, 200 rpm) was spread plated on Muller Hinton agar (Himedia, Mumbai, India). After about 30 min of inoculum soaking, susceptibility discs for various antibiotics (Himedia) were placed on plates and incubated overnight in dark at 28 °C. The observed zone of inhibition was measured as diameters (= extent of bacterial susceptibility to the antibiotic) otherwise depicting antibiotic resistance. Results were expressed as means of the means from three replicates each arriving from three trials per antibiotic assayed. A. rhizogenes strain A532 was used for reference. Antibiotic sensitivity data of the isolate was compared with that reported for AP strain NRCPB10 T and MB17-a 30,33 . Molecular identification of the isolate. Pure culture of the bacterial isolate JAS1 was sent to MTCC, CSIR-IMTECH, Chandigarh for 16S rRNA sequence-based identification. For further validation, JAS1 genomic DNA was used as a template for PCR amplification of two housekeeping genes, recA (with primers recAF: 5′-ATG GCA CAA AAT TCT TTG CGT CTC GTA GAG and recAR: TCAVCCT TCG TCA CCR TCG CCG TCA TCG C) and atpD (with primers atpDF: ATG GCT AAG GCA GCT ACC CCMAAG AAA ACC and atpDR: TCA GGC AGC YTC GGC AGC CAG CTT CTTSGC). PCR reactions consisted of 200 mM PCR buffer, 1.5 mM MgCl 2 , 10 pmol µl −1 each primer (Bioserve Biotechnologies India Pvt. Ltd., Hyderabad, India), 10 mM dNTPs, 5 units of Taq-DNA polymerase (Himedia, Mumbai, India) and molecular biology grade water (Himedia, Mumbai, India) up to 50 µl total volume per reaction. PCR program (run on BIORAD S1000™ thermal cycler) included steps with initial denaturation (5 min, 95 °C) followed by 32 cycles each with intermittent denaturation (1 min, 95 °C), annealing (1.5 min, 60 °C) and extension (1 min, 72 °C); altogether followed with a final extension step (10 min, 72 °C). Amplicons were gel extracted (Himedia, Mumbai, India) and ligated to pGEM-T easy vector (Promega, New Delhi, India) which was then transformed to DH5-α competent bacteria following the heat-shock protocol. Recombinant clones were screened using blue-white screening and validated for inserts using colony PCR and/ or restriction enzyme digestions of the plasmids. Plasmid isolates with respective gene inserts were sequenced using universal M13 primers (Eurofins Genomics India Pvt., Bangalore). Sequences were screened and trimmed for vector backbone sequences using the VecScreen tool (https:// www. ncbi. nlm. nih. gov/ tools/ vecsc reen/) and then analyzed for chromatograms using the CHROMAS software (version 2.6.6, Technelysium Pty Ltd). BLAST Scientific Reports | (2022) 12:21330 | https://doi.org/10.1038/s41598-022-25225-y www.nature.com/scientificreports/ for sequence similarity search was carried out with GenBank (https:// www. ncbi. nlm. nih. gov/ genba nk/) and EzBioCloud databases (https:// www. ezbio cloud. net/), respectively. Clustal W program was used to align highly similar sequences and MEGA (version 11.0.11, https:// www. megas oftwa re. net/ downl oads/ dload_ win_ gui) 98 for constructing phylogenetic trees based on various algorithms. All outlined kit-based procedures follow the manufacturer's recommendations.
EPS production and purification. Crude EPS was isolated from the oozing spread of JAS1 that developed over 10 days on MSA plates (inoculated with a revived single colony on NA, 28 °C). Drooping volume (~ 10 ml directly from the lid of the inverted plate) was collected into a falcon tube. To ease bacterial separation, crude EPS was double diluted with SDW. Near clear supernatant was resolved after centrifugation (10,000 rpm, 30 min, 4 °C on REMI-CPR-24 PLUS, Remi, Mumbai, India), and further clarified through 0.45 µm filter discs on an ultra-filtration assembly (Tarsons, Kolkata, India). EPS was precipitated with two volumes of ice-cold 100% absolute ethanol and overnight stored at −20 °C. Later, EPS was spool-collected into a fresh falcon tube and centrifuged (10,000 rpm, 30 min, 4 °C). Pellet was left for drying at 60 °C and later mixed in 5-10 ml SDW and dialyzed through cellulose membrane (10-14 KDa MWCO; Himedia, Mumbai, India) for 48 h at constant stirring in 2 l ultra-filtered SDW (at 22 °C). EPS was recovered and lyophilized in a freeze dryer (Allied-Frost, New Delhi, India) to result in a powdered preparation. This was weighed to account for the total EPS recovered from 10 ml of droop collected from the MSA plate. The resulting EPS was also estimated using a standard acid hydrolysis test 99 . Other than this small-scale EPS production regime used inoculating 50 µl JAS1 starter (overnight raised on NB using a NA raised single colony; 28 °C) in 50 ml MSB in several 250 ml vol. flasks for a month's incubation (28 °C, 120 rpm). At definite intervals, flasks were removed from incubations and instantly processed for EPS recovery and purification using the same steps outlined before with proportionate volumetric adjustments and stringencies to avoid any volume loss. Freeze-dried weights were measured in respective purifications to account for final EPS yield (w/v) at various day intervals post-inoculation. All tests were repeated thrice and the results were shown as means of the means. These EPS preparations were further used in FTIR analysis and other essays when required. Based on the recovery from small-scale EPS production trials (above), a liter's scale batch production was attempted in MSB inoculated with 1 ml overnight starter culture of JAS1 (raised from a single colony over NA) in a 5-l Erlenmeyer flask and incubated for 10 days in an orbital shaker (120 rpm, 28 °C on REMI CIS-18plus, Kolkata, India). The whole (highly viscous) culture was doubly diluted with ultra-filtered SDW and centrifuged thrice (10, Plant pathogenicity test. Carrot roots were availed fresh from a village (Gharuan) farm in the close vicinity of Chandigarh University and were followed with standard surface sterilization and bacterization protocol 104 . In brief, surface sterile 1 cm thin, cross-sectioned root discs were established on NA overlaid with a spread of JAS1 (1 × 10 4 CFU ml −1 ) and incubated in a PTC room with standard conditions mentioned before. Root discs were observed for 2 weeks for any pathological symptoms. All trials were performed thrice each with three replicates per trial.
PGP characterizations. Phosphate solubilization. JAS1 isolate was tested for phosphate solubilization using a standard Pikovskaya's agar plate protocol 105 with slight modifications. JAS1 isolate was spot inoculated on Pikovskaya's agar (Himedia, Mumbai, India) supplemented with tricalcium phosphate as an insoluble P source. The culture was incubated at 28 °C for up to 10 days for the development of a clear halo around the colonies which infer positive phosphate solubilization measurable as an index using the following formula Qualitative analysis of soluble phosphate was followed using standard methods [105][106][107] with some modifications. In brief, JAS1 isolate was cultured in 250 ml flasks with 50 ml of National Botanical Research Institute's Phosphate (NBRIP) medium (supplementary file). The culture was incubated in an orbital shaker (28 °C, 180 rpm) for 10 days followed with an intermittent assay for soluble phosphates released in the NBRIP medium. Briefly, 2 ml culture aliquots were withdrawn at 48, 120, 160, and 216 hourly intervals and centrifuged (10,000 rpm, 10 min). Cell-free supernatant (1 ml) was then mixed well with 2 ml of 1% boric acid and 3 ml of freshly prepared molybdate reagent (supplementary file) and incubated undisturbed (RT, 40 min). Samples were read for OD 700 values spectrophotometer (UV-1800, Shimadzu, New Delhi, India). Quantification of available phosphates followed with plotting OD 700 values on a standard curve (potassium dihydrogen phosphate in the range of 2-30 µg ml −1 ). All tests were repeated thrice with four replicates per test.
ACC deaminase production. Production of ACC deaminase by JAS1 isolate was detected with its growth on DF minimal salt medium (supplementary file) supplemented with 2 g l −1 (NH 4 ) 2 SO 4 and incubated for 72 h at 28 °C 108 .
Siderophore production. Screening and evaluation of siderophore production used freshly prepared Chrome Azurol S (CAS) reagent. As per the norm, any trace iron in all culture and test vessels were removed by rinsing them overnight in 3 mol l −1 HCl and washing later with SDW 109 . Qualitative test for siderophore production followed the standard CAS agar plate method 110 . Briefly, nutrient agar (NA) plates supplemented with 10% CAS reagent were streaked with the bacterial isolate and incubated at 28 °C for a week. Formation of a yellow to orange halo around the streak infers a positive result. Quantitation of siderophore production used a modified microtiter plate method 111 . Hundred microliter aliquots were withdrawn from an NB grown culture of the isolate at various time intervals (48, 120, 160, and 216 h), centrifuged (at 10,000 rpm, 10 min), and respectively mixed with 100 µl freshly prepared CAS reagent. After 20 min of incubation, formation of a yellow to orange colored complex in test samples infers a positive siderophore production. The OD 630 values of the test samples were recorded on a microplate reader (RT2100C, Rayto, Guangming New District, China). The experiment was performed thrice with four replicates per test. Siderophore production was quantified using the standard formula 112 : Here: A r is the absorbance of reference (uninoculated broth and CAS reagent); A s is the absorbance of sample (inoculated broth and CAS reagent).
Indole-3-acetic acid (IAA) production. IAA production was assayed using a standard method 113 with slight modifications. Bacterial isolate was cultured in NB supplemented with 0.1% tryptophan and incubated for 10 days (at 28 °C, 120 rpm). Culture aliquots were withdrawn at different time intervals (48,120,160, and 216th h) and centrifuged to remove the pellet (10,000 rpm, 15 min). The supernatant (1 ml) was properly mixed with 2 ml of freshly prepared Salkowski reagent (1 ml FeCl 3 and 50 ml 35% HClO 4 ) 114 and allowed to stand in dark for 30 min. Pink coloration in the test samples infers IAA production which was quantified spectrophotometrically (OD 530 ) using a standard curve for commercially purchased IAA (Sigma-Aldrich, USA). All tests were repeated thrice with four replicates per test.
Gibberellic acid (GA) production. GA production by JAS1 was elucidated by employing a standard spectrophotometric assay 115 . In brief, 2 ml supernatants from culture (at 28 °C, 120 rpm) aliquots were withdrawn at various time intervals (48,120,160, and 216 h) after centrifugation (10,000 rpm, 10 min) and mixed with 280 µl of 1 M zinc acetate and later with 280 µl of 10.6% of potassium ferrocyanide solution under vigorous vortexing. Centrifugation (4500 rpm, 10 min) led supernatant was equally mixed (v/v) with 30% HCl (added slowly) and incubated (at RT for 75 min). Absorbance was recorded in a spectrophotometer (OD 254 ) against a blank containing 5% HCl and was fitted to a standard curve for commercial purchased GA (20-200 μg ml −1 , Himedia, Mumbai, India).
Zinc solubilization. For the preliminary inference on the zinc solubilization activity of JAS1, a standard plate assay was employed 116 that requires spot inoculation of the isolate over a semisolid basal media (supplementary file) mixed with 0.1% zinc oxide (insoluble, Himedia, Mumbai, India) and incubation at 28 °C for a week to observe any clear zones around the spot colony inferring zinc solubilization which can be measured as below Alternatively, quantification of solubilized zinc in JAS1 inoculated broth (media as above) was performed using a standard Atomic Absorption Spectrometry (AAS) method 116 . Samples were withdrawn at different time intervals (48,120,160, and 216th h) and supernatants (10,000 rpm, 15 min) were fed to a Perkin Elmer AAS (Jeeva Labs Pvt. Ltd., Nalagarh, Himachal Pradesh).
Potassium solubilization. Potassium solubilization of the bacterial isolate was studied on Aleksandrow agar media (supplementary file) by a standard spot plate assay 117 inoculated with a loop-full overnight grown culture and incubated at 28 °C. Clear zones around the colonies confirm positive potassium solubilization. The diameter of clear zones deduced the Khandeparkar's selection ratio as below: Nitrogen fixation. The nitrogen-fixing ability of the bacterial isolate was verified using a standard method based on the ability of bacteria to grow on nitrogen-free media 118 . In brief, bacterial isolate was streaked on Jensen media (supplementary file) and incubated (at 28 °C) for a week. The growth of bacteria with glistening colonies and/or streaks on the above media infers positive nitrogen fixation. The test was repeated thrice.
Hydrogen cyanide production. Production of hydrogen cyanide by bacteria was ascertained using a standard method 119 . Bacterial isolate was streaked over NA plates supplemented with glycine (4.4 g l −1 ) and was fixed under a suitably sized, autoclaved Whatman filter paper disc (No. 1) freshly soaked in 2% sodium carbonate solution (prepared in 0.5% picric acid). The plates were incubated in dark (at 28 °C for 4-5 days), and when developing a dark orange to red coloration on filter paper, inferred positive HCN production by the isolate. The assay was repeated thrice.
Biofilm formation. Congo red agar (CRA) method is a qualitative method for screening biofilm formation (BF) ability in microorganisms. Post streak inoculation of the bacterial isolate, CRA plates (supplementary file) were incubated at 28 °C for 24 h. Crystalline blackening of colonies with dry consistency would vouch for the positive bio-film forming by bacteria 120 . In vitro effects of JAS1 on ST leaf explants. ST leaf explants were established in jam jars following our standardized PTC protocol 88 . Post root formation (at 3 weeks after ST explant establishment), a test set of explants were primed with JAS1 (1 × 10 4 CFU ml −1 raised in NB) while those in control were treated with sterile NB. Cultures were maintained under PTC room conditioning discussed before. Observations were recorded post 5 weeks of JAS1 treatments. The experiment was repeated thrice with five replicates each within the test and control sets.
Effects of JAS1 priming on ex vitro wheat growth. PGP effects of the isolate were validated experimentally in a controlled glasshouse setting (25-27 °C, 70-90% relative humidity and under natural photoperiods) following a completely randomized design (CRD). Seeds (10 g) of a commercial wheat variety (UNNAT PBW 343) were purchased locally (Grain market, Mohali, Punjab, India) and soaked overnight in 100 ml of ultra-filtered (0.22 μm pore sized, nylon filter paper discs, Millipore, Bengaluru, India) tap water (TW). Seeds were divided into test and control sets (100 seeds each). The test set was primed with 100 ml of TW resuspended JAS1 culture (1 × 10 8 CFU ml −1 ; raised overnight from a single colony on NB; 28 °C, 120 rpm, dark). The control set (uninoculated with JAS1) was treated with just the same volume of TW. The seeds (70 numbers per set) were then sown directly (1.0 cm deep) into a soil bedding (prepared with mesh sorted and autoclaved garden soil) layered (soil depth 4 cm) onto plastic trays (43 × 34 × 7 cm). Soil beds were spray watered on the same day post www.nature.com/scientificreports/ sowing with ca. 150 ml TW, thereafter every day with 50 ml (TW). This included as well booster dosing test soil beds with spray inoculation of JAS1 (50 ml TW with 1 × 10 8 CFU ml −1 ) once per week for only initial three weeks while control beds were treated only with 50 ml TW. Seedling growth was monitored and recorded periodically. After 8 weeks, various growth paraments viz., overall fresh and dry weight (FW and DW respectively), and primordial length were compared following soil drain-out. In another similarly designed trial, post 4 weeks' treatments as in above, soil beds were left unwatered for a week to observe seedling performance under a soil drying regime. Each of the above trials was repeated thrice.
Effects of JAS1 on in vitro wheat growth. In vitro trials were designed each comprising a test set with three replicates of sterile PTC jam jars each with two surface-sterilized wheat seeds (var. UNNAT PBW-343) established over 50 ml semisolid MSA media carrying 1 ml fresh overlay spread of SDW resuspended bacterial pellet (1 × 10 4 CFU) derived from an overnight grown starter culture of JAS1. In the control set, the JAS1 spread was replaced with SDW. Culture incubations were done for a month in the PTC room followed by finally recording growth parameters viz., shoot and root length, root branching, and root hair density and length. Standard PTC room conditions were maintained as mentioned earlier. Trials were run thrice following the above experimental regime.
Effect of JAS1 on chickpea. Like in wheat, we also attempted to characterize the PGP effects of JAS1 on a legume crop. For this, seeds of a locally available commercial variety L550 of chickpea (Cicer arietinum L) were in transparent plastic pots (1 l) containing ca. 0.7 kg of finely sieved and autoclaved garden soil (as detailed before with wheat tray trial). About 25 g seeds were cleaned for 30 min with two drops of Tween-20 under running tap water followed by three rinses with ample TW. For the JAS1 test sets, seed priming involved imbibing and co-cultivating them together in 100 ml of TW resuspended JAS1 inoculum (1 × 10 8 CFU ml −1 , overnight, 28 °C) raised using an overnight starter culture grown from a single colony (in NB, 28 °C, 120 rpm, dark). Control sets were similarly imbibed in an equal volume of TW (devoid of JAS1). Each of the control and JAS1 treatment sets consisted of five pots (replicates) with three seeds sown per pot. On the 15th day post sowing, a booster dose of JAS1 (10 ml of TW resuspended 1 × 10 8 CFU ml −1 ) was applied to each pot (except the control sets which were irrigated with 10 ml sterile TW) following a soil drench method 34 . All plants were irrigated with 30 ml of TW every third day till the 27th day (3 days before the drain out for final observations of roots). The experiment was repeated thrice with each trial scheduled to run for a month to record the effects on the shoot and root development.
Soil water retention. Independent trials were attempted using 150 g of autoclaved and oven-dried garden soil-beddings prepared on plastic plates and drenched with 30 ml of either (i) MSB resuspended overnight grown JAS1 inoculum (1 × 10 8 CFU ml −1 ), or (ii) EPS preparation derived by SDW resuspending the dried powdered ethanol precipitate from the centrifuged supernatant of a 10-day MSB grown JAS1 culture (= EPS treatment), or (iii) SDW (= control treatment). The respective plates were left in a laminar airflow hood at 28 °C and the blower speed was set to 0.45 ms −1 . Water retention percentage was derived by periodically weighing the plates and evaluating water retained after a periodic water loss in each replicate put under the three different treatments. All experimental trials were repeated thrice with three replicates per treatment.
Biochemical estimates of plant growth. Tray and pot trials with and without JAS1 priming treatments respectively carried out for wheat and chickpea (see above) were randomly sampled for standard biochemical assays as under.
Total proline content. As per a standard method (referred in 121 ), 100 mg of shoot biomass was thoroughly crushed and mixed with 5 ml of 3% sulfosalicylic acid. From the supernatant retrieved after centrifugation (at 10,000 rpm, 4 °C, 10 min), 2 ml was mixed subsequently with 2 ml of each ninhydrin reagent and glacial acetic acid. This reaction mixture was heated at 100 °C for 1 h and cooled on ice, followed by the addition to it of 4 ml toluene. Brief vortexing and then stilling (15 min) of this mixture developed a light pink chromophore layer, which was retrieved for spectrophotometry (OD 520 ) against a toluene blank. The resulting values were plotted over a standard curve using a commercially purchased proline (Himedia, Mumbai, India) to estimate proline concentration in the test samples.
Total chlorophyll and carotenoids. As per a standard protocol 122 , 100 mg of shoot biomass was crushed in 2 ml DMSO, and supernatant derived after centrifugation (at 5000 rpm, 4 °C, 15 min) was spectrophotometrically read respectively for chlorophyll a (OD 665 ), chlorophyll b (OD 649 ) and carotenoids (OD 480 ). The resultant concentrations were computed and expressed into mg pigment per gram fresh weight as per recommendations 123 .
Total phenols and flavonoids. Respective samples of shoot biomass were subjected to air drying (24 h., 35 °C), and from each ca. 200 mg was crushed and mixed with 5 ml of 100% methanol. Supernatants were retrieved post centrifugation (10,000 rpm, 15 min, 4 °C) and stored for further use. Protocols each for phenol and carotenoid estimations observed slight modifications to methods referred elsewhere 124 . For estimating total phenols, 1 ml of this supernatant from each plant-derived sample and separate 1 ml aliquots each corresponding to a gallic acid standard (ranging from 10-100 µg ml −1 ) were individually resuspended into 5 ml SDW. Thereafter, 0.5 ml of Folin-Ciocalteu's reagent was added to each solution and mixed by shaking. By 5 min later, these were added with 1.5 ml of 20% sodium carbonate with finally volumetric makeup to 10 ml using SDW. Post incubation for www.nature.com/scientificreports/ 2 h at RT, blue color developed in each reaction vessel which was read spectrophotometrically (OD 750 ). Resultant total phenols in tests were derived from the gallic acid standard plot and were expressed as mg of gallic acid equivalents per gram of dry biomass. For total flavonoids, as before 1 ml of supernatant from methanol extract of each plant sample and separate 1 ml aliquots, each corresponding to a quercetin standard (ranging from 100 to 1000 µg ml −1 ) were individually resuspended into 4 ml SDW. To these, 0.3 ml of 5% sodium nitrate was mixed and kept for 5 min at RT. Thereafter, 0.3 ml of 10% aluminium chloride was added to these. Abruptly at the 6th min, 2 ml of 1 M NaOH was added to each of the reactions, all of which then were volumetrically adjusted to 10 ml using SDW. A yellowish-orange color developed in each reaction vessel, which was read spectrophotometrically (OD 510 ). The resultant total flavonoids in tests were derived from the quercetin standard plot and were expressed as mg of quercetin equivalents per gram of dry biomass.
Statistical and computational analyses. All

Results and discussions
Isolation of JAS1 from ST PTC leaf explants. In our aseptic plant tissue culture facility, we routinely attempt callus induction and other whole plant regeneration trials with ST leaf segments. We recently reported a protocol for quick and efficient whole plant regeneration using IBA rooted leaf explants cultured at relatively high-temperature incubations 88 . In many of these and other trials, alongside the growth of intact callus, callus suspension, and the differentiating meristemoids, we also recorded 5-15% of our culture replicates showing extensive growth of some bacteria. On liquid media (MSB) it exhibited a peculiar highly viscous material sticking to the walls of the PTC vessels ( Fig. 1a-c) while on semisolid media (MSA) it showed a distinct oozy bacterial growth (Fig. 1d,e). These did not negatively influence the in vitro plant propagation but favored morphogenesis with more root and shoot primordia over the ST explant (see further). Upon subculture, to MSA the isolate www.nature.com/scientificreports/ reproduced colonies overgrowing with time which drooped on the lid (Fig. 1f,g). From these instances as well as following a standard endophyte isolation protocol 89 over healthy ST leaf segments, we could isolate similarly morphed bacterial clones and scrutinized them variously for their growth, molecular identity, and other characteristics following standard assays (discussed ahead).
Biochemical and enzyme activity assays. Based on the morphological and biochemical characterizations (Table S1, supplementary file) the isolate JAS1 is a gram-negative, rod-shaped bacteria and it tested positive for catalase, methyl red, indole, and motility assays. Its ferments various carbohydrate substrates except lactose and gelatin and can limitedly grow in NaCl supplementation of up to 1%. Endophytes are known to produce and secrete various extracellular enzymes which significantly regulate key processes viz., nutrient acquisition, motility, symbiosis, commensalism, pathogenesis, etc. JAS1, however, tested positive only for protease production while remaining negative for pectinase, lipase, cellulase, and amylase. This may supportively hint at JAS1 co-cultivating with ST in vitro without influencing plant growth per se negatively testifying for enzymes known to degrade essential plant cell wall components such as cellulose and starch. It was intriguing otherwise as to why only protease activity could be detected in assays. Literature supports many of the endophytes positive for proteases implicated in equipping with extended antimicrobial defense 126 . We further plan to explore these effects in JAS1.
In vitro growth of JAS1: EPS secretion, swimming, and swarming motility. Within two days of growth on MSA, the streak plated inoculum of JAS1 formed pale white irregularly shaped colonies with a peculiar bulge or ooze which on further incubation drooped over the petri-dish lid (Fig. 2a,b). Streaks on the NA plate, however, grew to form peculiar fingerlike outgrowths covering the entire media surface within two days post-inoculation (Fig. 2c,d). These projections characterize a multicellularity trait called 'swarming motil- . In (e) shows an EPS front following right after the lane with higher bacterial density, however in (f-l) is shown the growth of JAS1 forming swarms, In (f), shown three superposed layers or fronts with different bacterial (density) patterns, wherein the upper-front with probably the older and excessively denser growth, followed then with the approaching middle-front with newly produced swarms (with spindles pointing outwards, probably yet to form a spiral and wavy swarms), and then the lower-front with well-formed snake-like wavy swarms (with spiral spindles in tufts); Of these the middle front is magnified sequentially (in g-i) to show the quorum spindles. In (j-l), well-formed wavy swarms with spindles (as the lower front in f) when mechanically disturbed show bacteria dislodged from the spindle formation. www.nature.com/scientificreports/ ity' witnessed, in some bacteria under certain in vitro conditions of growth 127 . The motility of JAS1 isolate was priorly confirmed using two standard methods. On semi-solid SIM Medium, it grew along the visible stab line rendering cloudiness in the butt. Alternatively, under light microscopy with wet mounts, swimming motility in discreet directions was evidenced. Oozy growth (as on MSA) is commonly seen with rhizobacteria and is known to effectuate from the synthesis and secretion of certain exopolysaccharide (EPS) complexes. EPS synthesis is influenced majorly by both the concentration and types of sugars and nitrogenous species in the media 128 as also witnessed for JAS1 (data not shown). EPS complexes may render such bacteria to develop mucoid (or slimy) and ropy colonies 129 . JAS1 however indicated only slimy EPS secretion on MSA (Table S1, supplementary file). Hence, the JAS1 isolate showed apparent variability in growth patterns on two different media, which respectively reveal its EPS production and swarming motility features.
Further, under the light microscope the JAS1 growth on both MSA and NA was closely observed (Fig. 2e-l). Compared to that on MSA, where the streak margins always exhibited a peculiar EPS front backed by a population gradient of JAS1 (Fig. 2e), the swarm margins on NA however showed distinguished formations (Fig. 2f). Further on NA, most of the emerging swarm outgrowths appeared exclusively in wavy and/or spiral fashion. Each of the waves or spiral assemblies showed a well-defined length within the swarms (Fig. 2g-i). Freely moving bacteria were more apparent at the swarm fronts than in the back (Supplementary Video 1). Upon disturbing some of the bundled spirals by gently pressing with a fine aseptic needle, which probably dislodged individual bacterium off the spirals (Fig. 2j-l). Dislodged bacteria surprisingly showcased their swimming motility over the area (notched wet) with the pressed agar (Supplementary Video 2). These observations indicate that JAS1 equips both swarming and swimming motility which might be variously showcased in vitro depending on the media composition and substratum factors. Such growth and motility attributes have been shown in many bacteria including as well AP strains 38,130 . Swarming also exemplifies quorum sensing in bacteria and helps bacteria to variously cling/attach facilitating their easy and speedy escape from local stresses, migration to favored and preferred localities, and/or the efficient invasion of the host 127 . In some plant-associated bacteria, swarming motility is known to aid virulence while other many are implicated in use as biocontrol agents as they deliver protection to their plant host(s) from other competing bacterial and fungal phytopathogens 127 . Both swarming and swimming activities facilitate root attachment, and colonization 131,132 . Answers to how JAS1 orchestrates these features, especially in light of its PGP attributes (discussed ahead) demand further studies.  30 as both share a common ancestor (Fig. 3a). These results confidently position JAS1 within the genus Agrobacterium (considering as well the revised inclusion of Rhizobium pusense strains into agrobacteria) 31,32 . Further, we deduced the complete sequences of two housekeeping genes (atpD and recA) PCR amplified from JAS1 (GenBank accessions MZ741443 and MZ741444 respectively), and analyzed their phylogenetic relatedness with other known species, after multiple sequence alignment. Based on sequence alignments, atpD in JAS1 could only sparsely relate to a few A. tumefaciens strains (B6, 93.89%; and C58, 93.33%) and R. endophyticum strain (CCGE2052, 91.03%), but surprisingly did not align to any of the reported A. pusense strains. Also, Agrobacterium strains showing maximum sequence similarity were distantly oriented in the phylogenetic tree (Fig. 3b). In recA (Fig. 3c) . Overall, the complete coding sequence of JAS1-RecA did not align completely with any agrobacterial strains. Notably, even with 79% of its sequence coverage, it still could not align identically with the typed strain NRCPB10 T (99.54% sequence identity). These results thus largely infer that JAS1 is a novel strain A. pusense which probably had co-evolved with/from other agrobacterial species.
Antibiotic sensitivity tests. JAS1 was found sensitive to many tested antibiotics (Table S2, supplementary file). In respect to those reported for the A. pusense typed strain, NRCPB10 T30 , it differed in showing resistance only to ampicillin, and trimethoprim while being susceptible to nalidixic acid, tetracycline, rifampicin, neomycin, kanamycin, streptomycin, and spectinomycin. EPS production, purification, and total yield. EPS from various microbial sources have found multifaceted applications 133,134 and have been variously studied considering EPS biosynthetic potential of each strain(s) and the influence over it from various parameters viz., physical factors, media components especially the carbohydrate and nitrogenous sources, use of elicitors and so on 129,135,136 . EPS yield also depends on the extent of its recovery from losses in various downstream extraction and purification processes, all of which vary from simple centrifugation to complex hydrolysis, precipitation, and other recovery steps. At the time of writing the manuscript, no elaborate studies could be found documenting EPS production amongst the known AP strains, except that of a commercial β-glucan (marketed as Salecan) released by the strain ZX09 137 . Other AP strains were limitedly reported to date with mucoid colonies and/or milky white streaks on agar media.
Our chanceful findings with the strain JAS1 hail from its enormous EPS secretion in PTC media which mandatorily contains 3% sucrose. We manifested a simple approach of diluting the resulting culture volume enough  www.nature.com/scientificreports/ to separate cell biomass using centrifugation followed by ultrafiltration to assure cell-free, near pure extraction. Additionally, to cater to purity requisites in FTIR and SEM (next sections) we further dialyzed and lyophilized EPS preparations. On average, 10 days' old culture of JAS1 spread plated on 20 ml semi-solid media MSA over a 100 mm diameter petri-dish yielded ca. 56 ± 2 mgs of EPS. Small scaled batch culture in 50 ml of MSB inoculated with JAS1 for 10 days offered a greater EPS yield of ca. 360 ± 2 mg. Surprisingly, however, batch cultures of JAS1 in a liter volume of MSB within the same duration resulted in an averaged total recoverable EPS yield of 7252 ± 2 mg l −1 . This is a fairly appreciable yield reported over that known from the AP strain ZX09 137 and is also comparable to that from other EPS-producing Agrobacterium species. Yield from the small scale (50 ml) batch correlated well with that resulting from a liter's scale interpreting an as minimal loss of only about 14 mg during the downstream processing with scaled-up produce.
FTIR and XRD analysis of EPS. The functional groups of EPS-JAS1 were studied with the use of ATR-FTIR (Fig. 4a) which depicted an intense broad peak at 3306.21 cm −1 corresponding to the presence of hydroxyl groups 138 . A small band witnessed at 2916.88 cm −1 unique to that for carbohydrates infers the presence of asymmetric C-H stretching vibration 139 . Furthermore, an asymmetric stretching peak was observed at 1632.92 cm −1 , corresponding to stretching vibrations from carbonyl groups (C=O) in CONH moieties 140 and may also interpret ring stretching vibrations in galactose and mannose 141 . The bands detected at 1416.03 cm −1 detects symmetrical stretching of COO− groups 138 . The smaller peaks at the 1267.07 cm −1 band are due to C-O stretching   142 . Absorption peaks around 800 and 1200 cm −1 would derive from sugars as well as β-glycosidic bonds among their monomers. The absorption peak at 1046.19 cm −1 is designated to C-O-H, C-O-C, and C-O, inferring polysaccharides in the sample 140,141,143 . The peaks at 895.44 and 815.97 cm −1 correspondingly indicate the α-glycosidic and β-glycosidic bonds 144 . The small peaks observed at 559.11 and 623.55 cm −1 depict glycosidic linkages in the polysaccharide 145 . The absorption peaks between 550 and 539 cm −1 match with stretching vibrations of the alkyl-halide groups 143 . The appearance of these functional groups enables JAS1-produced EPS (JAS1-EPS) to find potential use in numerous applications. For example, the water-loving nature of EPS is attributed to the presence of hydroxyls 146 which vouches the emulsifying 138 or biosurfactant property 147 for this EPS. Carboxylate groups ensure affinity to opposite charge molecules such as heavy metals 148 . The anionic nature and ability to chelate metals and ions ensure anticorrosive effects when applied to metals 149 . The above findings on functional groups from EPS secreted by JAS1 were found almost similar to those mentioned for exopolysaccharides called curdlan known to be secreted by many bacteria [150][151][152] . Phase identification of materials is purposefully gathered using XRD. XRD pattern of EPS (Fig. 4b) with broad peaks revealed prominent amorphous nature (83.4%) and sharp narrow peaks depicted low crystallinity (16.6%) profile.

SEM of EPS and JAS1
. SEM has been used to account for surface characteristics of polymers 153 . SEM micrographs depict that EPS from JAS1 has both smooth and rough patches at discreet locations on its surface exhibiting as well with irregular folds (Fig. 4c) which may impart compactness to the otherwise dispersive configuration. Small and variously sized pores were also witnessed which (along with folds) may suffice permeability and water-holding features qualifying JAS1-EPS for use in viscosifiers, thickeners, and preservatives for new-age foods 154 . Porosity may also help bacterial dynamics in plants, soil, and other factors as well within biofilms 155,156 . JAS1 can be seen as peculiar rods bathing in EPS (Fig. 4c).

EPS solubility in different solvents. Dried exopolysaccharide was found completely soluble in water
availing a homogenous and transparent solution with concentration-dependent increments in viscosity. EPS was however found insoluble in other tested solvents viz., ethanol, methanol, butanol, ethanediol; acetone, ethyl acetate, DMSO, chloroform, hexane, and benzene. These results are in congruence with the FTIR results and those reported for EPS known elsewhere, supportive in that its solubility exclusively in aqueous solvent correlates with the predominant hydroxyl groups in EPS 157-159 . Plant pathogenicity testing of JAS1. As mentioned before, JAS1 occurred in many of our PTC attempts with ST leaf explants where it did not negatively affect the vegetative growth of ST. However, many species in the agrobacterial genera are reported as plant pathogens while also some exist as symbionts and/or commensals in their host(s). Surprisingly, plant pathogenicity amongst AP strains has only recently been reported from Lawson Cyprus tree isolate, KH1 104 , known to cause tumors over carrot root discs 104 . Upon similar tests, JAS1 however failed to indicate any pathological symptoms on carrot root discs. Unfortunately, such pathogenicity assessments have not been made for other known AP strains as also only a few of these are studied for their PGP attributes. Some clinically reported AP strains are also known to cause sepsis in humans 160 , which warrants safety assessments over proclaimed PGP AP strains, their finished bioproducts (as also on those many known from various other microbes) including as well EPS, many have seen commercial translations with the consideration of generally regarded as safe (GRAS) 161 . Studies elsewhere as well as ours hint that AP strains may have evolved multiply and independently in natural extremities to showcase either a PGP and/or otherwise a pathogenic behavior.

PGP characterizations. Phosphate solubilization.
Phosphate is an important but also most limiting macronutrient in soil, with poor bioavailability to plants due to its insoluble forms mostly occurring as rock phosphates. To quench the deficit, most plants rely on endophytes capable of phosphate solubilization by soil acidification 162 . JAS1 was detected positive for phosphate solubilization, with a peculiar halo zone developed around its colonies on Pikovskaya's agar and a phosphate solubilization index (PSI) of 1.27 cm on the 9th day post-inoculation (Table 1). The highest solubilization potential (17.6 µg ml −1 ) could be achieved within 48 h in liquid NBRIP medium. Like JAS1, another AP strain MB-17a isolated from mung bean roots is also reported with slightly higher phosphate solubilization (PSI of 2.67 cm and solubilization potential of up to 53 μg ml −1 on the 6th day) 33 , while two other studied isolates from soybean and tomato are reported negative for phosphate solubilization potential 38,43 .
ACC deaminase production. 1-aminocyclopropane-1-carboxylic acid (ACC) is a precursor of ethylene, the phytohormone that under low titers enhances seed germination, root length, and root hair growth 163 . The successful growth of JAS1 tested over DF media inferred positive ACC deaminase production of ( Table 1). This PGP screen evaluates the ability of bacteria to control deleterious ethylene levels allowing their plant hosts to better manage various biotic and abiotic stressors 163,164 . ACC deaminase is reported in three other AP strains viz., MB-17a, RJG6, and IC59 33,34,165 .
Siderophore production. Siderophores are secondary metabolites with low molecular weight and have ironchelating potential 166 . It is involved in growth and development, and in restricting the pathogens proliferating in plants. JAS1 showed a peculiar yellow halo zone around its streaks over CAS agar inferring a positive siderophore production profile, quantifiably of 30.1% (%SU) at the 120 th hour of growth (Table 1) Indole-3-acetic acid (IAA) production. IAA is an important auxin that principally enhances root growth in plants 167 . JAS1 displayed appreciable IAA production (max. 86.95 µg ml −1 ) as determined by the Salkowski assay (Table 1) and is comparable to that in the AP strain MB-17a (reportedly producing 110.5 µg/ml IAA) 33 . Other reported AP strains such as YP3, YP4 and IC59 produced low IAA (59.2, 43.8, and 21.9 µg ml −1 , respectively) 34,40 , and few others were reported merely as positive for this PGP trait 38,40,43 .
Ammonia production. Ammonia variously sequesters plant growth development by a build-up of soil assimilable nitrogen resources, inhibition of pathogen attack, and biomass production among others 168,169 . JAS1 was found positively producing ammonia with the maximum release (42.66 ± 0.15 µmol ml −1 ) detected on the 5th day of assay incubations (Table 1). Only a few other AP strains viz., MB-17a, AM-4, and MS-1 have been tested positive for this attribute.
Gibberellic acid production. GA regulates root and stem growth and also improves seed germination in plants 170 . The highest GA production of 172.98 µg ml −1 was seen by JAS1 at the 120th h of the incubation regime (Table 1). This property has not been ever reported for any AP strain.
Zinc solubilization. Zinc deficiency in plants is considered a critical factor limiting crop productivity. Its sensitivity is more pronounced during drought stress and studies as well suggest that water use efficiency is critically dependent on plants' zinc profile 171 . Many soil zinc forms occur in complex with oxides, silicates, and carbonates while microorganisms are reported to augment soil fertility by solubilizing these forms 172 . Zinc solubilization effect JAS1 was preliminarily ascertained over insoluble zinc oxide in plate and broth assay. In plate assay, a zinc solubilization index of 2.3 cm was recorded at the 216th h of incubation, which continued to increase beyond the observation schedule (Table 1). This apparent finding was also confirmed quantitatively by AAS wherein JAS1 could offer almost 200 μg ml −1 week −1 solubilization of insoluble ZnO. This PGP trait is reported for only one AP strain IHCP2 solely using the plate method inferring a lower solubilization index (1.87 cm) 39 .
Nitrogen fixation. Nitrogen is a vital nutrient for organismic growth. However, plants are unable to utilize atmospheric ample of nascent nitrogen and for the supply of its assimilable forms rely on certain nitrogen-fixing microbes 175 . N 2 -fixing bacteria thus can grow over nitrogen-free media in vitro. Our JAS1 isolate showed growth over nitrogen-free Jensen's media and showcased glistening colonies 7 days post-inoculation (Table 1), thus indicating a positive N 2 -fixing PGP trait. Similarly, TMV2-6, YP3, YP4, and PR1 strains of AP are also known to confer this ability 40,176 .
Hydrogen cyanide production. Hydrogen cyanide is a volatile secondary metabolite synthesized by several PGPRs and is implicated as a biocontrol agent causing cytotoxic death of various plant pathogens 177 . However, the JAS1 isolate did not show HCN production ability. This trait is reported in only a few AP strains 34,37,39,43 . Recently, a group had shown that HCN production does not necessarily suppress plant pathogens, however, suffices phosphate availability in soil 178 . Given this, the JAS1 isolate already shows phosphate solubility trait www.nature.com/scientificreports/ (discussed before) while lacking the HCN production ability. This might suggest that HCN production and phosphate solubilization traits are independently governed in PGPRs, however, this needs extensive studies.
Biofilm formation. The biofilm formation (BF) property of JAS1 was assayed using the Congo red agar method. The isolated strain JAS1 showed strong BF after 24 h (Table 1) and produced black-colored colonies with a dry consistency. BF could be obviated already during sampling of JAS1 isolate in MSB cultures of ST depicting the biomass aggregates attached to the interior of the flasks forming a thin lining of JAS1 (Fig. 1). This BF is due to the EPS, as generally reported in many plant-associated bacteria which helps in adhesion to plant surfaces, and mitigating various abiotic and biotic stresses 23,[179][180][181] .

Effects of JAS1 on ST regeneration in vitro.
We tested the effect of bacterial isolate JAS1 on in vitro rooted ST leaf explants using our previously standardized PTC protocol for ST whole plant regeneration 88 . After 5 weeks, the JAS1 co-cultivated explants resulted in significantly enhanced shoot and root growth responses (Fig. 5a). In addition, roots in the JAS1 treatments showcased changes in texture from green to pale white (Fig. 5b), significant increments in girth, and lengthier root hairs compared to those in the control treatment (Fig. 5b,c). Roots from field-grown ST, however, did not exhibit these observations (Fig. 5d). These comparisons support that PGP prospects with JAS1 were delivered to its host plant (ST) while it's standardized in vitro regeneration regime. No pathological symptoms could be recorded in any of the replicates, all of which subsequently resulted in productive whole ST regenerants.
JAS1 enhances ex vitro growth of wheat seedlings. We studied the growth performance of wheat seedlings under the effect of JAS1. This used a seed priming approach for a commercial wheat variety which germinated and grew over well-watered tray soil-beds, and was observed all through for 8 weeks. With respect to (wrt) the unprimed seeds in the control sets where shoot emergence was withheld up to the 4th day, the JAS1 treated seeds showed early germination with clear emergence of shoot primordia above the soil within 3 days of sowing (Fig. 6a). Seed germination enhancement is a common prospect of PGPRs probably as also seen here with JAS1. The shoot primordia sustained higher growth increments in lengths wrt those in control throughout the 8-week trials (Figs. 6a,b, 7a-c). Furthering germination, in these trials we observed other various effects from JAS1 that enhanced the wheat seedling developmental profiles. For example, at 5 weeks post sowing, the first leaf easily uncurled and split up from the seedlings (Fig. 7b insets), while this was delayed by ca. 1 week www.nature.com/scientificreports/ in the untreated control sets. Followed after this shoot in the JAS1 treated beds showed healthy upright stature than those in the control set(s), the latter which with a distinct bent and with older leaves showcasing a peculiar yellowing at their tips extending towards the base, however, without any signs of interveinal chlorosis (Fig. 7b).
No such detriments could be recorded in any of the JAS1 treated test sets. Such leaf tip yellowing reportedly corresponds to a temporary nitrogen deficiency in wheat which either entails from the soil low in nitrogen and/or often its waterlogging 182 . Obviously, throughout these experimental trials, soil profiles were maintained equally well-watered and in addition, no seepage was provisioned. It is also to note that the soil beds with JAS1 primed seeds were booster dosed intermittently thrice. In addition, soil testing also indicated wheat-productive improvement of its fertility by JAS treatment (data not shown). This supports the successful translation of the www.nature.com/scientificreports/ PGP attributes of JAS1 in improving both soil and plant health. Data recorded post drain-out inferred that JAS1 treatment enhanced shoot and root biomass (Fig. 6c,d), and their lengths (Fig. 6e,f). In addition to this, comparative speculation of the root formations revealed that almost all roots in the JAS1 treatments had more branching points than on those in the untreated set (Fig. 7d), as well as that root branching is evident even at sites closer to the pod or that lay closer to topsoil profile (Fig. 7d,e). When observed under a stereomicroscopic magnification, roots from seedlings in the JAS1 treatment showcased denser and lengthier root hairs than those in the control treatments (Fig. 7e). Soil particles also adhered better to the roots under JAS1 treatment (Fig. 7d,e). Presumably, they form aggregates on the rhizosheath due to the viscous EPS secretion by JAS1 as also shown with many other rhizobacteria 183 . Enhanced branching, hair length, and density in roots are also established outcomes of PGPR application in plants.
JAS1 maintained plant and soil health under intermittent soil drought. While maintaining the above experimental design we also separately tested the effect of intermittent soil drying over JAS1-treated seedling growth. Watering of soil beds was completely withheld post 6 weeks of sowing for 10 days. We witnessed gradual leaf squinting and drooping in shoots in the control sets (Fig. 8a) as opposed to their normal growth in the same period under well-watered regimes. Shoots in the JAS1 treatment trays however maintained their overall growth stature at all times till the 10 days (Fig. 8a). Upon rewatering most seedlings in the control sets could not regain the healthy stature but wilted while those in JAS1 treatment continued to propagate normally without any signs of growth detriments (data not shown). In addition to this, we also noted a peculiar soil cracking phenomenon, usually witnessed in farm fields under conditions of drought. Close speculation of the tray trials depicted that soil beds with JAS1 treatment had developed comparatively lesser cracking extent than the beds in the control treatment (Fig. 8b). These results indicate that JAS1 treatment of wheat could rescue plants from intermittent soil drought. EPS releasing PGPRs are known to protect plants from drought tolerance by the formation of rhizosheath around the roots and as well by enhancing the soil water uptake 183,184 . These notions warranted testing the effect of JAS1 and its EPS on soil water retention. Soil beds prepared in plastic Petri-dishes were completely drenched with either MSB resuspended JAS1 pellet or water resuspended EPS or just water and were followed with a continuous drying regime. Under these trials, water-drenched soil beds showed complete drying within a week (Fig. 8c), however at this time point soils in the EPS and JAS1 treatment dishes still retained ca. 8% and 16% water and with their complete drying scenes delayed to ca. 11th and 13th day, respectively. These results indicate that JAS1 is effectively able to enhance water retention of soil and that water-soluble EPS released by JAS1 could be the possible governing factor. Soil cracking extent was well reduced in JAS1 and EPS treatment than in control treatment (data not shown).
These results support our notion of reduced soil cracking effect in JAS1-dosed soil beds. Higher water retention in JAS1 treated soil bed wrt that with EPS treatment was obviously due to the presence of MSB supportive to JAS1 growth and EPS secretion. The presence of MSB may be analogous to the presence of wheat plants in tray trials which might accordingly govern growth and EPS release in soil beds from JAS1 (dosed actually as water resuspended inoculum). Besides, positive growth and EPS secretion in JAS1 on agar solely supplemented with extracted ST, wheat biomass, and/or coconut water were also verified from other ongoing studies (data not shown). JAS1 growth and/or productive EPS release in soil beds otherwise treated exclusively with waterresuspended inoculum was negligible (data not shown).
The water solubility of the EPS may allow its expansive reach, deposition, and coverage as well as that of the releasing bacteria also abreast by various other mechanisms (bacterial swimming and swarming motility) to other portions within the soil profile. The ability of such bacteria to proliferate, within roots and/or on rhizoplane would allow extended cushioning besides the other PGP effects delivered consistently to the plant host, especially under abiotic stresses such as drought. In bigger field settings, how this may translate into crop productivity, and soil performance, otherwise also under various stressors is yet under testing for JAS1 (data not shown). Many PGP bacteria are already in commercial use as bioinoculants and biofertilizers 185-188 . JAS1 also enhances in vitro growth of wheat seedlings. To correlate with the PGP effects of JAS1 in tray trials (discussed above), we also attempted it's in vitro co-cultivation with surface sterile wheat seeds, established for a month on MSA in PTC jam jars (Fig. 9a,b). As expected, wrt that in controls, profound lengthening of shoots (Fig. 9c), roots (Fig. 9d), and an apparent increase in root branching (Fig. 9e) were evident. Other than these, bacterization also enhanced root hair growth both in its length and density (insets in Fig. 9e) as also seen respectively with tray trials. The root branching effect from JAS1 (in vitro), however, was prominent throughout the length on lateral (feeder) roots (Fig. 9e) as opposed to the lateral root branching points occurring immediately close to the root-shoot interface (in tray trials). These prominently branching feeder roots themselves appeared hovering over the culture media surface and rarely penetrating the media themselves except for their new branch outgrowths and otherwise also the primary roots (Fig. 9a,b,e). This may seek its support in that feeder (lateral) roots could be affinitive to EPS secreted readily by JAS1 overgrowing on the upper media profile (Fig. 9b) wrt control (Fig. 9a). Also, possibly branching could hail from the high IAA (biosynthesis from JAS1 as shown before) released along with the EPS. Other than these, gradually expanding EPS overlay on the media surface obviates more surface fluidity offering lesser relays, especially to the extending roots in agar (compared to that understandable with 0.8% gel strength or compactness).
In the case of tray trials, such compactness would be drastically higher in silica-contained soils, the latter which also contracts during soil drying phases showcased with the cracking phenomenon (Fig. 8b). In such scenarios, feeder roots might prefer extending more at the upper gel (and analogously also in topsoil) profile than traversing steep depths provided EPS overlaying is provisioned from JAS1. More prominent length enhancement and branching frequency in these feeders would allow for easy and quick acquisition of readily available surface  www.nature.com/scientificreports/ shoot-root interfaces or more appropriately the topsoil profile could effectuate much proportionately possibly due to locally higher IAA sequestration following the booster dosing of JAS1 hence, it may obviate higher PGP effects of the endophytes localized in this region. A better shoot growth would corroborate in light of the above effects on root and/or other PGP activities in the root, soil, and whole plant delivered by the endophyte, its EPS, and/or both 189 . Additional growth enhancement from JAS1 treatment in contrast to control sets seen on in vitro wheat seedlings propagated over a widely used plant-specific MS media composition would thus vouch for the positive PGP properties sufficed by JAS1.
JAS1 enhances the growth of chickpea. Like in wheat we uncovered similar PGP effects of JAS1 upon trials also with a commercially grown legume crop, chickpea. JAS1 treatments offered significantly better growth, witnessed in the higher shoot length increments compared to plants in the control sets (Fig. 10a,b). This effect was evident right after two days from the sowing date and the trend (higher shoot length in JAS1 than in controls) remained consistent till the end of the experiment (Fig. 10a). Within about 15 days post sowing, shoots (in sets under JAS1 treatment) were seen with an averagely doubled length than those in the control treatment ( Fig. 10a,b). JAS1 led growth enhancement was also evident with the roots which reached the bottom of the pot more rapidly (in almost all pots wrt control; Fig. 10c). Both these effects correlate with those seen with JAS1 trialed over wheat and could hence be more supportively attributed to the positive PGP effects of JAS1 (assayed biochemically before). As surmised from Fig. 10d, where a scoop was randomly taken from the top soils (from pots under trials respectively on the 30th day while the watering regime was halted beforehand on the 27th day), JAS1 treated soils remained moist even after the end of the watering regime (tightly held soil particles maintaining the scoop shape) as compared to the dried-out control soils (loosely held soil which could not maintain the scoop shape). Justifiably, JAS1 could productively synthesize EPS (as already assayed and shown before) while being attached to plants throughout the trial (as well as when applied with a booster dose) and helped the soil by retaining enough water. Observations post soil drain-out (Fig. 10e,f) suggest significant changes to root architecture in JAS1 treated chickpea wrt the control treatment. Comparatively, most of the roots under JAS1 treatment (except the primary) were slightly thinner (Fig. 10e,f), however, primary root length (Fig. 10e), as well as both secondary roots' number and length, exhibited significant increments (Fig. 10e). Additionally, as seen in other plant trials (Figs. 5, 7, and 9), JAS1 treatment with chickpea also offered a substantial increase in root hair density and length (Fig. 10f, magnified root mid-sections in upper panels and sections close to and including the tip). Nodule formation, however, could not be seen in any chickpea replicates in any of the trials as opposed to that reported to effect with an IC59 strain of AP 34 .

Plant physiological profiles under JAS1 treatment.
To validate growth enhancement in both wheat and chickpea seedlings under independent treatments with JAS1, changes in the standard physiological plant growth parameters viz., total chlorophylls, carotenoids, proline, phenols, flavonoids, and sugars were analyzed (Fig. 11). Overall chlorophyll content was significantly enhanced by 50.03% and 61.71% in JAS1 treated wheat and chickpea seedlings, respectively. Chlorophyll a and b contents were also shown to increase in both wheat (69.65% and 69.56% res.) and chickpea (150.64% and 67.19% res.). Similarly, wrt the control treatments, JAS1 www.nature.com/scientificreports/ affected significantly enhanced total carotenoids in wheat (8.35%) and with a drastic increase in chickpea (42.98%). Such enhancements in the photosynthetic pigments' profile would understandably corroborate to overall productive physiology in plants 190 . Proline is an amino acid and a key osmolyte that offers macromolecular stability to organellar structures, signal factors, enzymes, and membranes within the cells and is vital to various housekeeping tasks such as redox balancing for stress adaptations 191,192 . JAS1 treatment significantly enhanced total proline content in both wheat (74.6%), and chickpea (27.03%). Phenols protect plants under abiotic stressors by deactivating ROS and effectuating anti-oxidation 193 , and as such total phenols' content was witnessed significantly incremented in both wheat (25.23%) and chickpea (22.98%) under JAS1 treatment as compared to controls. Nonetheless, total flavonoids as well increased in wheat (57.68%) and chickpea (61.47%) under JAS1 treatments. These include specialized secondary metabolites which accumulate to counteract various plant stresses 194 . Enhanced physiological profiles under PGPR treatments would well verse the plants' photosynthetic performance, which should interpret an increase in total leaf sugar content 195 , as significantly accounted in both wheat (53.89%) and chickpea (48.02%) under JAS1 treatments. Sugar accumulation also fosters osmoregulatory requisites in plants, especially under stress 196 . Taken together, these (Fig. 11) and the outcomes from various plant treatments (Figs. 5, 6, 7, 8, 9 and 10) support the assayed PGP credentials (Table 1) of the novel AP strain, JAS1, while its EPS production attribute either exclusively improves soil health by optimizing water retention, reducing desiccation-cracking (Fig. 8b,c); and/or also, understandably by virtue of the PGP activities, that contribute to soil health variously via mineralization, ammonification and other processes.
We show that JAS1 priming enhances the root branching and root hair density in ST, wheat, and chickpea. Root hairs appear as single tubular cells, commissioned for upsizing the rhizospheric area and thereby enhancing water and mineral uptake. They are the preferred sites for entry and colocalization by PGPRs, deeming particularly advantageous for plants thriving in low phosphorus soils [197][198][199][200] . Rock phosphates are insoluble and hence immobile components in soil and pose a criticality for plants by being such in a non-assimilable form of the microelement. Some plants secrete exudates to acidify soils and solubilize such phosphate forms 201 and/or otherwise recruit their phosphate solubilizing endophytes for this [202][203][204] . Many of these endophytes also concomitantly ease nutrient acquisitions in plants by productive remodeling of root architecture and its development viz., growth in size and number of roots, their branching, and root hairs 202,[205][206][207][208] . We glimpsed JAS1 colocalizing in root hairs of JAS1-primed PTC-raised ST explants (Supplementary Video 3) and intriguingly are exploring its transmissibility further up beyond the root-shoot interface (data not shown). Nonetheless, root hair length www.nature.com/scientificreports/ and density may correlate positively to high auxin content in the root tip, the latter has been shown recently to stimulate Arabidopsis growing over high Cd and As soils 209 . As shown, JAS1 delivers phosphate solubilization, and high IAA content and enhances root hair length and density besides other root growth parameters (see sections with ST, wheat, and chickpea treatments with JAS1). Surprisingly, by a mechanism yet unknown, ST roots are reported to show a high absorption and low translocation of soil Cd 70 . Possibly PGP endophytes contribute to this and the above features in such plants by eliciting auxin levels 210 . Reckoning that Sansevieria succulents can survive well in low fertility soils and bear the dexterity to withstand various biotic and abiotic cues, presumably endophyte(s) like JAS1 and others may suffice it and other Sansevieria species in sustenance as resilient succulents.

Conclusions
This manuscript stands as the first report on the isolation of a novel AP strain, JAS1, from the genus Sansevieria and as the second to any reported successful attempts on isolation, characterization, and bioprospecting of any bacterial endophyte(s) from Sansevieria plants. The first study reported a PGP (IAA producing and ACC deaminating)-cum-TMA resistant bacterial endophyte, Bacillus cereus strain EN1, isolated from S. kirkii, which was shown to enhance the TMA removal potential of its host 87 . In our study, the JAS1 strain of AP (isolated from ST) tested positive for multiple PGP properties, besides also for enormously producing EPS. As depicted by various morphological and physiological parameters, JAS1 leads to growth enhancement in wheat and chickpea seedlings, as also apparent in its priming trials with the ST host. Besides plants' growth requisites, water is essential for soil dexterity to enable mineralization, bio-fertility processes, and productive microbial dynamics within it. Conventional cropping systems have always faced the dilemma of low irrigated soils due to uneven rainfall, quick water seepage down to the underground table, and only a small fraction of it is available to crops and for a limited time, especially in sandy soils and/or areas under drought. Under an intermittent soil drying Figure 12. A compelling view of JAS1's prospects: PGP properties (shown in grey boxes on the right) JAS1 (shown as yellow rods) positively influence shoot and root growth parameters (positive influences without JAS1 treatment shown with green arrow-heads, yellow for control treatment) as well as by its EPS production enhance soil water holding capacity and negates soil cracking, a usual symptom of low soil moisture which variously impacts plant performance and soil microflora. Water retention and soil holding capacity would also be greatly enhanced with a JAS1-induced increase in root branching, root hair length, and density especially in the topsoil region, thus collectively obviating enhanced and prompt absorption of minerals and growth-related materials from the soil. JAS1 and its secreted EPS may work (as other PGPRs) to enrich the soil micro and macroflora by restricting and allowing the growth of pathogenic invasive microbes and attracting other species with symbiotic interests.
Scientific Reports | (2022) 12:21330 | https://doi.org/10.1038/s41598-022-25225-y www.nature.com/scientificreports/ regime, JAS1 and its EPS could minimize desiccation-cracking by enhancing soil water retention. This presumably also rescued and rejuvenated wheat seedlings from drought stress. These multitudes of attributes in JAS1 which collectively improve both plant and soil health are summarized in a compelling infographic (Fig. 12). Bioprospecting of PGP endophytes isolated from Sansevieria and other succulent plants would prove a boon as newer bio-inoculants for improving commercial crop and soil performances. Other than these, heightened production of bioactives like EPS in these endophytes would allow considering cost-effective alternatives for industries relevant to its plethora of applications.

Data availability
All data generated or analyzed during this study are included in this published article [and its supplementary information files]. All deduced nucleotide sequences in the bacterial isolate corresponding to its 16S rRNA, recA and atpD genes were deposited in NCBI's GenBank (with publicly available accession numbers MW827601, MZ741443 and MZ741444, respectively).