Maize rhizosphere soil was collected at a depth of 5–15 cm randomly, from five locations on a maize field at the Agricultural Research Council (ARC) Potchefstroom South Africa (Latitude 26° 43' 49.2” and Longitude 27° 3' 43.3”). Large particles were removed by sieving with a 2 mm sieve, and a composite soil sample made. The physicochemical characteristic of the soil was analyzed by the Agricultural Research Council (ARC) analytical laboratory, Africa (Supplementary Table 1). Soil organic carbon and organic matter were determined using the Walkley-Black titration method (Walkley and Black, 1934; Motsara and Roy, 2008). The method of Olsen and Sommers (1982) was used for determination of available phosphorus. Available potassium was determined by ICP-OES on an ammonium acetate extraction of the soil (Toth and Prince, 1949; Motsara and Roy, 2008), while total nitrogen was determined by the Kjeldahl method (Bremner, 1960). Soil moisture was measured using the method of Motsara and Roy (2008).

Bacteria strains were obtained using a pour plate method on nutrient agar (NA; Merck, South Africa) according to the method of Abiala et al. (2015), and incubated for 24 h at 28 ± 2°C. Pure cultures of strains were obtained which were subsequently characterized using morphological features and biochemical tests. The method of Cheesbrough (2006) was used for Gram stain, catalase activity, oxidase, nitrate reduction and starch hydrolysis tests. Hydrogen cyanide (HCN) was determined using the method of Kavamura et al. (2013). Development of orange color after 48–72 h at 28°C incubation was indicative of HCN production. Ammonia production was also determined using the method of Kavamura et al. (2013) by inoculating freshly grown cultures (18–24 h) of each isolate in 10 mL of peptone water and then incubated at 28°C for 48 h. Ammonia production was assessed by the development of faint yellow to deep yellow color by the strains in different proportions in 1.5 μL Eppendorf tubes containing 50 μL of Nessler's reagent (Dey et al., 2004).

Three bacterial strains Pseudomonas sp. MRBP4, Pseudomonas sp. MRBP13, and Bacillus sp. MRBP10 were selected for greenhouse study based on their plant growth-promoting traits and ability to withstand low water activity in vitro. 1-aminoccylopropane-1-carboxylate (ACC) deaminase activity was determined in accordance with the method of Ali et al. (2013) using tryptone soy broth.

The method of Kavamura et al. (2013) was used to determine indole-3-acetic acid (IAA) production by culturing the strains in triplicate in tryptophan nutrient broth (Oxoid Chemicals). The cultures were incubated with constant shaking for 48 h at 28 ± 2°C. After centrifugation of turbid cultures for 20 min at 10,000 rpm, 2 mL of the supernatant was mixed with 4 mL of Salkowsky reagent. After 20 min of the mixture kept in the dark, light absorption at 530 nm was measured using a spectrophotometer (Themospectronic Merck SA). The IAA production by each isolate in mg L⁻¹ was determined by comparing the light absorption to a standard curve prepared from pure IAA solutions (5, 10, 20, 50, and 100 mg L⁻¹).

Strains were grown in TSB medium amended with sucrose (10%) at pH 7.5 containing discs of sterile 5 mm filter paper in petri plates. Each disc was inoculated with 2 mL culture of each isolate for EPS production and incubated at 28°C for 48 h. The presence of EPS was confirmed from the precipitation observed after mixing a part of the mucoid substance formed around the discs in 2 mL of absolute ethanol (Paulo et al., 2012; Kavamura et al., 2013).

Each isolate was screened for phosphate solubilization on Pikovskaya's agar (PKA) medium according to the method of Paul and Sinha (2017). The strains were obtained using pour plate method and incubated for 48 h at 28 ± 2°C. Distinct colonies with halo zones were identified and the halo zone surrounding the colonies measured. Phosphate solubilization index was calculated using the formula of Premono et al. (1996).

The rhizobacteria strains were tested in vitro for the ability to grow on a medium with low water activity (0.919Aw; Kavamura et al., 2013). This was achieved by inoculating TSA medium (10%) supplemented with 450 g L⁻¹ D. sorbitol (Sigma, France), with 24 h culture of each isolate, incubated at 40°C. Strains were also tested on their ability to withstand high temperature of 50°C by inoculating 10 mL of sterilized TSB with 24 h culture of each isolate. The inoculated test tubes were incubated at 120 rpm for 24 h. Subsequently the optical density (OD) was measured at 600 nm using a UV spectrophotometer (Thermo Spectronic; Merck, South Africa). The strains were also evaluated for water-deficit stress tolerance by inoculating each isolate in 10 mL of sterilized TSB broth containing 20% polyethylene glycol (PEG) 8000 and subsequently incubated for 24 h at 28°C on a rotatory shaker. The OD was measured at 600 nm using a UV spectrophotometer (Thermo Spectronic; Merck, South Africa).

A ZR Soil Bacterial DNA MiniPrep extraction kit (Zymo Research, Irvine, CA, USA) was used for DNA extraction in accordance with the manufacturer's instructions. Inqaba Biotechnical Industrial (Pty) Ltd, Pretoria, South Africa sequenced the purified PCR products using PRISMTM Ready Reaction Dye Terminator Cycle Sequencing Kit. The partial 16S rRNA gene of each bacterial isolate was amplified Universal bacteria primers 341F and 907R (Fukuda et al., 2016). A 25-μl volume cocktail was made composed of 12.5 μL of 2x PCR Master Mix (0.05 U/μl Taq DNA polymerase, 4 mM MgCl₂ and 0.4 mM dNTPs (Fermentas), 0.5 μL of forward primer (341F), 0.5 μL of reverse primer (907R), 2.0 μL of template DNA, and 9.5 μL of nuclease free water. The PCR cocktail was subjected to 35 cycles in a Bio-Rad C-1000 thermocylcer beginning with an initial denaturation temperature of 94°C for 2 min, then 94°C for 30 s followed by an annealing temperature of 59°C for 1 min, extension at 72°C for 2 min and a final extension at 72°C for 8 min.

The chromatographs obtained were subjected to quality check using Chromas Lite version 6.5 (Technelysium, 2012). The cleaned chromatographs were edited using Bio Edit Sequence Alignment Editor (Hall, 1999). Subsequently, consensus sequences were generated and a Blast search made on the NCBI database (www.ncbi.nlm.nih.gov/Blast.cgi), using the Basic Alignment Search Tool (BLASTn) in order to identify the closest representative strains of the bacterial strains. Sequences of the strains were deposited in the GenBank and accession numbers given by the GenBank (Supplementary Table 2). The evolutionary history was inferred using the Neighbor-Joining method (Saitou and Nei, 1987) and evolutionary distances computed using the Tamura-Nei method (Tamura and Nei, 1993). Evolutionary analyses was conducted in MEGA11 (Tamura et al., 2021).

A pot experiment was conducted in the greenhouse at the North West University to assess the effectiveness of the rhizobacterial strains for enhancement of maize growth promotion under water-deficit stress. Field soil was used for the experiment with two maize varieties: a drought tolerant genotype MR44 and a recombinant inbreed line S0/8/W/I137TNW//CML550. The seeds of both genotypes were surface sterilized using 70% ethanol for 5 min, treated with 2% sodium hypochlorite for 10 min with a drop of Tween 20, and rinsed at least thrice with sterile distilled water. The bacteria strains MRBP4, MRBP10, and MRBP13 were prepared by growing each isolate in 500 mL of sterilized TSB on an orbital shaker at a speed of 120 rpm for 72 h at 28°C. Subsequently, bacterial cells were harvested by centrifugation for 15 min at 10,000 rpm, and the pellets washed twice with sterile distilled water and re-suspended in phosphate buffer (0.01 M, pH 7.0). The OD was adjusted to an absorbance of 1.4 at 600 nm using a spectrophotometer (Thermo Spectronic, Merck, SA). Pre-germinated maize seeds of each genotype were inoculated by placing them in a suspension of each bacterium alone and co-inoculated (10⁸ CFU mL⁻¹) for 12 h. Afterwards, the inoculated seeds were air dried in a laminar flow cabinet for 1 h. Control seeds were suspended in sterile distilled water for the same period, and air dried. Three inoculated and control seeds were sown in 36 cm pots containing 10 kg of sterile soil and thinned to one plant per pot 2 weeks after germination. The experiment was conducted between October and December 2018 and 2019. The average maximum day temperature ranged between 32 and 40°C as this was the summer period. The experiment was set up as a 2 × 2 × 8 factorial in a completely randomized design with three replications (Figure 1). The factors were:

Two maize genotypes-M₁-Genotype S0/8/W /I137TNW/ /CML550 an inbreed line, M₂-Genotype MR44 a drought tolerant variety.

Two levels of water field capacity partially watered at 50% FC (450 mL of water daily), and well-watered at 100% FC (900 mL of water daily).

Eight levels of bacterial inoculation-B₀, No inoculation; B₁, inoculation with Pseudomonas sp. MRBP4; B₂, inoculation with Pseudomonas sp. MRBP13; B₃, inoculation with Bacillus sp. MRBP10; B₁B₂, co-inoculation with Pseudomonas sp. strain MRBP4 and Pseudomonas sp. strain MRBP13. B₁B₃, co-inoculation with Pseudomonas sp. MRBP4 and Bacillus sp. MRBP10; B₂B₃, co-inoculation with Pseudomonas sp. MRBP13 and Bacillus sp. MRBP10; and B₁B₂B₃, inoculation with the three strains Pseudomonas sp. MRBP4 + Pseudomonas sp. MRBP13 + Bacillus sp. MRBP10. Two weeks after germination, water stress was imposed. 50 mL of each bacteria inoculum was used for root inoculation of seedlings at the 2nd and 4th week after commencement of water stress.

Plants were harvested 49 days after sowing. Weekly data was collected on the leaf area, number of leaves and shoot length. The shoot and root fresh weights and the shoot and root lengths were measured at the end of the 5th week of drought imposition. Shoots of each plant were cut at the base and dried at 72°C for 48 h, and dry weight taken using an electronic scale. The roots of the same plants with soil were harvested, washed carefully to separate roots, dried at 72°C for 48 h, and weighed using an electronic scale.

The leaf relative water content (RWC) for each treatment was measured according to the method described by Naveed et al. (2014) using the Flag leaves. The fresh weights of cut leaves from each treatment were measured and placed in distilled water in a 50 mL tube for 24 h at 4°C in the dark. Subsequently, the soaked leaves were carefully blotted with tissue paper and fully turgid weight measured. The dry weight was measured after the leaf samples were oven dried at 72°C for 24 h (Naveed et al., 2014). RWC was computed using the equation described by Naveed et al. (2014).

RWC=[Fresh weight of leaves-Dry weight of leavesFully turgid weight of leaves-Dry weight of leaves]×100 The total soluble sugar content was estimated by the method of Dey (1990). The absorbance was read at 485 nm using a spectrophotometer (Thermo Spectronic, Merck, SA) and the equivalent concentration of total soluble sugar determined against a standard curve prepared by using pure glucose solution. The amount of sugar was expressed as mg g⁻¹ DW⁻¹.

A dimethyl sulphoxide (DMSO) method was used to determine the chlorophyll pigment of the leave samples after harvest following the method of Hiscox and Israelstam (1979). 0.5 g leaf tissue of each sample was cut into little pieces and placed in test tubes containing 7 mL DMSO. The test tubes were incubated in a water bath at 65°C for 20 min. The supernatant obtained was decanted into 15 mL graduated tubes and made up to 10 mL with 3 mL DMSO and assayed immediately. The optical density was read at 480, 645, and 663 nm using DMSO as blank. Chlorophyll a, b, total chlorophyll, and carotenoid contents were determined according to the method of Arnon (1949).

Data collected were checked for test of normality using the “proc univariate” program of SAS software 9.4 (SAS, 2014) and square-root transformed if not in a normal distribution curve before being subjected to analysis of variance (ANOVA). Significantly different means were separated using Tukey's Studentized Range (HSD) and Duncan Multiple Range post-hoc test (p ≤ 0.05). Data were analyzed using the Statistical Analysis System software version 9.4 (SAS, 2014).

The rhizobacteria strains used in this study were identified using biochemical and morphological traits and RNA sequencing. Two of the strains were gram-negative rod-shaped bacteria of the genus Pseudomonas (MRBP4 and MRBP13) while the third MRBP10 was of the genus Bacillus (Table 1). Ammonia production was high in MRBP4 while MRBP10 had a very high catalase response (Table 1). Hydrogen cyanide was only produced by MRBP13. The Maximum Likelihood method used for phylogenetic tree reconstruction based on the Tamura-Nei method (Tamura and Nei, 1993) corresponded with the BLAST results obtained from the National Center for Biotechnology Information (NCBI) database. The Strains had a very high evolutionary relationship with the reference strains, with a bootstrap value of 100% (Figure 1). MRBP4 had a very high similarity with Pseudomonas sp. (KX254973, KJ831556, and MH156648), MRBP13 had extremely high similarity with Pseudomonas sp. (MN94409) as well as Pseudomonas mediterranea (MN 712327) and Pseudomonas lini (MK883138). The third strain MRBP10 had high similarity with Bacillus sp. (HM566589).

The strains were screened in vitro for PGP traits. Exopolysaccharide (EPS) production was highest in Bacillus sp. MRBP10 (0.87 ± 0.03 mm) but was not significantly different from EPS production by Pseudomonas sp. MRBP4 and Pseudomonas sp. MRBP13 (Table 2). However, the ability to solubilize phosphate in the form of tricalcium phosphate, synthesize indole-3-acetic acid and ACC deaminase activity were highest in Pseudomonas sp. MRBP13 (Table 2).

The three strains Pseudomonas sp. MRBP4 (MG953559), Pseudomonas sp. MRBP13 (MG953568), and Bacillus sp. MRBP10 (MG953564) were evaluated for their ability to grow on a medium amended with 20% PEG 8000 and could grow in nutrient broth incubated at temperatures of 28, 45, and 50°C with 24 h bacteria cultures. The strains grew on medium containing 405 g L⁻¹ sorbitol with lower water activity of 0.919Aw incubated at 40°C. Bacillus sp. MRBP10 had the highest growth in medium amended with 20% PEG (0.62 ± 0.01 nm) and also had the highest growth at a temperature of 50°C (Table 3). Since the strains were able to grow with an OD > 0.5 on medium with 20% PEG and grew at a high temperature of 50°C with OD > 0.1, they were regarded as zero-tolerant and temperature tolerant bacteria strains.

From the combined analysis of variance for the 2-year evaluation of maize genotypes under drought stress (2018 and 2019), the main effect of bacterial inoculation had a significant effect (p < 0.05) on the root fresh weight (RFW) of maize seedlings under mild drought stress but a non-significant effect on the shoot fresh weight (SFW) as presented in Figure 2A. Nevertheless, SFW was highest in maize seedlings co-inoculated with Pseudomonas sp. MRBP4 + Pseudomonas sp. MRBP 13 (6.68 ± 0.09 g). Inoculation with Pseudomonas sp. MRBP4 produced the highest RFW which was 15.98% higher than the un-inoculated control. Likewise, co-inoculation of maize seedlings with Pseudomonas sp. MRBP 4 + Pseudomonas sp. MRBP 13 increased the RFW by 6.97% over the un-inoculated control. Root dry weight (RDW) and total plant biomass (TPB) were also significantly highest in maize seedlings inoculated with Pseudomonas sp. MRBP 4 which was increased by 44.19 and 17.69% respectively, over the un-inoculated control. An increment of RDW and TPB by 37.21 and 10.47% was also observed over the un-inoculated control by co-inoculating the seedlings with Pseudomonas sp. MRBP 4 + Pseudomonas sp. MRBP 13 (Figure 2B). Nonetheless, the shoot dry weight (SDW) was highest in maize seedlings inoculated with Pseudomonas sp. MRBP 13 (3.17 ± 0.07 g) which was not significantly different from the co-inoculated plants (3.15 ± 0.07 g).

Table 4 shows the interactive effect of the treatments on biomass production of maize seedlings under mild drought stress (50% FC) and well-watered condition (100% FC). At 50% FC, the co-inoculated treatments had lower SFWs. SFW was significantly higher (p ≤ 0.05) in genotype MR44 sole inoculated with Pseudomonas sp. MRBP4 and then MR44 inoculated with Pseudomonas sp. MRBP13 which were significantly higher than the un-inoculated control by 59.86 and 43.81%, respectively. Similarly, the RFW was highest in maize genotype MR44 sole inoculated with Pseudomonas sp. MRBP4 followed by MR44 inoculated with Pseudomonas sp. MRBP 13. This was higher than the un-inoculated control of MR44 by 80.97 and 45.11%, respectively (Table 4). Co-inoculation with the three rhizobacteria strains; Pseudomonas sp. MRBP 4 + Pseudomonas sp. MRBP13 + Bacillus sp. MRBP 10 (B_IB₂B₃) increased the SFW under well-watered condition in CML550 by 10.02% while co-inoculation with Pseudomonas sp. MRBP 4 + Pseudomonas sp. MRBP13 increased it by 9.98% over the un-inoculated control.

A similar trend was observed in the SDW, RDW and TPB in both genotypes CML550 and MR44 under mild drought stress. RDW and TPB were significantly highest in Pseudomonas sp. MRBP4 under drought stress which was greater than the un-inoculated control by 84.87 and 85.24%, respectively (Table 4). Treatment MR44+B₂ (MR44 inoculated with Pseudomonas sp. MRBP13 under drought stress) also had high SDW (3.23 g), RDW (1.72 g), and TPB (3.08 g). SFW was significantly higher in genotype MR44 by 7.43% compared to CML 550 (Figure 3A). Likewise, the SDW and RDW were also higher in genotype MR44 by 6.29 and 8.39%, respectively over CML550 (Figure 3B).

Bacterial inoculation had no significant effect on the number of leaves, number of roots and shoot length of inoculated plants. However, it had a significant effect (p ≤ 0.05) on the root length of inoculated plants (Figure 4A). Root length was significantly highest in maize genotypes co-inoculated with Pseudomonas sp. MRBP 4 + Pseudomonas sp. MRBP13 + Bacillus sp. MRBP10 (B₁B₂B₃-which was 5.63% higher than the un-inoculated control. No significant difference was observed in the growth parameters in both genotypes (Figure 4B). Nonetheless, CML550 had higher root length (9.56 cm), number of roots (16.40), and shoot length (44.70 cm) compared to MR44 (9.54, 15.54, and 44.67 cm), respectively. Figure 5 shows the leaf area of inoculated plants which ranged from 150.69 ± 0.77 cm² (B₁B₃- Pseudomonas sp. MRBP4 + Bacillus sp. MRBP10) to 212.45 ± 0.87 cm² (B₁B₂- Pseudomonas sp. MRBP4 + Pseudomonas sp. MRBP13).

The combined analysis of variance for both years (2018 and 2019) revealed that bacterial inoculation had a highly significant (p < 0.001) effect on relative water content (Figure 6A). Inoculation of maize seedlings with Bacillus sp. MRBP 10 (B₃) and its co-inoculation with Pseudomonas sp. MRBP4 (B₁B₃) and Pseudomonas sp. MRBP13 (B₁B₂B₃) had higher relative water contents than any of the other treatments. Relative water content increased under drought stressed condition (58.63%) compared to well-watered condition (49.96%). A similar trend was observed for the total soluble sugar (TSS) produced in maize plants inoculated with the rhizobacteria strains. Bacterial inoculation had a significant (p ≤ 0.05) effect on TSS production (Figure 6B). Bacillus sp. MRBP10 (B₃) had the highest production of total soluble sugar which exceeded that produced by the un-inoculated control by 26.13% (Figure 6B). The TSS produced was not significantly different from the co-inoculated treatments B₁B₃ (Pseudomonas sp. MRBP 4 + Bacillus sp. MRBP10) and B₁B₂B₃ (Pseudomonas sp. MRBP 4+ Pseudomonas sp. MRBP 13+ Bacillus sp. MRBP 10). Bacillus sp. MRBP 10 significantly increased the RWC and the TSS in inoculated plants under drought stress (Figures 6A, B).

Bacterial inoculation had a significant effect (p ≤ 0.01) on the soil moisture content (SMC) of inoculated maize plants (Figure 6C). SMC was significantly (p ≤ 0.05) highest in the soil of maize plants co-inoculated (B₁B₂B₃) with Pseudomonas sp. MRBP4 + Pseudomonas sp. MRBP13 + Bacillus sp. MRBP10 (10.21%) which was higher than the un-inoculated control by 37.05%. Sole inoculation with Bacillus sp. MRBP10 (B₃) also had high SMC of 10.13% (Figure 6C).

Combined analysis of variance for the 2018 and 2019 planting season revealed significant differences in the chlorophyll content of inoculated maize plants (Figure 7). Bacterial inoculation had a significant effect (p ≤ 0.05) on the carotenoid contents of the maize plants. Carotenoid content ranged from 0.97 ± 0.09 mg g ⁻¹ fresh weight (Pseudomonas sp. MRBP4 + Pseudomonas sp. MRBP13) to 1.33 ± 0.09 mg g ⁻¹ fresh weight (Pseudomonas sp. MRBP13). Carotenoid content increased by 18.75 and 15.19% more than the un-inoculated control in maize seedlings inoculated with Pseudomonas sp. MRBP13, and co-inoculated with Pseudomonas sp. MRBP13 + Bacillus sp. MRBP10, respectively. Likewise, co-inoculation with Pseudomonas sp. MRBP13 + Bacillus sp. MRBP10 produced significantly (p ≤ 0.01) higher chlorophyll a content in maize plants which was 25.54% higher than the un-inoculated control, but not significantly different from the chlorophyll a produced by maize plants inoculated with Pseudomonas sp. MRBP13 (15.30 ± 0.13 mg g⁻¹ dry weight) or co-inoculated with the three strains; Pseudomonas sp. MRBP4 + Pseudomonas sp. MRBP13 + Bacillus sp. MRBP10 -B₁B₂B₃ (14.59 ± 0.14 mg g⁻¹ dry weight). Chlorophyll b content ranged from 4.05 ± 0.14 mg g⁻¹ dry weight in plants co-inoculated with Pseudomonas sp. MRBP4 + Pseudomonas sp. MRBP13 to 5.66 ± 0.09 mg g⁻¹ dry weight in maize plants inoculated with Pseudomonas sp. MRBP4. The total chlorophyll (Tchl) content significantly increased by 18.92% in maize plants inoculated with Pseudomonas sp. MRBP4 and by 17.57 and 18.62% in plants inoculated with Pseudomonas sp. MRBP13 and that co-inoculated with Pseudomonas sp. MRBP13 + Bacillus sp. MRBP10, respectively (Figure 7).

Evaluation of the physiological response of inoculated maize seeds at 50% FC (mild drought stress) and 100% FC (well-watered) revealed significant differences in the production of TSS and the chlorophyll contents (Table 5). Under drought stress, TSS was significantly increased in treatments CML550 + B₃ inoculated with Bacillus sp. MRBP10, and CML550 + B₁B₃ co-inoculated with Pseudomonas sp. MRBP4 and Bacillus sp. MRBP10, by 48.93 and 54.13% respectively, over the un-inoculated control. Treatment CML550 + B₁B₂ co-inoculated with Pseudomonas sp. MRBP4 and Pseudomonas sp. MRBP13 had the lowest TSS production (130.51 mg g⁻¹ dw⁻¹). Under well-watered conditions TSS was increased in treatments CML550 + B₁B₂B₃ and CML550 + B₃ by 47.72 and 45.65% respectively, over the un-inoculated control.

Carotenoid content increased by 156.25 and 155.20% in maize genotype CML550 inoculated with Bacillus sp. MRBP10 (B₃) and co-inoculated with Pseudomonas sp. MRBP13 + Bacillus sp. MRBP10 (B₂B₃) respectively, over the un-inoculated control under drought stress (Table 5). As observed for the TSS production, treatment CML550 + B₁B₂ co-inoculated with Pseudomonas sp. MRBP4 and Pseudomonas sp. MRBP13 had the lowest carotenoid content (0.33 mg g⁻¹ DW). Under well-watered condition, carotenoid content was significantly higher than other treatments, and 76.10% higher than the un-inoculated control in treatment CML550 + B₂ sole inoculated with Pseudomonas sp. MRBP13. Likewise, treatment CML550 + B₃ produced the highest chlorophyll a (19.26 mg g⁻¹ DW) and total chlorophyll (24.97 mg g⁻¹ DW) contents compared to the other treatments, and were significantly higher than the un-inoculated control plants by 24.74 and 25.60% respectively, under drought stress. Treatment CML550 + B₂B₃ also had increased levels of chlorophyll a and total chlorophyll by 20.47 and 20.12% respectively, over the un-inoculated control under drought stress (Table 5). Under well-watered conditions, treatment CML550 + B₂ consistently gave the highest level of chlorophyll a, chlorophyll b, and total chlorophyll contents compared to the other treatments. Genotype CML550 was more responsive than genotype MR44.