Variovorax sp. strain P1R9 applied individually or as part of bacterial consortia enhances wheat germination under salt stress conditions

Endophytes isolated from extremophile plants are interesting microbes for improving the stress tolerance of agricultural plants. Here, we isolated and characterized endophytic bacteria showing plant growth-promoting (PGP) traits from plants in two extreme Chilean biomes (Atacama Desert and Chilean Patagonia). Forty-two isolates were characterized as both halotolerant auxin producers (2–51 mg L−1) and 1-aminocyclopropane-1-carboxylate (ACC)-degrading bacteria (15–28 µmol αKB mg protein−1 h−1). The most efficient isolates were tested as single strains, in dual and triple consortia, or in combination with previously reported PGP rhizobacteria (Klebsiella sp. 27IJA and 8LJA) for their impact on the germination of salt-exposed (0.15 M and 0.25 M NaCl) wheat seeds. Interestingly, strain P1R9, identified as Variovorax sp., enhanced wheat germination under salt stress conditions when applied individually or as part of bacterial consortia. Under salt stress, plants inoculated with dual consortia containing the strain Variovorax sp. P1R9 showed higher biomass (41%) and reduced lipid peroxidation (33–56%) than uninoculated plants. Although the underlying mechanisms remain elusive, our data suggest that the application of Variovorax sp. P1R9, alone or as a member of PGP consortia, may improve the salt stress tolerance of wheat plants.

Due to long-term selection and coevolution of their bacterial communities, plants thriving in extreme habitats may represent an untapped source for biotechnologically relevant bacteria 13 .Previous studies have showed that the inoculation of rhizosphere bacteria from native plants grown in extreme environments (Atacama Desert and the Chilean Andes) increased the growth and stress tolerance of wheat plants exposed to salinity and water shortage conditions 14,15 .However, to date, mostly endophytes of crops have been investigated regarding their function in and importance for plant growth under stress conditions.
In agriculture, PGPB have been primarily applied as inoculants consisting of individual strains.However, there are increasing attempts to develop tailored bacterial consortia to harness synergetic beneficial effects not obtained by using single species only 16 , although the mechanisms leading to such synergetic PGP effects remain vastly elusive.For instance, several studies have demonstrated that two-or three-member consortia enable beneficial plant physiological and biochemical responses to abiotic stress 14,15,17,18 .In another study, Sun et al. 19 described synergistic activities between Bacillus velezensis SQR9 and an endophytic Pseudomonas stutzeri strain leading to increased plant growth and salt stress tolerance of cucumber plant seedlings.Therefore, bacterial interactions contribute to the effectiveness of bacterial consortia.
Here, we hypothesized that endophytic bacteria associated with extreme Chilean biomes (Atacama Desert and Patagonian) can be applied individually or as bacterial consortia to improve the germination of seeds and growth of wheat plants exposed to varying salt stress conditions (0.15 M and 0.25 M NaCl) under greenhouse and field conditions.In this study, wheat was chosen because it is highly relevant for Chilean agriculture, which is being significantly impacted by climatic events 20,21 , including higher temperatures and droughts, with concomitant lower moisture and higher concentrations of salts in soils.
Partial sequencing analysis of the 16S rRNA gene indicated that the 42 endophytic strains with assayed PGP traits belonged to the genera Bacillus (23 strains), Serratia (8), Staphylococcus (4), Pantoea (2), Acinetobacter Table 1.Number of isolates on LB and NM-1 minimal medium during sequential selection of putative plant growth-promoting (PGP) bacteria from plants in the Atacama Desert (Distichlis spicata and Pluchea absinthoides) and Chilean Patagonia (Gaultheria mucronata and Hieracium pilosella).PGP is based on the detection of representative plant growth-promoting traits.ERIC-PCR genotyping of strains by enterobacterial repetitive intergenic consensus, NBRIP National Botanical Research Institute's phosphate growth medium supplemented with insoluble (inorganic) tricalcium phosphate as sole P source, PVK Pikovskaya's medium supplemented with insoluble tricalcium phosphate as sole P source, PSM phytate-screening medium supplemented with insoluble (organic) phytic acid dodecasodium salt hydrate as the sole phosphorus source, DF-ACC growth in DF medium supplemented with 1-aminocyclopropane-1-carboxylic acid (ACC) as the sole source of nitrogen and carbon, auxin production of tryptophan-dependent auxins revealed in Luria-Bertani medium by Salkowski reagent.a Isolates showing inorganic and organic phosphate utilization.4).

Effects on physiological and biochemical responses of salt-exposed wheat plants
Greenhouse experiments were conducted to further test the effects of triple consortia 4 and 8 containing Variovorax sp.P1R9 on wheat seedlings by analyzing shoot and root biomass, leaf chlorophyll contents, stomatal conductance, lipid peroxidation (TBARS) and antioxidant enzymes (SOD and CAT) under salt stress conditions (Fig. 1, Table 4).The analyses showed that both consortia generally had beneficial impacts on the growth and well-being of plants exposed to salt stress.

Plant biomass
Uninoculated wheat plants showed significantly reduced (P < 0.05) root and shoot biomass upon exposure to 0.25 M NaCl salt compared with the salt-free control (Fig. 1A).Consortium 4 significantly increased (P < 0.05) shoot biomass (7.9 g) relative to uninoculated controls at 0.25 M NaCl (3.3 g).Similarly, Consortium 4 further caused higher root biomass at 0.25 M NaCl (15.3 g) relative to uninoculated controls at 0.25 M NaCl (3.6 g) and with the salt-free control (9.3 g).Compared with uninoculated controls (3.6 g), Consortium 8 also significantly promoted (P < 0.05) root growth at 0.25 M NaCl (9.1 g) but did not lead to beneficial effects on the shoots of wheat exposed to salt.

SPAD chlorophyll content
Leaves of uninoculated wheat plants exposed to salt showed lower, but not significant (P < 0.05), SPAD chlorophyll contents (4.3 and 3.8 SPAD units) relative to NaCl-free controls (4.8 SPAD units).Plants inoculated with either of the two triple consortia had similar SPAD indices compared to uninoculated control plants (Fig. 1B).

Stomatal conductance
Exposure of seedlings to 0.25 M NaCl resulted in an ~ 20% decrease in stomatal conductance in wheat leaves (Fig. 1C), which could be counteracted by the presence of Consortium 4 (but not of Consortium 8).However, both triple consortia had no significant (P < 0.05) beneficial impact on stomatal conductance at 0.15 M NaCl.

Lipid peroxidation
The presence of either of the triple consortia led to significantly (P < 0.05) lower TBARS levels (~ 9-17 nmol MDA g −1 FW) than that in uninoculated controls (~ 22 nmol MDA g −1 FW), indicating a stress-reducing effect of the inocula (Table 5).

Superoxide dismutase
Exposure of uninoculated seedlings to salt (0.15 M NaCl) led to twofold higher SOD activity than that in saltfree controls (Table 5).Plants inoculated with Consortium 4 showed higher SOD activity (33% and 121%) than uninoculated control plants at 0.15 M and 0.25 M NaCl, respectively.In contrast, similar SOD activity as that in uninoculated controls was observed in the presence of Consortium 8 at both salt levels.This indicates that bacterial inoculation induced the regulation of antioxidative enzymes against salt stress.
Sequencing proposed similar abundance-based coverage estimates (ACE); however, it revealed no differences between controls and salt-exposed plants regardless of treatment.ACE values ranged from 4160 to 4976 (0.25 M NaCl), 4314 to 4841 (0.15 M NaCl) and 4306 to 4565 (salt-free controls) (Fig. 3A).The Chao1 abundance index, as an indicator of richness changes across samples, was similar for all treatments.Likewise, sequencing coverage values (at 97% similarity) showed no differences.
The Shannon index, a measure of alpha diversity, did not show significant differences (P < 0.05) among treatments, except in plants treated with Consortium 8 at 0.15 M NaCl (Fig. 3A).Similarly, no differences in the Simpson diversity index were found among any of the plant treatments.Finally, nonmetric multidimensional scaling (NMDS) analysis revealed a highly heterogeneous composition of rhizosphere bacterial communities of wheat seedlings (Fig. 3B).Analysis of similarity (ANOSIM) thereby confirmed a separation between salt-free controls and salt treatments, where NaCl explained 51.5% of bacterial community variations (P < 0.001).However, no clustering in response to inoculated and control treatments was found (Fig. 3B).

Discussion
In this study, we isolated and characterized putative endophytic phosphobacteria with multiple PGPB traits associated with the roots of plants growing in the arid Atacama Desert and semiarid Patagonian regions in Chile.Isolates with the most promising PGP traits were tested as single strains, in consortia, and in combination with previously isolated PGP rhizosphere bacteria for their effects on the germination of wheat plants exposed to varying salt stress conditions in the laboratory.Of the initial 68 endophytic phosphobacteria selected and evaluated, 35 showed combined halotolerance (Table 2) with potential PGP traits (Table 1), confirming previous reports on combined halotolerance and ACC deaminase activity in endophytes from extreme habitats 14,22 and the potential of endophytic bacteria as beneficial plant inoculants 9 .ACC deaminase activity may play a crucial role in plant stress tolerance by lowering ethylene levels through ACC deamination 23 .It has been described that environmental bacteria might express wide variability in ACC-degrading activity 24 .Our reported ACC deaminase activity was higher (~ 5-28 µM α-ketobutyric acid mg −1 h −1 ) than that in avocado plant endophytes (~ 0.2-3.6 µM α-ketobutyric acid mg −1 h −1 ) 18 but was lower than that in plant isolates derived from the Atacama Desert or Antarctica (~ 13-40 mmol α-ketobutyric acid mg protein −1 h −1 ) 12,14 .The studied endophytes also produced auxin via the tryptophan-based pathway (Table 2), while only four isolates produced IAA at elevated concentrations in the range of 0.2-1.3mg L −1 (Table 2), i.e., values similar to those in endophytes of Thymus vulgaris 25 but lower than those in endophytes of rice seedling roots (34.2 mg L −1 ) 26 .Hypothesizing that endophyte bacteria of wild plants in extreme environments possess PGP effects on, e.g., the germination of salt-exposed weed seedlings, we selected five highly halotolerant strains (10% NaCl) with high ACC deaminase activity for further testing.16S rRNA gene sequencing characterized them as species of Variovorax, Staphylococcus, Bacillus, and Curtobacterium (Table 3), all genera that have been previously described as symbionts of the rhizo-and endospheres of wild plants 27,28 .All five strains were tested individually, and the three strains with the highest GI at 0.15 M NaCl were used in dual and triple consortia.Although fractions of seeds germinating under salt exposure were lower than in salt-free controls, the presence of Variovorax sp.P1R9, Staphylococcus sp.P1R11, Bacillus sp.P1R13, or Curtobacterium sp.P2H47 led to increased GI (112-163%; Table 3) and hence to positive effects on seed germination at 0.15 M NaCl.www.nature.com/scientificreports/Interestingly, Variovorax sp.P1R9 thereby showed the highest GI at both salt levels (Table 3).This finding supports previous descriptions of PGP by Variovorax on PGP 29 and its association with plant species, such as Beta vulgaris 30 , Gossypium hirsutum 31 , and Solanum lycopersicum 32 .Comparative genomic analysis suggests that members of the genus Variovorax can use a wide range of organic compounds, including toxic compounds such as 4-hydroxybenzoate and BTEX 33,34 .Such metabolic versatility may foster its symbiotic lifestyle with diverse plant taxa and allow it to cope with multiple stress conditions.Variovorax sp. has been used to study microbe-microbe and microbe-plant interactions in model systems 35 .For instance, Chen et al. 36 found that the Variovorax paradoxus 5C-2 strain with ACC deaminase activity enhanced leaf growth and flowering of Arabidopsis thaliana.Moreover, Bessadok et al. 37 reported that endophytic Variovorax sp.CT7.15 improved the in vitro seed germination, nodulation and growth of Calicotome villosa plants.They also found more plant growth promotion (e.g., phosphate solubilization and ACC deaminase activity) and nodulation when plants were coinoculated with wild-type rhizobia in arid Tunisian soils.In our study, we also assessed the possible synergetic effects of dual consortia of Variovorax sp.P1R9 with either Staphylococcus sp.P1R11 or Curtobacterium sp.P2H47 (consortia 1-3) as well as a triple consortium of all three strains (Consortium 4) on wheat seed germination (Table 3).We found that the consortia containing Variovorax sp.P1R9 consistently exhibited the highest GI values, suggesting that Variovorax sp.P1R9 had a beneficial effect on the germination of seeds exposed to salt stress (Table 3).However, competence or synergisms of Variovorax sp.P1R9 with other members of the consortia need to be demonstrated in further studies.
Given the beneficial role of Variovorax sp.P1R9 in endophytic consortia, we also tested its possible synergetic PGP effects in consortia with two known stress-alleviating PGP Klebsiella sp.strains isolated from a prior Atacama Desert altiplano 14 .Single or joint application (Consortium 7) of Klebsiella sp.strains resulted in intermediate to low salt stress-alleviating effects, and the combination with Variovorax sp.P1R9 strains in dual (consortia 5 & 6) or triple consortia (Consortium 8) led to a higher GI (Table 3) than the presence of only Klebsiella sp.Although the mechanism of such synergistic effects remains unclear, it may be associated with the ability of Variovorax strains to convert various substrates produced by other biota (e.g., acyl homoserine lactones (AHLs) and alkyl/aryl-sulfonates) into cell biomass 34 .This metabolic capacity also suggests that Variovorax may also exert significant roles in plant nutrient acquisition and natural biogeochemical cycling of soil nutrients.For instance, studies have shown that V. paradoxus 5C-2 led to increased soil nitrogen contents and increased plant stress, lowering abscisic acid concentrations in Pisum sativa plants 38,39 .Other studies have also shown that the production of ACC deaminase and IAA by Variovorax strains improve plant salt stress tolerance 36,40,41 .PGP effects of Variovorax sp.HRRK 170 were also linked to an optimal inoculum cell density and subsequent colonization of roots of Chinese cabbage and green pepper 30 .
Hence, we further studied the effects of the Variovorax sp.strain P1R9 within consortia 4 and 8 on the physiological and biochemical responses of salt-exposed wheat plants in greenhouse experiments.Endophytic Consortium 4 increased the biomass of shoots and roots in seedlings exposed to 0.25 M NaCl, while mixed Consortium 8 only had stress-alleviating effects on root biomass (Fig. 1).This is in agreement with the results of Barra et al. 18 , which showed stronger PGP effects of endophytic than of rhizosphere bacterial consortia upon inoculation of salt-exposed avocado plants.The close innate interactions of endophytes with host plants seem to be a driver for the beneficial inoculation of plants exposed to salt stress.Endophytic bacteria have been reported to be more efficient in inducing antioxidant activity responses against stresses than rhizosphere microbes 14,22 .
Higher stomatal conductance in plants inoculated with Consortium 4 would support this hypothesis.Additionally, Flores-Duarte et al. 28 reported that the endophytic Variovorax gossypii JM-310T enhanced the nodulation and chlorophyll and nitrogen contents of Medicago sativa plants, ameliorating the physiological status of plants in response to moderate and high levels of contamination.They also found improved plant growth and nodulation after inoculation with V. gossypii JM-310T, Ensifer medicae MA11 and V. paradoxus S110T.
The possible beneficial effects of endophytes were further followed by analyzing SOD, CAT and TBARS activity, i.e., antioxidant plant responses to salt stress.PGPB have been reported to enhance enzymatic antioxidant reactions in plants 1,8,18,28 , thereby contributing to stress mitigation.As shown in Table 5, TBARS levels were similar in uninoculated plants independent of the salt level.However, inoculation significantly decreased TBARS levels in controls and salt-exposed plants, with the highest decrease occurring in the presence of Consortium 4 at 0.15 M NaCl.This also confirms a previous observation that PGPB decrease TBARS levels in plant tissues 18 .Chandra et al. 42 further showed that inoculation of wheat plants with Variovorax paradoxus RAA3, Pseudomonas palleroniana DPB16 or Pseudomonas sp.UW4 decreased TBARS and H 2 O 2 contents and increased enzyme activities (SOD and CAT, among others) of wheat plants under field conditions with variable water supply.Similar to our results (Table 5), these authors found that the presence of a Variovorax strain (V.paradoxus RAA3) led to the highest increase in antioxidant enzymes under drought stress conditions compared to controls.
On the other hand, analysis of the diversity of the wheat seedling rhizobiome revealed clear differences between salt-exposed and control consortia yet minor differences upon inoculation with both consortia in both salt-free controls and salt-exposed plants (Fig. 3).Salt-induced changes in rhizosphere microbial community structure have also been reported by Xu et al. 43 .

Conclusions
This study demonstrated the association of wild plants (Distichlis spicata, Pluchea absinthoides, Gaultheria mucronata and Hieracium pilosella) grown in extreme Chilean biomes with potential PGP endophytic bacteria.Fortytwo isolates were characterized as nonredundant halotolerant auxin-and ACC deaminase-producing bacteria of the genera Variovorax, Bacillus, Staphylococcus and Curtobacterium, with strain Variovorax sp.P1R9 showing the best mitigation of salt-induced toxicity on wheat (T.aestivum var.Fritz) seed germination.However, the highest seed germination rates under salt stress were observed when strain P1R9 was coinoculated in a triple Vol:.(1234567890  8).The endophyte consortium also showed the best PGP effects on the physiological and biochemical responses of wheat seedlings when exposed to salt stress.Although the underlying mechanisms still need to be clarified, our data suggest that the application of Variovorax sp.strain P1R9 as a part of bacterial consortia may counteract the negative effects of soil salinity on wheat crops.Finally, future agricultural techniques for the promotion of plant growth should consider microbial interactions in plant metaorganisms.

Plant collection and isolation of culturable plant endophytes
Three wild specimens of each extremophyte species, Distichlis spicata (Poaceae) and Pluchea absinthoides (Asteraceae) from the Atacama Desert (AD; 23° 1ʹ 59ʹʹ S, 68° 11ʹ 59ʹʹ W) and Gaultheria mucronata (Ericaceae) and Hieracium pilosella (Asteraceae) from Chilean Patagonia (PAT; 53° 28ʹ 0ʹʹ S, 71° 0ʹ 59ʹʹ W), were collected from public lands according to the Chilean legislation.The specimens were randomly sampled from a 10 m transect by using a clean spade to remove intact roots from the soil, placed in plastic bags and then immediately transported on ice to the Applied Microbial Ecology Laboratory (EMALAB) at Universidad de La Frontera (Temuco, Chile) for microbiological analyses.Culturable endophytic bacteria were isolated as described previously by Rilling et al. 7 .Briefly, pieces of roots and leaves were separated and surface sterilized by repeated immersion in 70% (v/v) ethanol for 3 min, followed by 2.5% (v/v) sodium hypochlorite (NaOCl) for 5 min.After disinfection, the root and leaf samples were exhaustively rinsed with sterile distilled water (SDW), and tissue portions (2 g) were dissected and aseptically macerated with a sterile mortar and pestle.Root and leaf tissue homogenates were placed in sterile plastic tubes, mixed into 50 mL of sterile saline solution (0.85% NaCl), serially diluted, and plated onto 1.5% agar plates containing tenfold diluted Luria-Bertani (LB) medium and NM-1 minimal medium 7 .Both culture media were amended with 10 μg mL −1 cycloheximide to prevent fungal growth.
Colonies grown on agar plates were counted after 4 days of incubation at 30 °C.Colonies with different phenotypes (color, sizes, shapes, brightness, and elevation) were then randomly chosen and transferred to and grown in fresh culture media.Three hundred seventy-six isolates were purified on agar plates by streaking.Isolates were stored at − 80 °C in LB:glycerol (7:3) and used for further assays.

Screening for PGP activities
Cereal production in Chilean relays is implemented on ash-derived volcanic soils (Andisol), which are recognized by a high content of total phosphorus (P) and organic matter, and P-releasing ability is the main trait in the study and selection of potential PGPB, also known as phosphobacteria 44 .Thus, all isolates were first tested for their ability to use insoluble forms of inorganic (Pi) and organic (Po) phosphorus as described by Jorquera et al. 43 .All isolates were grown at 30 °C for 2-4 days on National Botanical Research Institute's phosphate growth medium (NBRIP) and Pikovskaya's medium (PVK) agar plates supplemented with insoluble tricalcium phosphate (Ca 3 [PO 4 ] 2 ) as the sole Pi source.Phytate-screening medium (PSM) supplemented with insoluble phytic acid dodecasodium salt hydrate (C 6 H 18 O 24 P 6 × 12Na × xH 2 O) as the sole Po source was also used.Isolates that grew with clear halos on agar as a result of bacterial growth were considered Pi-and Po-utilizers and selected for additional testing of putative ACC deaminase activity and the production of tryptophan-dependent auxins 45 .Isolates were grown in 5 mL DF minimal medium supplemented with (NH 4 ) 2 SO 4 as the sole source of nitrogen and incubated at 30 °C for 2 days 46 .Bacterial cells were collected by centrifugation (3000×g for 5 min) and exhaustively rinsed with 1 mL of sterile salt solution, and then 0.1 mL aliquots were transferred to fresh DF broth supplemented with 3 mM 1-aminocyclopropane-1-carboxylic acid (ACC) as the sole source of nitrogen (N).Isolates growing in ACC-supplemented DF broth were considered ACC deaminase positive.
The production of tryptophan-dependent auxins was colorimetrically determined using Salkowski's reagent (150 mL of concentrated H 2 SO 4 , 250 mL of distilled H 2 O, 7.5 mL of 0.5 M FeCl 3 × 6H 2 O) as described by Acuña et al. 45 .Each isolate was grown in LB broth supplemented with 5 mM L −1 tryptophan.After incubation at 30 °C for 36 h, bacterial cells were centrifuged (10,000×g for 10 min), and the supernatants were vigorously mixed 1:2 with Salkowski's reagent.Supernatants showing a red coloration after incubation at room temperature for 30 min were considered positive for tryptophan-dependent auxin production.For dereplication of isolates, enterobacterial repetitive intergenic consensus polymerase chain reaction (ERIC-PCR) was performed with the 1R (5ʹ-ATG TAA GCT CCT GGG GAT TCA C-3ʹ) and 2F (5ʹ-AAG TAA GTG ACT GGG GTG AGC G-3ʹ) primer sets as described by Cid et al. 47 .

Quantification of PGP activities and identification of putative PGP isolates
The ACC deaminase activity of the putative PGP endophytes was quantified by α-ketobutyrate production that was quantified on a microplate reader at 540 nm using an α-ketobutyrate (≥ 97%) calibration curve (0.1-1.8 µM) according to Penrose and Glick 23 .Tryptophan-dependent auxins were quantified at 280 nm by HPLC using a DAD Shimadzu, LC20AT pump, CTO 20AC furnace, a DAD SPD M20A detector and a C18 reversed-phase column (5 μm, 4.6 × 100 mm −2 ) based on calibration with pure auxin indole acetic acid (IAA) using concentrations ranging from 0 to 75 μg mL −1 .
Halotolerance of putative PGP endophytes was assayed as described by Barra et al. 18 .Isolates were grown in fresh LB broth and plated onto LB agar plates supplemented with 2.5, 5.0, 7.5 and 10.0% NaCl.Compared with the control (0.5% NaCl), colonies grown and showing the same size in media supplemented with NaCl were considered halotolerant.Halotolerant colonies with PGP activities were characterized by partial sequencing of their 16S rRNA genes.Partial amplification of the 16S rRNA gene was performed by endpoint PCR using the 27f (5ʹ-AGA GTT TGA TCC TGG CTC AG-3ʹ) and 1492r (5ʹ-TAC GGY TAC CTT GTT ACG ACT T-3ʹ)

Effects on the germination of salt-exposed wheat seeds
A germination toxicity assay was conducted using isolates with high ACC deaminase activity, auxin production and salt tolerance on commercial wheat seeds (T.aestivum var.Fritz  48,49 ).The seeds were surface sterilized by repeated immersion in 70% (v/v) ethanol for 3 min, followed by 5% (v/v) NaOCl for 5 min.The seeds were then exhaustively rinsed with SDW, dried over sterile filter paper on Petri dishes in a laminar flow hood for 2 h (n = 30 seeds per plate), wetted with SDW and placed in a plant growth incubation chamber for three days.All treatments were performed in quadruplicate.On Day 3, bacterial cultures were prepared in LB tubes, incubated for 24 h at 30 °C, pelletized via centrifugation (3000×g for 3 min), and resuspended in sterile 0 M SDW (control) or 0.15 and 0.25 M NaCl SDW.Then, the filter paper of each plate was irrigated with 10 mL of each suspension and incubated for 7 days at 30 °C.After 10 days, germinated seeds were counted, the radicle mean length was quantified, and stress-induced toxicity was measured via the germination index (GI) 50 , as derived from Eqs. (1)-( 3), with RSG being the relative seed germination (%) and RG the relative growth (%) as follows: where GI ≥ 80% indicates the absence of salt stress, 80% < GI < 50% intermediate salt stress and GI < 50% high salt stress.The three best-performing strains were selected and tested for effects in consortia application using the following consortia: In a second set of experiments, the effects of coinoculation of Variovorax sp.P1R9 with known halotoleranceinducing rhizosphere bacteria from the Atacama Desert (Klebsiella strains 8LJA and 27IJA) 14 on GI were tested as single-strain inoculation and in consortia as follows: Consortium 5: Variovorax sp.P1R9 and Klebsiella sp.8LJA; Consortium 6: Variovorax sp.P1R9 and Klebsiella sp.27IJA; Consortium 7: Klebsiella sp.8LJA and Klebsiella sp.27IJA; and Consortium 8: Variovorax sp.P1R9, Klebsiella sp.8LJA, and Klebsiella sp.27IJA.

Effects on physiological and biochemical responses of salt-exposed wheat plants
The consortia with the highest salt stress toxicity alleviation effects on wheat seed germination (consortia 4 & 8) were tested for their effects on wheat plants (T.aestivum var.Fritz) growing for 45 days under greenhouse conditions.One hundred commercial wheat seeds were sown in plastic pots containing 600 g of 3:1 soil:perlite PP8® and incubated in an open greenhouse during summer.The pots were regularly irrigated with DW.At Days 5, 15, and 30, plants were treated with 100 mL of the corresponding bacterial consortia in a 1:1:1 mixture of the individual strains (~ 1 × 10 8 cells mL −1 ), and each plant was supplemented with NaCl treatment (0.15 and 0.25 M NaCl).Inoculation by irrigation was chosen because it is the most common strategy used by Chilean farmers to introduce microbial inoculants in Chilean crops.Untreated pots (salt stress and bacteria) served as controls.After 45 days, the chlorophyll content and stomatal conductance were measured in the leaves of three plants per pot by using an MC-100 chlorophyll concentration meter (Apogee®, Instruments, Logan, UT, USA) and an SC-1 porometer (Decagon® Devices, Pullman, WA, USA), respectively.Immediately thereafter, roots and shoots were harvested, carefully washed, and split into two subsamples.One subset was dried (60 °C for 48 h) to determine the dry weight of biomass 18 , while the other fraction was frozen at − 80 °C for later quantification of thiobarbituric acid-reactive substances (TBARS) and superoxide dismutase (SOD) and catalase (CAT) activities [51][52][53] .TBARS were quantified using 0.03 g of fresh leaves macerated in 500 µL of 0.2% trichloroacetic acid and centrifuged at 10,000×g for 5 min (4 °C).The level of lipid peroxide quantified by the formation of malondialdehyde (MDA) was determined by measuring absorbances at 440 nm, 532 nm and 600 nm.In addition, SOD and CAT activities were evaluated using 0.1 g of fresh leaf tissue.Total crude proteins from the samples were extracted using 50 mM potassium phosphate buffer (pH 7.0), incubated for 5 min, and centrifuged at 10,000×g for 15 min (4 °C).SOD was quantified using the inhibition of the photochemical reduction of nitro blue tetrazolium by photochemically generated superoxide radicals, whereas the consumption of H 2 O 2 was used to estimate CAT activity.Protein contents were determined by the Bradford method to normalize antioxidant activities and lipid peroxidation.

Figure 1 .
Figure 1.Effect of salt-induced stress on the biomass (A), chlorophyll content (B) and stomatal conductance (C) of wheat seedlings inoculated with Variovorax sp.strain P1R9 as part of the root endosphere (Consortium 4) and a rhizosphere consortium (Consortium 8) (Tables1, 2).Bars represent mean values, while error bars represent standard deviation.Upper-case letters denote statistical differentiation within each salt (NaCl) treatment.Lower-case letters denote statistical differentiation between all samples (P ≤ 0.05; Tukey's test).

Figure 2 .
Figure 2.Effect of inoculation on wheat seedling rhizobacterial community size (measured as 16S rRNA) inoculated with Variovorax sp.strain P1R9 as part of the root endosphere (Consortium 4) and a rhizosphere consortium (Consortium 8) (Tables1, 2).Bars represent mean values, while error bars represent standard deviation.Upper-case letters denote statistical differentiation within each salt (NaCl) treatment.Lower-case letters denote statistical differentiation between all samples (P ≤ 0.05; Tukey's test).

Figure 3 .
Figure 3. Alpha and beta diversity indices of rhizobacterial communities associated with wheat seedlings inoculated with Variovorax sp.strain P1R9 as part of the root endosphere (Consortium 4) and a rhizosphere consortium (Consortium 8) (Tables 1, 2).(A) Diversity metrics (ACE, Chao1, Coverage, Shannon, Simpson and Sobs) were calculated using a subset of 25,000 reads per sample.Horizontal bars in the box plots indicate median proportional values.The lower and upper edges of the boxes represent the approximate 1st and 3rd quartiles, respectively.Different lower-case letters indicate significant differences (P < 0.05) among treatments.(B) Nonmetric multidimensional scaling (NMDS) based on Bray-Curtis dissimilarity showing the structures of the rhizobacterial community from salt-stressed wheat plant samples.

Table 2 .
Quantification of ACC deaminase activity and production of tryptophan-dependent auxins in PGP endophytic bacterial isolates from plants in the Atacama Desert (D. spicata and P. absinthoides) and Chilean Patagonia (G.mucronata and H. pilosella).ACC 1-aminocyclopropane-1-carboxylic acid deaminase, IAA indole acetic acid, N.D. not detected.† The values represent the means ± standard deviations from n = 4. ‡ The values represent the media of two technical measurements of the composite samples by HPLC.*Isolates in bold were further used for effects on wheat plants.

Table 3 .
Effect of putative endophytic isolates and consortia on germination of wheat seeds as quantified by the percentage (%) of seed germination and the germination index (GI).