Do bacterial root endophytes contribute to growth in saline conditions? A pre-reintroduction cultivation study of threatened saltmarsh Limonium species

Do bacterial root endophytes contribute to growth in saline conditions? 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A pre-reintroduction cultivation study of threatened saltmarsh Limonium species Amaia Nogales, Maria Cristina Simões Costa, Salvadora Navarro-Torre, and 2 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-4738414/v1 This work is licensed under a CC BY 4.0 License Status: Posted Version 1 posted You are reading this latest preprint version Abstract Background and Aims Highly specialized halophilic flora, such as the threatened endemic sea-lavender species Limonium algarvense and Limonium daveaui , are adapted to grow in saline environments. Plant-associated microorganisms associate with halophytes promoting their survival and growth, namely stress tolerance. In this study, we compared the effects of halophilic bacterial inoculants and characterized seed germination and plant growth under saline conditions. Methods A new protocol was developed for in vitro seed germination with bacterial inoculation. The experimental set up included three treatments: non-inoculation, inoculation with a single bacterial inoculum ( Pantoea sp., LDR15) or a consortium of halotolerant bacteria ( Pantoea genus). We assessed plants’ physiological status, biomass, and leaves characteristics under saline irrigation. Results Exposure to NaCl (200 mM) along with inoculation using either LDR15 strain or the bacterial consortium negatively affected seed germination. The inoculated bacteria were localized in root cortex and phloem. Under non-saline conditions, bacterial inoculation had no effect in leaf number and fresh biomass, being leaf reflectance values higher in L. algarvense than in L. daveaui . Salinity significantly reduced both leaf number and size, succulence and biomass, being this effect more pronounced in L. algarvense than in L. daveaui . The bacterial consortium negatively affected plant survival, but plants inoculated with the LDR15 strain had higher biomass than the non-inoculated ones. Conclusion We concluded that even though halotolerant bacteria did not improve seed germination upon salinity exposure, the bacterial inoculation with LDR15 strain in germinated plantlets can be a suitable strategy for promoting plant development in saline environments. halophytes Pantoea plant growth promoting bacteria seed germination salinity sea-lavenders Figures Figure 1 Figure 2 Figure 3 Figure 4 Introduction Soil biology has frequently been overlooked in plant reintroduction and practice, despite the crucial role played by soil microbial communities in species restoration (Farrell et al. 2020 ). Specifically, plants that thrive in saline habitats, such as salt-tolerant plants (halophytes), exhibit a high degree of habitat specialization and host a characteristic microbiome consisting of halophilic or halotolerant microorganisms (Navarro-Torre et al. 2023a ; Nogales et al. 2023 ). Halotolerant/halophilic bacteria either tolerate or require saline conditions for their growth (Ventosa et al. 1998 ), since they possess mechanisms that include changes in cell wall and/or plasmatic membrane to prevent salt entry (e.g., Na and K pumps and antiporter channels), accumulate osmoprotectants (e.g., ectoine, betaine, trehalose), and/or secrete of exopolysaccharides to help in biofilm formation (Etesami and Beattie 2018 ). Some of these bacteria present plant growth promoting traits essential for plant nutrient uptake (e.g., N, P, Fe) as well as for improving host tolerance to biotic and abiotic stresses, including salinity stress (Trivedi et al. 2020 ; Navarro-Torre et al. 2023a ). As a result, microbial inoculation emerges as a valuable strategy in the agricultural management of saline soils (Navarro-Torre et al. 2023a ), phytoremediation in saline saltmarshes (Paredes-Páliz et al. 2017 ), and coastal restoration (Farrer et al. 2022 ), although neglected in plant conservation schemes. The Mediterranean halophytic flora and vegetation are characterized by a high degree of specialization, with species specifically adapted to saline environments, as exemplified by the genus Limonium Mill. (sea-lavenders), which harbors numerous endemic species (Caperta et al. 2020 ; Salazar-Mendías and Lendínez 2020 ). As other halophytes, their tolerance to salinity relies on many adaptive mechanisms like controlled uptake and compartmentalization of Na + , K + and Cl − , osmolytes synthesis (Flowers and Colmer 2008 ), and/or salt excretion through salt glands (Caperta et al. 2020 ). Many Limonium coastal endemics are threatened, such as Limonium perplexum in eastern Spain, established on deep crevices in native habitat, in which translocation works were developed to conserve this species (Laguna et al. 2016 ). In mainland Portugal, extinction risk assessments indicate that Limonium algarvense Erben is classified as Near Threatened, while Limonium daveaui Erben is deemed Critically Endangered according to extinction risk categories by the International Union for Conservation of Nature (IUCN) (Carapeto et al. 2020 ). While L. algarvense is distributed in Iberian Peninsula and in Morocco (Caperta et al. 2017 ; Carapeto et al. 2020 ), L. daveaui , which previously had a broader range, is now restricted to a few populations along the west coast of Portugal (Caperta and Carapeto 2020 ; Carapeto et al. 2020 ). Pre-reintroduction cultivation studies are essential for understanding the most effective methods for propagating such species and for making informed decisions to minimize the risks associated with translocating individuals into recipient habitats in species recovery programs (Godefroid et al. 2016 ). Both L. daveaui and L. algarvense can be cultivated using saline soil and brackish, estuarine water at least at 100 mM NaCl (Cortinhas et al. 2020 , 2021 ; Rodrigues et al. 2020 ). Previous greenhouse studies using microbial inoculation treatments in L. algarvense showed that plant growth promoting bacteria (PGPB) isolated from Arthrocnemum macrostachyum (Navarro-Torre et al. 2017 ) led to better plant physiological status under saline conditions (Nogales et al. 2023 ). However, an alternative approach is the use of bacterial inoculum sourced from reference ecosystems (e.g. saltmarshes), as it offers a specific microbial community. In some restoration projects PGPB were tested as biofertilizers, both as pure cultures and as consortia, to enhance the growth of Spartina alterniflora , a common wetland grass. The results of those projects indicated a significant growth improvement under saline conditions compared to freshwater under greenhouse conditions (Bledsoe and Boopathy 2016 ). In Limonium species, a great diversity of microorganisms has been isolated from Limonium sinense (Qin et al. 2018 ), L. daveaui , Limonium multiflorum Erben and Limonium vulgare roots (Navarro-Torre et al. 2023b ). In the last three species, the root endosphere was mainly formed by Gram negative bacteria, being the most representative genera Pantoea and Erwinia . In the presence of 0.2 M NaCl, these bacterial strains formed biofilms and the amount of IAA (indole-3-acetic acid, phytohormone auxin) produced was enhanced, being the highest increase found in the strain LDR15 isolated from L. daveaui (Navarro-Torre et al. 2023b ). In this framework, the hypothesis of our study was that endophytic bacteria isolated from autochthonous Limonium species could promote sea-lavenders’ tolerance to salinity, enabling them to withstand adverse saline cultivation environments. Using halotolerant bacteria isolated from Limonium sp. grown in saltmarshes and selected for their superior plant growth-promoting traits and NaCl tolerance (Navarro-Torre et al. 2023b ), the goals of this study were to: 1) investigate the impact of selected bacteria on Limonium sp. seed germination; 2) evaluate their effects on the leaf epidermis, including the density of salt glands, stomata, and pavement cells, in expanded leaves; and 3) assess their potential benefits for plant growth under saline conditions in relation to freshwater. Materials and Methods Germination assay A seed germination assay was performed to assess the impact of different PGPB inoculants, including both single species and a consortium, derived from bacterial strains isolated from L. daveaui and L. vulgare (Navarro-Torre et al. 2023b ). The effect of the bacterial strain LDR15 ( Pantoea sp.) on the germination of field-collected Limonium sp. seeds was compared to that of a consortium of P. anthophila , P. agglomerans , and Pantoea sp. (strains LDR2, LDR25 and LVR13). Pantoea sp. LDR15 was chosen as a single species-based inoculum because it presented remarkable plant growth promoting traits (IAA and siderophore production, phosphate solubilization, biofilm formation and nitrogen fixation). The bacterial strains LDR2, LDR25 and LVR13 were selected to be used as a consortium-based inoculum as they collectively presented highly interesting and complementary plant growth promoting traits. Specifically, LDR2 and LDR25 exhibited notable production of IAA, while LVR13 demonstrated high phosphate solubilization capability, particularly in saline conditions (0.2 M NaCl) (Navarro-Torre et al. 2023b ). To prepare the bacterial inocula (single species or consortium-based), each strain was incubated separately in 5 ml sterilized tryptic soy broth (TSB) medium for 24 h at 28 ˚C in continuous shaking (115 rpm). After that, cultures containing 10 8 cells·ml − 1 were centrifuged at 8000 rpm for 5 min. Then, pellets were washed with sterile 0.9% saline solution and centrifuged again. Finally, they were resuspended in 1 ml sterile 0.9% saline solution, and the strains that conform the consortium were mixed in the same tube (Navarro-Torre et al. 2017 ). A new protocol was developed for seed disinfection, in which seeds of each species were washed with tap water for 20 min, with shaking every 5 min. Seed surface disinfection was then carried out in a horizontal laminar flow air chamber (INC Gelaire®HF48). Three sterilising solutions were used for seed disinfection: hydrogen peroxide at 18% (g/v) for 5 min; alcohol 70% for 1 min and commercial bleach at 15% (with 5% sodium hypochlorite) with 3 drops of teepol / 100 ml, for 20 min. After immersion in each disinfectant solution, the seeds were collected on sterile filter paper, in a funnel, and rinsed five times with sterile distilled water. Seeds were left in distilled water for approximately 48 hours with agitation, in a growth chamber at 26/22 ± 1 ºC day/night, with 16 h / 8 h photoperiod. After 48 h, the disinfected seeds were bacterized for 1 h under shaking in 1 ml of the respective inoculum (LDR15 or the consortium). Seeds in the control treatment were incubated with sterile 0.9% saline solution. Then, 50 seeds of each experimental treatment (control, bacterized with either LDR15 or the consortium) were deposited in Petri plates containing a culture medium consisting of the medium described by Rossini et al. (2010) and supplemented with 0 M NaCl (w/v) or 0.2 M NaCl (w/v) before the addition of 7 g/L agar (Duchefa Biochemie). Four plates were considered per experimental treatment (200 seeds per treatment), which were placed in a growth chamber under the same temperature and light conditions as described above. Germination was checked every day for 20 days and the kinetic of germination percentage was recorded. Experimental set-up To determine the effects of the two inoculants (LDR15 bacteria or consortium) on L. daveaui and L. algarvense plant growth, twenty seedlings per species were surface-disinfected and germinated in in vitro conditions as described before. The seedlings were transferred to jiffy pots (peat substrate), placed in a growth chamber (23/18 ± 1 ºC day/night, 18/6 h photoperiod) and inoculated weekly with 1 mL of each bacterial inoculant or distilled water (in the control treatment). Four weeks later, plants were transplanted into individual pots with a mixture of peat and vermiculite (1:2 v/v). They were then divided into six different experimental groups: non-inoculated, LDR15-inoculated, and consortium-inoculated plants, each subjected to either saline irrigation (100 mM NaCl) or freshwater irrigation (tap water). Inoculum was prepared as described before, and each plant was inoculated 5 mL of the corresponding inoculum (LDR15 or the consortium of LDR2, LDR25 and LVR13). Non-inoculated plants were watered with 5 mL of saline solution. This procedure was repeated fortnightly. Irrigation was managed via an automatic watering system with regulated flow rates. The selection of 100 mM NaCl as the salinity concentration was based on prior research indicating favourable development of Limonium species under these conditions (Cortinhas et al. 2020 , 2021 ). Plants were maintained in greenhouse conditions with natural light for five months. Plant physiology and growth Plants physiological status was evaluated by reflectance index assessment (Normalized Difference Vegetation Index -NDVI and Photochemical Reflectance Index-PRI). The NDVI and PRI were measured using PlantPen NDVI 300 (PSI, Czech Republic) and PlantPen PRI 200 (PSI, Czech Republic) portable devices, respectively. Photochemical Reflectance Index is indicative of photosynthesis efficiency and plant stress levels (Garbulsky et al. 2011 ) while NDVI reflects plant vigor and, indirectly, chlorophyll status, phosphorus, and nitrogen nutrition (Sembiring et al. 1998 ). Measurements were performed four months after the first bacterial inoculation was done. Those were conducted in three young leaves per plant of each experimental group. At the end of the experiment, the number of leaves per plant was counted and the fresh biomass was determined. Pavement cells, stomatal and salt glands characteristics Leaf imprints from the abaxial/adaxial surfaces of young, expanded leaves from three L. algarvense and L. daveaui plants were taken from each treatment (species x saline condition x type of inoculation) following Balasooriya et al. ( 2009 ). The imprints were made using colourless nail polish and transparent adhesive tape. Then, they were fixed on a microscope slide and observed under light microscope (Leitz Dialux 20EB) at 125 x 10 magnification. Images of five fields per imprint were captured using a digital camera (Leica EC3) and image software (LAS V.2.13). The density of glands, stomata, and pavement cells were calculated per mm 2 leaf area using the software ImageJ v. 1 49s. Bacterial colonization A parallel experiment was conducted to study the localization of bacterial strain colonization. For this purpose, transformed LDR15, LDR2, LDR25 and LVR13 bacterial strains containing the plasmid pMP7604 that codifies for the mCherry fluorescent protein were used. Transformation procedure was conducted by conjugation as described in Navarro-Torre et al. ( 2023b ). For four months five plants were inoculated with each of the four individual fluorescent bacterial strains, and another set of five plants were watered with saline solution (0.09%). Each inoculum was prepared in the same way as described previously for the wild-type strains. Root bacterial colonization was analysed as described previously (Navarro-Torre et al. 2023b ). In brief, root samples from four-month-old plants of each species were collected and fixed using a 4% formaldehyde acetic solution in 70% ethanol for 48 h. Then, the roots were washed in a 70% ethanol solution and stored at 4 ºC until used. Samples were initially prepared for observation under light microscopy. They were first impregnated with DP1500 polyethylene glycol as described by Barbosa et al. ( 2010 ). Then, 17–20 µm thick transverse sections were cut using a sliding microtome (Leica SM 2400), stained with Safranin/Astrablue (1% aqueous solution) and mounted with a glycerine/water solution for temporary slide preparation. Images were captured using analysis image software (Leica Qwin) coupled to a microscope (Leica DMLA). For visualizing bacterial colonization, the preparations were observed under a fluorescence microscope (Zeiss Axioskop 2), equipped with a digital camera (Zeiss AxioCam) with a 10x objective. Image acquisition was performed using the Carl Zeiss/AxioVision 4.8 software. Statistical analyses Statistical analyses were performed using SPSS Statistics vs. 23 (IBM) program and in all cases data normality and variance homogeneity were tested prior to the analyses. Germination percentage data were analyzed by a three-way ANOVA where time, salinity and inoculation treatment were considered as main factors. Following this, to delve deeper into the impact of bacterial and salinity treatments, the influence of both factors was examined at each individual week by a two-way ANOVA. In the experiment of plant growth response to halotolerant bacteria inoculation and salinity, due to missing values in one experimental group (salinity-subjected plants), the number of leaves, fresh biomass, NDVI, PRI, density of glands, stomata and leaf pavements cells data were analyzed separately under freshwater and saline conditions. At each soil condition, data were investigated by a two-way ANOVA where species and inoculation treatment were considered as main factors. Results Effects of plant growth promoting bacteria in seed germination The results of the three-way ANOVA conducted to study the effects of bacterial inoculation, time, and salinity on Limonium species seed germination is shown in Supplementary Table S1 . As expected, time had a significant effect on germination percentage but did not interact with the other factors. Overall, salinity had a negative effect, but the inoculation factor did not, although the significance of this factor was p = 0.08. The interaction between both factors was nonsignificant, but it approached significance at a p-value of 0.098. To unveil potential patterns in the effects of both factors on seed germination that might be masked by cumulative effects over time, the data were analyzed separately for each week. When data were analyzed on a weekly basis, we observed a significant negative effect of NaCl on seed germination from week four onwards (Supplementary Table S2 , Fig. 1 ). On the other hand, bacterial inoculation became significant starting from week eight: non-inoculated seeds had higher germination percentage than those inoculated with the consortium, and the ones inoculated with LDR15 had intermediate values. From week 10 onwards the germination percentage of non-inoculated seeds under saline conditions continued to rise, while in the other experimental groups it approached its peak, which was consistently reached by week 15 across all groups. From this point onwards, the p-value for the interaction between both factors was 0.051, which was reflected by a pronounced impact of salinity in LDR15-inoculated and consortium-inoculated seeds, but not in the non-inoculated ones (Fig. 1 , Supplementary Table 1). Study of plant response to the inoculation with halotolerant bacteria and to salinity In plants grown for four months under freshwater conditions, the number of leaves and fresh biomass were not influenced by the bacterial inoculation treatment (non-inoculation, or inoculation with either LDR15 or the consortium) in any of the Limonium species. Moreover, while the number of leaves was similar in L. algarvense and L. daveaui , the fresh biomass was higher in L. daveaui than in L. algarvense (Fig. 2 ). In such conditions, the PRI was solely influenced by the species factor, with higher values in L. algarvense plants. In the case of NDVI, there was a significant interaction between species and inoculation treatment factors. While L. algarvense plants inoculated with LDR15 had significantly higher values than non-inoculated plants or plants inoculated with the consortium, the inoculation treatment did not have a significant effect in NDVI in L. daveaui (Fig. 2 ). Under saline conditions, both the number of leaves and fresh biomass were reduced, and all plants inoculated with the bacterial consortium died. The fresh weight was almost five times lower in both L. algarvense and L. daveaui plants growing in the salinized substrate, and the number of leaves was reduced from 11.3 ± 0.31 in plants grown under freshwater conditions to 3.6 ± 0.30 in plants growing in the salinized substrate. Besides, plants grown under salinity had very small and succulent leaves that did not allow us to conduct NDVI and PRI measurements. On the other hand, the inoculation factor had a significant effect, and plants inoculated with LDR15 tended to have higher biomass than the non-inoculated ones, although the multiple mean comparison test conducted to explore differences among all experimental groups did not show significant differences between non-inoculated and LDR15-inoculated plants. Influence of bacterial inoculation and saline treatment on salt glands, stomata, and pavement cells In both Limonium species, under non-saline or saline conditions, and regardless of the inoculation treatment, salt glands and stomata (Fig. 3 A) were noticeable on the surface of the two leaf blades, and adjacent salt glands and stomata were separated by at least two pavement cells. The salt glands were sunken into the epidermis, and stomata were at the same level as the pavement cells (Fig. 3 A). Each salt gland contained 16 cells arranged in quadrants, with four cells in each quadrant (Fig. 3 A). In L. algarvense , each salt gland was encircled by 4–7 pavement cells, while in L. daveaui , it was surrounded by 4–8 cells. Under freshwater conditions, the density of glands was not affected by the species or inoculation treatment factors (Fig. 3 B). However, the density of stomata and pavement cells showed a different pattern, and a significant interaction was found between both factors. Whereas L. algarvense plants had a consistent density of stomata and pavement cells across the three inoculation treatments, L. daveaui plants inoculated with the consortium had significantly lower values than the other two inoculation treatments (non-inoculated and inoculated with LDR15) (Fig. 3 B). In the salinized substrate, the density of stomata was influenced by the species, with L. daveaui exhibiting the lowest density (Fig. 3 B). The effect of the inoculation was not significant in this parameter when non-inoculated and LDR15-inoculated plants were considered, but as mentioned earlier, all plants inoculated with the consortium died. Contrastingly, pavement cell density was not affected by either the species or the inoculation treatment. Root bacterial colonization The blue autofluorescence due to compounds like lignin and suberin, usually deposited in the xylem tissue as well as in the Caspary bands, was visualized in both the PGPB-inoculated and non-inoculated root samples (Fig. 4 ). In the PGPB-inoculated samples, the fluorescence from the mCherry protein was recorded within epidermal, cortical parenchyma, and phloem cells, indicating that the bacteria were present in these tissues. Discussion Plant microorganisms present in the rhizosphere are essential for proper plant growth and adaptation to stress factors (Trivedi 2020) justifying their increasing use as a tool in sustainable agriculture and environmental management of saline soils (Navarro-Torre et al. 2023a ). However, there are limited studies available on the use of PGPB in the restoration of endangered endemic species, especially from coastal habitats (Farrer et al. 2022 ). Furthermore, some reports reveal contrasting results (Michaelis and Diekmann 2018 ). In the current study, we evaluated the effect of two PGPB inocula based on bacterial strains isolated from the plant rhizosphere of endangered halophyte Limonium species thriving in coastal saltmarshes, based on the hypothesis that halotolerant PGPB inoculations could possibly increase seed germination and plant adaptation to saline conditions. Inoculation with native halotolerant bacteria did not improve seed germination Halophyte seeds can remain viable after long periods of exposure to salinity and begin the germination process when salinity levels decrease (low osmotic potential) (Khan and Ungar 1996 ). The osmotic stress delays seed germination since salinity reduces seed imbibition (Muñoz-Rodríguez et al. 2017 ). For example, L. daveaui seed germination percentage was higher in seeds germinated in distilled water than in a saline Fluvisol (Cortinhas et al. 2021 ). Since in the present study we wanted to test the influence of bacterial inoculation in seed germination in vitro , it was essential to develop a new protocol for seed disinfection that allows both germination in aseptically conditions, as well as the development of bacterial communities. The disinfection procedure softened the seed coats possibly contributing to better seed imbibition. One of the sterilising solutions used was hydrogen peroxide (H 2 O 2 , a common reactive oxygen species) that is a regulator of developmental processes like dormancy release, cell-wall loosening, and reserve mobilization as found in non-halophytes like Arabidopsis (Liu et al. 2010 ; Wojtyla et al. 2016 ). In germinating seeds of halophytes Arthrocnemum macrostachyum , Arthrocnemum indicum , Suaeda fruticosa and Limonium stocksii the H 2 O 2 content enhanced with increases in NaCl concentration in both latter two species (Hameed et al. 2014 ; Nisar et al. 2019 ). Previous works have demonstrated that inoculation with PGPB can improve seed germination in saline conditions (Yousefi et al. 2017 ; Navarro-Torre et al. 2017 ). In this last study, bacterial pre-inoculation in a halophyte species (e.g. A. macrostachyum ) considerably enhanced the kinetics of germination and final germination percentage. Contrastingly, in our study, inoculation with halotolerant PGPB either alone (LDR15) or in consortium reduced the germination percentage in the presence of NaCl. Although the PGPB employed proved to be tolerant to 200 mM NaCl in the germination medium (Navarro-Torre et al. 2023b ), in general they did not have a seed germination promoting effect at 200 mM NaCl. Other studies also point towards the same scenario. Fouladvand and SoltaniIn (2024) compared the germination of non-inoculated and inoculated wheat seeds with different bacterial endophytes under 250 mM NaCl and most of these endophytes led to a lower percentage of germination than control seeds. In the same way, some of the single species or consortia inoculations tested in Petrillo et al. ( 2022 ) showed similar or negative effects on the germination of Spinacia oleracea seeds compared to the control seeds. Although the exact reasons for the negative effect of the halotolerant bacterial inoculation on seed germination remain uncertain, several factors could be involved, such as a poor bacterial adhesion on the seed surface, or an excess of bacterial cells in the inoculum. The simultaneous demands placed on the seed, including combating salinity while accommodating the proliferating symbionts, might have also overwhelmed its capacity to germinate effectively. Ultimately, this scenario reflects a complex interplay of factors, that may ultimately hinder its ability to allocate energy efficiently towards germination. Benefits of halotolerant bacteria inoculation in plant physiological status and growth In the present study, the Gram-negative bacteria used for plant inoculation were localized in the root endosphere of both L. algarvense and L. daveaui , regardless of whether the plants were grown in freshwater or saline conditions. Root colonization by different PGPB has also been reported in other Limonium species such as Limonium sinense and Limonium vulgare subjected to salinity (Qin et al. 2014 , 2018 ; Wang et al. 2017 ; Xiong et al. 2020 ). In our study, those bacteria had a specific tissue localization pattern, restricted to living cells and absent in the xylem vessels, as also observed in a non-halophyte species, i.e. grapevine, inoculated with the same bacterial inoculum (Navarro-Torre et al. 2023b ). In general, halophytes can survive under 200 mM NaCl or approximately 20 dS/m EC) (Flowers and Colmer 2008 ) and their growth can be stimulated within a salinity range of 15–25 dS/m EC (Rozema and Schats 2013 ). In our study, a differential species performance was observed, with the salinity effects being more pronounced in L. algarvense . The impact of salinity led to a reduction in both leaf number and size, leaf succulence, and biomass, with a greater effect observed in L. algarvense compared to L. daveaui. Succulence is an adaptive mechanism enabling halophytes to grow for long periods of time under high salinity (Dassanayake and Larkin 2017 ; Caperta et al. 2020 ). Moreover, both species presented salt glands and stomata in both leaf blades, with the salt glands displaying the previously identified complex 16-celled structure observed in various Limonium species (Caperta et al. 2020 ). In both Limonium species studied in the present work, the distribution patterns of salt glands in the leaf blades were similar, while in other Limonium species salt gland density varied in the adaxial and abaxial epidermis. For example, L. bicolor and L. franchetii exhibited greater salt gland density in the abaxial epidermis than in adaxial one, whereas L. gmelinii showed the opposite trend (Xin et al. 2012 ; Leng et al. 2018 ), indicating that saline gland distribution varies among Limonium species. By contrast, in L. aureum , L. gmelinii , L. otolepsis and L. sinuatum salt gland densities enhanced with increasing NaCl concentration (Mi et al. 2021 ). Moreover, under the same NaCl concentration, salt gland and stomata densities of L. bicolor were significantly higher than in L. gmelinii (Leng et al. 2018 ). In our study, the density of stomata was influenced by the species factor, with L. daveaui exhibiting the lowest density in saline conditions. As for plant growth, in previous studies, L. daveaui plants showed lower development as well as reduced values of photosynthetic indexes and biomass production when grown in a salinized substrate (100 mM NaCl) compared to the non-saline one (Cortinhas et al. 2021 ). A similar trend was observed for L. algarvense , where freshwater-irrigated plants exhibited greater growth while saline irrigation (100 mM NaCl) decreased plant growth in terms of number of leaves and flowers (Cortinhas et al. 2020 ; Rodrigues et al. 2020 ). By contrast, in other Limonium species ( L. aureum , L. gmelinii , L. otolepis , L. sinuatum ) the addition of 100 mM NaCl to the growth medium significantly increased plant biomass and leaf area (Mi et al. 2021 ). Altogether these findings point to species specific responses to salinity, probably related with the coastal saltmarsh habitats in which the studied species grow. Although both species thrive in priority habitat 1510* Mediterranean salt steppes ( Limonietalia ) (European C. 2013 ), they inhabit soils periodically flooded by saline water and experiencing extreme summer drying, often characterized by salt efflorescence (Pena et al. 2020 , 2021 ), they occupy different positions in saltmarshes. Limonium algarvense thrives in the middle marsh where the debris brought in by the high tide is usually deposited, whereas L. daveaui grows in the high marsh on the margins of slopes, on the banks of embankments, channels, salt marsh walls of Salinas and removed soils (Costa 2001 ; Costa et al. 2014 ), which have drier conditions, and possibly, higher salinity levels. On the other hand, under saline conditions, although the bacterial inoculation factor did not have a significant effect on the number of leaves compared to non-inoculated conditions, it did significantly impact biomass. Overall (considering both species together), plants inoculated with LDR15 exhibited higher biomass than non-inoculated plants. This confirmsother studies, where Limonium sinense plants inoculated with PGPB had increased fresh weight, root length, leaf length and total chlorophyll and proline contents under salinity conditions (Qin et al. 2018 ). In fact, many PGPB have evolved a series of mechanisms which may lead to the reduction of salt concentration in their hosts tissues, such as the formation of biofilms (Navarro-Torre et al. 2023a ), or the induction of changes in root exudation of some compounds (e.g., polysaccharides and organic acids), that in turn may alter other microorganisms growth, composition, and activity, ultimately improving soil/substrate properties and plant development. Contrastingly, inoculation with a consortium of PGPB not only failed to promote plant growth, but also resulted in the death of all inoculated plants. This contrasts with previous findings, where the same consortium was applied in grapevine roots in a salinized substrate with positive results (Navarro-Torre et al. 2023b ). Furthermore, Nogales et al. ( 2023 ) also demonstrated the beneficial effects of halotolerant PGPB consortium inoculation isolated from the root endosphere of A. macrostachyum (Navarro-Torre et al. 2016 ) in L. algarvense , that improved plant growth in an amended saline Fluvisol. Although pinpointing the exact cause for this effect is not possible in the current study, several factors could have potentially contributed to this phenomenon. The complex interactions between different bacterial species, their compatibility with the host plant, and their responses to saline conditions can lead to varied outcomes when plants are inoculated with a consortium versus a single bacterium. These outcomes highlight the importance of understanding the specific mechanisms underlying plant-microbe interactions in different environmental contexts (Ciccillo et al. 2002 ; Flores-Duarte et al. 2023 ). Conclusions In summary, the use of native PGPB inoculation using Pantoea strains as single-species inoculum and as consortium yielded different results in halophyte Limonium species. Seed germination was the most salt sensitive characteristic, and the inoculation with our two selected halotolerant PGPB inocula negatively affected it. However, while the inoculation with the consortium led to plant death under saline conditions, inoculation with LDR15 promoted a general growth increase in Limonium species. Those results indicate that a careful selection of PGPB bacteria to be included within the inoculum is imperative, since positive outcomes observed in specific plant species may not necessarily generalize across all species. Furthermore, the identification of additional functional bacterial groups within the native soil could prove advantageous for facilitating the establishment of Limonium plants in saline conditions, as well as for counteracting the effect of any potential pathogens that may pose a threat to transplantation efforts. Declarations The authors declare that they have no conflict of interest. Funding This work was funded by the National Funds through Foundation for Science and Technology (FCT) under the Project UIDB/04129/2020 BACHALOPH funded by the Research Unit LEAF Linking Landscape, Environment, Agriculture and Food Research Center (Instituto Superior de Agronomia). S.N.-T. thanks the Federation of European Microbiological Societies (FEMS) (FEMS-GO-2020-203) and University of Sevilla (Spain; Plan Propio de Investigación y Tranferencia 2021 Ayuda A1-I.3A1) for grants to support the stay at ISA-ULisboa (Portugal). VS acknowledges the funding of a research contract (DL57/2016/CP1382/CT0004). Author Contributions Conceptualization, A.D.C. and A.N.; methodology, A.N. and A.D.C.; software, A.N.; validation, A.D.C. and A.N.; investigation S.N.-T., M.C.S.C. and V.S.; writing—original draft preparation, A.N. and A.D.C.; writing—review and editing, A.N., M.C.S.C.; V.S. and A.D.C.; supervision, A.N. and A.D.C.; project administration, A.D.C.; funding acquisition, A.D.C. All authors have read and agreed to the published version of the manuscript. References Balasooriya BLWK, Samson R, Mbikwa F, Boeckx P, Van Meirvenne M (2009) Biomonitoring of urban habitat quality by anatomical and chemical leaf characteristics. EEB 65:386–394. 10.1016/j.envexpbot.2008.11.009 Barbosa ACF, Pace MR, Witovisk L, Angyalossy V (2010) A new method to obtain good anatomical slides of heterogeneous plant parts. 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J Sustain For 36:107–120. 10.1080/10549811.2016.1256220 Statements & Declarations The authors declare that they have no conflict of interest Supplementary Files SupplementaryTableS1.docx SupplementaryTableS2.docx Cite Share Download PDF Status: Posted Version 1 posted You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. As a division of Research Square Company, we’re committed to making research communication faster, fairer, and more useful. We do this by developing innovative software and high quality services for the global research community. Our growing team is made up of researchers and industry professionals working together to solve the most critical problems facing scientific publishing. Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-4738414","acceptedTermsAndConditions":true,"allowDirectSubmit":true,"archivedVersions":[],"articleType":"Research Article","associatedPublications":[],"authors":[{"id":331676810,"identity":"45a9a6e7-6c94-4181-8d21-e19bccbfdfd3","order_by":0,"name":"Amaia Nogales","email":"","orcid":"","institution":"","correspondingAuthor":false,"prefix":"","firstName":"Amaia","middleName":"","lastName":"Nogales","suffix":""},{"id":331676811,"identity":"d1ef80b8-8456-41e8-b72b-376211d17872","order_by":1,"name":"Maria Cristina Simões Costa","email":"","orcid":"","institution":"","correspondingAuthor":false,"prefix":"","firstName":"Maria","middleName":"Cristina Simões","lastName":"Costa","suffix":""},{"id":331676812,"identity":"dd5455e1-65cb-4b15-a7c8-48ec6553b203","order_by":2,"name":"Salvadora Navarro-Torre","email":"","orcid":"","institution":"","correspondingAuthor":false,"prefix":"","firstName":"Salvadora","middleName":"","lastName":"Navarro-Torre","suffix":""},{"id":331676813,"identity":"664b87b2-7504-470c-b184-6a0ea884a950","order_by":3,"name":"Vicelina Sousa","email":"","orcid":"","institution":"","correspondingAuthor":false,"prefix":"","firstName":"Vicelina","middleName":"","lastName":"Sousa","suffix":""},{"id":331676814,"identity":"5c43d31e-6905-4a5b-8b53-68023332f662","order_by":4,"name":"Ana Caperta","email":"data:image/png;base64,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","orcid":"https://orcid.org/0000-0003-0142-8351","institution":"","correspondingAuthor":true,"prefix":"","firstName":"Ana","middleName":"","lastName":"Caperta","suffix":""}],"badges":[],"createdAt":"2024-07-14 12:49:45","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-4738414/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-4738414/v1","draftVersion":[],"editorialEvents":[],"editorialNote":"","failedWorkflow":false,"files":[{"id":63052951,"identity":"231b4a96-c2cb-4c8c-b711-f5024b9838ec","added_by":"auto","created_at":"2024-08-22 14:32:00","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":259248,"visible":true,"origin":"","legend":"\u003cp\u003eGermination percentage (%) of \u003cem\u003eLimonium \u003c/em\u003esp. seeds. The seeds were inoculated with \u003cem\u003ePantoea \u003c/em\u003esp. LDR15, or with a consortium of bacteria (Consort) of three bacteria (\u003cem\u003eP. anthophila\u003c/em\u003e LDR2, \u003cem\u003eP. agglomerans\u003c/em\u003e LDR25, and \u003cem\u003ePantoea\u003c/em\u003e sp. LVR13) or were left as non-inoculated controls. The germination percentage was recorded in culture media (tryptic soy agar) with or without 200 mM NaCl.\u003c/p\u003e","description":"","filename":"Fig.1.png","url":"https://assets-eu.researchsquare.com/files/rs-4738414/v1/578b1216434b21063c0305e4.png"},{"id":63052956,"identity":"4d30379a-ef73-4c69-b4e3-219dd3861375","added_by":"auto","created_at":"2024-08-22 14:32:01","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":115794,"visible":true,"origin":"","legend":"\u003cp\u003eFresh biomass, number of leaves, normalized difference vegetation index (NDVI) and photochemical reflectance index (PRI) in \u003cem\u003eLimonium algarvense\u003c/em\u003e and \u003cem\u003eL. daveaui\u003c/em\u003e plants inoculated or non-inoculated with LDR15 bacteria (\u003cem\u003ePantoea \u003c/em\u003esp.) or with a consortium (Consort) of three bacteria (\u003cem\u003eP.\u003c/em\u003e \u003cem\u003eanthophila \u003c/em\u003eLDR2, \u003cem\u003eP.\u003c/em\u003e \u003cem\u003eagglomerans \u003c/em\u003eLDR25, and \u003cem\u003ePantoea\u003c/em\u003e sp. LVR13). Bars indicate the average values of five plants ± standard error, and the letters above the bars indicate significant differences among group means according to Duncan or Dunn test (low-case letters for the groups under non-saline conditions, and capital letters for the groups under saline conditions). The boxes above the bars represent the results of the two-way ANOVA test for species and inoculation factors. The asterisk (*) indicates significance at p=0.05 and “ns” non-significant effects.\u003c/p\u003e","description":"","filename":"Fig.2.png","url":"https://assets-eu.researchsquare.com/files/rs-4738414/v1/6173f4de810bbc9241db404c.png"},{"id":63052953,"identity":"630d4daa-2e96-4436-9c7c-915970897aaf","added_by":"auto","created_at":"2024-08-22 14:32:00","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":3927121,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eEpidermal cells of the studied \u003c/strong\u003e\u003cem\u003e\u003cstrong\u003eLimonium \u003c/strong\u003e\u003c/em\u003e\u003cstrong\u003especies. A. \u003c/strong\u003eLeaf epidermis of \u003cem\u003eLimonium algarvense \u003c/em\u003eand \u003cem\u003eL. daveaui\u003c/em\u003e\u003cstrong\u003e \u003c/strong\u003efrom non-inoculated plants under non-saline treatment.\u003cstrong\u003e \u003c/strong\u003eScale\u003cstrong\u003e \u003c/strong\u003eBar= 100 µm. Salt gland (SG), stomata (S) and pavement cells (PC).\u003cstrong\u003e B\u003c/strong\u003e. Density of glands, stomata, and pavement cells in the two \u003cem\u003eLimonium \u003c/em\u003especies. \u003cem\u003eLimonium algarvense\u003c/em\u003e and \u003cem\u003eL. daveaui\u003c/em\u003e plants were inoculated or non-inoculated with LDR15 bacteria (\u003cem\u003ePantoea \u003c/em\u003esp.) or with a consortium (Cons) of three bacteria (\u003cem\u003eP.\u003c/em\u003e \u003cem\u003eanthophila \u003c/em\u003eLDR2, \u003cem\u003eP.\u003c/em\u003e \u003cem\u003eagglomerans \u003c/em\u003eLDR25, and \u003cem\u003ePantoea\u003c/em\u003e sp. LVR13). Bars indicate the average values of 5 plants ± standard error, and the letters above the bars indicate significant differences among the groups means according to Duncan or Dunn test (low-case letters for the groups under non-saline conditions, and capital letters for the groups under saline conditions). The boxes above the bars represent the results of the two-way ANOVA test for species and inoculation factors. The asterisk (*) indicates significance at p=0.05 and “ns” non-significant effects.\u003c/p\u003e","description":"","filename":"Fig.3.png","url":"https://assets-eu.researchsquare.com/files/rs-4738414/v1/6cbd107df7258adfffa7b183.png"},{"id":63052955,"identity":"5503462d-b70a-4e0d-ab6a-49554803734c","added_by":"auto","created_at":"2024-08-22 14:32:00","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":3047412,"visible":true,"origin":"","legend":"\u003cp\u003eRoot transverse sections of \u003cem\u003eLimonium \u003c/em\u003esp. plants non-inoculated or inoculated with bacterial inoculum. The images of root transverse sections in \u003cstrong\u003eA\u003c/strong\u003e and \u003cstrong\u003eB\u003c/strong\u003ewere obtained using a fluorescence microscope and the image in \u003cstrong\u003eC \u003c/strong\u003ewas obtained using light microscopy with a 10x objective.\u003cstrong\u003e A.\u003c/strong\u003e Non-inoculated roots of \u003cem\u003eL. algarvense\u003c/em\u003e. Intense blue lignin autofluorescence is visualized in the exodermis and stelle and in the Caspary bands. \u003cstrong\u003eB.\u003c/strong\u003e \u003cem\u003eLimonium daveaui \u003c/em\u003eplants inoculated with a bacterial consortium. The red fluorescence (mCherry marker) is mainly distributed in the cortex and phloem living tissues. Note absence of red labelling in the non-inoculated root. \u003cstrong\u003eC.\u003c/strong\u003e Root transverse section of \u003cem\u003eL. algarvense \u003c/em\u003eshowing the epidermis (Ep), cortex parenchyma (Co), stele (St) or vascular cylinder, endodermis (En) and xylem vessels (arrows). Cell walls unlignified will appear blue (stained with astra blue) while those lignified will appear red (stained with safranin-O).\u003c/p\u003e","description":"","filename":"Fig.4.png","url":"https://assets-eu.researchsquare.com/files/rs-4738414/v1/92e7b92c9939bad9e67eda35.png"},{"id":66434305,"identity":"11566400-fe08-4e90-a332-49cf8c25d076","added_by":"auto","created_at":"2024-10-11 22:31:03","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":12035360,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-4738414/v1/1de20144-41a9-4618-b223-e46a31a57ced.pdf"},{"id":63052954,"identity":"47896b93-82c4-40bd-8b26-41bae0abf791","added_by":"auto","created_at":"2024-08-22 14:32:00","extension":"docx","order_by":1,"title":"","display":"","copyAsset":false,"role":"supplement","size":14562,"visible":true,"origin":"","legend":"","description":"","filename":"SupplementaryTableS1.docx","url":"https://assets-eu.researchsquare.com/files/rs-4738414/v1/0a4d257d3d43d8873d8a7291.docx"},{"id":63052952,"identity":"657f769a-53fc-4935-9467-cbe9c268a63f","added_by":"auto","created_at":"2024-08-22 14:32:00","extension":"docx","order_by":2,"title":"","display":"","copyAsset":false,"role":"supplement","size":17138,"visible":true,"origin":"","legend":"","description":"","filename":"SupplementaryTableS2.docx","url":"https://assets-eu.researchsquare.com/files/rs-4738414/v1/ac4e6ae837bda28a4c30d0d7.docx"}],"financialInterests":"","formattedTitle":"Do bacterial root endophytes contribute to growth in saline conditions? A pre-reintroduction cultivation study of threatened saltmarsh Limonium species","fulltext":[{"header":"Introduction","content":"\u003cp\u003eSoil biology has frequently been overlooked in plant reintroduction and practice, despite the crucial role played by soil microbial communities in species restoration (Farrell et al. \u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e2020\u003c/span\u003e). Specifically, plants that thrive in saline habitats, such as salt-tolerant plants (halophytes), exhibit a high degree of habitat specialization and host a characteristic microbiome consisting of halophilic or halotolerant microorganisms (Navarro-Torre et al. \u003cspan citationid=\"CR34\" class=\"CitationRef\"\u003e2023a\u003c/span\u003e; Nogales et al. \u003cspan citationid=\"CR37\" class=\"CitationRef\"\u003e2023\u003c/span\u003e). Halotolerant/halophilic bacteria either tolerate or require saline conditions for their growth (Ventosa et al. \u003cspan citationid=\"CR50\" class=\"CitationRef\"\u003e1998\u003c/span\u003e), since they possess mechanisms that include changes in cell wall and/or plasmatic membrane to prevent salt entry (e.g., Na and K pumps and antiporter channels), accumulate osmoprotectants (e.g., ectoine, betaine, trehalose), and/or secrete of exopolysaccharides to help in biofilm formation (Etesami and Beattie \u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e2018\u003c/span\u003e). Some of these bacteria present plant growth promoting traits essential for plant nutrient uptake (e.g., N, P, Fe) as well as for improving host tolerance to biotic and abiotic stresses, including salinity stress (Trivedi et al. \u003cspan citationid=\"CR49\" class=\"CitationRef\"\u003e2020\u003c/span\u003e; Navarro-Torre et al. \u003cspan citationid=\"CR34\" class=\"CitationRef\"\u003e2023a\u003c/span\u003e). As a result, microbial inoculation emerges as a valuable strategy in the agricultural management of saline soils (Navarro-Torre et al. \u003cspan citationid=\"CR34\" class=\"CitationRef\"\u003e2023a\u003c/span\u003e), phytoremediation in saline saltmarshes (Paredes-P\u0026aacute;liz et al. \u003cspan citationid=\"CR38\" class=\"CitationRef\"\u003e2017\u003c/span\u003e), and coastal restoration (Farrer et al. \u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e2022\u003c/span\u003e), although neglected in plant conservation schemes.\u003c/p\u003e \u003cp\u003eThe Mediterranean halophytic flora and vegetation are characterized by a high degree of specialization, with species specifically adapted to saline environments, as exemplified by the genus \u003cem\u003eLimonium\u003c/em\u003e Mill. (sea-lavenders), which harbors numerous endemic species (Caperta et al. \u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e2020\u003c/span\u003e; Salazar-Mend\u0026iacute;as and Lend\u0026iacute;nez \u003cspan citationid=\"CR46\" class=\"CitationRef\"\u003e2020\u003c/span\u003e). As other halophytes, their tolerance to salinity relies on many adaptive mechanisms like controlled uptake and compartmentalization of Na\u003csup\u003e+\u003c/sup\u003e, K\u003csup\u003e+\u003c/sup\u003e and Cl\u003csup\u003e\u0026minus;\u003c/sup\u003e, osmolytes synthesis (Flowers and Colmer \u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e2008\u003c/span\u003e), and/or salt excretion through salt glands (Caperta et al. \u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e2020\u003c/span\u003e). Many \u003cem\u003eLimonium\u003c/em\u003e coastal endemics are threatened, such as \u003cem\u003eLimonium perplexum\u003c/em\u003e in eastern Spain, established on deep crevices in native habitat, in which translocation works were developed to conserve this species (Laguna et al. \u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e2016\u003c/span\u003e). In mainland Portugal, extinction risk assessments indicate that \u003cem\u003eLimonium algarvense\u003c/em\u003e Erben is classified as Near Threatened, while \u003cem\u003eLimonium daveaui\u003c/em\u003e Erben is deemed Critically Endangered according to extinction risk categories by the International Union for Conservation of Nature (IUCN) (Carapeto et al. \u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e2020\u003c/span\u003e). While \u003cem\u003eL. algarvense\u003c/em\u003e is distributed in Iberian Peninsula and in Morocco (Caperta et al. \u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e2017\u003c/span\u003e; Carapeto et al. \u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e2020\u003c/span\u003e), L. \u003cem\u003edaveaui\u003c/em\u003e, which previously had a broader range, is now restricted to a few populations along the west coast of Portugal (Caperta and Carapeto \u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e2020\u003c/span\u003e; Carapeto et al. \u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e2020\u003c/span\u003e).\u003c/p\u003e \u003cp\u003ePre-reintroduction cultivation studies are essential for understanding the most effective methods for propagating such species and for making informed decisions to minimize the risks associated with translocating individuals into recipient habitats in species recovery programs (Godefroid et al. \u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e2016\u003c/span\u003e). Both \u003cem\u003eL. daveaui\u003c/em\u003e and \u003cem\u003eL. algarvense\u003c/em\u003e can be cultivated using saline soil and brackish, estuarine water at least at 100 mM NaCl (Cortinhas et al. \u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e2020\u003c/span\u003e, \u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e2021\u003c/span\u003e; Rodrigues et al. \u003cspan citationid=\"CR44\" class=\"CitationRef\"\u003e2020\u003c/span\u003e). Previous greenhouse studies using microbial inoculation treatments in \u003cem\u003eL. algarvense\u003c/em\u003e showed that plant growth promoting bacteria (PGPB) isolated from \u003cem\u003eArthrocnemum macrostachyum\u003c/em\u003e (Navarro-Torre et al. \u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e2017\u003c/span\u003e) led to better plant physiological status under saline conditions (Nogales et al. \u003cspan citationid=\"CR37\" class=\"CitationRef\"\u003e2023\u003c/span\u003e). However, an alternative approach is the use of bacterial inoculum sourced from reference ecosystems (e.g. saltmarshes), as it offers a specific microbial community.\u003c/p\u003e \u003cp\u003eIn some restoration projects PGPB were tested as biofertilizers, both as pure cultures and as consortia, to enhance the growth of \u003cem\u003eSpartina alterniflora\u003c/em\u003e, a common wetland grass. The results of those projects indicated a significant growth improvement under saline conditions compared to freshwater under greenhouse conditions (Bledsoe and Boopathy \u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e2016\u003c/span\u003e). In \u003cem\u003eLimonium\u003c/em\u003e species, a great diversity of microorganisms has been isolated from \u003cem\u003eLimonium sinense\u003c/em\u003e (Qin et al. \u003cspan citationid=\"CR43\" class=\"CitationRef\"\u003e2018\u003c/span\u003e), L. \u003cem\u003edaveaui\u003c/em\u003e, \u003cem\u003eLimonium multiflorum\u003c/em\u003e Erben and \u003cem\u003eLimonium vulgare\u003c/em\u003e roots (Navarro-Torre et al. \u003cspan citationid=\"CR35\" class=\"CitationRef\"\u003e2023b\u003c/span\u003e). In the last three species, the root endosphere was mainly formed by Gram negative bacteria, being the most representative genera \u003cem\u003ePantoea\u003c/em\u003e and \u003cem\u003eErwinia\u003c/em\u003e. In the presence of 0.2 M NaCl, these bacterial strains formed biofilms and the amount of IAA (indole-3-acetic acid, phytohormone auxin) produced was enhanced, being the highest increase found in the strain LDR15 isolated from \u003cem\u003eL. daveaui\u003c/em\u003e (Navarro-Torre et al. \u003cspan citationid=\"CR35\" class=\"CitationRef\"\u003e2023b\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eIn this framework, the hypothesis of our study was that endophytic bacteria isolated from autochthonous \u003cem\u003eLimonium\u003c/em\u003e species could promote sea-lavenders\u0026rsquo; tolerance to salinity, enabling them to withstand adverse saline cultivation environments. Using halotolerant bacteria isolated from \u003cem\u003eLimonium\u003c/em\u003e sp. grown in saltmarshes and selected for their superior plant growth-promoting traits and NaCl tolerance (Navarro-Torre et al. \u003cspan citationid=\"CR35\" class=\"CitationRef\"\u003e2023b\u003c/span\u003e), the goals of this study were to: 1) investigate the impact of selected bacteria on \u003cem\u003eLimonium\u003c/em\u003e sp. seed germination; 2) evaluate their effects on the leaf epidermis, including the density of salt glands, stomata, and pavement cells, in expanded leaves; and 3) assess their potential benefits for plant growth under saline conditions in relation to freshwater.\u003c/p\u003e"},{"header":"Materials and Methods","content":"\u003cdiv id=\"Sec3\" class=\"Section2\"\u003e \u003ch2\u003eGermination assay\u003c/h2\u003e \u003cp\u003eA seed germination assay was performed to assess the impact of different PGPB inoculants, including both single species and a consortium, derived from bacterial strains isolated from \u003cem\u003eL. daveaui\u003c/em\u003e and \u003cem\u003eL. vulgare\u003c/em\u003e (Navarro-Torre et al. \u003cspan citationid=\"CR35\" class=\"CitationRef\"\u003e2023b\u003c/span\u003e). The effect of the bacterial strain LDR15 (\u003cem\u003ePantoea\u003c/em\u003e sp.) on the germination of field-collected \u003cem\u003eLimonium\u003c/em\u003e sp. seeds was compared to that of a consortium of \u003cem\u003eP. anthophila\u003c/em\u003e, \u003cem\u003eP. agglomerans\u003c/em\u003e, and \u003cem\u003ePantoea\u003c/em\u003e sp. (strains LDR2, LDR25 and LVR13). \u003cem\u003ePantoea\u003c/em\u003e sp. LDR15 was chosen as a single species-based inoculum because it presented remarkable plant growth promoting traits (IAA and siderophore production, phosphate solubilization, biofilm formation and nitrogen fixation). The bacterial strains LDR2, LDR25 and LVR13 were selected to be used as a consortium-based inoculum as they collectively presented highly interesting and complementary plant growth promoting traits. Specifically, LDR2 and LDR25 exhibited notable production of IAA, while LVR13 demonstrated high phosphate solubilization capability, particularly in saline conditions (0.2 M NaCl) (Navarro-Torre et al. \u003cspan citationid=\"CR35\" class=\"CitationRef\"\u003e2023b\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eTo prepare the bacterial inocula (single species or consortium-based), each strain was incubated separately in 5 ml sterilized tryptic soy broth (TSB) medium for 24 h at 28 ˚C in continuous shaking (115 rpm). After that, cultures containing 10\u003csup\u003e8\u003c/sup\u003e cells\u0026middot;ml\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e were centrifuged at 8000 rpm for 5 min. Then, pellets were washed with sterile 0.9% saline solution and centrifuged again. Finally, they were resuspended in 1 ml sterile 0.9% saline solution, and the strains that conform the consortium were mixed in the same tube (Navarro-Torre et al. \u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e2017\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eA new protocol was developed for seed disinfection, in which seeds of each species were washed with tap water for 20 min, with shaking every 5 min. Seed surface disinfection was then carried out in a horizontal laminar flow air chamber (INC Gelaire\u0026reg;HF48). Three sterilising solutions were used for seed disinfection: hydrogen peroxide at 18% (g/v) for 5 min; alcohol 70% for 1 min and commercial bleach at 15% (with 5% sodium hypochlorite) with 3 drops of teepol / 100 ml, for 20 min. After immersion in each disinfectant solution, the seeds were collected on sterile filter paper, in a funnel, and rinsed five times with sterile distilled water. Seeds were left in distilled water for approximately 48 hours with agitation, in a growth chamber at 26/22\u0026thinsp;\u0026plusmn;\u0026thinsp;1 \u0026ordm;C day/night, with 16 h / 8 h photoperiod.\u003c/p\u003e \u003cp\u003eAfter 48 h, the disinfected seeds were bacterized for 1 h under shaking in 1 ml of the respective inoculum (LDR15 or the consortium). Seeds in the control treatment were incubated with sterile 0.9% saline solution. Then, 50 seeds of each experimental treatment (control, bacterized with either LDR15 or the consortium) were deposited in Petri plates containing a culture medium consisting of the medium described by Rossini et al. (2010) and supplemented with 0 M NaCl (w/v) or 0.2 M NaCl (w/v) before the addition of 7 g/L agar (Duchefa Biochemie). Four plates were considered per experimental treatment (200 seeds per treatment), which were placed in a growth chamber under the same temperature and light conditions as described above. Germination was checked every day for 20 days and the kinetic of germination percentage was recorded.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec4\" class=\"Section2\"\u003e \u003ch2\u003eExperimental set-up\u003c/h2\u003e \u003cp\u003eTo determine the effects of the two inoculants (LDR15 bacteria or consortium) on \u003cem\u003eL. daveaui\u003c/em\u003e and \u003cem\u003eL. algarvense\u003c/em\u003e plant growth, twenty seedlings per species were surface-disinfected and germinated in \u003cem\u003ein vitro\u003c/em\u003e conditions as described before. The seedlings were transferred to jiffy pots (peat substrate), placed in a growth chamber (23/18\u0026thinsp;\u0026plusmn;\u0026thinsp;1 \u0026ordm;C day/night, 18/6 h photoperiod) and inoculated weekly with 1 mL of each bacterial inoculant or distilled water (in the control treatment).\u003c/p\u003e \u003cp\u003eFour weeks later, plants were transplanted into individual pots with a mixture of peat and vermiculite (1:2 v/v). They were then divided into six different experimental groups: non-inoculated, LDR15-inoculated, and consortium-inoculated plants, each subjected to either saline irrigation (100 mM NaCl) or freshwater irrigation (tap water).\u003c/p\u003e \u003cp\u003eInoculum was prepared as described before, and each plant was inoculated 5 mL of the corresponding inoculum (LDR15 or the consortium of LDR2, LDR25 and LVR13). Non-inoculated plants were watered with 5 mL of saline solution. This procedure was repeated fortnightly.\u003c/p\u003e \u003cp\u003eIrrigation was managed via an automatic watering system with regulated flow rates. The selection of 100 mM NaCl as the salinity concentration was based on prior research indicating favourable development of \u003cem\u003eLimonium\u003c/em\u003e species under these conditions (Cortinhas et al. \u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e2020\u003c/span\u003e, \u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e2021\u003c/span\u003e). Plants were maintained in greenhouse conditions with natural light for five months.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec5\" class=\"Section2\"\u003e \u003ch2\u003ePlant physiology and growth\u003c/h2\u003e \u003cp\u003ePlants physiological status was evaluated by reflectance index assessment (Normalized Difference Vegetation Index -NDVI and Photochemical Reflectance Index-PRI). The NDVI and PRI were measured using PlantPen NDVI 300 (PSI, Czech Republic) and PlantPen PRI 200 (PSI, Czech Republic) portable devices, respectively. Photochemical Reflectance Index is indicative of photosynthesis efficiency and plant stress levels (Garbulsky et al. \u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e2011\u003c/span\u003e) while NDVI reflects plant vigor and, indirectly, chlorophyll status, phosphorus, and nitrogen nutrition (Sembiring et al. \u003cspan citationid=\"CR48\" class=\"CitationRef\"\u003e1998\u003c/span\u003e). Measurements were performed four months after the first bacterial inoculation was done. Those were conducted in three young leaves per plant of each experimental group. At the end of the experiment, the number of leaves per plant was counted and the fresh biomass was determined.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec6\" class=\"Section2\"\u003e \u003ch2\u003ePavement cells, stomatal and salt glands characteristics\u003c/h2\u003e \u003cp\u003eLeaf imprints from the abaxial/adaxial surfaces of young, expanded leaves from three \u003cem\u003eL. algarvense\u003c/em\u003e and \u003cem\u003eL. daveaui\u003c/em\u003e plants were taken from each treatment (species x saline condition x type of inoculation) following Balasooriya et al. (\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e2009\u003c/span\u003e). The imprints were made using colourless nail polish and transparent adhesive tape. Then, they were fixed on a microscope slide and observed under light microscope (Leitz Dialux 20EB) at 125 x 10 magnification. Images of five fields per imprint were captured using a digital camera (Leica EC3) and image software (LAS V.2.13). The density of glands, stomata, and pavement cells were calculated per mm\u003csup\u003e2\u003c/sup\u003e leaf area using the software ImageJ v. 1 49s.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec7\" class=\"Section2\"\u003e \u003ch2\u003eBacterial colonization\u003c/h2\u003e \u003cp\u003eA parallel experiment was conducted to study the localization of bacterial strain colonization. For this purpose, transformed LDR15, LDR2, LDR25 and LVR13 bacterial strains containing the plasmid pMP7604 that codifies for the \u003cem\u003emCherry\u003c/em\u003e fluorescent protein were used. Transformation procedure was conducted by conjugation as described in Navarro-Torre et al. (\u003cspan citationid=\"CR35\" class=\"CitationRef\"\u003e2023b\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eFor four months five plants were inoculated with each of the four individual fluorescent bacterial strains, and another set of five plants were watered with saline solution (0.09%). Each inoculum was prepared in the same way as described previously for the wild-type strains.\u003c/p\u003e \u003cp\u003eRoot bacterial colonization was analysed as described previously (Navarro-Torre et al. \u003cspan citationid=\"CR35\" class=\"CitationRef\"\u003e2023b\u003c/span\u003e). In brief, root samples from four-month-old plants of each species were collected and fixed using a 4% formaldehyde acetic solution in 70% ethanol for 48 h. Then, the roots were washed in a 70% ethanol solution and stored at 4 \u0026ordm;C until used.\u003c/p\u003e \u003cp\u003eSamples were initially prepared for observation under light microscopy. They were first impregnated with DP1500 polyethylene glycol as described by Barbosa et al. (\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2010\u003c/span\u003e). Then, 17\u0026ndash;20 \u0026micro;m thick transverse sections were cut using a sliding microtome (Leica SM 2400), stained with Safranin/Astrablue (1% aqueous solution) and mounted with a glycerine/water solution for temporary slide preparation. Images were captured using analysis image software (Leica Qwin) coupled to a microscope (Leica DMLA).\u003c/p\u003e \u003cp\u003eFor visualizing bacterial colonization, the preparations were observed under a fluorescence microscope (Zeiss Axioskop 2), equipped with a digital camera (Zeiss AxioCam) with a 10x objective. Image acquisition was performed using the Carl Zeiss/AxioVision 4.8 software.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec8\" class=\"Section2\"\u003e \u003ch2\u003eStatistical analyses\u003c/h2\u003e \u003cp\u003eStatistical analyses were performed using SPSS Statistics vs. 23 (IBM) program and in all cases data normality and variance homogeneity were tested prior to the analyses. Germination percentage data were analyzed by a three-way ANOVA where time, salinity and inoculation treatment were considered as main factors. Following this, to delve deeper into the impact of bacterial and salinity treatments, the influence of both factors was examined at each individual week by a two-way ANOVA.\u003c/p\u003e \u003cp\u003eIn the experiment of plant growth response to halotolerant bacteria inoculation and salinity, due to missing values in one experimental group (salinity-subjected plants), the number of leaves, fresh biomass, NDVI, PRI, density of glands, stomata and leaf pavements cells data were analyzed separately under freshwater and saline conditions. At each soil condition, data were investigated by a two-way ANOVA where species and inoculation treatment were considered as main factors.\u003c/p\u003e \u003c/div\u003e"},{"header":"Results","content":"\u003cdiv id=\"Sec10\" class=\"Section2\"\u003e \u003ch2\u003eEffects of plant growth promoting bacteria in seed germination\u003c/h2\u003e \u003cp\u003eThe results of the three-way ANOVA conducted to study the effects of bacterial inoculation, time, and salinity on \u003cem\u003eLimonium\u003c/em\u003e species seed germination is shown in Supplementary Table \u003cspan refid=\"MOESM1\" class=\"InternalRef\"\u003eS1\u003c/span\u003e. As expected, time had a significant effect on germination percentage but did not interact with the other factors. Overall, salinity had a negative effect, but the inoculation factor did not, although the significance of this factor was p\u0026thinsp;=\u0026thinsp;0.08. The interaction between both factors was nonsignificant, but it approached significance at a p-value of 0.098. To unveil potential patterns in the effects of both factors on seed germination that might be masked by cumulative effects over time, the data were analyzed separately for each week.\u003c/p\u003e \u003cp\u003eWhen data were analyzed on a weekly basis, we observed a significant negative effect of NaCl on seed germination from week four onwards (Supplementary Table \u003cspan refid=\"MOESM2\" class=\"InternalRef\"\u003eS2\u003c/span\u003e, Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003e). On the other hand, bacterial inoculation became significant starting from week eight: non-inoculated seeds had higher germination percentage than those inoculated with the consortium, and the ones inoculated with LDR15 had intermediate values. From week 10 onwards the germination percentage of non-inoculated seeds under saline conditions continued to rise, while in the other experimental groups it approached its peak, which was consistently reached by week 15 across all groups. From this point onwards, the p-value for the interaction between both factors was 0.051, which was reflected by a pronounced impact of salinity in LDR15-inoculated and consortium-inoculated seeds, but not in the non-inoculated ones (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003e, Supplementary Table\u0026nbsp;1).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec11\" class=\"Section2\"\u003e \u003ch2\u003eStudy of plant response to the inoculation with halotolerant bacteria and to salinity\u003c/h2\u003e \u003cp\u003eIn plants grown for four months under freshwater conditions, the number of leaves and fresh biomass were not influenced by the bacterial inoculation treatment (non-inoculation, or inoculation with either LDR15 or the consortium) in any of the \u003cem\u003eLimonium\u003c/em\u003e species. Moreover, while the number of leaves was similar in \u003cem\u003eL. algarvense\u003c/em\u003e and \u003cem\u003eL. daveaui\u003c/em\u003e, the fresh biomass was higher in \u003cem\u003eL. daveaui\u003c/em\u003e than in \u003cem\u003eL. algarvense\u003c/em\u003e (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003e). In such conditions, the PRI was solely influenced by the species factor, with higher values in \u003cem\u003eL. algarvense\u003c/em\u003e plants. In the case of NDVI, there was a significant interaction between species and inoculation treatment factors. While \u003cem\u003eL. algarvense\u003c/em\u003e plants inoculated with LDR15 had significantly higher values than non-inoculated plants or plants inoculated with the consortium, the inoculation treatment did not have a significant effect in NDVI in \u003cem\u003eL. daveaui\u003c/em\u003e (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eUnder saline conditions, both the number of leaves and fresh biomass were reduced, and all plants inoculated with the bacterial consortium died. The fresh weight was almost five times lower in both \u003cem\u003eL. algarvense\u003c/em\u003e and \u003cem\u003eL. daveaui\u003c/em\u003e plants growing in the salinized substrate, and the number of leaves was reduced from 11.3\u0026thinsp;\u0026plusmn;\u0026thinsp;0.31 in plants grown under freshwater conditions to 3.6\u0026thinsp;\u0026plusmn;\u0026thinsp;0.30 in plants growing in the salinized substrate. Besides, plants grown under salinity had very small and succulent leaves that did not allow us to conduct NDVI and PRI measurements. On the other hand, the inoculation factor had a significant effect, and plants inoculated with LDR15 tended to have higher biomass than the non-inoculated ones, although the multiple mean comparison test conducted to explore differences among all experimental groups did not show significant differences between non-inoculated and LDR15-inoculated plants.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec12\" class=\"Section2\"\u003e \u003ch2\u003eInfluence of bacterial inoculation and saline treatment on salt glands, stomata, and pavement cells\u003c/h2\u003e \u003cp\u003eIn both \u003cem\u003eLimonium\u003c/em\u003e species, under non-saline or saline conditions, and regardless of the inoculation treatment, salt glands and stomata (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eA) were noticeable on the surface of the two leaf blades, and adjacent salt glands and stomata were separated by at least two pavement cells. The salt glands were sunken into the epidermis, and stomata were at the same level as the pavement cells (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eA). Each salt gland contained 16 cells arranged in quadrants, with four cells in each quadrant (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eA). In \u003cem\u003eL. algarvense\u003c/em\u003e, each salt gland was encircled by 4\u0026ndash;7 pavement cells, while in \u003cem\u003eL. daveaui\u003c/em\u003e, it was surrounded by 4\u0026ndash;8 cells.\u003c/p\u003e \u003cp\u003eUnder freshwater conditions, the density of glands was not affected by the species or inoculation treatment factors (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eB). However, the density of stomata and pavement cells showed a different pattern, and a significant interaction was found between both factors. Whereas \u003cem\u003eL. algarvense\u003c/em\u003e plants had a consistent density of stomata and pavement cells across the three inoculation treatments, \u003cem\u003eL. daveaui\u003c/em\u003e plants inoculated with the consortium had significantly lower values than the other two inoculation treatments (non-inoculated and inoculated with LDR15) (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eB).\u003c/p\u003e \u003cp\u003eIn the salinized substrate, the density of stomata was influenced by the species, with \u003cem\u003eL. daveaui\u003c/em\u003e exhibiting the lowest density (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eB). The effect of the inoculation was not significant in this parameter when non-inoculated and LDR15-inoculated plants were considered, but as mentioned earlier, all plants inoculated with the consortium died. Contrastingly, pavement cell density was not affected by either the species or the inoculation treatment.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec13\" class=\"Section2\"\u003e \u003ch2\u003eRoot bacterial colonization\u003c/h2\u003e \u003cp\u003eThe blue autofluorescence due to compounds like lignin and suberin, usually deposited in the xylem tissue as well as in the Caspary bands, was visualized in both the PGPB-inoculated and non-inoculated root samples (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003e). In the PGPB-inoculated samples, the fluorescence from the mCherry protein was recorded within epidermal, cortical parenchyma, and phloem cells, indicating that the bacteria were present in these tissues.\u003c/p\u003e \u003c/div\u003e"},{"header":"Discussion","content":"\u003cp\u003ePlant microorganisms present in the rhizosphere are essential for proper plant growth and adaptation to stress factors (Trivedi 2020) justifying their increasing use as a tool in sustainable agriculture and environmental management of saline soils (Navarro-Torre et al. \u003cspan citationid=\"CR34\" class=\"CitationRef\"\u003e2023a\u003c/span\u003e). However, there are limited studies available on the use of PGPB in the restoration of endangered endemic species, especially from coastal habitats (Farrer et al. \u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e2022\u003c/span\u003e). Furthermore, some reports reveal contrasting results (Michaelis and Diekmann \u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e2018\u003c/span\u003e). In the current study, we evaluated the effect of two PGPB inocula based on bacterial strains isolated from the plant rhizosphere of endangered halophyte \u003cem\u003eLimonium\u003c/em\u003e species thriving in coastal saltmarshes, based on the hypothesis that halotolerant PGPB inoculations could possibly increase seed germination and plant adaptation to saline conditions.\u003c/p\u003e \u003cdiv id=\"Sec15\" class=\"Section2\"\u003e \u003ch2\u003eInoculation with native halotolerant bacteria did not improve seed germination\u003c/h2\u003e \u003cp\u003eHalophyte seeds can remain viable after long periods of exposure to salinity and begin the germination process when salinity levels decrease (low osmotic potential) (Khan and Ungar \u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e1996\u003c/span\u003e). The osmotic stress delays seed germination since salinity reduces seed imbibition (Mu\u0026ntilde;oz-Rodr\u0026iacute;guez et al. \u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e2017\u003c/span\u003e). For example, \u003cem\u003eL. daveaui\u003c/em\u003e seed germination percentage was higher in seeds germinated in distilled water than in a saline Fluvisol (Cortinhas et al. \u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e2021\u003c/span\u003e). Since in the present study we wanted to test the influence of bacterial inoculation in seed germination \u003cem\u003ein vitro\u003c/em\u003e, it was essential to develop a new protocol for seed disinfection that allows both germination in aseptically conditions, as well as the development of bacterial communities. The disinfection procedure softened the seed coats possibly contributing to better seed imbibition. One of the sterilising solutions used was hydrogen peroxide (H\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e, a common reactive oxygen species) that is a regulator of developmental processes like dormancy release, cell-wall loosening, and reserve mobilization as found in non-halophytes like \u003cem\u003eArabidopsis\u003c/em\u003e (Liu et al. \u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e2010\u003c/span\u003e; Wojtyla et al. \u003cspan citationid=\"CR53\" class=\"CitationRef\"\u003e2016\u003c/span\u003e). In germinating seeds of halophytes \u003cem\u003eArthrocnemum macrostachyum\u003c/em\u003e, \u003cem\u003eArthrocnemum indicum\u003c/em\u003e, \u003cem\u003eSuaeda fruticosa\u003c/em\u003e and \u003cem\u003eLimonium stocksii\u003c/em\u003e the H\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e content enhanced with increases in NaCl concentration in both latter two species (Hameed et al. \u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e2014\u003c/span\u003e; Nisar et al. \u003cspan citationid=\"CR36\" class=\"CitationRef\"\u003e2019\u003c/span\u003e).\u003c/p\u003e \u003cp\u003ePrevious works have demonstrated that inoculation with PGPB can improve seed germination in saline conditions (Yousefi et al. \u003cspan citationid=\"CR56\" class=\"CitationRef\"\u003e2017\u003c/span\u003e; Navarro-Torre et al. \u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e2017\u003c/span\u003e). In this last study, bacterial pre-inoculation in a halophyte species (e.g. \u003cem\u003eA. macrostachyum\u003c/em\u003e) considerably enhanced the kinetics of germination and final germination percentage. Contrastingly, in our study, inoculation with halotolerant PGPB either alone (LDR15) or in consortium reduced the germination percentage in the presence of NaCl. Although the PGPB employed proved to be tolerant to 200 mM NaCl in the germination medium (Navarro-Torre et al. \u003cspan citationid=\"CR35\" class=\"CitationRef\"\u003e2023b\u003c/span\u003e), in general they did not have a seed germination promoting effect at 200 mM NaCl. Other studies also point towards the same scenario. Fouladvand and SoltaniIn (2024) compared the germination of non-inoculated and inoculated wheat seeds with different bacterial endophytes under 250 mM NaCl and most of these endophytes led to a lower percentage of germination than control seeds. In the same way, some of the single species or consortia inoculations tested in Petrillo et al. (\u003cspan citationid=\"CR41\" class=\"CitationRef\"\u003e2022\u003c/span\u003e) showed similar or negative effects on the germination of \u003cem\u003eSpinacia oleracea\u003c/em\u003e seeds compared to the control seeds. Although the exact reasons for the negative effect of the halotolerant bacterial inoculation on seed germination remain uncertain, several factors could be involved, such as a poor bacterial adhesion on the seed surface, or an excess of bacterial cells in the inoculum. The simultaneous demands placed on the seed, including combating salinity while accommodating the proliferating symbionts, might have also overwhelmed its capacity to germinate effectively. Ultimately, this scenario reflects a complex interplay of factors, that may ultimately hinder its ability to allocate energy efficiently towards germination.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec16\" class=\"Section2\"\u003e \u003ch2\u003eBenefits of halotolerant bacteria inoculation in plant physiological status and growth\u003c/h2\u003e \u003cp\u003eIn the present study, the Gram-negative bacteria used for plant inoculation were localized in the root endosphere of both \u003cem\u003eL. algarvense\u003c/em\u003e and \u003cem\u003eL. daveaui\u003c/em\u003e, regardless of whether the plants were grown in freshwater or saline conditions. Root colonization by different PGPB has also been reported in other \u003cem\u003eLimonium\u003c/em\u003e species such as \u003cem\u003eLimonium sinense\u003c/em\u003e and \u003cem\u003eLimonium vulgare\u003c/em\u003e subjected to salinity (Qin et al. \u003cspan citationid=\"CR42\" class=\"CitationRef\"\u003e2014\u003c/span\u003e, \u003cspan citationid=\"CR43\" class=\"CitationRef\"\u003e2018\u003c/span\u003e; Wang et al. \u003cspan citationid=\"CR52\" class=\"CitationRef\"\u003e2017\u003c/span\u003e; Xiong et al. \u003cspan citationid=\"CR55\" class=\"CitationRef\"\u003e2020\u003c/span\u003e). In our study, those bacteria had a specific tissue localization pattern, restricted to living cells and absent in the xylem vessels, as also observed in a non-halophyte species, i.e. grapevine, inoculated with the same bacterial inoculum (Navarro-Torre et al. \u003cspan citationid=\"CR35\" class=\"CitationRef\"\u003e2023b\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eIn general, halophytes can survive under 200 mM NaCl or approximately 20 dS/m EC) (Flowers and Colmer \u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e2008\u003c/span\u003e) and their growth can be stimulated within a salinity range of 15\u0026ndash;25 dS/m EC (Rozema and Schats \u003cspan citationid=\"CR45\" class=\"CitationRef\"\u003e2013\u003c/span\u003e). In our study, a differential species performance was observed, with the salinity effects being more pronounced in \u003cem\u003eL. algarvense\u003c/em\u003e. The impact of salinity led to a reduction in both leaf number and size, leaf succulence, and biomass, with a greater effect observed in \u003cem\u003eL. algarvense\u003c/em\u003e compared to \u003cem\u003eL. daveaui.\u003c/em\u003e Succulence is an adaptive mechanism enabling halophytes to grow for long periods of time under high salinity (Dassanayake and Larkin \u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e2017\u003c/span\u003e; Caperta et al. \u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e2020\u003c/span\u003e). Moreover, both species presented salt glands and stomata in both leaf blades, with the salt glands displaying the previously identified complex 16-celled structure observed in various \u003cem\u003eLimonium\u003c/em\u003e species (Caperta et al. \u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e2020\u003c/span\u003e). In both \u003cem\u003eLimonium\u003c/em\u003e species studied in the present work, the distribution patterns of salt glands in the leaf blades were similar, while in other \u003cem\u003eLimonium\u003c/em\u003e species salt gland density varied in the adaxial and abaxial epidermis. For example, \u003cem\u003eL. bicolor\u003c/em\u003e and \u003cem\u003eL. franchetii\u003c/em\u003e exhibited greater salt gland density in the abaxial epidermis than in adaxial one, whereas \u003cem\u003eL. gmelinii\u003c/em\u003e showed the opposite trend (Xin et al. \u003cspan citationid=\"CR54\" class=\"CitationRef\"\u003e2012\u003c/span\u003e; Leng et al. \u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e2018\u003c/span\u003e), indicating that saline gland distribution varies among \u003cem\u003eLimonium\u003c/em\u003e species. By contrast, in \u003cem\u003eL. aureum\u003c/em\u003e, \u003cem\u003eL. gmelinii\u003c/em\u003e, \u003cem\u003eL. otolepsis\u003c/em\u003e and \u003cem\u003eL. sinuatum\u003c/em\u003e salt gland densities enhanced with increasing NaCl concentration (Mi et al. \u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e2021\u003c/span\u003e). Moreover, under the same NaCl concentration, salt gland and stomata densities of \u003cem\u003eL. bicolor\u003c/em\u003e were significantly higher than in \u003cem\u003eL. gmelinii\u003c/em\u003e (Leng et al. \u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e2018\u003c/span\u003e). In our study, the density of stomata was influenced by the species factor, with \u003cem\u003eL. daveaui\u003c/em\u003e exhibiting the lowest density in saline conditions.\u003c/p\u003e \u003cp\u003eAs for plant growth, in previous studies, \u003cem\u003eL. daveaui\u003c/em\u003e plants showed lower development as well as reduced values of photosynthetic indexes and biomass production when grown in a salinized substrate (100 mM NaCl) compared to the non-saline one (Cortinhas et al. \u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e2021\u003c/span\u003e). A similar trend was observed for \u003cem\u003eL. algarvense\u003c/em\u003e, where freshwater-irrigated plants exhibited greater growth while saline irrigation (100 mM NaCl) decreased plant growth in terms of number of leaves and flowers (Cortinhas et al. \u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e2020\u003c/span\u003e; Rodrigues et al. \u003cspan citationid=\"CR44\" class=\"CitationRef\"\u003e2020\u003c/span\u003e). By contrast, in other \u003cem\u003eLimonium\u003c/em\u003e species (\u003cem\u003eL. aureum\u003c/em\u003e, \u003cem\u003eL. gmelinii\u003c/em\u003e, \u003cem\u003eL. otolepis\u003c/em\u003e, \u003cem\u003eL. sinuatum\u003c/em\u003e) the addition of 100 mM NaCl to the growth medium significantly increased plant biomass and leaf area (Mi et al. \u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e2021\u003c/span\u003e). Altogether these findings point to species specific responses to salinity, probably related with the coastal saltmarsh habitats in which the studied species grow. Although both species thrive in priority habitat 1510* Mediterranean salt steppes (\u003cem\u003eLimonietalia\u003c/em\u003e) (European C. \u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e2013\u003c/span\u003e), they inhabit soils periodically flooded by saline water and experiencing extreme summer drying, often characterized by salt efflorescence (Pena et al. \u003cspan citationid=\"CR39\" class=\"CitationRef\"\u003e2020\u003c/span\u003e, \u003cspan citationid=\"CR40\" class=\"CitationRef\"\u003e2021\u003c/span\u003e), they occupy different positions in saltmarshes. \u003cem\u003eLimonium algarvense\u003c/em\u003e thrives in the middle marsh where the debris brought in by the high tide is usually deposited, whereas \u003cem\u003eL. daveaui\u003c/em\u003e grows in the high marsh on the margins of slopes, on the banks of embankments, channels, salt marsh walls of \u003cem\u003eSalinas\u003c/em\u003e and removed soils (Costa \u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e2001\u003c/span\u003e; Costa et al. \u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e2014\u003c/span\u003e), which have drier conditions, and possibly, higher salinity levels.\u003c/p\u003e \u003cp\u003eOn the other hand, under saline conditions, although the bacterial inoculation factor did not have a significant effect on the number of leaves compared to non-inoculated conditions, it did significantly impact biomass. Overall (considering both species together), plants inoculated with LDR15 exhibited higher biomass than non-inoculated plants. This confirmsother studies, where \u003cem\u003eLimonium sinense\u003c/em\u003e plants inoculated with PGPB had increased fresh weight, root length, leaf length and total chlorophyll and proline contents under salinity conditions (Qin et al. \u003cspan citationid=\"CR43\" class=\"CitationRef\"\u003e2018\u003c/span\u003e). In fact, many PGPB have evolved a series of mechanisms which may lead to the reduction of salt concentration in their hosts tissues, such as the formation of biofilms (Navarro-Torre et al. \u003cspan citationid=\"CR34\" class=\"CitationRef\"\u003e2023a\u003c/span\u003e), or the induction of changes in root exudation of some compounds (e.g., polysaccharides and organic acids), that in turn may alter other microorganisms growth, composition, and activity, ultimately improving soil/substrate properties and plant development.\u003c/p\u003e \u003cp\u003eContrastingly, inoculation with a consortium of PGPB not only failed to promote plant growth, but also resulted in the death of all inoculated plants. This contrasts with previous findings, where the same consortium was applied in grapevine roots in a salinized substrate with positive results (Navarro-Torre et al. \u003cspan citationid=\"CR35\" class=\"CitationRef\"\u003e2023b\u003c/span\u003e). Furthermore, Nogales et al. (\u003cspan citationid=\"CR37\" class=\"CitationRef\"\u003e2023\u003c/span\u003e) also demonstrated the beneficial effects of halotolerant PGPB consortium inoculation isolated from the root endosphere of \u003cem\u003eA. macrostachyum\u003c/em\u003e (Navarro-Torre et al. \u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e2016\u003c/span\u003e) in \u003cem\u003eL. algarvense\u003c/em\u003e, that improved plant growth in an amended saline Fluvisol. Although pinpointing the exact cause for this effect is not possible in the current study, several factors could have potentially contributed to this phenomenon. The complex interactions between different bacterial species, their compatibility with the host plant, and their responses to saline conditions can lead to varied outcomes when plants are inoculated with a consortium versus a single bacterium. These outcomes highlight the importance of understanding the specific mechanisms underlying plant-microbe interactions in different environmental contexts (Ciccillo et al. \u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e2002\u003c/span\u003e; Flores-Duarte et al. \u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e2023\u003c/span\u003e).\u003c/p\u003e \u003c/div\u003e"},{"header":"Conclusions","content":"\u003cp\u003eIn summary, the use of native PGPB inoculation using \u003cem\u003ePantoea\u003c/em\u003e strains as single-species inoculum and as consortium yielded different results in halophyte \u003cem\u003eLimonium\u003c/em\u003e species. Seed germination was the most salt sensitive characteristic, and the inoculation with our two selected halotolerant PGPB inocula negatively affected it. However, while the inoculation with the consortium led to plant death under saline conditions, inoculation with LDR15 promoted a general growth increase in \u003cem\u003eLimonium\u003c/em\u003e species. Those results indicate that a careful selection of PGPB bacteria to be included within the inoculum is imperative, since positive outcomes observed in specific plant species may not necessarily generalize across all species. Furthermore, the identification of additional functional bacterial groups within the native soil could prove advantageous for facilitating the establishment of \u003cem\u003eLimonium\u003c/em\u003e plants in saline conditions, as well as for counteracting the effect of any potential pathogens that may pose a threat to transplantation efforts.\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003eThe authors declare that they have no conflict of interest.\u003c/p\u003e\n\u003cp\u003e\u003cbr\u003e\u003c/p\u003e\u003ch2\u003eFunding\u003c/h2\u003e \u003cp\u003eThis work was funded by the National Funds through Foundation for Science and Technology (FCT) under the Project UIDB/04129/2020 BACHALOPH funded by the Research Unit LEAF Linking Landscape, Environment, Agriculture and Food Research Center (Instituto Superior de Agronomia). S.N.-T. thanks the Federation of European Microbiological Societies (FEMS) (FEMS-GO-2020-203) and University of Sevilla (Spain; Plan Propio de Investigaci\u0026oacute;n y Tranferencia 2021 Ayuda A1-I.3A1) for grants to support the stay at ISA-ULisboa (Portugal). VS acknowledges the funding of a research contract (DL57/2016/CP1382/CT0004).\u003c/p\u003e\u003ch2\u003eAuthor Contributions\u003c/h2\u003e \u003cp\u003eConceptualization, A.D.C. and A.N.; methodology, A.N. and A.D.C.; software, A.N.; validation, A.D.C. and A.N.; investigation S.N.-T., M.C.S.C. and V.S.; writing\u0026mdash;original draft preparation, A.N. and A.D.C.; writing\u0026mdash;review and editing, A.N., M.C.S.C.; V.S. and A.D.C.; supervision, A.N. and A.D.C.; project administration, A.D.C.; funding acquisition, A.D.C. All authors have read and agreed to the published version of the manuscript.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\u003cli\u003e\u003cspan\u003eBalasooriya BLWK, Samson R, Mbikwa F, Boeckx P, Van Meirvenne M (2009) Biomonitoring of urban habitat quality by anatomical and chemical leaf characteristics. EEB 65:386\u0026ndash;394. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003e10.1016/j.envexpbot.2008.11.009\u003c/span\u003e\u003cspan address=\"10.1016/j.envexpbot.2008.11.009\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eBarbosa ACF, Pace MR, Witovisk L, Angyalossy V (2010) A new method to obtain good anatomical slides of heterogeneous plant parts. 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J Sustain For 36:107\u0026ndash;120. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003e10.1080/10549811.2016.1256220\u003c/span\u003e\u003cspan address=\"10.1080/10549811.2016.1256220\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eStatements \u0026amp; Declarations\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eThe authors declare that they have no conflict of interest\u003c/span\u003e\u003c/li\u003e\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":true,"highlight":"","institution":"","isAcceptedByJournal":false,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true},"keywords":"halophytes, Pantoea, plant growth promoting bacteria, seed germination, salinity, sea-lavenders","lastPublishedDoi":"10.21203/rs.3.rs-4738414/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-4738414/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003e\u003cb\u003eBackground and Aims\u003c/b\u003e\u003c/p\u003e \u003cp\u003eHighly specialized halophilic flora, such as the threatened endemic sea-lavender species \u003cem\u003eLimonium algarvense\u003c/em\u003e and \u003cem\u003eLimonium daveaui\u003c/em\u003e, are adapted to grow in saline environments. Plant-associated microorganisms associate with halophytes promoting their survival and growth, namely stress tolerance. In this study, we compared the effects of halophilic bacterial inoculants and characterized seed germination and plant growth under saline conditions.\u003c/p\u003e\u003cp\u003e\u003cb\u003eMethods\u003c/b\u003e\u003c/p\u003e \u003cp\u003eA new protocol was developed for \u003cem\u003ein vitro\u003c/em\u003e seed germination with bacterial inoculation. The experimental set up included three treatments: non-inoculation, inoculation with a single bacterial inoculum (\u003cem\u003ePantoea\u003c/em\u003e sp., LDR15) or a consortium of halotolerant bacteria (\u003cem\u003ePantoea\u003c/em\u003e genus). We assessed plants\u0026rsquo; physiological status, biomass, and leaves characteristics under saline irrigation.\u003c/p\u003e\u003cp\u003e\u003cb\u003eResults\u003c/b\u003e\u003c/p\u003e \u003cp\u003eExposure to NaCl (200 mM) along with inoculation using either LDR15 strain or the bacterial consortium negatively affected seed germination. The inoculated bacteria were localized in root cortex and phloem. Under non-saline conditions, bacterial inoculation had no effect in leaf number and fresh biomass, being leaf reflectance values higher in \u003cem\u003eL. algarvense\u003c/em\u003e than in \u003cem\u003eL. daveaui\u003c/em\u003e. Salinity significantly reduced both leaf number and size, succulence and biomass, being this effect more pronounced in \u003cem\u003eL. algarvense\u003c/em\u003e than in \u003cem\u003eL. daveaui\u003c/em\u003e. The bacterial consortium negatively affected plant survival, but plants inoculated with the LDR15 strain had higher biomass than the non-inoculated ones.\u003c/p\u003e\u003cp\u003e\u003cb\u003eConclusion\u003c/b\u003e\u003c/p\u003e \u003cp\u003eWe concluded that even though halotolerant bacteria did not improve seed germination upon salinity exposure, the bacterial inoculation with LDR15 strain in germinated plantlets can be a suitable strategy for promoting plant development in saline environments.\u003c/p\u003e","manuscriptTitle":"Do bacterial root endophytes contribute to growth in saline conditions? A pre-reintroduction cultivation study of threatened saltmarsh Limonium species","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2024-08-22 14:31:53","doi":"10.21203/rs.3.rs-4738414/v1","editorialEvents":[{"type":"communityComments","content":0}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"e09fa3e7-0c93-4ee2-91aa-59730ef2b285","owner":[],"postedDate":"August 22nd, 2024","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"posted","subjectAreas":[],"tags":[],"updatedAt":"2025-02-01T00:52:13+00:00","versionOfRecord":[],"versionCreatedAt":"2024-08-22 14:31:53","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-4738414","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-4738414","identity":"rs-4738414","version":["v1"]},"buildId":"qtupq5eGEP_6zYnWcrvyt","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}