Endophyte-Assisted Phytoremediation by the Halophyte Halocnemum strobilaceum Coping with Extreme Salinity and Hydrocarbon Pollution

Abstract

Hypersaline waters contaminated with crude oil represent a major obstacle for phytoremediation, as few plant species tolerate both high salinity and hydrocarbon toxicity. In this study, the halophyte Halocnemum strobilaceum (Pallas) M. Bieb. was grown hydroponically in hypersaline solutions (50 and 80 g L⁻¹ NaCl) containing crude oil (600 mg L⁻¹). The plant was inoculated with endophytic bacteria isolated in a previous step from its root and selected for salt tolerance and hydrocarbon-degrading potential. The plant behaviour was assessed through growth and photosynthetic performance, while the degradation of hydrocarbons (C < 12 and C > 12) was monitored over time. At 50 g L⁻¹ NaCl, crude oil reduced the plant growth by 60%, but inoculation with endophytic bacteria mitigated this decline, demonstrating their positive influence under combined salt and hydrocarbon stress. At 80 g L⁻¹ NaCl, neither plant biomass nor chlorophyll fluorescence was significantly affected by crude oil, with or without bacterial inoculation, consistent with the strong intrinsic salt tolerance of H. strobilaceum, which likely buffered additional stress inputs. Metagenomic analyses revealed distinct root-associated microbial communities under different treatments, suggesting synergistic plant–microbe interactions that enhanced photosynthetic efficiency and metabolic stability. The presence of endophytes accelerated the degradation of aliphatic hydrocarbons (C10–C40) at both salinity levels. These findings highlight the potential of endophytic bacteria to enhance resilience in H. strobilaceum and its phytoremediation capacity, offering a promising nature-based approach for the sustainable treatment of highly saline, crude oil-contaminated industrial waters.

1. Introduction

Hypersaline conditions—typically defined as salinity levels exceeding 35–40 g L⁻¹ of dissolved salts—are often found in coastal and arid areas, where evaporation exceeds precipitations. Since hypersaline ecosystems have existed throughout the Earth’s evolution, several species have developed natural adaptation mechanisms to withstand and thrive under these conditions. For instance, halophytic plants such as Halocnemum strobilaceum (Pallas) M. Bieb. and Suaeda fruticosa (L.) Forsk can absorb and accumulate salt from the substrate, and their survival can be related to the activity of endophytic bacteria [1]. This feature was proved to be applicable in the remediation of salt from soil and industrial wastewaters [2,3].

In addition to natural hypersaline environments, the activities involved in oil exploitation and production can lead to artificial hypersaline environments due to the discharge of highly saline wastewater [3]. In these settings, oil spills are also a likely source of contamination. Hence, the ecosystem could be simultaneously subjected to crude oil pollution and hypersaline conditions, making developing and applying effective phytoremediation techniques even more challenging. Such combined environmental challenges require biological solutions relying on the efficiency of plants in cleaning up ecosystems and on their capacity to grow, propagate and restore the damaged biodiversity.

Phytoremediation is a plant-based remediation strategy in which plants and their associated microbiota remove, transform, or stabilize environmental contaminants, including hydrocarbons, heavy metals, and excess salts [4,5]. Relevant studies report diverse phytoremediation protocols allowing the decontamination of soil and produced waters (PWs) from oil and gas plants [6,7].

Phytoremediation techniques are often based on the natural adaptation capabilities of plants and the effect of associated endophytic bacteria strains [8]. Endophytic bacteria support the plant growth, often inhibited by abiotic stresses, such as salt stress [9,10]. In particular, endophytic bacteria can alleviate salinity impact on plants, and enhance the chlorophyll content, nitrogen uptake, and growth, as observed in rice seedlings [11]. Some strains of endophytic bacteria are also able of directly degrading hydrocarbons and promoting plant growth under salt stress [12,13]. Another beneficial effect of endophytes is their capacity to restore soil fertility, promoting the release of key nutrients like nitrogen and phosphorous. For example, ref. [14] reported a positive effect on nitrogen fixation by various Saccharum species in Sugarcane, and [15] showed a similar effect on phosphorous solubilisation in wild poplar (Populus trichocarpa). Nevertheless, knowledge of the exact biological mechanisms allowing endophytic bacteria to promote plant growth and development under hypersalinity is limited [9].

The synergy between endophytic bacteria strains and halophytic plants could provide a powerful tool to remediate complex contaminated matrices, like the ones found when hypersaline oil production waters reach the surface. Despite the recognized stress tolerance of many halophytes, the mechanisms underpinning plant responses to the combined constraints of hypersalinity and crude-oil exposure remain poorly understood. In particular, the extent to which endophytic bacterial strains selected for salt tolerance and hydrocarbon degradation can enhance plant performance and contaminant decomposition/removal under such extreme conditions remains unclear. Previous experiments [3] demonstrated the ability of H. strobilaceum to withstand high salinity levels (50 g L⁻¹ or 856 mM NaCl in hydroponics). In the study by [16], endophytic bacteria were isolated and screened for plant growth-promoting rhizobacteria (PGPR) properties from the roots of H. strobilaceum cultivated under hypersaline conditions (up to 100 g L⁻¹) and exposed to petroleum hydrocarbons. Here, H. strobilaceum was inoculated with those selected endophytic bacteria strains with the aim to investigate the ability of this association in mitigating the stress induced by petroleum hydrocarbons in H. strobilaceum and enhancing hydrocarbon degradation, as a step forward from the findings of our previous studies [3,16].

This research has the potential to contribute to the rehabilitation of degraded coastal arid/saline ecosystems, and the phytoremediation endeavours of hydrocarbon-contaminated environments. Using a selected halophyte (Halocnemum strobilaceum Pallas M. Bieb.) inoculated with endophyte strains previously isolated from its roots, specific objectives of this work are to (i) investigate the tolerance of this halophytic plant to hypersalinity and hydrocarbon pollution, (ii) assess the role of endophytic bacteria in counteracting such stress conditions, (iii) evaluate the effectiveness of the plant–microbe interactions in mitigating the impact of saline hydrocarbon effluents from industry, and (iv) characterize the composition of the plant microbiome in response to hydrocarbon pollution and extreme saline conditions. At a basic research level, the study addresses the open question of whether, and to what extent, endophyte–host interactions remain functionally effective under simultaneous salinity and hydrocarbon stress—thereby clarifying the mechanisms by which endophytes modulate plant resilience and contaminant turnover in extreme environments.

2. Materials and Methods

2.1. Isolation and Characterization of Endophytic Bacteria

The halophytic plant species Halocnemum strobilaceum (Pallas) M. Bieb. was selected in previous screening tests for tolerance to hypersaline conditions and hydrocarbons contamination [3,16]. For the identification of specific endophytic bacteria useful in improving the plant resistance to both high salinity and hydrocarbons, H. strobilaceum was cultivated for 18 months in a hydroponic mesocosm system with solutions (synthetic waters, SW) containing 50 or 65 g L⁻¹ NaCl (SW50 and SW65) and 600 mg L⁻¹ of crude oil (SW50H and SW65H), as already described [16]. The endophytic bacteria were isolated from the roots of plants grown in each experimental condition (Figure 1A). The root surface was sterilized by treating it with 70% EtOH for 5 min, with NaClO for 2 min, and again with 70% EtOH for another 5 min. They were then thoroughly rinsed at least three times with sterile water. After being finely chopped with a sterile scalpel, the roots were placed in sterile flasks containing TYEG (Trypticase Yeast Extract Glucose) medium and incubated for 15 h at 30 °C. Then, 100 µL of serial dilutions were spread on R2A agar plates, and after 5 days, several colonies appeared. About twenty phenotypically different colonies for each root type were isolated, and pure cultures were used for DNA extraction and taxonomic classification [17]. The bacterial strains of Biohazard Class 1 were subsequently assessed for their plant growth-promoting properties, as described in [18]. Four isolates (Brevibacillus nitrificans SMV487-E8, Priestia aryabhattai SMV490-E20, Microbacterium paraoxydans SMV492-M27 and Sphingobacterium canadense SMV494-M31) exhibited the most promising in vitro features (Figure 1A) and were thus selected as inoculants prepared. All endophytes were grown in LB medium for 72 h at 30 °C before being pelleted and resuspended in a protective medium (1% sodium glutamate, 7% sucrose, 5% dextran). The cultures were separated in small volumes (2 mL), pelleted again, frozen at −80 °C for 24 h, and finally lyophilized through exposure to a vacuum for ease of conservation and transport [19]. The lyophilized bacteria contained in 2 mL tubes were resuspended in 1.5 mL of plant growth solution (see Section 2.2 for composition) and vigorously vortexed. The inoculum was applied by pipetting the bacterial suspension into the nutrient solution of each hydroponic pot, ensuring direct contact with the root zone.

2.2. Plant Materials and Experimental Design

Plants of H. strobilaceum (Pallas) M. Bieb. supplied by a commercial nursery in Villacidro (Cagliari, Italy), were grown in a greenhouse under semi-controlled temperature, humidity and light conditions for seven months (June–December) in the campus of the National Research Centre (CNR) of Pisa, Italy (43.719426 N, 10.420671 E). The experimental test was carried out in hydroponic trials, composed of cylindrical plastic pots (31.0 cm and 26.0 cm in top and bottom diameter, respectively, and 33.5 cm in height) containing 14 L of nutrient solution and equipped with an aeration system (Figure S1). The hydroponic nutrient solution was derived from a liquid fertilizer (FLORA Series, General Hydroponics Europe-GHE, France) diluted in tap water. This fertilizer consisted of three components, with the following composition: 2% total nitrogen (N), 1% phosphoric anhydride (P₂O₅), 6% potassium oxide (K₂O) and 0.8% magnesium oxide (MgO) [FloraGro 2-1-6], 5% N, 1% K₂O, 6% calcium oxide (CaO), 0.01% boron (B), 0.01% copper (Cu)-chelated EDTA, 6% iron (Fe)-chelated (EDDHA-11% and DPTA-0.12%), 0.07% manganese (Mn)-chelated EDTA, 0.002% molybdenum (Mo) and 0.02% zinc (Zn)-chelated EDTA [FloraMicro 5-0-1], 5% P₂O₅, 4% K₂O, 3% MgO and 5% sulphuric anhydride (SO₃) [FloraBloom 0-5-4].

Three plants per pot were posed, and two pots per treatment were set up, with a total of six biological replicates per treatment. Before plant transplanting in the hydroponic pot, roots were carefully washed with running tap water to remove residues of the growing substrate. The salinity was gradually increased in all pots with applications of 2.5–5.0 g L⁻¹ NaCl every 1–2 days until reaching the final concentration of 50 g L⁻¹ (856 mM) or 80 g L⁻¹ (1369 mM) NaCl. These levels were chosen on the basis of previous studies on the tolerance/resistance of H. strobilaceum to high NaCl concentrations [3,16]. The NaCl dose of 50 g L⁻¹ was the target used for salt-tolerance selection of H. strobilaceum among halophytes and associated root endophytes. The NaCl dose of 80 g L⁻¹ was higher than 65 g L⁻¹ NaCl concentration at which H. strobilaceum showed tolerance to both salt and hydrocarbons [16], but still below 100 g L⁻¹ NaCl—the threshold at which clear morphophysiological stress responses were reported [3].

All the pots were maintained at constant volume and randomly distributed to eight different growth conditions (Figure 1B): nutrient solution with 50 g L⁻¹ NaCl (S50); S50 inoculated with endophytes (S50E); S50 added with petroleum hydrocarbons (S50P); S50 with both endophytes and petroleum hydrocarbons (S50EP); nutrient solution with 80 g L⁻¹ NaCl (S80); S80 with endophytes (S80E); S80 with petroleum hydrocarbons (S80P); S80 with both endophytes and petroleum hydrocarbons (S80EP). The electrolytic conductivity (EC) of the initial 50 and 80 g L⁻¹ NaCl saline nutrient solutions, measured with a conductivity meter (Crison GLP31, Crison Instrument, Alella, Barcelona, Spain), was 78.7 ± 0.3 and 131.5 ± 0.6 dS m⁻¹, respectively.

The endophyte inoculation (E) was carried out twice: the first one at the end of June, three weeks after the plant transfer/acclimation to hydroponics, and the second after three months (September). Two months after the second endophyte inoculation, the petroleum hydrocarbons (as crude oil, P) were added at the concentration of 600 mg L⁻¹ (November). The volume of nutrient solution and pH (~6) were adjusted weekly, using a pH meter (Hanna Instrument, Woonsocket, RI, USA). The first inoculation was carried out after plant acclimation to promote effective colonization; the second inoculation acted as a booster to reinforce endophyte establishment. The timing was defined based on protocols previously developed for H. strobilaceum and other halophyte–endophyte systems [16], together with empirical observations collected throughout the whole time-course of this experiment. The two month interval before applying crude oil was chosen to ensure both a stable plant–endophyte association and further plant growth, thereby improving plant resilience to the combined stress of high salinity and hydrocarbons.

Samples of solution (200 mL) were collected 3 h, 1 day, 3, 7, 14 and 30 days after the P addition using the specific tap installed in the growth pot. At each time point these liquid samples were analyzed for hydrocarbons with C < 12 and C > 12. The destructive sampling of plants was carried out 30 days after the addition of P (December).

2.3. Plant Growth and Biomass Partitioning

Throughout the duration of the experiment, plants underwent daily monitoring to visually evaluate stress symptoms such as chlorosis spots, wilting, and crumpled leaves. After seven months of growing and 30 days after the addition of crude oil, plants were removed from the pots and washed with tap water. The leaves, stems, and roots of each plant were carefully separated, rinsed with deionised water, and dried between layers of filtered paper. The fresh weight (FW) of each plant part was determined using an analytical balance (Mettler Toledo ME204, Columbus, OH, USA) before washing, and then all the organs were freeze-dried in a lyophiliser (Freeze Dryer LIO-5PDGT, Cinquepascal s.r.l., Trezzano sul Naviglio, Milan, Italy). After freeze-drying, the weight of each organ was recorded; when necessary, samples were dried at 60 °C in an oven (Memmert UN series, Memmert GmbH, Schwabach, Germany) until stable weight was obtained and the dry weight (DW) determined.

2.4. Chlorophyll Fluorescence Measurements

The fluorescence of chlorophyll a was measured using a pulse amplitude-modulated fluorometer (Mini-PAM; Heinz Walz GmbH, Effeltrich, Germany), as previously described [20]. Plants were pre-darkened for 30 min, and the maximum quantum efficiency of photosystem II (PSII) photochemistry (F_v/F_m) was evaluated as (F_m − F₀)/F_m, where F_m and F₀ are the maximum and the minimum fluorescence yield emitted by the leaves in the dark-adapted state respectively. The photosynthetic photon flux density (PPFD) of the saturating light flash used to determine F_m was about 8000 µmol m⁻² s⁻¹. Subsequently, measurements of the steady-state effective (ΦPSII) and maximum (F_v′/F_m′) photochemical yield of PSII in the light and the coefficient of photochemical fluorescence quenching (q_p) were performed on well-exposed leaves at green-house environmental conditions: PPFD of 200 μmol m⁻² s⁻¹, CO₂ concentration of 400 μmol mol⁻¹ and temperature of 16–30 °C daytime range. The ΦPSII value was determined as (F_m′ − F′)/F_m′, where F_m′ is the maximum fluorescence yield with all PSII reaction centres in the reduced state obtained superimposing a saturating light flash during exposition to actinic light and F′ is the fluorescence at the actual state of PSII reaction centres during actinic illumination. The F_v′/F_m′ and q_p values were calculated as (F_m′ − F₀′)/F_m′ and (F_m′ − F′)/(F_m′ − F₀′) respectively, where F₀′ is the minimum fluorescence yield in the light-adapted state obtained immediately after switching off the actinic light and applying a weak far-red light to excite PSI and force electrons to drain from PSII preferentially. The electron transport rate (ETR) was calculated by multiplying ΦPSII by the incident PPFD (200 μmol m⁻² s⁻¹) and then correcting for the actual fraction of absorbed light and the relative distribution of absorbed light between the two photosystems, assumed as 0.84 and 0.5, respectively [21].

2.5. Hydrocarbons Analysis

Collected liquid samples were analyzed for total petroleum hydrocarbons (TPH). For determining the TPH C < 12 fraction, liquid samples were filtered through a 0.45 µm syringe filter. Then, 5 mL of the neat sample were sealed in 10 mL crimp vials with PTFE-lined septa for headspace analysis. For the TPH C > 12 fraction, after filtration the liquid samples were purified using a Bond Elut Florisil SPE cartridge (1 g, 6 mL; Agilent Technologies, Santa Clara, CA, USA) to remove polar interferences; the purified extract was then mixed 9:1 with n-hexane in 2 mL crimp vials with PTFE-lined septa for direct injection. The TPH fraction C < 12 content was determined according to EPA 5021A [22] and EPA 8015C [23], and fraction C > 12 was performed according to the EPA 3510C [24] and EPA 8015C [23] methodology. Samples were analyzed by the GC-MS system using a gas-chromatograph Agilent 5977 (Santa Clara, CA, USA) coupled with a mass spectrometer Agilent 8890 (Santa Clara, CA, USA).

The petroleum hydrocarbon’s degradation-efficiency percentage degree (DE%) was calculated following Equation (1) [25], where C₀ is the initial concentration, and C_t is the concentration at time t for the analyzed fraction:

DE% = ((C₀ − C_t)/C₀) × 100

(1)

2.6. DNA Extraction, Quantification and Identification of Root Microbiomes from H. strobilaceum

Genomic DNA was extracted from 500 mg of roots from each soil sample using the Fast DNA^® Spin Kit for Soil (MP Biomedicals, Solon, OH, USA). DNA was quantified by Qubit^® 2.0 fluorometer (Invitrogen, Thermo Fisher Scientific, Eugine, OR, USA) and amplified following the programme previously used by [25] in a study aimed to analyze the root-associated microbiota response to water stress. Briefly, approx. 3 ng of the genomic DNA was amplified with the Thermo Scientific 16S Metagenomics Kit (Thermo Fisher Scientific, Carlsbad, CA, USA). The amplification programme used involved an initial denaturation step at 95 °C for 10 min, followed by 25 cycles at 95 °C per 30 s, 58 °C for 30 s, 72 °C for 20 s, a final hold time for 7 min at 72 °C, and a cooling step at 4 °C [26].

The preparation and purification of amplicon, and sequencing of the libraries followed the standard protocols for the Ion GeneStudio S5 systems (i.e., Ion Chef ™ System and Ion GeneStudio S5 Sequencer; Thermo Fisher Scientific, Carlsbad, CA, USA) provided by the manufacturer. The sequence alignment was carried out as described by [26], to obtain representative sequences with a cut-off of 97%. The Operational Taxonomic Units (OTUs) generated this way were classified and the alpha/beta diversity calculated. The output was elaborated to obtain each OTU’s relative abundance (%) in the total amounts of the entire sample. The relative abundance calculated was used downstream to evaluate the diversity of the microbial community through bioinformatic analysis, in order to gain a general understanding of the community structure of the roots from the H. strobilaceum plant species.

2.7. Statistical Analyses

The experiment was set up in a completely randomized design with six replicates (n = 6) for treatment. Differences among the groups were assessed by ANOVA (p ≤ 0.05). The salt (NaCl) concentration (S), crude oil or petroleum hydrocarbons presence (P), and endophytic bacteria (E) inoculation were considered three explanatory factors with different levels, and their effects and combined interactions on growth and photosynthetic performance of H. strobilaceum were evaluated with the application of three-way ANOVA (p ≤ 0.05). The results were further analyzed with Fisher’s least significant difference (LSD) test (p ≤ 0.05). Variations in the concentrations of C > 12 and C < 12 hydrocarbon fractions of the nutrient solutions during the time-course of the experiment were evaluated with the application of one-way ANOVA (p ≤ 0.05) and Student’s t-test. All analyses were conducted using STATISTICA 8.0 software package (StatSoft Inc., Tulsa, OK, USA).

For microbiome analyses, bacterial community composition associated with H. strobilaceum roots was assessed using 16S rRNA gene sequences (Section 2.6). Sequencing data were processed using the Metagenomics 16S workflow with the curated microSEQ^®16S database (Reference Library 2013.1) within Torrent Suite^TM Software (version 5.8). Reads were quality filtered according to Ion Torrent recommendations (AQ17–AQ20 thresholds) to reduce homopolymer-associated sequencing errors, and sequences were clustered into operational taxonomic units (OTUs) at 97% similarity. OTU relative abundances were used to calculate diversity metrics.

Changes in microbial community structure among treatments were evaluated using principal coordinate analysis (PCoA) based on Bray–Curtis dissimilarity matrices calculated from OTU relative abundances. Statistical significance of group separation was assessed using PERMANOVA with pseudo-F ratios implemented in the ADONIS function of the vegan package. Post hoc pairwise comparisons were performed with Bonferroni correction. Abundance plots and ordinations were generated using the ggplot2 package [27]. All bioinformatic and statistical analyses were performed in R v. 4.4.0 [28] using RStudio version 2024.04.1+748 (Windows) [29].

3. Results and Discussion

3.1. Plant Growth and Photosynthetic Performance

Halocnemum strobilaceum (Pallas) M. Bieb. is a halophytic plant species widespread in deserts from northern Africa to western Asia, able to grow in severe saline and waterlogging conditions; its survival and development is often associated with the activity of endophytic bacteria [1,3,30]. Under our experimental conditions, H. strobilaceum showed high tolerance to 50 g L⁻¹ NaCl (856 mM) maintaining total biomass and photosynthetic performance, confirming previous studies on halophytic adaptation [3,16]. Under exposure to 80 g L⁻¹ NaCl (1369 mM), the overall growth was inhibited if compared with that reached at 50 g L⁻¹ NaCl, but it remained stable with the addition of crude oil, further indicating the plant’s ability to withstand severe stress. Inoculation with selected root endophytic bacteria was hypothesized to enhance stress tolerance [12,31,32].

The production of total biomass in H. strobilaceum was influenced by the saline levels (S), the presence of crude oil (P), the interaction of these two factors (SxP), and by the combined interaction of SxP with the endophytes inoculation (SxPxE) (Figure 2). The biomass produced by H. strobilaceum in the presence of 80 g L⁻¹ NaCl was lower (−30.0%) than at 50 g L⁻¹ NaCl, mainly due to the reduction in leaf weight (Figure 2). This confirms our previous findings on the identification of 50 g L⁻¹ NaCl as the salt tolerance threshold for the growth of this halophytic plant species [3,16]. Indeed, the DW of plants grown under 80 g L⁻¹ NaCl was generally lower than under 50 g L⁻¹ NaCl, independently of P addition or E inoculation. However, the inoculation with selected E never directly affected the plant biomass or its partitioning among different organs, but it revealed a combined effect with S (SxE) and with both S and P (SxPxE), especially in the stem growth (Figure 2). The root development was impacted only by the P factor reaching low levels in S50P and S80P, although not significantly different from S50 and S80, respectively (Figure 2).

The addition of petroleum hydrocarbons (P) to the nutrient solution containing 50 g L⁻¹ NaCl (S50P) reduced the total DW by about 60% compared to plants grown in the presence of only salt (S50, Figure 2). The endophyte-supplemented plants treated with petroleum hydrocarbons (S50EP) showed a 41.8% increase in total biomass compared to petroleum-treated plants (S50P; p-value: 0.048), although still reduced compared to S50 (−32.4%; p: 0.004) or S50E (−34.0%; p: 0.027), as evidenced by LSD test in Figure 2. In such conditions (S50EP), the inoculated endophytes supported the plant growth (Figure 2), and maintained an adequate state of leaf hydration and photosynthetic efficiency (Figure 3 and Figure 4). The specific significance levels (p-value) of the three way-ANOVA applied to biometric and physiological measurements are shown in Table S1.

The chlorophyll fluorescence parameters F_v/F_m, ΦPSII and F_v′/F_m′ were directly influenced by the endophytic inoculation E, which interacted differently with S or P in determining their values; the SxPxE interaction was never significant (Figure 4). In particular, the ΦPSII was reduced (−9.0%) by P addition at 50 g L⁻¹ NaCl (S50P) compared with the salt control (50S), and it was partially recovered (+17.7%; p: < 0.001) in the inoculated treatment (S50EP) compared with S50P. In addition, the F_v′/F_m′ and ETR parameters, after being inhibited at S50P, were restored with the inoculation (S50EP) to the levels observed in the absence of petroleum (S50E, Figure 4). This beneficial effect was not observed in the presence of 80 g L⁻¹ NaCl, although in this case no differences in biomass production were found between treatments with petroleum hydrocarbons—with and without endophytes (S80P and S80EP, Figure 2). However, 80 g L⁻¹ NaCl did not significantly affect the photosynthetic efficiency, as well as the shoot to root biomass allocation, when compared to 50 g L⁻¹ NaCl (Figure 3 and Figure 4). This behaviour of H. strobilaceum under extreme salinity relies on its strong intrinsic tolerance mechanisms (ion compartmentalization, osmotic adjustment, and maintenance of photosynthetic stability) [3,16,30], which likely operate near their physiological limits. Indeed, all the morphophysiological parameters analyzed here were stable under 80 g L⁻¹ NaCl but lower than those measured at 50 g L⁻¹ NaCl. Under these conditions, salinity becomes the dominant stress factor, potentially overriding or masking additional contributions of endophytes. These results further support that depending on the type of stress, complex relationships among bacteria, and between bacteria and the host plant may be established [33]. Such high NaCl levels (80 g L⁻¹ NaCl) may constrain the metabolic activity of the inoculated strains, reducing their ability to exert measurable plant-growth-promoting effects. In addition, the endophytic bacteria can influence plant growth and development using different mechanisms depending on their nature (rhizoremediators, phytostimulators, stress controllers) and the environmental conditions. For example, in [32] the authors found that the line of Bacillus subtilis 26D + n stimulated the growth of wheat seedlings exposed to crude oil pollution when compared to both control and plants inoculated with the strain B. subtilis 26D. Moreover, it is noteworthy that in our study the selected endophytes mitigated the reduction in biomass and photosynthetic efficiency due to hydrocarbons in plants grown at 50 g L⁻¹ NaCl, but there was no stimulation/increase in growth, as is often found for the action of PGPR [12,33,34]. Similarly, the addition of root endophytes to H. strobilaceum plants grown at the highest salinity of 80 g L⁻¹ NaCl did not affect plant performance in the presence or absence of hydrocarbons.

The endophytes inoculum could contribute to the maintenance of plant development conditions and favour the hydrocarbon degradation capacity, which was high in the presence of both 50 g and 80 g L⁻¹ NaCl (Figure 5). This effect is also supported by the maintenance not only of the biomass produced, but also of its distribution in the various plant organs and of the shoot to root ratio, which in similar cases can be altered by the action/interaction between endophytic and rhizospheric bacteria [35].

The selected root endophytic bacteria exerted a beneficial effect on the host plant survival and development, preventing excessive biomass loss and probably facilitating the degradation of hydrocarbon pollutants.

To better understand the nature of the responses observed to the presence of both salt and hydrocarbons, a more in-depth analysis of the biodiversity of the microbial populations involved was performed.

3.2. Effects on Petroleum Hydrocarbon Contents

In the context of hydrocarbon contaminated soils, given the complex nature of the contaminant, it is common practice to determine the content of total petroleum hydrocarbons (TPH) as a proxy to quantify the amount of numerous chemical compounds originating from crude oil that could interact with the present biota. The classification of total petroleum hydrocarbons (TPH) into C < 12 and C > 12 fractions is commonly adopted to distinguish hydrocarbons with different physicochemical properties and environmental behaviour. The C < 12 compounds are generally more volatile, more mobile and more rapidly degradable in soil and groundwater, and thus potentially more critical from a human health perspective. In contrast, C > 12 fractions are less volatile, less mobile, and tend to persist longer in soils and waters. This distinction, also incorporated into Italian environmental legislation, provides a more targeted risk assessment and the selection of appropriate remediation strategies. In the context of our experiment, this subdivision is relevant because the plant performance and the effect of endophytes on hydrocarbon removal can differ according to the relative proportion and degradability of these two fractions [13].

In this study, the degradation of the two cited fractions was determined for both the salinity levels tested (i.e., 50 and 80 g L⁻¹ NaCl). As shown in Figure 5, the pots inoculation with selected endophytes seemed to enhance the biodegradation of the C > 12 hydrocarbons after 14 days. In general, the trend of hydrocarbon degradation (progressive reduction over time) appeared very similar for both NaCl doses. However, the initial decrease in hydrocarbons was higher in the presence of 50 g L⁻¹ than 80 g L⁻¹ NaCl from the beginning of monitoring (3 h or 0.13 days after the addition of crude oil, Figure 5). This suggests a higher initial degradation rate under the S50 salinity condition compared with S80 and reveals a relatively faster biodegradation rate of hydrocarbons in the S50 growth saline condition compared with S80. To distinguish biological from abiotic processes, an additional control without plants and without bacterial inoculum was included. This treatment did not show significant changes in hydrocarbon concentrations over the entire experimental period; therefore, these data were not included in Figure 5. The degradation observed in S50P and S80P is therefore attributable to the activity of the microbial community naturally developing under aerated conditions. This aspect is further explored in Section 3.2 through metagenomic characterization of root-associated bacteria. After 30 days, the mean residual hydrocarbon C > 12 concentrations in pots without E was about 30 mg L⁻¹ and 35 mg L⁻¹ for the pots with 50 and 80 g L⁻¹ NaCl, respectively. On the other hand, in the E-inoculated pots residual hydrocarbon concentrations were about 11 mg L⁻¹ in presence of 50 g L⁻¹ NaCl and 21 mg L⁻¹ at 80 g L⁻¹ NaCl conditions (Figure 5). Even if the trend of hydrocarbon degradation was similar in the E-inoculated and non-inoculated pots, it appeared to be stimulated by the bacteria inoculation after 1 day from the P addition in both 50 and 80 g L⁻¹ NaCl salinity levels.

This trend is more evident if the data are calculated as petroleum hydrocarbon’s degradation-efficiency percentage degree (DE%). The obtained data revealed that the inoculation with selected E positively influences the hydrocarbon degradation, with a final (30 days) increase of about 81% (without E inoculation) to about 88% (with E), both in the cases of 50 g L⁻¹ and 80 g L⁻¹ NaCl salinity levels.

The differences observed between the non-inoculated and E-inoculated pots are less noticeable in the lighter fraction C < 12, which is present in much lower concentrations (Figure 5C,D). In this case the degradation rate was about 96% and there was a 98% reduction in the original content with and without the addition of E in the pots with 50 and 80 g L⁻¹ NaCl. As stated before, these compounds are more easily subject to degradation, so processes such as the system’s aeration is already sufficient for their notable reduction.

The results showed that the plant root inoculation with selected endophytes might contribute significantly to the degradation rates of petroleum hydrocarbons, as well as counteracting the detrimental effects of high salinity environments. Results are in agreement with a study conducted with the addition of PGPR on saline soil contaminated with 400 mg kg⁻¹ of phenanthrene in which Trigonella foenum-graceum and Brassica juncea showed a reduction of 99% of the hydrocarbon in 60 days [36]. Hence, the results obtained support that the inoculation with the selected endophytes accelerated the natural degradation of hydrocarbons in our system made of synthetic water simulating the saline condition and hydrocarbon levels of PWs.

3.3. Microbial Diversity

The 16S rRNA data from the roots taken at the end of the experiment were used to evaluate the diversity of the samples through the Simpson Diversity Index. As shown in Figure 6, at 50 g L⁻¹ NaCl samples containing petroleum hydrocarbons (S50P) exhibited the highest average Simpson Diversity Index values. At 80 g L⁻¹ NaCl, the highest averages were observed in samples containing both endophytes and petroleum hydrocarbons (S80EP). These differences were numerical only and not statistically significant, suggesting that neither salinity level nor treatment produced a measurable effect on microbial diversity. This lack of significant variations in diversity indexes for root-associated communities is consistent with the previous observations reported in [37].

The metagenomic results obtained allowed the characterization of the composition and the abundance of the microbial communities developed on the H. strobilaceum roots subjected to the different treatments (S50, S50E, S50P, S50EP, S80, S80E, S80P, S80EP) after 30 days from P (600 mg L⁻¹ crude oil) application. The barplots presented in Figure 7 summarize the total relative abundances of the different genera identified in each sample (n = 4 per treatment) associated with the different treatments performed.

The diversity barplot in Figure 7 (where only the top 25 genera were considered) sheds light on the lack of statistically significant differences for the Simpson Diversity Index (Figure 6). All samples, regardless of the salt concentration, are dominated by members of the Halomonas genus, with very slight differences in the presence of generas like Paracoccus, Pseudomonas, unclassified Phormidiaceae, Oceanicola or Chromohalobacter, depending on the treatment. The Halomonas genus includes bacterial strains able to grow over a wide range of temperatures and salt concentrations, with members also able to effectively degrade aliphatic and aromatic hydrocarbons in hypersaline, oil-polluted sediments, and waters [38,39]. Another important aspect emerging from the metagenomic analysis concerns the detection of the inoculated endophytic genera. Although the inoculum promoted the growth of H. strobilaceum, the introduced genera were not consistently detected in root samples at the end of the experiment. As previously mentioned, the overall community structure was strongly dominated by native halophilic taxa such as Halomonas, Aequorivita, Idiomarina, Thalassospira, and Muricauda, all of which thrive under the hypersaline conditions applied. In such highly selective environments, introduced endophytes often persist at very low relative abundances, frequently below the detection threshold of 16S rRNA-based sequencing, particularly when competing with well-adapted resident communities [40]. Limited taxonomic resolution and phylogenetic overlap with indigenous strains may further hinder the assignment of inoculated genera [41,42]. This lack of detection does not necessarily imply functional inactivity: several studies have shown that inoculated endophytes can influence plant physiology and microbiome dynamics even when present at low, non-dominant levels [43]. In our study, the physiological responses of the plants and the treatment-specific shifts observed under moderate salinity (50 g L⁻¹ NaCl) suggest that the inoculated strains interacted with the root microbiome despite not emerging as detectable dominant taxa in the metagenomic profiles.

The Operational Taxonomic Units, OTUs, from the genera of known endophytes of halophytes were only detected in treatments that had the addition of endophytes, either with petroleum hydrocarbons (E + P, EP) or without (E) under both salinity conditions (S50 and S80), in particular genera like Kushneria and Rhizobium (Figure 7). Several authors have reported that the presence of Kushneria strains like K. phyllosphaerae and K. endophytica can aid plant growth through the production of siderophores, indoleacetic acid and ethylene, as well as the promotion of nitrogen fixation and phosphate solubilisation [44,45,46,47]. Members of this genus are also phylogenetically related to Chromohalobacter, consistent with the presence of both genera in samples from treatment S80EP. Similar growth-promoting capabilities under saline conditions have been reported for strains like Rhizobium and Mesorhizobium [48,49,50], further supporting their presence in these samples. The only genus that seems to be present exclusively in samples where petroleum hydrocarbons were added (S50P, S50EP and S80EP) is Alcanivorax (S50P: 3.61%; S50EP: 3.89%; S80EP: 3.37%). This result was expected, since this group of marine bacteria has been commonly found in petroleum-impacted areas because it exclusively uses petroleum hydrocarbons as sources of carbon and energy, with some strains growing even at NaCl concentrations of up to 15% [51,52,53,54].

The other genus present throughout most of our samples was Thalassospira. Strains of this genus are strictly halophilic, unable to grow without added Na⁺, and thrive in salinities ranging from 2 to 10% [55]. Members of this genus are also abundant in marine environments exposed to polycyclic aromatic hydrocarbons (PAHs) and can degrade naphthalene and phenanthrene [56,57,58]. This feature could explain its presence in treatments where petroleum hydrocarbons were added (S50P and S50EP), even with a lower relative abundance. Several strains, like T. tepidiphila or Thalassospira sp. strain GO-4, have been isolated from petroleum-contaminated sites. It has been proposed that Thalassospira could occupy niches that utilize single-ring aromatic acids released from more complex, hydrophobic aromatic hydrocarbon pollutants, growing thanks to the presence of other pioneering members that have the enzymatic capabilities for degrading PAHs upstream like Pseudomonas or Marinobacter [56]. Like the other genera described, Muricauda requires saline conditions to grow. Members of this genus are also able to degrade complex carbon sources; however, they do not show the ability to break down high-molecular-mass carbohydrates like starch [59,60,61,62]. Hence, their presence is at the root of all our treatments.

The metagenomic results can be summarized as follows: (1) while for the 50 g L⁻¹ NaCl condition, the contribution of the unclassified Halomonadaceae was below 2% in the 80 g L⁻¹ NaCl, this group could represent, together with Halomonas, almost 50% of all the genera present in some of the treatments; (2) the Alcanivorax genus gave a small contribution in the 80 g L⁻¹ NaCl treatment (S80) and it was relevant only for the S80EP treatment, while in the S50 condition it was absent or represented nearly 5% of the total relative abundance for the conditions S50EP and S50P; and (3) while Kushneira and Thalassospira were genera with a relevant contribution in half of the 50 g L⁻¹ NaCl treatment conditions, they were almost absent in treatments with 80 g L⁻¹ NaCl.

Considering these minor differences, a Principal Coordinate Analysis (PCoA) was performed to evaluate the possible influence of the different treatments on the microbial community composition at the end of the experiment. For this analysis, the relative abundance of each OTU was used and a dissimilarity matrix was calculated using the Bray–Curtis distance [63]. The first two axes of the PCoA explained almost 40% of the variance associated with the samples. Since PCoA is an ordination method, the farther apart the two points are, the more different the communities they represent are. Conversely, the closer the two points are, the more similar the communities they represent are (Figure S2).

In Figure 8, a clear separation was observed between the communities exposed to 50 g L⁻¹ NaCl and those exposed to 80 g L⁻¹ NaCl, indicating a strong salinity-driven shift in microbial composition. This separation is particularly evident when comparing the S50P condition with all treatments at 80 g L⁻¹ NaCl, which cluster together and occupy a distinct region of the ordination space. Within the 50 g L⁻¹ NaCl group, the communities associated with endophyte inoculation (S50E), endophyte plus petroleum (S50EP), and petroleum alone (S50P) form clearly differentiated clusters, suggesting that under moderate salinity the microbial community remains responsive to both endophyte addition and hydrocarbon presence. In contrast, all treatments at 80 g L⁻¹ NaCl (S80, S80E, S80EP, and S80P) overlap extensively and intersect with the area occupied by S50E. This convergence indicates that at high salinity the microbial community collapses toward a similar composition regardless of endophyte inoculation or petroleum addition, reinforcing the conclusion that salinity is the dominant structuring force under these conditions.

In Figure S2 it is confirmed that the genera Halomonas, Aequorivita, Idiomarina, Thalassospira, and Muricauda are the main contributors to the distribution of our samples within the PCoA ordination, all of which require salty conditions to thrive. The presence of Halomonas within the roots of our plants is expected given the hypersaline conditions used throughout the experiment, since several authors have isolated different strains of this genus from solar salterns, salt lakes, and saline and alkaline soils [64,65,66,67,68]. A similar requirement has been associated with both Aequorivita and Idiomarina genera, since their strains are capable of growing under slightly saline (up to 10% NaCl and between 0.6 and 25% NaCl, respectively) and strictly aerobic conditions [69,70]. In particular, some strains of Aequorovita, such as A. antarctica, A. lipolytica, and A. viscosa, require Na for growth [71], while strains like A. capsosiphonis have been isolated from marine algae Capsosiphon [68]. Finally, members of the Idiomarina genus have been isolated from saline environments, with strains like I. salinarium, I. seosinensis and I. insulisalsae isolated from the sediments or the hypersaline water of solar salterns in Korea [72,73,74].

These results, and the bacterial community composition represented in Figure 8, indicate that the stress produced by high salinity seems to be the controlling parameter over the diversity of the microbial community, despite the positive effect of the endophytic bacteria over the growth of H. strobilaceum.

Since the PCoA is only an ordination method, an ADONIS2 multiple comparison test was performed to evaluate the significance of the differences observed [75]. The ADONIS2 (perm = 9999) test confirmed the statistical significance of the differences observed on the PCoA representation. This test revealed a significant effect of the treatment over the community composition (F = 2.93, R² = 0.46, p-value < 0.05), indicating that treatment explained 46% of the variation in multivariate distances. We also tested the homogeneity of multivariate dispersion using the betadisper test (F = 0.69; p = 0.68), indicating that the PERMANOVA results reflect differences in community composition rather than being affected by unequal within-group variability. The differences observed between each treatment were further tested through post hoc pairwise comparisons (Table S2). Under moderate salinity (50 g L⁻¹ NaCl), several treatments differed significantly from one another (α ≈ 0.028–0.030), indicating that both petroleum and endophyte additions produced detectable shifts in community composition. However, when comparing 50 g L⁻¹ with 80 g L⁻¹ NaCl treatments, the S50 control differed significantly from S80, S80E, and S80P (α = 0.0248–0.0308), while the only non-significant comparison was S50 vs. S80EP (α = 0.05). This pattern shows that the transition to 80 g L⁻¹ NaCl consistently generated stronger compositional changes than the presence or absence of endophytes, supporting the conclusion that high salinity is the dominant driver shaping the bacterial community and can override endophyte-mediated effects.

These results could imply that in the presence of such high salt contents, the main driving force controlling the composition of bacterial community is the salinity. In fact, Figure 7 shows that in both S80 treatments that included petroleum hydrocarbons (S80EP and S80P), instead of two genera able to degrade such substances (i.e., Alcanivorax and Thalassospira), just one (Thalassospira) was detected in a percentage similar to the one observed in the S50 treatments. These findings are in agreement with the biomass production, photosynthetic efficiency, and the results on petroleum hydrocarbons degradation reported previously, also in the non-inoculated pots. We hypothesize that the community characterized at the end of the experiment is the main one responsible for the degradation trends of TPH fractions (C < 12 and C > 12) observed during the 30 days of monitoring.

4. Conclusions

Our results evidenced how root inoculation with endophytic bacteria positively promoted the ability of the halophyte Halocnemum strobilaceum to tolerate and/or degrade petroleum hydrocarbons under hypersalinity conditions (50 and 80 g L⁻¹ NaCl). This was demonstrated by several items of evidence.

-

Endophyte-assisted tolerance and function at the lower salinity level. The inoculation with selected endophytic bacteria strains ameliorated the plant performance in the presence of 50 g L⁻¹ NaCl and crude oil (P), recovering total and stem biomass and photosynthetic efficiency (S50EP versus S50P). This shows a practical strategy to sustain productivity and function of H. strobilaceum in saline, petroleum contaminated waters.

-

The higher salinity tested can override endophyte benefits. Under 80 g L⁻¹ NaCl, plants preserved biomass production and partitioning, leaf water status, and photosynthetic activity despite treatment with petroleum hydrocarbons (S80EP versus S80P), but endophyte mediated gains were not detectable. This indicated that extreme salinity may limit measurable endophyte effects in such conditions.

-

Accelerated hydrocarbon removal under lower salinity. Endophytes improved final hydrocarbon degradation at both salinities. Endophyte inoculation improved the final hydrocarbon degradation by approximately 7–10% at both salinity levels (50 and 80 g L⁻¹ NaCl). Moreover, under the lower salinity (50 g L⁻¹ NaCl), hydrocarbon removal proceeded more rapidly throughout the time course, resulting in a final residual hydrocarbon concentration about half of that observed under 80 g L⁻¹ NaCl (11 mg L⁻¹ vs. 21 mg L⁻¹).

-

The metagenomic analysis gave a precious insight into the community composition of the bacteria present on the roots of H. strobilaceum. The microbiome profiling evidenced that despite the presence of petroleum hydrocarbons at relatively high concentrations, salinity was the dominant factor controlling the root-associated microbial community composition and functioning. While in the presence of 50 g L⁻¹ NaCl the distribution of the microbial community was clearly distinguished in the four treatment conditions tested; a substantial overlap occurred at 80 g L⁻¹ NaCl among all the treatments (S80, S80E, S80P, S80EP) and the ones with the 50 g L⁻¹ NaCl treatment with endophytes inoculation (S50E). Thus, the endophytic bacteria play a crucial role in supporting the halophytic plants of H. strobilaceum to thrive and maintain their yield under conditions of extreme salinity and hydrocarbon pollution.

-

Implications for remediation of saline environments and industrial effluents. The integration of traditional ecophysiological experiments with NGS tests represents a modern and innovative approach to address organic pollution in saline environments where phytoremediation using halophytes and their associated endophytes can be applied. Implementing modern techniques and essential measures is crucial for effectively reclaiming saline and contaminated lands and maximizing their benefits. Thus, this type of approach can be used to identify the best combination of plants and bacteria in optimizing phytoremediation processes that are environmentally friendly, biological methods with high effectiveness and sustainability. In this context, results highlight the potential of halophytes and associated root-endophytic bacteria as promising candidates for remediating highly saline and polluted soils and waters, especially in arid-semiarid regions and drylands. In these areas, the flora development is limited due to hypersalinity and hydrocarbon pollution related to crude oil exploitation near petroleum oil fields. These results may be translated into tangible applied technologies, supporting the clean-up of anthropogenic contaminants, facilitating ecosystem recovery, and restoring the biodiversity of areas neighbouring oil plants.