Biostimulation of Mesembryanthemum crystallinum L. (The Common Ice Plant) by Plant Growth-Promoting Rhizobacteria: Implication for Cadmium Phytoremediation

Abstract

Plant growth-promoting rhizobacteria (PGPR) employ various mechanisms to enhance plant development and growth as well as to mitigate environmental stress, including heavy metal contamination. Cadmium is a particularly severe stressor, toxic to both plants and soil microbiota. Mesembryanthemum crystallinum L. (the common ice plant), a fast-growing semi-halophyte, was previously investigated for phytoremediation potential towards saline environments and toxic metals, especially cadmium and chromium. The study was aimed at assessing whether bacteria isolated from the rhizosphere of M. crystallinum treated with Cd reveal growth-promoting traits and if the plant tolerance to Cd results from a synergistic action of the Cd/salt-resistant strains. The isolates demonstrated PGP characteristics, including nitrogen fixation, phosphate solubilization, and production of ammonia, indolyl-3-acetic acid (IAA), and siderophores. A microbial consortium consisting of these strains was developed and applied to pots with M. crystallinum. After a 14-day experiment, plant growth and Cd-accumulation potential were evaluated upon treatment with 1 mM or 10 mM Cd, either in the presence or absence of NaCl. Plant inoculation with the consortium stimulated Cd accumulation both by roots and shoots at 10 mM Cd under saline conditions. The results suggest that bioaugmentation of M. crystallinum with the bacterial community can be used as an effective, sustainable phytoremediation method for cadmium-contaminated soils.

1. Introduction

Mesembryanthemum crystallinum L., known as the common ice plant, is a fast-growing succulent species of the Aizoaceae family. It is native to southern Europe as well as South and East Africa [1,2]. The plant is known for its low nutritional requirements, resistance to low temperatures, and tolerance to high saline concentrations in soil. Thanks to its metabolic plasticity, mainly the ability to switch from C3 to CAM (crassulacean acid metabolism) photosynthesis, the plant can improve its water use efficiency and carbon fixation capacity, which contributes to its adaptation to abiotic stresses. One of the plant responses to high salinity and water deficit relies on promotion of the formation of bladder cells, which are involved not only in water storage but also in UV protection and plant defense mechanisms [3,4,5,6].

M. crystallinum has been shown to have exceptional tolerance to elevated saline level in soil and to be capable of accumulation of this substance in shoots, making it a promising model for NaCl phytoremediation [7,8]. Furthermore, recent findings indicate its considerable potential for phytoextraction or phytostabilization of heavy metals such as chromium (Cr), cadmium (Cd) [9,10], nickel (Ni) [11], or Cu and Zn [12].

Soil salinization is primarily driven by climate change-related factors, including prolonged droughts, increasing aridity, tsunamis, and sea-level rise. Nevertheless, anthropogenic influence is also considerable and includes mining activities, industrial pollution, extensive application of de-icing agents such as road salts, and unsustainable land and water management practices, i.e., irrigation with seawater, deforestation, or excessive use of fertilizers [8,13]. Globally, saline-affected soils cover over 1380 million ha, representing nearly 11% of the total land area; for Europe and Eurasia, the affected area is estimated at 238 million ha (8.8%). In the near future, an increased risk of soil salinization is projected in Central Africa, Eastern Europe, parts of North and South America, China, and Kazakhstan [13,14]. Sustainable agricultural management of salt-affected soils involves both mitigation and adaptation strategies. Mitigation focuses on reducing soil salinity through practices such as leaching, improved drainage systems, mulching, organic matter amendments, deep plowing, crop system management, bioremediation, and phytoremediation. Adaptation, in turn, aims to maintain productivity under existing salinity levels and includes the use of salt-tolerant plant species (e.g., halophytes), halopriming, or bioinoculation with PGP microorganisms [13].

Cadmium pollution has become a severe environmental issue over recent decades due to anthropogenic activities involving the use of batteries, dyes, paints, fertilizers, electroplating, and fossil fuel combustion. In Poland, the mean Cd content in soils is approximately 0.33 mg kg⁻¹, with 13.2% of croplands exceeding 1 mg kg⁻¹. For comparison, the average Cd content in EU croplands is 0.17 mg kg⁻¹, with 4.1% of samples surpassing this threshold [15]. The metal is highly toxic to all living components of ecosystems [16,17,18,19]. In humans, exposure to Cd disrupts steroidogenesis, causes damage in pulmonary, nervous, and renal systems; problems with the reproductive hormones and menstrual cycle; leads to delays in puberty and menarche and may result in premature birth or pregnancy loss [20,21]. In plants, even low Cd concentrations can induce chlorosis, necroses, chloroplast damage, water stress, root elongation inhibition, impaired gas exchange, wilting, and disruption in macro- and micronutrient uptake [22,23,24]. In areas identified as hotspots of elevated soil cadmium contamination, the application of fertilizers with low Cd impurities is strongly recommended [15].

Phytoremediation is an environmentally friendly, sustainable, and cost-effective method that utilizes plants to remove, immobilize, or detoxify contaminants from the soil or water environment [25]. This technique is often favored over conventional physicochemical procedures such as soil washing or chemical oxidation, which are known to alter biological properties of soil or generate secondary pollutants [25,26,27]. Increasing attention is now directed toward the synergistic interactions between plants and their rhizosphere-associated microorganisms, as mounting evidence indicates that remediation efficiency depends not only on plant physiology but also on the abundance, composition, and functional capacity of the microbial community [28,29]. Phytostimulation, a key plant–microbe interaction mechanism, involves the enhancement of indigenous microbiota activity through root exudates, which stimulate microbial metabolism and accelerate contaminant degradation [30,31]. However, in heavily polluted environments, native microbial populations may be insufficient to achieve efficient remediation, which results either from insufficient abundance or poor metabolic potential. In such cases biostimulation, understood as the introduction of specific microbial strains with targeted metabolic capabilities, can significantly improve contaminant removal and plant growth [32,33].

Plant-associated microorganisms, particularly those inhabiting the rhizosphere, have been evidenced for their crucial role in mitigating adverse effects of soil contamination [25,34,35]. These microbes are involved in nitrogen fixation, nutrient solubilization, and also the production of various bioactive compounds such as legume nodulators, antibiotics, plant hormones, signaling molecules, vitamins, as well as several other compounds that promote plant growth. They have been categorized as plant growth-promoting microorganisms (PGPMs), among which are free-living rhizobacteria (PGPR), endophytic bacteria, cyanobacteria as well as mycorrhizal fungi. PGPMs can support plant growth at various stages of development, especially by increasing root length, root hair number, plant biomass, and chlorophyll content [36] through different mechanisms, including biofertilization, phytostimulation, and biocontrol [35,37].

Cadmium contamination and high salinity in soils pose severe challenges to plant growth and survival. While Mesembryanthemum crystallinum exhibits tolerance to such extreme environmental conditions, the mechanisms underlying this resilience, particularly the potential contribution of the rhizosphere-associated, salt- and cadmium-tolerant bacteria, remain insufficiently understood. The presence of such microorganisms in the root zone may facilitate the plant’s adaptation to physiological stressors [2]. In the previous study by Supel et al. [38], several cadmium-resistant bacterial strains were isolated from the rhizosphere of the common ice plant. Five isolates demonstrated intense growth in liquid media containing up to 10 mM Cd and 0.5 M NaCl. The aim of the present work was to assess the plant growth-promoting traits of these salt- and cadmium-tolerant bacterial strains associated with M. crystallinum and to verify the hypothesis that they possess biostimulatory capabilities that act synergistically with the plant’s inherent tolerance mechanisms, thereby influencing plant growth and development as well as enhancing its capacity for phytoremediation in extreme environments.

2. Materials and Methods

2.1. Tested Strains

Five bacterial strains—Rhodococcus erythropolis strains S4 and S10, Peanibacillus glucanolyticus strain S7, and Providencia rettgeri strains W6 and W7—were isolated and identified in our previous work [38]. Bacteria were long-term cryo-preserved in the microbial collection using the Microbank™ system (Pro-Lab Diagnostics, Richmond Hill, ON, Canada). After reanimation of the cells, bacterial suspensions were streaked onto Petri dishes containing optimal growth media solidified with 2.5% bacteriological agar (enriched agar, Biomaxima, Lublin, Poland) to obtain microbiologically pure cultures. The solid cultures were used for plant growth-promoting (PGP) assays. For the plant-growth experiment, liquid cultures were pre-grown in a Standard Nutrient Broth (SNB, Biomaxima, Lublin, Poland) medium. The monocultures of each strain were typically cultivated in Erlenmeyer flasks containing 50 mL of medium and rotary-shaken (150 rpm) on orbital laboratory shakers at room temperature with passive aeration. After five days of incubation, all cultures were combined to form a mixed microbial consortium, referred to as Mc-mix, and applied to the plants following 2–3 h of co-cultivation. The consortium was prepared de novo prior to each application to ensure the presence of all strains in a viable state at a defined, high density.

2.2. Plant Growth-Promotion Characteristics of the Strains

To evaluate the plant growth-promoting potential of the tested bacterial strains, a series of standard tests were conducted. These included the determination of indolyl-3-acetic acid (IAA) production, atmospheric nitrogen fixation, phosphate solubilization, and the production of siderophores, proteases, and ammonia.

IAA production was assessed using a colorimetric method described by Lelapalli et al. [39], following 5 days of cultivation in nutrient broth supplemented with 1 mg·mL⁻¹ of tryptophan. A positive result was indicated by a color change from yellow to orange or red.

Nitrogen fixation capability was evaluated by bacterial growth on three selective media: Burk medium [40], Ashby medium [41] and standard DSM Azotobacter medium n^o3 [42]. The growth of nitrogen-fixing strains was observed as whitish or transparent colonies.

Siderophore production was analyzed with Chrome Azurol S (CAS) medium, as described by Louden et al. [43]. A positive reaction was noted when a color shift in the medium from blue to yellow was observed, due to Fe³⁺ ion chelation.

Phosphate solubilization was determined by the presence of halo zones around colonies after growth on Pikovskaya (PVK) medium [44] and National Botanical Research Institute’s Phosphate (NBRIP) medium [45], formed as a result of calcium phosphate solubilization.

Ammonia production was assessed using Nessler’s reagent after bacterial cultivation in peptone water [46]. The change in color to brown indicated a positive reaction.

Proteolytic activity was evaluated on the protease medium with skimmed milk [47]. Protease-producing strains formed clear zones around colonies, indicating skimmed milk protein hydrolysis.

2.3. Plant Material and Growth Conditions

Mesembryanthemum crystallinum L. seeds were sown onto the substrate composed of 1 kg of sand mixed with 7.5 L of a commercial growing medium, Hartmann (Hartmann Poland, Poznań, Poland) of pH = 5.9, containing white peat, clay, CaCO₃, and a slow-release fertilizer to provide 150 mg·dm⁻³ of N, 150 mg·dm⁻³ of P, 250 mg·dm⁻³ of K, and 130 mg·dm⁻³ of Mg. To eliminate autochthonous microbiota, the soil mixture was sterilized by autoclaving (30 min at 1.2 atm).

Two-week-old Mesembryanthemum crystallinum seedlings with fully developed second leaf pairs were transplanted individually into 0.5 L pots (one plant per pot) containing the substrate described above and irrigated every third day. In the experimental group (designated as M), 50 mL of the Mc-mix bacterial consortium was applied per pot at three time points: at the beginning of the experiment and then after one and two weeks. In the control group (C), the same amount of a sterilized Mc-mix suspension was used. Starting from the third day of the experiment, cadmium (Cd) and sodium chloride (NaCl) treatments were introduced. These treatments began on the third day after plant transplantation and were then applied daily during the first week and every second day during the second week. Depending on the treatment variant, 10 mL of either 1 mM or 10 mM Cd solution was applied per pot, yielding calculated final amounts of 0.07 and 0.7 mmol of Cd per pot. For the salinity treatment, 50 mL of 0.4 M NaCl solution was used. Variants not receiving Cd or NaCl were supplemented with distilled water (volume of 10, 50, or 10 + 50 mL, regarding the variant).

Soil samples were collected every 2–4 days for analysis of microbial abundance, suspended in sterile distilled water at a ratio of 1:9, and incubated on a rotary shaker for 4 h. Serial dilutions were prepared and plated onto enriched agar (2.5%, Biomaxima, Lublin, Poland) using a modified Koch surface-plating method [48]. Microbial abundance was expressed as a number of colony-forming units (CFU) per gram of soil dry mass (d.m.).

After 15 days of experiment, when the plants had reached the mature growth phase, they were harvested. Soil residues were carefully removed from the roots. The following growth parameters were recorded: fresh weight of shoots and roots (g) and number of fully developed leaves. For dry mass determination, roots and shoots were oven-dried at 105 °C for 24 h.

2.4. Determination of Elemental Content and Evaluation of Cd-Accumulation Factors

The plant samples were mineralized and analyzed for the Cd and Na content. The mineralization protocol involved digestion with 5 mL of 65% nitric acid for 30 min at room temperature, followed by heating at the boiling point for 1.5 h. After cooling for 5 min, 1.65 mL of 30% H₂O₂ was added, and the samples were heated again for 20–30 min. After final cooling, the resultant extracts were filtered and diluted with deionized water to a final volume of 25 mL. Elemental analysis (Cd and Na) was performed using Inductively Coupled Plasma Mass Spectrometry (ICP-MS) with a Prodigy + Spectrometer (Leeman Labs, New Hampshire, MA, USA).

The soil samples were also collected after the experiment and subjected to analysis for Cd and Na content. Ten grams of oven-dried soil samples were digested with 100 mL of 1 M HCl for one hour, followed by filtration. The filtrates were analyzed with ICP-MS as described above.

Mesembryanthemum crystallinum cadmium bioaccumulation factors (BAFs) and translocation factors (TFs) were calculated as follows. The BAF was defined as Cd concentration [mg kg⁻¹ d.w.] in plant organs (roots or shoots) per concentration in the soil substrate [mg kg⁻¹ soil d.w.]. The TF was calculated as the ratio of Cd accumulated in shoots compared to that determined in roots [mg kg⁻¹ d.w.].

2.5. Statisitcal Analysis

Statistical analyses were performed using Statistica 13.5 software (Statsoft Inc., Tulsa, OC, USA). The significance of differences between treatments was determined with one-way analysis of variance (ANOVA) followed by Tukey’s post hoc test at p < 0.05.

3. Results and Discussion

3.1. Plant Growth-Promotion Characteristics of the Strains

The isolated bacteria were characterized by diverse plant growth-promoting (PGP) traits (

Table 1. PGP test results for rhizosphere bacterial isolates.

Tested Strain	Nitrogen Fixation			IAA Production	Phosphate Solubilization		Ammonia Production	Protease Production	Siderophore Production
	DSMZ	Burk	Ashby		PVK	NBRIP
R. erythropolis S4	+	+	+	+	−	−	+	−	−
P. glucanolyticus S7	+	+	+	+	−	−	+	−	+
R. erythropolis S10	+	+	+	+/−	−	−	+	−	+
P. rettgeri W6	+	−	−	−	−	−	+	−	++
P. rettgeri W7	−	−	−	−	+	+	+	−	+

The “−”—negative reaction, “+/−”—weak positive reaction, “+”—positive reaction, “++”—strong positive reaction. Explanations of the abbreviations are given in Methods Section 2.3.

). Each strain demonstrated at least two properties that may be considered beneficial for plant growth and development. None of the isolates produced proteolytic enzymes.

Two strains (S4 and S7) revealed extensive production of IAA. Bacterially produced indole-3-acetic acid (IAA) enhances the transcription of ACC deaminase, the enzyme that catalyzes the breakdown of 1-aminocyclopropane-1-carboxylic acid (ACC), the immediate precursor of ethylene [49]. The presence of this enzyme is beneficial for plants under stress conditions such as high salinity, heavy metal contamination, or drought. Plants exposed to stress factors typically produce elevated levels of ethylene, a phytohormone responsible for the inhibition of vegetative growth. Plants living in symbiosis with IAA-producing bacteria that also exhibit elevated ACC deaminase activity are more resistant to ethylene-induced stress due to the reduction in ethylene levels [49]. Weselowski et al. [50] described a plant-associated species of Peanibacillus that could directly influence plant growth by producing IAA and other phytohormones. Therefore, it is very likely that P. glucanolyticus S7 may also be beneficial for plants.

Phosphorus deficiency, particularly of its bioavailable forms, is a common phenomenon in nature and leads to growth inhibition and reduced productivity in plants [51]. Phosphate-solubilizing bacteria (PSB), including genera such as Agrobacterium, Azotobacter, Azospirillum, Bacillus, Pseudomonas, and Rhizobium, convert insoluble inorganic phosphate compounds to soluble forms, thereby increasing phosphorus availability to plants [39]. Among the tested strains, only Providencia rettgeri W7 demonstrated a positive reaction on both specific media, i.e., PVK and NBRIP.

Biological nitrogen fixation by bacteria can meet approximately 12–70% of the nitrogen requirements of cultivated plants under conditions of limited nitrogen availability. Several PGPB strains were shown to contribute to enhanced plant growth and yield through symbiotic nitrogen fixation [52]. In this study, all the tested strains except W7 showed growth on the N-free media, whereas all the isolates could produce ammonia. These findings prove the biological nitrogen fixation and transformation capabilities of strains. It should be emphasized here that the application of PGPB in agriculture represents a sustainable and environmentally friendly strategy that promotes plant development and productivity while significantly reducing crop dependency on synthetic nitrogen fertilizers [53].

Iron is an essential soil micronutrient required for the growth of both plants and microorganisms. However, its bioavailability in the soil is limited, leading to intense microbial competition for this element. Many PGPB species can regulate iron availability through the release of siderophores—iron-chelating compounds with high affinity for ferric ions [54]. Under iron-deficient conditions, PGPB can secrete siderophores that bind iron in the rhizosphere, securing it for their own metabolic needs as well as for plant uptake. The resulting iron–siderophore complexes are accessible only to the producing bacteria and the associated plant, while pathogenic microorganisms are unable to utilize them. Consequently, under iron scarcity, the activity and population of phytopathogens are reduced [55]. Siderophore production was identified in four tested strains, except for Rhodococcus erythropolis S4. The strongest positive reaction, that is, the CAS medium color change, was observed for the strain W6 (Figure 1).

3.2. Growth Experiment

PGPR have been successfully utilized in the field of phytoremediation [30,34,45]. Given the individual differences in PGP characteristics of the tested strains, it was assumed that the most effective impact on plants should be achieved through the application of a microbial consortium comprising all five bacterial isolates. The findings of Flores-Duarte et al. [56] indicate that model consortia, developed under laboratory conditions using strains with confirmed PGP properties, could be successfully applied to enhance the common ice plant growth under stress conditions resulting from the heavy metal presence. Moreover, beneficial bacteria were shown to modulate gene expression of plants to enhance their tolerance to heavy metals [57]. The unique aspect of the present study lies in the use of indigenous microorganisms previously isolated from the rhizosphere of M. crystallinum cultivated under combined Cd and NaCl stress [38]. The selected strains were chosen based on their demonstrated plant growth-promoting (PGP) traits and high tolerance to both cadmium and salinity. These characteristics distinguish them from microbial species or synthetic consortia applied in other phytoremediation studies. The strains were propagated to obtain high biomass and assembled into a carefully composed consortium, specifically designed to exploit their complementary PGP functions and stress-resistance mechanisms, thereby enhancing the plant’s tolerance and metal accumulation capacity.

Microbial frequency monitoring revealed that after two days of the adaptation phase, the number of bacteria in soil increased from the initial density of 10¹⁰ CFU·g⁻¹ d.m. in Mc-mix-treated variants and 10⁹ CFU·g⁻¹ d.m. in control variants to 10¹¹ CFU·g⁻¹ d.m. of soil, reached on day 4. Later analyses showed no significant changes in the abundance of bacteria in soil. No differences in the CFU counts were observed between the experimental groups, which suggests the capability of microbial adaptation to saline and cadmium stresses.

The application of the PGPR consortium had no effect on Mesembryanthemum crystallinum growth in all the experimental conditions tested. The plants exposed to elevated cadmium concentrations and saline stress exhibited reduced size compared to control (untreated) variants, even in the presence of the Mc-mix. However, this effect was only observed on fresh mass analysis of shoots. A quantitative analysis of plant dry mass revealed that differences between the experimental groups were mostly statistically not significant (Figure 2). The data suggest that the reduction in plant size resulted from the combined effect of salt stress and Cd presence rather than solely from elevated cadmium concentration. In the case of Mesembryanthemum crystallinum, Amari et al. [11] showed tolerance of this plant to the elevated level of NaCl, reaching 40% of dry mass, without visible symptoms of toxic effect. Similarly, Agarie et al. [8] proved the plant could accumulate NaCl at the level of 31.8% of dry mass. For the analyses carried out in our work, the sodium concentration in plant tissues and soil revealed a 10-times higher concentration of Na in above-ground parts as compared to the roots. The content of this element reached 10.2% of shoot dry mass for the experimental variant treated with 10 mM Cd, subjected to NaCl stress, and inoculated with the Mc-mix consortium (Figure 3). Crystallization of salt on plant leaves was also observed (Figure 4), which confirms that the shoots were exposed to extreme salinity conditions. Note that Agarie et al. [3] and Xia et al. [58] reported an increase in plant dry mass after exposure to saline solutions ranging from 0.1 to 0.5 M; however, these findings may be related to the less harsh conditions, specifically to the absence of heavy metal, less frequent application of saline solution, and the use of fully matured plants. Both studies mentioned [3,58] employed 7–8-week-old plants and monitored physiological changes for 21 days after the treatment. In contrast, the experiment presented in this work was focused on the maturation and development processes, with young, 2-week-old seedlings exposed to saline and cadmium stresses. The experiment was finished when the plants entered the mature stage of growth, i.e., after 15 days. This shorter growth period, beginning from an early developmental stage, was chosen deliberately to capture the establishment of stress responses and to assess the influence of PGPR strains during the most dynamic phase of plant growth.

Inoculation with the PGPR bacterial consortium seemingly positively influenced the common ice plant’s resistance to increased Cd levels. It was reported that concentrations of Cd ranging from 1 to 8 mg·kg⁻¹ of soil had a toxic effect on most plants [18,23]. In this study, efficient growth of M. crystallinum was observed in all the experimental groups, and the symptoms like chlorosis or necrosis were observed only in a few plants regarding the variant subjected to 10 mM Cd solution.

Beneficial microorganisms can significantly affect mobilization of metals in the soil and their accumulation in plants through such processes as the production of organic acids and metallophores, adsorption to bacterial exopolysaccharides, complexation, and redox reactions [56]. In this study, considerably higher cadmium accumulation was observed in variants treated with the Mc-mix consortium compared to respective control variants, either exposed or unexposed to NaCl stress (Figure 5). However, this effect was only statistically significant for higher cadmium concentrations, i.e., treatment with a 10 mM solution. The highest cadmium accumulation, both in the above-ground parts (332 µg·g⁻¹ d.m.) and in roots (368 µg·g⁻¹ d.m.), was observed for the experimental group of M. crystallinum inoculated with the Mc-Mix consortium, watered with NaCl solution, and treated with 10 mM of Cd (compared with the respective control values of 181 and 129 µg·g⁻¹ d.m., obtained for objects uninoculated with the PGPR consortium). These findings demonstrate that the tested five-strain consortium effectively stimulated the Cd accumulation in plant tissues, which proves its potential application for phytoremediation. Notably, the plants were subjected to cadmium stress at an early developmental stage, yet no significant adverse effect on the maturation was observed. This is an important observation when compared to the well-documented negative impact of cadmium on plant development, including alteration of root architecture and inhibition of growth [4]. In the studies of other authors, inoculation with PGPB was also shown to increase heavy metal accumulation in alfalfa and to improve the accumulation of desirable metabolites in wheat and tobacco [56]. Among PGP strains, Providencia rettgeri, also used by us as an element of the Mc-Mix consortium, proved to have a beneficial effect in decreasing cadmium toxicity for rice with Cd applied at a concentration of 100 mg·kg⁻¹ [59].

M. crystallinum proved to have high tolerance towards cadmium, revealing the excluder’s strategy [5,9,60]. To evaluate the Cd sequestration and internal distribution within the plant system, two indices were employed: bioaccumulation factor (BAF) (Figure 6) and translocation factor (TF) (

Table 2. Translocation factors (TF) calculated upon treatment of the M. crystallinum in a pot test.

TF (Shoot/Root)	With Microorganisms		Control
	+NaCl	+Water	+NaCl	+Water
1 mM Cd	0.73	3.29	1.85	0.49
10 mM Cd	0.91	1.89	1.46	2.14

). The BAF parameter quantifies the efficiency of metal absorption from the soil and can be calculated separately for the roots and shoots as the ratio of Cd content in the tissue and soil. The results of the BAF value increase indicate that the highest Cd bioaccumulation capacity was revealed both by shoots and roots of plants inoculated with the Mc-mix consortium in the presence of NaCl. The effect was observed for both variants treated with 1 mM and 10 mM Cd, and it was statistically significant. In shoots, however, bioaccumulation seems to be dependent rather on saline stress than on the inoculation with the PGP consortium.

The cadmium transport from the root system to the above-ground parts of the plant was illustrated with the TF and calculated as the ratio of a heavy metal accumulated in shoots compared to the amount in roots. Values > 1 suggest efficient translocation of the metal to the shoots and are regarded as vital for plants selected for phytoremediation since the harvest and removal of shoots from the soil is relatively easy. As compared to the results of other studies [5,9,61], in this present work, M. crystallinum revealed the exclusion strategy only in the experimental groups treated with Mc-Mix consortium under saline stress (TF 0.73 and 0.91 for treatment with 1 mM and 10 mM Cd, respectively) and in control, uninoculated plants not subjected to the salt stress and treated with 1 mM Cd (0.49). The highest value (TF = 3.29) was observed for the plants inoculated with the Mc-Mix consortium treated with 1 mM Cd solution in the absence of added NaCl.

Here, it should be stressed that a significant fraction of Cd admixed to the soil, up to 0.1 mM, may not be available for the plant and thus not taken up [5]. It was shown that this metal can be involved in various processes and reactions in soils, which results in its immobilization and decreased bioavailability [62,63]. However, the introduction of NaCl to the soil environment may promote cadmium mobility and uptake [5,64,65], which may explain higher BAF values obtained for the experimental group subjected to the salt stress.

The application of PGPMs as bioaugmentation agents in cadmium (Cd) phytoremediation offers promising potential but faces significant limitations. A key challenge is the variability of PGPMs performance under field conditions, where soil type, native microbiota competition, and environmental fluctuations can reduce their effectiveness. Strain survival, colonization rate, and functional stability remain critical elements for large-scale implementation [28]. Moreover, the mechanisms of Cd mobilization and uptake, such as siderophore production, organic acid secretion, or metal transporter gene expression, are not fully elucidated for many PGPMs, making optimization a challenging task [30,31]. Nevertheless, PGPM inoculation can enhance plant tolerance, root growth, and metal translocation, particularly when using consortia with complementary traits. Future research should focus on multi-strain formulations adapted to site-specific and plant-specific conditions [30]. Furthermore, integrating PGPM bioaugmentation with agronomic practices such as crop rotation or organic amendments may improve remediation stability and efficacy in real-scale applications. The combined use of PGPMs and hyperaccumulator plants remains a promising but still undervalued strategy for sustainable remediation of Cd-contaminated sites.

4. Conclusions

The tested bacterial strains revealed diverse PGP traits considered beneficial for plant growth and development. After application as a consortium, the constituent bacteria enabled unhindered Mesembryanthemum crystallinum growth under stress conditions, especially exposure to elevated cadmium concentrations.

The common ice plant proved its extreme tolerance towards cadmium as well as its halophytic physiology as shown by high Na accumulation in shoots under saline conditions. The Cd accumulation in plants inoculated with the Mc-Mix PGPR consortium was substantially higher than in respective control variants, especially in the experimental groups treated with 10 mM Cd solution. Thus, the results of this study suggest considerable Mc-Mix potential for enhancing heavy metal uptake, supporting its application in development of sustainable methods of efficient phytoremediation.

However, for real-scale implementation, further studies are necessary to validate the effectiveness of the consortium under field conditions. Such trials should account for the complex interactions with the native soil microbiota which may compete with or suppress the introduced strains, as well as shed more light on the potential influence of long-term cadmium contamination on microbial persistence and function. Moreover, environmental heterogeneity, seasonal variability, and site-specific physicochemical soil properties could significantly affect the plant–microbe performance. Long-term monitoring will be essential to assess the stability of inoculant populations, ecological safety, and reproducibility of remediation outcomes.