Piriformospora indica (P. indica) is a basidiomycete of the Sebacinales order. It is widely found as a harmless root endophyte and is extremely versatile due to its mycorrhizal association to promote plant growth (Kundu and Vadassery, 2022). It impacts growth and development through its imitation of root Arbuscular Mycorrhizal Fungi (AMF) functions such as bioregulation, biofertilization (nutrient exchange), and bioprotection (tolerance and resistance to abiotic and biotic stresses). The property that P. indica has a broad range of hosts led to the assumption regarding its potential application as a symbiotic microbe essential for enhancing plant yield and productivity in sustainable agriculture. The mechanisms behind its beneficial effects have been interpreted using different model interaction systems such as P. indica–Arabidopsis thaliana and P. indica–Hordeum vulgare (Achatz et al., 2010; Qiang et al., 2012). In contrast to AMF, P. indica can be easily cultivated in axenic culture, where it asexually produces a large number of thick-walled, pear-shaped spores called chlamydospores, which have a longer shelf-life.

P. indica can efficiently coexist with plants threatened by salinity stress. The concentration of harmful Na⁺ ions in plants increases due to excess of salt in the root zone, which limits the water uptake. This increases damage to the membrane integrity, causes reactive oxygen species (ROS) build-up, and results in imbalanced nutrient uptake. Consequently, it negatively impacts the photosynthetic capacity and alters cellular metabolism. This reduces growth and crop yield, as well as causes senescence (Li et al., 2022b). Plants use a variety of composite sensing, signaling, and response mechanism to regulate their tolerance for alleviating the negative damages of salt stress (Hanin et al., 2016).

These mechanisms include the ion compartmentalization in vacuoles, release of compatible solutes, and counterbalancing of ROS. Another pathway that plants have developed to cope with increased levels of tissue and cellular Na⁺ ions is homeostasis through Na⁺ transport regulation. To achieve ion homeostasis, sodium extrusion at the root–soil interface, as well as some level of Na⁺ efflux in every other cell type, is presumed to be of critical importance for improving the salt tolerance of glycophytes (Pardo et al., 2006). Sodium extrusion at the root surface is not the only mechanism through which plants counter sodium accumulation in the cell cytosol. In fact, sodium sequestration into the vacuole is aided by the tonoplast Na⁺/H⁺ antiporter (NHX)driven by the vacuolar H⁺-ATPase and H⁺-pyrophosphatase. These are important regulators of intracellular ion homeostasis that attenuate the toxic levels of cytoplasmic Na⁺, enhance K⁺ uptake, and are critical for cell expansion and plant stress acclimation (Bassil et al., 2019). High-affinity K⁺ transporter (HKT) is involved in encoding for Na⁺ and/or K⁺ transporters at the cell plasma membrane. These transporters control the movement of Na⁺ ions across tissues and are essential for improving salt tolerance in several plant species (Ali et al., 2021).

Symbiotic associations between plants and beneficial fungus such as P. indica alters plant secondary metabolism, promotes plant growth, and reduces the damages of abiotic stresses and enhances plants fitness by modulating their stress tolerance mechanisms (Li et al., 2022a). However, cell wall act as a barrier for beneficial microorganism’s entry into plant tissues, which is overcome through the secretion of cell wall-destroying enzymes (CWDE). The activity of P. indica CWDE destroys plant cell wall polysaccharides for an entry pathway to plant cells (Boorboori and Zhang, 2022). In extremely arid conditions, date palm, Phoenix dactyliferaremains viable in saline soils and soils with low water supply (Müller et al., 2017; Hazzouri et al., 2020). They can grow in soils irrigated with 12 dS m^-1 salinity level, exhibiting low to no symptoms of salt stress (Ramoliya and Pandey, 2003). However, date palms have been reported to encounter a 3.6% reduction in their yield with every unit increase in salinity above 4 dS m^-1 (Maas and Grattan, 1999). This has caused a reduction in productivity, crop failures, and abandonment of farms in severe salt-affected areas. Thus, P. indica can play a mutualistic role with date palm to improve the latter’s productivity in high salt-stressed soils.

The impact of salinity stress on date palm is similar to other plants. Al Kharusi et al. (2017) demonstrated a high concentration of Na⁺ ions in roots and shoots, reduced K⁺ ions in the shoot, declined leaf relative water content, and increased electrolyte leakage in sensitive varieties. The function of date palm salinity responsive genes was studied by Patankar et al. (2019), who showed that the expression of the P. dactylifera metallothionein gene (PdMT2A) in Saccharomyces cerevisiae, a salinity sensitive yeast mutant, enhanced its drought, oxidative, and salinity stresses tolerance. Moreover, the induced expression of PdMT2A in transgenic Arabidopsis plants reduced the accumulation of toxic Na⁺ ions and maintained higher K⁺/Na⁺ ratio in comparison to wild type. These changes were attributed to the regulatory role of the transgene on HKT transporters. P. indica triggers various salt-responsive genes associated with stress adaptation and cellular growth improvement during salt stress. Discovering genes and pathways involved in metabolic and molecular processes, such as ion homeostasis, antioxidant activities, and cell wall integrity are of particular importance to unravel the mechanism by which P. indica can trigger salt tolerance in date palm and to elucidate the molecular mechanisms related to stress adaptation and functional genomics studies.

The P. Indica’s influence on date palm salt stress tolerance mechanism have not been examined so far. Thus, the present study was aimed to investigate the potential role of P. indica in promoting the growth and ameliorating the salinity tolerance of date palm. This study was designed to assess the growth-related parameters of date palm seedlings colonized or not with P. indica under normal or salt stress conditions. The Na⁺/K⁺ ratio, which is an index to discriminate between sodium and potassium, was exploited as a selection tool to screen for salinity tolerance. Furthermore, antioxidant activity was explored as the tolerance mechanisms that P. indica triggers under salinity stress. In addition, the current study analysed the transcription level of several salt stress responsive genes in roots and leaves of inoculated and non-inoculated date palm seedlings.

P. indica is known to enhance the growth of plant and its tolerance to biotic and abiotic stresses (Gill et al., 2016; Lin et al., 2019). Its role in increasing abiotic stress tolerance has been reported in various plant species (Baltruschat et al., 2008; Alikhani et al., 2013; Ghabooli et al., 2013), but not in date palm. Most studies have confirmed a mutualistic relationship between plants and P. indica after pre-cultivation that enabled the plants to tolerate stress. Prolonged exposure to salt stress causes ion toxicity due to sodium and chloride ion build up within the cytosol. This leads to a decline in potassium uptake, which affects the activity of various enzymes and impedes plant growth. Peskan-Berghöfer et al. (2015) demonstrated that increasing ABA triggered by osmotic stress promotes fungal colonization of the roots, without impairing plant fitness. This shows that ABA can strengthen plant interaction with P. indica as a consequence of its effect on plant innate immunity. However, the basis of the mechanism through which plant growth is improved by P. indica is not clear. Hence, in this study, P. indica’s ability to enhance date palm seedling growth is investigated through its impact on plant Na⁺ and K⁺ ion concentrations and expression of some genes and ion channels that maintain the Na⁺/K⁺ homeostasis and the nuclear-localized calcium-sensing and signaling pathway, which is also an essential mechanism of plant stress tolerance.

The date palm seedlings’ growth was impaired by the inclusion of salt in the growth media. However, the addition of P. indica countered these effects in the seedlings, resulting in increased biomass and lateral root density under control and salinity stress conditions. These results are in concurrence with previous studies of plant growth stimulation with P. indica inoculation under salinity (Ghorbani et al. 2018; Abdelaziz et al., 2019; Bertolazi et al., 2019; Atia et al., 2020). Likewise, under drought stress, it was found that P. indica improved the biomass and morphological characteristics of Solanum melongena (Swetha and Padmavathi, 2020). Furthermore, P. indica enhanced the chlorophyll content of inoculated date palm, as exhibited by their improved fresh and dry weight, both under control and stressed conditions. Similarly, enhanced chlorophyll content was recorded in P. indica-colonized Arabidopsis thaliana and rice plants (Jogawat et al., 2013; Abdelaziz et al., 2017). The colonization with P. indica improves the root architecture of plants that increases root surface area, which leads to improved plant biomass and chlorophyll content due to the efficient absorption of several ions such as the K⁺ and Ca²⁺ (Liu et al., 2019).

The leaves and roots of salt-stressed non-colonized date palm seedlings displayed a higher Na⁺/K⁺ ratio, possessing higher Na⁺ and lower K⁺ contents. On the other hand, P. indica colonization of salt-stressed date palm seedlings altered Na⁺/K⁺ homeostasis and resulted in a decline in the Na⁺/K⁺ ratio in leaves and roots. In accordance with the current results, it has also been reported that P. indica-colonized barley cultivated under 300 mM NaCl had enhanced K⁺ and lower Na⁺ ion content in the leaves, as compared with non-colonized plants (Deshmukh et al., 2006). Furthermore, decline in the Na⁺ content with the P. indica co-cultivation of Arabidopsis and barley were also reported under salt stress (Lanza et al., 2019; Sepehri et al., 2021). In relation to the present study, increased K⁺ ion content in the shoots of tomato plants inoculated with P. indica was also associated with its salinity tolerance (Yun et al., 2018). Cytosolic K⁺ homeostasis is considered an essential adaptation mechanism for salt stress in various plant species (Zhao et al., 2020). Thus, it can be suggested that the favorable Na⁺/K⁺ ratio modulation in colonized plant leaves and roots is associated with P. indica’s ability to preserve K⁺ retention under salinity.

Salt stress causes an increase in free radicals and ROS, which damages plant cells (Ma et al., 2022). The present findings show that P. indica-inoculated date palm seedlings had remarkably higher levels of CAT, SOD, and POD enzymes under salt stress conditions in comparison with non-inoculated seedling’s roots and shoots. These results suggest that P. indica inoculation enhances the triggering of ROS scavengers, which contributes to ameliorated salinity stress tolerance in date palm seedlings. In addition to ROS scavengers, it was further reported by Mohsenifard et al. (2017) that two miRNAs (miRNA396 and miRNA159) involved in the regulation of ABA, were significantly upregulated after inoculation of rice plants with P. indica under drought stress condition. It is known that miRNAs are key factors in regulating many biological processes by degradation or translation inhibition of stress-related genes. Similarly, under salt stress, P. indica can significantly increase ABA that stimulates the stomatal closure and prevents damages to the plants. Furthermore, previous studies have also shown that P. indica inoculation causes a rise in detoxifying and antioxidant enzymes level in the host plant species (Baltruschat et al., 2008; Matsuo et al., 2015). As observed in this study, P. indica also contributed to the reduced symptoms of stress in the date palm seedlings under salinity stress. Consistent with present study’s results, Lin et al. (2019) reported that CAT, SOD, and POD activities, which are crucial to the antioxidant activity of plant cells, were enhanced by the fungus. Moreover, under water stress condition, similar effects of P. indica were found in rice plants, with an increase of CAT enzymatic activity (Tsai et al., 2020).

The mineral analysis profile indicated that the symbiotic association between P. indica and date palm seedlings enhanced the P and Zn content under normal and salt stress conditions. The current study focused on the P and Zn uptake due to their essential role in maintaining plant growth and development under salinity. P. indica solubilizes organic P from the soil to make it readily available as Pi, that constitutes an indirect Phosphate source for the plants. In exchange, P. indica derives photosynthates (carbon), which is essential for its metabolic processes and survival, from the plants (Aslam et al., 2019). The increase in plant rhizosphere macro- and micronutrients due to P. indica association has been described in several plants (Gil et al., 2016; Su et al., 2017). Adequate amount of P in the soil enhances plant tolerance to excess salts by instigating a deeper rooting system and providing sufficient inorganic P supply for the absorption of carbon in leaves. It also plays a role in preserving optimal leaf relative water content under high salinity (Tariq et al., 2017). Comparably, Zn is also crucial for date palm’s growth under saline conditions due to its role in regulating antioxidative enzymes such as CAT and SOD. Thus, higher Zn concentration in the soil also helps plants to avoid ROS damage under salinity stress. The impact of P. indica colonization on P and Zn levels detected in the current study has also been expressed in lettuce plant species (Padash et al., 2016). PHOSPHATE1 (PHO1) is a high-affinity Pi transporter expressed predominantly in the roots, and the gene is up-regulated under low Pi conditions (Hamburger et al., 2002). Bakshi et al. (2015) reported that P. indica promotes PHO1 expression under Pi limitation.

This study found that P. indica inoculation increased seedling biomass and the number of lateral roots, altered Na⁺/K⁺ homeostasis, enhanced the activation of ROS scavenging enzymes and allowed for efficient mineral uptake in salt-stressed date palm seedlings. Furthermore, in a recent study, RNA-Seq experiment was performed for gene expression differences in Arabidopsis plants inoculated with P. indica. It was reported that in response to salt stress, the number of downregulated genes were higher in comparison to the upregulated genes. The de novo assembly of P. indica transcriptome generated a total of 15,410 unigenes, out of which, there were 661 genes differentially expressed against the salinity stress (Nivedita et al., 2021). To understand the molecular mechanisms through which P. indica enhances plant’s stress tolerance, a transcriptome analysis using RNAseq technology was performed on the roots of colonized and non-colonized date palm seedlings under salt stress and control conditions (data not shown). The up-regulated genes identified by RNAseq and those that were significantly enriched with transcripts involved in membrane polarization, ion transportation, and ROS signaling were investigated by real-time PCR. The results displayed that P. indica inoculation led to a three-fold increase in HKT1;5, SOS1, and RSA1 transcript levels in the roots of inoculated plants as compared with non-inoculated plants under salt stress. The high HKT1;5 and SOS1 expression allows for the maintenance of a balanced Na+ and K+ ion homeostasis under salinity, which improves plant growth. Similar enhanced expression of HKT1 and SOS1 were found in Arabidopsis thaliana and Lycopersicon esculentum plants colonized with P. indica (Abdelaziz et al., 2017; Ghorbani et al., 2019). The HKT1;5 gene encodes a plasma membrane Na⁺ transporter expressed in the root cells surrounding xylem vessels (stele and epidermis), which is involved in Na⁺ retrieval from the xylem sap upon salt stress to reduce Na⁺ transfer to the leaves (Kiani et al., 2021). In rice, the Na⁺ transporter, OsHKT1;5, was expressed in vascular tissues (essentially in root xylem parenchyma), that retained Na⁺ ions in roots upon salt stress and enabled the shoot tissues to maintain K⁺ homeostasis. While the importance of SOS1 antiporter in plant salt tolerance is linked to Na⁺ homeostasis, that removes Na⁺ out of the cytosol into the apoplastic space and controls the root-to-shoot Na⁺ transport for the maintenance of Na⁺/K⁺ homeostasis (Shi et al., 2002; Olías et al., 2009). In addition, RSA1 was also highly expressed in the colonized roots challenged with salt stress. RSA1 and its partner, RITF1, govern the transcription level of various genes that play a role in ROS detoxification under salt stress. RSA1 further regulates the SOS1 gene that encodes for a plasma membrane Na⁺/H⁺ antiporter crucial for salinity tolerance (Guan et al., 2013).

LEA2 genes are of particular importance in date palm, and they are speculated to retain water molecules and prevent crystallization of cellular components under water deficiency resulting from drought, heat, and high salinity stresses. These genes are opulent in the P. dactylifera genome assembly; there are 62 LEA2 members in P. dactylifera as compared with 52 and 46 in rice and sorghum, respectively (Al-Mssallem et al., 2013). According to the genome assembly and the transcriptomic data generated from RNAseq carried out on date palm roots (data not shown), the current study analyzed the date palm’s LEA2 genes expressions that regulate the plant’s responses to abiotic stress. The results indicated that P. indica inoculated led to a roughly three-fold higher LEA2 transcript levels in leaves but not in inoculated plants roots contrast to non-inoculated plants under salt stress. In accordance with this result, Azizi et al. (2021) had reported that the interaction effect of drought and colonization of tomato seedlings with P. indica significantly enhanced the TAS14 gene expression, one of the essential groups of LEA2 genes.

The salinity stress negatively impacts the agronomical traits of the date palm due to the accumulation of toxic Na⁺ ions in the plant cytosol. In fact, Na⁺ accumulation can damage membrane systems and cytosolic proteins. To eliminate these toxic Na⁺ ions and maintain ion homeostasis at the plasma membrane, efficient Na+ and/or K+ transport systems are required. The current study revealed that the colonization of date palm seedlings with the beneficial endophyte P. indica significantly reduced the detrimental effects of salt stress by exhibiting enhanced growth through ions homeostasis and nutrients uptake, antioxidant activity, and several upregulated stress responsive genes. Date palm root colonization by P. indica is a remarkable example of a beneficial microbial symbiosis and the underlying molecular mechanisms by which it establishes itself and exerts its beneficial effects are poorly understood. To increase our understanding of the mechanisms underlying the ability of this endophyte to promote growth and increase salt stress tolerance, we need to advance our understanding of the colonization mechanism and how the host responds transcriptionally to colonization. The profound symbiotic association between the P. indica and date palm seedling found in the present study suggests that this endophytic fungus can be exploited as an essential plant symbiont for improving date palm production for economic sustainability in marginal environments.

The original contributions presented in the study are included in the article/supplementary material. Further inquiries can be directed to the corresponding author.

KM, MA, and MS conceived and wrote the original manuscript. SKM, MS, and MA performed the experiments. KM, MA, and MS analyzed the data. KM, MS, MA, SKM, FB, and HR reviewed and edited the draft. All the authors have read and approved to the submitted version of the manuscript.

This research work was supported by funding from the United Arab Emirates University, the Research Office to K.M. under grant number 31R203.

The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.

All claims expressed in this article are solely those of the authors and do not necessarily represent those of their affiliated organizations, or those of the publisher, the editors and the reviewers. Any product that may be evaluated in this article, or claim that may be made by its manufacturer, is not guaranteed or endorsed by the publisher.