Endophytic fungi are able to induce tolerance to salt stress in date palm seedlings (Phoenix dactylifera L.)

Abstract

Date palm, typically considered a salinity-resistant plant, grows in arid and semi-arid regions worldwide, and experiences decreased growth and yields under salt stress. This study investigates the efficacy of endophytic fungi (EF) in enhancing the salinity tolerance of date palm seedlings. In this experiment, EF were isolated from date tree roots and identified morphologically. Following molecular identification, superior strains were selected to inoculate date palm seedlings (Phoenix dactylifera L., cv. Mazafati). The seedlings were subjected to varying levels of salinity stress for 4 months, utilizing a completely randomized factorial design with two factors: fungal strain type (six levels) and salinity stress (0, 100, 200, and 300 mM sodium chloride). The diversity analysis of endophytic fungi in date palm trees revealed that the majority of isolates belonged to the Ascomycota family, with Fusarium and Alternaria being the most frequently isolated genera. In this research, the application of fungal endophytes resulted in increased dry weight of roots, shoots, root length, plant height, and leaf number. Additionally, EF symbiosis with date palm seedling roots led to a reduction in sodium concentration and an increase in potassium and phosphorus concentrations in aerial parts under salt-stress conditions. While salinity elevated lipid peroxidation, consequently increasing malondialdehyde (MDA) levels, EF mitigated damage from reactive oxygen species (ROS) by enhancing antioxidant enzyme activity, including superoxide dismutase (SOD), catalase (CAT), and ascorbate peroxidase (APX), while promoting proline and total soluble sugar (TSS) accumulation. The colonization percentage generally increased with salinity stress intensity in most strains. According to the results, the application of EF can alleviate the adverse effects of salinity stress and enhance the growth of date palm seedlings under saline conditions.

Introduction

Date palm (Phoenix dactylifera L.), a monocotyledonous and dioecious plant from the Arecaceae family, is often affected by soil fertility, drought, and salinity [5]. Despite these challenges, the date palm is recognized for its high salinity tolerance [49], with certain cultivars thriving near coastal shores exposed to seawater from tidal currents [54]. This resilience has led to the perception of date palm as an exceptional halophytic plant [84]. Plants employ various strategies to tolerate salt, showcasing remarkable models of evolutionary changes [29]. Halophytes, including the date palm, have developed special molecular mechanisms or cell structures to tolerate high salt concentrations [88]. Rodriguez et al. [71] describe symbiotically conferred stress tolerance as “habitat adapted symbiosis,” proposing its role in establishing plants in high-stress habitats. This suggests that plants may associate with endophytes to enhance tolerance to environmental stress, with these endophytes potentially conferring similar stress tolerance to genetically distant plants [43]. Studies indicate that endophytes from plants in natural and saline environments can enhance agricultural activities in arid regions [13]. The diversity within plant root-associated microbes encompasses tens of thousands of species. Recent advances in research on plant–microbe interactions have shown that plants can shape their rhizosphere microbiome because different plant species are the host of specific microbial communities when grown in the same soil [17]. The colonization of plant root tissue by fungal endophytes provides the greatest benefits in abiotically stressed environments [57]. Rhizosphere microorganisms, responding to plant root signal molecules, release diverse signals that enhance biotic and abiotic stress resistance, root development, and plant growth [87]. Symbiont fungi play crucial roles in modern agriculture and can be applied in short- and medium-term scenarios [73]. According to new views, microorganisms residing in healthy plant tissues, causing no clear signs of infection, are termed endophytes. A very low proportion of endophytes are shown to become pathogenic to their host plant at specific conditions, and the more typical lifestyle is harmless or undefined [77]. When interacting with endophytic fungi (EF), plants exhibit potential reactions to various abiotic stresses such as oxidative stress, drought, flooding, salinity, heat stress, and metal exposure (Al, Ni, Cd, Cu, Zn, Pb) [28]. EF are known to produce secondary metabolites with diverse biological activities, including anticancer, antimicrobial, antioxidant, antiviral, antifungal, and anti-inflammatory properties [36]. These fungi find applications in producing biopesticides, biofertilizers, and various industrial products such as dyes, lubricants, drugs, fuels, insecticides, wood preservatives, and cosmetics [58]. Fungal association enhances the uptake capacity of phosphorous by plants [10]. Phosphorus-solubilizing microbial technology is a sustainable solution for soil heavy metal remediation [22]. Taxonomically, EF belong to Ascomycota, Basidiomycota, and Zygomycota [21]. While the number of endophyte taxa isolated from a host species is typically large, only a few assemblages are host species-specific, with composition and frequencies significantly influenced by site conditions [64]. Saline soils represent unique ecological niches hosting extremophile microorganisms with specific adaptation strategies [61]. Research into the role of salt-resistant microorganisms in enhancing crop growth under saline conditions indicates that endophytic fungi (EF) can induce systemic tolerance in plants [35]. Soil salinity at extreme levels can strongly inhibit plant and fungal growth, but halophyte plant species, coexisting with specific endophytes, thrive in such environments [74]. Geographic specialization of certain fungal genera is suggested by the more frequent presence of endophytic isolates from tropical hosts compared to rare records from temperate regions [64]. Differential recruitment of endophytes in populations of plants growing in the same location indicates a coordinated evolution with host plants, highlighting a high level of host specificity [80]. This research operates on the assumption that EF within date palm roots contribute to increasing the tolerance of date palm trees to high salinity levels. EF were isolated from date palm roots across different regions of Iran and utilized to enhance the salinity tolerance of date seedlings.

Materials and methods

Sampling

To collect and identify isolates of the date palm EF, we conducted sampling on date palms cultivated in 40 date-growing areas. These areas varied in altitude from 25 to 1757 m and spanned from 48° 40′ longitude and 31° 19′ latitude to 57° 1′ longitude and 30° 16′ latitude. We collected approximately 1600 root samples from healthy date palms, free from signs of diseases and pests, across various date palm varieties such as Mazafati, Rabi, Qasb, Khenizi, Khasowi, Almehtri, Kloteh, Barhi, Pyaram, Negar, Khasi, Medjool, Zahedi, Shahabi, and Kabkab. These root samples were carefully placed in plastic bags, transferred to the laboratory, and stored at + 4 °C.

Isolation, propagation, and identification of fungal isolates

To ensure the contamination of roots with EF, the root samples from the lateral root system were carefully washed, cleared with 10% KOH, and stained with 0.05% trypan blue in lactic acid (v/v) [65]. Colonization frequency percentage (CF%) of date palm roots was determined by the technique described by Suryanarayanan et al. [75] (Formula 1). Fungal isolation followed Arnold [11] but with minor modifications. Samples were rinsed with running water and processed as follows. Samples were immersed in 75% ethanol for 1 min and in NaOCl (3%) for 5 min, washed three times with sterile distilled water, and then allowed to surface-dry on sterilized filter paper. Finally, samples were cut into 0.5–1-cm pieces and placed in Petri dishes (9 cm in diameter) on potato dextrose agar (PDA) medium and cultured at 25 °C in the dark for 1–2 weeks [52]. All fungal isolates were identified through a method of morphological analysis, which involved examining both macroscopic and microscopic features. The microscopic slides were prepared using lactophenol and lactophenol-cotton blue staining solutions and viewed under a light microscope (BH2, Olympus, Japan) at magnifications of 200 × and 400 × . The fungal isolates were then named and identified [14].

$$\begin{array}{ccc}\mathrm{Formula}1.& & \mathrm{Colonization}\mathrm{frequency}\mathrm{percentage}(\mathrm{CF}\%)=\frac{\mathrm{Number}\mathrm{of}\mathrm{segments}\mathrm{colonized}\mathrm{by}\mathrm{an}\mathrm{endophyte}}{\mathrm{Total}\mathrm{number}\mathrm{of}\mathrm{segments}\mathrm{analyzed}}\times 100\end{array}$$

Examining the non-pathogenicity of EF isolated from date palm roots on tomato plant

To assess the non-pathogenicity of EF isolated from date palm roots, all isolates were tested on tomato plants, known for their susceptibility to fungal diseases, within a greenhouse. The study utilized tomato seeds (Super Chief cultivar), which were planted and subsequently transplanted into sterile pots (10-cm diameter, 14-cm height) containing a soil mixture (sand:soil in a 3:1 ratio, 350 g) that had undergone three rounds of sterilization in an autoclave at 121 °C and 1.5 atmospheric pressure for 20 min each. In EF-inoculated treatments, four pieces of 5-mm plugs excised from the edge of an actively growing colony on culture medium were positioned 1 cm away from the roots of tomato seedlings. Control treatments received plugs excised from the PDA medium without fungus. All inoculation and residue addition processes were conducted on an ultraclean workbench. The potted plants were maintained in a growth chamber with a 14-h light/10-h dark photoperiod, a temperature of 27 °C/22 °C (day/night), and a mean air relative humidity of 65%. Out of the 120 isolates, 12 strains were selected based on their confirmed non-pathogenicity and observed enhancement of tomato plant biomass [24]. The roots of inoculated plants were stained following the method outlined by Phillips and Hayman [65].

Evaluating the effect of fungal isolates on corn plants

The evaluation of EF’s impact on corn plants was conducted due to the monocotyledonous nature of date trees. Twelve strains, previously identified for enhancing tomato plant biomass, were used to inoculate corn roots. Subsequently, five superior strains were selected based on biomass measurements (results not published).

Molecular identification of fungal isolates

Molecular identification of the isolates after DNA extraction was performed using polymerase chain reaction (PCR) and amplification of rDNA-ITS regions. For the mass production of fungal mycelium needed for DNA extraction, a piece of the fungus (5-mm diameter) was sampled from the edge of the culture and transferred to 50 ml of PDB dextrose potato liquid culture medium. The solution was maintained in an incubator with a shaker that rotated at 100 rpm (25 °C). After 1 week, a mycelium pellet formed and was used for DNA extraction. First, the cannon ball-like fungal mycelium mass in the PDB culture medium was transferred onto a sterile sieve and rinsed with sterile distilled water, so that the culture medium was completely removed from the fungal mycelium mass. The mass of mycelium was placed on a sterile filter paper to be dried. Then 1 g of dried fungal mycelium was ground to powder using liquid nitrogen and cold sterile mortar and transferred to a sterile vial. To extract the fungal DNA, a CTAB extraction buffer (2%) was used according to Doyle and Doyle’s method [26] with slight modifications [6]. To observe the quantity and quality of the DNA, spectrophotometry and electrophoresis were used with 0.7% agarose gel. To amplify the ITS region from nuclear rDNA, a combination of primers ITS1 (5′-TCCTCCGCTTATTGATATGC-3′) and ITS4 (5′-TCCTCCGCTTATTGATATGC-3′) were used [82]. Then the polymerase chain reaction (PCR) was performed [37].

The information of the sequenced samples was reviewed with Chromas 2.23 software and the desired DNA fragment was aligned by Clustal W software [78]. To compare the similarity and match the amplified sequences with similar regions of 18 s rRNA in the gene bank (NCBI), the blast protocol was used. To draw the phylogenetic tree, first similar components were aligned with Mega7 software. The phylogenetic tree was drawn based on the Neighbor-Joining method. The results of molecular identification were compared to the morphological identification of the strains. The ITS sequences of 4-B, 21-A, 22-C, 14-A, 39-D, 16-A, 8-B, 10-D, 11-C, and 22-E were deposited in GenBank with accession numbers OR016712, OR016714, OR016711, OR016713, OR020606, OR853709, OR853710, OR853711, OR853712, and OR853713 respectively. To ensure the availability of the fungal strains for future research, all strains that demonstrated the most promising effects have been deposited in the culture collection of the Horticultural Department, Agri-College, Vali-e-Asr University of Rafsangan.

Investigating the effects of EF strains on date seedlings under saline conditions

To prepare date seedlings, date seeds of the “Mazafati” cultivar (Phoenix dactylifera L., cv. Mazafati) were collected from a 20-year-old Mazafati date tree (Bam, Iran) that had good signs of growth. The seeds were similar in weight and size. The seeds were first disinfected with 0.5% sodium hypochlorite for 5 min, and then with 70% ethanol for 30 s. They were rinsed three times with sterile distilled water and submerged in distilled water in the dark at 30 °C for 2 weeks. Subsequently, the seeds were planted in plastic pots (14 cm in height and 10 cm in diameter). In each pot, approximately 350 g of soil mixture was used (sand:soil at a ratio of 3:1), which had been sterilized three times with an autoclave at 121 °C and 1.5 atmospheric pressure for 20 min. The physicochemical properties of the soil mixture comprised 0.82% organic carbon, 0.01% P, 0.01% K, 0.02% Na, EC 1.01 dS m⁻¹, and pH 7.3. Plants were grown for 3 months under controlled conditions in a greenhouse (at the Vali-e-Asr University, Rafsanjan, Iran) under natural light (photon flux density ranged from 500 to 750 μmol m⁻² s⁻¹) at a daytime temperature of 27 ± 2 °C and a night temperature of 18 ± 2 °C, with a relative humidity of 60–70%. The plants were irrigated once every 5 days with 200 ml of distilled water and next time with the same amount of half-strength Hoagland’s solution [40]. In this experiment, the top five strains obtained from the previous experiments on corn and tomato were used for inoculating date seedlings. To prepare fungal spore suspension, each fungal strain was cultured on potato dextrose agar (PDA) medium for 1 week in an incubator (24 ± 1 °C). The spores of each fungus were collected slowly from Petri dishes with a spatula, using 10 ml of 20% Tween-water solution. This suspension was passed through filter paper twice and centrifuged (3000 rpm for 7 min). Then using a Neobar slide, the spores in each petri dish were counted, and the fungal spores were prepared for each treatment at a concentration of about 1 × 10⁶ ml (ml⁻¹ colony-forming units (CFU)) [25]. After 3 months, date seedlings were transferred to 4-kg pots (two seedlings per pot). Approximately 1 kg of the described soil mixture was poured into 4 kg pots and 100 ml of each fungal strain suspension with the same concentration was added to the soil. Then the seedlings were placed on the soil impregnated with fungal strains so that the roots were in contact with impregnated soil and the area surrounding the seedlings’ roots was filled with the described soil mixture. The plants were placed in the greenhouse (at the Vali-e-Asr University, Rafsanjan, Iran) under natural light (photon flux density ranged from 500 to 750 μmol m⁻² s⁻¹) at a daytime temperature of 27 ± 2 °C and a night temperature of 18 ± 2 °C, with a relative humidity of 60–70%. The plants were irrigated once every 5 days with 400 ml of distilled water and next time with the same amount of half-strength Hoagland’s solution [40]. After 5 months, the salinity treatments were applied. A factorial experiment in the form of a completely randomized design was set up with two factors, including the type of fungal strain at 6 levels (4-B, 14-A, 21-A, 22-C, 39-D, and control) and the salinity treatment at 4 levels (0, 100, 200 and 300 mM of sodium chloride). There were 4 replications in total, comprising 96 pots. Each pot contained 2 date seedlings. During 4 months, the salinity treatments were applied once every 4 days. At 0 level of salt, 400 ml of distilled water was given to each pot, and as for the three salinity levels, the same volume (400 ml) was provided as salt solutions. To avoid salt accumulation, after every 4 rounds of irrigation with salt solution and in the fifth round, the plants were irrigated with distilled water until drainage was observed at the bottom of pots. At the end of the experiment, several parameters were measured in the date seedlings.

Measurement of vegetative parameters, photosynthetic pigments, and nutrients in date palm seedlings

Following the stress period, date seedlings were uprooted, washed with tap water, and distilled water. The shoot and root systems were then separated, and growth parameters including root length, number of leaves, and plant height were measured. Dry weight was determined by placing samples in an oven at 72 °C for 48 h. Chlorophyll a, b, total chlorophyll, and total carotenoids were measured [53]. Sodium and potassium amounts were assessed using flame photometry after converting the samples into ash [43]. Phosphorus levels were measured by ammonium molybdate and ammonium vanadate (yellow) methods, with spectrophotometry at a wavelength of 470 nm [60]. Additionally, the relative water content (RWC) of the leaves was calculated [81].

Measurement of total soluble proteins and enzyme activities in date seedlings

Root samples (0.1 g) were frozen in liquid nitrogen and the powder was extracted in 4 ml of 1 M phosphate buffer (pH 7) containing 5% polyvinylpolypyrrolidone (PVPP). The homogenate was centrifuged at 18,000 × g for 15 min at 4 °C and the supernatant was used for measuring antioxidant enzyme activities. Total soluble proteins (TSPs) were determined according to the method described by Bradford [18] using bovine serum albumin (BSA) as a standard. Superoxide dismutase (SOD, EC 1.15.1.1) enzyme activity was determined by measuring the ability of SOD to inhibit the photochemical reduction of nitro blue tetrazolium chloride [16]. The 3-ml reaction mixture contained 50 mM phosphate buffer, pH 7–8, 13 mM methionine, 75 µmol nitro blue tetrazolium (NBT), 2 µM riboflavin, and 0–1 mM ethylenediaminetetraacetic acid (EDTA), and 0–50 µmol enzyme extract riboflavin was added last and the tubes were shaken and placed 30 cm below a light bank consisting of two 15-W fluorescent lamps. The reaction was started by switching on the light and was allowed to run for 10 min during which time it was found earlier to be linear. The reaction was stopped by switching off the light and the tubes were covered with a black cloth. The absorbance by the reaction mixture at 560 nm was read. A non-irradiated reaction mixture did not develop color and served as control. One unit of SOD is equal to the amount of enzyme required to cause 50% inhibition of the NBT photo‐reduction rate. Ascorbate peroxidase (APX, EC 1.11.1.11) was assayed by the ascorbate oxidation method at 260 nm. The activity was determined as described by Nakano and Asada [59] with some modifications. A reaction mixture containing 0.1 ml of extract, 1 ml 0.1 M ascorbate, and 1 ml 0.5 mM H₂O₂ was prepared, and then the absorbance was determined. A calibration curve was set with ascorbate concentrations [59]. The reaction mixture consisted of 0.1 ml of extract, 1 ml 0.1 M ascorbate, and 1 ml 0.5 mM H₂O₂. The absorbance was determined. A calibration curve was set with ascorbate concentrations. Catalase (CAT, EC 1.11.1.6) activity was determined by monitoring the decrease in absorbance at 240 nm for 3 min following the consumption of H₂O₂ [3]. The reaction mixture consisted of 0.1 molar (M) potassium phosphate buffer (pH 7.0), 0.1 mM EDTA, 20 mM H₂O₂, and 100 μl of enzyme extract in a 2-ml volume.

Measurement of TSS, proline, and MDA in date seedlings

The amount of TSS in the leaves was measured according to a relevant method in the available literature [42]. TSSs were quantified in 50 mM potassium phosphate buffer (pH 7.5) extract of fresh leaves (1 g). The extract was filtered and centrifuged at 38,000 × g for 10 min at 4 °C. The supernatant was collected. For TSS analysis, 0.1 ml of potassium phosphate buffer (pH 7.5) extract was mixed with 3 ml freshly prepared anthrone (200 mg anthrone with 100 ml 72% H₂SO₄) and placed in a boiling water bath for 10 min. After cooling, the absorbance at 620 nm was determined. The calibration curve was made using glucose in the range of 20–400 µg ml⁻¹. The amount of proline was determined (μg/g fresh weight) according to a common method as described by Paquin and Lechasseur [63]. Free proline determination was estimated by reacting 5 ml of ninhydrin (3.125 g ninhydrin dissolved in 50 ml of phosphoric acid 6 M and 75 ml of glacial acetic acid) and placed in boiling water for 45 min. The absorbance was read in a spectrophotometer at 515 nm. Free proline concentration was calculated from a calibration curve using proline as a standard. Lipid peroxidation was measured in terms of MDA content according to Heath and Packer [38]. Leaf samples (0.5 g) were homogenized in 10 ml of 0.1% trichloroacetic acid (TCA) and centrifuged at 18,000 × g for 10 min. Two-milliliter aliquot of the supernatant was mixed with 2 ml of 20% TCA containing 0.5% tertiary butyl alcohol (TBA). The mixture was heated at 100 °C for 30 min, cooled, and the absorbance of the supernatant was read at 532 nm (A532). The unspecific turbidity was corrected by A600 subtracting from A532.

Evaluation of root fungi colonization

At the end of the experiment, the percentage and extent of root colonization in date seedlings were measured for the five fungal endophyte strains. The evaluations were conducted using formula 1 [75], with roots stained following the method described by Phillips and Hayman [65].

Treatment groups with endophytic fungi were denoted as + EF, while those without endophytic fungi were represented as − EF.

Data analysis

For data analysis, SPSS 20 software was utilized. Prior to statistical analyses, all data underwent tests for normality and homogeneity of variance. Mean differences among various treatments were compared using Duncan’s multiple-range tests at a significance level of P < 0.05. Graphs were drawn using Microsoft Excel.

Results

Identification of fungal strains

A total of 120 fungal strains were isolated from date palm roots and were classified into five main genera (Fig. 1). Based on the morphological results, the 5 EF strains in this experiment (Table 1) were related to Fusarium sp. (strains 4-B, 21-A, 22-C), Alternaria sp. (strain 14-A), and Acrocalymma sp. (strain 39-D). The results of morphological identification of EF strains from date palm roots that were applied in this experiment are shown in Fig. 2, and the results of molecular identification of EF isolated from date palm roots are shown in Fig. 3. The phylogenetic tree describes ten fungal strains that increased growth parameters in the initial experiments on symbiosis with tomato and corn roots. In this experiment, the top 5 strains that applied best in corn and tomato (4-B, 14-A, 21-A, 22-C, 39-D) were selected. According to ITS sequence analysis, EF species were identified (Fig. 3).

Fig. 1.

Frequency percentage of symbiotic fungal isolates of the roots of the studied palm trees

Table 1.

Location and soil characteristics of groves where date palm tree root samples were collected

Isolated code	Gathering location	Longitude	Latitude	Height Above sea level (m)	Soil pH	Soil EC (ds/m)
4-B	Rigan (Kerman, Iran)	59° 3'	28° 38'	611	7.9	4.36
14-A	Rudan (Hormozgan, Iran)	57° 11'	27° 27'	190	7.8	3.9
21-A	Molla-sani (Khoozestan,Iran)	48° 53'	31° 35'	29	7.9	11.47
22-C	Ahvaz (Khoozestan,Iran)	48° 40'	31° 19'	25	8	9.62
39-D	Borazjan (Bushehr,Iran)	51° 13'	29° 6'	68	7.8	18.74

Fig. 2.

The results of morphological identification of EF strains isolated from date palm cultivars roots and applied in this experiment. In each row from left to right: top and bottom view of colonies on media and light microscopy image of EF (400 ×) respectively. a (4-B) Fusarium sp., b (14-A) Alternaria sp., c (21-A) Fusarium sp., d (22-C) Fusarium sp., and e (39-D) Acrocalymma sp. respectively

Fig. 3.

Phylogenetic relationships of a selection of species isolated from date palm roots based on Neighbor Joining analysis of rDNA ITS sequences in MEGA 7.0. The bootstrap support from 1000 replication is indicated above the branches. Codes used for sequences of the used isolates are explained in the results section under “Identification of fungal strains”

Vegetative parameters

The results showed that EF had a significant effect on the growth parameters of the studied plants. As the salinity level increased, there was a decrease in shoot dry weight (Fig. 4a), root dry weight (Fig. 4b), root length (Fig. 4c), leaves number (Table 2), and plant height (Table 2). At zero salinity, except for strain 4-B, all strains caused an increase in shoot dry weight, compared to the control, and strain 39-D caused the highest increase (11.4%) (Fig. 4a). Despite the increase in salinity at different levels, all strains were able to maintain the dry weight of the shoots to some extent, compared to the decrease in the control. At 100 and 200 mM sodium chloride, strain 21-A caused the highest increase in shoot dry weight (12 and 20.7%, respectively), compared to the corresponding controls (− EF). At 300 mM, strain 39-D caused the highest increase in shoot dry weight (24.3%), compared to the corresponding control (− EF).

Fig. 4.

Interaction effect of EF including 4-B (Fusarium. solani), 14-A (Alternaria sp.), 21-A (Fusarium sp.), 22-C (Fusarium sp.), and 39-D (Acrocalymma sp.) and salinity levels (0, 100, 200, and 300 mM NaCl) as well as Cont. (Control) on shoot dry weight (a), root dry weight (b), and root length (c) of 1-year-old Mazafati date seedlings (Phoenix dactylifera) 4 months after salt treatment. Values with the same letter are not significantly different (p ≤ 0.05) according to Duncan’s multiple range test (n = 4)

Table 2.

Interaction effect of EF including 4-B (Fusarium. solani), 14-A (Alternaria sp.), 21-A (Fusarium sp.), 22-C (Fusarium sp.), and 39-D (Acrocalymma sp.) and salinity levels (0, 200, 100, and 300 mM NaCl) as well as Cont. (Control) on number of leaves, plant height, photosynthetic pigments (Chla, Chlb, Chlt, Car), relative water content (RWC), and catalase enzyme activity (CAT) of 1-year-old Mazafati date seedlings leaves (Phoenix dactylifera L.) 4 months after salt treatment

EF	salinity levels (mM NaCl)	Number of leaves	Plant height (cm)	RWC (%)	Chla (mg/g FW)	Chlb (mg/g FW)	Chlt (mg/g FW)	Cart (mg/g FW)	CAT (μmol H2O2/mg prot./min)
4-B	0 (mM) 100 (mM) 200 (mM) 300 (mM)	5.5a−c 5.12b−f 5.00c−f 4.37 g−i	33.12b−f 31.75c−i 28.5f−i 26.57i	87.55ab 83.83bc 79.14d−e 70.98f	21.9bc 18.43d−f 15.28i−l 10.44op	6.45bc 5.09de 4.68 fg 3.9f−h	28.35b 23.52 fg 19.96j 14.35 m	8.05b−d 6.85e−i 6.62f−i 4.23 mn	0.87 g 1.92be 2.61b 3.59a
14-A	0 (mM) 100 (mM) 200 (mM) 300 (mM)	5.62ab 5.37a−d 4.87d−g 4.62f−h	36.68ab 33.5a−f 28.87f−i 27.62ghi	87.08ab 81.32c−d 78.07de 70.52f	20.39 cd 17.52e−h 15.56 h−k 13.31 lm	5.63 cd 5.12de 3.66 g−i 2.75ij	26.03b−e 22.64gh 19.23j 16.06 lm	7.73 cd 7.26d−g 5.66j−l 5.41kl	0.94 fg 1.97b−e 2.39bc 3.31a
21-A	0 (mM) 100 (mM) 200 (mM) 300 (mM)	5.37a−d 4.75e−h 4.75e−h 4.25hi	38.37a 34.5a−e 32.00c−h 26.93hi	88.55a 84.49bc 81.66 cd 72.1f	19.04de 16.82f−i 14.25 k−m 10.97mo	4.89d−f 3.98f−h 3.24hi 2.87ij	23.94e−g 20.8 h−j 17.5kl 13.85 m	7.35d−f 6.38 g−j 5.53j−l 4.99 lm	1.38dg 1.83c−e 2.57bc 3.4a
22-C	0 (mM) 100 (mM) 200 (mM) 300 (mM)	5.87a 5.25b−e 5.00c−f 4.75e−h	36.00a−c 32.5b−g 29.5e−i 27.06hi	87.64ab 84.29bc 80.8c−e 73.58f	22.57ab 20.49b−d 17.88e−g 13.53 k−m	8.2a 6.63b 4.55 fg 3.93f−h	30.82a 27.13bd 22.43 g−i 17.47 km	8.79ab 8.12b−d 7.00e−h 5.34kl	0.93 fg 1.86c−e 2.51bc 3.57a
39-D	0 (mM) 100 (mM) 200 (mM) 300 (mM)	5.5a−c 5.25b−e 4.75e−h 4.62f−h	38.37a 35.31a−d 30.68d−i 28.25f−i	86.96ab 84.59bc 81.45c−e 71.25f	24.07a 21.36bc 20.54b−d 13.68 k−m	7.7a 6.73b 5.32de 3.65 g−i	31.77a 28.09bc 25.87c−f 17.33kl	9.25a 8.38bc 7.98b−d 6.01i−k	1.03 g 2.07b−d 2.51bc 3.53a
Control	0 (mM) 100 (mM) 200 (mM) 300 (mM)	5.00c−f 4.62f−h 4.00i 4.00i	37.5ab 30.5d−i 22.00j 18.00j	84.09bc 77.74e 73.21f 66.44j	20.14 cd 16.24 g−j 12.69 mn 8.6p	5.13de 3.95f−h 3.08 h−g 2.15j	25.27c−f 20.20ij 15.78 lm 10.76n	7.71c−e 6.25 h−k 5.41kl 3.65n	0.7b−e 1.28e−g 1.63d−f 1.92a
p-value S E S × E		** ** ns	** ** ns	** ** ns	** ** ns	** ** ns	** ** ns	** ** ns	** ** ns

S and E: salinity (mM NaCl) and EF isolates, respectively; ns not significant

**Significant at p ≤ 0.01. In each column, values with the same letter are not significantly different (p ≤ 0.05) according to Duncan’s multiple range test (n = 4)

At all salinity levels, the strains caused plants to have more root dry weights, compared to the control. At 0, 100, 200, and 300 mM salinity, the strains 39-D, 14-A, 21-A, and 14-A caused increases in root dry weight (29.4%, 18.3%, 20%, and 49.3%, respectively), compared to the corresponding controls (Fig. 4b). Symbiosis with the EF at high salinity levels caused a significant increase in root length, compared to the respected controls (− EF). Strain 4-B caused the highest increase (40.4%) at the 300 mM salinity level, compared to the same salt-affected control (− EF) (Fig. 4c). All EF increased the number of leaves and the height of date palms compared to the control. However, there was no significant difference between the effectiveness of the various strains at each salinity level (Table 2).

Photosynthetic pigments

Applying salt stress (NaCl) caused a significant decrease in chlorophyll a, chlorophyll b, total chlorophyll, and carotenoid contents in plants (Table 2). The inoculation of plants with fungal strains significantly improved the amounts of different photosynthetic pigments. However, at each salinity level, the effectiveness of each strain was not significantly different from that of another in increasing the photosynthetic pigments of the seedlings (Table 2).

Relative leaf water content

As the salinity level increased, the RWC of the leaves decreased significantly (Table 2). All strains caused an increase in the RWC of the leaves, compared to the control. However, there was no significant difference in the effectiveness of the various strains in this respect at each salinity level (Table 2).

Enzyme activities

Under salt stress, there was a significant decrease in TSP, whereas the activities of SOD, CAT, and APX enzymes increased significantly, compared to the control. In symbiosis with fungal strains, leaf TSP content was less affected by salinity in comparison with control while the activities of SOD, CAT, and APX enzymes were increased more than control by increasing salinity level. The lowest TSP occurred in the control treatment in response to the highest salinity level. At the highest salinity level, strain 21-A caused an increase (12%) in TSP, compared to the control (Fig. 5a). SOD activity increased in stressed and non-stressed plants in response to + EF, compared to the − EF control plants (except 39-D). The highest amount of SOD activity was caused by the 4-B strain (51%) at the highest salinity level, compared to the control (Fig. 5b). All strains increased the CAT enzyme activity, compared to the control, although there was no significant difference in the effectiveness of the various strains in increasing the CAT activity at each salinity level (Table 2). At the highest level of salinity, the APX enzyme activity increased maximally in response to strains 39-D (31%), 21-A (28%), and 22-C (26%), compared to the control (Fig. 5c).

Fig. 5.

Interactions effect of EF including 4-B (Fusarium. solani), 14-A (Alternaria sp.), 21-A (Fusarium sp.), 22-C (Fusarium sp.), and 39-D (Acrocalymma sp.) and salinity level (0, 100, 200, and 300 mM NaCl) as well as Cont. (Control) on total soluble protein) TSP ((a), superoxide dismutase (SOD) (b), and ascorbate peroxidase (APX) (c) content of 1-year-old Mazafati date seedling leaf (Phoenix dactylifera) 4 months after salt treatment. Values with the same letter are not significantly different (p ≤ 0.05) according to Duncan’s multiple range test (n = 4)

Elements

The increase in salinity level caused a significant increase in sodium and a significant decrease in potassium and phosphorus contents in the aerial parts of the plant. At the highest salinity level, strain 22-C caused the lowest sodium absorption (23% decrease), and the − EF treatment caused the highest sodium absorption in shoot organs (Fig. 6a). There was no significant difference in potassium absorption under zero salinity conditions compared to + EF, but a significant increase in potassium absorption was observed compared to the − EF treatment (Fig. 6b). Strain 22-C and 4-B caused a higher concentration of phosphorus than other treatments. Strain 14-A and the − EF treatment led to the lowest phosphorus absorption (Fig. 6c).

Fig. 6.

Interactions effect of EF including 4-B (Fusarium. solani), 14-A (Alternaria sp.), 21-A (Fusarium sp.), 22-C (Fusarium sp.), and 39-D (Acrocalymma sp.) and salinity levels (0, 100, 200, and 300 mM NaCl) as well as Cont. (Control) on sodium (a), potassium (b), and phosphorus (c) of 1-year-old Mazafati date seedlings leaf (Phoenix dactylifera) 4 months after salt treatment. Values with the same letter are not significantly different (p ≤ 0.05) according to Duncan’s multiple range test (n = 4)

TSS and proline

In all treatment groups, TSS increased significantly as the salinity level increased (Fig. 7a). TSS content was higher in + EF than in − EF at each salinity level. At the highest salinity level, strain 39-D (37.8%) and strain 4-B (39.2%) caused a significant increase compared to the − EF treatment. By increasing the salinity levels, the amount of proline also increased significantly in all treatment groups (Fig. 7b). At each salinity level, the amount of proline in the shoots was higher in + EF than in − EF plants. At the highest salinity level, strain 39-D caused a significant increase in proline compared to the − EF treatment (51.7%). At 100 mM salinity, no statistical difference was observed between the + EF treatment groups in terms of proline content.

Fig. 7.

Interactions effect of EF including 4-B (Fusarium. solani), 14-A (Alternaria sp.), 21-A (Fusarium sp.), 22-C (Fusarium sp.), and 39-D (Acrocalymma sp.) and salinity levels (0, 100, 200, and 300 mM NaCl) as well as Cont. (Control) on, total soluble sugar (TSS) (a), Prolin (b), and malondialdehyde (MDA) (c) content of 1-year-old Mazafati date seedlings leaf (Phoenix dactylifera) 4 months after salt treatment. Values with the same letter are not significantly different (p ≤ 0.05) according to Duncan’s multiple range test (n = 4)

MDA

The maximum amount of MDA occurred in the control (14.17 µmol g⁻¹ fresh weight) by the effect of 300 mM salinity. This amount of MDA was higher than the amounts observed in treatment groups 14-A, 22-C, 4-B, 21-A, and 39-D at the same salinity level which, respectively, caused increases in MDA levels by 15.5, 29.2, 31.1, 37.6, and 44.3% (Fig. 7c).

Colonization

Root colonization was not observed in − EF plants. With the increase in salinity level, the percentage of root colonization was increased significantly, so that at 200 mM NaCl, 17% more colonization was recorded in comparison with control. The highest colonization percentage was observed in the 39-D treatment at 200 mM (59%) and 300 mM (57%) salinity levels (Fig. 8).

Fig. 8.

Interactions effect of EF including 4-B (Fusarium. solani), 14-A (Alternaria sp.), 21-A (Fusarium sp.), 22-C (Fusarium sp.), and 39-D (Acrocalymma sp.) and salinity levels (0, 100, 200, and 300 mM NaCl) as well as Cont. (Control) on colonization percentage of 1-year-old Mazafati date seedlings root (Phoenix dactylifera) 4 months after salt treatment commencement. Values with the same letter are not significantly different (p ≤ 0.05) according to Duncan’s multiple range test (n = 4)

At the isolation stage, a high degree of diversity was observed in EF isolates extracted from date trees of different locations (Fig. 9). Figure 10 indicates colonized roots by some strains in this experiment.

Fig. 9.

Microscopic observation of EF within roots of Phoenix dactylifera

Fig. 10.

Colonization of EF strains in the roots of one-year-old Mazafati date seedlings) Phoenix dactylifera (4 months after salt treatments commencement. A–E Roots inoculated with 21-A, 22-C, 4-B (Fusarium sp.), 14-A (Alternaria sp.), and 39-D (Acrocalymma sp.) respectively

Discussion

EF, residing within the inner tissues of living plants [89], are likely less influenced by environmental fluctuations due to the relatively stable internal plant tissue environments [20]. The composition of endophytic communities is affected by geographic and host variations. Host plant adaptation, influenced by factors such as climate and genetic diversity, plays a crucial role [34]. The host species has the strongest correlation with the composition of endophyte communities, while altitude, soil parameters, or season have relatively minor influences [19]. In our study, we observed the presence of two highly abundant fungal genera, Alternaria and Fusarium, in the roots of date palm trees. Similarly, a study on EF in medicinal plants identified Alternaria and Fusarium as prominent genera [7]. Additional investigations into the endophytic microbiome of date palm roots (Phoenix dactylifera) consistently reported frequent occurrences of the Fusarium genus [54, 83]. In a study examining the composition and diversity of the endophytic community in six stress-tolerant desert plants within a hot desert region, Fusarium emerged as the most frequently encountered genus, being hosted by all six studied plants [9]. According to reports on the identification and classification of Taxus globosa endophytes [70] and seven native tree species in the Atlantic rainforest [19], the Alternaria genus was identified as one of the dominant endophytes.

In the present study, we investigated five strains of EF. Despite Fusarium or Alternaria species being commonly recognized as plant pathogens, research by Bonfim [19] indicates that certain fungal species may induce disease symptoms in specific host plants while remaining neutral or even beneficial to others. Recent studies highlight the ability of some species to establish symbiotic relationships with plants, especially under biotic and abiotic stresses [7, 37, 83]. Results from Attia et al. [12] revealed that EF, Aspergillus flavus, Aspergillus niger, Mucor circinelloides, and Pencillium oxalicum significantly reduced Fusarium wilt disease in tomato plants. Furthermore, an EF isolate (Fs-K), identified as a Fusarium solani strain, demonstrated the capability to colonize root tissue and protect tomato plants against the root pathogen Fusarium oxysporum [46]. Paparu [62] demonstrated that inoculating non-pathogenic endophytic Fusarium oxysporum into banana (Musa spp.) tissue culture plants provided protection against banana weevils (Cosmopolites sordidus) and nematodes (Radopholus similis). Moreover, Fusarium oxysporum EF119, isolated from healthy tissues of vegetable plants, exhibited potent anti-oomycete activity against the Phytophthora infestans pathogen in tomato plants [48]. In the study of Damankeshan et al. [24], fungal endophyte strains Fusarium solani 4-B and Penicillium expansum 11-C demonstrated no pathogenic effects on the growth of tomato (Lycopersicon esculentum) plants. Alternaria sp. SZMC 23772 displayed high inhibitory activity in antimicrobial tests against selected bacteria [85]. Fungi associated with plants manifest a broad range of lifestyles, being either pathogenic or mutualistic depending on the host. However, the mechanisms governing the shift in their lifestyles remain incompletely understood [39]. In specific geographic locations, a single fungus can cause disease in some plant species while providing beneficial effects, such as disease resistance, protection against drought, and promotion of growth in other plant species. The lifestyle of fungi and the outcome of symbiosis are influenced by the plant, which can be attributed to differences in fungal gene expression in response to the plant or variations in the plant’s ability to respond to the fungus. Even slight genetic variations among cultivars of a single plant species can significantly impact the outcome of fungal/plant symbioses [69]. The lifestyle of fungi and the outcome of symbiosis are influenced by the plant. This can be due to differences in fungal gene expression in response to the plant, or differences in the plant’s ability to respond to the fungus. Even slight genetic variations among cultivars of a single plant species can significantly impact the outcome of fungal/plant symbioses [69]. Hiruma et al. [39] discovered that a cluster of fungal metabolic pathways enables infectious fungi to modulate their lifestyles along the parasitic-mutualistic continuum in response to changing environmental conditions.

Total biomass is usually evaluated as an indicator of a plant’s ability to tolerate salinity. In the current research, the application of fungal endophytes increased the dry weight of roots, shoots, root length, and the number of leaves. Phoenix dactylifera seedlings colonized with Piriformospora indica exhibited elevated plant biomass, root branching, and lateral root density. This improvement in plant growth strongly suggests that P. indica colonization enhances salt stress tolerance in date palms [72]. Additionally, a thermophilic endophytic (CpE) fungus isolated from the heat-adapted delile (Cullen plicata) mitigated the adverse effects of heat stress on cucumber plants, notably by increasing root length compared to untreated plants [8]. Inoculating maize plants with Penicillium commune EP-5, isolated from healthy leaves of E. pachyclada, resulted in increased root lengths and improved vegetative growth [47]. Furthermore, EF symbiosis, such as with P. indica, led to greater root volume and biomass in corn plants (Zea mays) under saline conditions [86]. The changes in root architecture observed in EF symbiosis serves as a crucial adaptive trait in desert ecosystems, enabling plants to better penetrate and extract nutrients and soil moisture under limited water conditions [8]. Colonized plants exhibit greater root length and relative water content under stressful conditions, attributed to increased absorption sites for water and nutrients compared to non-colonized plants [41].

In the current study, salinity led to a decrease in the concentration of photosynthetic pigments and the Relative Water Content (RWC) of leaves. Salinity typically induces a reduction in D1 and D2 proteins in the photosystem II reaction center, crucial for protein phosphorylation and electron flow [44]. The activity of enzymes synthesizing pigments diminishes under salinity [23], leading to the destruction of chloroplasts [90]. Additionally, salinity reduces water availability in plants, causing water stress as salt immobilizes water, making it inaccessible for plant roots [30]. This condition also results in the closure of stomatal openings, impeding CO₂ absorption and disrupting photosynthetic activities [79]. The decrease in chlorophyll content may be attributed to low mineral absorption, especially magnesium, in saline conditions [32]. In contrast, the present study indicates that the symbiosis of date seedlings with fungal strains improved the RWC of leaves (except 14-A) and the concentration of photosynthetic pigments in both saline and non-saline conditions. Despite the decline in pigment concentration and water absorption with increasing salinity, the symbiosis with fungal strains demonstrated a reduction in the adverse effects of salinity. The net chlorophyll content of Phoenix dactylifera seedlings showed a significant increase in plants inoculated with P. indica compared to non-inoculated plants [72]. Similarly, the inoculation of tomato plants with A.niger, M. circinelloides, A. flavus, and P. oxalicum resulted in an improvement in photosynthetic pigment levels [12]. Proline accumulation in inoculated plants may play a role in protecting pigment-protein complexes and enhancing photosynthetic parameters [5, 76]. Fungal endophytes (EF) have the potential to increase the water potential of leaves by promoting root growth and enhancing water absorption in saline conditions. This, in turn, prevents stomatal closure, increases CO₂ absorption, and enhances photosynthetic capacity. The main objective of the study was to evaluate the levels of photosynthetic pigments as an indication of the photosynthetic process. However, to better understand the role of this symbiosis in photosynthesis, it is essential to measuring the photosynthesis rate and other related parameters directly.

In our study, as salinity levels increased, leaf total soluble proteins (TSP) decreased, and the levels of superoxide dismutase (SOD), catalase (CAT), and ascorbate peroxidase (APX) enzymes increased in the + EF treatments compared to the control. Salt stress typically elevates reactive oxygen species (ROS) in plants, leading to oxidative stress. SOD, an initial defense enzyme, converts superoxide radicals into hydrogen peroxide, which is further detoxified into water molecules by enzymes like CAT, APX, and POD [57]. Fungal endophytes (EF) demonstrated an improvement in antioxidant systems in date palms [72] and cucumbers [8]. Similar findings were reported by other researchers under conditions of salinity stress [5] or prolonged drought [15] in fungal symbiosis with date palms. Inoculating blueberry seedlings with T010 (EF) increased the antioxidant activities of blueberry roots. Studies indicated that VabZIP12, acting as a transcription activator, could bind both G-Box 1 and G-Box 2 motifs. These results suggest that the induction of VabZIP12 contributes to enhancing the salt stress tolerance of blueberries through T010 inoculation [67].

In our current research, sodium absorption increased with the rising salinity levels in all treatments, but the rate of increase was significantly lower in the + EF treatment compared to the − EF treatment. Concurrently, potassium and phosphorus absorptions were higher in the + EF treatment group. EF played a crucial role in reducing sodium absorption by plants while promoting increased potassium and phosphorus absorption. The accumulation of salt induces competition in nutrient absorption and transfer, leading to an imbalance in the ionic composition of plants and impacting their physiological characteristics. Fungal endophytes enhance gene expression and increase transcript levels in the roots of salt-stressed plants, regulating various cation transporters. By modulating ion accumulation, EF contribute to maintaining ionic homeostasis, limiting the transfer of Na⁺ to leaves, and regulating the proper cytosolic ratio of sodium to potassium. This ultimately leads to improved nutrient absorption and the preservation of ionic homeostasis [35]. Phoenix dactylifera seedlings colonized by P. indica exhibited lower concentrations of Na⁺ ions in their roots and leaves compared to K⁺ ions. This reduced Na⁺/K⁺ ratio under saline conditions in the presence of P. indica indicates its role in enhancing plant salinity stress tolerance [72]. Inoculated maize plants with P. indica under salt stress displayed higher stomatal conductance, shoot K⁺ content, and lower root K⁺ efflux compared to non-inoculated plants [86]. The utilization of A. niger, M. circinelloides, A. flavus, and P. oxalicum resulted in a significant increase in various growth parameters of tomato plants due to their ability to solubilize phosphate from the soil [12]. Similarly, the growth results of soybean plants under salt stress, coupled with the phosphate solubilization ability of Fusarium verticillioides RK01, suggest the favorable effects of exogenous application of F. verticillioides RK01 EF [68].

In our study, proline and total soluble solids (TSS) levels increased with the elevation of salinity levels. Under normal conditions, proline typically comprises less than 5% of amino acids, but under various stress conditions, including salinity, its concentration can rise up to 80% of the total amino acid content [56]. Plants synthesize proline, soluble sugars, glycine betaine, and other osmolytes to maintain osmotic balance at the cellular level [51]. Increasing the levels of these compounds is a crucial strategy employed by plants to tolerate and adapt to stressful conditions [31]. The elevation of proline and TSS levels may represent a mechanism through which plants mitigate the effects of salinity, facilitating osmotic regulation, scavenging free radicals [45], and maintaining cellular balance [66]. In our study, plants colonized with all strains demonstrated higher TSS and proline levels than non-colonized plants under saline conditions. The use of the endophyte Aspergillus fumigatus increased proline and phenol levels in corn plants [1]. While many reports indicate an increase in proline during salinity stress [35], heat stress [8], and colonization with endophytes, there are exceptions. One report highlighted a decrease in proline under salt stress conditions after the colonization of tomato plants with the endophyte Piriformospora indica [2].

In the present study, as salinity levels increased, MDA content increased, indicating oxidative damage resulting from the oxidation of unsaturated fatty acids. This aligns with previous research where increasing salinity led to elevated MDA levels and decreased plant biomass [4]. Notably, + EF plants exhibited lower MDA amounts compared to − EF plants, attributed to the ability of EF to enhance proline, TSS, and antioxidant production, consequently reducing ROS content [1, 50].

Certain strains in the experiment displayed higher colonization rates, demonstrating the efficiency of EF even under salt stress conditions. The study revealed that salinity had no negative impact on root colonization percentage, and in fact, with increasing salinity levels (up to 200 mM sodium chloride), the colonization percentage increased, highlighting the efficacy of EF in salt stress conditions, a tolerance observed by other researchers as well [27].

In conclusion, date palms are commonly cultivated in regions with elevated salinity levels in both soil and water, which can limit plant growth and physiological parameters. This study suggests that the symbiosis between EF strains and date palm tree roots can improve the growth parameters of date seedlings under saline conditions. Specific strains, such as 39-D (Acrocalymma sp.), demonstrated enhanced accumulation of proline, TSS, and antioxidant activity, mitigating the harmful effects of ROS and reducing MDA content, thereby increasing the efficiency of photosynthetic pigments. Strains 4-B, 22-C, and 21-A (Fusarium sp.) contributed to improved essential element absorption, prevented an increase in Na⁺ in aerial plant parts, and increased leaf RWC. Moreover, the percentage of root colonization increased with salinity levels for most strains, reinforcing the beneficial effects of EF symbiosis on date seedlings grown in saline conditions. This finding suggests the potential use of these fungi in palm groves facing saline soil or salty irrigation water.

The datasets generated during and/or analysed during the current study are available from the corresponding author on reasonable request.

Data availability

Data availability are available from the corresponding author upon reasonable request.

Declarations

Conflict of interest

The authors declare no competing interests.