Beneficial root endophyte Piriformospora indica reduces plant sodium uptake and enhances cell hydraulic conductivity and salt tolerance of maize seedlings

Beneficial root endophyte Piriformospora indica reduces plant sodium uptake and enhances cell hydraulic conductivity and salt tolerance of maize seedlings | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Research Article Beneficial root endophyte Piriformospora indica reduces plant sodium uptake and enhances cell hydraulic conductivity and salt tolerance of maize seedlings Seong Hee Lee, Yi Wang, Janusz Zwiazek This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-7095632/v1 This work is licensed under a CC BY 4.0 License Status: Posted Version 1 posted You are reading this latest preprint version Abstract Background and aims Fungal root endophyte Piriformospora indica has been implicated in enhancing plant tolerance to salt stress. This study aimed to understand the role of root cell hydraulic conductivity in enhancing salt tolerance of maize plants by this fungal endophyte. Methods Maize seedlings inoculated with P. indica were compared with non-inoculated control plants following exposure to 0, 60, and 120 mM NaCl for 24 days and their growth and physiological parameters including root cell hydraulic conductivity examined. Results The shoot dry weights were significantly higher in P. indica- inoculated seedlings compared with non-inoculated plants regardless of the NaCl concentration treatment. Compared to 60 mM NaCl, the 120 mM NaCl treatment further decreased shoot dry weights or shoot to root dry weight ratios in the non-inoculated seedlings, but not in plants inoculated with P. indica . The 120 mM NaCl treatment reduced the root cell hydraulic conductivity, net photosynthetic rates, transpiration rates, and leaf chlorophyll in the non-inoculated plants compared to the inoculated plants. Following the 120 mM Nacl treatment, P. indica- inoculated seedlings had lower root and shoot Na concentrations compared with the non-inoculated seedlings. Both 60 mM and 120 mM NaCl treatments affected the final seed yield less in the inoculated compared with the non-inoculated plants. Conclusions The results demonstrate that the enhancement of salt tolerance in maize plants by P. indica involves reductions in root and shoot Na uptake and maintenance of the transmembrane root water transport which helped alleviate the effects of NaCl on gas exchange and growth. cell hydraulic conductivity growth maize nutrient utilization Piriformospora indica salt yield Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 Figure 7 Introduction Maize is one of the most important cereal crops, but its productivity is threatened by soil salinization which affects many semi-arid and arid areas in the world where maize is cultivated. Maize plants are considered moderately salt-sensitive, and their growth is sharply reduced by the soil salinity levels exceeding 3 dS m − 1 (Cao et al. 2023 ; Vennam et al. 2024 ). Salt tolerance of plants is enhanced in the presence of mycorrhizal fungi as demonstrated for ectomycorrhizal (Muhsin and Zwiazek 2002 ; Lee et al. 2010 ), ericoid mycorrhizal (Fadaei et al. 2020 ) and arbuscular mycorrhizal (Klinsukon et al. 2021 ; Wang et al. 2021 ) associations. Although the processes contributing to this tolerance enhancement are not fully understood, they may include salt exclusion by roots (Muhsin and Zwiazek 2002 ), reduced Na transfer from roots to shoots (Zwiazek et al. 2019 ), Na sequestration into vacuoles (Pehlivan et al. 2016 ; Evelin et al 2019 ; Wang et al. 2023 ), improved K/Na homeostasis (Chen et al. 2017 ), and enhanced aquaporin (AQP)-mediated water transport (Lee et al. 2010 ). Similar explanations have been offered to explain the alleviation of salt stress in plants by the root endophytic fungus Piriformospora indica (Abdelaziz et al. 2017 ; Yun et al. 2018 ; Ghorbani et al. 2019 ). Fungal root endophytes include taxonomically diverse fungi which, unlike mycorrhizal fungi, do not form defined exchange structures (Schulz and Boyle 2005 ; Barberis et al. 2021 ). P. indica establishes the arbuscular-like symbiotic relationship with a wide range of plant species including the members of Brassicaceae family (Waller et al. 2005 ; Abdelazziz et al. 2017; Ghorbani et al. 2019 ) and enhances plant growth. However, its taxonomic classification, structural interactions, and the ability to grow outside of the host plants make it distinct from the arbuscular mycorrhizal fungi (Verma et al. 1998 ; Varma et al. 1999 ). P. indica , also classified as Serendipita indica , belongs to the order Sebacinales within the phylum Basidiomycota (Verma et al. 1998 ; Hibbett et al. 2007 ; Qiang et al. 2012 ). The hyphae of this root endophyte colonize plant roots and form chlamydospores both inside the root tissues and in the external environment (Verma et al. 1998 ; Varma et al. 1999 ). Similarly to mycorrhizal fungi, the hyphae extend from the roots and enlarge the root’s surface area to facilitate the uptake of water and mineral nutrients (Sherameti et al. 2005 ; Ghorbani et al. 2019 ; Tsai et al. 2020 ). Plant growth enhancement was found to occur even before P. indica hyphae extended outside of the root system (Peškan-Berghöfer et al. 2004 ). In Arabidopsis seedlings, inoculation with P. indica resulted in larger, more abundant leaves, faster growth, and earlier flowering (Peškan-Berghöfer et al. 2004 ). Since the function of AQPs in glycophytes is sensitive to Na (Lee and Zwiazek 2015 ; Vaziriyeganeh et al. 2022 , 2023 ), some of the early effects of NaCl on plants include a sharp decrease of cell hydraulic conductivity in roots (L pc ), which increases root hydraulic resistance that triggers stomatal closure (Lee and Zwiazek 2015 ; Vaziriyeganeh et al. 2018 ). High L pc is a fundamental requirement for plant growth and development since efficient water delivery from roots to leaves helps maintain water balance and enhances gas exchange through the stomatal opening (Lee and Zwiazek 2015 ). Increasing gene expression of major water-transporting AQPs is an effective mechanism of alleviating the effects of Na on root water transport (Lee and Zwiazek 2015 ), especially in halophytic plants in which some of AQPs have modified protein structure that makes them insensitive to Na (Vaziriyeganeh et al. 2023 ). Enhanced gene expression of AQPs has been reported in the roots of ectomycorrhizal (Xu et al. 2015 ) and arbuscular mycorrhizal (Porcel et al. 2006 ; Li et al. 2013 ; Ni et al. 2024 ) plants, as well as for plant roots colonized with P. indica (Ghorbani et al. 2019 ). Improved plant salt tolerance by P. indica has been also attributed to the increased expression of NHXs, SOS1 and CNGC15 genes as well as the genes encoding the high affinity K transporter 1 and the inward-rectifying K channels KAT1 and KAT2, resulting in a lower Na/K ratio and shoot Na concentration (Abdelaziz et al. 2017 ; Yun et al. 2018 ; Ghorbani et al. 2019 ). The function of AQPs, which control cell-to-cell water transport, is also affected by nutrient balance (Wang et al. 2016 ). A well-documented effect of P. indica is its enhancement of nutrient uptake by plants, including nitrogen (N) and phosphorus (P) (Gill et al. 2016 ; Su et al. 2017 ; Saddique et al. 2018 ). In addition to the improved access to N and P outside of the depletion zone (Sherameti et al. 2005 ; Ngwene et al. 2016 ), an increased acquisition of N and P in Brassica napus by P. indica was attributed to the increased activity of key metabolic enzymes including N-acetyl-gamma-glutamyl-phosphate reductase, aminoacylase, and adenylosuccinate synthetase (Shrivastava et al. 2018 ). P. indica also improved K, Ca, Mg, and Zn uptake by plants exposed to salt and osmotic stresses (Su et al. 2017 ; Saddique et al. 2018 ; Ghorbani et al. 2019 ). In this study, we compared the responses to NaCl of maize seedling inoculated with the root fungal endophyte P. indica with non-inoculated plants. The main objectives of the study were to determine: 1) the effects of P. indica on salt tolerance of maize, and 2) the contributions of root cell water transport and ion homeostasis to the salt tolerance processes. We examined the hypothesis that the enhancement of salt tolerance in maize by P. indica would involve a reduction in root Na uptake and enhancement of L pc , which would result in lessening the effects of salt on gas exchange, nutrient uptake and, ultimately, plant growth. Materials and methods Plant material, growth conditions, fungal inoculation, and NaCl treatment Sweet corn ( Zea mays , Honey select) was surface sterilized for 20 minutes with 1% sodium hypochlorite containing 0.01% Tween 20 followed by two rinses with distilled water. The seeds were then sterilized again for 5 minutes with 70% ethanol for 5 minutes and rinsed six times with sterilized distilled water. The seeds were germinated in the Petri dish, and the germinants transferred after 3 days to 3 L sterilized pots (one seedling per pot) filled with the autoclaved potting mix (Sunshine Professional Growing LA4 mix, Sun Gro Horticulture, Saba Beach, AB, Canada). The pots were placed on the bench in the growth room at 23/18 o C (day/night) temperature, 60 ± 10% relative humidity, and 16-h photoperiod with 400 µmol m − 2 s − 1 photosynthetic photon flux density (PPFD) at the top of the seedlings provided by the full-spectrum fluorescent bulbs (Philips high output, Markham, ON, Canada). Piriformospora indica was cultured in a modified Melin-Norkans (MMN) liquid medium for 4 weeks at 23 o C (Sherameti et al. 2005 ). For inoculation (Bertolazi et al. 2019 ), the mycelium was filtered through the cheesecloth to remove the excess medium and washed three times with distilled water. After the washing step, the mycelium was collected by centrifuging at 4000 g for 7 min. The pellet was suspended in distilled water and 20 mL of the blended mycelium (1% w/v) was injected with a syringe into the soil near each seedling. The same amount of the autoclaved P. indica fungal inoculum was used as an inoculation control. Ten days after the inoculation, inoculated and non-inoculated seedlings were divided into three groups and treated with 0 (NaCl control), 60, and 120 mM NaCl. NaCl treatments were carried out by immersing the 2–3 cm bottoms of the pots in the NaCl solutions (water for NaCl control) for one day and then draining the pots on the bench for the next 2 day, for the total of 24 days. For the seed yield measurements, the plants were allowed to grow after the 24 days of treatments for another 70 days before being harvested. During that time, the plants were irrigated daily and provided weekly with modified 100% Hoagland’s solution (Epstein 1972 ). Root fungal colonization Roots of six non-inoculated seedlings and seedlings inoculated with P. indica were randomly selected to determine fungal colonization after 24 days of NaCl treatments. After removal of the growth medium, roots were washed with tap water. Distal, 50-mm long, root segments were excised from each of the six seedlings per treatment and preserved in FAA (formalin: acetic acid: alcohol, 5%: 5%: 90%). For microscopic observations, root samples were rinsed several times with distilled water and cleared with 10% KOH for 30 min at 70 o C. The cleared root samples were then rinsed and then stained with 5% black ink-vinegar solution for 1 h at room temperature (Vierheilig et al. 1998 ). The stained roots were rinsed with distilled water and mounted in 50% polyvinyl-lacto-glycerol for viewing with a microscope (Zeiss, Göttingen, Germany). Plant growth and seed yield After measuring stem heights and diameters, roots and shoots of plants treated with NaCl for 24 days were excised and dried in an oven at 70 o C for 3 days to determine dry weights (DW) (n = 7). Seed yield was determined in plants treated with NaCl for 24 days and allowed to grow in the absence of NaCl for additional 70 days before the harvest. The harvested maize ears were air dried, and the seeds were extracted to determine their weights and numbers per plant (n = 6). Measurements of cell hydraulic conductivity Distal root segments (about 50 mm long) were fixed using a small magnetic bar on a metal sledge which was covered with a paper tissue (Lee and Zwiazek 2015 ). A gentle stream of 100% Hoagland’s solution with 120 mM NaCl (for plants treated for 24 days with 120 mM NaCl) or 100% Hoagland’s solution without NaCl (for control plants grown in 100% Hoagland’s solution) was flowed along the roots during the measurements. A single root cortical cell was punctured at about 25- to 30-mm above the root tip with a silicon oil-filled microcapillary (10 µm tip diameter). After a meniscus was formed between cell sap and oil, cell turgor (P) was rebuilt by gently pushing the meniscus to a position close to the root surface. Once P became steady, half-times of water exchange (T 1/2 ) and cell elastic modulus were determined (Lee and Zwiazek 2015 ). The dimensions of the root cells obtained from cross and longitudinal sections were used to determine the cell volume and cell surface area and for the calculation of cell hydraulic conductivity (L p ) as earlier described (Lee and Zwiazek 2015 ). Six inoculated and non-inoculated plants from the control and NaCl treatment groups were used for the measurements (n = 6). Tissue elemental analysis Roots and leaves of plants treated for 24 days with NaCl were freeze-dried for 48 h and then pulverized using the Wiley Mill. The samples containing 50 mg of ground tissue were extracted using the sulfuric acid-hydrogen peroxide wet digestion, and Na content was determined with an atomic absorption spectrophotometer (Spectra AA880; Varian, Inc., Mississauga, ON, Canada). Chloride (Cl) concentrations were measured in roots and shoots after hot water extraction, and then the extracts were analyzed using a DI 300 ion chromatograph (Dionex; Sunnyvale, CA). Other tissue elements, including K, P, Ca, Mg, Fe, B, Mn, and Zn, were extracted with 5 ml 70% HNO 3 and diluted with Milli-Q water to 20 ml. The extracts were then filtered and analyzed by inductively coupled plasma-optical emission spectrometry (iCap 6000, Thermo Fisher Scientific Inc, Waltham, MA, USA) (Zarcinas et al. 1987 ). Total N was determined by the dry combustion method with the Thermo Flash 2000 Organic Elemental Analyzer (Thermo Fisher Scientific Inc., Bremen, Germany) (Tabatabai and Bremner 1991 ). Gas exchange and leaf chlorophyll concentrations After 24 days of NaCl treatments, seven maize plants (n = 7) were randomly taken form each treatment for the measurements of net photosynthesis (P n ) and transpiration (E) rates. Fully expanded leaves were inserted in the leaf chamber of an infrared gas analyzer (LI- 6400, LI-COR, Lincoln, NE, USA) and measured as previously described (Xu et al. 2015 ). The reference CO 2 concentration was 400 µmol and the PPFD was set to 400 µmol m − 2 s − 1 in the leaf chamber. The measurements were conducted between 9:00–12:00 h. To determine the chlorophyll (Chl a + b) concentrations, fully expanded leaves from seven plants per treatment were freeze-dried for 48 h and pulverized using the Wiley Mill (n = 7). Chlorophyll was extracted with dimethyl sulfoxide (DMSO) and the absorbance of the filtered DMSO extracts was measured with a spectrophotometer (Genesys 10 S-UV0VIS, Thermo Fisher Scientific Inc., NJ, USA) at 648 and 665 nm, respectively (Barnes et al. 1992 ). Experimental design and statistical analysis The experiment was a 2×3 complete randomized factorial design with two fungal treatments (F; P. indica -inoculated and non-inoculated) and three NaCl levels (S; 0, 60, 120 mM NaCl). The data were analyzed by ANOVA followed by the Fisher’s least significant differences (LSD) comparison test. The two-way ANOVA p -values used to evaluate the effects of fungal inoculation (F), NaCl treatment (S), and fungal inoculation × NaCl treatment (F × S) interaction are shown in Table 1 , Table 2 and Table 3 . Statistical analyses were carried out using the SigmaPlot version 11.0 (Systat Software Inc, California, USA) at p ≤ 0.05 level. Results Root colonization and plant growth After 24 days of treatments, fungal hyphae and spores were observed in all examined roots of plants inoculated with P. indica , and none were present in the non-inoculated plants in both the control and NaCl treatments (Fig. 1 d, e). At the end of the treatments, maize seedlings inoculated with P. indica were taller and had more leaves compared with the non-inoculated plants (Fig. 1 a, b). Most of the leaves in the inoculated plants remained green even in the highest, 120 mM NaCl treatment which caused leaf chlorosis in the non-inoculated plants (Fig. 1 c). Shoot DW, shoot to root DW ratios, stem heights and stem diameters, but not root DW, were significantly higher in P. indica- inoculated seedlings in all NaCl treatments compared with those in non-inoculated seedlings (Figs. 1 a, b, Fig. 2 a-e). There were no differences in root DW, shoot DW, shoot to root DW ratios, stem heights and stem diameter between 0 and 60 mM NaCl in non-inoculated and P. indica -inoculated seedlings, respectively (Fig. 2 a-e). Compared to 0 mM NaCl, the120 mM NaCl treatment significantly decreased root and shoot DW and stem heights in inoculated seedlings, and reduced shoot DW, the shoot to root ratios and stem heights in non-inoculated seedlings (Fig. 2 a-d). There were no significant differences in shoot DW or shoot/root ratios between 60 and 120 mM NaCl in P. indica -inoculated seedlings (Fig. 2 b, c). However, the 120 mM NaCl treatment further decreased shoot DW, shoot/root ratios and stem heights in non-inoculated seedlings compared to the 60 mM NaCl treatment (Fig. 2 b-d). A relatively small, but statistically significant, increase in stem diameters was observed as a result of inoculation with P. indica in all NaCl treatments (Fig. 2 e). However, no significant F × S interaction was observed for root DW, shoot DW, stem height, or stem diameter (Table 1 ). Table 1 ANOVA p -values for the main factors and their interactions Variance Root DW Shoot DW Stem height Stem diameter Shoot/Root L pc Root Na Leaf Na Root Cl Leaf Cl F 0.036 ˂0.001 ˂0.001 0.257 ˂0.001 0.313 0.057 0.008 0.108 0.038 S 0.003 ˂0.001 ˂0.001 0.001 0.035 0.035 ˂0.001 ˂0.001 ˂0.001 ˂0.001 F × S 0.249 0.411 0.802 0.057 0.017 0.034 0.032 0.005 0.422 0.078 The main factors are fungal inoculation (F; non-inoculated, inoculated with P. indica ) and NaCl (S; 0, 60 120 mM). The measured parameters include root DW, shoot DW, stem height, stem diameter, shoot/root ratio, L pc, Root Na and Cl and Leaf Na and Cl. Half-times of water exchange (T 1/2 ) and cell hydraulic conductivity (L pc ) The T 1/2 values in root cortical cells varied from 2.3 to 2.8 s in the non-inoculated seedlings and between 2.9 and 4.2 s in the inoculated plants grown in the 0 mM NaCl for 24 days (Fig. 3 a, b). In the non-inoculated plants treated with 120 mM NaCl for 24 days, T 1/2 was higher by 4-fold and L pc lower by 3-4-fold compared with control plants (0 mM NaCl) (Fig. 3 c, e). However, in the inoculated seedlings the T 1/2 and L pc values were similar in NaCl treated and untreated plants (Fig. 3 d, e). A significant F × S interaction was observed for L pc ( p = 0.034) (Table 1 ). Tissue elemental concentrations The 120 mM NaCl treatment significantly increased the Na concentrations in roots and leaves of both non-inoculated and inoculated seedlings, but the increase was of a lower magnitude in seedlings inoculated with P. indica (Fig. 4 a). The concentrations of Cl in roots and leaves were elevated by a similar extent in both groups of plants treated with 120 mM NaCl (Fig. 4 b). A significant F × S interaction was observed for Na in root and leaf ( p ≤ 0.05), but not for Cl (Table 1 ). The non-inoculated seedlings had higher root and leaf concentrations of N and P compared to P. indica -inoculated seedlings (Fig. 5 a, b). The 120 mM NaCl treatment significantly decreased the N and P concentrations in roots and leaves of the non-inoculated seedlings but had little effect on the N and P concentrations in the inoculated plants (Fig. 5 a, b, Table 2 ). The root K concentrations were higher in the non-inoculated compared with inoculated plants (Fig. 5 c). The 120 mM NaCl treatment significantly declined the root K concentrations while the leaf K, Ca, and Mg concentrations as well as root Mg concentration remained unchanged in both inoculated and non-inoculated seedlings (Fig. 5 c-e, Table 2 ). A significant F × S interaction was observed for N, P, K and Ca in root, and for P in leaf ( p ≤ 0.05) (Table 2 ) There were little differences in the leaf and root Fe concentrations between the non-inoculated and inoculated seedlings, regardless of NaCl concentraion (Fig. 5 f). Root B concentrations were significantly lower in the inoculated compared to non-inoculated plants (Fig. 5 g, Table 2 ). The 120 mM NaCl treatment did not affect B concentrations in roots and leaves and had no effect on Zn concentration in the leaves of both inoculation groups (Fig. 5 g, j, Table 2 ). The root and leaf Mn concentrations were increased by the 120 mM NaCl tratement in both non-inoculation and inoculation groups (Fig. 5 h). The non-inoculated seedlings had higher leaf concentrations of Cu compared to P. indica -inoculated seedlings (Fig. 5 i). The 120 mM NaCl treatment significantly decreased the Cu concentrations in leaves of non-inoculated seedlings, but root Cu concentration remained unchanged in both seedlings regardless of NaCl concentration (Fig. 5 i, Table 2 ). A significant F × S interaction was observed for Cu and Zn in roots ( p ≤ 0.05) (Table 2 ). The 120 mM NaCl treatment significantly decreased root Zn concentrations in non-inoculated seedlings but had no effect on root Zn concentrations in the inoculated plants (Fig. 5 j, Table 2 ). Table 2 ANOVA p -values for the main factors and their interactions Variance N P K Ca Mg Fe B Mn Cu Zn Root F 0.004 0.006 0.010 0.359 0.160 0.642 ˂0.001 0.539 0.383 ˂0.001 S ˂0.001 0.001 ˂0.001 0.014 0.057 0.725 0.140 0.007 0.789 0.001 F × S 0.002 0.031 0.007 0.028 0.526 0.121 0.469 0.260 0.012 ˂0.001 Leaf F 0.023 ˂0.001 0.388 0.143 0.198 0.783 0.451 0.510 0.034 0.329 S 0.005 0.019 0.047 0.681 0.887 0.012 0.025 0.001 0.038 0.594 F × S 0.137 0.007 0.200 0.488 0.595 0.384 0.366 0.242 0.147 0.178 The main factors are fungal inoculation (F; non-inoculated, inoculated with P. indica ) and NaCl (S; 0, 60 120 mM). The measured parameters include root and leaf N, P, K, Ca, Mg, Fe, B, Mn, Cu and Zn. Net photosynthesis rates (P n ), transpiration rates (E), and leaf chlorophyll (Chl) concentrations The 60 mM NaCl treatment did not affect P n and E in the non-inoculated and inoculated seedlings (Fig. 7 a, b). The 120 mM NaCl treatment did not affect both parameters in the inoculated plants but reduced both P n and E in the non-inoculated plants (Fig. 6 a, b). Chl concentrations did not significantly vary between the inoculated and non-inoculated plants subjected to 0 and 60 mM NaCl treatments (Fig. 6 c). However, the 120 mM NaCl treatment resulted in a sharp decrease in the non-inoculated plants while having little effect in plants inoculated with P. indica (Fig. 6 c, Table 3 ). Table 3 ANOVA p -values for the main factors and their interactions Variance P n E Chl Seed number Seed weight F 0.613 0.149 0.003 0.010 0.017 S 0.020 0.007 ˂0.001 ˂0.001 ˂0.001 F × S 0.106 0.192 ˂0.001 0.145 0.116 The main factors are fungal inoculation (F; non-inoculated, inoculated with P. indica ) and NaCl (S; 0, 60 120 mM). The measured parameters include P n, E, Chl, seed number and seed weight. Seed weight and seed number Plant seed weights and seed numbers in control (0 mM NaCl) and both NaCl treatments, were significantly greater in the inoculated compared with non-inoculated plants (Fig. 7 a, b, Table 3 ). Both 60 mM and 120 mM NaCl inhibited seed development and maturation in the non-inoculated plants and, therefore, the seed number and seed weight could not be determined for the non-inoculated seedlings treated with 60 and 120 mM NaCl since no mature seeds were produced in these treatments during the duration of the experiment (Fig. 7 a, b). Discussion Our study sheds new light on the processes of enhanced salt tolerance conferred to maize plants by Piriformospora indica . At the end of the 24 days of treatments, plants inoculated with P. indica were taller, produced more leaves, and developed cobs earlier compared with the non-inoculated plants. Also, their shoot dry weights were significantly higher in the control (0 mM NaCl) and in both NaCl treatments. Enhanced salt tolerance of plants has been documented for several types of mycorrhizal associations including the ectomycorrhizal (Muhsin and Zwiazek 2002 ; Calvo-Polanco et al. 2008 ; Lee et al. 2010 ; Xu et al. 2015 ), arbuscular mycorrhizal (Chen et al. 2017 ; Evelin et al. 2019 ; Klinsukon et al. 2021 ), and ericoid mycorrhizal (Fadaei et al. 2020 ) associations as well as in plants colonized by root fungal endophytes (Abdelaziz et al. 2017 ; Yun et al. 2018 ; Ghorbani et al. 2019 ). However, the processes involved in this enhancement remain poorly understood. Our results indicate that the inoculation of maize roots with P. indica , affected cell water relations and nutrient uptake of plants exposed to NaCl. Maintenance of plant water transport and water balance plays an important role in salt tolerance of plants. Interestingly, in our study, 120 mM NaCl had no effect on L pc in plants inoculated with P. indica , but in 0 mM NaCl (NaCl control), L pc values were similar in the inoculated and non-inoculated plants. These results indicate that the effect of the fungus on L pc was its protection against NaCl, which could have been due to different indirect or direct factors influencing L pc under salt conditions. Since cell-to-cell water transport is regulated by AQPs, the effect of fungal inoculation on L pc was likely due to its effects on the activity and/or expression levels of root AQPs as previously reported for the ectomycorrhizal (Lee et al. 2010 ; Xu et al. 2015 ) and arbuscular mycorrhizal (Bitterlich et al. 2018 ; Pauwels et al. 2023 ) associations. Even low concentrations of Na strongly inhibit the AQP-mediated water transport in salt-sensitive glycophytic plants (Lee and Zwiazek 2015 ). However, depending on their tolerance level, salt-tolerant grasses can either maintain or increase L pc when exposed to 100 mM NaCl, and this effect was attributed to the overexpression of some of the plasma membrane AQPs (Vaziriyeganeh et al. 2018 , 2021 , 2022 ). It was also found that both the gene expression and water transport through PnuPIP2;2, one of the major water-conducting AQPs in the halophytic grass Puccinellia nuttalliana , were enhanced by NaCl (Vaziriyeganeh et al. 2023 ). Maintenance of the AQP-mediated transport has been proposed to play a major role in the enhancement of plant salt tolerance by the ectomycorrhizal (Lee et al. 2010 ; Xu et al. 2015 ) and arbuscular mycorrhizal (Liang et al. 2022 ; Asadollahi et al. 2023 ). Therefore, our results strengthen the notion that cell water transport is an important factor contributing to salt tolerance enhancement by beneficial root fungi. In addition to its effect on water transport, root inoculation with P. indica reduced Na accumulation in roots and leaves of plants exposed to NaCl. Reduced root Na uptake and its transfer to shoots has been well documented in ectomycorrhizal and arbuscular mycorrhizal plants (Smith and Read 2008 ; Lehto and Zwiazek 2011 ). Therefore, it appears that this may be a common response for plants with the roots colonized by beneficial fungi. Although the processes involved in this response are still largely unknown, it has been demonstrated for ectomycorrhizal plants that most of the Na in plants treated with NaCl remained either in the extraradical hyphae or in the root cortex reducing its transfer to the photosynthetic tissues (Muhsin and Zwiazek 2002 ; Bandou et al. 2006 ; Guerrero-Galán et al. 2019 ). In contrast, arbuscular mycorrhizal fungi primarily enhance plant tolerance to salinity by improving nutrient uptake and maintaining favorable ion balances, such as increased K/Na ratios, rather than by sequestering Na within their structures (Pooja et al. 2024 ). Also, arbuscular mycorrhizal inoculation has been shown to lower Na concentrations in roots, enhance stomatal conductance, and increase net photosynthetic rates (Zong et al. 2023 ). While direct evidence for Na sequestration within P. indica hyphae or root cells is limited, its colonization of plant roots has been commonly associated with improved ion regulation and reduced Na accumulation in shoots, suggesting the presence of mechanisms that mitigate sodium stress (Abdelaziz et al. 2017 ; Yun et al. 2018 ; Ghorbani et al. 2019 ). Inoculation with P. indica significantly enhanced maize growth and yield as a results of enhanced salt tolerance. Despite the increases in growth parameters in all treatments, plant inoculation with P. indica resulted in higher E and P n in plants subjected to 120 mM NaCl but had little effect on E and P n in control plants or in the plants treated with 60 mM NaCl little effect on E and P n in control plants. Therefore, our results suggest that in the absence of NaCl, P. indica enhanced plant growth through other mechanisms than cell water transport or gas exchange, potentially involving improved nutrient acquisition, or altered hormonal regulation (Smith and Read 2008 ; Contreras-Cornejo et al. 2009 ). In our study, roots of maize plants colonized by P. indica tended to develop more root aerenchyma (data not shown) under optimal conditions compared with the non-inoculated plants, suggesting that root cortical aerenchyma enhances nutrient acquisition (Saengwilai et al. 2014 ). Interestingly, in the present study, N and P concentrations in both roots and leaves were lower in the P. indica -inoculated plants compared to non-inoculated seedlings in both NaCl-treated and untreated plants. Likewise, root B and Zn concentrations were significantly lower in inoculated seedlings. However, an increase of nutrient elements by P. indica -inoculated in Brassica napus (Su et al. 2017 ) and in tomato seedling (Ghorbani et al. 2019 ) have also been reported. These large discrepancies remain unclear, but the decreased nutrient concentrations relative to the rapid growth of maize seedlings may be attributed to the nutrient dilution effect caused by increased growth (Kaspari and Welti 2024 ). This phenomenon has been often reported for plants due to environmental factors which lead to an enhanced synthesis of carbohydrates resulting in increased growth and, consequently, the dilution of nutrient concentrations (Jarrell and Beverly 1981 ; Kaspari and Welti 2024 ). Our study demonstrated that P. indica enhanced the L pc in seedlings subjected to NaCl. Since L pc is regulated by the AQPs (Lee et al. 2010 , 2015; Xu et al. 2015 ), and the AQP function is affected by the mineral nutrition (Carvajal et al. 1996 ; Wang et al. 2001 ), it is plausible that the effects of P. indica on L pc could involve its effects on nutrient uptake. However, a possible link between the mineral nutrition and root water transport in plants colonized by the mycorrhizal and endophytic fungi has not been clearly established. In conclusion, our study demonstrated that the enhancement of salt tolerance in maize plants by P. indica involves reductions in root and shoot Na uptake and maintenance of the transmembrane root water transport which helped alleviate the effects of NaCl on gas exchange, growth, and seed yield. Possible links between the mineral nutrition and AQP-mediated transport in roots of plants colonized by endophytic fungi should be further explored. Declarations Funding Funding for this study was provided by the Natural Sciences and Engineering Discover Research Grant to JJZ. We also gratefully acknowledge financial support from National Natural Science Foundation Project (32360379), Guizhou Provincial Basic Research Program (Natural Science) (qjhe-MS[2025]211), and China Scholarship Council for the scholarship awarded to YW. Competing interests The authors have no conflicts of interest to declare. Author’s contributions SL and JZ designed the study, SL and YW conducted the experiments, all authors contributed to writing and editing the manuscript. All authors approved the submitted version of the manuscript. Acknowledgements We thank Dr. Wenqing Zhang, University of Alberta, Edmonton, Canada, for assisting with tissue preparation for element analysis. Data availability The data that support the finding of this study are available from the corresponding author upon reasonable request. 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Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-7095632","acceptedTermsAndConditions":true,"allowDirectSubmit":true,"archivedVersions":[],"articleType":"Research Article","associatedPublications":[],"authors":[{"id":486582642,"identity":"c7b8e665-1a1f-437c-9e24-200d49f1551c","order_by":0,"name":"Seong Hee Lee","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAAA1ElEQVRIiWNgGAWjYNCCAhsGBnYekrQYpDEwMIO0JBCv5TAJWuTbm489/GJwPrG/mffg48ofdgz87QcImH/mWLqxjMHtxBmH+ZINzyQkM0icIWCVgUSOmbQEUEvDYR4zyYYEZgYDQq6Tn/8GpOVc4vzDPOY/GxLqGQz4HxDwzA2g4R8MDiRuANrC2JBwGGgvIYedSUuTZjBINt4I9ItkQ9pxHokbBGyRbz98TPJHhZ3svOO9Bz822FTL8fcTsAUEmJHjnbg0wPiDKGWjYBSMglEwYgEA5GI/sgE6wqAAAAAASUVORK5CYII=","orcid":"","institution":"University of Alberta","correspondingAuthor":true,"prefix":"","firstName":"Seong","middleName":"Hee","lastName":"Lee","suffix":""},{"id":486582643,"identity":"74f8ce49-94c5-4115-b2af-132be866a1fe","order_by":1,"name":"Yi Wang","email":"","orcid":"","institution":"","correspondingAuthor":false,"prefix":"","firstName":"Yi","middleName":"","lastName":"Wang","suffix":""},{"id":486582644,"identity":"41c00831-e776-4abd-9183-7ef0fef5070e","order_by":2,"name":"Janusz Zwiazek","email":"","orcid":"","institution":"","correspondingAuthor":false,"prefix":"","firstName":"Janusz","middleName":"","lastName":"Zwiazek","suffix":""}],"badges":[],"createdAt":"2025-07-10 18:52:36","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-7095632/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-7095632/v1","draftVersion":[],"editorialEvents":[],"editorialNote":"","failedWorkflow":false,"files":[{"id":87187556,"identity":"cfd53eac-ef9d-464e-94f7-c76c26015950","added_by":"auto","created_at":"2025-07-21 10:43:01","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":279792,"visible":true,"origin":"","legend":"\u003cp\u003eEffects of\u003cem\u003e P. indica\u003c/em\u003e on plant growth and leaves colors. The pictures show the growth of non-inoculated (a) and inoculated with \u003cem\u003eP. indica\u003c/em\u003e (b) maize seedlings as well as leaves color (c) after 24 days of 0, 60 and 120 mM NaCl treatments. Roots of non-inoculated (d) and \u003cem\u003eP. indica\u003c/em\u003e-inoculated seedlings (e) treated with 120 mM NaCl were viewed after stain of ink-vinegar using the light-microscope. The arrows and asterisks show chlamydospores and hyphae, respectively. The symbol of - means non-inoculated, of + means inoculated with \u003cem\u003eP. indica\u003c/em\u003e.\u003c/p\u003e","description":"","filename":"1.png","url":"https://assets-eu.researchsquare.com/files/rs-7095632/v1/4dfa04c9f87b59c1df31264b.png"},{"id":87189088,"identity":"552ec26a-1436-43aa-b855-0f31faee62d0","added_by":"auto","created_at":"2025-07-21 10:59:01","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":18213,"visible":true,"origin":"","legend":"\u003cp\u003eEffects of \u003cem\u003eP. indica\u003c/em\u003e on root and shoot dry weight (DW), shoot to root ratio, stem height and stem diameter. The root DW (a), shoot DW (b), shoot to root ratio (c), stem height (d) and stem diameter (e) were measured in the non-inoculated and inoculated with \u003cem\u003eP. indica\u003c/em\u003e maize seedlings after 24 days of 0, 60 and 120 mM NaCl. The symbol - means non-inoculated, + means inoculated with \u003cem\u003eP. indica\u003c/em\u003e. Different letters above the bars indicate significant differences (\u003cem\u003ep \u003c/em\u003e≤ 0.05) between treatments in both seedlings by ANOVA followed by Fisher LSD test. Means ± SE (n = 7 plants) are shown.\u003c/p\u003e","description":"","filename":"2.png","url":"https://assets-eu.researchsquare.com/files/rs-7095632/v1/ecd1798496bc3e7e27edaa44.png"},{"id":87187549,"identity":"1bc240bd-fc2f-4f35-95d9-7580d34ee0bb","added_by":"auto","created_at":"2025-07-21 10:43:01","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":18233,"visible":true,"origin":"","legend":"\u003cp\u003eEffects of \u003cem\u003eP. indica\u003c/em\u003e on half times of water exchange (T\u003csub\u003e1/2\u003c/sub\u003e)and hydraulic conductivity (L\u003csub\u003epc\u003c/sub\u003e) of individual root cortical cells of non-inoculated and inoculated with \u003cem\u003eP. indica\u003c/em\u003e maize seedlings subjected to 0 and 120 mM NaCl for 24 days. T\u003csub\u003e1/2\u003c/sub\u003e measured in the non-inoculated with \u003cem\u003eP. indica\u003c/em\u003e roots treated with\u0026nbsp; 0 mM NaCl (\u003cem\u003e-P. indica\u003c/em\u003e: 0 mM NaCl) (a) ; T\u003csub\u003e1/2\u003c/sub\u003e measured in the inoculated with \u003cem\u003eP. indica\u003c/em\u003e roots treated with 0 mM NaCl (\u003cem\u003e+P. indica\u003c/em\u003e: 0 mM NaCl) (b); T\u003csub\u003e1/2\u003c/sub\u003e measured in the non-inoculated with \u003cem\u003eP. indica\u003c/em\u003e roots treated with 120 mM NaCl\u0026nbsp; (-\u003cem\u003eP.indica\u003c/em\u003e:120 mM) (c); T\u003csub\u003e1/2\u003c/sub\u003e measured in the inoculated with \u003cem\u003eP. indica\u003c/em\u003e roots treated with 120 mM NaCl (+\u003cem\u003eP.indica\u003c/em\u003e:120 mM NaCl) (d). On the right side of the traces (a, b, c, d), quick changes in pressure were imposed to determine cell elasticity. Cell hydraulic conductivity (L\u003csub\u003epc\u003c/sub\u003e) calculated in the non-inoculated and inoculated with \u003cem\u003eP. indica\u003c/em\u003e roots under 0 and 120 mM NaCl (e). The symbol - means non-inoculated, + means inoculated with \u003cem\u003eP. indica\u003c/em\u003e. Different letters above the bars indicate significant differences (\u003cem\u003ep \u003c/em\u003e≤ 0.05) between treatments in both seedlings by ANOVA followed by Fisher LSD test. Means ± SE (n = 6 plants) are shown.\u003c/p\u003e","description":"","filename":"3.png","url":"https://assets-eu.researchsquare.com/files/rs-7095632/v1/3108ec36fa68d08d569f918c.png"},{"id":87187550,"identity":"ac439173-86a8-4c0d-9a85-a15c248162f5","added_by":"auto","created_at":"2025-07-21 10:43:01","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":17048,"visible":true,"origin":"","legend":"\u003cp\u003eEffect of \u003cem\u003eP. indica\u003c/em\u003e on Na and Cl accumulation in the roots and leaves. The concentrations of Na (a) and Cl (b) were analyzed in the non-inoculated and inoculated with \u003cem\u003eP. indica \u003c/em\u003emaize seedlings after 24 days of 0 and 120 mM NaCl treatment. The symbol - means non-inoculated, + means inoculated with \u003cem\u003eP. indica\u003c/em\u003e. Different letters above the bars indicate significant differences (\u003cem\u003ep \u003c/em\u003e≤ 0.05) within treatments for each tissue type as determined by ANOVA followed by Fisher LSD test. Means ± SE (n = 6 plants) are shown.\u003c/p\u003e","description":"","filename":"4.png","url":"https://assets-eu.researchsquare.com/files/rs-7095632/v1/4642bd6ac2d076904ba4e9f1.png"},{"id":87187914,"identity":"28647e09-8763-4242-b4dc-ddd01b8a7b53","added_by":"auto","created_at":"2025-07-21 10:51:01","extension":"png","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":61664,"visible":true,"origin":"","legend":"\u003cp\u003eEffect of \u003cem\u003eP. indica\u003c/em\u003e on concentrations of N (a), P (b), K (c), Ca (d), Mg (e), Fe (f), B (g), Mn (h), Cu (i), and Zn (j) in the roots and leaves. The concentrations of these elements in tissue were analyzed in the non-inoculated and inoculated with \u003cem\u003eP. indica \u003c/em\u003emaize seedlings after 24 days of 0 and 120 mM NaCl. The elements measured in the non-inoculated with \u003cem\u003eP. indica\u003c/em\u003e roots treated with 0 (\u003cem\u003e-P. indica\u003c/em\u003e: 0 mM NaCl) and 120 mM NaCl (\u003cem\u003e-P. indica\u003c/em\u003e: 120 mM NaCl), and in the inoculated with \u003cem\u003eP. indica\u003c/em\u003e roots treated with 0 (\u003cem\u003e+P. indica\u003c/em\u003e: 0 mM NaCl) and 120 mM NaCl (\u003cem\u003e+P. indica\u003c/em\u003e: 120 mM NaCl). The symbol - means non-inoculated, + means inoculated with \u003cem\u003eP. indica\u003c/em\u003e. Different letters above the bars indicate significant differences (\u003cem\u003ep \u003c/em\u003e≤ 0.05) within treatments for each tissue type as determined by ANOVA followed by Fisher LSD test. Means ± SE (n = 6 plants) are shown.\u003c/p\u003e","description":"","filename":"5.png","url":"https://assets-eu.researchsquare.com/files/rs-7095632/v1/dd420528d7bf093716e42284.png"},{"id":87187552,"identity":"3ca231d5-235f-445a-8190-bd582af87836","added_by":"auto","created_at":"2025-07-21 10:43:01","extension":"png","order_by":6,"title":"Figure 6","display":"","copyAsset":false,"role":"figure","size":13267,"visible":true,"origin":"","legend":"\u003cp\u003eEffects of\u003cem\u003e P. indica\u003c/em\u003e on net photosynthesis rate (P\u003csub\u003en\u003c/sub\u003e), transpiration (E) and chlorophyll (Chl). The P\u003csub\u003en\u003c/sub\u003e (a), E (b) and Chl (c) were measured in the non-inoculated and inoculated with \u003cem\u003eP. indica\u003c/em\u003e maize seedlings after 24 days of 0, 60 and 120 mM NaCl treatment. The symbol - means non-inoculated, + means inoculated with \u003cem\u003eP. indica\u003c/em\u003e. Different letters above the bars indicate significant differences (\u003cem\u003ep \u003c/em\u003e≤ 0.05) between treatments in both seedlings by ANOVA followed by Fisher LSD test. Means ± SE (n = 7 plants) are shown.\u003c/p\u003e","description":"","filename":"6.png","url":"https://assets-eu.researchsquare.com/files/rs-7095632/v1/34684d25fbdcb43f49ac7f70.png"},{"id":87187555,"identity":"d5f23428-abec-48de-afc4-77927b602a8f","added_by":"auto","created_at":"2025-07-21 10:43:01","extension":"png","order_by":7,"title":"Figure 7","display":"","copyAsset":false,"role":"figure","size":10789,"visible":true,"origin":"","legend":"\u003cp\u003eEffects of \u003cem\u003eP. indica\u003c/em\u003e on seed number and seed weight. The seed number (a) and seed weight (b) were measured in the non-inoculated and inoculated with \u003cem\u003eP. indica\u003c/em\u003e maize seedlings treated with 0, 60 and 120 mM NaCl for 24 days and then grown to water and nutrients without any NaCl treatments until harvest. The symbol - means non-inoculated, + means inoculated with \u003cem\u003eP. indica\u003c/em\u003e. The n.mindicates that the values were not measurable due to the presence of mostly premature seeds. Different letters above the bars indicate significant differences (\u003cem\u003ep \u003c/em\u003e≤ 0.05) between treatments in both seedlings by ANOVA followed by Fisher LSD test. Means ± SE (n = 6 plants) are shown.\u003c/p\u003e","description":"","filename":"7.png","url":"https://assets-eu.researchsquare.com/files/rs-7095632/v1/61493f88fc45fb6f27e7b748.png"},{"id":91791008,"identity":"36d34ae3-6873-45b9-befb-4b59ca9646fb","added_by":"auto","created_at":"2025-09-21 12:10:00","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":1169502,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-7095632/v1/905c86f1-8941-424e-a519-1acfd17ca392.pdf"}],"financialInterests":"","formattedTitle":"Beneficial root endophyte Piriformospora indica reduces plant sodium uptake and enhances cell hydraulic conductivity and salt tolerance of maize seedlings","fulltext":[{"header":"Introduction","content":"\u003cp\u003eMaize is one of the most important cereal crops, but its productivity is threatened by soil salinization which affects many semi-arid and arid areas in the world where maize is cultivated. Maize plants are considered moderately salt-sensitive, and their growth is sharply reduced by the soil salinity levels exceeding 3 dS m\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e (Cao et al. \u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e2023\u003c/span\u003e; Vennam et al. \u003cspan citationid=\"CR51\" class=\"CitationRef\"\u003e2024\u003c/span\u003e).\u003c/p\u003e\u003cp\u003eSalt tolerance of plants is enhanced in the presence of mycorrhizal fungi as demonstrated for ectomycorrhizal (Muhsin and Zwiazek \u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e2002\u003c/span\u003e; Lee et al. \u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e2010\u003c/span\u003e), ericoid mycorrhizal (Fadaei et al. \u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e2020\u003c/span\u003e) and arbuscular mycorrhizal (Klinsukon et al. \u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e2021\u003c/span\u003e; Wang et al. \u003cspan citationid=\"CR56\" class=\"CitationRef\"\u003e2021\u003c/span\u003e) associations. Although the processes contributing to this tolerance enhancement are not fully understood, they may include salt exclusion by roots (Muhsin and Zwiazek \u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e2002\u003c/span\u003e), reduced Na transfer from roots to shoots (Zwiazek et al. \u003cspan citationid=\"CR63\" class=\"CitationRef\"\u003e2019\u003c/span\u003e), Na sequestration into vacuoles (Pehlivan et al. \u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e2016\u003c/span\u003e; Evelin et al \u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e2019\u003c/span\u003e; Wang et al. \u003cspan citationid=\"CR57\" class=\"CitationRef\"\u003e2023\u003c/span\u003e), improved K/Na homeostasis (Chen et al. \u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e2017\u003c/span\u003e), and enhanced aquaporin (AQP)-mediated water transport (Lee et al. \u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e2010\u003c/span\u003e). Similar explanations have been offered to explain the alleviation of salt stress in plants by the root endophytic fungus \u003cem\u003ePiriformospora indica\u003c/em\u003e (Abdelaziz et al. \u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e2017\u003c/span\u003e; Yun et al. \u003cspan citationid=\"CR60\" class=\"CitationRef\"\u003e2018\u003c/span\u003e; Ghorbani et al. \u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e2019\u003c/span\u003e).\u003c/p\u003e\u003cp\u003eFungal root endophytes include taxonomically diverse fungi which, unlike mycorrhizal fungi, do not form defined exchange structures (Schulz and Boyle \u003cspan citationid=\"CR39\" class=\"CitationRef\"\u003e2005\u003c/span\u003e; Barberis et al. \u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e2021\u003c/span\u003e). \u003cem\u003eP. indica\u003c/em\u003e establishes the arbuscular-like symbiotic relationship with a wide range of plant species including the members of Brassicaceae family (Waller et al. \u003cspan citationid=\"CR54\" class=\"CitationRef\"\u003e2005\u003c/span\u003e; Abdelazziz et al. 2017; Ghorbani et al. \u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e2019\u003c/span\u003e) and enhances plant growth. However, its taxonomic classification, structural interactions, and the ability to grow outside of the host plants make it distinct from the arbuscular mycorrhizal fungi (Verma et al. \u003cspan citationid=\"CR52\" class=\"CitationRef\"\u003e1998\u003c/span\u003e; Varma et al. \u003cspan citationid=\"CR46\" class=\"CitationRef\"\u003e1999\u003c/span\u003e). \u003cem\u003eP. indica\u003c/em\u003e, also classified as \u003cem\u003eSerendipita indica\u003c/em\u003e, belongs to the order \u003cem\u003eSebacinales\u003c/em\u003e within the phylum \u003cem\u003eBasidiomycota\u003c/em\u003e (Verma et al. \u003cspan citationid=\"CR52\" class=\"CitationRef\"\u003e1998\u003c/span\u003e; Hibbett et al. \u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e2007\u003c/span\u003e; Qiang et al. \u003cspan citationid=\"CR36\" class=\"CitationRef\"\u003e2012\u003c/span\u003e). The hyphae of this root endophyte colonize plant roots and form chlamydospores both inside the root tissues and in the external environment (Verma et al. \u003cspan citationid=\"CR52\" class=\"CitationRef\"\u003e1998\u003c/span\u003e; Varma et al. \u003cspan citationid=\"CR46\" class=\"CitationRef\"\u003e1999\u003c/span\u003e). Similarly to mycorrhizal fungi, the hyphae extend from the roots and enlarge the root\u0026rsquo;s surface area to facilitate the uptake of water and mineral nutrients (Sherameti et al. \u003cspan citationid=\"CR40\" class=\"CitationRef\"\u003e2005\u003c/span\u003e; Ghorbani et al. \u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e2019\u003c/span\u003e; Tsai et al. \u003cspan citationid=\"CR45\" class=\"CitationRef\"\u003e2020\u003c/span\u003e). Plant growth enhancement was found to occur even before \u003cem\u003eP. indica\u003c/em\u003e hyphae extended outside of the root system (Peškan-Bergh\u0026ouml;fer et al. \u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e2004\u003c/span\u003e). In \u003cem\u003eArabidopsis\u003c/em\u003e seedlings, inoculation with \u003cem\u003eP. indica\u003c/em\u003e resulted in larger, more abundant leaves, faster growth, and earlier flowering (Peškan-Bergh\u0026ouml;fer et al. \u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e2004\u003c/span\u003e).\u003c/p\u003e\u003cp\u003eSince the function of AQPs in glycophytes is sensitive to Na (Lee and Zwiazek \u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e2015\u003c/span\u003e; Vaziriyeganeh et al. \u003cspan citationid=\"CR47\" class=\"CitationRef\"\u003e2022\u003c/span\u003e, \u003cspan citationid=\"CR49\" class=\"CitationRef\"\u003e2023\u003c/span\u003e), some of the early effects of NaCl on plants include a sharp decrease of cell hydraulic conductivity in roots (L\u003csub\u003epc\u003c/sub\u003e), which increases root hydraulic resistance that triggers stomatal closure (Lee and Zwiazek \u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e2015\u003c/span\u003e; Vaziriyeganeh et al. \u003cspan citationid=\"CR50\" class=\"CitationRef\"\u003e2018\u003c/span\u003e). High L\u003csub\u003epc\u003c/sub\u003e is a fundamental requirement for plant growth and development since efficient water delivery from roots to leaves helps maintain water balance and enhances gas exchange through the stomatal opening (Lee and Zwiazek \u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e2015\u003c/span\u003e).\u003c/p\u003e\u003cp\u003eIncreasing gene expression of major water-transporting AQPs is an effective mechanism of alleviating the effects of Na on root water transport (Lee and Zwiazek \u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e2015\u003c/span\u003e), especially in halophytic plants in which some of AQPs have modified protein structure that makes them insensitive to Na (Vaziriyeganeh et al. \u003cspan citationid=\"CR49\" class=\"CitationRef\"\u003e2023\u003c/span\u003e). Enhanced gene expression of AQPs has been reported in the roots of ectomycorrhizal (Xu et al. \u003cspan citationid=\"CR59\" class=\"CitationRef\"\u003e2015\u003c/span\u003e) and arbuscular mycorrhizal (Porcel et al. \u003cspan citationid=\"CR35\" class=\"CitationRef\"\u003e2006\u003c/span\u003e; Li et al. \u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e2013\u003c/span\u003e; Ni et al. \u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e2024\u003c/span\u003e) plants, as well as for plant roots colonized with \u003cem\u003eP. indica\u003c/em\u003e (Ghorbani et al. \u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e2019\u003c/span\u003e). Improved plant salt tolerance by \u003cem\u003eP. indica\u003c/em\u003e has been also attributed to the increased expression of \u003cem\u003eNHXs, SOS1 and CNGC15\u003c/em\u003e genes as well as the genes encoding the high affinity K transporter 1 and the inward-rectifying K channels KAT1 and KAT2, resulting in a lower Na/K ratio and shoot Na concentration (Abdelaziz et al. \u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e2017\u003c/span\u003e; Yun et al. \u003cspan citationid=\"CR60\" class=\"CitationRef\"\u003e2018\u003c/span\u003e; Ghorbani et al. \u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e2019\u003c/span\u003e).\u003c/p\u003e\u003cp\u003eThe function of AQPs, which control cell-to-cell water transport, is also affected by nutrient balance (Wang et al. \u003cspan citationid=\"CR55\" class=\"CitationRef\"\u003e2016\u003c/span\u003e). A well-documented effect of \u003cem\u003eP. indica\u003c/em\u003e is its enhancement of nutrient uptake by plants, including nitrogen (N) and phosphorus (P) (Gill et al. \u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e2016\u003c/span\u003e; Su et al. \u003cspan citationid=\"CR43\" class=\"CitationRef\"\u003e2017\u003c/span\u003e; Saddique et al. \u003cspan citationid=\"CR37\" class=\"CitationRef\"\u003e2018\u003c/span\u003e). In addition to the improved access to N and P outside of the depletion zone (Sherameti et al. \u003cspan citationid=\"CR40\" class=\"CitationRef\"\u003e2005\u003c/span\u003e; Ngwene et al. \u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e2016\u003c/span\u003e), an increased acquisition of N and P in \u003cem\u003eBrassica napus\u003c/em\u003e by \u003cem\u003eP. indica\u003c/em\u003e was attributed to the increased activity of key metabolic enzymes including N-acetyl-gamma-glutamyl-phosphate reductase, aminoacylase, and adenylosuccinate synthetase (Shrivastava et al. \u003cspan citationid=\"CR41\" class=\"CitationRef\"\u003e2018\u003c/span\u003e). \u003cem\u003eP. indica\u003c/em\u003e also improved K, Ca, Mg, and Zn uptake by plants exposed to salt and osmotic stresses (Su et al. \u003cspan citationid=\"CR43\" class=\"CitationRef\"\u003e2017\u003c/span\u003e; Saddique et al. \u003cspan citationid=\"CR37\" class=\"CitationRef\"\u003e2018\u003c/span\u003e; Ghorbani et al. \u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e2019\u003c/span\u003e).\u003c/p\u003e\u003cp\u003eIn this study, we compared the responses to NaCl of maize seedling inoculated with the root fungal endophyte \u003cem\u003eP. indica\u003c/em\u003e with non-inoculated plants. The main objectives of the study were to determine: 1) the effects of \u003cem\u003eP. indica\u003c/em\u003e on salt tolerance of maize, and 2) the contributions of root cell water transport and ion homeostasis to the salt tolerance processes. We examined the hypothesis that the enhancement of salt tolerance in maize by \u003cem\u003eP. indica\u003c/em\u003e would involve a reduction in root Na uptake and enhancement of L\u003csub\u003epc\u003c/sub\u003e, which would result in lessening the effects of salt on gas exchange, nutrient uptake and, ultimately, plant growth.\u003c/p\u003e"},{"header":"Materials and methods","content":"\u003cp\u003ePlant material, growth conditions, fungal inoculation, and NaCl treatment\u003c/p\u003e\u003cp\u003eSweet corn (\u003cem\u003eZea mays\u003c/em\u003e, Honey select) was surface sterilized for 20 minutes with 1% sodium hypochlorite containing 0.01% Tween 20 followed by two rinses with distilled water. The seeds were then sterilized again for 5 minutes with 70% ethanol for 5 minutes and rinsed six times with sterilized distilled water. The seeds were germinated in the Petri dish, and the germinants transferred after 3 days to 3 L sterilized pots (one seedling per pot) filled with the autoclaved potting mix (Sunshine Professional Growing LA4 mix, Sun Gro Horticulture, Saba Beach, AB, Canada). The pots were placed on the bench in the growth room at 23/18\u003csup\u003eo\u003c/sup\u003eC (day/night) temperature, 60\u0026thinsp;\u0026plusmn;\u0026thinsp;10% relative humidity, and 16-h photoperiod with 400 \u0026micro;mol m\u003csup\u003e\u0026minus;\u0026thinsp;2\u003c/sup\u003es\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e photosynthetic photon flux density (PPFD) at the top of the seedlings provided by the full-spectrum fluorescent bulbs (Philips high output, Markham, ON, Canada).\u003c/p\u003e\u003cp\u003e\u003cem\u003ePiriformospora indica\u003c/em\u003e was cultured in a modified Melin-Norkans (MMN) liquid medium for 4 weeks at 23\u003csup\u003eo\u003c/sup\u003eC (Sherameti et al. \u003cspan citationid=\"CR40\" class=\"CitationRef\"\u003e2005\u003c/span\u003e). For inoculation (Bertolazi et al. \u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e2019\u003c/span\u003e), the mycelium was filtered through the cheesecloth to remove the excess medium and washed three times with distilled water. After the washing step, the mycelium was collected by centrifuging at 4000 g for 7 min. The pellet was suspended in distilled water and 20 mL of the blended mycelium (1% w/v) was injected with a syringe into the soil near each seedling. The same amount of the autoclaved \u003cem\u003eP. indica\u003c/em\u003e fungal inoculum was used as an inoculation control. Ten days after the inoculation, inoculated and non-inoculated seedlings were divided into three groups and treated with 0 (NaCl control), 60, and 120 mM NaCl. NaCl treatments were carried out by immersing the 2\u0026ndash;3 cm bottoms of the pots in the NaCl solutions (water for NaCl control) for one day and then draining the pots on the bench for the next 2 day, for the total of 24 days. For the seed yield measurements, the plants were allowed to grow after the 24 days of treatments for another 70 days before being harvested. During that time, the plants were irrigated daily and provided weekly with modified 100% Hoagland\u0026rsquo;s solution (Epstein \u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e1972\u003c/span\u003e).\u003c/p\u003e\u003cp\u003eRoot fungal colonization\u003c/p\u003e\u003cp\u003eRoots of six non-inoculated seedlings and seedlings inoculated with \u003cem\u003eP. indica\u003c/em\u003e were randomly selected to determine fungal colonization after 24 days of NaCl treatments. After removal of the growth medium, roots were washed with tap water. Distal, 50-mm long, root segments were excised from each of the six seedlings per treatment and preserved in FAA (formalin: acetic acid: alcohol, 5%: 5%: 90%). For microscopic observations, root samples were rinsed several times with distilled water and cleared with 10% KOH for 30 min at 70\u003csup\u003eo\u003c/sup\u003eC. The cleared root samples were then rinsed and then stained with 5% black ink-vinegar solution for 1 h at room temperature (Vierheilig et al. \u003cspan citationid=\"CR53\" class=\"CitationRef\"\u003e1998\u003c/span\u003e). The stained roots were rinsed with distilled water and mounted in 50% polyvinyl-lacto-glycerol for viewing with a microscope (Zeiss, G\u0026ouml;ttingen, Germany).\u003c/p\u003e\u003cp\u003ePlant growth and seed yield\u003c/p\u003e\u003cp\u003eAfter measuring stem heights and diameters, roots and shoots of plants treated with NaCl for 24 days were excised and dried in an oven at 70\u003csup\u003eo\u003c/sup\u003eC for 3 days to determine dry weights (DW) (n\u0026thinsp;=\u0026thinsp;7). Seed yield was determined in plants treated with NaCl for 24 days and allowed to grow in the absence of NaCl for additional 70 days before the harvest. The harvested maize ears were air dried, and the seeds were extracted to determine their weights and numbers per plant (n\u0026thinsp;=\u0026thinsp;6).\u003c/p\u003e\u003cp\u003eMeasurements of cell hydraulic conductivity\u003c/p\u003e\u003cp\u003eDistal root segments (about 50 mm long) were fixed using a small magnetic bar on a metal sledge which was covered with a paper tissue (Lee and Zwiazek \u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e2015\u003c/span\u003e). A gentle stream of 100% Hoagland\u0026rsquo;s solution with 120 mM NaCl (for plants treated for 24 days with 120 mM NaCl) or 100% Hoagland\u0026rsquo;s solution without NaCl (for control plants grown in 100% Hoagland\u0026rsquo;s solution) was flowed along the roots during the measurements. A single root cortical cell was punctured at about 25- to 30-mm above the root tip with a silicon oil-filled microcapillary (10 \u0026micro;m tip diameter). After a meniscus was formed between cell sap and oil, cell turgor (P) was rebuilt by gently pushing the meniscus to a position close to the root surface. Once P became steady, half-times of water exchange (T\u003csub\u003e1/2\u003c/sub\u003e) and cell elastic modulus were determined (Lee and Zwiazek \u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e2015\u003c/span\u003e). The dimensions of the root cells obtained from cross and longitudinal sections were used to determine the cell volume and cell surface area and for the calculation of cell hydraulic conductivity (L\u003csub\u003ep\u003c/sub\u003e) as earlier described (Lee and Zwiazek \u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e2015\u003c/span\u003e). Six inoculated and non-inoculated plants from the control and NaCl treatment groups were used for the measurements (n\u0026thinsp;=\u0026thinsp;6).\u003c/p\u003e\u003cp\u003eTissue elemental analysis\u003c/p\u003e\u003cp\u003eRoots and leaves of plants treated for 24 days with NaCl were freeze-dried for 48 h and then pulverized using the Wiley Mill. The samples containing 50 mg of ground tissue were extracted using the sulfuric acid-hydrogen peroxide wet digestion, and Na content was determined with an atomic absorption spectrophotometer (Spectra AA880; Varian, Inc., Mississauga, ON, Canada). Chloride (Cl) concentrations were measured in roots and shoots after hot water extraction, and then the extracts were analyzed using a DI 300 ion chromatograph (Dionex; Sunnyvale, CA). Other tissue elements, including K, P, Ca, Mg, Fe, B, Mn, and Zn, were extracted with 5 ml 70% HNO\u003csub\u003e3\u003c/sub\u003e and diluted with Milli-Q water to 20 ml. The extracts were then filtered and analyzed by inductively coupled plasma-optical emission spectrometry (iCap 6000, Thermo Fisher Scientific Inc, Waltham, MA, USA) (Zarcinas et al. \u003cspan citationid=\"CR61\" class=\"CitationRef\"\u003e1987\u003c/span\u003e). Total N was determined by the dry combustion method with the Thermo Flash 2000 Organic Elemental Analyzer (Thermo Fisher Scientific Inc., Bremen, Germany) (Tabatabai and Bremner \u003cspan citationid=\"CR44\" class=\"CitationRef\"\u003e1991\u003c/span\u003e).\u003c/p\u003e\u003cp\u003eGas exchange and leaf chlorophyll concentrations\u003c/p\u003e\u003cp\u003eAfter 24 days of NaCl treatments, seven maize plants (n\u0026thinsp;=\u0026thinsp;7) were randomly taken form each treatment for the measurements of net photosynthesis (P\u003csub\u003en\u003c/sub\u003e) and transpiration (E) rates. Fully expanded leaves were inserted in the leaf chamber of an infrared gas analyzer (LI- 6400, LI-COR, Lincoln, NE, USA) and measured as previously described (Xu et al. \u003cspan citationid=\"CR59\" class=\"CitationRef\"\u003e2015\u003c/span\u003e). The reference CO\u003csub\u003e2\u003c/sub\u003e concentration was 400 \u0026micro;mol and the PPFD was set to 400 \u0026micro;mol m\u003csup\u003e\u0026minus;\u0026thinsp;2\u003c/sup\u003e s\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e in the leaf chamber. The measurements were conducted between 9:00\u0026ndash;12:00 h.\u003c/p\u003e\u003cp\u003eTo determine the chlorophyll (Chl a\u0026thinsp;+\u0026thinsp;b) concentrations, fully expanded leaves from seven plants per treatment were freeze-dried for 48 h and pulverized using the Wiley Mill (n\u0026thinsp;=\u0026thinsp;7). Chlorophyll was extracted with dimethyl sulfoxide (DMSO) and the absorbance of the filtered DMSO extracts was measured with a spectrophotometer (Genesys 10 S-UV0VIS, Thermo Fisher Scientific Inc., NJ, USA) at 648 and 665 nm, respectively (Barnes et al. \u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e1992\u003c/span\u003e).\u003c/p\u003e\u003cp\u003eExperimental design and statistical analysis\u003c/p\u003e\u003cp\u003eThe experiment was a 2\u0026times;3 complete randomized factorial design with two fungal treatments (F; \u003cem\u003eP. indica\u003c/em\u003e-inoculated and non-inoculated) and three NaCl levels (S; 0, 60, 120 mM NaCl). The data were analyzed by ANOVA followed by the Fisher\u0026rsquo;s least significant differences (LSD) comparison test. The two-way ANOVA \u003cem\u003ep\u003c/em\u003e-values used to evaluate the effects of fungal inoculation (F), NaCl treatment (S), and fungal inoculation \u0026times; NaCl treatment (F \u0026times; S) interaction are shown in Table\u0026nbsp;\u003cspan refid=\"Tab1\" class=\"InternalRef\"\u003e1\u003c/span\u003e, Table\u0026nbsp;\u003cspan refid=\"Tab2\" class=\"InternalRef\"\u003e2\u003c/span\u003e and Table\u0026nbsp;\u003cspan refid=\"Tab3\" class=\"InternalRef\"\u003e3\u003c/span\u003e. Statistical analyses were carried out using the SigmaPlot version 11.0 (Systat Software Inc, California, USA) at \u003cem\u003ep\u003c/em\u003e\u0026thinsp;\u0026le;\u0026thinsp;0.05 level.\u003c/p\u003e"},{"header":"Results","content":"\u003cp\u003eRoot colonization and plant growth\u003c/p\u003e\u003cp\u003eAfter 24 days of treatments, fungal hyphae and spores were observed in all examined roots of plants inoculated with \u003cem\u003eP. indica\u003c/em\u003e, and none were present in the non-inoculated plants in both the control and NaCl treatments (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e1\u003c/span\u003ed, e).\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003cp\u003eAt the end of the treatments, maize seedlings inoculated with \u003cem\u003eP. indica\u003c/em\u003e were taller and had more leaves compared with the non-inoculated plants (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e1\u003c/span\u003ea, b). Most of the leaves in the inoculated plants remained green even in the highest, 120 mM NaCl treatment which caused leaf chlorosis in the non-inoculated plants (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e1\u003c/span\u003ec).\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003cp\u003eShoot DW, shoot to root DW ratios, stem heights and stem diameters, but not root DW, were significantly higher in \u003cem\u003eP. indica-\u003c/em\u003einoculated seedlings in all NaCl treatments compared with those in non-inoculated seedlings (Figs.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e1\u003c/span\u003ea, b, Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e2\u003c/span\u003ea-e). There were no differences in root DW, shoot DW, shoot to root DW ratios, stem heights and stem diameter between 0 and 60 mM NaCl in non-inoculated and \u003cem\u003eP. indica\u003c/em\u003e-inoculated seedlings, respectively (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e2\u003c/span\u003ea-e). Compared to 0 mM NaCl, the120 mM NaCl treatment significantly decreased root and shoot DW and stem heights in inoculated seedlings, and reduced shoot DW, the shoot to root ratios and stem heights in non-inoculated seedlings (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e2\u003c/span\u003ea-d). There were no significant differences in shoot DW or shoot/root ratios between 60 and 120 mM NaCl in \u003cem\u003eP. indica\u003c/em\u003e-inoculated seedlings (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e2\u003c/span\u003eb, c). However, the 120 mM NaCl treatment further decreased shoot DW, shoot/root ratios and stem heights in non-inoculated seedlings compared to the 60 mM NaCl treatment (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e2\u003c/span\u003eb-d). A relatively small, but statistically significant, increase in stem diameters was observed as a result of inoculation with \u003cem\u003eP. indica\u003c/em\u003e in all NaCl treatments (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e2\u003c/span\u003ee). However, no significant F \u0026times; S interaction was observed for root DW, shoot DW, stem height, or stem diameter (Table\u0026nbsp;\u003cspan refid=\"Tab1\" class=\"InternalRef\"\u003e1\u003c/span\u003e).\u003c/p\u003e\u003cp\u003e\u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab1\" border=\"1\"\u003e\u003ccaption language=\"En\"\u003e\u003cdiv class=\"CaptionNumber\"\u003eTable 1\u003c/div\u003e\u003cdiv class=\"CaptionContent\"\u003e\u003cp\u003eANOVA \u003cem\u003ep\u003c/em\u003e-values for the main factors and their interactions\u003c/p\u003e\u003c/div\u003e\u003c/caption\u003e\u003ccolgroup cols=\"11\"\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e\u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e\u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e\u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c4\" colnum=\"4\"\u003e\u003c/div\u003e\u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c5\" colnum=\"5\"\u003e\u003c/div\u003e\u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c6\" colnum=\"6\"\u003e\u003c/div\u003e\u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c7\" colnum=\"7\"\u003e\u003c/div\u003e\u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c8\" colnum=\"8\"\u003e\u003c/div\u003e\u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c9\" colnum=\"9\"\u003e\u003c/div\u003e\u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c10\" colnum=\"10\"\u003e\u003c/div\u003e\u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c11\" colnum=\"11\"\u003e\u003c/div\u003e\u003cthead\u003e\u003ctr\u003e\u003cth align=\"left\" colname=\"c1\"\u003e\u003cp\u003eVariance\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c2\"\u003e\u003cp\u003eRoot DW\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c3\"\u003e\u003cp\u003eShoot DW\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c4\"\u003e\u003cp\u003eStem height\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c5\"\u003e\u003cp\u003eStem diameter\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c6\"\u003e\u003cp\u003eShoot/Root\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c7\"\u003e\u003cp\u003eL\u003csub\u003epc\u003c/sub\u003e\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c8\"\u003e\u003cp\u003eRoot Na\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c9\"\u003e\u003cp\u003eLeaf Na\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c10\"\u003e\u003cp\u003eRoot Cl\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c11\"\u003e\u003cp\u003eLeaf Cl\u003c/p\u003e\u003c/th\u003e\u003c/tr\u003e\u003c/thead\u003e\u003ctbody\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eF\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e\u003cp\u003e0.036\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e\u003cp\u003e˂0.001\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e\u003cp\u003e˂0.001\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e\u003cp\u003e0.257\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e\u003cp\u003e˂0.001\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c7\"\u003e\u003cp\u003e0.313\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c8\"\u003e\u003cp\u003e0.057\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c9\"\u003e\u003cp\u003e0.008\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c10\"\u003e\u003cp\u003e0.108\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c11\"\u003e\u003cp\u003e0.038\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eS\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e\u003cp\u003e0.003\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e\u003cp\u003e˂0.001\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e\u003cp\u003e˂0.001\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e\u003cp\u003e0.001\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e\u003cp\u003e0.035\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c7\"\u003e\u003cp\u003e0.035\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c8\"\u003e\u003cp\u003e˂0.001\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c9\"\u003e\u003cp\u003e˂0.001\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c10\"\u003e\u003cp\u003e˂0.001\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c11\"\u003e\u003cp\u003e˂0.001\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eF \u0026times; S\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e\u003cp\u003e0.249\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e\u003cp\u003e0.411\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e\u003cp\u003e0.802\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e\u003cp\u003e0.057\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e\u003cp\u003e0.017\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c7\"\u003e\u003cp\u003e0.034\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c8\"\u003e\u003cp\u003e0.032\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c9\"\u003e\u003cp\u003e0.005\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c10\"\u003e\u003cp\u003e0.422\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c11\"\u003e\u003cp\u003e0.078\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003c/tbody\u003e\u003c/colgroup\u003e\u003c/table\u003e\u003c/div\u003e\u003c/p\u003e\u003cp\u003eThe main factors are fungal inoculation (F; non-inoculated, inoculated with \u003cem\u003eP. indica\u003c/em\u003e) and NaCl (S; 0, 60 120 mM). The measured parameters include root DW, shoot DW, stem height, stem diameter, shoot/root ratio, L\u003csub\u003epc,\u003c/sub\u003e Root Na and Cl and Leaf Na and Cl.\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003cp\u003eHalf-times of water exchange (T\u003csub\u003e1/2\u003c/sub\u003e) and cell hydraulic conductivity (L\u003csub\u003epc\u003c/sub\u003e)\u003c/p\u003e\u003cp\u003eThe T\u003csub\u003e1/2\u003c/sub\u003e values in root cortical cells varied from 2.3 to 2.8 s in the non-inoculated seedlings and between 2.9 and 4.2 s in the inoculated plants grown in the 0 mM NaCl for 24 days (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e3\u003c/span\u003ea, b). In the non-inoculated plants treated with 120 mM NaCl for 24 days, T\u003csub\u003e1/2\u003c/sub\u003e was higher by 4-fold and L\u003csub\u003epc\u003c/sub\u003e lower by 3-4-fold compared with control plants (0 mM NaCl) (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e3\u003c/span\u003ec, e). However, in the inoculated seedlings the T\u003csub\u003e1/2\u003c/sub\u003e and L\u003csub\u003epc\u003c/sub\u003e values were similar in NaCl treated and untreated plants (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e3\u003c/span\u003ed, e). A significant F \u0026times; S interaction was observed for L\u003csub\u003epc\u003c/sub\u003e (\u003cem\u003ep\u003c/em\u003e\u0026thinsp;=\u0026thinsp;0.034) (Table\u0026nbsp;\u003cspan refid=\"Tab1\" class=\"InternalRef\"\u003e1\u003c/span\u003e).\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003cp\u003eTissue elemental concentrations\u003c/p\u003e\u003cp\u003eThe 120 mM NaCl treatment significantly increased the Na concentrations in roots and leaves of both non-inoculated and inoculated seedlings, but the increase was of a lower magnitude in seedlings inoculated with \u003cem\u003eP. indica\u003c/em\u003e (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e4\u003c/span\u003ea). The concentrations of Cl in roots and leaves were elevated by a similar extent in both groups of plants treated with 120 mM NaCl (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e4\u003c/span\u003eb). A significant F \u0026times; S interaction was observed for Na in root and leaf (\u003cem\u003ep\u003c/em\u003e\u0026thinsp;\u0026le;\u0026thinsp;0.05), but not for Cl (Table\u0026nbsp;\u003cspan refid=\"Tab1\" class=\"InternalRef\"\u003e1\u003c/span\u003e).\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003cp\u003eThe non-inoculated seedlings had higher root and leaf concentrations of N and P compared to \u003cem\u003eP. indica\u003c/em\u003e-inoculated seedlings (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e5\u003c/span\u003ea, b). The 120 mM NaCl treatment significantly decreased the N and P concentrations in roots and leaves of the non-inoculated seedlings but had little effect on the N and P concentrations in the inoculated plants (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e5\u003c/span\u003ea, b, Table\u0026nbsp;\u003cspan refid=\"Tab2\" class=\"InternalRef\"\u003e2\u003c/span\u003e). The root K concentrations were higher in the non-inoculated compared with inoculated plants (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e5\u003c/span\u003ec). The 120 mM NaCl treatment significantly declined the root K concentrations while the leaf K, Ca, and Mg concentrations as well as root Mg concentration remained unchanged in both inoculated and non-inoculated seedlings (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e5\u003c/span\u003ec-e, Table\u0026nbsp;\u003cspan refid=\"Tab2\" class=\"InternalRef\"\u003e2\u003c/span\u003e). A significant F \u0026times; S interaction was observed for N, P, K and Ca in root, and for P in leaf (\u003cem\u003ep\u003c/em\u003e\u0026thinsp;\u0026le;\u0026thinsp;0.05) (Table\u0026nbsp;\u003cspan refid=\"Tab2\" class=\"InternalRef\"\u003e2\u003c/span\u003e)\u003c/p\u003e\u003cp\u003eThere were little differences in the leaf and root Fe concentrations between the non-inoculated and inoculated seedlings, regardless of NaCl concentraion (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e5\u003c/span\u003ef). Root B concentrations were significantly lower in the inoculated compared to non-inoculated plants (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e5\u003c/span\u003eg, Table\u0026nbsp;\u003cspan refid=\"Tab2\" class=\"InternalRef\"\u003e2\u003c/span\u003e). The 120 mM NaCl treatment did not affect B concentrations in roots and leaves and had no effect on Zn concentration in the leaves of both inoculation groups (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e5\u003c/span\u003eg, j, Table\u0026nbsp;\u003cspan refid=\"Tab2\" class=\"InternalRef\"\u003e2\u003c/span\u003e). The root and leaf Mn concentrations were increased by the 120 mM NaCl tratement in both non-inoculation and inoculation groups (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e5\u003c/span\u003eh). The non-inoculated seedlings had higher leaf concentrations of Cu compared to \u003cem\u003eP. indica\u003c/em\u003e-inoculated seedlings (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e5\u003c/span\u003ei). The 120 mM NaCl treatment significantly decreased the Cu concentrations in leaves of non-inoculated seedlings, but root Cu concentration remained unchanged in both seedlings regardless of NaCl concentration (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e5\u003c/span\u003ei, Table\u0026nbsp;\u003cspan refid=\"Tab2\" class=\"InternalRef\"\u003e2\u003c/span\u003e). A significant F \u0026times; S interaction was observed for Cu and Zn in roots (\u003cem\u003ep\u003c/em\u003e\u0026thinsp;\u0026le;\u0026thinsp;0.05) (Table\u0026nbsp;\u003cspan refid=\"Tab2\" class=\"InternalRef\"\u003e2\u003c/span\u003e). The 120 mM NaCl treatment significantly decreased root Zn concentrations in non-inoculated seedlings but had no effect on root Zn concentrations in the inoculated plants (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e5\u003c/span\u003ej, Table\u0026nbsp;\u003cspan refid=\"Tab2\" class=\"InternalRef\"\u003e2\u003c/span\u003e).\u003c/p\u003e\u003cp\u003e\u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab2\" border=\"1\"\u003e\u003ccaption language=\"En\"\u003e\u003cdiv class=\"CaptionNumber\"\u003eTable 2\u003c/div\u003e\u003cdiv class=\"CaptionContent\"\u003e\u003cp\u003eANOVA \u003cem\u003ep\u003c/em\u003e-values for the main factors and their interactions\u003c/p\u003e\u003c/div\u003e\u003c/caption\u003e\u003ccolgroup cols=\"12\"\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e\u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e\u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c4\" colnum=\"4\"\u003e\u003c/div\u003e\u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c5\" colnum=\"5\"\u003e\u003c/div\u003e\u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c6\" colnum=\"6\"\u003e\u003c/div\u003e\u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c7\" colnum=\"7\"\u003e\u003c/div\u003e\u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c8\" colnum=\"8\"\u003e\u003c/div\u003e\u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c9\" colnum=\"9\"\u003e\u003c/div\u003e\u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c10\" colnum=\"10\"\u003e\u003c/div\u003e\u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c11\" colnum=\"11\"\u003e\u003c/div\u003e\u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c12\" colnum=\"12\"\u003e\u003c/div\u003e\u003cthead\u003e\u003ctr\u003e\u003cth align=\"left\" colname=\"c1\"\u003e\u0026nbsp;\u003c/th\u003e\u003cth align=\"left\" colname=\"c2\"\u003e\u003cp\u003eVariance\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c3\"\u003e\u003cp\u003eN\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c4\"\u003e\u003cp\u003eP\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c5\"\u003e\u003cp\u003eK\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c6\"\u003e\u003cp\u003eCa\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c7\"\u003e\u003cp\u003eMg\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c8\"\u003e\u003cp\u003eFe\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c9\"\u003e\u003cp\u003eB\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c10\"\u003e\u003cp\u003eMn\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c11\"\u003e\u003cp\u003eCu\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c12\"\u003e\u003cp\u003eZn\u003c/p\u003e\u003c/th\u003e\u003c/tr\u003e\u003c/thead\u003e\u003ctbody\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\" morerows=\"2\" rowspan=\"3\"\u003e\u003cp\u003eRoot\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003eF\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e\u003cp\u003e0.004\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e\u003cp\u003e0.006\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e\u003cp\u003e0.010\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e\u003cp\u003e0.359\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c7\"\u003e\u003cp\u003e0.160\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c8\"\u003e\u003cp\u003e0.642\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c9\"\u003e\u003cp\u003e˂0.001\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c10\"\u003e\u003cp\u003e0.539\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c11\"\u003e\u003cp\u003e0.383\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c12\"\u003e\u003cp\u003e˂0.001\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003eS\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e\u003cp\u003e˂0.001\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e\u003cp\u003e0.001\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e\u003cp\u003e˂0.001\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e\u003cp\u003e0.014\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c7\"\u003e\u003cp\u003e0.057\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c8\"\u003e\u003cp\u003e0.725\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c9\"\u003e\u003cp\u003e0.140\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c10\"\u003e\u003cp\u003e0.007\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c11\"\u003e\u003cp\u003e0.789\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c12\"\u003e\u003cp\u003e0.001\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003eF \u0026times; S\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e\u003cp\u003e0.002\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e\u003cp\u003e0.031\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e\u003cp\u003e0.007\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e\u003cp\u003e0.028\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c7\"\u003e\u003cp\u003e0.526\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c8\"\u003e\u003cp\u003e0.121\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c9\"\u003e\u003cp\u003e0.469\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c10\"\u003e\u003cp\u003e0.260\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c11\"\u003e\u003cp\u003e0.012\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c12\"\u003e\u003cp\u003e˂0.001\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\" morerows=\"2\" rowspan=\"3\"\u003e\u003cp\u003eLeaf\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003eF\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e0.023\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e˂0.001\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003e0.388\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u003cp\u003e0.143\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c7\"\u003e\u003cp\u003e0.198\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c8\"\u003e\u003cp\u003e0.783\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c9\"\u003e\u003cp\u003e0.451\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c10\"\u003e\u003cp\u003e0.510\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c11\"\u003e\u003cp\u003e0.034\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c12\"\u003e\u003cp\u003e0.329\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003eS\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e\u003cp\u003e0.005\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e\u003cp\u003e0.019\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e\u003cp\u003e0.047\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e\u003cp\u003e0.681\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c7\"\u003e\u003cp\u003e0.887\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c8\"\u003e\u003cp\u003e0.012\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c9\"\u003e\u003cp\u003e0.025\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c10\"\u003e\u003cp\u003e0.001\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c11\"\u003e\u003cp\u003e0.038\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c12\"\u003e\u003cp\u003e0.594\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003eF \u0026times; S\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e\u003cp\u003e0.137\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e\u003cp\u003e0.007\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e\u003cp\u003e0.200\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e\u003cp\u003e0.488\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c7\"\u003e\u003cp\u003e0.595\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c8\"\u003e\u003cp\u003e0.384\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c9\"\u003e\u003cp\u003e0.366\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c10\"\u003e\u003cp\u003e0.242\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c11\"\u003e\u003cp\u003e0.147\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c12\"\u003e\u003cp\u003e0.178\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003c/tbody\u003e\u003c/colgroup\u003e\u003c/table\u003e\u003c/div\u003e\u003c/p\u003e\u003cp\u003eThe main factors are fungal inoculation (F; non-inoculated, inoculated with \u003cem\u003eP. indica\u003c/em\u003e) and NaCl (S; 0, 60 120 mM). The measured parameters include root and leaf N, P, K, Ca, Mg, Fe, B, Mn, Cu and Zn.\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003cp\u003eNet photosynthesis rates (P\u003csub\u003en\u003c/sub\u003e), transpiration rates (E), and leaf chlorophyll (Chl) concentrations\u003c/p\u003e\u003cp\u003eThe 60 mM NaCl treatment did not affect P\u003csub\u003en\u003c/sub\u003e and E in the non-inoculated and inoculated seedlings (Fig.\u0026nbsp;\u003cspan refid=\"Fig8\" class=\"InternalRef\"\u003e7\u003c/span\u003ea, b). The 120 mM NaCl treatment did not affect both parameters in the inoculated plants but reduced both P\u003csub\u003en\u003c/sub\u003e and E in the non-inoculated plants (Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e6\u003c/span\u003ea, b).\u003c/p\u003e\u003cp\u003eChl concentrations did not significantly vary between the inoculated and non-inoculated plants subjected to 0 and 60 mM NaCl treatments (Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e6\u003c/span\u003ec). However, the 120 mM NaCl treatment resulted in a sharp decrease in the non-inoculated plants while having little effect in plants inoculated with \u003cem\u003eP. indica\u003c/em\u003e (Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e6\u003c/span\u003ec, Table\u0026nbsp;\u003cspan refid=\"Tab3\" class=\"InternalRef\"\u003e3\u003c/span\u003e).\u003c/p\u003e\u003cp\u003e\u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab3\" border=\"1\"\u003e\u003ccaption language=\"En\"\u003e\u003cdiv class=\"CaptionNumber\"\u003eTable 3\u003c/div\u003e\u003cdiv class=\"CaptionContent\"\u003e\u003cp\u003eANOVA \u003cem\u003ep\u003c/em\u003e-values for the main factors and their interactions\u003c/p\u003e\u003c/div\u003e\u003c/caption\u003e\u003ccolgroup cols=\"7\"\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e\u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e\u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e\u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c4\" colnum=\"4\"\u003e\u003c/div\u003e\u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c5\" colnum=\"5\"\u003e\u003c/div\u003e\u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c6\" colnum=\"6\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c7\" colnum=\"7\"\u003e\u003c/div\u003e\u003cthead\u003e\u003ctr\u003e\u003cth align=\"left\" colname=\"c1\"\u003e\u003cp\u003eVariance\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c2\"\u003e\u003cp\u003eP\u003csub\u003en\u003c/sub\u003e\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c3\"\u003e\u003cp\u003eE\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c4\"\u003e\u003cp\u003eChl\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c5\"\u003e\u003cp\u003eSeed number\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c6\"\u003e\u003cp\u003eSeed weight\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c7\"\u003e\u0026nbsp;\u003c/th\u003e\u003c/tr\u003e\u003c/thead\u003e\u003ctbody\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eF\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e\u003cp\u003e0.613\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e\u003cp\u003e0.149\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e\u003cp\u003e0.003\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e\u003cp\u003e0.010\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e\u003cp\u003e0.017\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c7\" morerows=\"2\" rowspan=\"3\"\u003e\u0026nbsp;\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eS\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e\u003cp\u003e0.020\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e\u003cp\u003e0.007\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e\u003cp\u003e˂0.001\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e\u003cp\u003e˂0.001\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e\u003cp\u003e˂0.001\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eF \u0026times; S\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e\u003cp\u003e0.106\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e\u003cp\u003e0.192\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e\u003cp\u003e˂0.001\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e\u003cp\u003e0.145\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e\u003cp\u003e0.116\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003c/tbody\u003e\u003c/colgroup\u003e\u003c/table\u003e\u003c/div\u003e\u003c/p\u003e\u003cp\u003eThe main factors are fungal inoculation (F; non-inoculated, inoculated with \u003cem\u003eP. indica\u003c/em\u003e) and NaCl (S; 0, 60 120 mM). The measured parameters include P\u003csub\u003en,\u003c/sub\u003e E, Chl, seed number and seed weight.\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003cp\u003eSeed weight and seed number\u003c/p\u003e\u003cp\u003ePlant seed weights and seed numbers in control (0 mM NaCl) and both NaCl treatments, were significantly greater in the inoculated compared with non-inoculated plants (Fig.\u0026nbsp;\u003cspan refid=\"Fig8\" class=\"InternalRef\"\u003e7\u003c/span\u003ea, b, Table\u0026nbsp;\u003cspan refid=\"Tab3\" class=\"InternalRef\"\u003e3\u003c/span\u003e). Both 60 mM and 120 mM NaCl inhibited seed development and maturation in the non-inoculated plants and, therefore, the seed number and seed weight could not be determined for the non-inoculated seedlings treated with 60 and 120 mM NaCl since no mature seeds were produced in these treatments during the duration of the experiment (Fig.\u0026nbsp;\u003cspan refid=\"Fig8\" class=\"InternalRef\"\u003e7\u003c/span\u003ea, b).\u003c/p\u003e\u003cp\u003e\u003c/p\u003e"},{"header":"Discussion","content":"\u003cp\u003eOur study sheds new light on the processes of enhanced salt tolerance conferred to maize plants by \u003cem\u003ePiriformospora indica\u003c/em\u003e. At the end of the 24 days of treatments, plants inoculated with \u003cem\u003eP. indica\u003c/em\u003e were taller, produced more leaves, and developed cobs earlier compared with the non-inoculated plants. Also, their shoot dry weights were significantly higher in the control (0 mM NaCl) and in both NaCl treatments. Enhanced salt tolerance of plants has been documented for several types of mycorrhizal associations including the ectomycorrhizal (Muhsin and Zwiazek \u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e2002\u003c/span\u003e; Calvo-Polanco et al. \u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e2008\u003c/span\u003e; Lee et al. \u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e2010\u003c/span\u003e; Xu et al. \u003cspan citationid=\"CR59\" class=\"CitationRef\"\u003e2015\u003c/span\u003e), arbuscular mycorrhizal (Chen et al. \u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e2017\u003c/span\u003e; Evelin et al. \u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e2019\u003c/span\u003e; Klinsukon et al. \u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e2021\u003c/span\u003e), and ericoid mycorrhizal (Fadaei et al. \u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e2020\u003c/span\u003e) associations as well as in plants colonized by root fungal endophytes (Abdelaziz et al. \u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e2017\u003c/span\u003e; Yun et al. \u003cspan citationid=\"CR60\" class=\"CitationRef\"\u003e2018\u003c/span\u003e; Ghorbani et al. \u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e2019\u003c/span\u003e). However, the processes involved in this enhancement remain poorly understood.\u003c/p\u003e\u003cp\u003eOur results indicate that the inoculation of maize roots with \u003cem\u003eP. indica\u003c/em\u003e, affected cell water relations and nutrient uptake of plants exposed to NaCl. Maintenance of plant water transport and water balance plays an important role in salt tolerance of plants. Interestingly, in our study, 120 mM NaCl had no effect on L\u003csub\u003epc\u003c/sub\u003e in plants inoculated with \u003cem\u003eP. indica\u003c/em\u003e, but in 0 mM NaCl (NaCl control), L\u003csub\u003epc\u003c/sub\u003e values were similar in the inoculated and non-inoculated plants. These results indicate that the effect of the fungus on L\u003csub\u003epc\u003c/sub\u003e was its protection against NaCl, which could have been due to different indirect or direct factors influencing L\u003csub\u003epc\u003c/sub\u003e under salt conditions.\u003c/p\u003e\u003cp\u003eSince cell-to-cell water transport is regulated by AQPs, the effect of fungal inoculation on L\u003csub\u003epc\u003c/sub\u003e was likely due to its effects on the activity and/or expression levels of root AQPs as previously reported for the ectomycorrhizal (Lee et al. \u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e2010\u003c/span\u003e; Xu et al. \u003cspan citationid=\"CR59\" class=\"CitationRef\"\u003e2015\u003c/span\u003e) and arbuscular mycorrhizal (Bitterlich et al. \u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e2018\u003c/span\u003e; Pauwels et al. \u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e2023\u003c/span\u003e) associations. Even low concentrations of Na strongly inhibit the AQP-mediated water transport in salt-sensitive glycophytic plants (Lee and Zwiazek \u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e2015\u003c/span\u003e). However, depending on their tolerance level, salt-tolerant grasses can either maintain or increase L\u003csub\u003epc\u003c/sub\u003e when exposed to 100 mM NaCl, and this effect was attributed to the overexpression of some of the plasma membrane AQPs (Vaziriyeganeh et al. \u003cspan citationid=\"CR50\" class=\"CitationRef\"\u003e2018\u003c/span\u003e, \u003cspan citationid=\"CR48\" class=\"CitationRef\"\u003e2021\u003c/span\u003e, \u003cspan citationid=\"CR47\" class=\"CitationRef\"\u003e2022\u003c/span\u003e). It was also found that both the gene expression and water transport through PnuPIP2;2, one of the major water-conducting AQPs in the halophytic grass \u003cem\u003ePuccinellia nuttalliana\u003c/em\u003e, were enhanced by NaCl (Vaziriyeganeh et al. \u003cspan citationid=\"CR49\" class=\"CitationRef\"\u003e2023\u003c/span\u003e). Maintenance of the AQP-mediated transport has been proposed to play a major role in the enhancement of plant salt tolerance by the ectomycorrhizal (Lee et al. \u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e2010\u003c/span\u003e; Xu et al. \u003cspan citationid=\"CR59\" class=\"CitationRef\"\u003e2015\u003c/span\u003e) and arbuscular mycorrhizal (Liang et al. \u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e2022\u003c/span\u003e; Asadollahi et al. \u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2023\u003c/span\u003e). Therefore, our results strengthen the notion that cell water transport is an important factor contributing to salt tolerance enhancement by beneficial root fungi.\u003c/p\u003e\u003cp\u003eIn addition to its effect on water transport, root inoculation with \u003cem\u003eP. indica\u003c/em\u003e reduced Na accumulation in roots and leaves of plants exposed to NaCl. Reduced root Na uptake and its transfer to shoots has been well documented in ectomycorrhizal and arbuscular mycorrhizal plants (Smith and Read \u003cspan citationid=\"CR42\" class=\"CitationRef\"\u003e2008\u003c/span\u003e; Lehto and Zwiazek \u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e2011\u003c/span\u003e). Therefore, it appears that this may be a common response for plants with the roots colonized by beneficial fungi. Although the processes involved in this response are still largely unknown, it has been demonstrated for ectomycorrhizal plants that most of the Na in plants treated with NaCl remained either in the extraradical hyphae or in the root cortex reducing its transfer to the photosynthetic tissues (Muhsin and Zwiazek \u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e2002\u003c/span\u003e; Bandou et al. \u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e2006\u003c/span\u003e; Guerrero-Gal\u0026aacute;n et al. \u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e2019\u003c/span\u003e). In contrast, arbuscular mycorrhizal fungi primarily enhance plant tolerance to salinity by improving nutrient uptake and maintaining favorable ion balances, such as increased K/Na ratios, rather than by sequestering Na within their structures (Pooja et al. \u003cspan citationid=\"CR34\" class=\"CitationRef\"\u003e2024\u003c/span\u003e). Also, arbuscular mycorrhizal inoculation has been shown to lower Na concentrations in roots, enhance stomatal conductance, and increase net photosynthetic rates (Zong et al. \u003cspan citationid=\"CR62\" class=\"CitationRef\"\u003e2023\u003c/span\u003e). While direct evidence for Na sequestration within \u003cem\u003eP. indica\u003c/em\u003e hyphae or root cells is limited, its colonization of plant roots has been commonly associated with improved ion regulation and reduced Na accumulation in shoots, suggesting the presence of mechanisms that mitigate sodium stress (Abdelaziz et al. \u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e2017\u003c/span\u003e; Yun et al. \u003cspan citationid=\"CR60\" class=\"CitationRef\"\u003e2018\u003c/span\u003e; Ghorbani et al. \u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e2019\u003c/span\u003e).\u003c/p\u003e\u003cp\u003eInoculation with \u003cem\u003eP. indica\u003c/em\u003e significantly enhanced maize growth and yield as a results of enhanced salt tolerance. Despite the increases in growth parameters in all treatments, plant inoculation with \u003cem\u003eP. indica\u003c/em\u003e resulted in higher E and P\u003csub\u003en\u003c/sub\u003e in plants subjected to 120 mM NaCl but had little effect on E and P\u003csub\u003en\u003c/sub\u003e in control plants or in the plants treated with 60 mM NaCl little effect on E and P\u003csub\u003en\u003c/sub\u003e in control plants. Therefore, our results suggest that in the absence of NaCl, \u003cem\u003eP. indica\u003c/em\u003e enhanced plant growth through other mechanisms than cell water transport or gas exchange, potentially involving improved nutrient acquisition, or altered hormonal regulation (Smith and Read \u003cspan citationid=\"CR42\" class=\"CitationRef\"\u003e2008\u003c/span\u003e; Contreras-Cornejo et al. \u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e2009\u003c/span\u003e). In our study, roots of maize plants colonized by \u003cem\u003eP. indica\u003c/em\u003e tended to develop more root aerenchyma (data not shown) under optimal conditions compared with the non-inoculated plants, suggesting that root cortical aerenchyma enhances nutrient acquisition (Saengwilai et al. \u003cspan citationid=\"CR38\" class=\"CitationRef\"\u003e2014\u003c/span\u003e).\u003c/p\u003e\u003cp\u003eInterestingly, in the present study, N and P concentrations in both roots and leaves were lower in the \u003cem\u003eP. indica\u003c/em\u003e-inoculated plants compared to non-inoculated seedlings in both NaCl-treated and untreated plants. Likewise, root B and Zn concentrations were significantly lower in inoculated seedlings. However, an increase of nutrient elements by \u003cem\u003eP. indica\u003c/em\u003e-inoculated in \u003cem\u003eBrassica napus\u003c/em\u003e (Su et al. \u003cspan citationid=\"CR43\" class=\"CitationRef\"\u003e2017\u003c/span\u003e) and in tomato seedling (Ghorbani et al. \u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e2019\u003c/span\u003e) have also been reported. These large discrepancies remain unclear, but the decreased nutrient concentrations relative to the rapid growth of maize seedlings may be attributed to the nutrient dilution effect caused by increased growth (Kaspari and Welti \u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e2024\u003c/span\u003e). This phenomenon has been often reported for plants due to environmental factors which lead to an enhanced synthesis of carbohydrates resulting in increased growth and, consequently, the dilution of nutrient concentrations (Jarrell and Beverly \u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e1981\u003c/span\u003e; Kaspari and Welti \u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e2024\u003c/span\u003e).\u003c/p\u003e\u003cp\u003eOur study demonstrated that \u003cem\u003eP. indica\u003c/em\u003e enhanced the L\u003csub\u003epc\u003c/sub\u003e in seedlings subjected to NaCl. Since L\u003csub\u003epc\u003c/sub\u003e is regulated by the AQPs (Lee et al. \u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e2010\u003c/span\u003e, 2015; Xu et al. \u003cspan citationid=\"CR59\" class=\"CitationRef\"\u003e2015\u003c/span\u003e), and the AQP function is affected by the mineral nutrition (Carvajal et al. \u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e1996\u003c/span\u003e; Wang et al. \u003cspan citationid=\"CR58\" class=\"CitationRef\"\u003e2001\u003c/span\u003e), it is plausible that the effects of \u003cem\u003eP. indica\u003c/em\u003e on L\u003csub\u003epc\u003c/sub\u003e could involve its effects on nutrient uptake. However, a possible link between the mineral nutrition and root water transport in plants colonized by the mycorrhizal and endophytic fungi has not been clearly established.\u003c/p\u003e\u003cp\u003eIn conclusion, our study demonstrated that the enhancement of salt tolerance in maize plants by \u003cem\u003eP. indica\u003c/em\u003e involves reductions in root and shoot Na uptake and maintenance of the transmembrane root water transport which helped alleviate the effects of NaCl on gas exchange, growth, and seed yield. Possible links between the mineral nutrition and AQP-mediated transport in roots of plants colonized by endophytic fungi should be further explored.\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003ch2\u003eFunding\u003c/h2\u003e\u003cp\u003eFunding for this study was provided by the Natural Sciences and Engineering Discover Research Grant to JJZ. We also gratefully acknowledge financial support from National Natural Science Foundation Project (32360379), Guizhou Provincial Basic Research Program (Natural Science) (qjhe-MS[2025]211), and China Scholarship Council for the scholarship awarded to YW.\u003c/p\u003e\u003c/p\u003e\u003cp\u003e\u003cstrong\u003eCompeting interests\u003c/strong\u003e\u003cp\u003eThe authors have no conflicts of interest to declare.\u003c/p\u003e\u003c/p\u003e\u003ch2\u003eAuthor\u0026rsquo;s contributions\u003c/h2\u003e\u003cp\u003eSL and JZ designed the study, SL and YW conducted the experiments, all authors contributed to writing and editing the manuscript. All authors approved the submitted version of the manuscript.\u003c/p\u003e\u003ch2\u003eAcknowledgements\u003c/h2\u003e\u003cp\u003eWe thank Dr. Wenqing Zhang, University of Alberta, Edmonton, Canada, for assisting with tissue preparation for element analysis.\u003c/p\u003e\u003ch2\u003eData availability\u003c/h2\u003e\u003cp\u003eThe data that support the finding of this study are available from the corresponding author upon reasonable request.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\n\u003cli\u003eAbdelaziz ME, Kim D, Ali S, Fedoroff NV, Al-Babili S (2017) The endophytic fungus \u003cem\u003ePiriformospora indica \u003c/em\u003eenhances \u003cem\u003eArabidopsis thaliana \u003c/em\u003egrowth and modulates Na\u003csup\u003e+\u003c/sup\u003e/K\u003csup\u003e+\u003c/sup\u003e homeostasis under salt stress conditions. 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Mycorrhiza 29: 303-312.\u003c/li\u003e\n\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":true,"highlight":"","institution":"","isAcceptedByJournal":false,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true},"keywords":"cell hydraulic conductivity, growth, maize, nutrient utilization, Piriformospora indica, salt, yield","lastPublishedDoi":"10.21203/rs.3.rs-7095632/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-7095632/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003eBackground and aims\u003c/p\u003e\u003cp\u003eFungal root endophyte \u003cem\u003ePiriformospora indica\u003c/em\u003e has been implicated in enhancing plant tolerance to salt stress. This study aimed to understand the role of root cell hydraulic conductivity in enhancing salt tolerance of maize plants by this fungal endophyte.\u003c/p\u003e\u003cp\u003eMethods\u003c/p\u003e\u003cp\u003eMaize seedlings inoculated with \u003cem\u003eP. indica\u003c/em\u003e were compared with non-inoculated control plants following exposure to 0, 60, and 120 mM NaCl for 24 days and their growth and physiological parameters including root cell hydraulic conductivity examined.\u003c/p\u003e\u003cp\u003eResults\u003c/p\u003e\u003cp\u003eThe shoot dry weights were significantly higher in \u003cem\u003eP. indica-\u003c/em\u003einoculated seedlings compared with non-inoculated plants regardless of the NaCl concentration treatment. Compared to 60 mM NaCl, the 120 mM NaCl treatment further decreased shoot dry weights or shoot to root dry weight ratios in the non-inoculated seedlings, but not in plants inoculated with \u003cem\u003eP. indica\u003c/em\u003e. The 120 mM NaCl treatment reduced the root cell hydraulic conductivity, net photosynthetic rates, transpiration rates, and leaf chlorophyll in the non-inoculated plants compared to the inoculated plants. Following the 120 mM Nacl treatment, \u003cem\u003eP. indica-\u003c/em\u003einoculated seedlings had lower root and shoot Na concentrations compared with the non-inoculated seedlings. Both 60 mM and 120 mM NaCl treatments affected the final seed yield less in the inoculated compared with the non-inoculated plants.\u003c/p\u003e\u003cp\u003eConclusions\u003c/p\u003e\u003cp\u003eThe results demonstrate that the enhancement of salt tolerance in maize plants by \u003cem\u003eP. indica\u003c/em\u003e involves reductions in root and shoot Na uptake and maintenance of the transmembrane root water transport which helped alleviate the effects of NaCl on gas exchange and growth.\u003c/p\u003e","manuscriptTitle":"Beneficial root endophyte Piriformospora indica reduces plant sodium uptake and enhances cell hydraulic conductivity and salt tolerance of maize seedlings","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2025-07-21 10:42:56","doi":"10.21203/rs.3.rs-7095632/v1","editorialEvents":[{"type":"communityComments","content":0}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"9d605586-48c2-4505-896b-feae656ea7e1","owner":[],"postedDate":"July 21st, 2025","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"posted","subjectAreas":[],"tags":[],"updatedAt":"2025-12-24T06:24:04+00:00","versionOfRecord":[],"versionCreatedAt":"2025-07-21 10:42:56","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-7095632","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-7095632","identity":"rs-7095632","version":["v1"]},"buildId":"8U1c8b4HqxoKbykW_rLl7","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}