Mycorrhization of Quercus dentata seedlings with Laccaria bicolor enhances salt tolerance of plants only under relatively moderate soil salinity level

Mycorrhization of Quercus dentata seedlings with Laccaria bicolor enhances salt tolerance of plants only under relatively moderate soil salinity level | 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 Mycorrhization of Quercus dentata seedlings with Laccaria bicolor enhances salt tolerance of plants only under relatively moderate soil salinity level Wenlong Sun, Luyu Qi, Haonan Chen, Yixin Song, Jiaqi Jiang, Puyi Zhang, and 8 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-5474395/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 Soil salinization is a growing global concern in many ecosystems. Although ectomycorrhizal fungi have been shown to alleviate the effects of salinity in some tree species, uncertainties persist concerning their effectiveness when plants are exposed to different salinity levels that are commonly present in salt-affected soils. Objective This study explored the effects of Laccaria bicolor on Quercus dentata seedlings under different levels of salt stress. Methods Q. dentata seedlings were grown in pots and were either non-inoculated (mycorrhizal control) or inoculated with the ectomycorrhizal fungus L. bicolor . The seedlings were then treated with three NaCl concentrations (0, 0.4%, and 0.8%), that was added to the soil for 30 days, after which plant physiological, stoichiometric, and growth characteristics were examined. Results NaCl exposure significantly impaired growth and physiological parameters in all seedling groups. The influence of L. bicolor on Q. dentata seedlings varied with salt concentration. Under moderate salinity, the seedlings colonized by L. bicolor exhibited an enhancement in root biomass and leaf chlorophyll concentrations, concomitant with a reduction in leaf Na + concentrations and the Na + /K + ratios. Conversely, under relatively high salinity, colonization of L. bicolor was associated with a reduction in leaf water content and fluorescence parameters, as well as an elevation in leaf Na + concentrations. Conclusions The effect of ectomycorrhizal fungus L. bicolor on Q. dentata seedlings was dependent on NaCl concentration, and our results indicate that the use of L. bicolor in afforestation efforts with Q. dentata would only be effective under relatively low soil salinity levels. ectomycorrhiza Laccaria bicolor phytoremediation Quercus dentata salt tolerance Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 Figure 7 Figure 8 Figure 9 Introduction Increasing soil salinization is one of the major and widespread soil factors that threatens plant survival and affects their growth and yield in many ecosystems (Kopittke et al. 2019 ; Singh et al. 2021). Combined with progressing global climate changes and human activities, major disturbances of the ecosystems could be expected and cause catastrophic collapse of the ecosystem states and services (Negacz et al. 2022 ). To achieve sustainability of agricultural production and stability of ecosystems, it is essential to prevent further salinization of soils and to develop knowledge required to manage salt-affected sites to restore their productivity (Ullah et al. 2017 ; Mukhopadhyay et al. 2021 ). It is generally recognized that phytoremediation and revegetation of salinized land are appropriate, but challenging, tasks that can mitigate soil salinization (Crooks et al. 2018 ; Litalien et al. 2020). For the revegetation processes, species selection and remediation methods are especially critical and may be combined with an enhancement of plant salt tolerance by beneficial microorganisms (Van Zelm et al. 2020 ; Hao et al. 2021 ). Plant species commonly used in saline phytoremediation are mainly halophytes (Ruan et al. 2010 ; Manousaki et al. 2011b; Stavi et al. 2021 ), which have been employed to restore landscapes damaged by a variety of contaminants and disturbances (Manousaki et al. 2011a; Liang et al. 2017 ). However, other salt-tolerant plant species have been also considered for their suitability of restoration of moderately saline areas. Quercus is considered to be a tree genus with a successful evolutionary history of adaptation to changing and complex environments (Kremer et al. 2019; Sork et al. 2022 ). Studies have shown that some species of Quercus , including Quercus robur (Alaoui-Sossé et al. 1998 ), Q. virginiana (Wang et al. 2014 )d ilex (Gugliuzza et al. 2023 ), exhibit tolerance of moderate salt levels. Therefore, oaks should be considered as potential tree species for restoration of salt-affected areas. In addition to selecting halophytes with superior salt tolerance (Ruan et al. 2010 ; Litalien et al. 2020), appropriate management techniques corresponding to soil regeneration are also required to improve ecosystem stability (Jesus et al. 2015 ). And phytoremediation with mycorrhizal fungi that can live in symbiosis with their host plants is one of the effective and sustainable methods. Mycorrhizal fungi have been long known to contribute to stress tolerance of plants, including salinity in various plants (Muhsin and Zwiazek, 2002 ; Li et al. 2012 ; Wen et al. 2022 ; Rose et al. 2024 ), including oak species (Arai et al. 2017 ; Guerrero-Galán et al. 2019 ). Studies have shown that inoculation with ectomycorrhizal fungi (EMF) can alleviate the toxic effects of salinity in Q. mongolica seedlings by promoting the uptake of K + and Ca 2+ , which helps regulate ionic balance by increasing K + /Na + and Ca 2+ /Na + ratios (Guo et al. 2022 ). Seedling height increased following inoculation, but the magnitude of the effect of different species of EMF varied (Guo et al. 2022 ). Another study has also shown that inoculation with EMF improved salt tolerance in Q. mongolica seedlings by increasing in plants the contents of soluble sugars, soluble proteins, and chlorophyll, in addition to enhancing leaf stomatal conductance (g s ) and intercellular CO 2 concentrations (C i ), suggesting that different species of EMF may improve salt tolerance in Quercus species via different mechanisms (Bai et al. 2021 ). Within the range of salt stress tolerated by the fungi, the host plants could be able to survive better. Moreover, the stabilization of the cooperation in mutualisms might be limited by the severity of environmental stress. Fungi themselves vary in salt tolerance (Bois et al. 2006 a). There has been, for example, some research published for different EMF cultivated on plates (Tang et al. 2009 ). Then if salt stress in the environment is exacerbated, the survival of the fungus is bound to suffer. The contested point we need to focus on is if mycorrhizas will offer any protection to plants when they are exposed to moderate and high salt concentrations. Also, maintaining mycorrhizas is costly to the plant since the fungi drain the plant from its carbon resources. The question is, if the plants are severely stressed with salt, will the fungus be still helpful or will it become a burden for the plant weakened by the salt. In the present study, we examined effects of the EMF L . bicolor on the responses of Japanese emperor oak ( Quercus dentata ) to relatively moderate (0.4% soil DW) and high (0.8% soil DW) NaCl concentrations. Q . dentata has been reported to have high genetic differentiation and diversity (Zhou et al. 2022 ) as well as superior salt tolerance even at the seedling stage (Hao et al. 2020 ). The trees of this species are broadly distributed across temperate zone of the northern hemisphere (Sork et al. 2022 ), and form symbiotic relationships with a wide range of EMF (Arai et al. 2017 ). Q . dentata has been widely used for vegetation restoration in fragile environments, especially in temperate regions (Qiu et al. 2023 ). Since EMF vary in their effectiveness in enhancing salt tolerance to trees, the choice of an appropriate fungal species for mycorrhization of Q. dentata could help with improving the restoration efforts of areas affected by salinity. As the first sequenced ectomycorrhizal fungus (Martin et al. 2008 ), L. bicolor forms ectomycorrhizal associations with a wide range of host tree species and has been successfully used to improve salinity tolerance in multiple host plants (Bois et al. 2006 a; Polanco et al. 2008 ; Xu et al. 2015 ). L. bicolor , therefore, can form symbioses with multiple host plants including oaks (Shi et al. 2017 ). However, most of the studies examined the effects of mycorrhization on plant responses to relatively low salinity levels and it is presently little known how effective the ectomycorrhizal fungi are in enhancing salt tolerance in plants facing the salinity levels in the soils that are classified as saline. We, therefore, carried out experiments with Q. dentata seedlings that were either non-inoculated or inoculated with the ectomycorrhizal fungus L. bicolor and exposed the seedlings to relatively moderate and high soil NaCl levels. We hypothesized that an inoculation of Q. dentata seedlings with L. bicolor will improve the salt tolerance of seedlings under both relatively moderate and high soil salinity conditions, but the effectiveness of the mycorrhization will be more pronounced under the lower level of soil salinity. Materials and methods Seed collection and germination Q. dentata acorns were collected from Zhongshan Park in Tsingtao, Shandong province, China and stored at 4℃. Large, healthy acorns were selected and soaked in 5% H 2 O 2 for 5 minutes, washed with water, and germinated in sand in a greenhouse (70% light transmittance, 25.1℃ average temperature, 80.4% average relative humidity during the experimental period). The growth substrate was composed of equal volumes of river sand and organic cultivation soil that had been sterilized twice using high-pressure steam (121℃, 20 min). After germination, the seedlings with a radicle of approximately the same length (2–3 cm) were selected and individually transferred into the separate pots (24 × 26 cm (diameter × height) filled with 5 kg soil) and watered daily with distilled water to sustain vigorous growth. The whole experiment had been performed under the cited greenhouse conditions. Fungal culture and inoculation The fungal strain L . bicolor UAMH8232 provided by the University of Alberta, Microfungus Collection and Herbarium, Edmonton, Canada., was cultured with modified Melin-Norkran’s medium (MMN medium) (Bois et al. 2006 a). After 2 weeks of cultivation in a shaking incubator (25℃, 120 rpm), the filtered and homogenized mycelia were diluted with distilled water to 0.5 mg dry mycelia/ml (Polanco et al. 2008 ) to make an inoculum. One week after germination, the potted plants were divided into two equal groups. The mycorrhizal group was inoculated with L. bicolor by adding 10 mL of mycelial suspension to the soil ( L.b ). The soil of the second group was injected with 10 mL distilled water to serve as the control check (CK). For the inoculation, four holes were made in the soil near the plants and the inoculum (2.5 mL) was added to each hole. The inoculation treatments were repeated every other week for the total of four times. NaCl treatments NaCl treatments started two months after the fourth root mycorrhizal inoculation. Referring to the classification criteria of saline soils in China, soil salinity over 0.6% is classified as saline soil (Wang et al. 1993). Therefore, in addition to the NaCl control (0% NaCl), we subjected the seedlings to two NaCl soil concentrations below (0.4% NaCl, moderate salinity treatment) and above (0.8% NaCl, high salinity treatment) this level. To avoid seedling shock response, salt solution was added gradually to the soil (the soil salinity was incrementally adjusted by adding NaCl solution eight times to ultimately achieve the predetermined salinity level). Overwatering and spilling were avoided during watering over the one month of treatment duration to ensure that the NaCl did not leach from the soil. There were ten replicated seedlings in each treatment group (one seedling per pot), for the total of sixty seedlings (two inoculation treatments × three salinity levels × ten replicates) in the experiment. Mycorrhizal colonization rate determination After one month of NaCl treatments, three seedlings were randomly selected from each treatment group. The first-order fine roots were carefully excised, washed with slow running water, and cut into ~ 1 cm long root segments. 20 root segments were randomly selected from each seedling to examine for the presence of fungal hyphae and mycorrhizal structures. The roots were stained (Phillips and Hayman 1970 ) and examined under the light microscope. The proportion of mycorrhizal root segments in the total number of observed roots was considered as the mycorrhizal colonization rate. Plant measurements At the end of the NaCl treatments, plant heights (H) and base stem diameters (BD) were measured. Plant height was measured between the base of the plant and the shoot tip and base stem diameter was measured with a Vernier caliper 1 cm above the soil surface. Before harvest, mature and fully expanded functional leaves were selected for instantaneous measurements of gas exchange parameters using a portable gas exchange measurement system (Li-6800, Li-Cor, Lincoln, NE, USA). Measurements were taken between 9:00 and 12:00 h on a sunny day. Net photosynthetic rate (A), transpiration rate (E), and stomatal conductance (g s ) were measured, and instantaneous water use efficiency (iWUE) was calculated as the ratio of A to E. Leaf chamber parameters were maintained at: leaf temperature, 28℃; relative air humidity, 70%; light intensity, 1000 µmol·m − 2 ·s − 1 ; and CO 2 concentration, 400 µmol·mol − 1 . Chlorophyll fluorescence parameters and P700 were measured simultaneously using a portable chlorophyll fluorometer (Dual-PAM-100, Walz, Effeltrich, Germany) set to dual-channel mode. After allowing leaves to adapt to the dark for 30 minutes, original fluorescence (Fo) was measured, then a saturating pulse (20000 µmol∙m − 2 ·s − 1 ) was performed to obtain the maximum fluorescence in the dark-adapted state (Fm) and the maximum absorption change in the dark-adapted state (Pm). After that actinic light (400 µmol·m − 2 ·s − 1 ) was turned on to drive photosynthesis for 40 s, following which, a saturation pulse was performed to record the maximum fluorescence intensity of PSII (Fm') and the maximum absorption change of PSI (Pm'). The steady-state fluorescence intensity (Ft) of PSII and steady-state absorption changes of PSI were recorded under continuous background light. Next, maximum quantum yield of PS II (Fv/Fm), photochemical quenching (qP), electron transport rate (ETR) and actual quantum yield (Y) of photosystem Ⅰ and II were calculated according to the following formula (Lu et al. 1999; Maxwell and Johnson, 2000 ): \(F_{O}^{\prime }=1/(1/{F_O} - 1/{F_M}+1/F_{M}^{\prime })\) \({F_V}/{F_M}=({F_M} - {F_O})/{F_M}\) \(qP=(F_{M}^{\prime } - Ft)/(F_{M}^{\prime } - F_{O}^{\prime })\) \(Y(II)=1 - Y(NPQ) - Y(NO)=(F_{M}^{\prime } - F)/F_{M}^{\prime }\) \(Y(I)=1 - Y(ND) - Y(NA)\) \(ETR(I)=0.5 \times Y(I) \times 0.84 \times PAR\) \(ETR(II)=0.5 \times Y(II) \times 0.84 \times PAR\) Where Y(NPQ) and Y(NO) correspond to the quantum yield of regulatory and non-regulatory energy dissipation, they can be obtained by the following formula. \(Y(NPQ)=\frac{F}{{F_{M}^{\prime }}} - \frac{F}{{{F_M}}}\) \(Y(NO)=\frac{F}{{{F_M}}}\) And Y(ND) corresponds to the fraction of P700 that is already oxidized by actinic light; Y(NA) corresponds to the fraction of P700 that are closed owing to acceptor side limitation, they can be obtained by the following formula. \(Y(ND)=\frac{{P{{700}_{OX}}}}{{{P_M}}}\) \(Y(NA)=\frac{{{P_M}}}{{P_{M}^{\prime }}}\) 0.5 is the fraction of absorbed light reaching PSI or PSII; and PAR is photosynthetically active radiation, we take 0.84 of the PAR as absorbed irradiance. The leaf blades were collected as above for chlorophyll concentration measurements. The leaf samples were weighed to determine fresh weights and soaked in 10 mL 95% ethanol in the dark until chlorophyll was fully leached out. The absorbance value of the extract was then determined by an ultraviolet-visible spectrophotometer (UV-9000s, Metash, Shanghai, China) at wavelengths of 649 and 665 nm, with 95% ethanol used as a blank control. The chlorophyll content was calculated using the formula (Lichtenthaler et al. 1983) as follows. \({\text{Chlorophyll a concentration (Chl a)}}={\text{13}}{\text{.95}} \times {\text{D665}} - {\text{6}}{\text{.88}} \times {\text{D649}}\) \({\text{Chlorophyll b concentration (Chl b)}}={\text{24}}{\text{.96}} \times {\text{D649}} - {\text{7}}{\text{.32}} \times {\text{D665}}\) \({\text{Chlorophyll content}}=\frac{{{\text{Chl}} \times {{\text{V}}_{{\text{leach liquor}}}}}}{{{\text{fresh weight}}}}\) \({\text{Total chlorophyll content (Chl t)}}={\text{Chl a content}}+{\text{Chl b content}}\) Where D649 and D665 correspond to the absorbance value of the extract measured at 649 and 665 nm wavelengths, respectively. Harvested plant material was sorted into leaves, stems, and roots. Leaf fresh weight was recorded and the leaf area of 10 mature and fully unfolded leaves was measured using a scanner (LiDE120, Canon, Tokyo, Japan). Leaf dry weight was measured after drying and used to calculate specific leaf area (SLA; leaf area/leaf dry weight) and leaf water content (LWC; (leaf fresh weight-leaf dry weight)/leaf dry weight). We present these two indicators together, rather than separately, in the growth indicators section. All plant fractions were heat treated at 105℃ for 30 min and oven-dried at 65℃ for 48 h, after which dry weight was collected for each portion. Seedling biomass allocation was assessed as the proportion of each part to total biomass and included root mass ratio (RMR), stem mass ratio (SMR), and leaf mass ratio (LMR). The root-shoot ratio (R/S) was calculated as the ratio of below- and above-ground plant dry weight. Next, all dried leaf, stem, and root tissues were powdered and packaged. 0.2 g from each fraction was digested by mixing with 30 mL HClO 4 and HNO 3 solution (v/v = 1:5) and heating to 70℃ for 10 min, followed by 40 min at 153℃ and 70 min at 190℃. After smoke in digestion tubes dispersed and the liquid had cooled, the transparent liquid was transferred to a 100 mL volumetric flask and diluted to volume with deionized water. CsCl solution was added to determine the concentration of sodium and potassium ions, and LaCl 3 solution was to determine concentrations of calcium and magnesium ions. Ion concentrations were measured using an atomic absorption spectrophotometer (AA-7000, Shimadzu, Kyoto, Japan) and calculated according to corresponding dilution ratios. Nitrogen content was determined for each fraction using the Kjeldahl method. 0.2 g of each fraction was mixed with catalyst catalyst (CuSO 4 , 0.2 g; K 2 SO 4 , 3 g) and 8 mL concentrated sulfuric acid, after which it was digested by heating to 200℃ for 40min followed by 400℃ for 40min. The liquid was transferred to a 100 mL volumetric flask and diluted with deionized water to volume. 10 mL of the liquid under test was taken to determined total nitrogen content of each part of the plant organs by a Kjeldahl apparatus (K9860, Hanon, Jinan, China). And another 5 mL liquid under test was transferred to a volumetric flask (50 mL) and diluted to volume in order to determine the phosphorus content using a spectrophotometer (UV-9000s, Metash, Shanghai, China) by ascorbic acid-molybdophosphate blue method. Statistical analysis Normality test and variance homogeneity test were performed on all data before testing analysis. The effects of fungal inoculation and salt treatment on plant physiological and growth indexes of Q. dentata seedlings were analyzed by two-way analysis of variance (ANOVA) using SPSS 25.0 (SPSS Inc., Chicago, USA). The responses of seedlings to different treatment combinations were analyzed by one-way analysis of variance (ANOVA). Before ANOVA analysis, data were tested for normality and homogeneity of variance first, and were log- or sqrt- transformed when necessary. Multiple comparisons were performed with chi-square test, and the significance levels were set at α = 0.05. There were five to six biological replicates for each measurement. Plotting was performed using Origin 2019 (OriginLab Co., MA, USA). Results Inoculation and mycorrhiza formation There were no ectomycorrhizal structures or hyphae detected in the control group (CK). The root colonization rates were similar in the NaCl control and 0.4% NaCl treatment. However, the root colonization rate was drastically reduced by the 0.8 NaCl treatment (Table 1). Table 1. Root colonization of Q. dentata seedlings in control group (CK) and inoculated seedlings ( L.b ) subjected to treatments with different NaCl concentrations. Different letters indicate significant differences between treatments ( p < 0.05, n = 3) determined by chi-square test. Treatment Soil salinity Root colonization rate (%) CK 0 0 0.4% NaCl 0 0.8% NaCl 0 L. b 0 48 ± 8 a 0.4% NaCl 50 ± 5 a 0.8% NaCl 22 ± 3 b Effects of L. bicolor inoculation on growth indicators under salt stress Prior to multiple comparisons, the effects of fungal inoculation, salt treatment, and interactions between the two was assessed using two-way ANOVA (Table S1). Multiple comparison revealed that there was no significant difference in H and BD of fungus-inoculated and non-inoculated seedlings under different salt stress conditions (Fig. 1 a; b). The leaf water content of both inoculated and CK seedlings under salt stress was significantly lower than that of CK without salt stress, and leaf water content decreased by more than 50% under high salt stress (Fig. 1 c). Compared to CK, leaf water content of inoculated seedlings was significantly lower under high salt stress, but was not significantly different compared to other salt treatments (Fig. 1 c). SLA increased with increasing salinity in both inoculated and non-inoculated seedlings. SLA was higher in the inoculated group compared to the non-inoculated group, but this difference was only significant under moderate salt stress conditions (Fig. 1 d). Generally, root, stem, leaf, and total plant biomass decreased with increasing salinity in both inoculated and non-inoculated seedlings. Inoculation significantly increased root biomass in CK seedlings under CK and moderate salt stress by 44.5% and 27.8%, respectively, compared to non-inoculated seedlings. There was no significant difference in root biomass between inoculated and CK seedlings under high salt stress (Fig. 2 a). Inoculation did not affect stem or leaf biomass under different salt stress treatments (Fig. 2 b; c). Total seedling biomass increased by 29.8% in the inoculated group under no salt stress (Fig. 2 d). Increased biomass in the inoculated group under no salt stress was mainly due to differences in root biomass. RMR decreased in both control and inoculated seedlings under salt stress. Inoculation increased RMR significantly, but only under no salt stress (Fig. 3 a). Neither inoculation nor NaCl treatment had a significant impact on seedling SMR (Fig. 3 b). In contrast to RMR, LMR was significantly higher under salt stress, but inoculation did not have an effect, regardless of salt stress treatment (Fig. 3 c). R/S followed the same trend as RMR, and inoculation significantly increased the root-shoot ratio of seedlings, but only those in the no salt stress group (Fig. 3 d). Effects of L. bicolor on physiological indicators under salt stress Two-way ANOVA revealed that inoculation did not significantly affect gas exchange parameters in seedlings (Table S1), while salt stress significantly affected most gas exchange parameters (Fig. 4 a ;b; d). Our findings suggest that plant physiological activity was increasingly restricted as salinity increased. We also noted that iWUE did not exhibit the same degree of change with increasing salinity as other gas exchange parameters (Fig. 4 c). Inoculation reduced stomatal conductance only in the no salt stress group (Fig. 4 d). Salt treatment significantly reduced chlorophyll a and total chlorophyll content. Both increased in inoculated seedlings, but this increase was significant only in the moderate salt stress treatment group (Fig. 5 a; c). Under salt stress, chlorophyll b content of inoculated seedlings was slightly higher than that of the non-inoculated control group, but the difference was not significant (Fig. 5 b). Furthermore, inoculation did not have a significant effect on seedlings’ chlorophyll a/b under different levels of salt stress. Chlorophyll a/b decreased with increasing soil salinity in both inoculated and control groups (Fig. 5 d). ETR and Y varied similarly. ETR and Y of PS II were not significantly affected by inoculation, but gradually decreased with increasing salinity (Fig. 6 c; d). ETR(I) and Y(I) both declined with increasing salinity, but the decline was exacerbated by inoculation under high-salt conditions (Fig. 6 a; b). Whereas Fv/Fm and qP declined only under high salt stress and, an effect that was exacerbated by inoculation, there were no significant changes in no-salt and low-salt environments (Fig. 6 e; f). With increasing soil salinity, Na + content of all seedling organs increased significantly. Inoculation did not significantly affect root and stem Na + content; compared to CK, however, leaf Na + content was significantly lower under moderate salt stress and significantly higher under high salt stress conditions (Fig. 7 a). Salt stress did not have a significant effect on root K + content; stem K + decreased with increasing salinity, while leaf K + content increased (Fig. 7 b). Fungal inoculation did not affect root or stem K + content, but increased the leaf K + content significantly under moderate salt stress conditions (Fig. 7 b). Na + /K + ratios increased with increasing salinity in all organs, and fungal inoculation lowered leaf Na + /K + ratios significantly only in low-salt environments (Fig. 7 e). There was no significant difference in root, stem, or leaf Ca 2+ and Mg 2+ content between inoculated and control seedlings under different salt treatments. Stem Ca 2+ only increased significantly in inoculated seedlings (Fig. 7 c; d). Except for root N concentration, which increased in CK seedlings but decreased in inoculated seedlings in high salt environments, neither salt treatment nor fungal inoculation had a significant effect on plant organ N (Fig. 8 a; c; e). Plant organ P content did not change significantly in response to NaCl treatment or fungal inoculation (Fig. 8 b; d; f). Discussion Response of Q. dentata seedlings to NaCl treatment The NaCl treatment appeared to have a greater impact on Q. dentata seedlings than inoculation with L. bicolor (Table S1), both in terms of growth and physiological indicators. Most parameters change monotonically with increasing salinity. Results also show that one-year old Q. dentata seedlings were resistant to relatively low salt stress and that increasing concentrations of NaCl exacerbated negative effects on seedling growth (Fig. 9 ). This is consistent with previous work reporting salt tolerance in oaks, even at the seedling stage (Wang et al. 2014 ; Gugliuzza et al. 2023 ). Other research found that Q. dentata is also resistant to NaCl stress within a certain range (Hao et al. 2020 ). Whether they were inoculated or not, seedlings were able to survive in saline environments (0-0.8%), exhibiting intermediate and considerable NaCl tolerance compared with salt-tolerant species planted in afforestation projects, such as Q. virginiana , Q. acutissima (Wang et al. 2014 ), and Robinia pseudoacacia (Mao et al. 2016 ). Our findings showing that Q. dentata has a certain degree of salt tolerance is promising for saline-alkaline land restoration. With increasing salinity (0.4%), the negative effects of NaCl on seedling growth caused a decrease in plant biomass (Fig. 2 ). Seedlings altered nutrient and biomass distribution ratios by increasing leaf biomass ratio and by decreasing root biomass ratio and root to shoot ratio (Fig. 3 ) to reduce salt uptake and upward transport by roots and to prioritize resource allocation to leaves for photosynthesis. This pattern of biomass distribution has also been observed in Ceriops tagal (Patel et al. 2010 ), a mangrove halophyte. An opposite, upward trend has been reported in Picea glauca and Pinus banksiana (Bois et al. 2006 a), even in individuals inoculated with L. bicolor . As soil salt content increases further (0.8%), sodium ion toxicity occurs, plant water-salt balance is disrupted, leaf tissue water loss is more serious, and leaves wither or senesce (Horie et al. 2009 ; Hammer et al. 2011 ; Heng et al. 2022 ). As a result, osmotic stress and ionic toxicity become stronger. Total chlorophyll content decreased significantly with increasing salt stress (Fig. 5 c). This could be caused by two reasons. On the one hand, ion toxicity resulting from excess Na + restricts binding between chlorophyll and pigment proteins and destroys chloroplast structures ( e.g ., thylakoid membranes), causing chloroplast damage and dysfunction and reducing chlorophyll synthesis (Pardo et al. 2010), on the other hand, our analysis revealed equivalent changes between chlorophyll a and total chlorophyll content, with both decreasing as salt stress intensified (Fig. 5 ). NaCl could have increased the activity of chlorophyllase and accelerated the degradation of chlorophyll a under the action of reactive oxygen species (Li et al. 2015 ). The greater sensitivity to salt stress of chlorophyll a relative to chlorophyll b in seedlings is reflected in lower ratios of chlorophyll a/b (Akram, 2014 ). In addition, plant photosynthetic capacity was lower under NaCl treatment. This was reflected not only in lower chlorophyll content, but also in stomatal limitation and altered electron transport (Van Zelm et al. 2020 ). Gas exchange parameters, such as net photosynthetic rate, transpiration rate, and stomatal conductance, decreased significantly, decreased under all salt stress treatments (Fig. 4 ), indicating that seedling photosynthesis was sensitive to increasing soil salt (Sudhir et al. 2004). The decrease in photosynthetic efficiency under salt stress can be attributed to stomatal limitation occurring because of decreased stomatal conductance (Bose et al. 2017 ), which could be caused by decreased turgor pressure in guard cells resulting from water deficits in leaf tissues (Akram, 2014 ; Tombesi et al. 2015 ). Salt stress can also reduce chlorophyll thylakoid membrane stacking and stability, restricting electron flow from PS II to PS I and blocking photosynthesis (Zahra et al. 2022 ). Here, ETR and Y of both PS I and PS II decreased with increasing salt concentration (Fig. 6 ), suggesting that salt stress attenuates the absorption and conversion efficiency of light energy by leaves, inhibits electron transfer in photosystem reaction centers, and weakens photosynthesis (Gururani et al. 2015 ). Declines in qP and Fv/Fm were not significant under relatively low salt conditions, indicating that PS II has a certain salt tolerance for the conversion capacity of photosynthesis. However, declines were significant under high salt conditions, while the photodamage occurring under saline conditions results in poor PS II function (Hameed et al. 2021). Positive effects of L. bicolor on Q. dentata seedlings under moderate salinity EMF partner with many woody plants and usually maintain reciprocal symbiosis (Genre et al. 2020 ). Plants provide fungi with a portion of the carbohydrate fixed by photosynthesis and fungi help the plant absorb water and nutrients in return, improving resilience (Bahadur et al. 2019 ). Here, inoculation worked in Q. dentata seedlings and played a beneficial role in non-hypersaline environments. We found that inoculation under salt-free conditions significantly increased root biomass of Q. dentata seedlings, resulting in higher total biomass for the inoculated group compared to CK. Root-shoot ratio was also significantly higher among inoculated seedlings, indicating that colonization by EMF promotes root growth in Q. dentata seedlings and increases the proportion of resources allocated belowground, enhancing plant uptake and storage of water and nutrients (Wang et al. 2008 ). In addition, we also found that symbiosis with L. bicolor improves seedling salt tolerance under moderate salinity. Under relatively low NaCl stress, L. bicolor induced host plants to expand their roots (Fig. 2 a) to alleviate osmotic stress caused and to supply leaves with adequate water (Fig. 1 c), representing a trade-off between its own water absorption capacity and the osmotic pressure caused by salt stress. Likewise, ion toxicity to plant leaves decreased proportionally with decreasing sodium-potassium ratios in relation to sodium ion concentrations (Fig. 7 a; e), which resulted from increased water supply to leaves. As a result, the total chlorophyll content of Q. dentata seedlings increased with increasing chlorophyll a content under relatively low salt stress (Fig. 5 a; c) to mitigate the degradative effects of salt stress (Al-Khaliel et al. 2010). This is consistent with previous research and is probably due to the fact that the symbiotic fungus secretes growth regulators such as cytokinins, thereby increasing leaf chlorophyll content (Tilak et al. 2006 ). Our findings suggest that symbiosis with L. bicolor has a positive effect on Q. dentata seedling growth and can promote seedling growth under moderate salt stress. Negative effects of L. bicolor on Q. dentata seedlings under high salinity Symbiotic relationships between mycorrhizal fungi and host plants are not unbreakable (Douglas et al. 2008), and cooperation becomes uneven when resource exchange is diminished or stops in response to environmental stress (Bonfante et al. 2010; Kiers et al. 2011 ). As soil salinity gradually exceeds the normal tolerance range of plants, the relationship between symbiotic mycorrhiza and host plant becomes more subtle (Johnson et al. 1997 ; Kaldorf et al. 2005 ). The effects of EMF inoculation on various indicators like tissue biomass, gas exchange parameters, chlorophyll content, chlorophyll fluorescence parameters, and sodium-potassium ratio were weaker under high salt stress. Regardless, mycorrhizal association did not provide any benefit to host plants when seedlings were unable to withstand the damage caused by salt stress (Fig. 9 ). At the same time, we found that moderate NaCl promotes the growth of L. bicolor mycelium, the colony diameters on the seventh day were 33.3 ± 0.7 mm, 45.7 ± 1.2 mm, and 44.9 ± 2.1 mm at 0, 0.4%, and 0.8% medium salinity, respectively (Table S2), consistent with previous research demonstrating the salt tolerance of L. bicolor (Bois et al. 2006 b). As salinity increases, the ability of the fungus to generate its own growth as well as its vigour to colonise and live in symbiosis with the host plant decreases, in particular mycorrhizal colonisation rates are significantly lower under high salt conditions than they are under no-salt and medium-salt conditions (Table 1 ). The high salinity environment could raise the threshold of fungal competitiveness to host plants, L. bicolor that colonise Q. dentata seedlings not only become less numerous, but also play a negative role in plant growth. In relatively harsh environments, where fungus is restricted in its own growth and its protective effect on plants from NaCl is lost, the limited available resources made by the seedlings face intense competition from both the fungus and the host plant, and the fungus will rob the survival resources from the plants thereby inhibiting there growth. Thus, host plants are unable to meet mycorrhizal demand for resources, which remain high under saline conditions, and exacerbated nutrient and water stress reduce root nitrogen (Fig. 8 a) and leaf water content (Fig. 1 c). As the main photosynthetic organ, leaves accumulate sodium when water is lost (Flowers et al. 2015 ) and experience worsening ion toxicity (Fig. 7 a). Chlorophyll fluorescence parameters, which are sensitive to ion penetration and water-salt balance, also decrease (Fig. 6 ). Notably, our results suggest that leaf Na + content is a sensitive and critical indicator of fungal colonization under different salt stress conditions. That is, when plant growth is limited primarily by environmental salt stress, the physiological changes induced by a plant’s association with symbiotic mycorrhizal fungi are most obvious in leaf Na + content (Fig. 7 ). Inoculation with L. bicolor reduces photosystem efficiency in highly saline soil. Moreover, the effect of salt stress on photosynthetic efficiency is most apparent in changes in ETR and Y of photosystem I, which are more sensitive to the effects of fungal inoculation than those of photosystem II (Fig. 6 ). Severe salinity decreases PSII stability and donor side intactness (Zahra et al. 2022 ) which were maintained under relatively low salt stress (Fig. 6 e; f). Fungal inoculation further jeopardizes the stability of the photosynthetic apparatus under high salt conditions (Fig. 6 ). These results suggest that symbiosis with L. bicolor can help Q. dentata seedlings maintain photosynthesis and manage oxidative stress under moderate salt stress, but that it fails under high salt stress. Indicators such as chlorophyll fluorescence parameters and metal ion content, therefore, should be monitored when mycorrhizal symbiosis is part of afforestation strategies in saline-alkali soil. Implications for afforestation activities in saline areas Many oak species are tolerant to salt stress and have potential applications in land afforestation in saline-alkali soil (Li et al. 2015 ; Gugliuzza et al. 2023 ). Q. dentata , which acts as a pioneer during naturalized developmental vegetation restoration (Qiu et al. 2023 ), exhibits somewhat improved salt tolerance when associated with EMF (Fig. 9 ). Mycorrhizal fungi have a strong regulatory effect on root exudation and nitrogen uptake in temperate tree species (Liese et al. 2018 ). Afforestation studies suggest that EMF symbiosis during silviculture can enhance carbon sequestration and improve soil properties (Hong et al. 2018 ; Yuan et al. 2023 ). Although it is possible that afforestation, combined with carefully selected EMF symbionts, can improve saline-alkali soil (Huang et al. 2008 ; Qin et al. 2017 ), the mechanisms underpinning these improvements and the potential use of EMF, including L. bicolor , are still poorly understood. It appears that in this study, L. bicolor was effective only under the moderate salinity level because the fungus itself could not tolerate higher salt concentration as shown by a reduction in root colonization under the high salt concentration (Table 1 ). It’s proved that the survival and mycelial extension of L. bicolor cultured in vitro were not significantly affected under higher salt concentration (Table S2). It’ a fact that L. bicolor is one kind of mycorrhizal fungi with strong saline-alkali tolerance (Kernaghan et al. 2002 ) and even certain salinity could promote the proliferation of fungi in vitro (Table S2). while the higher salinity level might restrict the inoculation of EMF as shown by less colonization density on the same individual (Table 1 ). Because of the decline in co-benefits, the probability of L. bicolor to refuse to inoculate plants increase under high salinity concentration. Therefore, the effectiveness of the fungus in conferring salt tolerance to plants can be only up to the salt concentration that the fungus can tolerate itself. It is the salt concentration in vitro that may not only be too high for the mycorrhizal fungi combined with plants in afforestation to be survival, but also determines the activeness and effectiveness in conferring salt protection to plants. Therefore, it is necessary to consider the effect of fungal cooperative afforestation according to the requirements and adaptability of applied fungi to the soil environment. Furthermore, a deeper understanding is needed of the long-term effects of salinity on Q. dentata - L. bicolor symbionts and the underlying mechanisms. Conclusions In our study, mycorrhization with L. bicolor had either positive or negative impact on plant responses to NaCl depending on the soil salt. Here, this association helped plants cope with salt stress by promoting root growth, increasing light capture area, promoting chlorophyll accumulation, and regulating ion balance under moderate salt stress. In contrast, EMF association under high salt stress exacerbated nutrient stress, water stress, and Na + toxicity, leading to a decrease in plant photosynthetic activity, rate, and yield. Therefore, mycorrhizal seedlings inoculated with EMF can be considered for vegetation restoration in mildly saline soil, which supports Quercus growth. However, when the soil salinity is high beyond a certain range, it is necessary to fully consider not only the salt tolerance of Quercus themselves, but also the survival of symbiotic fungi in saline environment and the actual effect of mycorrhizal symbiosis, under which mycorrhizal means may not be applicable. Collectively, afforestation with Q. dentata seedlings combined with symbiosis with L. bicolor is a reasonable choice with broad application prospects. However, additional research investigating the genetic and molecular mechanisms driving enhanced salt tolerance is needed to maximize the benefits of the interaction between L. bicolor and Q. dentata . Declarations Competing Interests The authors declare they have no relevant financial or non-financial interests to disclose. Funding This study was funded by National Natural Science Foundation of China (No. 32471580), Natural Science Foundation of Shandong Province, China (No. ZR2020MC035; ZR2024MC012), Shandong Province "Double-Hundred Talent Plan" Project (No. WSG2018023), Seed Funding for International Scientific Research Cooperation of Shandong University (No. WSG2018023) and Key Research and Development Program of Shandong Province, China(No. 2021CXGC010803). Author Contribution All authors contributed to the study conception and design. Material preparation, data collection and analysis were performed by Wenlong Sun, Haonan Chen, Yixin Song and Luyu Qi. 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Plant Physiol Bioch 178: 55-69. https://doi.org/10.1016/j.plaphy.2022.03.003 Zhou BF, Shi Y, Chen XY, Yuan S, Liang YY, Wang BS (2022) Linked selection, ancient polymorphism, and ecological adaptation shape the genomic landscape of divergence in Quercus dentata . J Syst Evol 60(6): 1344-1357. https://doi.org/10.1111/jse.12817 Additional Declarations No competing interests reported. Supplementary Files SupplementalInformation.docx Cite Share Download PDF Status: Posted Version 1 posted You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. As a division of Research Square Company, we’re committed to making research communication faster, fairer, and more useful. We do this by developing innovative software and high quality services for the global research community. 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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-5474395","acceptedTermsAndConditions":true,"allowDirectSubmit":true,"archivedVersions":[],"articleType":"Research Article","associatedPublications":[],"authors":[{"id":381078918,"identity":"139525cb-167b-445e-8371-91825c85acf3","order_by":0,"name":"Wenlong Sun","email":"","orcid":"","institution":"Shandong University","correspondingAuthor":false,"prefix":"","firstName":"Wenlong","middleName":"","lastName":"Sun","suffix":""},{"id":381078919,"identity":"661b2df0-d646-4c1c-b751-199ea61cd5cc","order_by":1,"name":"Luyu Qi","email":"","orcid":"","institution":"Shandong University","correspondingAuthor":false,"prefix":"","firstName":"Luyu","middleName":"","lastName":"Qi","suffix":""},{"id":381078920,"identity":"c04188f2-bea0-4271-9602-39a65bfff8a0","order_by":2,"name":"Haonan Chen","email":"","orcid":"","institution":"Shandong University","correspondingAuthor":false,"prefix":"","firstName":"Haonan","middleName":"","lastName":"Chen","suffix":""},{"id":381078921,"identity":"73160d16-1147-4f0f-a914-12a2f6f7c1a3","order_by":3,"name":"Yixin Song","email":"","orcid":"","institution":"Shandong University","correspondingAuthor":false,"prefix":"","firstName":"Yixin","middleName":"","lastName":"Song","suffix":""},{"id":381078922,"identity":"6fd6e344-940e-463a-b006-f54829f88581","order_by":4,"name":"Jiaqi Jiang","email":"","orcid":"","institution":"Shandong University","correspondingAuthor":false,"prefix":"","firstName":"Jiaqi","middleName":"","lastName":"Jiang","suffix":""},{"id":381078923,"identity":"725b3023-822a-402c-ae72-c8301ca6e9cd","order_by":5,"name":"Puyi Zhang","email":"","orcid":"","institution":"Shandong University","correspondingAuthor":false,"prefix":"","firstName":"Puyi","middleName":"","lastName":"Zhang","suffix":""},{"id":381078924,"identity":"f3e20fba-08b7-4496-a4a5-a1d13332efc1","order_by":6,"name":"Bojian Wang","email":"","orcid":"","institution":"","correspondingAuthor":false,"prefix":"","firstName":"Bojian","middleName":"","lastName":"Wang","suffix":""},{"id":381078925,"identity":"78fcf609-51dc-4ea8-826e-539a8c655b00","order_by":7,"name":"Qiang Wang","email":"","orcid":"","institution":"","correspondingAuthor":false,"prefix":"","firstName":"Qiang","middleName":"","lastName":"Wang","suffix":""},{"id":381078926,"identity":"9320e8f4-b877-4299-bf0f-6c731421e77a","order_by":8,"name":"Gaode Meng","email":"","orcid":"","institution":"Shandong University","correspondingAuthor":false,"prefix":"","firstName":"Gaode","middleName":"","lastName":"Meng","suffix":""},{"id":381078927,"identity":"78fb9cdd-370a-4779-a261-51a646d84080","order_by":9,"name":"Tianyu Ji","email":"","orcid":"","institution":"Shandong University","correspondingAuthor":false,"prefix":"","firstName":"Tianyu","middleName":"","lastName":"Ji","suffix":""},{"id":381078928,"identity":"d9c57cbb-0043-4289-bd7c-fb8d80958ba1","order_by":10,"name":"Xinke Sun","email":"","orcid":"","institution":"Shandong University","correspondingAuthor":false,"prefix":"","firstName":"Xinke","middleName":"","lastName":"Sun","suffix":""},{"id":381078929,"identity":"7262425d-12f3-47fb-b5b4-da6377fa3212","order_by":11,"name":"Weihua Guo","email":"","orcid":"","institution":"Shandong University","correspondingAuthor":false,"prefix":"","firstName":"Weihua","middleName":"","lastName":"Guo","suffix":""},{"id":381078930,"identity":"0969b098-2d1b-4784-a86c-e4aea24140fb","order_by":12,"name":"Ning Du","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAAAyElEQVRIiWNgGAWjYDCCA2AEYjAfOPChgjQtbIkHZ5whUguUwWN8mLeFCB18N3IPHi74xWDPdyPnwwHeBgZ5frED+LVI3shLODyzjyFx5o3cDQckdzAYzpydgF+LwY0cg8O8PQwJBiAthmeAjNtEarEHMh4cSGwjVgvPDwbGDTdyGA4cJEaL5Jk3QFsagH4588zgYMMZCcJ+4TueY/yZ5w8wxI4nP/78p8JGnl+agBYwYGz7D2NKEKEcDP4Qq3AUjIJRMApGJAAAPKVRFtCAynEAAAAASUVORK5CYII=","orcid":"","institution":"Shandong University","correspondingAuthor":true,"prefix":"","firstName":"Ning","middleName":"","lastName":"Du","suffix":""},{"id":381078931,"identity":"1c88bd2c-8cf9-4e08-89f5-25e919ae966a","order_by":13,"name":"Janusz J. Zwiazek","email":"","orcid":"","institution":"University of Alberta","correspondingAuthor":false,"prefix":"","firstName":"Janusz","middleName":"J.","lastName":"Zwiazek","suffix":""}],"badges":[],"createdAt":"2024-11-18 09:08:09","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-5474395/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-5474395/v1","draftVersion":[],"editorialEvents":[],"editorialNote":"","failedWorkflow":false,"files":[{"id":70466238,"identity":"fe34b3dc-0d9e-49f8-b9cc-b4ddf31605b3","added_by":"auto","created_at":"2024-12-03 12:35:45","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":557528,"visible":true,"origin":"","legend":"\u003cp\u003eGrowth traits and leaf water content of \u003cem\u003eQ. dentata\u003c/em\u003eseedlings in the control (CK) and inoculation (\u003cem\u003eL.b\u003c/em\u003e)groups subjected to different NaCl treatment concentrations. Bars represent means of six replicates (±SE). For each parameter, bars with different letters indicate significant differences among treatments (\u003cem\u003ep\u003c/em\u003e ≤ 0.05) determined by one-way analysis of variance (ANOVA).\u003c/p\u003e","description":"","filename":"floatimage1.png","url":"https://assets-eu.researchsquare.com/files/rs-5474395/v1/98d714625f985306596f615d.png"},{"id":70466243,"identity":"6396d75f-f3f1-43bd-86bd-9125f1524221","added_by":"auto","created_at":"2024-12-03 12:35:45","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":482178,"visible":true,"origin":"","legend":"\u003cp\u003eBiomass of \u003cem\u003eQ. dentata\u003c/em\u003eseedlings in the control (CK) and inoculation (\u003cem\u003eL.b\u003c/em\u003e) groups subjected to different NaCl treatment concentrations. Bars represent means of six replicates (±SE). For each parameter, bars with different letters indicate significant differences among treatments (\u003cem\u003ep\u003c/em\u003e ≤ 0.05) determined by one-way ANOVA.\u003c/p\u003e","description":"","filename":"floatimage2.png","url":"https://assets-eu.researchsquare.com/files/rs-5474395/v1/9697a01553097531aeea96d8.png"},{"id":70467509,"identity":"5cff5d47-c7a3-4820-beb7-2ba5db15e7dd","added_by":"auto","created_at":"2024-12-03 12:43:45","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":515801,"visible":true,"origin":"","legend":"\u003cp\u003eBiomass allocation of \u003cem\u003eQ. dentata\u003c/em\u003e seedlings in the control (CK) and inoculation (\u003cem\u003eL.b\u003c/em\u003e) groups subjected to different NaCl treatment concentrations. (a) root mass ratio, (b) stem mass ratio, (c) leaf mass ratio, (d) root-shoot ratio. Bars represent means of six replicates (±SE). For each parameter, bars with different letters indicate significant differences among treatments (\u003cem\u003ep\u003c/em\u003e ≤ 0.05) determined by one-way ANOVA.\u003c/p\u003e","description":"","filename":"floatimage3.png","url":"https://assets-eu.researchsquare.com/files/rs-5474395/v1/b0574511cde9d080e809f386.png"},{"id":70467508,"identity":"6b24ebb9-8a3a-4813-aa3d-369024bb3dea","added_by":"auto","created_at":"2024-12-03 12:43:45","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":546016,"visible":true,"origin":"","legend":"\u003cp\u003eGas exchange parameters of \u003cem\u003eQ. dentata\u003c/em\u003e seedlings in the control (CK) and inoculation (\u003cem\u003eL.b\u003c/em\u003e) groups subjected to different NaCl treatment concentrations. Bars represent means of five replicates (±SE). For each parameter, bars with different letters indicate significant differences among treatments (\u003cem\u003ep\u003c/em\u003e ≤ 0.05) determined by one-way ANOVA.\u003c/p\u003e","description":"","filename":"floatimage4.png","url":"https://assets-eu.researchsquare.com/files/rs-5474395/v1/0340d9f306122a881dbbcdcc.png"},{"id":70467510,"identity":"5779dc2b-1a9b-4bc0-a616-a6ad2634a329","added_by":"auto","created_at":"2024-12-03 12:43:45","extension":"png","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":555554,"visible":true,"origin":"","legend":"\u003cp\u003eLeaf chlorophyll content of \u003cem\u003eQ. dentata\u003c/em\u003e seedlings in the control (CK) and inoculation (\u003cem\u003eL.b\u003c/em\u003e) groups subjected to different NaCl treatment concentrations. Bars represent means of five replicates (±SE). For each parameter, bars with different letters indicate significant differences among treatments (\u003cem\u003ep\u003c/em\u003e ≤ 0.05) determined by one-way ANOVA.\u003c/p\u003e","description":"","filename":"floatimage5.png","url":"https://assets-eu.researchsquare.com/files/rs-5474395/v1/191fbe43b0f2b634eb5bb293.png"},{"id":70466241,"identity":"d8cb20a7-b6e4-43be-833a-06e32337d881","added_by":"auto","created_at":"2024-12-03 12:35:45","extension":"png","order_by":6,"title":"Figure 6","display":"","copyAsset":false,"role":"figure","size":743750,"visible":true,"origin":"","legend":"\u003cp\u003eChlorophyll fluorescence parameters of \u003cem\u003eQ. dentata\u003c/em\u003eseedlings in the control (CK) and inoculation (\u003cem\u003eL.b\u003c/em\u003e) groups subjected to different NaCl treatment concentrations. Bars represent means of five replicates (±SE). For each parameter, bars with different letters indicate significant differences among treatments (\u003cem\u003ep\u003c/em\u003e ≤ 0.05) determined by one-way ANOVA.\u003c/p\u003e","description":"","filename":"floatimage6.png","url":"https://assets-eu.researchsquare.com/files/rs-5474395/v1/fcc68627cf9de6208fe5ba2f.png"},{"id":70466246,"identity":"a8ba93c0-0079-4cf4-8b4b-69a42370a894","added_by":"auto","created_at":"2024-12-03 12:35:45","extension":"png","order_by":7,"title":"Figure 7","display":"","copyAsset":false,"role":"figure","size":1053168,"visible":true,"origin":"","legend":"\u003cp\u003eMetal ion content and ratio of \u003cem\u003eQ. dentata\u003c/em\u003e seedlings in the control (CK) and inoculation (\u003cem\u003eL.b\u003c/em\u003e) groups subjected to different NaCl treatment concentrations. Bars represent means of five replicates (±SE). For each parameter, bars with different letters indicate significant differences among treatments (\u003cem\u003ep\u003c/em\u003e ≤ 0.05) determined by one-way ANOVA.\u003c/p\u003e","description":"","filename":"floatimage7.png","url":"https://assets-eu.researchsquare.com/files/rs-5474395/v1/4fd19ca5b9f8f4b5a10f4bcf.png"},{"id":70466247,"identity":"21fd2109-5d44-44b5-b3bc-ec5082bb7833","added_by":"auto","created_at":"2024-12-03 12:35:46","extension":"png","order_by":8,"title":"Figure 8","display":"","copyAsset":false,"role":"figure","size":877299,"visible":true,"origin":"","legend":"\u003cp\u003eNutrient concentration of \u003cem\u003eQ. dentata\u003c/em\u003e seedlings in the control (CK) and inoculation (\u003cem\u003eL.b\u003c/em\u003e) groups subjected to different NaCl treatment concentrations. Bars represent means of five replicates (±SE). For each parameter, bars with different letters indicate significant differences among treatments (\u003cem\u003ep\u003c/em\u003e ≤ 0.05) determined by one-way ANOVA.\u003c/p\u003e","description":"","filename":"floatimage8.png","url":"https://assets-eu.researchsquare.com/files/rs-5474395/v1/1c8342300cf96eda3bee846c.png"},{"id":70466244,"identity":"e3e41a6c-835c-4295-a7f1-556f130f9359","added_by":"auto","created_at":"2024-12-03 12:35:45","extension":"png","order_by":9,"title":"Figure 9","display":"","copyAsset":false,"role":"figure","size":529355,"visible":true,"origin":"","legend":"\u003cp\u003eConceptual model summarizing the effects of \u003cem\u003eL. bicolor\u003c/em\u003e inoculation on \u003cem\u003eQ. dentata\u003c/em\u003e seedlings under saline conditions\u003c/p\u003e","description":"","filename":"floatimage9.png","url":"https://assets-eu.researchsquare.com/files/rs-5474395/v1/ed0c918b926a1b1591c12e32.png"},{"id":70466239,"identity":"d43ba25e-2c06-430a-b52e-26fb16ccb74b","added_by":"auto","created_at":"2024-12-03 12:35:45","extension":"docx","order_by":1,"title":"","display":"","copyAsset":false,"role":"supplement","size":1008712,"visible":true,"origin":"","legend":"","description":"","filename":"SupplementalInformation.docx","url":"https://assets-eu.researchsquare.com/files/rs-5474395/v1/dd4d58ffbb178601574eb94c.docx"}],"financialInterests":"No competing interests reported.","formattedTitle":"Mycorrhization of Quercus dentata seedlings with Laccaria bicolor enhances salt tolerance of plants only under relatively moderate soil salinity level","fulltext":[{"header":"Introduction","content":"\u003cp\u003eIncreasing soil salinization is one of the major and widespread soil factors that threatens plant survival and affects their growth and yield in many ecosystems (Kopittke et al. \u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e2019\u003c/span\u003e; Singh et al. 2021). Combined with progressing global climate changes and human activities, major disturbances of the ecosystems could be expected and cause catastrophic collapse of the ecosystem states and services (Negacz et al. \u003cspan citationid=\"CR49\" class=\"CitationRef\"\u003e2022\u003c/span\u003e). To achieve sustainability of agricultural production and stability of ecosystems, it is essential to prevent further salinization of soils and to develop knowledge required to manage salt-affected sites to restore their productivity (Ullah et al. \u003cspan citationid=\"CR66\" class=\"CitationRef\"\u003e2017\u003c/span\u003e; Mukhopadhyay et al. \u003cspan citationid=\"CR48\" class=\"CitationRef\"\u003e2021\u003c/span\u003e). It is generally recognized that phytoremediation and revegetation of salinized land are appropriate, but challenging, tasks that can mitigate soil salinization (Crooks et al. \u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e2018\u003c/span\u003e; Litalien et al. 2020). For the revegetation processes, species selection and remediation methods are especially critical and may be combined with an enhancement of plant salt tolerance by beneficial microorganisms (Van Zelm et al. \u003cspan citationid=\"CR67\" class=\"CitationRef\"\u003e2020\u003c/span\u003e; Hao et al. \u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e2021\u003c/span\u003e).\u003c/p\u003e \u003cp\u003ePlant species commonly used in saline phytoremediation are mainly halophytes (Ruan et al. \u003cspan citationid=\"CR57\" class=\"CitationRef\"\u003e2010\u003c/span\u003e; Manousaki et al. 2011b; Stavi et al. \u003cspan citationid=\"CR61\" class=\"CitationRef\"\u003e2021\u003c/span\u003e), which have been employed to restore landscapes damaged by a variety of contaminants and disturbances (Manousaki et al. 2011a; Liang et al. \u003cspan citationid=\"CR37\" class=\"CitationRef\"\u003e2017\u003c/span\u003e). However, other salt-tolerant plant species have been also considered for their suitability of restoration of moderately saline areas. \u003cem\u003eQuercus\u003c/em\u003e is considered to be a tree genus with a successful evolutionary history of adaptation to changing and complex environments (Kremer et al. 2019; Sork et al. \u003cspan citationid=\"CR60\" class=\"CitationRef\"\u003e2022\u003c/span\u003e). Studies have shown that some species of \u003cem\u003eQuercus\u003c/em\u003e, including \u003cem\u003eQuercus robur\u003c/em\u003e (Alaoui-Soss\u0026eacute; et al. \u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e1998\u003c/span\u003e), Q. \u003cem\u003evirginiana\u003c/em\u003e (Wang et al. \u003cspan citationid=\"CR69\" class=\"CitationRef\"\u003e2014\u003c/span\u003e)d \u003cem\u003eilex\u003c/em\u003e (Gugliuzza et al. \u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e2023\u003c/span\u003e), exhibit tolerance of moderate salt levels. Therefore, oaks should be considered as potential tree species for restoration of salt-affected areas.\u003c/p\u003e \u003cp\u003eIn addition to selecting halophytes with superior salt tolerance (Ruan et al. \u003cspan citationid=\"CR57\" class=\"CitationRef\"\u003e2010\u003c/span\u003e; Litalien et al. 2020), appropriate management techniques corresponding to soil regeneration are also required to improve ecosystem stability (Jesus et al. \u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e2015\u003c/span\u003e). And phytoremediation with mycorrhizal fungi that can live in symbiosis with their host plants is one of the effective and sustainable methods. Mycorrhizal fungi have been long known to contribute to stress tolerance of plants, including salinity in various plants (Muhsin and Zwiazek, \u003cspan citationid=\"CR47\" class=\"CitationRef\"\u003e2002\u003c/span\u003e; Li et al. \u003cspan citationid=\"CR34\" class=\"CitationRef\"\u003e2012\u003c/span\u003e; Wen et al. \u003cspan citationid=\"CR71\" class=\"CitationRef\"\u003e2022\u003c/span\u003e; Rose et al. \u003cspan citationid=\"CR56\" class=\"CitationRef\"\u003e2024\u003c/span\u003e), including oak species (Arai et al. \u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e2017\u003c/span\u003e; Guerrero-Gal\u0026aacute;n et al. \u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e2019\u003c/span\u003e). Studies have shown that inoculation with ectomycorrhizal fungi (EMF) can alleviate the toxic effects of salinity in \u003cem\u003eQ. mongolica\u003c/em\u003e seedlings by promoting the uptake of K\u003csup\u003e+\u003c/sup\u003e and Ca\u003csup\u003e2+\u003c/sup\u003e, which helps regulate ionic balance by increasing K\u003csup\u003e+\u003c/sup\u003e/Na\u003csup\u003e+\u003c/sup\u003e and Ca\u003csup\u003e2+\u003c/sup\u003e/Na\u003csup\u003e+\u003c/sup\u003e ratios (Guo et al. \u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e2022\u003c/span\u003e). Seedling height increased following inoculation, but the magnitude of the effect of different species of EMF varied (Guo et al. \u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e2022\u003c/span\u003e). Another study has also shown that inoculation with EMF improved salt tolerance in \u003cem\u003eQ. mongolica\u003c/em\u003e seedlings by increasing in plants the contents of soluble sugars, soluble proteins, and chlorophyll, in addition to enhancing leaf stomatal conductance (g\u003csub\u003es\u003c/sub\u003e) and intercellular CO\u003csub\u003e2\u003c/sub\u003e concentrations (C\u003csub\u003ei\u003c/sub\u003e), suggesting that different species of EMF may improve salt tolerance in \u003cem\u003eQuercus\u003c/em\u003e species via different mechanisms (Bai et al. \u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e2021\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eWithin the range of salt stress tolerated by the fungi, the host plants could be able to survive better. Moreover, the stabilization of the cooperation in mutualisms might be limited by the severity of environmental stress. Fungi themselves vary in salt tolerance (Bois et al. \u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e2006\u003c/span\u003ea). There has been, for example, some research published for different EMF cultivated on plates (Tang et al. \u003cspan citationid=\"CR63\" class=\"CitationRef\"\u003e2009\u003c/span\u003e). Then if salt stress in the environment is exacerbated, the survival of the fungus is bound to suffer. The contested point we need to focus on is if mycorrhizas will offer any protection to plants when they are exposed to moderate and high salt concentrations. Also, maintaining mycorrhizas is costly to the plant since the fungi drain the plant from its carbon resources. The question is, if the plants are severely stressed with salt, will the fungus be still helpful or will it become a burden for the plant weakened by the salt.\u003c/p\u003e \u003cp\u003eIn the present study, we examined effects of the EMF \u003cem\u003eL\u003c/em\u003e. \u003cem\u003ebicolor\u003c/em\u003e on the responses of Japanese emperor oak (\u003cem\u003eQuercus dentata\u003c/em\u003e) to relatively moderate (0.4% soil DW) and high (0.8% soil DW) NaCl concentrations. \u003cem\u003eQ\u003c/em\u003e. \u003cem\u003edentata\u003c/em\u003e has been reported to have high genetic differentiation and diversity (Zhou et al. \u003cspan citationid=\"CR75\" class=\"CitationRef\"\u003e2022\u003c/span\u003e) as well as superior salt tolerance even at the seedling stage (Hao et al. \u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e2020\u003c/span\u003e). The trees of this species are broadly distributed across temperate zone of the northern hemisphere (Sork et al. \u003cspan citationid=\"CR60\" class=\"CitationRef\"\u003e2022\u003c/span\u003e), and form symbiotic relationships with a wide range of EMF (Arai et al. \u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e2017\u003c/span\u003e). \u003cem\u003eQ\u003c/em\u003e. \u003cem\u003edentata\u003c/em\u003e has been widely used for vegetation restoration in fragile environments, especially in temperate regions (Qiu et al. \u003cspan citationid=\"CR55\" class=\"CitationRef\"\u003e2023\u003c/span\u003e). Since EMF vary in their effectiveness in enhancing salt tolerance to trees, the choice of an appropriate fungal species for mycorrhization of \u003cem\u003eQ. dentata\u003c/em\u003e could help with improving the restoration efforts of areas affected by salinity. As the first sequenced ectomycorrhizal fungus (Martin et al. \u003cspan citationid=\"CR45\" class=\"CitationRef\"\u003e2008\u003c/span\u003e), L. \u003cem\u003ebicolor\u003c/em\u003e forms ectomycorrhizal associations with a wide range of host tree species and has been successfully used to improve salinity tolerance in multiple host plants (Bois et al. \u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e2006\u003c/span\u003ea; Polanco et al. \u003cspan citationid=\"CR53\" class=\"CitationRef\"\u003e2008\u003c/span\u003e; Xu et al. \u003cspan citationid=\"CR72\" class=\"CitationRef\"\u003e2015\u003c/span\u003e). \u003cem\u003eL. bicolor\u003c/em\u003e, therefore, can form symbioses with multiple host plants including oaks (Shi et al. \u003cspan citationid=\"CR58\" class=\"CitationRef\"\u003e2017\u003c/span\u003e). However, most of the studies examined the effects of mycorrhization on plant responses to relatively low salinity levels and it is presently little known how effective the ectomycorrhizal fungi are in enhancing salt tolerance in plants facing the salinity levels in the soils that are classified as saline. We, therefore, carried out experiments with \u003cem\u003eQ. dentata\u003c/em\u003e seedlings that were either non-inoculated or inoculated with the ectomycorrhizal fungus \u003cem\u003eL. bicolor\u003c/em\u003e and exposed the seedlings to relatively moderate and high soil NaCl levels. We hypothesized that an inoculation of \u003cem\u003eQ. dentata\u003c/em\u003e seedlings with \u003cem\u003eL. bicolor\u003c/em\u003e will improve the salt tolerance of seedlings under both relatively moderate and high soil salinity conditions, but the effectiveness of the mycorrhization will be more pronounced under the lower level of soil salinity.\u003c/p\u003e"},{"header":"Materials and methods","content":"\u003cdiv id=\"Sec3\" class=\"Section2\"\u003e \u003ch2\u003eSeed collection and germination\u003c/h2\u003e \u003cp\u003e \u003cem\u003eQ. dentata\u003c/em\u003e acorns were collected from Zhongshan Park in Tsingtao, Shandong province, China and stored at 4℃. Large, healthy acorns were selected and soaked in 5% H\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e for 5 minutes, washed with water, and germinated in sand in a greenhouse (70% light transmittance, 25.1℃ average temperature, 80.4% average relative humidity during the experimental period). The growth substrate was composed of equal volumes of river sand and organic cultivation soil that had been sterilized twice using high-pressure steam (121℃, 20 min). After germination, the seedlings with a radicle of approximately the same length (2\u0026ndash;3 cm) were selected and individually transferred into the separate pots (24 \u0026times; 26 cm (diameter \u0026times; height) filled with 5 kg soil) and watered daily with distilled water to sustain vigorous growth. The whole experiment had been performed under the cited greenhouse conditions.\u003c/p\u003e \u003c/div\u003e\n\u003ch3\u003eFungal culture and inoculation\u003c/h3\u003e\n\u003cp\u003eThe fungal strain \u003cem\u003eL\u003c/em\u003e. \u003cem\u003ebicolor\u003c/em\u003e UAMH8232 provided by the University of Alberta, Microfungus Collection and Herbarium, Edmonton, Canada., was cultured with modified Melin-Norkran\u0026rsquo;s medium (MMN medium) (Bois et al. \u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e2006\u003c/span\u003ea). After 2 weeks of cultivation in a shaking incubator (25℃, 120 rpm), the filtered and homogenized mycelia were diluted with distilled water to 0.5 mg dry mycelia/ml (Polanco et al. \u003cspan citationid=\"CR53\" class=\"CitationRef\"\u003e2008\u003c/span\u003e) to make an inoculum.\u003c/p\u003e \u003cp\u003eOne week after germination, the potted plants were divided into two equal groups. The mycorrhizal group was inoculated with L. \u003cem\u003ebicolor\u003c/em\u003e by adding 10 mL of mycelial suspension to the soil (\u003cem\u003eL.b\u003c/em\u003e). The soil of the second group was injected with 10 mL distilled water to serve as the control check (CK). For the inoculation, four holes were made in the soil near the plants and the inoculum (2.5 mL) was added to each hole. The inoculation treatments were repeated every other week for the total of four times.\u003c/p\u003e\n\u003ch3\u003eNaCl treatments\u003c/h3\u003e\n\u003cp\u003eNaCl treatments started two months after the fourth root mycorrhizal inoculation. Referring to the classification criteria of saline soils in China, soil salinity over 0.6% is classified as saline soil (Wang et al. 1993). Therefore, in addition to the NaCl control (0% NaCl), we subjected the seedlings to two NaCl soil concentrations below (0.4% NaCl, moderate salinity treatment) and above (0.8% NaCl, high salinity treatment) this level. To avoid seedling shock response, salt solution was added gradually to the soil (the soil salinity was incrementally adjusted by adding NaCl solution eight times to ultimately achieve the predetermined salinity level). Overwatering and spilling were avoided during watering over the one month of treatment duration to ensure that the NaCl did not leach from the soil. There were ten replicated seedlings in each treatment group (one seedling per pot), for the total of sixty seedlings (two inoculation treatments \u0026times; three salinity levels \u0026times; ten replicates) in the experiment.\u003c/p\u003e\n\u003ch3\u003eMycorrhizal colonization rate determination\u003c/h3\u003e\n\u003cp\u003eAfter one month of NaCl treatments, three seedlings were randomly selected from each treatment group. The first-order fine roots were carefully excised, washed with slow running water, and cut into ~\u0026thinsp;1 cm long root segments. 20 root segments were randomly selected from each seedling to examine for the presence of fungal hyphae and mycorrhizal structures. The roots were stained (Phillips and Hayman \u003cspan citationid=\"CR52\" class=\"CitationRef\"\u003e1970\u003c/span\u003e) and examined under the light microscope. The proportion of mycorrhizal root segments in the total number of observed roots was considered as the mycorrhizal colonization rate.\u003c/p\u003e\n\u003ch3\u003ePlant measurements\u003c/h3\u003e\n\u003cp\u003eAt the end of the NaCl treatments, plant heights (H) and base stem diameters (BD) were measured. Plant height was measured between the base of the plant and the shoot tip and base stem diameter was measured with a Vernier caliper 1 cm above the soil surface.\u003c/p\u003e \u003cp\u003eBefore harvest, mature and fully expanded functional leaves were selected for instantaneous measurements of gas exchange parameters using a portable gas exchange measurement system (Li-6800, Li-Cor, Lincoln, NE, USA). Measurements were taken between 9:00 and 12:00 h on a sunny day. Net photosynthetic rate (A), transpiration rate (E), and stomatal conductance (g\u003csub\u003es\u003c/sub\u003e) were measured, and instantaneous water use efficiency (iWUE) was calculated as the ratio of A to E. Leaf chamber parameters were maintained at: leaf temperature, 28℃; relative air humidity, 70%; light intensity, 1000 \u0026micro;mol\u0026middot;m\u003csup\u003e\u0026minus;\u0026thinsp;2\u003c/sup\u003e\u0026middot;s\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e; and CO\u003csub\u003e2\u003c/sub\u003e concentration, 400 \u0026micro;mol\u0026middot;mol\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e.\u003c/p\u003e \u003cp\u003eChlorophyll fluorescence parameters and P700 were measured simultaneously using a portable chlorophyll fluorometer (Dual-PAM-100, Walz, Effeltrich, Germany) set to dual-channel mode. After allowing leaves to adapt to the dark for 30 minutes, original fluorescence (Fo) was measured, then a saturating pulse (20000 \u0026micro;mol∙m\u003csup\u003e\u0026minus;\u0026thinsp;2\u003c/sup\u003e\u0026middot;s\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e) was performed to obtain the maximum fluorescence in the dark-adapted state (Fm) and the maximum absorption change in the dark-adapted state (Pm). After that actinic light (400 \u0026micro;mol\u0026middot;m\u003csup\u003e\u0026minus;\u0026thinsp;2\u003c/sup\u003e\u0026middot;s\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e) was turned on to drive photosynthesis for 40 s, following which, a saturation pulse was performed to record the maximum fluorescence intensity of PSII (Fm') and the maximum absorption change of PSI (Pm'). The steady-state fluorescence intensity (Ft) of PSII and steady-state absorption changes of PSI were recorded under continuous background light. Next, maximum quantum yield of PS II (Fv/Fm), photochemical quenching (qP), electron transport rate (ETR) and actual quantum yield (Y) of photosystem Ⅰ and II were calculated according to the following formula (Lu et al. 1999; Maxwell and Johnson, \u003cspan citationid=\"CR46\" class=\"CitationRef\"\u003e2000\u003c/span\u003e):\u003c/p\u003e \u003cp\u003e \u003cspan class=\"InlineEquation\"\u003e \u003cspan class=\"mathinline\"\u003e\\(F_{O}^{\\prime }=1/(1/{F_O} - 1/{F_M}+1/F_{M}^{\\prime })\\)\u003c/span\u003e \u003c/span\u003e \u003c/p\u003e \u003cp\u003e \u003cspan class=\"InlineEquation\"\u003e \u003cspan class=\"mathinline\"\u003e\\({F_V}/{F_M}=({F_M} - {F_O})/{F_M}\\)\u003c/span\u003e \u003c/span\u003e \u003c/p\u003e \u003cp\u003e \u003cspan class=\"InlineEquation\"\u003e \u003cspan class=\"mathinline\"\u003e\\(qP=(F_{M}^{\\prime } - Ft)/(F_{M}^{\\prime } - F_{O}^{\\prime })\\)\u003c/span\u003e \u003c/span\u003e \u003c/p\u003e \u003cp\u003e \u003cspan class=\"InlineEquation\"\u003e \u003cspan class=\"mathinline\"\u003e\\(Y(II)=1 - Y(NPQ) - Y(NO)=(F_{M}^{\\prime } - F)/F_{M}^{\\prime }\\)\u003c/span\u003e \u003c/span\u003e \u003c/p\u003e \u003cp\u003e \u003cspan class=\"InlineEquation\"\u003e \u003cspan class=\"mathinline\"\u003e\\(Y(I)=1 - Y(ND) - Y(NA)\\)\u003c/span\u003e \u003c/span\u003e \u003c/p\u003e \u003cp\u003e \u003cspan class=\"InlineEquation\"\u003e \u003cspan class=\"mathinline\"\u003e\\(ETR(I)=0.5 \\times Y(I) \\times 0.84 \\times PAR\\)\u003c/span\u003e \u003c/span\u003e \u003c/p\u003e \u003cp\u003e \u003cspan class=\"InlineEquation\"\u003e \u003cspan class=\"mathinline\"\u003e\\(ETR(II)=0.5 \\times Y(II) \\times 0.84 \\times PAR\\)\u003c/span\u003e \u003c/span\u003e \u003c/p\u003e \u003cp\u003eWhere Y(NPQ) and Y(NO) correspond to the quantum yield of regulatory and non-regulatory energy dissipation, they can be obtained by the following formula.\u003c/p\u003e \u003cp\u003e \u003cspan class=\"InlineEquation\"\u003e \u003cspan class=\"mathinline\"\u003e\\(Y(NPQ)=\\frac{F}{{F_{M}^{\\prime }}} - \\frac{F}{{{F_M}}}\\)\u003c/span\u003e \u003c/span\u003e \u003c/p\u003e \u003cp\u003e \u003cspan class=\"InlineEquation\"\u003e \u003cspan class=\"mathinline\"\u003e\\(Y(NO)=\\frac{F}{{{F_M}}}\\)\u003c/span\u003e \u003c/span\u003e \u003c/p\u003e \u003cp\u003eAnd Y(ND) corresponds to the fraction of P700 that is already oxidized by actinic light; Y(NA) corresponds to the fraction of P700 that are closed owing to acceptor side limitation, they can be obtained by the following formula.\u003c/p\u003e \u003cp\u003e \u003cspan class=\"InlineEquation\"\u003e \u003cspan class=\"mathinline\"\u003e\\(Y(ND)=\\frac{{P{{700}_{OX}}}}{{{P_M}}}\\)\u003c/span\u003e \u003c/span\u003e \u003c/p\u003e \u003cp\u003e \u003cspan class=\"InlineEquation\"\u003e \u003cspan class=\"mathinline\"\u003e\\(Y(NA)=\\frac{{{P_M}}}{{P_{M}^{\\prime }}}\\)\u003c/span\u003e \u003c/span\u003e \u003c/p\u003e \u003cp\u003e0.5 is the fraction of absorbed light reaching PSI or PSII; and PAR is photosynthetically active radiation, we take 0.84 of the PAR as absorbed irradiance.\u003c/p\u003e \u003cp\u003eThe leaf blades were collected as above for chlorophyll concentration measurements. The leaf samples were weighed to determine fresh weights and soaked in 10 mL 95% ethanol in the dark until chlorophyll was fully leached out. The absorbance value of the extract was then determined by an ultraviolet-visible spectrophotometer (UV-9000s, Metash, Shanghai, China) at wavelengths of 649 and 665 nm, with 95% ethanol used as a blank control. The chlorophyll content was calculated using the formula (Lichtenthaler et al. 1983) as follows.\u003c/p\u003e \u003cp\u003e \u003cspan class=\"InlineEquation\"\u003e \u003cspan class=\"mathinline\"\u003e\\({\\text{Chlorophyll a concentration (Chl a)}}={\\text{13}}{\\text{.95}} \\times {\\text{D665}} - {\\text{6}}{\\text{.88}} \\times {\\text{D649}}\\)\u003c/span\u003e \u003c/span\u003e \u003c/p\u003e \u003cp\u003e \u003cspan class=\"InlineEquation\"\u003e \u003cspan class=\"mathinline\"\u003e\\({\\text{Chlorophyll b concentration (Chl b)}}={\\text{24}}{\\text{.96}} \\times {\\text{D649}} - {\\text{7}}{\\text{.32}} \\times {\\text{D665}}\\)\u003c/span\u003e \u003c/span\u003e \u003c/p\u003e \u003cp\u003e \u003cspan class=\"InlineEquation\"\u003e \u003cspan class=\"mathinline\"\u003e\\({\\text{Chlorophyll content}}=\\frac{{{\\text{Chl}} \\times {{\\text{V}}_{{\\text{leach liquor}}}}}}{{{\\text{fresh weight}}}}\\)\u003c/span\u003e \u003c/span\u003e \u003c/p\u003e \u003cp\u003e \u003cspan class=\"InlineEquation\"\u003e \u003cspan class=\"mathinline\"\u003e\\({\\text{Total chlorophyll content (Chl t)}}={\\text{Chl a content}}+{\\text{Chl b content}}\\)\u003c/span\u003e \u003c/span\u003e \u003c/p\u003e \u003cp\u003eWhere D649 and D665 correspond to the absorbance value of the extract measured at 649 and 665 nm wavelengths, respectively.\u003c/p\u003e \u003cp\u003eHarvested plant material was sorted into leaves, stems, and roots. Leaf fresh weight was recorded and the leaf area of 10 mature and fully unfolded leaves was measured using a scanner (LiDE120, Canon, Tokyo, Japan). Leaf dry weight was measured after drying and used to calculate specific leaf area (SLA; leaf area/leaf dry weight) and leaf water content (LWC; (leaf fresh weight-leaf dry weight)/leaf dry weight). We present these two indicators together, rather than separately, in the growth indicators section.\u003c/p\u003e \u003cp\u003eAll plant fractions were heat treated at 105℃ for 30 min and oven-dried at 65℃ for 48 h, after which dry weight was collected for each portion. Seedling biomass allocation was assessed as the proportion of each part to total biomass and included root mass ratio (RMR), stem mass ratio (SMR), and leaf mass ratio (LMR). The root-shoot ratio (R/S) was calculated as the ratio of below- and above-ground plant dry weight.\u003c/p\u003e \u003cp\u003eNext, all dried leaf, stem, and root tissues were powdered and packaged. 0.2 g from each fraction was digested by mixing with 30 mL HClO\u003csub\u003e4\u003c/sub\u003e and HNO\u003csub\u003e3\u003c/sub\u003e solution (v/v\u0026thinsp;=\u0026thinsp;1:5) and heating to 70℃ for 10 min, followed by 40 min at 153℃ and 70 min at 190℃. After smoke in digestion tubes dispersed and the liquid had cooled, the transparent liquid was transferred to a 100 mL volumetric flask and diluted to volume with deionized water. CsCl solution was added to determine the concentration of sodium and potassium ions, and LaCl\u003csub\u003e3\u003c/sub\u003e solution was to determine concentrations of calcium and magnesium ions. Ion concentrations were measured using an atomic absorption spectrophotometer (AA-7000, Shimadzu, Kyoto, Japan) and calculated according to corresponding dilution ratios.\u003c/p\u003e \u003cp\u003eNitrogen content was determined for each fraction using the Kjeldahl method. 0.2 g of each fraction was mixed with catalyst catalyst (CuSO\u003csub\u003e4\u003c/sub\u003e, 0.2 g; K\u003csub\u003e2\u003c/sub\u003eSO\u003csub\u003e4\u003c/sub\u003e, 3 g) and 8 mL concentrated sulfuric acid, after which it was digested by heating to 200℃ for 40min followed by 400℃ for 40min. The liquid was transferred to a 100 mL volumetric flask and diluted with deionized water to volume. 10 mL of the liquid under test was taken to determined total nitrogen content of each part of the plant organs by a Kjeldahl apparatus (K9860, Hanon, Jinan, China). And another 5 mL liquid under test was transferred to a volumetric flask (50 mL) and diluted to volume in order to determine the phosphorus content using a spectrophotometer (UV-9000s, Metash, Shanghai, China) by ascorbic acid-molybdophosphate blue method.\u003c/p\u003e \u003cdiv id=\"Sec8\" class=\"Section2\"\u003e \u003ch2\u003eStatistical analysis\u003c/h2\u003e \u003cp\u003eNormality test and variance homogeneity test were performed on all data before testing analysis. The effects of fungal inoculation and salt treatment on plant physiological and growth indexes of \u003cem\u003eQ. dentata\u003c/em\u003e seedlings were analyzed by two-way analysis of variance (ANOVA) using SPSS 25.0 (SPSS Inc., Chicago, USA). The responses of seedlings to different treatment combinations were analyzed by one-way analysis of variance (ANOVA). Before ANOVA analysis, data were tested for normality and homogeneity of variance first, and were log- or sqrt- transformed when necessary. Multiple comparisons were performed with chi-square test, and the significance levels were set at α\u0026thinsp;=\u0026thinsp;0.05. There were five to six biological replicates for each measurement. Plotting was performed using Origin 2019 (OriginLab Co., MA, USA).\u003c/p\u003e \u003c/div\u003e"},{"header":"Results","content":"\u003ch3\u003e\u003cstrong\u003eInoculation and mycorrhiza formation\u003c/strong\u003e\u003c/h3\u003e\n\u003cp\u003eThere were no ectomycorrhizal structures or hyphae detected in the control group (CK). The root colonization rates were similar in the NaCl control and 0.4% NaCl treatment. However, the root colonization rate was drastically reduced by the 0.8 NaCl treatment (Table 1).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eTable 1.\u003c/strong\u003e Root colonization of \u003cem\u003eQ. dentata\u003c/em\u003e seedlings in control group (CK) and inoculated seedlings (\u003cem\u003eL.b\u003c/em\u003e) subjected to treatments with different NaCl concentrations. Different letters indicate significant differences between treatments (\u003cem\u003ep\u003c/em\u003e \u0026lt; 0.05, n = 3) determined by chi-square test.\u003c/p\u003e\n\u003cdiv align=\"center\"\u003e\n \u003ctable border=\"1\" cellspacing=\"0\" cellpadding=\"0\" width=\"100%\"\u003e\n \u003ctbody\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 34px;\"\u003e\n \u003cp\u003e\u0026nbsp;Treatment\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 29px;\"\u003e\n \u003cp\u003eSoil salinity\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 36px;\"\u003e\n \u003cp\u003eRoot colonization rate (%)\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd rowspan=\"3\" style=\"width: 34px;\"\u003e\n \u003cp\u003eCK\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 29px;\"\u003e\n \u003cp\u003e0\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 36px;\"\u003e\n \u003cp\u003e0\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 29px;\"\u003e\n \u003cp\u003e0.4% NaCl\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 36px;\"\u003e\n \u003cp\u003e0\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 29px;\"\u003e\n \u003cp\u003e0.8% NaCl\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 36px;\"\u003e\n \u003cp\u003e0\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd rowspan=\"3\" style=\"width: 34px;\"\u003e\n \u003cp\u003eL. b\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 29px;\"\u003e\n \u003cp\u003e0\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 36px;\"\u003e\n \u003cp\u003e48 \u0026plusmn; 8 a \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 29px;\"\u003e\n \u003cp\u003e0.4% NaCl\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 36px;\"\u003e\n \u003cp\u003e50 \u0026plusmn; 5 a\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 29px;\"\u003e\n \u003cp\u003e0.8% NaCl\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 36px;\"\u003e\n \u003cp\u003e22 \u0026plusmn; 3 b\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003c/tbody\u003e\n \u003c/table\u003e\n\u003c/div\u003e\n\u003ch3\u003e\u003cstrong\u003eEffects of \u003cem\u003eL. bicolor\u003c/em\u003e inoculation on growth indicators under salt stress\u003c/strong\u003e\u003c/h3\u003e\n\u003cp\u003ePrior to multiple comparisons, the effects of fungal inoculation, salt treatment, and interactions between the two was assessed using two-way ANOVA (Table S1). Multiple comparison revealed that there was no significant difference in H and BD of fungus-inoculated and non-inoculated seedlings under different salt stress conditions (Fig. 1 a; b). The leaf water content of both inoculated and CK seedlings under salt stress was significantly lower than that of CK without salt stress, and leaf water content decreased by more than 50% under high salt stress (Fig. 1 c). Compared to CK, leaf water content of inoculated seedlings was significantly lower under high salt stress, but was not significantly different compared to other salt treatments (Fig. 1 c). SLA increased with increasing salinity in both inoculated and non-inoculated seedlings. SLA was higher in the inoculated group compared to the non-inoculated group, but this difference was only significant under moderate salt stress conditions (Fig. 1 d).\u003c/p\u003e\n\u003cp\u003eGenerally, root, stem, leaf, and total plant biomass decreased with increasing salinity in both inoculated and non-inoculated seedlings. Inoculation significantly increased root biomass in CK seedlings under CK and moderate salt stress by 44.5% and 27.8%, respectively, compared to non-inoculated seedlings. There was no significant difference in root biomass between inoculated and CK seedlings under high salt stress (Fig. 2 a). Inoculation did not affect stem or leaf biomass under different salt stress treatments (Fig. 2 b; c). Total seedling biomass increased by 29.8% in the inoculated group under no salt stress (Fig. 2 d). Increased biomass in the inoculated group under no salt stress was mainly due to differences in root biomass.\u003c/p\u003e\n\u003cp\u003eRMR decreased in both control and inoculated seedlings under salt stress. Inoculation increased RMR significantly, but only under no salt stress (Fig. 3 a). Neither inoculation nor NaCl treatment had a significant impact on seedling SMR (Fig. 3 b). In contrast to RMR, LMR was significantly higher under salt stress, but inoculation did not have an effect, regardless of salt stress treatment (Fig. 3 c). R/S followed the same trend as RMR, and inoculation significantly increased the root-shoot ratio of seedlings, but only those in the no salt stress group (Fig. 3 d).\u003c/p\u003e\n\u003ch3\u003e\u003cstrong\u003eEffects of \u003cem\u003eL. bicolor\u003c/em\u003e on physiological indicators under salt stress\u003c/strong\u003e\u003c/h3\u003e\n\u003cp\u003eTwo-way ANOVA revealed that inoculation did not significantly affect gas exchange parameters in seedlings (Table S1), while salt stress significantly affected most gas exchange parameters (Fig. 4 a ;b; d). Our findings suggest that plant physiological activity was increasingly restricted as salinity increased. We also noted that iWUE did not exhibit the same degree of change with increasing salinity as other gas exchange parameters (Fig. 4 c). Inoculation reduced stomatal conductance only in the no salt stress group (Fig. 4 d).\u003c/p\u003e\n\u003cp\u003eSalt treatment significantly reduced chlorophyll a and total chlorophyll content. Both increased in inoculated seedlings, but this increase was significant only in the moderate salt stress treatment group (Fig. 5 a; c). Under salt stress, chlorophyll b content of inoculated seedlings was slightly higher than that of the non-inoculated control group, but the difference was not significant (Fig. 5 b). Furthermore, inoculation did not have a significant effect on seedlings\u0026rsquo; chlorophyll a/b under different levels of salt stress. Chlorophyll a/b decreased with increasing soil salinity in both inoculated and control groups (Fig. 5 d).\u003c/p\u003e\n\u003cp\u003eETR and Y varied similarly. ETR and Y of PS II were not significantly affected by inoculation, but gradually decreased with increasing salinity (Fig. 6 c; d). ETR(I) and Y(I) both declined with increasing salinity, but the decline was exacerbated by inoculation under high-salt conditions (Fig. 6 a; b). Whereas Fv/Fm and qP declined only under high salt stress and, an effect that was exacerbated by inoculation, there were no significant changes in no-salt and low-salt environments (Fig. 6 e; f).\u003c/p\u003e\n\u003cp\u003eWith increasing soil salinity, Na\u003csup\u003e+\u003c/sup\u003e content of all seedling organs increased significantly. Inoculation did not significantly affect root and stem Na\u003csup\u003e+\u003c/sup\u003e content; compared to CK, however, leaf Na\u003csup\u003e+\u003c/sup\u003e content was significantly lower under moderate salt stress and significantly higher under high salt stress conditions (Fig. 7 a).\u003c/p\u003e\n\u003cp\u003eSalt stress did not have a significant effect on root K\u003csup\u003e+\u003c/sup\u003e content; stem K\u003csup\u003e+\u003c/sup\u003e decreased with increasing salinity, while leaf K\u003csup\u003e+\u003c/sup\u003e content increased (Fig. 7 b). Fungal inoculation did not affect root or stem K\u003csup\u003e+\u003c/sup\u003e content, but increased the leaf K\u003csup\u003e+\u003c/sup\u003e content significantly under moderate salt stress conditions (Fig. 7 b). Na\u003csup\u003e+\u003c/sup\u003e/K\u003csup\u003e+\u003c/sup\u003e ratios increased with increasing salinity in all organs, and fungal inoculation lowered leaf Na\u003csup\u003e+\u003c/sup\u003e/K\u003csup\u003e+\u003c/sup\u003e ratios significantly only in low-salt environments (Fig. 7 e). There was no significant difference in root, stem, or leaf Ca\u003csup\u003e2+\u003c/sup\u003e and Mg\u003csup\u003e2+\u003c/sup\u003econtent between inoculated and control seedlings under different salt treatments. Stem Ca\u003csup\u003e2+\u003c/sup\u003e only increased significantly in inoculated seedlings (Fig. 7 c; d).\u003c/p\u003e\n\u003cp\u003eExcept for root N concentration, which increased in CK seedlings but decreased in inoculated seedlings in high salt environments, neither salt treatment nor fungal inoculation had a significant effect on plant organ N (Fig. 8 a; c; e). Plant organ P content did not change significantly in response to NaCl treatment or fungal inoculation (Fig. 8 b; d; f).\u003c/p\u003e"},{"header":"Discussion","content":"\u003cp\u003e \u003cb\u003eResponse of\u003c/b\u003e \u003cb\u003eQ. dentata\u003c/b\u003e \u003cb\u003eseedlings to NaCl treatment\u003c/b\u003e\u003c/p\u003e \u003cp\u003eThe NaCl treatment appeared to have a greater impact on \u003cem\u003eQ. dentata\u003c/em\u003e seedlings than inoculation with \u003cem\u003eL. bicolor\u003c/em\u003e (Table S1), both in terms of growth and physiological indicators. Most parameters change monotonically with increasing salinity. Results also show that one-year old \u003cem\u003eQ. dentata\u003c/em\u003e seedlings were resistant to relatively low salt stress and that increasing concentrations of NaCl exacerbated negative effects on seedling growth (Fig.\u0026nbsp;\u003cspan refid=\"Fig9\" class=\"InternalRef\"\u003e9\u003c/span\u003e). This is consistent with previous work reporting salt tolerance in oaks, even at the seedling stage (Wang et al. \u003cspan citationid=\"CR69\" class=\"CitationRef\"\u003e2014\u003c/span\u003e; Gugliuzza et al. \u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e2023\u003c/span\u003e). Other research found that \u003cem\u003eQ. dentata\u003c/em\u003e is also resistant to NaCl stress within a certain range (Hao et al. \u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e2020\u003c/span\u003e). Whether they were inoculated or not, seedlings were able to survive in saline environments (0-0.8%), exhibiting intermediate and considerable NaCl tolerance compared with salt-tolerant species planted in afforestation projects, such as \u003cem\u003eQ. virginiana\u003c/em\u003e, \u003cem\u003eQ. acutissima\u003c/em\u003e (Wang et al. \u003cspan citationid=\"CR69\" class=\"CitationRef\"\u003e2014\u003c/span\u003e), and \u003cem\u003eRobinia pseudoacacia\u003c/em\u003e (Mao et al. \u003cspan citationid=\"CR44\" class=\"CitationRef\"\u003e2016\u003c/span\u003e). Our findings showing that \u003cem\u003eQ. dentata\u003c/em\u003e has a certain degree of salt tolerance is promising for saline-alkaline land restoration.\u003c/p\u003e \u003cp\u003eWith increasing salinity (0.4%), the negative effects of NaCl on seedling growth caused a decrease in plant biomass (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003e). Seedlings altered nutrient and biomass distribution ratios by increasing leaf biomass ratio and by decreasing root biomass ratio and root to shoot ratio (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003e) to reduce salt uptake and upward transport by roots and to prioritize resource allocation to leaves for photosynthesis. This pattern of biomass distribution has also been observed in \u003cem\u003eCeriops tagal\u003c/em\u003e (Patel et al. \u003cspan citationid=\"CR51\" class=\"CitationRef\"\u003e2010\u003c/span\u003e), a mangrove halophyte. An opposite, upward trend has been reported in \u003cem\u003ePicea glauca\u003c/em\u003e and \u003cem\u003ePinus banksiana\u003c/em\u003e (Bois et al. \u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e2006\u003c/span\u003ea), even in individuals inoculated with \u003cem\u003eL. bicolor\u003c/em\u003e. As soil salt content increases further (0.8%), sodium ion toxicity occurs, plant water-salt balance is disrupted, leaf tissue water loss is more serious, and leaves wither or senesce (Horie et al. \u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e2009\u003c/span\u003e; Hammer et al. \u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e2011\u003c/span\u003e; Heng et al. \u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e2022\u003c/span\u003e). As a result, osmotic stress and ionic toxicity become stronger.\u003c/p\u003e \u003cp\u003eTotal chlorophyll content decreased significantly with increasing salt stress (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003ec). This could be caused by two reasons. On the one hand, ion toxicity resulting from excess Na\u003csup\u003e+\u003c/sup\u003e restricts binding between chlorophyll and pigment proteins and destroys chloroplast structures (\u003cem\u003ee.g\u003c/em\u003e., thylakoid membranes), causing chloroplast damage and dysfunction and reducing chlorophyll synthesis (Pardo et al. 2010), on the other hand, our analysis revealed equivalent changes between chlorophyll a and total chlorophyll content, with both decreasing as salt stress intensified (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003e). NaCl could have increased the activity of chlorophyllase and accelerated the degradation of chlorophyll a under the action of reactive oxygen species (Li et al. \u003cspan citationid=\"CR35\" class=\"CitationRef\"\u003e2015\u003c/span\u003e). The greater sensitivity to salt stress of chlorophyll a relative to chlorophyll b in seedlings is reflected in lower ratios of chlorophyll a/b (Akram, \u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e2014\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eIn addition, plant photosynthetic capacity was lower under NaCl treatment. This was reflected not only in lower chlorophyll content, but also in stomatal limitation and altered electron transport (Van Zelm et al. \u003cspan citationid=\"CR67\" class=\"CitationRef\"\u003e2020\u003c/span\u003e). Gas exchange parameters, such as net photosynthetic rate, transpiration rate, and stomatal conductance, decreased significantly, decreased under all salt stress treatments (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003e), indicating that seedling photosynthesis was sensitive to increasing soil salt (Sudhir et al. 2004). The decrease in photosynthetic efficiency under salt stress can be attributed to stomatal limitation occurring because of decreased stomatal conductance (Bose et al. \u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e2017\u003c/span\u003e), which could be caused by decreased turgor pressure in guard cells resulting from water deficits in leaf tissues (Akram, \u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e2014\u003c/span\u003e; Tombesi et al. \u003cspan citationid=\"CR65\" class=\"CitationRef\"\u003e2015\u003c/span\u003e). Salt stress can also reduce chlorophyll thylakoid membrane stacking and stability, restricting electron flow from PS II to PS I and blocking photosynthesis (Zahra et al. \u003cspan citationid=\"CR74\" class=\"CitationRef\"\u003e2022\u003c/span\u003e). Here, ETR and Y of both PS I and PS II decreased with increasing salt concentration (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003e), suggesting that salt stress attenuates the absorption and conversion efficiency of light energy by leaves, inhibits electron transfer in photosystem reaction centers, and weakens photosynthesis (Gururani et al. \u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e2015\u003c/span\u003e). Declines in qP and Fv/Fm were not significant under relatively low salt conditions, indicating that PS II has a certain salt tolerance for the conversion capacity of photosynthesis. However, declines were significant under high salt conditions, while the photodamage occurring under saline conditions results in poor PS II function (Hameed et al. 2021).\u003c/p\u003e \u003cp\u003e \u003cb\u003ePositive effects of\u003c/b\u003e \u003cb\u003eL. bicolor\u003c/b\u003e \u003cb\u003eon\u003c/b\u003e \u003cb\u003eQ. dentata\u003c/b\u003e \u003cb\u003eseedlings under moderate salinity\u003c/b\u003e\u003c/p\u003e \u003cp\u003eEMF partner with many woody plants and usually maintain reciprocal symbiosis (Genre et al. \u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e2020\u003c/span\u003e). Plants provide fungi with a portion of the carbohydrate fixed by photosynthesis and fungi help the plant absorb water and nutrients in return, improving resilience (Bahadur et al. \u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e2019\u003c/span\u003e). Here, inoculation worked in \u003cem\u003eQ. dentata\u003c/em\u003e seedlings and played a beneficial role in non-hypersaline environments.\u003c/p\u003e \u003cp\u003eWe found that inoculation under salt-free conditions significantly increased root biomass of \u003cem\u003eQ. dentata\u003c/em\u003e seedlings, resulting in higher total biomass for the inoculated group compared to CK. Root-shoot ratio was also significantly higher among inoculated seedlings, indicating that colonization by EMF promotes root growth in \u003cem\u003eQ. dentata\u003c/em\u003e seedlings and increases the proportion of resources allocated belowground, enhancing plant uptake and storage of water and nutrients (Wang et al. \u003cspan citationid=\"CR68\" class=\"CitationRef\"\u003e2008\u003c/span\u003e). In addition, we also found that symbiosis with \u003cem\u003eL. bicolor\u003c/em\u003e improves seedling salt tolerance under moderate salinity. Under relatively low NaCl stress, \u003cem\u003eL. bicolor\u003c/em\u003e induced host plants to expand their roots (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003ea) to alleviate osmotic stress caused and to supply leaves with adequate water (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003ec), representing a trade-off between its own water absorption capacity and the osmotic pressure caused by salt stress. Likewise, ion toxicity to plant leaves decreased proportionally with decreasing sodium-potassium ratios in relation to sodium ion concentrations (Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003ea; e), which resulted from increased water supply to leaves. As a result, the total chlorophyll content of \u003cem\u003eQ. dentata\u003c/em\u003e seedlings increased with increasing chlorophyll a content under relatively low salt stress (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003ea; c) to mitigate the degradative effects of salt stress (Al-Khaliel et al. 2010). This is consistent with previous research and is probably due to the fact that the symbiotic fungus secretes growth regulators such as cytokinins, thereby increasing leaf chlorophyll content (Tilak et al. \u003cspan citationid=\"CR64\" class=\"CitationRef\"\u003e2006\u003c/span\u003e). Our findings suggest that symbiosis with \u003cem\u003eL. bicolor\u003c/em\u003e has a positive effect on \u003cem\u003eQ. dentata\u003c/em\u003e seedling growth and can promote seedling growth under moderate salt stress.\u003c/p\u003e \u003cp\u003e \u003cb\u003eNegative effects of\u003c/b\u003e \u003cb\u003eL. bicolor\u003c/b\u003e \u003cb\u003eon\u003c/b\u003e \u003cb\u003eQ. dentata\u003c/b\u003e \u003cb\u003eseedlings under high salinity\u003c/b\u003e\u003c/p\u003e \u003cp\u003eSymbiotic relationships between mycorrhizal fungi and host plants are not unbreakable (Douglas et al. 2008), and cooperation becomes uneven when resource exchange is diminished or stops in response to environmental stress (Bonfante et al. 2010; Kiers et al. \u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e2011\u003c/span\u003e). As soil salinity gradually exceeds the normal tolerance range of plants, the relationship between symbiotic mycorrhiza and host plant becomes more subtle (Johnson et al. \u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e1997\u003c/span\u003e; Kaldorf et al. \u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e2005\u003c/span\u003e). The effects of EMF inoculation on various indicators like tissue biomass, gas exchange parameters, chlorophyll content, chlorophyll fluorescence parameters, and sodium-potassium ratio were weaker under high salt stress. Regardless, mycorrhizal association did not provide any benefit to host plants when seedlings were unable to withstand the damage caused by salt stress (Fig.\u0026nbsp;\u003cspan refid=\"Fig9\" class=\"InternalRef\"\u003e9\u003c/span\u003e). At the same time, we found that moderate NaCl promotes the growth of \u003cem\u003eL. bicolor\u003c/em\u003e mycelium, the colony diameters on the seventh day were 33.3\u0026thinsp;\u0026plusmn;\u0026thinsp;0.7 mm, 45.7\u0026thinsp;\u0026plusmn;\u0026thinsp;1.2 mm, and 44.9\u0026thinsp;\u0026plusmn;\u0026thinsp;2.1 mm at 0, 0.4%, and 0.8% medium salinity, respectively (Table S2), consistent with previous research demonstrating the salt tolerance of \u003cem\u003eL. bicolor\u003c/em\u003e (Bois et al. \u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e2006\u003c/span\u003eb). As salinity increases, the ability of the fungus to generate its own growth as well as its vigour to colonise and live in symbiosis with the host plant decreases, in particular mycorrhizal colonisation rates are significantly lower under high salt conditions than they are under no-salt and medium-salt conditions (Table\u0026nbsp;\u003cspan refid=\"Tab1\" class=\"InternalRef\"\u003e1\u003c/span\u003e). The high salinity environment could raise the threshold of fungal competitiveness to host plants, \u003cem\u003eL. bicolor\u003c/em\u003e that colonise \u003cem\u003eQ. dentata\u003c/em\u003e seedlings not only become less numerous, but also play a negative role in plant growth.\u003c/p\u003e \u003cp\u003eIn relatively harsh environments, where fungus is restricted in its own growth and its protective effect on plants from NaCl is lost, the limited available resources made by the seedlings face intense competition from both the fungus and the host plant, and the fungus will rob the survival resources from the plants thereby inhibiting there growth. Thus, host plants are unable to meet mycorrhizal demand for resources, which remain high under saline conditions, and exacerbated nutrient and water stress reduce root nitrogen (Fig.\u0026nbsp;\u003cspan refid=\"Fig8\" class=\"InternalRef\"\u003e8\u003c/span\u003ea) and leaf water content (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003ec). As the main photosynthetic organ, leaves accumulate sodium when water is lost (Flowers et al. \u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e2015\u003c/span\u003e) and experience worsening ion toxicity (Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003ea). Chlorophyll fluorescence parameters, which are sensitive to ion penetration and water-salt balance, also decrease (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003e).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eNotably, our results suggest that leaf Na\u003csup\u003e+\u003c/sup\u003e content is a sensitive and critical indicator of fungal colonization under different salt stress conditions. That is, when plant growth is limited primarily by environmental salt stress, the physiological changes induced by a plant\u0026rsquo;s association with symbiotic mycorrhizal fungi are most obvious in leaf Na\u003csup\u003e+\u003c/sup\u003e content (Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003e). Inoculation with \u003cem\u003eL. bicolor\u003c/em\u003e reduces photosystem efficiency in highly saline soil. Moreover, the effect of salt stress on photosynthetic efficiency is most apparent in changes in ETR and Y of photosystem I, which are more sensitive to the effects of fungal inoculation than those of photosystem II (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003e). Severe salinity decreases PSII stability and donor side intactness (Zahra et al. \u003cspan citationid=\"CR74\" class=\"CitationRef\"\u003e2022\u003c/span\u003e) which were maintained under relatively low salt stress (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003ee; f). Fungal inoculation further jeopardizes the stability of the photosynthetic apparatus under high salt conditions (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003e). These results suggest that symbiosis with \u003cem\u003eL. bicolor\u003c/em\u003e can help \u003cem\u003eQ. dentata\u003c/em\u003e seedlings maintain photosynthesis and manage oxidative stress under moderate salt stress, but that it fails under high salt stress. Indicators such as chlorophyll fluorescence parameters and metal ion content, therefore, should be monitored when mycorrhizal symbiosis is part of afforestation strategies in saline-alkali soil.\u003c/p\u003e \u003cdiv id=\"Sec12\" class=\"Section2\"\u003e \u003ch2\u003eImplications for afforestation activities in saline areas\u003c/h2\u003e \u003cp\u003eMany oak species are tolerant to salt stress and have potential applications in land afforestation in saline-alkali soil (Li et al. \u003cspan citationid=\"CR35\" class=\"CitationRef\"\u003e2015\u003c/span\u003e; Gugliuzza et al. \u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e2023\u003c/span\u003e). \u003cem\u003eQ. dentata\u003c/em\u003e, which acts as a pioneer during naturalized developmental vegetation restoration (Qiu et al. \u003cspan citationid=\"CR55\" class=\"CitationRef\"\u003e2023\u003c/span\u003e), exhibits somewhat improved salt tolerance when associated with EMF (Fig.\u0026nbsp;\u003cspan refid=\"Fig9\" class=\"InternalRef\"\u003e9\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eMycorrhizal fungi have a strong regulatory effect on root exudation and nitrogen uptake in temperate tree species (Liese et al. \u003cspan citationid=\"CR39\" class=\"CitationRef\"\u003e2018\u003c/span\u003e). Afforestation studies suggest that EMF symbiosis during silviculture can enhance carbon sequestration and improve soil properties (Hong et al. \u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e2018\u003c/span\u003e; Yuan et al. \u003cspan citationid=\"CR73\" class=\"CitationRef\"\u003e2023\u003c/span\u003e). Although it is possible that afforestation, combined with carefully selected EMF symbionts, can improve saline-alkali soil (Huang et al. \u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e2008\u003c/span\u003e; Qin et al. \u003cspan citationid=\"CR54\" class=\"CitationRef\"\u003e2017\u003c/span\u003e), the mechanisms underpinning these improvements and the potential use of EMF, including \u003cem\u003eL. bicolor\u003c/em\u003e, are still poorly understood.\u003c/p\u003e \u003cp\u003eIt appears that in this study, \u003cem\u003eL. bicolor\u003c/em\u003e was effective only under the moderate salinity level because the fungus itself could not tolerate higher salt concentration as shown by a reduction in root colonization under the high salt concentration (Table\u0026nbsp;\u003cspan refid=\"Tab1\" class=\"InternalRef\"\u003e1\u003c/span\u003e). It\u0026rsquo;s proved that the survival and mycelial extension of \u003cem\u003eL. bicolor\u003c/em\u003e cultured in vitro were not significantly affected under higher salt concentration (Table S2). It\u0026rsquo; a fact that \u003cem\u003eL. bicolor\u003c/em\u003e is one kind of mycorrhizal fungi with strong saline-alkali tolerance (Kernaghan et al. \u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e2002\u003c/span\u003e) and even certain salinity could promote the proliferation of fungi in vitro (Table S2). while the higher salinity level might restrict the inoculation of EMF as shown by less colonization density on the same individual (Table\u0026nbsp;\u003cspan refid=\"Tab1\" class=\"InternalRef\"\u003e1\u003c/span\u003e). Because of the decline in co-benefits, the probability of \u003cem\u003eL. bicolor\u003c/em\u003e to refuse to inoculate plants increase under high salinity concentration. Therefore, the effectiveness of the fungus in conferring salt tolerance to plants can be only up to the salt concentration that the fungus can tolerate itself. It is the salt concentration in vitro that may not only be too high for the mycorrhizal fungi combined with plants in afforestation to be survival, but also determines the activeness and effectiveness in conferring salt protection to plants. Therefore, it is necessary to consider the effect of fungal cooperative afforestation according to the requirements and adaptability of applied fungi to the soil environment. Furthermore, a deeper understanding is needed of the long-term effects of salinity on \u003cem\u003eQ. dentata\u003c/em\u003e-\u003cem\u003eL. bicolor\u003c/em\u003e symbionts and the underlying mechanisms.\u003c/p\u003e \u003c/div\u003e"},{"header":"Conclusions","content":"\u003cp\u003eIn our study, mycorrhization with \u003cem\u003eL. bicolor\u003c/em\u003e had either positive or negative impact on plant responses to NaCl depending on the soil salt. Here, this association helped plants cope with salt stress by promoting root growth, increasing light capture area, promoting chlorophyll accumulation, and regulating ion balance under moderate salt stress. In contrast, EMF association under high salt stress exacerbated nutrient stress, water stress, and Na\u003csup\u003e+\u003c/sup\u003e toxicity, leading to a decrease in plant photosynthetic activity, rate, and yield. Therefore, mycorrhizal seedlings inoculated with EMF can be considered for vegetation restoration in mildly saline soil, which supports \u003cem\u003eQuercus\u003c/em\u003e growth. However, when the soil salinity is high beyond a certain range, it is necessary to fully consider not only the salt tolerance of \u003cem\u003eQuercus\u003c/em\u003e themselves, but also the survival of symbiotic fungi in saline environment and the actual effect of mycorrhizal symbiosis, under which mycorrhizal means may not be applicable. Collectively, afforestation with \u003cem\u003eQ. dentata\u003c/em\u003e seedlings combined with symbiosis with \u003cem\u003eL. bicolor\u003c/em\u003e is a reasonable choice with broad application prospects. However, additional research investigating the genetic and molecular mechanisms driving enhanced salt tolerance is needed to maximize the benefits of the interaction between \u003cem\u003eL. bicolor\u003c/em\u003e and \u003cem\u003eQ. dentata\u003c/em\u003e.\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003e \u003ch2\u003eCompeting Interests\u003c/h2\u003e \u003cp\u003eThe authors declare they have no relevant financial or non-financial interests to disclose.\u003c/p\u003e \u003c/p\u003e\u003ch2\u003eFunding\u003c/h2\u003e \u003cp\u003eThis study was funded by National Natural Science Foundation of China (No. 32471580), Natural Science Foundation of Shandong Province, China (No. ZR2020MC035; ZR2024MC012), Shandong Province \"Double-Hundred Talent Plan\" Project (No. WSG2018023), Seed Funding for International Scientific Research Cooperation of Shandong University (No. WSG2018023) and Key Research and Development Program of Shandong Province, China(No. 2021CXGC010803).\u003c/p\u003e\u003ch2\u003eAuthor Contribution\u003c/h2\u003e\u003cp\u003eAll authors contributed to the study conception and design. Material preparation, data collection and analysis were performed by Wenlong Sun, Haonan Chen, Yixin Song and Luyu Qi. The first draft of the manuscript was written by Wenlong Sun and all authors commented on previous versions of the manuscript. All authors read and approved the final manuscript.\u003c/p\u003e\u003ch2\u003eAcknowledgement\u003c/h2\u003e\u003cp\u003eWe thank Chengjia Zhang and Nannan Dong of the Core Facilities for Life and Environmental Sciences, State Key laboratory of Microbial Technology of Shandong University for metal ion assay and analysis.\u003c/p\u003e\u003ch2\u003eData availability\u003c/h2\u003e \u003cp\u003eData will be made available from the corresponding author on reasonable request.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\n\u003cli\u003eAkram M (2014) Effects of nitrogen application on chlorophyll content, water relations, and yield of maize hybrids under saline conditions. 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Sci Rep 5(1): 12449. https://doi.org/10.1038/srep12449\u003c/li\u003e\n\u003cli\u003eUllah S, Dahlawi S, Naeem A, Rengel Z, Naidu R (2017) Biochar application for the remediation of salt-affected soils: Challenges and opportunities. Sci Total Environ 625: 320-335. https://doi.org/10.1016/j.scitotenv.2017.12.257\u003c/li\u003e\n\u003cli\u003eVan Zelm EV, Zhang YX, Testerink C (2020) Salt tolerance mechanisms of plants. Annu Rev Plant Biol 71(1): 403-433. https://doi.org/10.1146/annurev-arplant-050718-100005\u003c/li\u003e\n\u003cli\u003eWang RZ, Chen L, Bai YG, Xiao CW (2008) Seasonal dynamics in resource partitioning to growth and storage in response to drought in a perennial rhizomatous grass, \u003cem\u003eLeymus chinensis\u003c/em\u003e. J Plant Growth Regul 27(01): 39-48. https://doi.org/10.1007/s00344-007-9029-0\u003c/li\u003e\n\u003cli\u003eWang SF, Hu YX, Sun HJ, Shi X, Pan HW, Chen YT (2014) Effects of salt stress on growth and root development of two oak seedlings. 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New Phytol 205(02): 757-770. https://doi.org/10.1111/nph.13098\u003c/li\u003e\n\u003cli\u003eYuan CX, Wu FZ, Peng Y, Wu QQ, Zhu GQ, Zhao ZM, Wang YQ, An NN, Ni XY, Yue K (2023) Pain or gain: the dual role of afforestation effects on soil pH at the global scale. Plant Soil 493: 617-628. https://doi.org/10.1007/s11104-023-06254-6\u003c/li\u003e\n\u003cli\u003eZahra N, Al Hinai MS, Hafeez MB, Rehman A, Wahid A, Siddique KHM, Farooq M (2022) Regulation of photosynthesis under salt stress and associated tolerance mechanisms. Plant Physiol Bioch 178: 55-69. https://doi.org/10.1016/j.plaphy.2022.03.003\u003c/li\u003e\n\u003cli\u003eZhou BF, Shi Y, Chen XY, Yuan S, Liang YY, Wang BS (2022) Linked selection, ancient polymorphism, and ecological adaptation shape the genomic landscape of divergence in \u003cem\u003eQuercus dentata\u003c/em\u003e. J Syst Evol 60(6): 1344-1357. https://doi.org/10.1111/jse.12817\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":false,"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":"ectomycorrhiza, Laccaria bicolor, phytoremediation, Quercus dentata, salt tolerance","lastPublishedDoi":"10.21203/rs.3.rs-5474395/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-5474395/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003ch2\u003eBackground\u003c/h2\u003e \u003cp\u003eSoil salinization is a growing global concern in many ecosystems. Although ectomycorrhizal fungi have been shown to alleviate the effects of salinity in some tree species, uncertainties persist concerning their effectiveness when plants are exposed to different salinity levels that are commonly present in salt-affected soils.\u003c/p\u003e\u003ch2\u003eObjective\u003c/h2\u003e \u003cp\u003eThis study explored the effects of \u003cem\u003eLaccaria bicolor\u003c/em\u003e on \u003cem\u003eQuercus dentata\u003c/em\u003e seedlings under different levels of salt stress.\u003c/p\u003e\u003ch2\u003eMethods\u003c/h2\u003e \u003cp\u003e \u003cem\u003eQ. dentata\u003c/em\u003e seedlings were grown in pots and were either non-inoculated (mycorrhizal control) or inoculated with the ectomycorrhizal fungus \u003cem\u003eL. bicolor\u003c/em\u003e. The seedlings were then treated with three NaCl concentrations (0, 0.4%, and 0.8%), that was added to the soil for 30 days, after which plant physiological, stoichiometric, and growth characteristics were examined.\u003c/p\u003e\u003ch2\u003eResults\u003c/h2\u003e \u003cp\u003eNaCl exposure significantly impaired growth and physiological parameters in all seedling groups. The influence of \u003cem\u003eL. bicolor\u003c/em\u003e on \u003cem\u003eQ. dentata\u003c/em\u003e seedlings varied with salt concentration. Under moderate salinity, the seedlings colonized by \u003cem\u003eL. bicolor\u003c/em\u003e exhibited an enhancement in root biomass and leaf chlorophyll concentrations, concomitant with a reduction in leaf Na\u003csup\u003e+\u003c/sup\u003e concentrations and the Na\u003csup\u003e+\u003c/sup\u003e/K\u003csup\u003e+\u003c/sup\u003e ratios. Conversely, under relatively high salinity, colonization of \u003cem\u003eL. bicolor\u003c/em\u003e was associated with a reduction in leaf water content and fluorescence parameters, as well as an elevation in leaf Na\u003csup\u003e+\u003c/sup\u003e concentrations.\u003c/p\u003e\u003ch2\u003eConclusions\u003c/h2\u003e \u003cp\u003eThe effect of ectomycorrhizal fungus \u003cem\u003eL. bicolor\u003c/em\u003e on \u003cem\u003eQ. dentata\u003c/em\u003e seedlings was dependent on NaCl concentration, and our results indicate that the use of \u003cem\u003eL. bicolor\u003c/em\u003e in afforestation efforts with \u003cem\u003eQ. dentata\u003c/em\u003e would only be effective under relatively low soil salinity levels.\u003c/p\u003e","manuscriptTitle":"Mycorrhization of Quercus dentata seedlings with Laccaria bicolor enhances salt tolerance of plants only under relatively moderate soil salinity level","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2024-12-03 12:35:40","doi":"10.21203/rs.3.rs-5474395/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":"1ae3a51c-9d2c-4c81-916a-28449928c4a2","owner":[],"postedDate":"December 3rd, 2024","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"posted","subjectAreas":[],"tags":[],"updatedAt":"2024-12-20T15:38:42+00:00","versionOfRecord":[],"versionCreatedAt":"2024-12-03 12:35:40","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-5474395","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-5474395","identity":"rs-5474395","version":["v1"]},"buildId":"qtupq5eGEP_6zYnWcrvyt","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}